The Social Amoeba Polysphondylium pallidum Loses Encystation and Sporulation, but Can Still Erect Fruiting Bodies in the Absence of Cellulose

PubMed Central

Du, Qingyou; Schaap, Pauline

2014-01-01

Amoebas and other freely moving protists differentiate into walled cysts when exposed to stress. As cysts, amoeba pathogens are resistant to biocides, preventing treatment and eradication. Lack of gene modification procedures has left the mechanisms of encystation largely unexplored. Genetically tractable Dictyostelium discoideum amoebas require cellulose synthase for formation of multicellular fructifications with cellulose-rich stalk and spore cells. Amoebas of its distant relative Polysphondylium pallidum (Ppal), can additionally encyst individually in response to stress. Ppal has two cellulose synthase genes, DcsA and DcsB, which we deleted individually and in combination. Dcsa- mutants formed fruiting bodies with normal stalks, but their spore and cyst walls lacked cellulose, which obliterated stress-resistance of spores and rendered cysts entirely non-viable. A dcsa-/dcsb- mutant made no walled spores, stalk cells or cysts, although simple fruiting structures were formed with a droplet of amoeboid cells resting on an sheathed column of decaying cells. DcsB is expressed in prestalk and stalk cells, while DcsA is additionally expressed in spores and cysts. We conclude that cellulose is essential for encystation and that cellulose synthase may be a suitable target for drugs to prevent encystation and render amoeba pathogens susceptible to conventional antibiotics. PMID:25113829

Xeroderma Pigmentosum Group A Promotes Autophagy to Facilitate Cisplatin Resistance in Melanoma Cells through the Activation of PARP1.

PubMed

Ge, Rui; Liu, Lin; Dai, Wei; Zhang, Weigang; Yang, Yuqi; Wang, Huina; Shi, Qiong; Guo, Sen; Yi, Xiuli; Wang, Gang; Gao, Tianwen; Luan, Qi; Li, Chunying

2016-06-01

Xeroderma pigmentosum group A (XPA), a key protein in the nucleotide excision repair pathway, has been shown to promote the resistance of tumor cells to chemotherapeutic drugs by facilitating the DNA repair process. However, the role of XPA in the resistance of melanoma to platinum-based drugs like cisplatin is largely unknown. In this study, we initially found that XPA was expressed at higher levels in cisplatin-resistant melanoma cells than in cisplatin-sensitive ones. Furthermore, the knockdown of XPA not only increased cellular apoptosis but also inhibited cisplatin-induced autophagy, which rendered the melanoma cells more sensitive to cisplatin. Moreover, we discovered that the increased XPA in resistant melanoma cells promoted poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) activation and that the inhibition of PARP1 could attenuate the cisplatin-induced autophagy. Finally, we proved that the inhibition of PARP1 and the autophagy process made resistant melanoma cells more susceptible to cisplatin treatment. Our study shows that XPA can promote cell-protective autophagy in a DNA repair-independent manner by enhancing the activation of PARP1 in melanoma cells resistant to cisplatin and that the XPA-PARP1-mediated autophagy process can be targeted to overcome cisplatin resistance in melanoma chemotherapy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin-resistant diabetes.

PubMed Central

Accili, D; Frapier, C; Mosthaf, L; McKeon, C; Elbein, S C; Permutt, M A; Ramos, E; Lander, E; Ullrich, A; Taylor, S I

1989-01-01

Insulin binds to a receptor on the cell surface, thereby triggering a biological response within the target cell. Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. We have studied a family in which two sisters have a genetic form of insulin-resistant diabetes mellitus. The technique of homozygosity mapping has been used to demonstrate that the mutation causing diabetes in this consanguineous family is genetically linked to the insulin receptor gene. The two insulin-resistant sisters are homozygous for a mutation encoding substitution of valine for phenylalanine at position 382 in the alpha-subunit of the insulin receptor. Transfection of mutant insulin receptor cDNA into NIH3T3 cells demonstrated that the Val382 mutation impaired post-translational processing and retarded transport of the insulin receptor to the plasma membrane. Thus, the mutation causes insulin resistance by decreasing the number of insulin receptors on the surface of the patients' cells. Images PMID:2573522

Preclinical Evaluation of MET Inhibitor INC-280 With or Without the Epidermal Growth Factor Receptor Inhibitor Erlotinib in Non–Small-Cell Lung Cancer

PubMed Central

Lara, Matthew S.; Holland, William S.; Chinn, Danielle; Burich, Rebekah A.; Lara, Primo N.; Gandara, David R.; Kelly, Karen; Mack, Philip C.

2018-01-01

The MET inhibitor INC-280 restored sensitivity to erlotinib and promoted apoptosis in non–small-cell lung cancer models rendered resistant to erlotinib by hepatocyte growth factor. Background Although the epidermal growth factor receptor (EGFR) inhibitor erlotinib is initially effective in non–small-cell lung cancer (NSCLC) patients with tumors harboring activating mutations of EGFR, most subsequently develop acquired resistance. One recognized resistance mechanism occurs through activation of bypass signaling via the hepatocyte growth factor (HGF)-MET pathway. INC-280 is a small molecule kinase inhibitor of MET. We sought to demonstrate the activity of INC-280 on select NSCLC cell lines both as a single agent and in combination with erlotinib using exogenous HGF to simulate MET up-regulation. Methods Four NSCLC cell lines (HCC827, PC9, H1666, and H358) were treated with either single-agent INC-280 or in combination with erlotinib with or without HGF. The activity of the drug treatments was measured by cell viability assays. Immunoblotting was used to monitor expression of EGFR/pEGFR, MET/pMET, GAB1/pGAB1, AKT/pAKT, and ERK/pERK as well as markers of apoptosis (PARP and capase-3 cleavage) in H1666, HCC827, and PC9. Results As a single agent, INC-280 showed minimal cytotoxicity despite potent inhibition of MET kinase activity at concentrations as low as 10 nM. Addition of HGF prevented erlotinib-induced cell death. The addition of INC280 to HGF-mediated erlotinib-resistant models restored erlotinib sensitivity for all cell lines tested, associated with cleavage of both PARP and caspase-3. In these models, INC-280 treatment was sufficient to restore erlotinib-induced inhibition of MET, GAB1, AKT, and ERK in the presence of HGF. Conclusion Although the MET inhibitor INC-280 alone had no discernible effect on cell growth, it was able to restore sensitivity to erlotinib and promote apoptosis in NSCLC models rendered erlotinib resistant by HGF. These data provide a preclinical rationale for an ongoing phase 1 clinical trial of erlotinib plus INC-280 in EGFR-mutated NSCLC. PMID:28038979

C/EBPβ LIP augments cell death by inducing osteoglycin.

PubMed

Wassermann-Dozorets, Rina; Rubinstein, Menachem

2017-04-06

Many types of tumor cell are devoid of the extracellular matrix proteoglycan osteoglycin (Ogn), but its role in tumor biology is poorly studied. Here we show that RNAi of Ogn attenuates stress-triggered cell death, whereas its overexpression increases cell death. We found that the transcription factor C/EBPβ regulates the expression of Ogn. C/EBPβ is expressed as a full-length, active form (LAP) and as a truncated, dominant-negative form (LIP), and the LIP/LAP ratio is positively correlated with the extent of cell death under stress. For example, we reported that drug-resistant tumor cells lack LIP altogether, and its supplementation abolished their resistance to chemotherapy and to endoplasmic reticulum (ER) stress. Here we further show that elevated LIP/LAP ratio robustly increased Ogn expression and cell death under stress by modulating the mitogen-activated protein kinase/activator protein 1 pathway (MAPK/AP-1). Our findings suggest that LIP deficiency renders tumor cell resistant to ER stress by preventing the induction of Ogn.

Enhancing the potency and specificity of engineered T cells for cancer treatment.

PubMed

Sukumaran, Sujita; Watanabe, Norihiro; Bajgain, Pradip; Raja, Kanchana; Mohammed, Somala; Fisher, William E; Brenner, Malcolm K; Leen, Ann M; Vera, Juan F

2018-06-07

The adoptive transfer of chimeric antigen receptor (CAR)-modified T cells has produced tumor responses even in patients with refractory diseases. However, the paucity of antigens that are tumor selective has resulted, on occasion, in "on-target, off-tumor" toxicities. To address this issue, we developed an approach to render T cells responsive to an expression pattern present exclusively at the tumor by using a trio of novel chimeric receptors. Using pancreatic cancer as a model, we demonstrate how T cells engineered with receptors that recognize PSCA, TGFβ, and IL4, and whose endodomains recapitulate physiologic T cell signaling by providing signals for activation, co-stimulation and cytokine support, produce potent anti-tumor effects selectively at the tumor site. In addition, this strategy has the benefit of rendering our cells resistant to otherwise immunosuppressive cytokines (TGFβ and IL4) and can be readily extended to other inhibitory molecules present at the tumor site (e.g. PD-L1, IL10, IL13). Copyright ©2018, American Association for Cancer Research.

Stem cell transplantation in the context of HIV--how can we cure HIV infection?

PubMed

Bauer, Gerhard; Anderson, Joseph S

2014-01-01

All HIV target cells are derived from hematopoietic stem cells. More than two decades ago, a hypothesis was postulated that a cure for HIV may be possible by performing a transplant with HIV-resistant hematopoietic stem cells that would allow for an HIV-resistant immune system to arise. HIV-resistant stem cells could be generated by genetically modifying them with gene therapy vectors transferring anti-HIV genes. First attempts of stem cell gene therapy for HIV were carried out in the USA in the 1990s demonstrating safety, but also little efficacy at that time. The first demonstration that the postulated hypothesis was correct was the cure of an HIV-infected individual in Berlin in 2009 who received an allogeneic bone marrow transplant from a donor who lacked the CCR5 chemokine receptor, a naturally arising mutation rendering HIV target cells resistant to infection with macrophage tropic strains of HIV. In 2013, reports were published about a possible cure of HIV-infected individuals who received allogeneic bone marrow transplants with cells not resistant to HIV. We will review these stem cell transplant procedures and discuss their utility to provide a cure for HIV infection, including efficacious future stem cell gene therapy applications.

AXL mediates resistance to cetuximab therapy

PubMed Central

Brand, Toni M.; Iida, Mari; Stein, Andrew P.; Corrigan, Kelsey L.; Braverman, Cara; Luthar, Neha; Toulany, Mahmoud; Gill, Parkash S.; Salgia, Ravi; Kimple, Randall J.; Wheeler, Deric L.

2014-01-01

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical problem. In this study we show that overexpression of the oncogenic receptor kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated and tightly associated with EGFR expression in cells resistant to cetuximab (CtxR cells). Using RNAi methods and novel AXL targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation and MAPK signaling in CtxR cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in CtxR cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft assays, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL targeting drugs to treat cetuximab-resistant cancers. PMID:25136066

Biophysical model of bacterial cell interactions with nanopatterned cicada wing surfaces.

PubMed

Pogodin, Sergey; Hasan, Jafar; Baulin, Vladimir A; Webb, Hayden K; Truong, Vi Khanh; Phong Nguyen, The Hong; Boshkovikj, Veselin; Fluke, Christopher J; Watson, Gregory S; Watson, Jolanta A; Crawford, Russell J; Ivanova, Elena P

2013-02-19

The nanopattern on the surface of Clanger cicada (Psaltoda claripennis) wings represents the first example of a new class of biomaterials that can kill bacteria on contact based solely on their physical surface structure. The wings provide a model for the development of novel functional surfaces that possess an increased resistance to bacterial contamination and infection. We propose a biophysical model of the interactions between bacterial cells and cicada wing surface structures, and show that mechanical properties, in particular cell rigidity, are key factors in determining bacterial resistance/sensitivity to the bactericidal nature of the wing surface. We confirmed this experimentally by decreasing the rigidity of surface-resistant strains through microwave irradiation of the cells, which renders them susceptible to the wing effects. Our findings demonstrate the potential benefits of incorporating cicada wing nanopatterns into the design of antibacterial nanomaterials. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A Novel SND1-BRAF Fusion Confers Resistance to c-Met Inhibitor PF-04217903 in GTL16 Cells though MAPK Activation

PubMed Central

Lee, Nathan V.; Lira, Maruja E.; Pavlicek, Adam; Ye, Jingjing; Buckman, Dana; Bagrodia, Shubha; Srinivasa, Sreesha P.; Zhao, Yongjun; Aparicio, Samuel; Rejto, Paul A.; Christensen, James G.; Ching, Keith A.

2012-01-01

Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling. PMID:22745804

ZitB (YbgR), a Member of the Cation Diffusion Facilitator Family, Is an Additional Zinc Transporter in Escherichia coli

PubMed Central

Grass, Gregor; Fan, Bin; Rosen, Barry P.; Franke, Sylvia; Nies, Dietrich H.; Rensing, Christopher

2001-01-01

The Escherichia coli zitB gene encodes a Zn(II) transporter belonging to the cation diffusion facilitator family. ZitB is specifically induced by zinc. ZitB expression on a plasmid rendered zntA-disrupted E. coli cells more resistant to zinc, and the cells exhibited reduced accumulation of 65Zn, suggesting ZitB-mediated efflux of zinc. PMID:11443104

AXL mediates resistance to cetuximab therapy.

PubMed

Brand, Toni M; Iida, Mari; Stein, Andrew P; Corrigan, Kelsey L; Braverman, Cara M; Luthar, Neha; Toulany, Mahmoud; Gill, Parkash S; Salgia, Ravi; Kimple, Randall J; Wheeler, Deric L

2014-09-15

The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical outcome. In this study, we show that overexpression of the oncogenic receptor tyrosine kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated, and tightly associated with EGFR expression in cells resistant to cetuximab (Ctx(R) cells). Using RNAi methods and novel AXL-targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation, and MAPK signaling in Ctx(R) cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in Ctx(R) cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft models, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL-targeting drugs to treat cetuximab-resistant cancers. Cancer Res; 74(18); 5152-64. ©2014 AACR. ©2014 American Association for Cancer Research.

Activation of RAS family members confers resistance to ROS1 targeting drugs

PubMed Central

Cargnelutti, Marilisa; Corso, Simona; Pergolizzi, Margherita; Mévellec, Laurence; Aisner, Dara L.; Dziadziuszko, Rafal; Varella-Garcia, Marileila; Comoglio, Paolo M.; Doebele, Robert C.; Vialard, Jorge; Giordano, Silvia

2015-01-01

The ROS1 tyrosine kinase is activated in lung cancer as a consequence of chromosomal rearrangement. Although high response rates and disease control have been observed in lung cancer patients bearing rearranged ROS1 tumors (ROS1+) treated with the kinase inhibitor crizotinib, many of these patients eventually relapse. To identify mechanisms of resistance to ROS1 inhibitors we generated resistant cells from HCC78 lung cancer cells bearing the SLC34A2-ROS1 rearrangement. We found that activation of the RAS pathway in the HCC78 cell model, due to either KRAS/NRAS mutations or to KRAS amplification, rendered the cells resistant to ROS1 inhibition. These cells were cross-resistant to different ROS1 inhibitors, but sensitive to inhibitors of the RAS signaling pathway. Interestingly, we identified focal KRAS amplification in a biopsy of a tumor from a patient that had become resistant to crizotinib treatment. Altogether our data suggest that the activation of members of the RAS family can confer resistance to ROS1 inhibitors. This has important clinical implications as: (i) RAS genetic alterations in ROS1+ primary tumors are likely negative predictors of efficacy for targeted drugs and (ii) this kind of resistance is unlikely to be overcome by the use of more specific or more potent ROS1 targeting drugs. PMID:25691052

Exosomal DNMT1 mediates cisplatin resistance in ovarian cancer.

PubMed

Cao, Ya-Lei; Zhuang, Ting; Xing, Bao-Heng; Li, Na; Li, Qin

2017-08-01

Ovarian cancer is the most common malignancy in women. Owing to late syndromic presentation and lack of efficient early detection, most cases are diagnosed at advanced stages. Surgery and platinum-based chemotherapy are still the standard care currently. However, resistance invoked often compromises the clinical value of the latter. Expression of DNA methyltransferase 1 (DNMT1) was analysed by gene array. Protein was determined by immunoblotting. Exosome was isolated with commercial kit. Cell proliferation was measured by CCK8 method. Annexin V-PI double staining was performed for apoptosis evaluation. Xenograft model was established and administrated with exosome. Tumour growth and overall survival were monitored. We demonstrated the upregulation of DNMT1 in both tumour and derived cell line. DNMT1 transcripts were highly enriched in exosomes from conditioned medium of ovarian cells. Co-incubation with exosomes stimulated endogenous expression and rendered host cell the resistance to cytotoxicity of cisplatin. In vivo administration of DNMT1-containing exosomes exacerbated xenograft progression and reduced overall survival significantly. Moreover, treatment with exosome inhibitor GW4869 almost completely restored sensitivity in resistant cells. Our data elucidated an unappreciated mechanism of exosomal DNMT1 in cisplatin resistance in ovarian cancer, also indicating the potential of the combination of exosome inhibitor with cisplatin in resistant patients. Copyright © 2017 John Wiley & Sons, Ltd.

Mn complex-mediated enhancement of antitumor response through modulating myeloid-derived suppressor cells in drug-resistant tumor.

PubMed

Das, Satyajit; Banerjee, Kaushik; Roy, Susmita; Majumder, Saikat; Chatterjee, Mitali; Majumdar, Subrata; Choudhuri, Soumitra Kumar

2014-01-01

The tumor microenvironment (TME) renders tumor cells more resistant to chemotherapy. However, effective immunomodulators for cancer therapy are still elusive. We hypothesized that Mn-N-(2-hydroxyacetophenone) glycinate (MnNG), reported to be an antitumor agent, can modulate the TME. Immunomodulatory effects of MnNG were performed through assessing Myeloid Derived Suppressor Cells (MDSCs), Interferon-γ (Ifnγ)- and Interleukin-4 (Il4)-secreting Cluster of Differentiation 4 (Cd4)(+) T-cells by annexin V-binding assay in drug-resistant TME and T-cell proliferation following in vitro co-culture assay by flow cytometry. MnNG induced infiltration of Ifnγ-secreting Cd4(+) T-cells and reduces MDSC numbers in vivo. Furthermore, it modulated differentiation of MDSCs towards dendritic cells with up-regulation of co-stimulatory molecules and reversed the suppressive function of MDSC's that enhances T-helper cell 1 (Th1) response. MnNG treatment resulted in reduced expression of IL4, but enhanced expression of Ifnγ when Cd4(+) T-cells were co-cultured with MDSCs. MnNG modulates MDSCs differentiaton towards dendritic cells and enhances Th1 response in drug-resistant TME, leading to immunomodulatory efficacy. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Progesterone resistance in endometriosis: origins, consequences and interventions.

PubMed

Patel, Bansari G; Rudnicki, Martin; Yu, Jie; Shu, Yimin; Taylor, Robert N

2017-06-01

Endometriosis is a common cause of pelvic pain and affects up to 10% of women of reproductive age. Aberrant progesterone signaling in the endometrium plays a significant role in impaired decidualization and establishment of ectopic endometrial implants. Eutopic endometrial cells from women with endometriosis fail to downregulate genes needed for decidualization, such as those involved in cell cycle regulation, leading to unbridled proliferation. Several causes of progesterone resistance in the endometrium have been postulated, including congenital "preconditioning", whereby the in utero environment renders infants susceptible to neonatal uterine bleeding and endometriosis. Progesterone action is crucial to decreasing inflammation in the endometrium, and deviant progesterone signaling results in a proinflammatory phenotype. Conversely, chronic inflammation can induce a progesterone-resistant state. Repetitive retrograde endometrial shedding begets chronic peritoneal inflammation, which further exacerbates progesterone resistance. Genetic causes of progesterone resistance include progesterone receptor gene polymorphisms, altered microRNA expression, and epigenetic modifications to progesterone receptors and their targets. Environmental toxins such as dioxin play a possible role in the genesis of endometriosis by permitting an inflammatory milieu. A consequence of impaired progesterone action is that hormonal therapy is rendered ineffective for a subset of women with endometriosis. Synthetic progestins, such as dienogest, may overcome this phenomenon by increasing progesterone receptor expression and decreasing proinflammatory cytokines. Other modalities include high dose depot formulations of progestins, medicated intrauterine devices and the likely advent of oral GnRH antagonists. Unearthing root causes of progesterone inaction in endometriosis will aid in the development of novel therapeutics geared toward prevention and treatment. © 2017 Nordic Federation of Societies of Obstetrics and Gynecology.

Up-regulation of HOXB cluster genes are epigenetically regulated in tamoxifen-resistant MCF7 breast cancer cells.

PubMed

Yang, Seoyeon; Lee, Ji-Yeon; Hur, Ho; Oh, Ji Hoon; Kim, Myoung Hee

2018-05-28

Tamoxifen (TAM) is commonly used to treat estrogen receptor (ER)-positive breast cancer. Despite the remarkable benefits, resistance to TAM presents a serious therapeutic challenge. Since several HOX transcription factors have been proposed as strong candidates in the development of resistance to TAM therapy in breast cancer, we generated an in vitro model of acquired TAM resistance using ER-positive MCF7 breast cancer cells (MCF7-TAMR), and analyzed the expression pattern and epigenetic states of HOX genes. HOXB cluster genes were uniquely up-regulated in MCF7-TAMR cells. Survival analysis of in slico data showed the correlation of high expression of HOXB genes with poor response to TAM in ER-positive breast cancer patients treated with TAM. Gain- and loss-of-function experiments showed that the overexpression of multi HOXB genes in MCF7 renders cancer cells more resistant to TAM, whereas the knockdown restores TAM sensitivity. Furthermore, activation of HOXB genes in MCF7-TAMR was associated with histone modifications, particularly the gain of H3K9ac. These findings imply that the activation of HOXB genes mediate the development of TAM resistance, and represent a target for development of new strategies to prevent or reverse TAM resistance.

Kaposi sarcoma associated herpesvirus (KSHV) induces AKT hyperphosphorylation, bortezomib-resistance and GLUT-1 plasma membrane exposure in THP-1 monocytic cell line.

PubMed

Gonnella, Roberta; Santarelli, Roberta; Farina, Antonella; Granato, Marisa; D'Orazi, Gabriella; Faggioni, Alberto; Cirone, Mara

2013-10-23

Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway regulates multiple cellular processes such as cell proliferation, evasion from apoptosis, migration, glucose metabolism, protein synthesis and proper differentiation in immune cells. Kaposi sarcoma-associated herpesvirus (KSHV), an oncogenic virus associated with several human malignancies, expresses a variety of latent and lytic proteins able to activate PI3K/AKT pathway, promoting the growth of infected cells and a successful viral infection. We found that KSHV latent infection of THP-1 cells, a human monocytic cell line derived from an acute monocytic leukemia patient, resulted in an increase of AKT phoshorylation, not susceptible to bortezomib-induced dephosphorylation, compared to the mock-infected THP-1. Accordingly, THP-1-infected cells displayed increased resistance to the bortezomib cytotoxic effect in comparison to the uninfected cells, which was counteracted by pre-treatment with AKT-specific inhibitors. Finally, AKT hyperactivation by KSHV infection correlated with plasma membrane exposure of glucose transporter GLUT1, particularly evident during bortezomib treatment. GLUT1 membrane trafficking is a characteristic of malignant cells and underlies a change of glucose metabolism that ensures the survival to highly proliferating cells and render these cells highly dependent on glycolysis. GLUT1 membrane trafficking in KSHV-infected THP-1 cells indeed led to increased sensitivity to cell death induced by the glycolysis inhibitor 2-Deoxy-D-glucose (2DG), further potentiated by its combination with bortezomib. KSHV confers to the THP-1 infected cells an oncogenic potential by altering the phosphorylation, expression and localization of key molecules that control cell survival and metabolism such as AKT and GLUT1. Such modifications in one hand lead to resistance to cell death induced by some chemotherapeutic drugs such as bortezomib, but on the other hand, offer an Achilles heel, rendering the infected cells more sensitive to other treatments such as AKT or glycolysis inhibitors. These therapeutic strategies can be exploited in the anticancer therapy of KSHV-associated malignancies.

Peptides Displayed as High Density Brush Polymers Resist Proteolysis and Retain Bioactivity

PubMed Central

2015-01-01

We describe a strategy for rendering peptides resistant to proteolysis by formulating them as high-density brush polymers. The utility of this approach is demonstrated by polymerizing well-established cell-penetrating peptides (CPPs) and showing that the resulting polymers are not only resistant to proteolysis but also maintain their ability to enter cells. The scope of this design concept is explored by studying the proteolytic resistance of brush polymers composed of peptides that are substrates for either thrombin or a metalloprotease. Finally, we demonstrate that the proteolytic susceptibility of peptide brush polymers can be tuned by adjusting the density of the polymer brush and offer in silico models to rationalize this finding. We contend that this strategy offers a plausible method of preparing peptides for in vivo use, where rapid digestion by proteases has traditionally restricted their utility. PMID:25314576

Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK

PubMed Central

Katayama, Ryohei; Khan, Tahsin M.; Benes, Cyril; Lifshits, Eugene; Ebi, Hiromichi; Rivera, Victor M.; Shakespeare, William C.; Iafrate, A. John; Engelman, Jeffrey A.; Shaw, Alice T.

2011-01-01

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene represents a molecular target in a small subset of non-small cell lung cancers (NSCLCs). This fusion leads to constitutive ALK activation with potent transforming activity. In a pivotal phase 1 clinical trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib (PF-02341066) demonstrated impressive antitumor activity in the majority of patients with NSCLC harboring ALK fusions. However, despite these remarkable initial responses, cancers eventually develop resistance to crizotinib, usually within 1 y, thereby limiting the potential clinical benefit. To determine how cancers acquire resistance to ALK inhibitors, we established a model of acquired resistance to crizotinib by exposing a highly sensitive EML4-ALK–positive NSCLC cell line to increasing doses of crizotinib until resistance emerged. We found that cells resistant to intermediate doses of crizotinib developed amplification of the EML4-ALK gene. Cells resistant to higher doses (1 μM) also developed a gatekeeper mutation, L1196M, within the kinase domain, rendering EML4-ALK insensitive to crizotinib. This gatekeeper mutation was readily detected using a unique and highly sensitive allele-specific PCR assay. Although crizotinib was ineffectual against EML4-ALK harboring the gatekeeper mutation, we observed that two structurally different ALK inhibitors, NVP-TAE684 and AP26113, were highly active against the resistant cancer cells in vitro and in vivo. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitor 17-AAG. Thus, we have developed a model of acquired resistance to ALK inhibitors and have shown that second-generation ALK TKIs or Hsp90 inhibitors are effective in treating crizotinib-resistant tumors harboring secondary gatekeeper mutations. PMID:21502504

Oligosaccharide ligands for NKR-P1 protein activate NK cells and cytotoxicity

NASA Astrophysics Data System (ADS)

Bezouška, Karel; Yuen, Chun-Ting; O'Brien, Jacqui; Childs, Robert A.; Chai, Wengang; Lawson, Alexander M.; Drbal, Karel; Fišerová, Anna; Posíšil, Miloslav; Feizi, Ten

1994-11-01

A diversity of high-affinity Oligosaccharide ligands are identified for NKR-P1, a membrane protein on natural killer (NK) cells which contains an extracellular Ca2+-dependent lectin domain. Interactions of such oligosaccharides on the target cell surface with NKR-P1 on the killer cell surface are crucial both for target cell recognition and for delivery of stimulatory or inhibitory signals linked to the NK cytolytic machinery. NK-resistant tumour cells are rendered susceptible by preincubation with liposomes expressing NKR-P1 ligands, suggesting that purging of tumour or virally infected cells in vivo may be a therapeutic possibility.

BAG3-dependent expression of Mcl-1 confers resistance of mutant KRAS colon cancer cells to the HSP90 inhibitor AUY922.

PubMed

Wang, Chun Yan; Guo, Su Tang; Croft, Amanda; Yan, Xu Guang; Jin, Lei; Zhang, Xu Dong; Jiang, Chen Chen

2018-02-01

Past studies have shown that mutant KRAS colon cancer cells are susceptible to apoptosis induced by the HSP90 inhibitor AUY922. Nevertheless, intrinsic and acquired resistance remains an obstacle for the potential application of the inhibitor in the treatment of the disease. Here we report that Mcl-1 is important for survival of colon cancer cells in the presence of AUY922. Mcl-1 was upregulated in mutant KRAS colon cancer cells selected for resistance to AUY922-induced apoptosis. This was due to its increased stability mediated by Bcl-2-associated athanogene domain 3 (BAG3), which was also increased in resistant colon cancer cells by heat shock factor 1 (HSF1) as a result of chronic endoplasmic reticulum (ER) stress. Functional investigations demonstrated that inhibition of Mcl-1, BAG3, or HSF1 triggered apoptosis in resistant colon cancer cells, and rendered AUY922-naïve colon cancer cells more sensitive to the inhibitor. Together, these results identify that the HSF1-BAG3-Mcl-1 signal axis is critical for protection of mutant KRAS colon cancer cells from AUY922-induced apoptosis, with potential implications for targeting HSF1/BAG3/Mcl-1 to improve the efficacy of AUY922 in the treatment of colon cancer. © 2017 Wiley Periodicals, Inc.

Tcf3 and cell cycle factors contribute to butyrate resistance in colorectal cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chiaro, Christopher, E-mail: cchiaro@tcmedc.org; Lazarova, Darina L., E-mail: dlazarova@tcmedc.org; Bordonaro, Michael, E-mail: mbordonaro@tcmedc.org

2012-11-09

Highlights: Black-Right-Pointing-Pointer We investigate mechanisms responsible for butyrate resistance in colon cancer cells. Black-Right-Pointing-Pointer Tcf3 modulates butyrate's effects on Wnt activity and cell growth in resistant cells. Black-Right-Pointing-Pointer Tcf3 modulation of butyrate's effects differ by cell context. Black-Right-Pointing-Pointer Cell cycle factors are overexpressed in the resistant cells. Black-Right-Pointing-Pointer Reversal of altered gene expression can enhance the anti-cancer effects of butyrate. -- Abstract: Butyrate, a fermentation product of dietary fiber, inhibits clonal growth in colorectal cancer (CRC) cells dependent upon the fold induction of Wnt activity. We have developed a CRC cell line (HCT-R) that, unlike its parental cell line, HCT-116,more » does not respond to butyrate exposure with hyperactivation of Wnt signaling and suppressed clonal growth. PCR array analyses revealed Wnt pathway-related genes, the expression of which differs between butyrate-sensitive HCT-116 CRC cells and their butyrate-resistant HCT-R cell counterparts. We identified overexpression of Tcf3 as being partially responsible for the butyrate-resistant phenotype, as this DNA-binding protein suppresses the hyperinduction of Wnt activity by butyrate. Consequently, Tcf3 knockdown in HCT-R cells restores their sensitivity to the effects of butyrate on Wnt activity and clonal cell growth. Interestingly, the effects of overexpressed Tcf3 differ between HCT-116 and HCT-R cells; thus, in HCT-116 cells Tcf3 suppresses proliferation without rendering the cells resistant to butyrate. In HCT-R cells, however, the overexpression of Tcf3 inhibits Wnt activity, and the cells are still able to proliferate due to the higher expression levels of cell cycle factors, particularly those driving the G{sub 1} to S transition. Knowledge of the molecular mechanisms determining the variable sensitivity of CRC cells to butyrate may assist in developing approaches that prevent or reverse butyrate resistance.« less

NF-κB RelA renders tumor-associated macrophages resistant to and capable of directly suppressing CD8+ T cells for tumor promotion.

PubMed

Li, Liwen; Han, Lei; Sun, Fan; Zhou, Jingjiao; Ohaegbulam, Kim C; Tang, Xudong; Zang, Xingxing; Steinbrecher, Kris A; Qu, Zhaoxia; Xiao, Gutian

2018-01-01

Activation of the inflammatory transcription factor NF-κB in tumor-associated macrophages (TAMs) is assumed to contribute to tumor promotion. However, whether and how NF-κB drives the antitumor macrophages to become pro-tumorigenic have not been determined in any cancer type yet. Similarly, how TAMs repress CD8 + cytotoxic T lymphocytes (CTLs) remains largely unknown, although their importance in regulatory T (Treg) cell regulation and tumor promotion has been well appreciated. Here, using an endogenous lung cancer model we uncover a direct crosstalk between TAMs and CTLs. TAMs suppress CTLs through the T-cell inhibitory molecule B7x (B7-H4/B7S1) in a cell-cell contact manner, whereas CTLs kill TAMs in a tumor antigen-specific manner. Remarkably, TAMs secrete the known T-cell suppressive cytokine interleukin-10 (IL-10) to activate, but not to repress, CTLs. Notably, one major role of cell-intrinsic NF-κB RelA is to drive TAMs to suppress CTLs for tumor promotion. It induces B7x expression in TAMs directly, and restricts IL-10 expression indirectly by repressing expression of the NF-κB cofactor Bcl3 and subsequent Bcl3/NF-κB1-mediated transcription of IL-10. It also renders TAMs resistant to CTLs by up-regulating anti-apoptotic genes. These studies help understand how immunity is shaped in lung tumorigenesis, and suggest a RelA-targeted immunotherapy for this deadliest cancer.

The C-type Lectin Langerin Functions as a Receptor for Attachment and Infectious Entry of Influenza A Virus

PubMed Central

Ng, Wy Ching; Londrigan, Sarah L.; Nasr, Najla; Cunningham, Anthony L.; Turville, Stuart; Brooks, Andrew G.

2015-01-01

ABSTRACT It is well established that influenza A virus (IAV) attachment to and infection of epithelial cells is dependent on sialic acid (SIA) at the cell surface, although the specific receptors that mediate IAV entry have not been defined and multiple receptors may exist. Lec2 Chinese hamster ovary (CHO) cells are SIA deficient and resistant to IAV infection. Here we demonstrate that the expression of the C-type lectin receptor langerin in Lec2 cells (Lec2-Lg) rendered them permissive to IAV infection, as measured by replication of the viral genome, transcription of viral mRNA, and synthesis of viral proteins. Unlike SIA-dependent infection of parental CHO cells, IAV attachment and infection of Lec2-Lg cells was mediated via lectin-mediated recognition of mannose-rich glycans expressed by the viral hemagglutinin glycoprotein. Lec2 cells expressing endocytosis-defective langerin bound IAV efficiently but remained resistant to IAV infection, confirming that internalization via langerin was essential for infectious entry. Langerin-mediated infection of Lec2-Lg cells was pH and dynamin dependent, occurred via clathrin- and caveolin-mediated endocytic pathways, and utilized early (Rab5+) but not late (Rab7+) endosomes. This study is the first to demonstrate that langerin represents an authentic receptor that binds and internalizes IAV to facilitate infection. Moreover, it describes a unique experimental system to probe specific pathways and compartments involved in infectious entry following recognition of IAV by a single cell surface receptor. IMPORTANCE On the surface of host cells, sialic acid (SIA) functions as the major attachment factor for influenza A viruses (IAV). However, few studies have identified specific transmembrane receptors that bind and internalize IAV to facilitate infection. Here we identify human langerin as a transmembrane glycoprotein that can act as an attachment factor and a bone fide endocytic receptor for IAV infection. Expression of langerin by an SIA-deficient cell line resistant to IAV rendered cells permissive to infection. As langerin represented the sole receptor for IAV infection in this system, we have defined the pathways and compartments involved in infectious entry of IAV into cells following recognition by langerin. PMID:26468543

Targeting the cancer initiating cell: the Achilles' heel of cancer.

PubMed

McCubrey, James A; Chappell, William H; Abrams, Stephen L; Franklin, Richard A; Long, Jacquelyn M; Sattler, Jennifer A; Kempf, C Ruth; Laidler, Piotr; Steelman, Linda S

2011-01-01

We have isolated cell with the cancer initiating cell (CIC) phenotype from PC3 cells. The PC3/(CIC) cells are more resistant than the PC3/(BC) cells to chemotherapeutic drugs such as docetaxel which is used to treat prostate cancer. Thus these prostate CICs could lay dormant and persist even after chemotherapeutic drug treatment. Then when the chemotherapeutic drug is removed, they could potentially repopulate the original tumor site or metastize to a distant site. However, the prostate CICs were not significantly more resistant to drugs which target EGFR, NF-κB, Smo and the natural product genistein. Interesting the prostate CICs could be rendered more sensitive to docetaxel by inclusion of suboptimal doses of genistein, cyclopamine, and EGFR inhibitors. In contrast, addition of suboptimal amounts of genistein, cyclopamine, or EGFR inhibitors did not increase the sensitivity of the PC/(BC) cells to docetaxel. Similar results were observed when combination experiments were performed with cyclopamine and suboptimal doses of either genistein or docetaxel. The BC cells are usually more rapidly proliferating than the CICs. Thus the CICs are not as sensitive to docetaxel which targets replication. In contrast, the CICs could be rendered sensitive to docetaxel or cyclopamine by co-treatment with certain other drugs, including the natural product genistein which is present in the human diet of many people, especially Asians. Genistein is by itself only weakly toxic to prostate and other cancer cells. That is probably one of the big reasons that it can be used as a dietary supplement for prostate and breast cancers. It is clear from our studies that low doses of genistein can increase the sensitivity of prostate CICs to drugs such as docetaxel and cyclopamine, two drugs either used or under consideration for prostate cancer therapy.

Apoptosis resistance in epithelial tumors is mediated by tumor-cell-derived interleukin-4.

PubMed

Todaro, M; Lombardo, Y; Francipane, M G; Alea, M Perez; Cammareri, P; Iovino, F; Di Stefano, A B; Di Bernardo, C; Agrusa, A; Condorelli, G; Walczak, H; Stassi, G

2008-04-01

We investigated the mechanisms involved in the resistance to cell death observed in epithelial cancers. Here, we identify that primary epithelial cancer cells from colon, breast and lung carcinomas express high levels of the antiapoptotic proteins PED, cFLIP, Bcl-xL and Bcl-2. These cancer cells produced interleukin-4 (IL-4), which amplified the expression levels of these antiapoptotic proteins and prevented cell death induced upon exposure to TRAIL or other drug agents. IL-4 blockade resulted in a significant decrease in the growth rate of epithelial cancer cells and sensitized them, both in vitro and in vivo, to apoptosis induction by TRAIL and chemotherapy via downregulation of the antiapoptotic factors PED, cFLIP, Bcl-xL and Bcl-2. Furthermore, we provide evidence that exogenous IL-4 was able to upregulate the expression levels of these antiapoptotic proteins and potently stabilized the growth of normal epithelial cells rendering them apoptosis resistant. In conclusion, IL-4 acts as an autocrine survival factor in epithelial cells. Our results indicate that inhibition of IL-4/IL-4R signaling may serve as a novel treatment for epithelial cancers.

Interferon-alpha and interferon-gamma modulate Fas-mediated apoptosis in mitomycin-C-resistant human Tenon's fibroblasts.

PubMed

Wang, Xiao Yang; Crowston, Jonathan G; White, Andrew J R; Zoellner, Hans; Healey, Paul R

2014-08-01

The aim of the study was to investigate, using a native mitomycin-C-resistant human Tenon's fibroblast cell line, the possibility that interferon-alpha and gamma could be used with Fas agonists as an alternative anti-fibrotic strategy to mitomycin-C in trabeculectomy. A clinically resistant and in vitro verified mitomycin-C-resistant human Tenon's fibroblast cell line was pretreated with interferon-alpha and interferon-gamma for 48 h before stimulation with an agonistic Fas antibody (CH11) for 2 days to induce cell death. Cell death assays were undertaken. Changes in apoptosis-related proteins were determined by flow cytometry and Western blot. Pretreatment with interferon-alpha or interferon-gamma for 48 h increased Fas, Fas-associated protein with death domain and caspase-8 expression. Protein expression was further increased by combined exposure to interferon-alpha and gamma. Pretreatment with cytokines had no effect on Fas-L and Bcl-2. Interferon-alpha alone did not change the rate of induced cell death. A combination of interferon-alpha and gamma synergistically increased the sensitivity of mitomycin-C-resistant human Tenon's fibroblast cell line to induced cell death. An antagonistic anti-Fas antibody (ZB4) completely blocked induced cell death. Broad caspase inhibitors specific for caspases-8 and -3 reduced induced deaths in interferon pretreated mitomycin-C-resistant human Tenon's fibroblast cell line in a dose-dependent manner. Interferon-alpha and interferon-gamma render mitomycin-C-resistant human Tenon's fibroblast cell line sensitive to Fas-mediated apoptosis. The mechanism involves increased death-inducing signalling complex formation by upregulation of Fas, Fas-associated protein with death domain and caspase-8 expression. © 2013 Royal Australian and New Zealand College of Ophthalmologists.

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor.

PubMed

Stahl, Sebastian; da Silva Mateus Seidl, Ana Rita; Ducret, Axel; Kux van Geijtenbeek, Sabine; Michel, Sven; Racek, Tomas; Birzele, Fabian; Haas, Alexander K; Rueger, Ruediger; Gerg, Michael; Niederfellner, Gerhard; Pastan, Ira; Brinkmann, Ulrich

2015-08-25

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.

The Transcription Factor Wilms Tumor 1 Confers Resistance in Myeloid Leukemia Cells against the Proapoptotic Therapeutic Agent TRAIL (Tumor Necrosis Factor α-related Apoptosis-inducing Ligand) by Regulating the Antiapoptotic Protein Bcl-xL*

PubMed Central

Bansal, Hima; Seifert, Theresea; Bachier, Carlos; Rao, Manjeet; Tomlinson, Gail; Iyer, Swaminathan Padmanabhan; Bansal, Sanjay

2012-01-01

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias. PMID:22898820

The transcription factor Wilms tumor 1 confers resistance in myeloid leukemia cells against the proapoptotic therapeutic agent TRAIL (tumor necrosis factor α-related apoptosis-inducing ligand) by regulating the antiapoptotic protein Bcl-xL.

PubMed

Bansal, Hima; Seifert, Theresea; Bachier, Carlos; Rao, Manjeet; Tomlinson, Gail; Iyer, Swaminathan Padmanabhan; Bansal, Sanjay

2012-09-21

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias.

An activating mutation of GNB1 is associated with resistance to tyrosine kinase inhibitors in ETV6-ABL1-positive leukemia

PubMed Central

Zimmermannova, O; Doktorova, E; Stuchly, J; Kanderova, V; Kuzilkova, D; Strnad, H; Starkova, J; Alberich-Jorda, M; Falkenburg, J H F; Trka, J; Petrak, J; Zuna, J; Zaliova, M

2017-01-01

Leukemias harboring the ETV6-ABL1 fusion represent a rare subset of hematological malignancies with unfavorable outcomes. The constitutively active chimeric Etv6-Abl1 tyrosine kinase can be specifically inhibited by tyrosine kinase inhibitors (TKIs). Although TKIs represent an important therapeutic tool, so far, the mechanism underlying the potential TKI resistance in ETV6-ABL1-positive malignancies has not been studied in detail. To address this issue, we established a TKI-resistant ETV6-ABL1-positive leukemic cell line through long-term exposure to imatinib. ETV6-ABL1-dependent mechanisms (including fusion gene/protein mutation, amplification, enhanced expression or phosphorylation) and increased TKI efflux were excluded as potential causes of resistance. We showed that TKI effectively inhibited the Etv6-Abl1 kinase activity in resistant cells, and using short hairpin RNA (shRNA)-mediated silencing, we confirmed that the resistant cells became independent from the ETV6-ABL1 oncogene. Through analysis of the genomic and proteomic profiles of resistant cells, we identified an acquired mutation in the GNB1 gene, K89M, as the most likely cause of the resistance. We showed that cells harboring mutated GNB1 were capable of restoring signaling through the phosphoinositide-3-kinase (PI3K)/Akt/mTOR and mitogen-activated protein kinase (MAPK) pathways, whose activation is inhibited by TKI. This alternative GNB1K89M-mediated pro-survival signaling rendered ETV6-ABL1-positive leukemic cells resistant to TKI therapy. The mechanism of TKI resistance is independent of the targeted chimeric kinase and thus is potentially relevant not only to ETV6-ABL1-positive leukemias but also to a wider spectrum of malignancies treated by kinase inhibitors. PMID:28650474

Structure-dependent Pseudoreceptor Intracellular Traffic of Adamantyl Globotriaosyl Ceramide Mimics*

PubMed Central

Saito, Mitsumasa; Mylvaganum, Murugespillai; Tam, Patty; Novak, Anton; Binnington, Beth; Lingwood, Clifford

2012-01-01

The verotoxin (VT) (Shiga toxin) receptor globotriaosyl ceramide (Gb3), mediates VT1/VT2 retrograde transport to the endoplasmic reticulum (ER) for cytosolic A subunit access to inhibit protein synthesis. Adamantyl Gb3 is an amphipathic competitive inhibitor of VT1/VT2 Gb3 binding. However, Gb3-negative VT-resistant CHO/Jurkat cells incorporate adaGb3 to become VT1/VT2-sensitive. CarboxyadaGb3, urea-adaGb3, and hydroxyethyl adaGb3, preferentially bound by VT2, also mediate VT1/VT2 cytotoxicity. VT1/VT2 internalize to early endosomes but not to Golgi/ER. AdabisGb3 (two deacyl Gb3s linked to adamantane) protects against VT1/VT2 more effectively than adaGb3 without incorporating into Gb3-negative cells. AdaGb3 (but not hydroxyethyl adaGb3) incorporation into Gb3-positive Vero cells rendered punctate cell surface VT1/VT2 binding uniform and subverted subsequent Gb3-dependent retrograde transport to Golgi/ER to render cytotoxicity (reduced for VT1 but not VT2) brefeldin A-resistant. VT2-induced vacuolation was maintained in adaGb3-treated Vero cells, but vacuolar membrane VT2 was lost. AdaGb3 destabilized membrane cholesterol and reduced Gb3 cholesterol stabilization in phospholipid liposomes. Cholera toxin GM1-mediated Golgi/ER targeting was unaffected by adaGb3. We demonstrate the novel, lipid-dependent, pseudoreceptor function of Gb3 mimics and their structure-dependent modulation of endogenous intracellular Gb3 vesicular traffic. PMID:22418442

Streptococcal cell wall-induced arthritis and adjuvant arthritis in F344----Lewis and in Lewis----F344 bone marrow chimeras

DOE Office of Scientific and Technical Information (OSTI.GOV)

van Bruggen, M.C.; van den Broek, M.F.; van den Berg, W.B.

1991-09-01

Streptococcal cell wall (SCW)-induced arthritis and adjuvant arthritis (AA) are rat models for chronic, erosive polyarthritis. Both models can be induced in susceptible Lewis rats, whereas F344 rats are resistant. In AA as well as in SCW arthritis, antigen-specific T lymphocytes have been demonstrated to be crucial for chronic disease. In this communication the authors describe their studies to probe the cellular mechanism responsible for the difference in susceptibility of Lewis and F344, using bone marrow chimeras. By transplanting bone marrow cells from F344 into lethally irradiated Lewis recipients, Lewis rats were rendered resistant to SCW arthritis induction. F344 ratsmore » reconstituted with Lewis bone marrow, i.e., Lewis----F344 chimeras, develop an arthritis upon SCW injection. For AA comparable results were obtained. These data suggest that both resistance and susceptibility to bacterium-induced chronic arthritis are mediated by hemopoietic/immune cells and that the recipiental environment does not influence the susceptibility to chronic joint inflammation.« less

Low abundance of mitochondrial DNA changes mitochondrial status and renders cells resistant to serum starvation and sodium nitroprusside insult.

PubMed

Lee, Sung Ryul; Heo, Hye Jin; Jeong, Seung Hun; Kim, Hyoung Kyu; Song, In Sung; Ko, Kyung Soo; Rhee, Byoung Doo; Kim, Nari; Han, Jin

2015-07-01

Mutation or depletion of mitochondrial DNA (mtDNA) can cause severe mitochondrial malfunction, originating from the mitochondrion itself, or from the crosstalk between nuclei and mitochondria. However, the changes that would occur if the amount of mtDNA is diminished are less known. Thus, we generated rat myoblast H9c2 cells containing lower amounts of mtDNA via ethidium bromide and uridine supplementation. After confirming the depletion of mtDNA by quantitative PCR and gel electrophoresis analysis, we investigated the changes in mitochondrial physical parameters by using flow cytometry. We also evaluated the resistance of these cells to serum starvation and sodium nitroprusside. H9c2 cells with diminished mtDNA contents showed decreased mitochondrial membrane potential, mass, free calcium, and zinc ion contents as compared to naïve H9c2 cells. Furthermore, cytosolic and mitochondrial reactive oxygen species levels were significantly higher in mtDNA-lowered H9c2 cells than in the naïve cells. Although the oxygen consumption rate and cell proliferation were decreased, mtDNA-lowered H9c2 cells were more resistant to serum deprivation and nitroprusside insults than the naïve H9c2 cells. Taken together, we conclude that the low abundance of mtDNA cause changes in cellular status, such as changes in reactive oxygen species, calcium, and zinc ion levels inducing resistance to stress. © 2015 International Federation for Cell Biology.

Glucose deprivation elicits phenotypic plasticity via ZEB1-mediated expression of NNMT

PubMed Central

Kanska, Justyna; Aspuria, Paul-Joseph P.; Taylor-Harding, Barbie; Spurka, Lindsay; Funari, Vincent; Orsulic, Sandra; Karlan, Beth Y.; Wiedemeyer, W. Ruprecht

2017-01-01

Glucose is considered the primary energy source for all cells, and some cancers are addicted to glucose. Here, we investigated the functional consequences of chronic glucose deprivation in serous ovarian cancer cells. We found that cells resistant to glucose starvation (glucose-restricted cells) demonstrated increased metabolic plasticity that was dependent on NNMT (Nicotinamide N-methyltransferase) expression. We further show that ZEB1 induced NNMT, rendered cells resistant to glucose deprivation and recapitulated metabolic adaptations and mesenchymal gene expression observed in glucose-restricted cells. NNMT depletion reversed metabolic plasticity in glucose-restricted cells and prevented de novo formation of glucose-restricted colonies. In addition to its role in glucose independence, we found that NNMT was required for other ZEB1-induced phenotypes, such as increased migration. NNMT protein levels were also elevated in metastatic and recurrent tumors compared to matched primary carcinomas, while normal ovary and fallopian tube tissue had no detectable NNMT expression. Our studies define a novel ZEB1/NNMT signaling axis, which elicits mesenchymal gene expression, as well as phenotypic and metabolic plasticity in ovarian cancer cells upon chronic glucose starvation. Understanding the causes of cancer cell plasticity is crucial for the development of therapeutic strategies to counter intratumoral heterogeneity, acquired drug resistance and recurrence in high-grade serous ovarian cancer (HGSC). PMID:28412735

Effect of Her-2/neu Signaling on Sensitivity to TRAIL in Prostate Cancer

DTIC Science & Technology

2005-06-01

cytokines (18), and matrix metalloprotease inhibitors (19) are able to render TRAIL-resistant tumor cells sensitive to TRAIL apoptosis. In recent...TRAIL-induced cytotoxicity. As DU-145 cells were treated with acetyl salicylic acid (ASA: aspirin), an inhibitor of IKK , we observed that TRAIL...sulfide (IC50 = 1.02 M for COX-1 and IC50 = 10.43 M for COX-2), NS-398 (a selective COX-2 inhibitor ; IC50 = 4.81 M for COX-1 and IC50 = 0.47 M for COX-2

3-Bromopyruvate induces endoplasmic reticulum stress, overcomes autophagy and causes apoptosis in human HCC cell lines.

PubMed

Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H; Kunjithapatham, Rani; Buijs, Manon; Syed, Labiq H; Rao, Pramod P; Ota, Shinichi; Kwak, Byung Kook; Loffroy, Romaric; Vali, Mustafa

2010-03-01

Autophagy, a cellular response to stress, plays a role in resistance to chemotherapy in cancer cells. Resistance renders systemic chemotherapy generally ineffective against human hepatocellular carcinoma (HCC). Recently, we reported that the pyruvate analog 3-bromopyruvate (3-BrPA) promoted tumor cell death by targeting GAPDH. In continuance, we investigated the intracellular response of two human HCC cell lines (Hep3B and SK-Hep1) that differ in their status of key apoptotic regulators, p53 and Fas. 3-BrPA treatment induced endoplasmic reticulum (ER) stress, translation inhibition and apoptosis based on Western blot and qPCR, pulse labeling, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and active caspase-3 in both the cell lines. However, electron microscopy revealed that 3-BrPA treated SK-Hep1 cells underwent classical apoptotic cell death while Hep3B cells initially responded with the protective autophagy that failed to prevent eventual apoptosis. 3-BrPA treatment promotes apoptosis in human HCC cell lines, irrespective of the intracellular response.

Prokaryotic arsenate reductase enhances arsenate resistance in Mammalian cells.

PubMed

Wu, Dan; Tao, Xuanyu; Wu, Gaofeng; Li, Xiangkai; Liu, Pu

2014-01-01

Arsenic is a well-known heavy metal toxicant in the environment. Bioremediation of heavy metals has been proposed as a low-cost and eco-friendly method. This article described some of recent patents on transgenic plants with enhanced heavy metal resistance. Further, to test whether genetic modification of mammalian cells could render higher arsenic resistance, a prokaryotic arsenic reductase gene arsC was transfected into human liver cancer cell HepG2. In the stably transfected cells, the expression level of arsC gene was determined by quantitative real-time PCR. Results showed that arsC was expressed in HepG2 cells and the expression was upregulated by 3 folds upon arsenate induction. To further test whether arsC has function in HepG2 cells, the viability of HepG2-pCI-ArsC cells exposed to arsenite or arsenate was compared to that of HepG2-pCI cells without arsC gene. The results indicated that arsC increased the viability of HepG2 cells by 25% in arsenate, but not in arsenite. And the test of reducing ability of stably transfected cells revealed that the concentration of accumulated trivalent arsenic increased by 25% in HepG2-pCI-ArsC cells. To determine the intracellular localization of ArsC, a fusion vector with fluorescent marker pEGFP-N1-ArsC was constructed and transfected into.HepG2. Laser confocal microscopy showed that EGFP-ArsC fusion protein was distributed throughout the cells. Taken together, these results demonstrated that prokaryotic arsenic resistant gene arsC integrated successfully into HepG2 genome and enhanced arsenate resistance of HepG2, which brought new insights of arsenic detoxification in mammalian cells.

Systemic Inflammatory Response Elicited by Superantigen Destabilizes T Regulatory Cells Rendering them Ineffective during Toxic Shock Syndrome 1

PubMed Central

Tilahun, Ashenafi Y.; Chowdhary, Vaidehi R.; David, Chella S.; Rajagopalan, Govindarajan

2014-01-01

Life-threatening infections caused by Staphylococcus aureus, particularly the community-acquired methicillin-resistant strains of S. aureus (CA-MRSA), continue to pose serious problems. Greater virulence and increased pathogenicity of certain S. aureus strains are attributed to higher prevalence of exotoxins. Of these exotoxins, the superantigens (SAg) are likely most pathogenic because of their ability to rapidly and robustly activate the T cells even in extremely small quantities. Therefore, countering SAg-mediated T cell activation using T regulatory cells (Tregs) might be beneficial in diseases such as toxic shock syndrome (TSS). As the normal numbers of endogenous Tregs in a typical host are insufficient, we hypothesized that increasing the Treg numbers by administration of IL2-anti-IL2 antibody complexes (IL2C) or by adoptive transfer of ex vivo expanded Tregs might be more effective in countering SAg-mediated immune activation. HLA-DR3 transgenic mice that closely recapitulate human TSS, were treated with IL2C to increase endogenous Tregs or received ex vivo expanded Tregs. Subsequently, they were challenged with SAg to induce TSS. Analyses of various parameters reflective of TSS (serum cytokine/chemokine levels, multiple organ pathology and SAg-induced peripheral T cell expansion) indicated that increasing the Tregs failed to mitigate TSS. On the contrary, serum IFN-γ levels were increased in IL2C treated mice. Exploration into the reasons behind the lack of protective effect of Tregs revealed IL-17 and IFN-γ-dependent loss of Tregs during TSS. In addition, significant upregulation of GITR on conventional T cells during TSS could render them resistant to Treg mediated suppression, contributing to failure of Treg-mediated immune regulation. PMID:25092888

The C-type Lectin Langerin Functions as a Receptor for Attachment and Infectious Entry of Influenza A Virus.

PubMed

Ng, Wy Ching; Londrigan, Sarah L; Nasr, Najla; Cunningham, Anthony L; Turville, Stuart; Brooks, Andrew G; Reading, Patrick C

2016-01-01

It is well established that influenza A virus (IAV) attachment to and infection of epithelial cells is dependent on sialic acid (SIA) at the cell surface, although the specific receptors that mediate IAV entry have not been defined and multiple receptors may exist. Lec2 Chinese hamster ovary (CHO) cells are SIA deficient and resistant to IAV infection. Here we demonstrate that the expression of the C-type lectin receptor langerin in Lec2 cells (Lec2-Lg) rendered them permissive to IAV infection, as measured by replication of the viral genome, transcription of viral mRNA, and synthesis of viral proteins. Unlike SIA-dependent infection of parental CHO cells, IAV attachment and infection of Lec2-Lg cells was mediated via lectin-mediated recognition of mannose-rich glycans expressed by the viral hemagglutinin glycoprotein. Lec2 cells expressing endocytosis-defective langerin bound IAV efficiently but remained resistant to IAV infection, confirming that internalization via langerin was essential for infectious entry. Langerin-mediated infection of Lec2-Lg cells was pH and dynamin dependent, occurred via clathrin- and caveolin-mediated endocytic pathways, and utilized early (Rab5(+)) but not late (Rab7(+)) endosomes. This study is the first to demonstrate that langerin represents an authentic receptor that binds and internalizes IAV to facilitate infection. Moreover, it describes a unique experimental system to probe specific pathways and compartments involved in infectious entry following recognition of IAV by a single cell surface receptor. On the surface of host cells, sialic acid (SIA) functions as the major attachment factor for influenza A viruses (IAV). However, few studies have identified specific transmembrane receptors that bind and internalize IAV to facilitate infection. Here we identify human langerin as a transmembrane glycoprotein that can act as an attachment factor and a bone fide endocytic receptor for IAV infection. Expression of langerin by an SIA-deficient cell line resistant to IAV rendered cells permissive to infection. As langerin represented the sole receptor for IAV infection in this system, we have defined the pathways and compartments involved in infectious entry of IAV into cells following recognition by langerin. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Function of Oxidative Cross-Linking of Cell Wall Structural Proteins in Plant Disease Resistance.

PubMed

Brisson, L. F.; Tenhaken, R.; Lamb, C.

1994-12-01

Elicitation of soybean cells causes a rapid insolubilization of two cell wall structural proteins, p33 and p100. Likewise, a short elicitation of 30 min rendered cell walls more refractory to enzyme digestion as assayed by the yield of protoplasts released. This effect could be ascribed to protein cross-linking because of its insensitivity to inhibitors of transcription (actinomycin D) and translation (cycloheximide) and its induction by exogenous H2O2. Moreover, the induced loss of protoplasts could be prevented by preincubation with DTT, which also blocks peroxidase-mediated oxidative cross-linking. The operation of protein insolubilization in plant defense was also demonstrated by its occurrence in the incompatible interaction but not in the compatible interaction between soybean and Pseudomonas syringae pv glycinea. Likewise, protein insolubilization was observed in bean during non-host hypersensitive resistance to the tobacco pathogen P. s. pv tabaci mediated by the hypersensitive resistance and pathogenicity (Hrp) gene cluster. Our data strongly suggest that rapid protein insolubilization leads to a strengthened cell wall, and this mechanism functions as a rapid defense in the initial stages of the hypersensitive response prior to deployment of transcription-dependent defenses.

Survival of Listeria monocytogenes strain H7762 and resistance to simulated gastric fluid following exposure to frankfurter exudate.

PubMed

Wonderling, Laura D; Bayles, Darrell O

2004-06-01

Listeria monocytogenes strain H7762, a frankfurter isolate, was tested to determine whether it was able to survive at 4 degrees C in frankfurter pack fluid (exudate) and to determine whether food exposure affects its acid sensitivity. Cultures were sampled and tested for acid sensitivity by challenge with simulated gastric fluid (SGF). SGF challenges performed immediately after inoculation revealed that between 20 and 26% of the cells survived the full 30 min of SGF challenge regardless of whether the cells were inoculated into brain heart infusion broth (BHI) or exudate. After 2 days of incubation, cells exposed to both exudate and BHI had significantly decreased SGF resistance; however, the cells exposed to exudate were significantly more SGF resistant than cells exposed to BHI (after 15 min of SGF treatment, 33% of the exudate-exposed cells survived and 12% of the BHI-exposed cells survived). L. monocytogenes exposed to exudate had greater SGF resistance at all challenge times compared with BHI-exposed cells from day 2 through day 4. From days 8 to 15, exudate-exposed cells continued to have greater SGF resistance than BHI-exposed cells up to 10 min of SGF challenge but were as sensitive as the BHI-exposed cells at 20 to 30 min of challenge. By day 25, cells exposed to exudate were significantly more sensitive to SGF challenge than BHI-exposed cells. The survivor data generated from SGF challenges were modeled by a nonlinear regression analysis to calculate the underlying distribution of SGF resistance found in the challenged populations. These analyses indicated that L. monocytogenes exposed to exudate at 4 degrees C had a broader distribution of resistance to SGF compared with cells exposed to BHI at 4 degrees C. In addition, the mean time of death during SGF treatment was greater after exposure to exudate, indicating that cells exposed to exudate were more resistant to killing by SGF These data suggest that exposure to frankfurter exudate might render L. monocytogenes more able to survive the stomach environment during the initial stages of infection.

Akt-dependent glucose metabolism promotes Mcl-1 synthesis to maintain cell survival and resistance to Bcl-2 inhibition.

PubMed

Coloff, Jonathan L; Macintyre, Andrew N; Nichols, Amanda G; Liu, Tingyu; Gallo, Catherine A; Plas, David R; Rathmell, Jeffrey C

2011-08-01

Most cancer cells utilize aerobic glycolysis, and activation of the phosphoinositide 3-kinase/Akt/mTOR pathway can promote this metabolic program to render cells glucose dependent. Although manipulation of glucose metabolism may provide a means to specifically eliminate cancer cells, mechanistic links between cell metabolism and apoptosis remain poorly understood. Here, we examined the role and metabolic regulation of the antiapoptotic Bcl-2 family protein Mcl-1 in cell death upon inhibition of Akt-induced aerobic glycolysis. In the presence of adequate glucose, activated Akt prevented the loss of Mcl-1 expression and protected cells from growth factor deprivation-induced apoptosis. Mcl-1 associated with and inhibited the proapoptotic Bcl-2 family protein Bim, contributing to cell survival. However, suppression of glucose metabolism led to induction of Bim, decreased expression of Mcl-1, and apoptosis. The proapoptotic Bcl-2/Bcl-xL/Bcl-w inhibitor, ABT-737, shows clinical promise, but Mcl-1 upregulation can promote resistance. Importantly, inhibition of glucose metabolism or mTORC1 overcame Mcl-1-mediated resistance in diffuse large B cell leukemic cells. Together these data show that Mcl-1 protein synthesis is tightly controlled by metabolism and that manipulation of glucose metabolism may provide a mechanism to suppress Mcl-1 expression and sensitize cancer cells to apoptosis.

Akt-Dependent Glucose Metabolism Promotes Mcl-1 Synthesis to Maintain Cell Survival and Resistance to Bcl-2 Inhibition

PubMed Central

Coloff, Jonathan L.; Macintyre, Andrew N.; Nichols, Amanda G.; Liu, Tingyu; Gallo, Catherine A.; Plas, David R.; Rathmell, Jeffrey C.

2011-01-01

Most cancer cells utilize aerobic glycolysis, and activation of the phosphatidyl-inositol 3-kinase (PI3K)/Akt/mTOR pathway can promote this metabolic program to render cells glucose-dependent. While manipulation of glucose metabolism may provide a means to specifically eliminate cancer cells, mechanistic links between cell metabolism and apoptosis remain poorly understood. Here we examine the role and metabolic regulation of the anti-apoptotic Bcl-2 family protein Mcl-1 in cell death upon inhibition of Akt-induced aerobic glycolysis. In the presence of adequate glucose, activated Akt prevented the loss of Mcl-1 expression and protected cells from growth factor-deprivation induced apoptosis. Mcl-1 associated with and inhibited the pro-apoptotic Bcl-2 family protein Bim, contributing to cell survival. However, suppression of glucose metabolism led to induction of Bim, decreased expression of Mcl-1, and apoptosis. The pro-apoptotic Bcl-2/Bcl-xL/Bcl-w inhibitor, ABT-737, shows clinical promise, but Mcl-1 upregulation can promote resistance. Importantly, inhibition of glucose metabolism or mTORC1 overcame Mcl-1-mediated resistance in diffuse large B cell leukemic cells. Together these data show that Mcl-1 protein synthesis is tightly controlled by metabolism and that manipulation of glucose metabolism may provide a mechanism to suppress Mcl-1 expression and sensitize cancer cells to apoptosis. PMID:21670080

Surface changes and polymyxin interactions with a resistant strain of Klebsiella pneumoniae.

PubMed

Velkov, Tony; Deris, Zakuan Z; Huang, Johnny X; Azad, Mohammad A K; Butler, Mark; Sivanesan, Sivashangarie; Kaminskas, Lisa M; Dong, Yao-Da; Boyd, Ben; Baker, Mark A; Cooper, Matthew A; Nation, Roger L; Li, Jian

2014-05-01

This study examines the interaction of polymyxin B and colistin with the surface and outer membrane components of a susceptible and resistant strain of Klebsiella pneumoniae. The interaction between polymyxins and bacterial membrane and isolated LPS from paired wild type and polymyxin-resistant strains of K. pneumoniae were examined with N-phenyl-1-naphthylamine (NPN) uptake, fluorometric binding and thermal shift assays, lysozyme and deoxycholate sensitivity assays, and by (1)H NMR. LPS from the polymyxin-resistant strain displayed a reduced binding affinity for polymyxins B and colistin in comparison with the wild type LPS. The outer membrane NPN permeability of the resistant strain was greater compared with the susceptible strain. Polymyxin exposure enhanced the permeability of the outer membrane of the wild type strain to lysozyme and deoxycholate, whereas polymyxin concentrations up to 32 mg/ml failed to permeabilize the outer membrane of the resistant strain. Zeta potential measurements revealed that mid-logarithmic phase wild type cells exhibited a greater negative charge than the mid-logarithmic phase-resistant cells. Taken together, our findings suggest that the resistant derivative of K. pneumoniae can block the electrostatically driven first stage of polymyxin action, which thereby renders the hydrophobically driven second tier of polymyxin action on the outer membrane inconsequential.

Natto (fermented soybean) extract extends the adult lifespan of Caenorhabditis elegans.

PubMed

Ibe, Sachie; Kumada, Kaoru; Yoshida, Keiko; Otobe, Kazunori

2013-01-01

We investigated the effects of a water extract of natto on the aging of the nematode Caenorhabditis elegans. The water extract significantly prolonged the adult lifespan of the wild-type worms and rendered them resistant to oxidative and thermal stress. In addition, treatment with natto extract significantly delayed the accumulation of lipofuscin, a characteristic of aging cells. Our findings suggest that components of natto have a beneficial anti-aging effect in vivo.

Osimertinib, a third-generation tyrosine kinase inhibitor targeting non-small cell lung cancer with EGFR T790M mutations.

PubMed

McCoach, C E; Jimeno, A

2016-10-01

Oncogenic driver mutations in the epidermal growth factor receptor (EGFR) gene have provided a focus for effective targeted therapy. Unfortunately, all patients eventually develop resistance to frontline therapy with EGFR tyrosine kinase inhibitors (TKIs). The majority of patients develop a large subclonal population of tumor cells with a T790M mutation that renders these cells resistant to first-generation TKIs. Osimertinib is a third-generation EGFR TKI that was designed to overcome resistance from T790M mutations. This agent has demonstrated strong preclinical activity, and in the clinic it has demonstrated a high objective response rate and progression-free survival in patients with EGFR double mutations (L858R/T790M and exon 19 deletion/T790M). It is now approved by the FDA for patients who have a documented T790M mutation and who have progressed on a prior TKI. Osimertinib is also approved in the E.U. and Japan. Copyright 2016 Prous Science, S.A.U. or its licensors. All rights reserved.

Androgen deprivation therapy sensitizes prostate cancer cells to T-cell killing through androgen receptor dependent modulation of the apoptotic pathway

PubMed Central

Ardiani, Andressa; Gameiro, Sofia R.; Kwilas, Anna R.; Donahue, Renee N.; Hodge, James W.

2014-01-01

Despite recent advances in diagnosis and management, prostrate cancer remains the second most common cause of death from cancer in American men, after lung cancer. Failure of chemotherapies and hormone-deprivation therapies is the major cause of death in patients with castration-resistant prostate cancer (CRPC). Currently, the androgen inhibitors enzalutamide and abiraterone are approved for treatment of metastatic CRPC. Here we show for the first time that both enzalutamide and abiraterone render prostate tumor cells more sensitive to T cell-mediated lysis through immunogenic modulation, and that these immunomodulatory activities are androgen receptor (AR)-dependent. In studies reported here, the NAIP gene was significantly down-regulated in human prostate tumor cells treated in vitro and in vivo with enzalutamide. Functional analysis revealed that NAIP played a critical role in inducing CTL sensitivity. Amplification of AR is a major mechanism of resistance to androgen-deprivation therapy (ADT). Here, we show that enzalutamide enhances sensitivity to immune-mediated killing of prostate tumor cells that overexpress AR. The immunomodulatory properties of enzalutamide and abiraterone provide a rationale for their use in combination with immunotherapeutic agents in CRPC, especially for patients with minimal response to enzalutamide or abiraterone alone, or for patients who have developed resistance to ADT. PMID:25344864

Combinatorial BTK and MALT1 inhibition augments killing of CD79 mutant diffuse large B cell lymphoma

PubMed Central

Nagel, Daniel; Bognar, Miriam; Eitelhuber, Andrea C.; Kutzner, Kerstin; Vincendeau, Michelle; Krappmann, Daniel

2015-01-01

Survival of activated B cell-subtype (ABC) of diffuse large B cell lymphoma (DLBCL) is driven by chronic B cell receptor (BCR) signaling that activates the canonical NF-κB pathway. Inhibition of BTK by Ibrutinib has been shown to kill ABC DLBCL cells that carry activating mutations in the BCR adaptor CD79. However, mutations in BTK or in downstream components such as CARMA1/CARD11 can render lymphomas Ibrutinib resistant. Therefore, we assessed here the simultaneous inhibition of BTK and the protease MALT1 that acts downstream of CARMA1 and is essential for ABC DLBCL tumor growth. We show that in CD79 mutant cells BTK is a crucial upstream regulator of MALT1, but dispensable in CARMA1 mutant ABC DLBCL. Combined inhibition of BTK by Ibrutinib and MALT1 by S-Mepazine additively impaired MALT1 cleavage activity and expression of NF-κB pro-survival factors. Thereby, combinatorial Ibrutinib and S-Mepazine treatment enhanced killing of CD79 mutant ABC DLBCL cells. Moreover, while expression of oncogenic CARMA1 in CD79 mutant cells conferred Ibrutinib resistance, double mutant cells were still sensitive to MALT1 inhibition by S-Mepazine. Thus, based on the genetic background combinatorial BTK and MALT1 inhibition may improve effectiveness of therapeutic treatment and reduce the chances for the development of drug resistances. PMID:26540570

Nitric oxide inhibits topoisomerase II activity and induces resistance to topoisomerase II-poisons in human tumor cells.

PubMed

Kumar, Ashutosh; Ehrenshaft, Marilyn; Tokar, Erik J; Mason, Ronald P; Sinha, Birandra K

2016-07-01

Etoposide and doxorubicin, topoisomerase II poisons, are important drugs for the treatment of tumors in the clinic. Topoisomerases contain several free sulfhydryl groups which are important for their activity and are also potential targets for nitric oxide (NO)-induced nitrosation. NO, a physiological signaling molecule nitrosates many cellular proteins, causing altered protein and cellular functions. Here, we have evaluated the roles of NO/NO-derived species in the activity/stability of topo II both in vitro and in human tumor cells, and in the cytotoxicity of topo II-poisons, etoposide and doxorubicin. Treatment of purified topo IIα with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of both the catalytic and relaxation activity in vitro, and decreased etoposide-dependent cleavable complex formation in both human HT-29 colon and MCF-7 breast cancer cells. PPNO treatment also induced significant nitrosation of topo IIα protein in these human tumor cells. These events, taken together, caused a significant resistance to etoposide in both cell lines. However, PPNO had no effect on doxorubicin-induced cleavable complex formation, or doxorubicin cytotoxicity in these cell lines. Inhibition of topo II function by NO/NO-derived species induces significant resistance to etoposide, without affecting doxorubicin cytotoxicity in human tumor cells. As tumors express inducible nitric oxide synthase and generate significant amounts of NO, modulation of topo II functions by NO/NO-derived species could render tumors resistant to certain topo II-poisons in the clinic. Published by Elsevier B.V.

Direct Downregulation of B-Cell Translocation Gene 3 by microRNA-93 Is Required for Desensitizing Esophageal Cancer to Radiotherapy.

PubMed

Cui, Hujun; Zhang, Shengqiang; Zhou, Hongbo; Guo, Ling

2017-08-01

Esophageal squamous carcinoma (ESC) is one of the most fatal malignancies worldwide with increasing occurrences yet poor outcome. MicroRNAs were reported to play roles in ESC. We aimed to understand how miRNAs affect the radiotherapy resistance of ESC. MicroRNA assays, real-time PCR, and Western blot were performed for expression analysis of miR-93 and BTG3. Luciferase activity assay was conducted with mutated B-cell translocation gene 3 (BTG3) 3'-UTR sequence in the 3' end of luciferase sequence with miR-93 inhibitor. ESC cells were treated with irradiation (IR) and clonogenic assay was utilized to detect the cell viability. Human ESC xenograft mouse model was established and subjected to target IR treatment followed by tumor size analysis. MiR-93 was decreased and BTG3 was increased in ESC cells, with negative correlation of their expression in ESC tissues. MiR-93 directly targeted BTG3 3'-UTR by luciferase activity assay. Either miR-93 inhibition or BTG3 overexpression decreased radiation resistance. Furthermore, miR-93 inhibition suppressed radiation resistance through BTG3. Direct downregulation of BTG3 by miR-93 is able to render ESC resistant to radiotherapy, and both BTG3 and miR-93 may potentially serve as clinical markers for ESC and contribute to the treatment of ESC.

A novel Smac mimetic APG-1387 demonstrates potent antitumor activity in nasopharyngeal carcinoma cells by inducing apoptosis.

PubMed

Li, Ning; Feng, Lin; Han, Hui-Qiong; Yuan, Jing; Qi, Xue-Kang; Lian, Yi-Fan; Kuang, Bo-Hua; Zhang, Yu-Chen; Deng, Cheng-Cheng; Zhang, Hao-Jiong; Yao, You-Yuan; Xu, Miao; He, Gui-Ping; Zhao, Bing-Chun; Gao, Ling; Feng, Qi-Sheng; Chen, Li-Zhen; Yang, Lu; Yang, Dajun; Zeng, Yi-Xin

2016-10-10

Despite advances in the development of radiation against nasopharyngeal carcinoma (NPC), the management of advanced NPC remains a challenge. Smac mimetics are designed to neutralize inhibitor of apoptosis (IAP) proteins, thus reactivating the apoptotic program in cancer cells. In this study, we investigated the effect of a novel bivalent Smac mimetic APG-1387 in NPC. In vitro, APG-1387 in combination with TNF-α potently decreased NPC cell viability by inducing apoptosis in majority of NPC cell lines. The in vitro antitumor effect was RIPK1-dependent, whereas it was independent on IAPs, USP11, or EBV. Of note, the inhibition of NF-κB or AKT pathway rendered resistant NPC cells responsive to the treatment of APG-1387/TNF-α. In vivo, APG-1387 displayed antitumor activity as a single agent at well-tolerated doses, even in an in vitro resistant cell line. In summary, our results demonstrate that APG-1387 exerts a potent antitumor effect on NPC. These findings support clinical evaluation of APG-1387 as a potential treatment for advanced NPC. Copyright © 2016. Published by Elsevier Ireland Ltd.

Photo-excitable hybrid nanocomposites for image-guided photo/TRAIL synergistic cancer therapy.

PubMed

Lin, Gan; Zhang, Yang; Zhu, Congqing; Chu, Chengchao; Shi, Yesi; Pang, Xin; Ren, En; Wu, Yayun; Mi, Peng; Xia, Haiping; Chen, Xiaoyuan; Liu, Gang

2018-05-22

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in cancer cells without toxicity to normal cells. However, the efficiency is greatly limited by its short half-life and wild resistance in various cancer cells. In this study, we reported a micellar hybrid nanoparticle to carry TRAIL ligand (denoted as IPN@TRAIL) for a novel photo-excited TRAIL therapy. These IPN@TRAIL offered increased TRAIL stability, prolonged half-life and enhanced tumor accumulation, monitored by dual mode imaging. Furthermore, IPN@TRAIL nanocomposites enhanced wrapped TRAIL therapeutic efficiency greatly towards resistant cancer cells by TRAIL nanovectorization. More importantly, when upon external laser, these nanocomposites not only triggered tumor photothermal therapy (PTT), but also upregulated the expression of death receptors (DR4 and DR5), resulting in a greater apoptosis mediated by co-delivered TRAIL ligand. Such photo/TRAIL synergistic effect showed its great killing effects in a controllable manner on TRAIL-resistant A549 tumor model bearing mice. Finally, these nanocomposites exhibited rapid clearance without obvious systemic toxicity. All these features rendered our nanocomposites a promising theranostic platform in cancer therapy. Copyright © 2018 Elsevier Ltd. All rights reserved.

Repair-Resistant DNA Lesions

PubMed Central

2017-01-01

The eukaryotic global genomic nucleotide excision repair (GG-NER) pathway is the major mechanism that removes most bulky and some nonbulky lesions from cellular DNA. There is growing evidence that certain DNA lesions are repaired slowly or are entirely resistant to repair in cells, tissues, and in cell extract model assay systems. It is well established that the eukaryotic DNA lesion-sensing proteins do not detect the damaged nucleotide, but recognize the distortions/destabilizations in the native DNA structure caused by the damaged nucleotides. In this article, the nature of the structural features of certain bulky DNA lesions that render them resistant to NER, or cause them to be repaired slowly, is compared to that of those that are good-to-excellent NER substrates. Understanding the structural features that distinguish NER-resistant DNA lesions from good NER substrates may be useful for interpreting the biological significance of biomarkers of exposure of human populations to genotoxic environmental chemicals. NER-resistant lesions can survive to replication and cause mutations that can initiate cancer and other diseases. Furthermore, NER diminishes the efficacy of certain chemotherapeutic drugs, and the design of more potent pharmaceuticals that resist repair can be advanced through a better understanding of the structural properties of DNA lesions that engender repair-resistance. PMID:28750166

Syringyl-rich lignin renders poplars more resistant to degradation by wood decay fungi.

PubMed

Skyba, Oleksandr; Douglas, Carl J; Mansfield, Shawn D

2013-04-01

In order to elucidate the effects of lignin composition on the resistance of wood to degradation by decay fungi, wood specimens from two transgenic poplar lines expressing an Arabidopsis gene encoding ferulate 5-hydroxylase (F5H) driven by the cinnimate-4-hydroxylase promoter (C4H::F5H) that increased syringyl/guaiacyl (S/G) monolignol ratios relative to those in the untransformed control wood were incubated with six different wood decay fungi. Alterations in wood weight and chemical composition were monitored over the incubation period. The results showed that transgenic poplar lines extremely rich in syringyl lignin exhibited a drastically improved resistance to degradation by all decay fungi evaluated. Lignin monomer composition and its distribution among cell types and within different cell layers were the sole wood chemistry parameters determining wood durability. Since transgenic poplars with exceedingly high syringyl contents were recalcitrant to degradation, where wood durability is a critical factor, these genotypes may offer improved performance.

Syringyl-Rich Lignin Renders Poplars More Resistant to Degradation by Wood Decay Fungi

PubMed Central

Skyba, Oleksandr; Douglas, Carl J.

2013-01-01

In order to elucidate the effects of lignin composition on the resistance of wood to degradation by decay fungi, wood specimens from two transgenic poplar lines expressing an Arabidopsis gene encoding ferulate 5-hydroxylase (F5H) driven by the cinnimate-4-hydroxylase promoter (C4H::F5H) that increased syringyl/guaiacyl (S/G) monolignol ratios relative to those in the untransformed control wood were incubated with six different wood decay fungi. Alterations in wood weight and chemical composition were monitored over the incubation period. The results showed that transgenic poplar lines extremely rich in syringyl lignin exhibited a drastically improved resistance to degradation by all decay fungi evaluated. Lignin monomer composition and its distribution among cell types and within different cell layers were the sole wood chemistry parameters determining wood durability. Since transgenic poplars with exceedingly high syringyl contents were recalcitrant to degradation, where wood durability is a critical factor, these genotypes may offer improved performance. PMID:23396333

Regulation of tamoxifen sensitivity by a PAK1–EBP1 signalling pathway in breast cancer

PubMed Central

Ghosh, A; Awasthi, S; Peterson, J R; Hamburger, A W

2013-01-01

Background: EBP1, an ErbB3-binding protein, sensitises breast cancer cells to tamoxifen in part by decreasing ErbB2 protein levels. The p21-regulated serine/threonine kinase PAK1, implicated in tamoxifen resistance, phosphorylates EBP1 in vitro and in vivo at T261. Phosphorylation of EBP1 at this site induces tamoxifen resistance. We thus postulated that inhibition of PAK1 activity, by restoring EBP1 function, could ameliorate the hormone refractory phenotype of ErbB2-overexpressing breast cancer cells. Methods: Effects of EBP1 on ErbB2 levels were measured by western blotting. Effects of EBP1 and IPA-3 on tamoxifen sensitivity were measured using a tetrazolium based cell viability assay. Results: Transient transfection studies indicated that an EBP1 T261E mutant, which mimics EPB1 phosphorylated by PAK1, increased ErbB2 protein levels. An EBP1 T261A mutant, unable to be phosphorylated by PAK1, ameliorated PAK1-induced tamoxifen resistance, suggesting that phosphorylation of EBP1 by PAK1 contributes to tamoxifen resistance. We then tested if pharmacological inhibition of PAK1 activity might render hormone resistant cells, which endogenously overexpress PAK1, tamoxifen sensitive. IPA-3, a specific small MW PAK1 inhibitor, sensitised cells to tamoxifen only when EBP1 was ectopically expressed. IPA had no effect on tamoxifen resistance in T47D cells in which EBP1 protein had been ablated by shRNA. The IPA-induced increase in tamoxifen sensitivity was accompanied by a decrease in ErbB2 levels only in EBP1-overexpressing cells. Conclusion: These studies suggest that phosphorylation of EBP1 may be one mechanism of PAK1-induced hormone resistance and that PAK1 inhibitors may be useful in cells in which EBP1 is overexpressed. PMID:23361053

Nasopharyngeal infection by Streptococcus pyogenes requires superantigen-responsive Vβ-specific T cells

PubMed Central

Zeppa, Joseph J.; Kasper, Katherine J.; Mohorovic, Ivor; Mazzuca, Delfina M.

2017-01-01

The globally prominent pathogen Streptococcus pyogenes secretes potent immunomodulatory proteins known as superantigens (SAgs), which engage lateral surfaces of major histocompatibility class II molecules and T-cell receptor (TCR) β-chain variable domains (Vβs). These interactions result in the activation of numerous Vβ-specific T cells, which is the defining activity of a SAg. Although streptococcal SAgs are known virulence factors in scarlet fever and toxic shock syndrome, mechanisms by how SAgs contribute to the life cycle of S. pyogenes remain poorly understood. Herein, we demonstrate that passive immunization against the Vβ8-targeting SAg streptococcal pyrogenic exotoxin A (SpeA), or active immunization with either wild-type or a nonfunctional SpeA mutant, protects mice from nasopharyngeal infection; however, only passive immunization, or vaccination with inactive SpeA, resulted in high-titer SpeA-specific antibodies in vivo. Mice vaccinated with wild-type SpeA rendered Vβ8+ T cells poorly responsive, which prevented infection. This phenotype was reproduced with staphylococcal enterotoxin B, a heterologous SAg that also targets Vβ8+ T cells, and rendered mice resistant to infection. Furthermore, antibody-mediated depletion of T cells prevented nasopharyngeal infection by S. pyogenes, but not by Streptococcus pneumoniae, a bacterium that does not produce SAgs. Remarkably, these observations suggest that S. pyogenes uses SAgs to manipulate Vβ-specific T cells to establish nasopharyngeal infection. PMID:28794279

Pokemon silencing leads to Bim-mediated anoikis of human hepatoma cell QGY7703.

PubMed

Liu, Kun; Liu, Feng; Zhang, Nannan; Liu, Shiying; Jiang, Yuyang

2012-01-01

Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer.

Pokemon Silencing Leads to Bim-Mediated Anoikis of Human Hepatoma Cell QGY7703

PubMed Central

Liu, Kun; Liu, Feng; Zhang, Nannan; Liu, Shiying; Jiang, Yuyang

2012-01-01

Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer. PMID:22754333

A Zn-Dependent Metallopeptidase Is Responsible for Sensitivity to LsbB, a Class II Leaderless Bacteriocin of Lactococcus lactis subsp. lactis BGMN1-5

PubMed Central

Uzelac, Gordana; Lozo, Jelena; Aleksandrzak-Piekarczyk, Tamara; Gabrielsen, Christina; Kristensen, Tom; Nes, Ingolf F.; Diep, Dzung B.; Topisirovic, Ljubisa

2013-01-01

Lactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors. PMID:24123824

GDP-mannose-4,6-dehydratase (GMDS) Deficiency Renders Colon Cancer Cells Resistant to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Receptor- and CD95-mediated Apoptosis by Inhibiting Complex II Formation*

PubMed Central

Moriwaki, Kenta; Shinzaki, Shinichiro; Miyoshi, Eiji

2011-01-01

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through binding to TRAIL receptors, death receptor 4 (DR4), and DR5. TRAIL has potential therapeutic value against cancer because of its selective cytotoxic effects on several transformed cell types. Fucosylation of proteins and lipids on the cell surface is a very important posttranslational modification that is involved in many cellular events. Recently, we found that a deficiency in GDP-mannose-4,6-dehydratase (GMDS) rendered colon cancer cells resistant to TRAIL-induced apoptosis, resulting in tumor development and metastasis by escape from tumor immune surveillance. GMDS is an indispensable regulator of cellular fucosylation. In this study, we investigated the molecular mechanism of inhibition of TRAIL signaling by GMDS deficiency. DR4, but not DR5, was found to be fucosylated; however, GMDS deficiency inhibited both DR4- and DR5-mediated apoptosis despite the absence of fucosylation on DR5. In addition, GMDS deficiency also inhibited CD95-mediated apoptosis but not the intrinsic apoptosis pathway induced by anti-cancer drugs. Binding of TRAIL and CD95 ligand to their cognate receptors primarily leads to formation of a complex comprising the receptor, FADD, and caspase-8, referred to as the death-inducing signaling complex (DISC). GMDS deficiency did not affect formation of the primary DISC or recruitment to and activation of caspase-8 on the DISC. However, formation of secondary FADD-dependent complex II, comprising caspase-8 and cFLIP, was significantly inhibited by GMDS deficiency. These results indicate that GMDS regulates the formation of secondary complex II from the primary DISC independent of direct fucosylation of death receptors. PMID:22027835

Resistance to Cell Death and Its Modulation in Cancer Stem Cells

PubMed Central

Safa, Ahmad R.

2017-01-01

Accumulating evidence has demonstrated that human cancers arise from various tissues of origin that initiate from cancer stem cells (CSCs) or cancer-initiating cells. The extrinsic and intrinsic apoptotic pathways are dysregulated in CSCs, and these cells play crucial roles in tumor initiation, progression, cell death resistance, chemo- and radiotherapy resistance, and tumor recurrence. Understanding CSC-specific signaling proteins and pathways is necessary to identify specific therapeutic targets that may lead to the development of more efficient therapies selectively targeting CSCs. Several signaling pathways—including the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), maternal embryonic leucine zipper kinase (MELK), NOTCH1, and Wnt/β-catenin—and expression of the CSC markers CD133, CD24, CD44, Oct4, Sox2, Nanog, and ALDH1A1 maintain CSC properties. Studying such pathways may help to understand CSC biology and lead to the development of potential therapeutic interventions to render CSCs more sensitive to cell death triggered by chemotherapy and radiation therapy. Moreover, recent demonstrations of dedifferentiation of differentiated cancer cells into CSC-like cells have created significant complexity in the CSCs hypothesis. Therefore, any successful therapeutic agent or combination of drugs for cancer therapy must eliminate not only CSCs but differentiated cancer cells and the entire bulk of tumor cells. This review article expands on the CSC hypothesis and paradigm with respect to major signaling pathways and effectors that regulate CSC apoptosis resistance. Moreover, selective CSC apoptotic modulators and their therapeutic potential for making tumors more responsive to therapy are discussed. The use of novel therapies, including small-molecule inhibitors of specific proteins in signaling pathways that regulate stemness, proliferation and migration of CSCs, immunotherapy, and noncoding microRNAs may provide better means of treating CSCs. PMID:27915972

Predominant mode of human immunodeficiency virus transfer between T cells is mediated by sustained Env-dependent neutralization-resistant virological synapses.

PubMed

Chen, Ping; Hübner, Wolfgang; Spinelli, Matthew A; Chen, Benjamin K

2007-11-01

Cell-free human immunodeficiency virus type 1 (HIV-1) can initiate infections, but contact between infected and uninfected T cells can enhance viral spread through intercellular structures called virological synapses (VS). The relative contribution of VS to cell-free viral transfer has not been carefully measured. Using an ultrasensitive, fluorescent virus transfer assay, we estimate that when VS between HIV-expressing Jurkat T cells and primary CD4(+) T cells are formed, cell-associated transfer of virus is 18,000-fold more efficient than uptake of cell-free virus. Furthermore, in contrast to cell-free virus uptake, the VS deposits virus rapidly into focal, trypsin-resistant compartments in target T cells. This massive virus internalization requires Env-CD4 receptor interactions but is resistant to inhibition by patient-derived neutralizing antisera that inhibit homologous cell-free virus. Deleting the Env cytoplasmic tail does not abrogate VS-mediated transfer, but it renders the VS sensitive to neutralizing antibodies, suggesting that the tail limits exposure of VS-neutralizing epitopes on the surface of infected cells. Dynamic live imaging of the VS reveals that HIV-expressing cells are polarized and make sustained, Env-dependent contacts with target cells through uropod-like structures. The polarized T-cell morphology, Env-CD4 coordinated adhesion, and viral transfer from HIV-infected to uninfected cells suggest that VS allows HIV-1 to evade antibody neutralization and to disseminate efficiently. Future studies will discern to what extent this massive viral transfer contributes to productive infection or viral dissemination through the migration of virus-carrying T cells.

Cell cycle control in acute myeloid leukemia

PubMed Central

Schnerch, Dominik; Yalcintepe, Jasmin; Schmidts, Andrea; Becker, Heiko; Follo, Marie; Engelhardt, Monika; Wäsch, Ralph

2012-01-01

Acute myeloid leukemia (AML) is the result of a multistep transforming process of hematopoietic precursor cells (HPCs) which enables them to proceed through limitless numbers of cell cycles and to become resistant to cell death. Increased proliferation renders these cells vulnerable to acquiring mutations and may favor leukemic transformation. Here, we review how deregulated cell cycle control contributes to increased proliferation in AML and favors genomic instability, a prerequisite to confer selective advantages to particular clones in order to adapt and independently proliferate in the presence of a changing microenvironment. We discuss the connection between differentiation and proliferation with regard to leukemogenesis and outline the impact of specific alterations on response to therapy. Finally, we present examples, how a better understanding of cell cycle regulation and deregulation has already led to new promising therapeutic strategies. PMID:22957304

Role of extracellular matrix protein CabA in resistance of Vibrio vulnificus biofilms to decontamination strategies.

PubMed

Park, Jin Hwan; Lee, Byungho; Jo, Youmi; Choi, Sang Ho

2016-11-07

Biofilms are recalcitrant and raise safety problems in the food industry. In this study, the role of CabA, an extracellular matrix protein, in the resistance of the biofilms of Vibrio vulnificus, a foodborne pathogen, to decontamination strategies was investigated. Biofilms of the cabA mutant revealed reduced resistance to detachment by vibration and disinfection by sodium hypochlorite compared to the biofilms of the parental wild type in vitro. The reduced resistance of the cabA mutant biofilms was complemented by introducing a recombinant cabA, indicating that the reduced resistance of the cabA mutant biofilms is caused by the inactivation of cabA. The expression of cabA was induced in cells bound to oyster, the primary vehicle of the pathogen. The cabA mutant biofilms on oyster are defective in biomass and resistance to detachment and disinfection. The bacterial cells in the wild-type biofilms are clustered by filaments which are not apparent in the cabA mutant biofilms. The combined results indicated that CabA contributes to the structural integrity of V. vulnificus biofilms possibly by forming filaments in the matrix and thus rendering the biofilms robust, suggesting that CabA could be a target to control V. vulnificus biofilms on oyster. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel association between p130Cas and resistance to the chemotherapeutic drug adriamycin in human breast cancer cells.

PubMed

Ta, Huy Q; Thomas, Keena S; Schrecengost, Randy S; Bouton, Amy H

2008-11-01

Resistance to chemotherapy remains a major obstacle for the treatment of breast cancer. Understanding the molecular mechanism(s) of resistance is crucial for the development of new effective therapies to treat this disease. This study examines the putative role of p130(Cas) (Cas) in resistance to the cytotoxic agent Adriamycin. High expression of Cas in primary breast tumors is associated with the failure to respond to the antiestrogen tamoxifen and poor prognosis, highlighting the potential clinical importance of this molecule. Here, we show a novel association between Cas and resistance to Adriamycin. We show that Cas overexpression renders MCF-7 breast cancer cells less sensitive to the growth inhibitory and proapoptotic effects of Adriamycin. The catalytic activity of the nonreceptor tyrosine kinase c-Src, but not the epidermal growth factor receptor, is critical for Cas-mediated protection from Adriamycin-induced death. The phosphorylation of Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) is elevated in Cas-overexpressing cells treated with Adriamycin, whereas expression of the proapoptotic protein Bak is decreased. Conversely, Cas depletion in the more resistant T47D and MDA-MB-231 cell lines increases sensitivity to Adriamycin. Based on these data, we propose that Cas activates growth and survival pathways regulated by c-Src, Akt, and ERK1/2 that lead to the inhibition of mitochondrial-mediated apoptosis in the presence of Adriamycin. Because Cas is frequently expressed at high levels in breast cancers, these findings raise the possibility of resensitizing Cas-overexpressing tumors to chemotherapy through perturbation of Cas signaling pathways.

Phosphatidyl inositol-3 kinase (PIK3CA) E545K mutation confers cisplatin resistance and a migratory phenotype in cervical cancer cells.

PubMed

Arjumand, Wani; Merry, Cole D; Wang, Chen; Saba, Elias; McIntyre, John B; Fang, Shujuan; Kornaga, Elizabeth; Ghatage, Prafull; Doll, Corinne M; Lees-Miller, Susan P

2016-12-13

The phosphatidylinositol-3 kinase (PI3K)/Akt/mTOR signaling pathway is activated in many human cancers. Previously, we reported that patients with early stage cervical cancer whose tumours harbour PIK3CA exon 9 or 20 mutations have worse overall survival in response to treatment with radiation and cisplatin than patients with wild-type PIK3CA. The purpose of this study was to determine whether PIK3CA-E545K mutation renders cervical cancer cells more resistant to cisplatin and/or radiation, and whether PI3K inhibition reverses the phenotype. We found that CaSki cells that are heterozygous for the PIK3CA-E545K mutation are more resistant to cisplatin or cisplatin plus radiation than either HeLa or SiHa cells that express only wild-type PIK3CA. Similarly, HeLa cells engineered to stably express PIK3CA-E545K were more resistant to cisplatin or cisplatin plus radiation than cells expressing only wild-type PIK3CA or with PIK3CA depleted. Cells expressing the PIK3CA-E545K mutation also had constitutive PI3K pathway activation and increased cellular migration and each of these phenotypes was reversed by treatment with the PI3K inhibitor GDC-0941/Pictilisib. Our results suggests that cervical cancer patients whose tumours are positive for the PIK3CA-E545K mutation may benefit from PI3K inhibitor therapy in concert with standard cisplatin and radiation therapy.

Cancer of the Pancreas: Molecular Pathways and Current Advancement in Treatment.

PubMed

Polireddy, Kishore; Chen, Qi

2016-01-01

Pancreatic cancer is one of the most lethal cancers among all malignances, with a median overall survival of <1 year and a 5-year survival of ~5%. The dismal survival rate and prognosis are likely due to lack of early diagnosis, fulminant disease course, high metastasis rate, and disappointing treatment outcome. Pancreatic cancers harbor a variety of genetic alternations that render it difficult to treat even with targeted therapy. Recent studies revealed that pancreatic cancers are highly enriched with a cancer stem cell (CSC) population, which is resistant to chemotherapeutic drugs, and therefore escapes chemotherapy and promotes tumor recurrence. Cancer cell epithelial to mesenchymal transition (EMT) is highly associated with metastasis, generation of CSCs, and treatment resistance in pancreatic cancer. Reviewed here are the molecular biology of pancreatic cancer, the major signaling pathways regulating pancreatic cancer EMT and CSCs, and the advancement in current clinical and experimental treatments for pancreatic cancer.

Effect of simvastatin on the resistance to EGFR tyrosine kinase inhibitors in a non-small cell lung cancer with the T790M mutation of EGFR.

PubMed

Hwang, Ki-Eun; Kwon, Su-Jin; Kim, Young-Suk; Park, Do-Sim; Kim, Byoung-Ryun; Yoon, Kwon-Ha; Jeong, Eun-Taik; Kim, Hak-Ryul

2014-05-01

Although non-small cell lung cancer (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs) including gefitinib and erlotinib, development of acquired resistance is almost inevitable. Statins show antitumor activity, but it is unknown whether they can reverse EGFR-TKIs resistance in NSCLC with the T790M mutation of EGFR. This study investigated overcoming resistance to EGFR-TKI using simvastatin. We demonstrated that addition of simvastatin to gefitinib enhanced caspase-dependent apoptosis in T790M mutant NSCLC cells. Simvastatin also strongly inhibited AKT activation, leading to suppression of β-catenin activity and the expression of its targets, survivin and cyclin D1. Both insulin treatment and AKT overexpression markedly increased p-β-catenin and survivin levels, even in the presence of gefitinib and simvastatin. However, inhibition of AKT by siRNA or LY294002 treatment decreased p-β-catenin and survivin levels. To determine the role of survivin in simvastatin-induced apoptosis of gefitinib-resistant NSCLC, we showed that the proportion of apoptotic cells following treatment with survivin siRNA and the gefitinib-simvastatin combination was greater than the theoretical additive effects, whereas survivin up-regulation could confer protection against gefitinib and simvastatin-induced apoptosis. Similar results were obtained in erlotinib and simvastatin-treated HCC827/ER cells. These findings suggest that survivin is a key molecule that renders T790M mutant NSCLC cells resistant to apoptosis induced by EGFR-TKIs and simvastatin. Overall, these data indicate that simvastatin may overcome EGFR-TKI resistance in T790M mutant NSCLCs via an AKT/β-catenin signaling-dependent down-regulation of survivin and apoptosis induction. Copyright © 2014 Elsevier Inc. All rights reserved.

Antitumor activity of pluripotent cell-engineered vaccines and their potential to treat lung cancer in relation to different levels of irradiation

PubMed Central

Zhang, Yan-na; Duan, Xiao-gang; Zhang, Wen-hui; Wu, Ai-ling; Yang, Huan-Huan; Wu, Dong-ming; Wei, Yu-Quan; Chen, Xian-cheng

2016-01-01

Cancer stem cells (CSCs) are critical for tumor initiation/maintenance and recurrence or metastasis, so they may serve as a potential therapeutic target. However, CSC-established multitherapy resistance and immune tolerance render tumors resistant to current tumor-targeted strategies. To address this, renewable multiepitope-integrated spheroids based on placenta-derived mesenchymal stem cells (pMSCs) were X-ray-modified, at four different irradiation levels, including 80, 160, 240, and 320 Gy, as pluripotent biologics, to inoculate hosts bearing Lewis lung carcinoma (LL2) and compared with X-ray-modified common LL2 cells as control. We show that the vaccines at the 160/240 Gy irradiation levels could rapidly trigger tumor cells into the apoptosis loop and evidently prolong the tumor-bearing host’s survival cycle, in contrast to vaccines irradiated at other levels (P<0.05), with tumor-sustaining stromal cell-derived factor-1/CXCR4 pathway being selectively blockaded. Meanwhile, almost no or minimal toxicity was detected in the vaccinated hosts. Importantly, 160/240 Gy-irradiated vaccines could provoke significantly higher killing of CSCs and non-CSCs, which may provide an access to developing a novel biotherapy against lung carcinoma. PMID:27042111

Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor

PubMed Central

Goldgof, Gregory M.; Durrant, Jacob D.; Ottilie, Sabine; Vigil, Edgar; Allen, Kenneth E.; Gunawan, Felicia; Kostylev, Maxim; Henderson, Kiersten A.; Yang, Jennifer; Schenken, Jake; LaMonte, Gregory M.; Manary, Micah J.; Murao, Ayako; Nachon, Marie; Stanhope, Rebecca; Prescott, Maximo; McNamara, Case W.; Slayman, Carolyn W.; Amaro, Rommie E.; Suzuki, Yo; Winzeler, Elizabeth A.

2016-01-01

The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity. PMID:27291296

NEU3 Sialidase Is Activated under Hypoxia and Protects Skeletal Muscle Cells from Apoptosis through the Activation of the Epidermal Growth Factor Receptor Signaling Pathway and the Hypoxia-inducible Factor (HIF)-1α

PubMed Central

Scaringi, Raffaella; Piccoli, Marco; Papini, Nadia; Cirillo, Federica; Conforti, Erika; Bergante, Sonia; Tringali, Cristina; Garatti, Andrea; Gelfi, Cecilia; Venerando, Bruno; Menicanti, Lorenzo; Tettamanti, Guido; Anastasia, Luigi

2013-01-01

NEU3 sialidase, a key enzyme in ganglioside metabolism, is activated under hypoxic conditions in cultured skeletal muscle cells (C2C12). NEU3 up-regulation stimulates the EGF receptor signaling pathway, which in turn activates the hypoxia-inducible factor (HIF-1α), resulting in a final increase of cell survival and proliferation. In the same cells, stable overexpression of sialidase NEU3 significantly enhances cell resistance to hypoxia, whereas stable silencing of the enzyme renders cells more susceptible to apoptosis. These data support the working hypothesis of a physiological role played by NEU3 sialidase in protecting cells from hypoxic stress and may suggest new directions in the development of therapeutic strategies against ischemic diseases, particularly of the cerebro-cardiovascular system. PMID:23209287

NEU3 sialidase is activated under hypoxia and protects skeletal muscle cells from apoptosis through the activation of the epidermal growth factor receptor signaling pathway and the hypoxia-inducible factor (HIF)-1α.

PubMed

Scaringi, Raffaella; Piccoli, Marco; Papini, Nadia; Cirillo, Federica; Conforti, Erika; Bergante, Sonia; Tringali, Cristina; Garatti, Andrea; Gelfi, Cecilia; Venerando, Bruno; Menicanti, Lorenzo; Tettamanti, Guido; Anastasia, Luigi

2013-02-01

NEU3 sialidase, a key enzyme in ganglioside metabolism, is activated under hypoxic conditions in cultured skeletal muscle cells (C2C12). NEU3 up-regulation stimulates the EGF receptor signaling pathway, which in turn activates the hypoxia-inducible factor (HIF-1α), resulting in a final increase of cell survival and proliferation. In the same cells, stable overexpression of sialidase NEU3 significantly enhances cell resistance to hypoxia, whereas stable silencing of the enzyme renders cells more susceptible to apoptosis. These data support the working hypothesis of a physiological role played by NEU3 sialidase in protecting cells from hypoxic stress and may suggest new directions in the development of therapeutic strategies against ischemic diseases, particularly of the cerebro-cardiovascular system.

Cancer resistance of SR/CR mice in the genetic knockout backgrounds of leukocyte effector mechanisms: determinations for functional requirements.

PubMed

Sanders, Anne M; Stehle, John R; Blanks, Michael J; Riedlinger, Gregory; Kim-Shapiro, Jung W; Monjazeb, Arta M; Adams, Jonathan M; Willingham, Mark C; Cui, Zheng

2010-03-31

Spontaneous Regression/Complete Resistant (SR/CR) mice are a colony of cancer-resistant mice that can detect and rapidly destroy malignant cells with innate cellular immunity, predominately mediated by granulocytes. Our previous studies suggest that several effector mechanisms, such as perforin, granzymes, or complements, may be involved in the killing of cancer cells. However, none of these effector mechanisms is known as critical for granulocytes. Additionally, it is unclear which effector mechanisms are required for the cancer killing activity of specific leukocyte populations and the survival of SR/CR mice against the challenges of lethal cancer cells. We hypothesized that if any of these effector mechanisms was required for the resistance to cancer cells, its functional knockout in SR/CR mice should render them sensitive to cancer challenges. This was tested by cross breeding SR/CR mice into the individual genetic knockout backgrounds of perforin (Prf-/-), superoxide (Cybb-/), or inducible nitric oxide (Nos2-/). SR/CR mice were bred into individual Prf-/-, Cybb-/-, or Nos2-/- genetic backgrounds and then challenged with sarcoma 180 (S180). Their overall survival was compared to controls. The cancer killing efficiency of purified populations of macrophages and neutrophils from these immunodeficient mice was also examined. When these genetically engineered mice were challenged with cancer cells, the knockout backgrounds of Prf-/-, Cybb-/-, or Nos2-/- did not completely abolish the SR/CR cancer resistant phenotype. However, the Nos2-/- background did appear to weaken the resistance. Incidentally, it was also observed that the male mice in these immunocompromised backgrounds tended to be less cancer-resistant than SR/CR controls. Despite the previously known roles of perforin, superoxide or nitric oxide in the effector mechanisms of innate immune responses, these effector mechanisms were not required for cancer-resistance in SR/CR mice. The resistance was functional when any one of these effector mechanisms was completely absent, except some noticeably reduced penetrance, but not abolishment, of the phenotype in the male background in comparison to female background. These results also indicate that some other effector mechanism(s) of granulocytes may be involved in the killing of cancer cells in SR/CR mice.

PI3K/Akt inhibitor LY294002 potentiates homoharringtonine antimyeloma activity in myeloma cells adhered to stromal cells and in SCID mouse xenograft.

PubMed

Chen, Ping; Wen, Xiaofang; Wang, Bin; Hou, Diyu; Zou, Hong; Yuan, Qin; Yang, Hui; Xie, Jieqiong; Huang, Huifang

2018-05-01

Homoharringtonine (HHT) is a known anti-leukemia drug that inhibits multiple myeloma (MM) cells both in vitro and in vivo. Our prior study demonstrated that the potency of HHT in MM cells was compromised significantly when myeloma cells were co-cultured with BM stromal cells. This study aimed to investigate whether PI3K/Akt inhibitor LY294002 could potentiate the antimyeloma activity of HHT against MM cells adhered to BM stromal cells and in vivo xenograft models. A co-culture system composed of MM cells and human stromal cells was employed to mimic MM cells in bone marrow niche. The inhibitory and pro-apoptotic effect of HHT and LY294002 was determined by CCK-8 assay or flow cytometry. Expression of PI3K/Akt signaling molecules and anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1) was assessed by western blot analysis and/or reverse transcription real-time quantitative PCR (RT-qPCR). MM xenografts were used to evaluate antitumor effect of combined therapy with HHT and LY294002. Adhesion to BM stromal cells rendered MM cells resistant to HHT whereas silencing Mcl-1 partly reversed the resistance. LY294002 induced apoptosis in MM cells and potentiated the antimyeloma effects of HHT by inhibiting the PI3K/Akt signal pathway which was abnormally activated during adhesion. LY294002 also enhanced the antimyeloma effect of HHT in in vivo xenograft models. These findings suggest that activation of PI3K/Akt signal pathway was responsible for the resistance to HHT in MM cells adhered to stromal cells. LY294002 can potentiate the antimyeloma activity of HHT both in vitro and in vivo, which may represent a new clinical treatment in MM.

Role and regulation of sigma S in general resistance conferred by low-shear simulated microgravity in Escherichia coli.

PubMed

Lynch, S V; Brodie, E L; Matin, A

2004-12-01

Life on Earth evolved in the presence of gravity, and thus it is of interest from the perspective of space exploration to determine if diminished gravity affects biological processes. Cultivation of Escherichia coli under low-shear simulated microgravity (SMG) conditions resulted in enhanced stress resistance in both exponential- and stationary-phase cells, making the latter superresistant. Given that microgravity of space and SMG also compromise human immune response, this phenomenon constitutes a potential threat to astronauts. As low-shear environments are encountered by pathogens on Earth as well, SMG-conferred resistance is also relevant to controlling infectious disease on this planet. The SMG effect resembles the general stress response on Earth, which makes bacteria resistant to multiple stresses; this response is sigma s dependent, irrespective of the growth phase. However, SMG-induced increased resistance was dependent on sigma s only in stationary phase, being independent of this sigma factor in exponential phase. sigma s concentration was some 30% lower in exponential-phase SMG cells than in normal gravity cells but was twofold higher in stationary-phase SMG cells. While SMG affected sigma s synthesis at all levels of control, the main reasons for the differential effect of this gravity condition on sigma s levels were that it rendered the sigma protein less stable in exponential phase and increased rpoS mRNA translational efficiency. Since sigma s regulatory processes are influenced by mRNA and protein-folding patterns, the data suggest that SMG may affect these configurations.

Allicin disrupts the cell's electrochemical potential and induces apoptosis in yeast.

PubMed

Gruhlke, Martin C H; Portz, Daniela; Stitz, Michael; Anwar, Awais; Schneider, Thomas; Jacob, Claus; Schlaich, Nikolaus L; Slusarenko, Alan J

2010-12-15

The volatile substance allicin gives crushed garlic (Allium sativum) its characteristic odor and is a pro-oxidant that undergoes thiol-disulfide exchange reactions with -SH groups in proteins and glutathione. The antimicrobial activity of allicin is suspected to be due to the oxidative inactivation of essential thiol-containing enzymes. We investigated the hypothesis that at threshold inhibitory levels allicin can shunt yeast cells into apoptosis by altering their overall redox status. Yeast cells were treated either with chemically synthesized, pure allicin or with allicin in garlic juice. Allicin-dependent cell oxidation was demonstrated with a redox-sensitive GFP construct and the shift in cellular electrochemical potential (E(hc)) from less than -215 to -181mV was calculated using the Nernst equation after the glutathione/glutathione disulfide couple (2GSH/GSSG) in the cell was quantified. Caspase activation occurred after allicin treatment, and yeast expressing a human antiapoptotic Bcl-XL construct was rendered more resistant to allicin. Also, a yeast apoptosis-inducing factor deletion mutant was more resistant to allicin than wild-type cells. We conclude that allicin in garlic juice can activate apoptosis in yeast cells through its oxidizing properties and that this presents an alternative cell-killing mechanism to the previously proposed specific oxidative inactivation of essential enzymes. Copyright © 2010 Elsevier Inc. All rights reserved.

Metabolic stability of some tachykinin analogues to cell-surface peptidases: roles for endopeptidase-24.11 and aminopeptidase N.

PubMed

Medeiros, M D; Turner, A J

1995-01-01

The metabolism of several tachykinin antagonists by membrane peptidases has been examined. [beta Ala8]NKA(4-10) was not stabilized against degradation by endopeptidase-24.11 and this was the major activity in renal brush border membranes hydrolyzing this peptide. The antagonist MEN 10263 was much more resistant to hydrolysis by endopeptidase-24.11, although hydrolysis of the C-terminal Leu-Phe bond was detectable. Three other tachykinin receptor antagonists (MEN 10208, MEN 10207, and MEN 10376), by virtue of D-Trp substitutions, were rendered resistant to endopeptidase-24.11 but were still susceptible to aminopeptidase action. These studies provide further insight into design features necessary to produce metabolically stable peptide analogues.

Hyper-O-GlcNAcylation induces cisplatin resistance via regulation of p53 and c-Myc in human lung carcinoma.

PubMed

Luanpitpong, Sudjit; Angsutararux, Paweorn; Samart, Parinya; Chanthra, Nawin; Chanvorachote, Pithi; Issaragrisil, Surapol

2017-09-06

Aberrant metabolism in hexosamine biosynthetic pathway (HBP) has been observed in several cancers, affecting cellular signaling and tumor progression. However, the role of O-GlcNAcylation, a post-translational modification through HBP flux, in apoptosis remains unclear. Here, we found that hyper-O-GlcNAcylation in lung carcinoma cells by O-GlcNAcase inhibition renders the cells to apoptosis resistance to cisplatin (CDDP). Profiling of various key regulatory proteins revealed an implication of either p53 or c-Myc in the apoptosis regulation by O-GlcNAcylation, independent of p53 status. Using co-immunoprecipitation and correlation analyses, we found that O-GlcNAcylation of p53 under certain cellular contexts, i.e. high p53 activation, promotes its ubiquitin-mediated proteasomal degradation, resulting in a gain of oncogenic and anti-apoptotic functions. By contrast, O-GlcNAcylation of c-Myc inhibits its ubiquitination and subsequent proteasomal degradation. Gene manipulation studies revealed that O-GlcNAcylation of p53/c-Myc is in part a regulator of CDDP-induced apoptosis. Accordingly, we classified CDDP resistance by hyper-O-GlcNAcylation in lung carcinoma cells as either p53 or c-Myc dependence based on their molecular targets. Together, our findings provide novel mechanisms for the regulation of lung cancer cell apoptosis that could be important in understanding clinical drug resistance and suggest O-GlcNAcylation as a potential target for cancer therapy.

Battling Btk Mutants With Noncovalent Inhibitors That Overcome Cys481 and Thr474 Mutations.

PubMed

Johnson, Adam R; Kohli, Pawan Bir; Katewa, Arna; Gogol, Emily; Belmont, Lisa D; Choy, Regina; Penuel, Elicia; Burton, Luciana; Eigenbrot, Charles; Yu, Christine; Ortwine, Daniel F; Bowman, Krista; Franke, Yvonne; Tam, Christine; Estevez, Alberto; Mortara, Kyle; Wu, Jiansheng; Li, Hong; Lin, May; Bergeron, Philippe; Crawford, James J; Young, Wendy B

2016-10-21

The Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown impressive clinical efficacy in a range of B-cell malignancies. However, acquired resistance has emerged, and second generation therapies are now being sought. Ibrutinib is a covalent, irreversible inhibitor that modifies Cys481 in the ATP binding site of Btk and renders the enzyme inactive, thereby blocking B-cell receptor signal transduction. Not surprisingly, Cys481 is the most commonly mutated Btk residue in cases of acquired resistance to ibrutinib. Mutations at other sites, including Thr474, a gatekeeper residue, have also been detected. Herein, we describe noncovalent Btk inhibitors that differ from covalent inhibitors like ibrutinib in that they do not interact with Cys481, they potently inhibit the ibrutinib-resistant Btk C481S mutant in vitro and in cells, and they are exquisitely selective for Btk. Noncovalent inhibitors such as GNE-431 also show excellent potency against the C481R, T474I, and T474M mutants. X-ray crystallographic analysis of Btk provides insight into the unique mode of binding of these inhibitors that explains their high selectivity for Btk and their retained activity against mutant forms of Btk. This class of noncovalent Btk inhibitors may provide a treatment option to patients, especially those who have acquired resistance to ibrutinib by mutation of Cys481 or Thr474.

Two complementary strategies to improve cell engraftment in mesenchymal stem cell-based therapy: Increasing transplanted cell resistance and increasing tissue receptivity.

PubMed

Ezquer, Fernando E; Ezquer, Marcelo E; Vicencio, Jose M; Calligaris, Sebastián D

2017-01-02

Over the past 2 decades, therapies based on mesenchymal stem cells (MSC) have been tested to treat several types of diseases in clinical studies, due to their potential for tissue repair and regeneration. Currently, MSC-based therapy is considered a biologically safe procedure, with the therapeutic results being very promising. However, the benefits of these therapies are not stable in the long term, and the final outcomes manifest with high inter-patient variability. The major cause of these therapeutic limitations results from the poor engraftment of the transplanted cells. Researchers have developed separate strategies to improve MSC engraftment. One strategy aims at increasing the survival of the transplanted MSCs in the recipient tissue, rendering them more resistant to the hostile microenvironment (cell-preconditioning). Another strategy aims at making the damaged tissue more receptive to the transplanted cells, favoring their interactions (tissue-preconditioning). In this review, we summarize several approaches using these strategies, providing an integral and updated view of the recent developments in MSC-based therapies. In addition, we propose that the combined use of these different conditioning strategies could accelerate the process to translate experimental evidences from pre-clinic studies to the daily clinical practice.

Corrosion-resistant metal surfaces

DOEpatents

Sugama, Toshifumi [Wading River, NY

2009-03-24

The present invention relates to metal surfaces having thereon an ultrathin (e.g., less than ten nanometer thickness) corrosion-resistant film, thereby rendering the metal surfaces corrosion-resistant. The corrosion-resistant film includes an at least partially crosslinked amido-functionalized silanol component in combination with rare-earth metal oxide nanoparticles. The invention also relates to methods for producing such corrosion-resistant films.

Induction of Autophagic Cell Death in Apoptosis-resistant Pancreatic Cancer Cells using Benzo[α]phenoxazines Derivatives, 10-methyl-benzo[α]phenoxazine-5-one and benzo[α]phenoxazine-5-one.

PubMed

Pal, Sanjima; Salunke-Gawalib, Sunita; Konkimallaa, V Badireenath

2017-01-01

Intrinsic resistance to apoptotic cell death due to co-occurrence of mutated KRAS and p53 is frequently reported in pancreatic cancer that renders them aggressive, highly proliferative and metastatic. In addition, these cancer types are less sensitive to apoptosis inducing drugs where promotion of autophagic cell death could be a viable option for treatment under such circumstances. In this study we examined the potential of three intrinsically fluorescent benzo[α]phenoxazines or BPZs (R=Cl, CH3, H) to induce cytotoxic autophagy in chemo and apoptosis-resistant, KRAS and p53 mutated pancreatic cancer model cell line, MIAPaCa-2. Cells were adapted at in vitro metabolically stressed condition (5% serum) to initiate intrinsic cell survival strategies within. Cell proliferation, colonogenicity, cellular uptake, retention, localization, cellular granularity and presence of both apoptosis and autophagy biomarkers were assessed in BPZ treated/untreated (solvent) cells to validate induction of concentration dependent cytotoxic autophagy and other consequences. For the first time, we report the ability of this class of compounds to accumulate within cells increasing its granularity, inducing death via autophagy. From different kinetics study, it was observed that the autophagic-cell death was dependent on the ligand type, duration of incubation or working concentrations. Among the three BPZ tested, both 3B (benzo[α]phenoxazine-5-one) and 2B (10-methyl-benzo[α] phenoxazine-5-one) induced pro-death autophagy in MIAPaCa-2 cells at an IC50 of 5 μM and 20 μM respectively. Such compounds would be of great interest to explore as novel cytotoxic autophagy inducing agents in apoptosisresistant cancer types.

Generation of herpesvirus entry mediator (HVEM)-restricted herpes simplex virus type 1 mutant viruses: resistance of HVEM-expressing cells and identification of mutations that rescue nectin-1 recognition.

PubMed

Uchida, Hiroaki; Shah, Waris A; Ozuer, Ali; Frampton, Arthur R; Goins, William F; Grandi, Paola; Cohen, Justus B; Glorioso, Joseph C

2009-04-01

Both initial infection and cell-to-cell spread by herpes simplex virus type 1 (HSV-1) require the interaction of the viral glycoprotein D (gD) with an entry receptor on the cell surface. The two major HSV entry receptors, herpesvirus entry mediator (HVEM) and nectin-1, mediate infection independently but are coexpressed on a variety of cells. To determine if both receptors are active in these instances, we have established mutant viruses that are selectively impaired for recognition of one or the other receptor. In plaque assays, these viruses showed approximately 1,000-fold selectivity for the matched receptor over the mismatched receptor. Separate assays showed that each virus is impaired for both infection and spread through the mismatched receptor. We tested several human tumor cell lines for susceptibility to these viruses and observed that HT29 colon carcinoma cells are susceptible to infection by nectin-1-restricted virus but are highly resistant to HVEM-restricted virus infection, despite readily detectable HVEM expression on the cell surface. HVEM cDNA isolated from HT29 cells rendered HSV-resistant cells permissive for infection by the HVEM-restricted virus, suggesting that HT29 cells lack a cofactor for HVEM-mediated infection or express an HVEM-specific inhibitory factor. Passaging of HVEM-restricted virus on nectin-1-expressing cells yielded a set of gD missense mutations that each restored functional recognition of nectin-1. These mutations identify residues that likely play a role in shaping the nectin-1 binding site of gD. Our findings illustrate the utility of these receptor-restricted viruses in studying the early events in HSV infection.

Inhibition of Fas (CD95) expression and Fas-mediated apoptosis by oncogenic Ras.

PubMed

Fenton, R G; Hixon, J A; Wright, P W; Brooks, A D; Sayers, T J

1998-08-01

The ras oncogene plays an important role in the multistep progression to cancer by activation of signal transduction pathways that contribute to aberrant growth regulation. Although many of these effects are cell autonomous, the ras oncogene also regulates the expression of genes that alter host/tumor interactions. We now extend the mechanisms through which ras promotes tumor survival by demonstrating that oncogenic Ras inhibits expression of the fas gene and renders Ras-transformed cells resistant to Fas-induced apoptosis. A panel of Ras-transformed clones exhibited a marked inhibition in fas mRNA and Fas cell surface expression as compared with untransformed parental cell lines. Fas expression was induced by culture in the presence of IFN-gamma + tumor necrosis factor alpha; however, the maximal level attained in Ras transformants was approximately 10-fold below the level of untransformed cells. Whereas untransformed cells were sensitive to apoptotic death induced by cross-linking surface Fas (especially after cytokine treatment), Ras-transformed cells were very resistant to Fas-induced death even under the most stringent assay conditions. To demonstrate that this resistance was mediated by oncogenic Ras and not secondary genetic events, pools of Ras-transformed cells were generated using a highly efficient retroviral transduction technique. Transformed pools were assayed 6 days after infection and demonstrated a marked decrease in fas gene expression and Fas-mediated apoptosis. Oncogenic Ras did not promote general resistance to apoptosis, because ectopic expression of a fas cDNA in Ras-transformed cells restored sensitivity to Fas-induced apoptosis. These data indicate that oncogenic Ras inhibits basal levels of expression of the fas gene, and although cytokine signal transduction pathways are functional in these cells, the level of surface Fas expression remains below the threshold required for induction of apoptosis. These data identify a mechanism by which Ras-transformed cells may escape from host-mediated immune destruction.

Vorinostat, a histone deacetylase (HDAC) inhibitor, promotes cell cycle arrest and re-sensitizes rituximab- and chemo-resistant lymphoma cells to chemotherapy agents.

PubMed

Xue, Kai; Gu, Juan J; Zhang, Qunling; Mavis, Cory; Hernandez-Ilizaliturri, Francisco J; Czuczman, Myron S; Guo, Ye

2016-02-01

Preclinical models of chemotherapy resistance and clinical observations derived from the prospective multicenter phase III collaborative trial in relapsed aggressive lymphoma (CORAL) study demonstrated that primary refractory/relapsed B cell diffuse large B cell lymphoma has a poor clinical outcome with current available second-line treatments. Preclinically, we found that rituximab resistance is associated with a deregulation on the mitochondrial potential rendering lymphoma cells resistant to chemotherapy-induced apoptotic stimuli. There is a dire need to develop agents capable to execute alternative pathways of cell death in an attempt to overcome chemotherapy resistance. Posttranscriptional histone modification plays an important role in regulating gene transcription and is altered by histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs regulate several key cellular functions, including cell proliferation, cell cycle, apoptosis, angiogenesis, migration, antigen presentation, and/or immune regulation. Given their influence in multiple regulatory pathways, HDAC inhibition is an attractive strategy to evaluate its anti-proliferation activity in cancer cells. To this end, we studied the anti-proliferation activity and mechanisms of action of suberoylanilide hydroxamic acid (SAHA, vorinostat) in rituximab-chemotherapy-resistant preclinical models. A panel of rituximab-chemotherapy-sensitive (RSCL) and rituximab-chemotherapy-resistant cell lines (RRCL) and primary tumor cells isolated from relapsed/refractory B cell lymphoma patients were exposed to escalating doses of vorinostat. Changes in mitochondrial potential, ATP synthesis, and cell cycle distribution were determined by Alamar blue reduction, Titer-Glo luminescent assays, and flow cytometric, respectively. Protein lysates were isolated from vorinostat-exposed cells, and changes in members of Bcl-2 family, cell cycle regulatory proteins, and the acetylation status of histone H3 were evaluated by Western blotting. Finally, cell lines were pre-exposed to vorinostat for 48 h and subsequently exposed to several chemotherapy agents (cisplatin, etoposide, or gemcitabine); changes in cell viability were determined by CellTiter-Glo(®) luminescence assay (Promega, Fitchburg, WI), and synergistic activity was evaluated using the CalcuSyn software. Vorinostat induced dose-dependent cell death in RRCL and in primary tumor cells. In addition, in vitro exposure of RRCL to vorinostat resulted in an increase in p21 and acetylation of histone H3 leading to G1 cell cycle arrest. Vorinostat exposure resulted in apoptosis in RSCL cell lines but not in RRCL. This finding suggests that in RRCL, vorinostat induces cell death by alternative pathways (i.e., irreversible cell cycle arrest). Of interest, vorinostat was found to reverse acquired chemotherapy resistance in RRCL. Our data suggest that vorinostat is active in RRCL with a known defective apoptotic machinery, it can active alternative cell death pathways. Given the multiple pathways affected by HDAC inhibition, vorinostat can potentially be used to overcome acquired resistant to chemotherapy in aggressive B cell lymphoma.

Host-Directed Antimicrobial Drugs with Broad-Spectrum Efficacy against Intracellular Bacterial Pathogens

PubMed Central

Czyż, Daniel M.; Potluri, Lakshmi-Prasad; Jain-Gupta, Neeta; Riley, Sean P.; Martinez, Juan J.; Steck, Theodore L.; Crosson, Sean; Gabay, Joëlle E.

2014-01-01

ABSTRACT We sought a new approach to treating infections by intracellular bacteria, namely, by altering host cell functions that support their growth. We screened a library of 640 Food and Drug Administration (FDA)-approved compounds for agents that render THP-1 cells resistant to infection by four intracellular pathogens. We identified numerous drugs that are not antibiotics but were highly effective in inhibiting intracellular bacterial growth with limited toxicity to host cells. These compounds are likely to target three kinds of host functions: (i) G protein-coupled receptors, (ii) intracellular calcium signals, and (iii) membrane cholesterol distribution. The compounds that targeted G protein receptor signaling and calcium fluxes broadly inhibited Coxiella burnetii, Legionella pneumophila, Brucella abortus, and Rickettsia conorii, while those directed against cholesterol traffic strongly attenuated the intracellular growth of C. burnetii and L. pneumophila. These pathways probably support intracellular pathogen growth so that drugs that perturb them may be therapeutic candidates. Combining host- and pathogen-directed treatments is a strategy to decrease the emergence of drug-resistant intracellular bacterial pathogens. PMID:25073644

ABCG2 transporter inhibitor restores the sensitivity of triple negative breast cancer cells to aminolevulinic acid-mediated photodynamic therapy.

PubMed

Palasuberniam, Pratheeba; Yang, Xue; Kraus, Daniel; Jones, Patrick; Myers, Kenneth A; Chen, Bin

2015-08-18

Photosensitizer protoporphyrin IX (PpIX) fluorescence, intracellular localization and cell response to photodynamic therapy (PDT) were analyzed in MCF10A normal breast epithelial cells and a panel of human breast cancer cells including estrogen receptor (ER) positive, human epidermal growth factor receptor 2 (HER2) positive and triple negative breast cancer (TNBC) cells after treatment with PpIX precursor aminolevulinic acid (ALA). Although PpIX fluorescence was heterogeneous in different cells, TNBC cells showed significantly lower PpIX level than MCF10A and ER- or HER2-positive cells. PpIX fluorescence in TNBC cells also had much less mitochondrial localization than other cells. There was an inverse correlation between PpIX fluorescence and cell viability after PDT. Breast cancer cells with the highest PpIX fluorescence were the most sensitive to ALA-PDT and TNBC cells with the lowest PpIX level were resistant to PDT. Treatment of TNBC cells with ABCG2 transporter inhibitor Ko143 significantly increased ALA-PpIX fluorescence, enhanced PpIX mitochondrial accumulation and sensitized cancer cells to ALA-PDT. Ko143 treatment had little effect on PpIX production and ALA-PDT in normal and ER- or HER2-positive cells. These results demonstrate that enhanced ABCG2 activity renders TNBC cell resistance to ALA-PDT and inhibiting ABCG2 transporter is a promising approach for targeting TNBC with ALA-based modality.

Antibacterial activity of 3-methylbenzo[d]thiazol-methylquinolinium derivatives and study of their action mechanism.

PubMed

Sun, Ning; Du, Ruo-Lan; Zheng, Yuan-Yuan; Guo, Qi; Cai, Sen-Yuan; Liu, Zhi-Hua; Fang, Zhi-Yuan; Yuan, Wen-Chang; Liu, Ting; Li, Xiao-Mei; Lu, Yu-Jing; Wong, Kwok-Yin

2018-12-01

The increasing incidence of multidrug resistant bacterial infection renders an urgent need for the development of new antibiotics. To develop small molecules disturbing FtsZ activity has been recognized as promising approach to search for antibacterial of high potency systematically. Herein, a series of novel quinolinium derivatives were synthesized and their antibacterial activities were investigated. The compounds show strong antibacterial activities against different bacteria strains including MRSA, VRE and NDM-1 Escherichia coli. Among these derivatives, a compound bearing a 4-fluorophenyl group (A2) exhibited a superior antibacterial activity and its MICs to the drug-resistant strains are found lower than those of methicillin and vancomycin. The biological results suggest that these quinolinium derivatives can disrupt the GTPase activity and dynamic assembly of FtsZ, and thus inhibit bacterial cell division and then cause bacterial cell death. These compounds deserve further evaluation for the development of new antibacterial agents targeting FtsZ.

Transient dominant host-range selection using Chinese hamster ovary cells to generate marker-free recombinant viral vectors from vaccinia virus.

PubMed

Liu, Liang; Cooper, Tamara; Eldi, Preethi; Garcia-Valtanen, Pablo; Diener, Kerrilyn R; Howley, Paul M; Hayball, John D

2017-04-01

Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses. Here, we describe a new method for the construction of rVACVs using the cowpox CP77 protein and unmodified VACV as the starting material. Our selection system will expand the range of tools available for positive selection of rVACV during vector construction, and it is substantially more high-fidelity than approaches based on selection for drug resistance.

Potential for Bacteriophage Endolysins to Supplement or Replace Antibiotics in Food Production and Clinical Care

PubMed Central

Love, Michael J.; Bhandari, Dinesh; Dobson, Renwick C. J.

2018-01-01

There is growing concern about the emergence of bacterial strains showing resistance to all classes of antibiotics commonly used in human medicine. Despite the broad range of available antibiotics, bacterial resistance has been identified for every antimicrobial drug developed to date. Alarmingly, there is also an increasing prevalence of multidrug-resistant bacterial strains, rendering some patients effectively untreatable. Therefore, there is an urgent need to develop alternatives to conventional antibiotics for use in the treatment of both humans and food-producing animals. Bacteriophage-encoded lytic enzymes (endolysins), which degrade the cell wall of the bacterial host to release progeny virions, are potential alternatives to antibiotics. Preliminary studies show that endolysins can disrupt the cell wall when applied exogenously, though this has so far proven more effective in Gram-positive bacteria compared with Gram-negative bacteria. Their potential for development is furthered by the prospect of bioengineering, and aided by the modular domain structure of many endolysins, which separates the binding and catalytic activities into distinct subunits. These subunits can be rearranged to create novel, chimeric enzymes with optimized functionality. Furthermore, there is evidence that the development of resistance to these enzymes may be more difficult compared with conventional antibiotics due to their targeting of highly conserved bonds. PMID:29495476

CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection

PubMed Central

Liu, Jingjing; Zhang, Di; Kimata, Jason T.; Zhou, Paul

2014-01-01

CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1. PMID:25541967

Susceptibility of ATM-deficient pancreatic cancer cells to radiation.

PubMed

Ayars, Michael; Eshleman, James; Goggins, Michael

2017-05-19

Ataxia telangiectasia mutated (ATM) is inactivated in a significant minority of pancreatic ductal adenocarcinomas and may be predictor of treatment response. We determined if ATM deficiency renders pancreatic cancer cells more sensitive to fractionated radiation or commonly used chemotherapeutics. ATM expression was knocked down in three pancreatic cancer cell lines using ATM-targeting shRNA. Isogenic cell lines were tested for sensitivity to several chemotherapeutic agents and radiation. DNA repair kinetics were analyzed in irradiated cells using the comet assay. We find that while rendering pancreatic cancer cells ATM-deficient did not significantly change their sensitivity to several chemotherapeutics, it did render them exquisitely sensitized to radiation. Pancreatic cancer ATM status may help predict response to radiotherapy.

Cigarette smoke induces aberrant EGF receptor activation which mediates lung cancer development and resistance to tyrosine kinase inhibitors

PubMed Central

Filosto, Simone; Becker, Cathleen R.; Goldkorn, Tzipora

2015-01-01

The EGF Receptor (EGFR) and its downstream signaling are implicated in lung cancer development. Therefore, much effort was spent in developing specific tyrosine kinase inhibitors (TKIs) that bind to the EGFR ATP-pocket, blocking EGFR phosphorylation/signaling. Clinical use of TKIs is effective in a subset of lung cancers with mutations in the EGFR kinase domain, rendering the receptor highly susceptible to TKIs. However, these benefits are limited, and emergence of additional EGFR mutations usually results in TKI resistance and disease progression. Previously, we demonstrated one mechanism linking cigarette smoke (CS) to EGFR-driven lung cancer. Specifically, exposure of lung epithelial cells to CS-induced oxidative stress stimulates aberrant EGFR phosphorylation/activation with impaired receptor ubiquitination/degradation. The abnormal stabilization of the activated receptor leads to uncontrolled cell growth and tumorigenesis. Here we describe for the first time a novel post-translational mechanism of EGFR resistance to TKIs. Exposure of airway epithelial cells to CS causes aberrant phosphorylation/activation of EGFR, resulting in a conformation that is different from that induced by the ligand EGF. Unlike EGF-activated EGFR, CS-activated EGFR binds c-Src and caveolin-1 and does not undergo canonical dimerization. Importantly, the CS-activated EGFR is not inhibited by TKIs (AG1478; Erlotinib; Gefitinib); in fact, the CS exposure induces TKI-resistance even in the TKI-sensitive EGFR mutants. Our findings demonstrate that CS exposure stimulates not only aberrant EGFR phosphorylation impairing receptor degradation, but also induces a different EGFR conformation and signaling that are resistant to TKIs. Together, these findings offer new insights into CS-induced lung cancer development and TKI resistance. PMID:22302097

Electrical properties of graphene film for counter electrode in dye sensitized solar cells

NASA Astrophysics Data System (ADS)

Khalifa, Ali; Shafie, S.; Hasan, W. Z. W.; Lim, H. N.; Rusop, M.; Samaila, Buda

2018-05-01

A graphene counter electrode for dye-sensitized solar cell was prepared simply by drop casting method on a conducting FTO glass at room temperature. Raman spectroscopy was used to study the defection in the graphene films. The sheet resistance was also measured and recoded minimum value of 7.04 Ω/□ at 22.19µm thickness. The casted films show good adhesion to substrates with low defects. A DSSC based on graphene counter electrode demonstrates reasonable conversion efficiency of 2.78% with short circuit current of 7.60mA, open circuit voltage of 0.69V and fill factor of 0.52. The high conductivity and low defects render the prepared graphene dispersion for DSSCs' CE application.

A Model to Estimate the Operating and Maintenance (O&M) Costs of the Mine Resistant Ambush Protected (MRAP) Vehicles

DTIC Science & Technology

2010-12-01

industry will yield a different learning curve slope. Table 4 ( Heizer & Render , PowerPoint presentation, 2008, slides 7–8) shows some examples of the...September 28, 2010 from http://www.globalsecurity.org/military/systems/ground/mrap-var.htm Heizer , J., & Render , B. (2008). Operations Management

Targeting cholesterol synthesis increases chemoimmuno-sensitivity in chronic lymphocytic leukemia cells

PubMed Central

2014-01-01

Background Cholesterol plays an important role in cancer development, drug resistance and chemoimmuno-sensitivity. Statins, cholesterol lowering drugs, can induce apoptosis, but also negatively interfere with CD-20 and rituximab-mediated activity. Our goal is to identify the alternative targets that could reduce cholesterol levels but do not interfere with CD-20 in chemo immunotherapy of chronic lymphocytic leukemia (CLL). Methods MEC-2 cells, a CLL cell line, and the peripheral blood mononuclear cells (PBMCs) from CLL patients were treated with cholesterol lowering agents, and analyzed the effect of these agents on cholesterol levels, CD-20 expression and distribution, and cell viability in the presence or absence of fludarabine, rituximab or their combinations. Results We found that MEC-2 cells treated with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) reduced 20% of total cellular cholesterol levels, but also significantly promoted CD-20 surface expression. Furthermore, treatment of cells with fludarabine, rituximab or their combinations in the presence of BIBB-515, YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity measured by cell viability. More importantly, these cholesterol lowering agents also significantly enhanced chemoimmuno-sensitivity of the PBMCs from CLL patients. Conclusion Our data demonstrate that BIBB-515, YM53601 and TAK-475 render chemoimmuno-therapy resistant MEC-2 cells sensitive to chemoimmuno-therapy and enhance CLL cell chemoimmuno-sensitivity without CD-20 epitope presentation or its downstream signaling. These results provide a novel strategy which could be applied to CLL treatment. PMID:25401046

Targeting cholesterol synthesis increases chemoimmuno-sensitivity in chronic lymphocytic leukemia cells.

PubMed

Benakanakere, Indira; Johnson, Tyler; Sleightholm, Richard; Villeda, Virgilio; Arya, Monika; Bobba, Ravi; Freter, Carl; Huang, Chunfa

2014-01-01

Cholesterol plays an important role in cancer development, drug resistance and chemoimmuno-sensitivity. Statins, cholesterol lowering drugs, can induce apoptosis, but also negatively interfere with CD-20 and rituximab-mediated activity. Our goal is to identify the alternative targets that could reduce cholesterol levels but do not interfere with CD-20 in chemo immunotherapy of chronic lymphocytic leukemia (CLL). MEC-2 cells, a CLL cell line, and the peripheral blood mononuclear cells (PBMCs) from CLL patients were treated with cholesterol lowering agents, and analyzed the effect of these agents on cholesterol levels, CD-20 expression and distribution, and cell viability in the presence or absence of fludarabine, rituximab or their combinations. We found that MEC-2 cells treated with cholesterol lowering agents (BIBB-515, YM-53601 or TAK-475) reduced 20% of total cellular cholesterol levels, but also significantly promoted CD-20 surface expression. Furthermore, treatment of cells with fludarabine, rituximab or their combinations in the presence of BIBB-515, YM-53601 or TAK-475 enhanced MEC-2 cell chemoimmuno-sensitivity measured by cell viability. More importantly, these cholesterol lowering agents also significantly enhanced chemoimmuno-sensitivity of the PBMCs from CLL patients. Our data demonstrate that BIBB-515, YM53601 and TAK-475 render chemoimmuno-therapy resistant MEC-2 cells sensitive to chemoimmuno-therapy and enhance CLL cell chemoimmuno-sensitivity without CD-20 epitope presentation or its downstream signaling. These results provide a novel strategy which could be applied to CLL treatment.

Microbial fuel cell characterisation and evaluation of Lysinibacillus macroides MFC02 electrigenic capability.

PubMed

Uma Vanitha, Murugan; Natarajan, Muthusamy; Sridhar, Harikrishnamoorthy; Umamaheswari, Sankaran

2017-05-01

Microbial fuel cell (MFC) is the most prominent research field due to its capability to generate electricity by utilizing the renewable sources. In the present study, Two MFC designs namely, H type-Microbial fuel cell (HT-MFC) and U type-Microbial fuel cell (UT-MFC) were constructed based on standardized H shaped anode and cathode compartment as well as U shaped anode and cathode compartments, respectively. In order to lower the cost for MFC construction, Pencil graphite lead was used as electrode and salt agar as Proton exchange membrane. Results inferred that newly constructed UT-MFC showed high electron production when compared to the HT-MFC. UT-MFC displayed an output of about 377 ± 18.85 mV (millivolts); whereas HT-MFC rendered only 237 ± 11.85 mV (millivolts) of power generation, which might be due to the low internal resistance. By increasing the number of cathode in UT-MFC, power production was increased upto 313 ± 15.65 mV in Open circuit voltage (OCV). Electrogenic bacteria namely, Lysinibacillus macroides (Acc. No. KX011879) rendered enriched power generation. The attachment of bacteria as a biofilm on pencil graphite lead was analyzed using fluorescent microscope and Scanning Electron Microscope (SEM). Based on our findings, it was observed that UT-MFC has a tendency to produce high electron generation using pencil graphite lead as the electrode material.

Plk1 is upregulated in androgen-insensitive prostate cancer cells and its inhibition leads to necroptosis

PubMed Central

Deeraksa, Arpaporn; Pan, Jing; Sha, Youbao; Liu, Xian-De; Eissa, N Tony; Lin, Sue-Hwa; Yu-Lee, Li-yuan

2012-01-01

Castration-resistant prostate cancer (PCa) is refractory to hormone therapy and new strategies for treatment are urgently needed. We found that androgen-insensitive (AI) PCa cells, LNCaP-AI, are reprogrammed to upregulate the mitotic kinase Plk1 and other M phase cell cycle proteins, which may underlie AI PCa growth. In androgen-depleted media, LNCaP-AI cells showed exquisite sensitivity to growth inhibition by subnanomolar concentrations of a small molecule inhibitor of Plk1, BI2536, suggesting that these cells are dependent on Plk1 for growth. In contrast, the androgen-responsive parental LNCaP cells showed negligible responses to BI2536 treatment under the same condition. BI2536 treatment of LNCaP-AI cells resulted in an increase in cell death marker PARP-1 but did not activate caspase-3, an apoptosis marker, suggesting that the observed cell death was caspase-independent. BI2536-treated LNCaP-AI cells formed multinucleated giant cells that contain clusters of nuclear vesicles indicative of mitotic catastrophe. Live-cell time-lapse imaging revealed that BI2536-treated giant LNCaP-AI cells underwent necroptosis, as evidenced by “explosive” cell death and partial reversal of cell death by a necroptosis inhibitor. Our studies suggest that LNCaP-AI cells underwent reprogramming in both their cell growth and cell death pathways, rendering them highly sensitive to Plk1 inhibition that induces necroptosis. Harnessing necroptosis through Plk1 inhibition may be explored for therapeutic intervention of castration-resistant PCa. PMID:22890325

NF-{kappa}B signaling is activated and confers resistance to apoptosis in three-dimensionally cultured EGFR-mutant lung adenocarcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakuma, Yuji, E-mail: ysakuma@gancen.asahi.yokohama.jp; Yamazaki, Yukiko; Nakamura, Yoshiyasu

2012-07-13

Highlights: Black-Right-Pointing-Pointer EGFR-mutant cells in 3D culture resist EGFR inhibition compared with suspended cells. Black-Right-Pointing-Pointer Degradation of I{kappa}B and activation of NF-{kappa}B are observed in 3D-cultured cells. Black-Right-Pointing-Pointer Inhibiting NF-{kappa}B enhances the efficacy of the EGFR inhibitor in 3D-cultured cells. -- Abstract: Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells in suspension undergo apoptosis to a greater extent than adherent cells in a monolayer when EGFR autophosphorylation is inhibited by EGFR tyrosine kinase inhibitors (TKIs). This suggests that cell adhesion to a culture dish may activate an anti-apoptotic signaling pathway other than the EGFR pathway. Since the microenvironment of cellsmore » cultured in a monolayer are substantially different to that of cells existing in three-dimension (3D) in vivo, we assessed whether two EGFR-mutant lung adenocarcinoma cell lines, HCC827 and H1975, were more resistant to EGFR TKI-induced apoptosis when cultured in a 3D extracellular matrix (ECM) as compared with in suspension. The ECM-adherent EGFR-mutant cells in 3D were significantly less sensitive to treatment with WZ4002, an EGFR TKI, than the suspended cells. Further, a marked degradation of I{kappa}B{alpha}, the inhibitor of nuclear factor (NF)-{kappa}B, was observed only in the 3D-cultured cells, leading to an increase in the activation of NF-{kappa}B. Moreover, the inhibition of NF-{kappa}B with pharmacological inhibitors enhanced EGFR TKI-induced apoptosis in 3D-cultured EGFR-mutant cells. These results suggest that inhibition of NF-{kappa}B signaling would render ECM-adherent EGFR-mutant lung adenocarcinoma cells in vivo more susceptible to EGFR TKI-induced cell death.« less

Novel therapeutic applications of nitric oxide donors in cancer: roles in chemo- and immunosensitization to apoptosis and inhibition of metastases.

PubMed

Bonavida, Benjamin; Baritaki, Stavroula; Huerta-Yepez, Sara; Vega, Mario I; Chatterjee, Devasis; Yeung, Kam

2008-09-01

The treatment of primary tumors results in an initial response to approved conventional therapeutics. However, recurrences and malignancies develop as a result of tumors' acquisition of anti-apoptotic mechanisms of resistance. Hence, there is an urgent need of novel therapeutics that can reverse resistance. One approach of interest is the inhibition of cell survival and anti-apoptotic pathways by sensitizing agents that can render resistant tumor cells sensitive to respond to various cytotoxic therapies. We have found that nitric oxide donors, similar to DETANONOate, inhibit cell survival anti-apoptotic pathways, such as the constitutively activated NF-kappaB and sensitize drug-resistant tumor cells to apoptosis by both chemotherapy and immunotherapy. Sensitization by DETANONOate was shown to inhibit the transcription repressor Yin Yang1 (YY1) shown to regulate resistance to both Fas ligand and TRAIL. In addition, DETANONOate-induced inhibition of NF-kappaB results downstream in the inhibition of several anti-apoptotic gene products, thus facilitating the activation of the apoptotic pathways with both chemotherapy and immunotherapy. In addition, DETANONOate induces the expression of the metastatic tumor suppressor gene product, Raf-1 Kinase Inhibitor Protein (RKIP), which inhibits the survival pathways induced by NF-kappaB and Raf-1/MEK which also contributes to the sensitizing activity. This indicates a novel finding that RKIP may also play an important role in the prevention of metastasis. Inhibition of NF-kappaB activation by DETANONOate results downstream in the inhibition of the RKIP transcription repressor Snail, resulting in upregulation of RKIP. Inhibition of Snail results in downstream inhibition of the metastatic cascade initiated by the epithelial-mesenchymal transition (EMT). Thus, nitric oxide donors have the dual functions of both sensitizing tumor cells to chemotherapy and immunotherapy and are also involved in the regulation and inhibition of metastasis.

Inhibiting oncogenic signaling by sorafenib activates PUMA via GSK3β and NF-κB to suppress tumor cell growth.

PubMed

Dudgeon, C; Peng, R; Wang, P; Sebastiani, A; Yu, J; Zhang, L

2012-11-15

Aberrant Ras/Raf/MEK/ERK signaling is one of the most prevalent oncogenic alterations and confers survival advantage to tumor cells. Inhibition of this pathway can effectively suppress tumor cell growth. For example, sorafenib, a multi-kinase inhibitor targeting c-Raf and other oncogenic kinases, has been used clinically for treating advanced liver and kidney tumors, and also has shown efficacy against other malignancies. However, how inhibition of oncogenic signaling by sorafenib and other drugs suppresses tumor cell growth remains unclear. In this study, we found that sorafenib kills cancer cells by activating PUMA (p53-upregulated modulator of apoptosis), a p53 target and a BH3-only Bcl-2 family protein. Sorafenib treatment induces PUMA in a variety of cancer cells irrespective of their p53 status. Surprisingly, the induction of PUMA by sorafenib is mediated by IκB-independent activation of nuclear factor (NF)-κB, which directly binds to the PUMA promoter to activate its transcription. NF-κB activation by sorafenib requires glycogen synthase kinase 3β activation, subsequent to ERK inhibition. Deficiency in PUMA abrogates sorafenib-induced apoptosis and caspase activation, and renders sorafenib resistance in colony formation and xenograft tumor assays. Furthermore, the chemosensitization effect of sorafenib is dependent on PUMA, and involves concurrent PUMA induction through different pathways. BH3 mimetics potentiate the anti-cancer effects of sorafenib, and restore sorafenib sensitivity in resistant cells. Together, these results demonstrate a key role of PUMA-dependent apoptosis in therapeutic inhibition of Ras/Raf/MEK/ERK signaling. They provide a rationale for manipulating the apoptotic machinery to improve sensitivity and overcome resistance to the therapies that target oncogenic kinase signaling.

Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition

PubMed Central

Tang, Yujie; Gholamin, Sharareh; Schubert, Simone; Willardson, Minde I.; Lee, Alex; Bandopadhayay, Pratiti; Bergthold, Guillame; Masoud, Sabran; Nguyen, Brian; Vue, Nujsaubnusi; Balansay, Brianna; Yu, Furong; Oh, Sekyung; Woo, Pamelyn; Chen, Spenser; Ponnuswami, Anitha; Monje, Michelle; Atwood, Scott X.; Whitson, Ramon J.; Mitra, Siddhartha; Cheshier, Samuel H.; Qi, Jun; Beroukhim, Rameen; Tang, Jean Y.; Wechsler-Reya, Rob; Oro, Anthony E.; Link, Brian A.; Bradner, James E.; Cho, Yoon-Jae

2014-01-01

Hedgehog signaling drives oncogenesis in several cancers and strategies targeting this pathway have been developed, most notably through inhibition of Smoothened. However, resistance to Smoothened inhibitors occurs via genetic changes of Smoothened or other downstream Hedgehog components. Here, we overcome these resistance mechanisms by modulating GLI transcription via inhibition of BET bromodomain proteins. We show the BET bromodomain protein, BRD4, regulates GLI transcription downstream of SMO and SUFU and chromatin immunoprecipitation studies reveal BRD4 directly occupies GLI1 and GLI2 promoters, with a substantial decrease in engagement of these sites upon treatment with JQ1, a small molecule inhibitor targeting BRD4. Globally, genes associated with medulloblastoma-specific GLI1 binding sites are downregulated in response to JQ1 treatment, supporting direct regulation of GLI activity by BRD4. Notably, patient- and GEMM-derived Hedgehog-driven tumors (basal cell carcinoma, medulloblastoma and atypical teratoid/rhabdoid tumor) respond to JQ1 even when harboring genetic lesions rendering them resistant to Smoothened antagonists. PMID:24973920

FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma.

PubMed

Suryo Rahmanto, Aldwin; Savov, Vasil; Brunner, Andrä; Bolin, Sara; Weishaupt, Holger; Malyukova, Alena; Rosén, Gabriela; Čančer, Matko; Hutter, Sonja; Sundström, Anders; Kawauchi, Daisuke; Jones, David Tw; Spruck, Charles; Taylor, Michael D; Cho, Yoon-Jae; Pfister, Stefan M; Kool, Marcel; Korshunov, Andrey; Swartling, Fredrik J; Sangfelt, Olle

2016-10-17

SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here, we show that SOX9 is a substrate of FBW7, a tumor suppressor, and a SCF (SKP1/CUL1/F-box)-type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase and consequently degraded by SCF FBW 7α Failure to degrade SOX9 promotes migration, metastasis, and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. FBW7 is either mutated or downregulated in medulloblastoma, and in cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Transcriptional profiling of medulloblastoma cells expressing a degradation-resistant SOX9 mutant reveals activation of pro-metastatic genes and genes linked to cisplatin resistance. Finally, we show that pharmacological inhibition of PI3K/AKT/mTOR pathway activity destabilizes SOX9 in a GSK3/FBW7-dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Paediatrics, insulin resistance and the kidney.

PubMed

Marlais, Matko; Coward, Richard J

2015-08-01

Systemic insulin resistance is becoming more prevalent in the young due to modern lifestyles predisposing to the metabolic syndrome and obesity. There is also evidence that there are critical insulin-resistant phases for the developing child, including puberty, and that renal disease per se causes systemic insulin resistance. This review considers the factors that render children insulin resistant, as well as the accumulating evidence that the kidney is an insulin-responsive organ and could be affected by insulin resistance.

Silencing of secretory clusterin sensitizes NSCLC cells to V-ATPase inhibitors by downregulating survivin.

PubMed

Kim, Young-Sun; Jin, Hyeon-Ok; Hong, Sung-Eun; Song, Jie-Young; Hwang, Chang-Sun; Park, In-Chul

2018-01-08

Secretory clusterin (sCLU) is a stress-associated protein that confers resistance to therapy when overexpressed. In this study, we observed that the V-ATPase inhibitors bafilomycin A1 and concanamycin A significantly stimulated sCLU protein expression. Knockdown of sCLU with siRNA sensitized non-small cell lung cancer (NSCLC) cells to bafilomycin A1, suggesting that sCLU expression renders cells resistant to V-ATPase inhibitors. The dual PI3K/AKT and mTOR inhibitor BEZ235 suppressed sCLU expression and enhanced cell sensitivity induced by bafilomycin A1. Notably, sCLU knockdown further decreased the expression of the survivin protein by bafilomycin A1, and the ectopic expression of survivin alleviated the cell sensitivity by bafilomycin A1 and sCLU depletion, suggesting that increased sensitivity to sCLU depletion in the cells with V-ATPase inhibitors is due, at least in part, to the down-regulation of survivin. Taken together, we demonstrated that the depletion of sCLU expression enhances the sensitivity of NSCLC cells to V-ATPase inhibitors by decreasing survivin expression. Inhibition of the PI3K/AKT/mTOR pathway enhances the sensitivity of NSCLC cells to V-ATPase inhibitors, leading to decreased sCLU and survivin expression. Thus, we suggest that a combination of PI3K/AKT/mTOR inhibitors with V-ATPase inhibitors might be an effective approach for NSCLC treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative glycoproteomics of stem cells identifies new players in ricin toxicity.

PubMed

Stadlmann, Johannes; Taubenschmid, Jasmin; Wenzel, Daniel; Gattinger, Anna; Dürnberger, Gerhard; Dusberger, Frederico; Elling, Ulrich; Mach, Lukas; Mechtler, Karl; Penninger, Josef M

2017-09-28

Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite the importance of glycosylation in biology, the identification and functional validation of complex glycoproteins has remained largely unexplored. Here we develop a novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing us not only to infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We apply this method to human and mouse embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method is confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin, and we discover proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins render cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our comparative glycoproteomics platform, SugarQb, enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems.

Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors

PubMed Central

McSharry, James J.; McDonough, Ann C.; Olson, Betty A.; Drusano, George L.

2004-01-01

A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses. PMID:14715540

Carpobrotus edulis methanol extract inhibits the MDR efflux pumps, enhances killing of phagocytosed S. aureus and promotes immune modulation.

PubMed

Ordway, Diane; Hohmann, Judit; Viveiros, Miguel; Viveiros, Antonio; Molnar, Joseph; Leandro, Clara; Arroz, Maria Jorge; Gracio, Maria Amelia; Amaral, Leonard

2003-05-01

Although alkaloids from the family Aizoaceae have anticancer activity, species of this family have received little attention. Because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, a methanol extract of Carpobrotus edulis, a common plant found along the Portuguese coast, was studied for properties normally associated with plasma membrane active compounds. The results of this study show that the extract is non-toxic at concentrations that inhibit a verapamil sensitive efflux pump of L5178 mouse T cell lymphoma cell line thereby rendering these multi-drug resistant cells susceptible to anticancer drugs. These non-toxic concentrations also prime THP-1 human monocyte-derived macrophages to kill ingested Staphylococcus aureus and to promote the release of lymphokines associated with cellular immune functions. The extract also induces the proliferation of THP-1 cells within 1 day of exposure to quantities normally associated with phytohaemagglutinin. The potential role of the compound(s) isolated from this plant in cancer biology is intriguing and is currently under investigation. It is supposed that the resistance modifier and immunomodulatory effect of this plant extract can be exploited in the experimental chemotherapy of cancer and bacterial or viral infections. Copyright 2003 John Wiley & Sons, Ltd.

Estimation of ovular fiber production in cotton

DOEpatents

Van't Hof, Jack

1998-09-01

The present invention is a method for rendering cotton fiber cells that are post-anthesis and pre-harvest available for analysis of their physical properties. The method includes the steps of hydrolyzing cotton fiber cells and separating cotton fiber cells from cotton ovules thereby rendering the cells available for analysis. The analysis of the fiber cells is through any suitable means, e.g., visual inspection. Visual inspection of the cells can be accomplished by placing the cells under an instrument for detection, such as microscope or other means.

In vitro transfer of multiple resistance observed in vivo during a Salmonella london epidemic.

PubMed

Lantos, J; Marjai, E

1980-01-01

Between 1976 and 1978, waves of Salmonella london infections conveyed by raw meat and meat products were observed. The strains isolated during the epidemic were first susceptible then developed multiple antibiotic resistance. The identical antibiotic resistance patterns of the strain and their more frequent occurrence in hospital environments indicated plasmid-mediated resistance. R-plasmid transfer, minimum inhibition concentration and resistance elimination were studied in representative strains. The resistant S. london strain and transconjugants of Escherichia coli rendered resistant were compared. The results proved that multiple resistance was plasmid-mediated.

Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1.

PubMed

Kvansakul, Marc; Wei, Andrew H; Fletcher, Jamie I; Willis, Simon N; Chen, Lin; Roberts, Andrew W; Huang, David C S; Colman, Peter M

2010-12-23

Epstein-Barr virus (EBV) is associated with human malignancies, especially those affecting the B cell compartment such as Burkitt lymphoma. The virally encoded homolog of the mammalian pro-survival protein Bcl-2, BHRF1 contributes to viral infectivity and lymphomagenesis. In addition to the pro-apoptotic BH3-only protein Bim, its key target in lymphoid cells, BHRF1 also binds a selective sub-set of pro-apoptotic proteins (Bid, Puma, Bak) expressed by host cells. A consequence of BHRF1 expression is marked resistance to a range of cytotoxic agents and in particular, we show that its expression renders a mouse model of Burkitt lymphoma untreatable. As current small organic antagonists of Bcl-2 do not target BHRF1, the structures of it in complex with Bim or Bak shown here will be useful to guide efforts to target BHRF1 in EBV-associated malignancies, which are usually associated with poor clinical outcomes.

Bacteriophage endolysins as novel antimicrobials

PubMed Central

Schmelcher, Mathias; Donovan, David M; Loessner, Martin J

2013-01-01

Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can access the peptidoglycan and destroy these organisms when applied externally, making them interesting antimicrobial candidates, particularly in light of increasing bacterial drug resistance. This article reviews the modular structure of these enzymes, in which cell wall binding and catalytic functions are separated, as well as their mechanism of action, lytic activity and potential as antimicrobials. It particularly focuses on molecular engineering as a means of optimizing endolysins for specific applications, highlights new developments that may render these proteins active against Gram-negative and intracellular pathogens and summarizes the most recent applications of endolysins in the fields of medicine, food safety, agriculture and biotechnology. PMID:23030422

Nitric oxide inhibits ATPase activity and induces resistance to topoisomerase II-poisons in human MCF-7 breast tumor cells.

PubMed

Sinha, Birandra K; Kumar, Ashutosh; Mason, Ronald P

2017-07-01

Topoisomerase poisons are important drugs for the management of human malignancies. Nitric oxide ( • NO), a physiological signaling molecule, induces nitrosylation (or nitrosation) of many cellular proteins containing cysteine thiol groups, altering their cellular functions. Topoisomerases contain several thiol groups which are important for their activity and are also targets for nitrosation by nitric oxide. Here, we have evaluated the roles of • NO/ • NO-derived species in the stability and activity of topo II (α and β) both in vitro and in human MCF-7 breast tumor cells. Furthermore, we have examined the effects of • NO on the ATPase activity of topo II. Treatment of purified topo IIα and β with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of the catalytic activity of topo II. Furthermore, PPNO significantly inhibited topo II-dependent ATP hydrolysis. • NO-induced inhibition of these topo II (α and β) functions resulted in a decrease in cleavable complex formation in MCF-7 cells in the presence of m-AMSA and XK469 and induced significant resistance to both drugs in MCF-7 cells. PPNO treatment resulted in the nitrosation of the topo II protein in MCF-7 cancer cells and inhibited both catalytic-, and ATPase activities of topo II. Furthermore, PPNO significantly affected the DNA damage and cytotoxicity of m-AMSA and XK469 in MCF-7 tumor cells. As tumors express nitric oxide synthase and generate • NO, inhibition of topo II functions by • NO/ • NO-derived species could render tumors resistant to certain topo II-poisons in the clinic.

Gene encoding acetyl-coenzyme A carboxylase

DOEpatents

Roessler, Paul G.; Ohlrogge, John B.

1996-01-01

A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

Resistance to diabetes-promoting lifestyle factors -- what is the mechanism?

PubMed

Kolb, Hubert

2012-08-01

Not all people exposed to diabetes-promoting lifestyle factors progress to overt type 2 diabetes. The emerging concept of hormesis provides an explanation for the resistance to metabolic stress. Hormesis requires limited stress or damage which elicits an adaptive repair and protective response which renders the organism resistant to further metabolic stress. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Hawaii natural compounds are promising to reduce ovarian cancer deaths.

PubMed

Fei-Zhang, David J; Li, Chunshun; Cao, Shugeng

2016-07-02

The low survival rate of patients with ovarian cancer largely results from the advanced ovarian tumors as well as tumor resistance to chemotherapy, leading to metastasis and recurrence. However, it is missing as to an effective therapeutic approach that focuses on these aspects to prolong progression-free survival and to decrease mortality in ovarian cancer patients. Here, based on our cancer drug discovery studies, we provide prospective insights into the development of a future line of drugs to effectively reduce ovarian cancer deaths. Pathways that increase the probability of cancer, such as the defective Fanconi anemia (FA) pathway, may render cancer cells more sensitive to new drug targeting.

Gene encoding acetyl-coenzyme A carboxylase

DOEpatents

Roessler, P.G.; Ohlrogge, J.B.

1996-09-24

A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

Zoledronic acid renders human M1 and M2 macrophages susceptible to Vδ2+ γδ T cell cytotoxicity in a perforin-dependent manner.

PubMed

Fowler, Daniel W; Copier, John; Dalgleish, Angus G; Bodman-Smith, Mark D

2017-09-01

Vδ2 + T cells are a subpopulation of γδ T cells in humans that are cytotoxic towards cells which accumulate isopentenyl pyrophosphate. The nitrogen-containing bisphosphonate, zoledronic acid (ZA), can induce tumour cell lines to accumulate isopentenyl pyrophosphate, thus rendering them more susceptible to Vδ2 + T cell cytotoxicity. However, little is known about whether ZA renders other, non-malignant cell types susceptible. In this study we focussed on macrophages (Mϕs), as these cells have been shown to take up ZA. We differentiated peripheral blood monocytes from healthy donors into Mϕs and then treated them with IFN-γ or IL-4 to generate M1 and M2 Mϕs, respectively. We characterised these Mϕs based on their phenotype and cytokine production and then tested whether ZA rendered them susceptible to Vδ2 + T cell cytotoxicity. Consistent with the literature, IFN-γ-treated Mϕs expressed higher levels of the M1 markers CD64 and IL-12p70, whereas IL-4-treated Mϕs expressed higher levels of the M2 markers CD206 and chemokine (C-C motif) ligand 18. When treated with ZA, both M1 and M2 Mϕs became susceptible to Vδ2 + T cell cytotoxicity. Vδ2 + T cells expressed perforin and degranulated in response to ZA-treated Mϕs as shown by mobilisation of CD107a and CD107b to the cell surface. Furthermore, cytotoxicity towards ZA-treated Mϕs was sensitive-at least in part-to the perforin inhibitor concanamycin A. These findings suggest that ZA can render M1 and M2 Mϕs susceptible to Vδ2 + T cell cytotoxicity in a perforin-dependent manner, which has important implications regarding the use of ZA in cancer immunotherapy.

Lapatinib-resistant cancer cells possessing epithelial cancer stem cell properties develop sensitivity during sphere formation by activation of the ErbB/AKT/cyclin D2 pathway.

PubMed

Ohnishi, Yuichi; Yasui, Hiroki; Kakudo, Kenji; Nozaki, Masami

2016-11-01

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/ErbB2, has antiproliferative effects and is used to treat patients with ErbB2-positive metastatic breast cancer. In the present study, we examined the effects of lapatinib on growth of oral and prostate cancer cells. Oral squamous cell carcinoma (OSCC) cell lines HSC3, HSC4 and Ca9-22 were sensitive to the antiproliferative effects of lapatinib in anchorage-dependent culture, but the OSCC cell lines KB and SAS and the prostate cancer cell line DU145 were resistant to lapatinib. Phosphorylation levels of EGFR in all cell lines decreased during lapatinib treatment in anchorage‑dependent culture. Furthermore, the phosphorylation levels of ErbB2, ErbB3 and Akt and the protein levels of cyclin D1 were decreased by lapatinib treatment of HSC3, HSC4 and Ca9-22 cells. ErbB3 was not expressed and cyclin D1 protein levels were not altered by lapatinib treatment in KB, DU145 and SAS cells. The phosphorylation of ErbB2 and AKT was not affected by lapatinib in SAS cells and was not detected in KB and DU145 cells. Lapatinib-resistant cell lines exhibited sphere-forming ability, and SAS cells developed sensitivity to lapatinib during sphere formation. The phosphorylation levels of ErbB2 and AKT and protein levels of cyclin D2 increased during sphere formation of SAS cells and decreased with lapatinib treatment. In addition, sphere formation of SAS cells was inhibited by the AKT inhibitor MK2206. AKT phosphorylation and cyclin D2 levels in SAS spheres were decreased by MK2206 treatment. SAS cells expressed E-cadherin, but not vimentin and KB cells expressed vimentin, but not E-cadherin. DU145 cells expressed vimentin and E-cadherin. These results suggested that phosphorylation of EGFR and ErbB2 by cell detachment from the substratum induces the AKT pathway/cyclin D2-dependent sphere growth in SAS epithelial cancer stem-like cells, thereby rendering SAS spheres sensitive to lapatinib treatment.

Mistranslation can enhance fitness through purging of deleterious mutations

PubMed Central

Bratulic, Sinisa; Toll-Riera, Macarena; Wagner, Andreas

2017-01-01

Phenotypic mutations are amino acid changes caused by mistranslation. How phenotypic mutations affect the adaptive evolution of new protein functions is unknown. Here we evolve the antibiotic resistance protein TEM-1 towards resistance on the antibiotic cefotaxime in an Escherichia coli strain with a high mistranslation rate. TEM-1 populations evolved in such strains endow host cells with a general growth advantage, not only on cefotaxime but also on several other antibiotics that ancestral TEM-1 had been unable to deactivate. High-throughput sequencing of TEM-1 populations shows that this advantage is associated with a lower incidence of weakly deleterious genotypic mutations. Our observations show that mistranslation is not just a source of noise that delays adaptive evolution. It could even facilitate adaptive evolution by exacerbating the effects of deleterious mutations and leading to their more efficient purging. The ubiquity of mistranslation and its effects render mistranslation an important factor in adaptive protein evolution. PMID:28524864

Estimation of ovular fiber production in cotton

DOEpatents

Van`t Hof, J.

1998-09-01

The present invention is a method for rendering cotton fiber cells that are post-anthesis and pre-harvest available for analysis of their physical properties. The method includes the steps of hydrolyzing cotton fiber cells and separating cotton fiber cells from cotton ovules thereby rendering the cells available for analysis. The analysis of the fiber cells is through any suitable means, e.g., visual inspection. Visual inspection of the cells can be accomplished by placing the cells under an instrument for detection, such as microscope or other means. 4 figs.

Estimation of ovular fiber production in cotton

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van`t Hof, J.

The present invention is a method for rendering cotton fiber cells that are post-anthesis and pre-harvest available for analysis of their physical properties. The method includes the steps of hydrolyzing cotton fiber cells and separating cotton fiber cells from cotton ovules thereby rendering the cells available for analysis. The analysis of the fiber cells is through any suitable means, e.g., visual inspection. Visual inspection of the cells can be accomplished by placing the cells under an instrument for detection, such as microscope or other means. 4 figs.

Sensitization of B16 tumor cells with a CXCR4 antagonist increases the efficacy of immunotherapy for established lung metastases

PubMed Central

Lee, Chih-hung; Kakinuma, Takashi; Wang, Julia; Zhang, Hong; Palmer, Douglas C.; Restifo, Nicholas P.; Hwang, Sam T.

2008-01-01

Expression of the chemokine receptor CXCR4 by tumor cells promotes metastasis, possibly by activating pro-survival signals that render cancer cells resistant to immune attack. Inhibition of CXCR4 with a peptide antagonist, T22, blocks metastatic implantation of CXCR4-transduced B16 (CXCR4-luc-B16) melanoma cells in lung, but not the outgrowth of established metastases, raising the question of how T22 can best be used in a clinical setting. Herein, whereas the treatment of CXCR4-luc-B16 cells in vitro with the CXCR4 ligand CXCL12 did not reduce killing induced by cisplatin or cyclophosphamide, CXCL12 markedly reduced Fas-dependent killing by gp100-specific (pmel-1) CD8+ T cells. T22 pretreatment restored sensitivity of CXCR4-luc-B16 cells to pmel-1 killing, even in the presence of CXCL12. Two immune-augmenting regimens were used in combination with T22 to treat experimental lung metastases. First, low-dose cyclophosphamide treatment (100 mg/kg) on day 5 in combination with T22 (days 4–7) yielded a ~70% reduction of B16 metastatic tumor burden in the lungs compared with cyclophosphamide treatment alone (P < 0.001). Furthermore, whereas anti–CTL antigen 4 (CTLA4) monoclonal antibody (mAb; or T22 treatment) alone had little effect on established B16 metastases, pretreatment with T22 (in combination with anti-CTLA4 mAb) resulted in a 50% reduction in lung tumor burden (P = 0.02). Thus, in vitro, CXCR4 antagonism with T22 renders B16 cells susceptible to killing by antigen-specific T cells. In vivo, T22 synergizes with cyclophosphamide or anti-CTLA4 mAb in the treatment of established lung metastases, suggesting a novel strategy for augmenting the efficacy of immunotherapy. PMID:17041104

Interfacing superhydrophobic silica nanoparticle films with graphene and thermoplastic polyurethane for wear/abrasion resistance.

PubMed

Naderizadeh, Sara; Athanassiou, Athanassia; Bayer, Ilker S

2018-06-01

Nanoparticle films are one of the most suitable platforms for obtaining sub-micrometer and nanometer dual-scale surface texture required for liquid repellency. The assembly of superhydrophobic nanoparticles into conformal and strongly adherent films having abrasion-induced wear resistance still poses a significant challenge. Various techniques have been developed over the years to render nanoparticle films with good liquid repellent properties and transparency. However, forming abrasion resistant superhydrophobic nanoparticle films on hard surfaces is challenging. One possibility is to partially embed or weld nanoparticles in thin thermoplastic primers applied over metals. Hexamethyldisilazane-functionalized fumed silica nanoparticle films spray deposited on aluminum surfaces were rendered abrasion resistant by thermally welding them into thermoplastic polyurethane (TPU) primer applied a priori over aluminum. Different solvents, nanoparticle concentrations and annealing temperatures were studied to optimize nanoparticle film morphology and hydrophobicity. Thermal annealing at 150 °C enhanced stability and wear resistance of nanoparticle films. A thin thermal interface layer of graphene nanoplatelets (GnPs) between the primer and the nanoparticle film significantly improved superhydrophobic wear resistance after annealing. As such, superhydrophobic nanocomposite films with the GnPs thermal interface layer displayed superior abrasion-induced wear resistance under 20 kPa compared to films having no GnPs-based thermal interface. Copyright © 2018 Elsevier Inc. All rights reserved.

Simultaneous Knockout of CXCR4 and CCR5 Genes in CD4+ T Cells via CRISPR/Cas9 Confers Resistance to Both X4- and R5-Tropic Human Immunodeficiency Virus Type 1 Infection.

PubMed

Yu, Songlin; Yao, Yongchao; Xiao, Hongkui; Li, Jiaojiao; Liu, Quan; Yang, Yijun; Adah, Dickson; Lu, Junnan; Zhao, Siting; Qin, Li; Chen, Xiaoping

2018-01-01

Previous research has proven that disruption of either the CCR5 or the CXCR4 gene confers resistance to R5-tropic or X4-tropic human immunodeficiency virus type 1 (HIV-1) infection, respectively. However, the urgent need to ablate both of the co-receptors in individual post-thymic CD4+ T cells for dual protection remains. This study ablated the CCR5 and CXCR4 genes in human CD4+ cell lines and primary CD4+ T cells simultaneously using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, a well-developed, highly efficient genetic engineering tool. The efficiency of gene modification is as high as 55% for CCR5 and 36% for CXCR4 in CD4+ cell lines through infection of a single lentiviral vector (LV-X4R5), which were markedly protected from both HIV-1 NL4-3 (X4-using strain) and HIV-1 YU-2 (R5-using strain) infection. Importantly, approximately 9% of the modified GHOST (3) CXCR4+CCR5+ cells harbor four bi-allelic gene disruptions in both the CXCR4 and CCR5 loci. Moreover, co-delivery of two single-guide RNAs loaded with Cas9: ribonucleoprotein (sgX4&R5 Cas9RNP) disrupted >12% of CCR5 and 10% of CXCR4 in primary human CD4+ T cells, which were rendered resistant to HIV-1 NL4-3 and HIV-1 YU-2 in vitro. Further, the modified cells do not show discernible mutagenesis in top-ranked off-target genes by the Surveyor assay and Sanger sequencing analysis. The results demonstrate the safety and efficacy of CRISPR/Cas9 in multiplex gene modification on peripherally circulating CD4+ T cells, which may promote a functional cure for HIV-1 infection.

Hierarchical and Parallelizable Direct Volume Rendering for Irregular and Multiple Grids

NASA Technical Reports Server (NTRS)

Wilhelms, Jane; VanGelder, Allen; Tarantino, Paul; Gibbs, Jonathan

1996-01-01

A general volume rendering technique is described that efficiently produces images of excellent quality from data defined over irregular grids having a wide variety of formats. Rendering is done in software, eliminating the need for special graphics hardware, as well as any artifacts associated with graphics hardware. Images of volumes with about one million cells can be produced in one to several minutes on a workstation with a 150 MHz processor. A significant advantage of this method for applications such as computational fluid dynamics is that it can process multiple intersecting grids. Such grids present problems for most current volume rendering techniques. Also, the wide range of cell sizes (by a factor of 10,000 or more), which is typical of such applications, does not present difficulties, as it does for many techniques. A spatial hierarchical organization makes it possible to access data from a restricted region efficiently. The tree has greater depth in regions of greater detail, determined by the number of cells in the region. It also makes it possible to render useful 'preview' images very quickly (about one second for one-million-cell grids) by displaying each region associated with a tree node as one cell. Previews show enough detail to navigate effectively in very large data sets. The algorithmic techniques include use of a kappa-d tree, with prefix-order partitioning of triangles, to reduce the number of primitives that must be processed for one rendering, coarse-grain parallelism for a shared-memory MIMD architecture, a new perspective transformation that achieves greater numerical accuracy, and a scanline algorithm with depth sorting and a new clipping technique.

High-Level Fosfomycin Resistance in Vancomycin-Resistant Enterococcus faecium

PubMed Central

Guo, Yan; Tomich, Adam D.; McElheny, Christi L.; Cooper, Vaughn S.; Tait-Kamradt, Amelia; Wang, Minggui; Hu, Fupin; Rice, Louis B.; Sluis-Cremer, Nicolas

2017-01-01

Of 890 vancomycin-resistant Enterococcus faecium isolates obtained by rectal screening from patients in Pittsburgh, Pennsylvania, USA, 4 had MICs >1,024 μg/mL for fosfomycin. These isolates had a Cys119Asp substitution in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase. This substitution increased the fosfomycin MIC >4-fold and rendered this drug inactive in biochemical assays. PMID:29048285

Wheat Gene TaATG8j Contributes to Stripe Rust Resistance.

PubMed

Mamun, Md Abdullah-Al; Tang, Chunlei; Sun, Yingchao; Islam, Md Nazrul; Liu, Peng; Wang, Xiaojie; Kang, Zhensheng

2018-06-05

Autophagy-related 8 (ATG8) protein has been reported to be involved in plant's innate immune response, but it is not clear whether such genes play a similar role in cereal crops against obligate biotrophic fungal pathogens. Here, we reported an ATG8 gene from wheat ( Triticum aestivum ), designated TaATG8j . This gene has three copies located in chromosomes 2AS, 2BS, and 2DS. The transcriptions of all three copies were upregulated in plants of the wheat cultivar Suwon 11, inoculated with an avirulent race (CYR23) of Puccinia striiformis f. sp. tritici ( Pst ), the causal fungal pathogen of stripe rust. The transient expression of TaATG8j in Nicotiana benthamiana showed that TaATG8j proteins were distributed throughout the cytoplasm, but mainly in the nucleus and plasma membrane. The overexpression of TaATG8j in N. benthamiana slightly delayed the cell death caused by the mouse apoptotic protein BAX (BCL2-associated X protein). However, the expression of TaATG8j in yeast ( Schizosaccharomyces pombe ) induced cell death. The virus-induced gene silencing of all TaATG8j copies rendered Suwon 11 susceptible to the avirulent Pst race CYR23, accompanied by an increased fungal biomass and a decreased necrotic area per infection site. These results indicate that TaATG8j contributes to wheat resistance against stripe rust fungus by regulating cell death, providing information for the understanding of the mechanisms of wheat resistance to the stripe rust pathogen.

Novel tumor suppressive function of Smad4 in serum starvation-induced cell death through PAK1–PUMA pathway

PubMed Central

Lee, S-H; Jung, Y-S; Chung, J-Y; Oh, A Y; Lee, S-J; Choi, D H; Jang, S M; Jang, K-S; Paik, S S; Ha, N-C; Park, B-J

2011-01-01

DPC4 (deleted in pancreatic cancer 4)/Smad4 is an essential factor in transforming growth factor (TGF)-β signaling and is also known as a frequently mutated tumor suppressor gene in human pancreatic and colon cancer. However, considering the fact that TGF-β can contribute to cancer progression through transcriptional target genes, such as Snail, MMPs, and epithelial–mesenchymal transition (EMT)-related genes, loss of Smad4 in human cancer would be required for obtaining the TGF-β signaling-independent advantage, which should be essential for cancer cell survival. Here, we provide the evidences about novel role of Smad4, serum-deprivation-induced apoptosis. Elimination of serum can obviously increase the Smad4 expression and induces the cell death by p53-independent PUMA induction. Instead, Smad4-deficient cells show the resistance to serum starvation. Induced Smad4 suppresses the PAK1, which promotes the PUMA destabilization. We also found that Siah-1 and pVHL are involved in PAK1 destabilization and PUMA stabilization. In fact, Smad4-expressed cancer tissues not only show the elevated expression of PAK1, but also support our hypothesis that Smad4 induces PUMA-mediated cell death through PAK1 suppression. Our results strongly suggest that loss of Smad4 renders the resistance to serum-deprivation-induced cell death, which is the TGF-β-independent tumor suppressive role of Smad4. PMID:22130069

4-(1-Ethyl-4-anisyl-imidazol-5-yl)-N-hydroxycinnamide - A new pleiotropic HDAC inhibitor targeting cancer cell signalling and cytoskeletal organisation.

PubMed

Mahal, Katharina; Kahlen, Philip; Biersack, Bernhard; Schobert, Rainer

2015-08-15

Histone deacetylases (HDAC) which play a crucial role in cancer cell proliferation are promising drug targets. However, HDAC inhibitors (HDACi) modelled on natural hydroxamic acids such as trichostatin A frequently lead to resistance or even an increased agressiveness of tumours. As a workaround we developed 4-(1-ethyl-4-anisyl-imidazol-5-yl)-N-hydroxycinnamide (etacrox), a hydroxamic acid that combines HDAC inhibition with synergistic effects of the 4,5-diarylimidazole residue. Etacrox proved highly cytotoxic against a panel of metastatic and resistant cancer cell lines while showing greater specificity for cancer over non-malignant cells when compared to the approved HDACi vorinostat. Like the latter, etacrox and the closely related imidazoles bimacroxam and animacroxam acted as pan-HDACi yet showed some specificity for HDAC6. Akt signalling and interference with nuclear beta-catenin localisation were elicited by etacrox at lower concentrations when compared to vorinostat. Moreover, etacrox disrupted the microtubule and focal adhesion dynamics of cancer cells and inhibited the proteolytic activity of prometastatic and proangiogenic matrix metalloproteinases. As a consequence, etacrox acted strongly antimigratory and antiinvasive against various cancer cell lines in three-dimensional transwell invasion assays and also antiangiogenic in vivo with respect to blood vessel formation in the chorioallantoic membrane assay. These pleiotropic effects and its water-solubility and tolerance by mice render etacrox a promising new HDACi candidate. Copyright © 2015 Elsevier Inc. All rights reserved.

Triptolide enhances the tumoricidal activity of TRAIL against renal cell carcinoma.

PubMed

Brincks, Erik L; Kucaba, Tamara A; James, Britnie R; Murphy, Katherine A; Schwertfeger, Kathryn L; Sangwan, Veena; Banerjee, Sulagna; Saluja, Ashok K; Griffith, Thomas S

2015-12-01

Renal cell carcinoma (RCC) is resistant to traditional cancer therapies, and metastatic RCC (mRCC) is incurable. The shortcomings in current therapeutic options for patients with mRCC provide the rationale for the development of novel treatment protocols. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has proven to be a potent inducer of tumor cell death in vitro and in vivo, and a number of TRAIL death receptor agonists (recombinant TRAIL or TRAIL death receptor-specific mAb) have been developed and tested clinically. Unfortunately the clinical efficacy of TRAIL has been underwhelming and is likely due to a number of possible mechanisms that render tumors resistant to TRAIL, prompting the search for drugs that increase tumor cell susceptibility to TRAIL. The objective of this study was to determine the effectiveness of combining the diterpene triepoxide triptolide, or its water-soluble prodrug, Minnelide, with TRAIL receptor agonists against RCC in vitro or in vivo, respectively. TRAIL-induced apoptotic death of human RCC cells was increased in the presence of triptolide. The triptolide-induced sensitization was accompanied by increased TRAIL-R2 (DR5) and decreased heat shock protein 70 expression. In vivo treatment of mice bearing orthotopic RCC (Renca) tumors showed the combination of Minnelide and agonistic anti-DR5 mAb significantly decreased tumor burden and increased animal survival compared to either therapy alone. Our data suggest triptolide/Minnelide sensitizes RCC cells to TRAIL-induced apoptosis through altered TRAIL death receptor and heat shock protein expression. © 2015 FEBS.

Triptolide Enhances the Tumoricidal Activity of TRAIL Against Renal Cell Carcinoma

PubMed Central

James, Britnie R.; Murphy, Katherine A.; Schwertfeger, Kathryn L.; Sangwan, Veena; Banerjee, Sulagna; Saluja, Ashok K.; Griffith, Thomas S.

2015-01-01

Renal cell carcinoma (RCC) is resistant to traditional cancer therapies, and metastatic RCC (mRCC) is incurable. The shortcomings in current therapeutic options for patients with mRCC provide the rationale for the development of novel treatment protocols. TNF-related apoptosis-inducing ligand (TRAIL) has proven to be a potent inducer of tumor cell death in vitro and in vivo, and a number of TRAIL death receptor agonists (recombinant TRAIL or TRAIL death receptor-specific mAb) has been developed and tested clinically. Unfortunately the clinical efficacy of TRAIL has been underwhelming and is likely due to a number of possible mechanisms that render tumors resistant to TRAIL, prompting the search for drugs that increase tumor cell susceptibility to TRAIL. The objective of this study was to determine the effectiveness of combining the diterpene triepoxide triptolide, or its water-soluble prodrug, Minnelide, with TRAIL receptor agonists against RCC in vitro or in vivo, respectively. TRAIL-induced apoptotic death of human RCC cells was increased in the presence of triptolide. The triptolide-induced sensitization was accompanied by increased TRAIL-R2 (DR5) and decreased HSP70 expression. In vivo treatment of mice bearing orthotopic RCC (Renca) tumors showed the combination of Minnelide and agonistic anti-DR5 mAb significantly decreased tumor burden and increased animal survival compared to either therapy alone. Our data suggest triptolide/Minnelide sensitizes RCC cells to TRAIL-induced apoptosis through altered TRAIL death receptor and heat shock protein expression. PMID:26426449

A Novel Route Controlling Begomovirus Resistance by the Messenger RNA Surveillance Factor Pelota

PubMed Central

Lapidot, Moshe; Karniel, Uri; Gelbart, Dana; Fogel, Doron; Evenor, Dalia; Kutsher, Yaarit; Makhbash, Zion; Nahon, Sahadia; Shlomo, Haviva; Chen, Lea; Reuveni, Moshe; Levin, Ilan

2015-01-01

Tomato yellow leaf curl virus (TYLCV) is a devastating disease of tomato (Solanum lycopersicum) that can be effectively controlled by the deployment of resistant cultivars. The TYLCV-resistant line TY172 carries a major recessive locus for TYLCV resistance, designated ty-5, on chromosome 4. In this study, the association between 27 polymorphic DNA markers, spanning the ty-5 locus, and the resistance characteristics of individual plants inoculated with TYLCV in 51 segregating recombinant populations were analyzed. These analyses localized ty-5 into a 425 bp region containing two transversions: one in the first exon of a gene encoding the tomato homolog of the messenger RNA surveillance factor Pelota (Pelo), and a second in its proximal promoter. Analyses of susceptible and resistant lines revealed that the relative transcript level of the gene remained unchanged, regardless of whether the plants were infected with TYLCV or not. This suggests that the polymorphism discovered in the coding region of the gene controls the resistance. Silencing of Pelo in a susceptible line rendered the transgenic plants highly resistant, while in the resistant line TY172 had no effect on symptom development. In addition, over-expression of the susceptible allele of the gene in the resistant TY172 line rendered it susceptible, while over-expression of the resistant allele in susceptible plants had no effect. These results confirm that Pelo is the gene controlling resistance at the ty-5 locus. Pelo, implicated in the ribosome recycling-phase of protein synthesis, offers an alternative route to promote resistance to TYLCV and other viruses. PMID:26448569

Arabidopsis ARF-GTP exchange factor, GNOM, mediates transport required for innate immunity and focal accumulation of syntaxin PEN1

PubMed Central

Nielsen, Mads Eggert; Feechan, Angela; Böhlenius, Henrik; Ueda, Takashi; Thordal-Christensen, Hans

2012-01-01

Penetration resistance to powdery mildew fungi, conferred by localized cell wall appositions (papillae), is one of the best-studied processes in plant innate immunity. The syntaxin PENETRATION (PEN)1 is required for timely appearance of papillae, which contain callose and extracellular membrane material, as well as PEN1 itself. Appearance of membrane material in papillae suggests secretion of exosomes. These are potentially derived from multivesicular bodies (MVBs), supported by our observation that ARA6-labeled organelles assemble at the fungal attack site. However, the trafficking components that mediate delivery of extracellular membrane material are unknown. Here, we show that the delivery is independent of PEN1 function. Instead, we find that application of brefeldin (BF)A blocks the papillary accumulation of GFP-PEN1–labeled extracellular membrane and callose, while impeding penetration resistance. We subsequently provide evidence indicating that the responsible BFA-sensitive ADP ribosylation factor–GTP exchange factor (ARF-GEF) is GNOM. Firstly, analysis of the transheterozygote gnomB4049/emb30-1 (gnomB/E) mutant revealed a delay in papilla formation and reduced penetration resistance. Furthermore, a BFA-resistant version of GNOM restored the BFA-sensitive papillary accumulation of GFP-PEN1 and callose. Our data, therefore, provide a link between GNOM and disease resistance. We suggest that papilla formation requires rapid reorganization of material from the plasma membrane mediated by GNOM. The papilla material is subsequently presumed to be sorted into MVBs and directed to the site of fungal attack, rendering the epidermal plant cell inaccessible for the invading powdery mildew fungus. PMID:22733775

Surface modification of Ni–Ti alloys for stent application after magnetoelectropolishing

PubMed Central

Musaramthota, Vishal; Munroe, Norman; Datye, Amit; Dua, Rupak; Haider, Waseem; McGoron, Anthony; Rokicki, Ryszard

2015-01-01

The constant demand for new implant materials and the multidisciplinary design approaches for stent applications have expanded vastly over the past decade. The biocompatibility of these implant materials is a function of their surface characteristics such as morphology, surface chemistry, roughness, surface charge and wettability. These surface characteristics can directly influence the material's corrosion resistance and biological processes such as endothelialization. Surface morphology affects the thermodynamic stability of passivating oxides, which renders corrosion resistance to passivating alloys. Magnetoelectropolishing (MEP) is known to alter the morphology and composition of surface films, which assist in improving corrosion resistance of Nitinol alloys. This work aims at analyzing the surface characteristics of MEP Nitinol alloys by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The wettability of the alloys was determined by contact angle measurements and the mechanical properties were assessed by Nanoindentation. Improved mechanical properties were observed with the addition of alloying elements. Cyclic potentiodynamic polarization tests were performed to determine the corrosion susceptibility. Further, the alloys were tested for their cytotoxicity and cellular growth with endothelial cells. Improved corrosion resistance and cellular viability were observed with MEP surface treated alloys. PMID:25746243

A study of building the hydrogeological apparent model with geoelectrical measurements for the Chia-Nan Coastal Plain of SW Taiwan

NASA Astrophysics Data System (ADS)

Chang, P. Y.; Tsai, J. P.; Chang, L. C.

2016-12-01

In the study we used the resistivity measurements collected in the Chia-Nan coastal plain of SW Taiwan to establish a three-dimensional (3D) hydrogeological apparent model. The resistivity measurements include data from half-Schlumberger surveys conducted during the year of 1990-2000 across the entire area and from the recent two-dimensional resistivity surveys for characterizing the recharge zone boundaries. Core records from monitoring wells in the area were used for the training data to help determining the resistivity ranges of the gavel, sand, and muddy sediments in the coastal plain. These resistivity measurements were inverted and converted into 1-D data form and interpolated for rendering a three dimensional resistivity volume that represents the general resistivity distribution in the coastal-plain systems. In addition we used water resistivity data from the observation wells to calculating the formation factors (FI) and to render the FI model. We then compared the FIs with indexed core records near some of the resistivity surveys sites, and concluded the range of the FIs for different materials in a statistical sense. Lastly we transfer the FI model into the gravel-sand-clay apparent model with the classification criteria from previous petrophysical analysis. Because there are more resistivity measurements than the limited geological boreholes, the apparent model is better to represent the detailed sedimentary structures than the traditional over-simplified conceptual models.

Rapid diagnosis of multidrug resistance in cancer by electrochemical sensor based on carbon nanotubes-drug supramolecular nanocomposites.

PubMed

Zhang, Haijun; Jiang, Hui; Sun, Feifei; Wang, Huangping; Zhao, Juan; Chen, Baoan; Wang, Xuemei

2011-03-15

The multidrug resistance (MDR) in cancer is a major chemotherapy obstacle, rendering many currently available chemotherapeutic drugs ineffective. The aim of this study was to explore the new strategy to early diagnose the MDR by electrochemical sensor based on carbon nanotubes-drug supramolecular interaction. The carbon nanotubes modified glassy carbon electrodes (CNTs/GCE) were directly immersed into the cells suspension of the sensitive leukemia cells K562 and/or its MDR cells K562/A02 to detect the response of the electrochemical probe of daunorubicin (DNR) residues after incubated with cells for 1h. The fresh evidence from the electrochemical studies based on CNTs/GCE demonstrated that the homogeneous, label-free strategy could directly measure the function of cell membrane transporters in MDR cancer cells, identify the cell phenotype (sensitive or MDR). When the different ratios of the sensitive leukemia cells K562 and its MDR ones K562/A02 were applied as a model of MDR levels to simulate the MDR occurrence in cancer, the cathodic peak current showed good linear response to the fraction of MDR with a correlation coefficient of 0.995. Therefore, the MDR fraction can be easily predicted based on the calibration curve of the cathodic peak current versus the fraction of MDR. These results indicated that the sensing strategy could provide a powerful tool for assessment of MDR in cancer. The new electrochemical sensor based on carbon nanotubes-drug supramolecular nanocomposites could represent promising approach in the rapid diagnosis of MDR in cancer. Copyright © 2011 Elsevier B.V. All rights reserved.

Use of bacteriophage to target bacterial surface structures required for virulence: a systematic search for antibiotic alternatives.

PubMed

Orndorff, Paul E

2016-11-01

Bacteriophages (phage) that infect pathogenic bacteria often attach to surface receptors that are coincidentally required for virulence. Receptor loss or modification through mutation renders mutants both attenuated and phage resistant. Such attenuated mutants frequently have no apparent laboratory growth defects, but in the host, they fail to exhibit properties needed to produce disease such as mucosal colonization or survival within professional phagocytic cells. The connection between attenuation and phage resistance has been exploited in experimental demonstrations of phage therapy. In such experiments, phage resistant mutants that arise naturally during therapy are inconsequential because of their attenuated status. A more contemporary approach to exploiting this connection involves identifying small effector molecules, identified in high-throughput screens, that inhibit one or more of the steps needed to produce a functioning phage receptor. Since such biosynthetic steps are unique to bacteria, inhibitors can be utilized therapeutically, in lieu of antibiotics. Also, since the inhibitor is specific to a particular bacterium or group of bacteria, no off-target resistance is generated in the host's commensal bacterial population. This brief review covers examples of how mutations that confer phage resistance produce attenuation, and how this coincidental relationship can be exploited in the search for the next generation of therapeutic agents for bacterial diseases.

Fine-structure mapping of the firA gene, a locus involved in the phenotypic expression of rifampin resistance in Escherichia.

PubMed

Lathe, R

1977-09-01

The firA (Ts)200 mutation not only eliminates the resistance to rifampin of certain genetically resistant strains, but, moreover, renders ribonucleic acid synthesis thermolabile. The firA gene has been mapped by P1 tranduction and is located extremely close to the structural gene for deoxyribonucleic acid polymerase III at 4 min on the Escherichia coli linkage map.

Hypoxic and Ras-transformed cells support growth by scavenging unsaturated fatty acids from lysophospholipids

PubMed Central

Kamphorst, Jurre J.; Cross, Justin R.; Fan, Jing; de Stanchina, Elisa; Mathew, Robin; White, Eileen P.; Thompson, Craig B.; Rabinowitz, Joshua D.

2013-01-01

Cancer cell growth requires fatty acids to replicate cellular membranes. The kinase Akt is known to up-regulate fatty acid synthesis and desaturation, which is carried out by the oxygen-consuming enzyme stearoyl-CoA desaturase (SCD)1. We used 13C tracers and lipidomics to probe fatty acid metabolism, including desaturation, as a function of oncogene expression and oxygen availability. During hypoxia, flux from glucose to acetyl-CoA decreases, and the fractional contribution of glutamine to fatty acid synthesis increases. In addition, we find that hypoxic cells bypass de novo lipogenesis, and thus, both the need for acetyl-CoA and the oxygen-dependent SCD1-reaction, by scavenging serum fatty acids. The preferred substrates for scavenging are phospholipids with one fatty acid tail (lysophospholipids). Hypoxic reprogramming of de novo lipogenesis can be reproduced in normoxic cells by Ras activation. This renders Ras-driven cells, both in culture and in allografts, resistant to SCD1 inhibition. Thus, a mechanism by which oncogenic Ras confers metabolic robustness is through lipid scavenging. PMID:23671091

Chronic treadmill exercise in rats delicately alters the Purkinje cell structure to improve motor performance and toxin resistance in the cerebellum.

PubMed

Huang, Tung-Yi; Lin, Lung-Sheng; Cho, Keng-Chi; Chen, Shean-Jen; Kuo, Yu-Min; Yu, Lung; Wu, Fong-Sen; Chuang, Jih-Ing; Chen, Hsiun-Ing; Jen, Chauying J

2012-09-01

Although exercise usually improves motor performance, the underlying cellular changes in the cerebellum remain to be elucidated. This study aimed to investigate whether and how chronic treadmill exercise in young rats induced Purkinje cell changes to improve motor performance and rendered the cerebellum less vulnerable to toxin insults. After 1-wk familiarization of treadmill running, 6-wk-old male Wistar rats were divided into exercise and sedentary groups. The exercise group was then subjected to 8 wk of exercise training at moderate intensity. The rotarod test was carried out to evaluate motor performance. Purkinje cells in cerebellar slices were visualized by lucifer yellow labeling in single neurons and by calbindin immunostaining in groups of neurons. Compared with sedentary control rats, exercised rats not only performed better in the rotarod task, but also showed finer Purkinje cell structure (higher dendritic volume and spine density with the same dendritic field). The exercise-improved cerebellar functions were further evaluated by monitoring the long-lasting effects of intraventricular application of OX7-saporin. In the sedentary group, OX7-saporin treatment retarded the rotarod performance and induced ∼60% Purkinje cell loss in 3 wk. As a comparison, the exercise group showed much milder injuries in the cerebellum by the same toxin treatment. In conclusion, exercise training in young rats increased the dendritic density of Purkinje cells, which might play an important role in improving the motor performance. Furthermore, as Purkinje cells in the exercise group were relatively toxin resistant, the exercised rats showed good motor performance, even under toxin-treated conditions.

Engineering T Cells to Functionally Cure HIV-1 Infection.

PubMed

Leibman, Rachel S; Riley, James L

2015-07-01

Despite the ability of antiretroviral therapy to minimize human immunodeficiency virus type 1 (HIV-1) replication and increase the duration and quality of patients' lives, the health consequences and financial burden associated with the lifelong treatment regimen render a permanent cure highly attractive. Although T cells play an important role in controlling virus replication, they are themselves targets of HIV-mediated destruction. Direct genetic manipulation of T cells for adoptive cellular therapies could facilitate a functional cure by generating HIV-1-resistant cells, redirecting HIV-1-specific immune responses, or a combination of the two strategies. In contrast to a vaccine approach, which relies on the production and priming of HIV-1-specific lymphocytes within a patient's own body, adoptive T-cell therapy provides an opportunity to customize the therapeutic T cells prior to administration. However, at present, it is unclear how to best engineer T cells so that sustained control over HIV-1 replication can be achieved in the absence of antiretrovirals. This review focuses on T-cell gene-engineering and gene-editing strategies that have been performed in efforts to inhibit HIV-1 replication and highlights the requirements for a successful gene therapy-mediated functional cure.

Cellular Homeostasis and Aging.

PubMed

Hartl, F Ulrich

2016-06-02

Aging and longevity are controlled by a multiplicity of molecular and cellular signaling events that interface with environmental factors to maintain cellular homeostasis. Modulation of these pathways to extend life span, including insulin-like signaling and the response to dietary restriction, identified the cellular machineries and networks of protein homeostasis (proteostasis) and stress resistance pathways as critical players in the aging process. A decline of proteostasis capacity during aging leads to dysfunction of specific cell types and tissues, rendering the organism susceptible to a range of chronic diseases. This volume of the Annual Review of Biochemistry contains a set of two reviews addressing our current understanding of the molecular mechanisms underlying aging in model organisms and humans.

RGS10 exerts a neuroprotective role through the PKA/c-AMP response-element (CREB) pathway in dopaminergic neuron-like cells

PubMed Central

Lee, Jae-Kyung; Chung, Jaegwon; Druey, Kirk M.; Tansey, Malú G.

2012-01-01

Regulator of G-protein signaling-10 (RGS10) is a GTPase activating protein (GAP) for Gαi/q/z subunits that is highly expressed in the immune system and in a broad range of brain regions including the hippocampus, striatum, dorsal raphe, and ventral midbrain. Previously, we reported that RGS10-null mice display increased vulnerability to chronic systemic inflammation-induced degeneration of nigral dopaminergic (DA) neurons. Given that RGS10 is expressed in DA neurons, we investigated the extent to which RGS10 regulates cell survival under conditions of inflammatory stress. Because of the inherent limitations associated with use of primary DA neurons for biochemical analyses, we employed a well-characterized ventral mesencephalon DA neuroblastoma cell line (MN9D) for our studies. We found that stable over-expression of RGS10 rendered them resistant to TNF-induced cytotoxicity; whereas MN9D cells expressing mutant RGS10-S168A (which is resistant to phosphorylation by protein kinase A (PKA) at a serine residue that promotes its nuclear translocation) showed similar sensitivity to TNF as the parental MN9D cells. Using biochemical and pharmacological approaches, we identified protein kinase A (PKA) and the downstream phospho-cAMP response element-binding (CREB) signaling pathway (and ruled out ERK 1/2, JNK, and NFkB) as key mediators of the neuroprotective effect of RGS10 against inflammatory stress. PMID:22564151

Cell wall-anchored nuclease of Streptococcus sanguinis contributes to escape from neutrophil extracellular trap-mediated bacteriocidal activity.

PubMed

Morita, Chisato; Sumioka, Ryuichi; Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

2014-01-01

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg(2+) and Ca(2+) for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.

Cell Wall-Anchored Nuclease of Streptococcus sanguinis Contributes to Escape from Neutrophil Extracellular Trap-Mediated Bacteriocidal Activity

PubMed Central

Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

2014-01-01

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg2+ and Ca2+ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression. PMID:25084357

The role of NK cells in HIV-1 protection: autologous, allogeneic or both?

PubMed

Hens, Jef; Jennes, Wim; Kestens, Luc

2016-01-01

Natural killer (NK) cells specialize in killing virally infected- or tumor cells and are part of the innate immune system. The activational state of NK cells is determined by the balance of incoming activating and inhibitory signals mediated by receptor-ligand binding with the target cell. These receptor-ligand bonds mainly consist of the killer immunoglobulin-like receptors (KIR), which are expressed at the cell surface of NK cells, and their ligands: the highly variable human leukocyte antigen -class I molecules (HLA). Absence of an inhibitory receptor-ligand bond lowers the NK cell activation threshold, whereas an activating receptor-ligand bond stimulates the cell, potentially overcoming this threshold and triggering NK cell activation. NK cells influence the course of infection as well as the acquisition of HIV-1. Several lines of evidence relate the activating NK cell receptor KIR3DS1, in the presence or absence of its putative ligand HLA-Bw4, with slower disease progression as well as resistance to HIV-1 infection. Overall, resistance to HIV-1 infection predominantly correlates with activating KIR/HLA profiles, consisting of e.g. activating KIRs, group B haplotypes, or inhibitory KIRs in absence of their ligands. Such a conclusion is less evident for studies of HIV-1 disease progression, with studies reporting beneficial as well as detrimental effects of activating KIR/HLA genotypes. It is likely that KIR/HLA association studies are complicated by the complexity of the KIR and HLA loci and their mutual interactions, as well as by additional factors like route of HIV exposure, immune activation, presence of co-infections, and the effect of anti-HIV-1 antibodies. One newly discovered NK cell activation pathway associated with resistance to HIV-1 infection involves the presence of an iKIR/HLA mismatch between partners. The absence of such an iKIR/HLA bond renders donor-derived allogeneic HIV-1 infected cells vulnerable to NK cell responses during HIV-1 transmission. Therefore, theoretically, HIV-1 would be eliminated before it has the chance to infect the autologous cells in the recipient. While this "alloreactive" NK cell mechanism is especially relevant to HIV transmission in monogamous couples, it would be interesting to investigate how it could influence resistance to HIV in other settings. The objective of this review is to summarize the knowledge about these autologous and alloreactive NK cell responses with regard to HIV-1 outcome.

Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine.

PubMed

Tran, Ngoc Q; Rezende, Lisa F; Qimron, Udi; Richardson, Charles C; Tabor, Stanley

2008-07-08

Bacteriophage T7 DNA polymerase efficiently incorporates dideoxynucleotides into DNA, resulting in chain termination. Dideoxythymidine (ddT) present in the medium at levels not toxic to Escherichia coli inhibits phage T7. We isolated 95 T7 phage mutants that were resistant to ddT. All contained a mutation in T7 gene 1.7, a nonessential gene of unknown function. When gene 1.7 was expressed from a plasmid, T7 phage resistant to ddT still arose; analysis of 36 of these mutants revealed that all had a single mutation in gene 5, which encodes T7 DNA polymerase. This mutation changes tyrosine-526 to phenylalanine, which is known to increase dramatically the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. DNA synthesis in cells infected with wild-type T7 phage was inhibited by ddT, suggesting that it resulted in chain termination of DNA synthesis in the presence of gene 1.7 protein. Overexpression of gene 1.7 from a plasmid rendered E. coli cells sensitive to ddT, indicating that no other T7 proteins are required to confer sensitivity to ddT.

Fine-structure mapping of the firA gene, a locus involved in the phenotypic expression of rifampin resistance in Escherichia.

PubMed Central

Lathe, R

1977-01-01

The firA (Ts)200 mutation not only eliminates the resistance to rifampin of certain genetically resistant strains, but, moreover, renders ribonucleic acid synthesis thermolabile. The firA gene has been mapped by P1 tranduction and is located extremely close to the structural gene for deoxyribonucleic acid polymerase III at 4 min on the Escherichia coli linkage map. PMID:330494

Purification and Properties of Bacteriophage T4-Induced RNA Ligase*

PubMed Central

Silber, Robert; Malathi, V. G.; Hurwitz, Jerard

1972-01-01

An enzyme, purified 300-fold from Escherichia coli infected with bacteriophage T4, catalyzes the conversion of 5′-termini of polyribonucleotides to internal phosphodiester bonds. The reaction requires ATP and Mg++. For every 5′-32P terminus rendered resistant to alkaline phosphatase, an equal amount of AMP and PPi are formed. Various polyribonucleotides are substrates in the reaction; to date, the best substrate is [5′-32P]polyriboadenylate. With the latter substrate, no evidence of intermolecular reaction was obtained. However, the 5′-32P termini of poly(A) rendered resistant to alkaline phosphatase are also resistant to attack by RNase II, polynucleotide phosphorylase, and low concentrations of venom phosphodiesterase. Since the product formed with poly(A) lacks 3′-hydroxyl ends, as measured with these exonucleases, the enzyme appears to convert linear molecules of polyriboadenylate to a circular form by the intramolecular covalent linkage of the 5′-phosphate end to the 3′-hydroxyl terminus. Images PMID:4342972

Systems biology of cisplatin resistance: past, present and future.

PubMed

Galluzzi, L; Vitale, I; Michels, J; Brenner, C; Szabadkai, G; Harel-Bellan, A; Castedo, M; Kroemer, G

2014-05-29

The platinum derivative cis-diamminedichloroplatinum(II), best known as cisplatin, is currently employed for the clinical management of patients affected by testicular, ovarian, head and neck, colorectal, bladder and lung cancers. For a long time, the antineoplastic effects of cisplatin have been fully ascribed to its ability to generate unrepairable DNA lesions, hence inducing either a permanent proliferative arrest known as cellular senescence or the mitochondrial pathway of apoptosis. Accumulating evidence now suggests that the cytostatic and cytotoxic activity of cisplatin involves both a nuclear and a cytoplasmic component. Despite the unresolved issues regarding its mechanism of action, the administration of cisplatin is generally associated with high rates of clinical responses. However, in the vast majority of cases, malignant cells exposed to cisplatin activate a multipronged adaptive response that renders them less susceptible to the antiproliferative and cytotoxic effects of the drug, and eventually resume proliferation. Thus, a large fraction of cisplatin-treated patients is destined to experience therapeutic failure and tumor recurrence. Throughout the last four decades great efforts have been devoted to the characterization of the molecular mechanisms whereby neoplastic cells progressively lose their sensitivity to cisplatin. The advent of high-content and high-throughput screening technologies has accelerated the discovery of cell-intrinsic and cell-extrinsic pathways that may be targeted to prevent or reverse cisplatin resistance in cancer patients. Still, the multifactorial and redundant nature of this phenomenon poses a significant barrier against the identification of effective chemosensitization strategies. Here, we discuss recent systems biology studies aimed at deconvoluting the complex circuitries that underpin cisplatin resistance, and how their findings might drive the development of rational approaches to tackle this clinically relevant problem.

Systems biology of cisplatin resistance: past, present and future

PubMed Central

Galluzzi, L; Vitale, I; Michels, J; Brenner, C; Szabadkai, G; Harel-Bellan, A; Castedo, M; Kroemer, G

2014-01-01

The platinum derivative cis-diamminedichloroplatinum(II), best known as cisplatin, is currently employed for the clinical management of patients affected by testicular, ovarian, head and neck, colorectal, bladder and lung cancers. For a long time, the antineoplastic effects of cisplatin have been fully ascribed to its ability to generate unrepairable DNA lesions, hence inducing either a permanent proliferative arrest known as cellular senescence or the mitochondrial pathway of apoptosis. Accumulating evidence now suggests that the cytostatic and cytotoxic activity of cisplatin involves both a nuclear and a cytoplasmic component. Despite the unresolved issues regarding its mechanism of action, the administration of cisplatin is generally associated with high rates of clinical responses. However, in the vast majority of cases, malignant cells exposed to cisplatin activate a multipronged adaptive response that renders them less susceptible to the antiproliferative and cytotoxic effects of the drug, and eventually resume proliferation. Thus, a large fraction of cisplatin-treated patients is destined to experience therapeutic failure and tumor recurrence. Throughout the last four decades great efforts have been devoted to the characterization of the molecular mechanisms whereby neoplastic cells progressively lose their sensitivity to cisplatin. The advent of high-content and high-throughput screening technologies has accelerated the discovery of cell-intrinsic and cell-extrinsic pathways that may be targeted to prevent or reverse cisplatin resistance in cancer patients. Still, the multifactorial and redundant nature of this phenomenon poses a significant barrier against the identification of effective chemosensitization strategies. Here, we discuss recent systems biology studies aimed at deconvoluting the complex circuitries that underpin cisplatin resistance, and how their findings might drive the development of rational approaches to tackle this clinically relevant problem. PMID:24874729

Blindsided by the Avatar: White Saviors and Allies out of Hollywood and in Education

ERIC Educational Resources Information Center

Cammarota, Julio

2011-01-01

This article discusses how the white savior syndrome renders the misrepresentation of the potential of people of color to resist and lead the transformation of oppressive conditions within their own social context. Indigenous resistance requires endogenous (internal) leadership such that all social justice actions derive from and continue to flow…

ROLE OF TOLL LIKE RECEPTORS ON PULMONARY INFLAMMATORY RESPONSES TO SIZE FRACTIONATED COMBUSTION AND AMBIENT AIR PARTICLES.

EPA Science Inventory

C3H/HeJ mice feature a single point mutation in the Toll like receptor 4 gene which renders these animals resistant to a number of pro-inflammatory agents including lipopolysaccharide and ozone. This study compared pulmonary inflammatory responses in endotoxin resistant (C3H/HeJ...

The function of yeast CAP family proteins in lipid export, mating, and pathogen defense.

PubMed

Darwiche, Rabih; El Atab, Ola; Cottier, Stéphanie; Schneiter, Roger

2018-04-01

In their natural habitat, yeast cells are constantly challenged by changing environmental conditions and a fierce competition for limiting resources. To thrive under such conditions, cells need to adapt and divide quickly, and be able to neutralize the toxic compounds secreted by their neighbors. Proteins like the pathogen-related yeast, Pry proteins, which belong to the large CAP/SCP/TAPS superfamily, may have an important role in this function. CAP proteins are conserved from yeast to man and are characterized by a unique αβα sandwich fold. They are mostly secreted glycoproteins and have been implicated in many different physiological processes including pathogen defense, virulence, venom toxicity, and sperm maturation. Yeast members of this family bind and export sterols as well as fatty acids, and they render cells resistant to eugenol, an antimicrobial compound present in clove oil. CAP family members might thus exert their various physiological functions through binding, sequestration, and neutralization of such small hydrophobic compounds. © 2017 Federation of European Biochemical Societies.

Alpha-lipoic acid blocks HIV-1 LTR-dependent expression of hygromycin resistance in THP-1 stable transformants.

PubMed

Merin, J P; Matsuyama, M; Kira, T; Baba, M; Okamoto, T

1996-09-23

Gene expression of human immunodeficiency virus (HIV) depends on a host cellular transcription factors including nuclear factor-kappaB (NF-kappaB). The involvement of reactive oxygen intermediates (ROI) has been implicated as intracellular messengers in the inducible activation of NF-kappaB. In this study, we compared the efficacy of two antioxidants, alpha-lipoic acid (LA) and N-acetylcysteine (NAC), which are widely recognized NF-kappaB inhibitors. Here, we demonstrate that LA has a more potent activity in inhibiting NF-KappaB-mediated gene expression in THP-1 cells that have been stably transfected with a plasmid bearing a hygromycin B resistance gene under the control of HIV-1 long terminal repeat (LTR) promoter. The spontaneous activation of NF-kappaB in this cell culture system leads to expression of the hygromycin phosphotransferase gene hence rendering the cells resistance to hygromycin B. In this study, the effect of the test compounds against transcriptional activity of HIV-1 LTR was evaluated based on the degree of cellular toxicity due to the inhibitory activity on the expression of hygromycin B resistance gene in the presence of hygromycin B. We also found that 0.2 mM LA could cause 40% reduction in the HIV-1 expression from the TNF-alpha-stimulated OM 10.1, a cell line latently infected with HIV-1. On the other hand, 10 mM NAC was required to elicit the same effect. Furthermore, the initiation of HIV-1 induction by TNF-alpha was completely abolished by 1 mM LA. These findings confirm the involvement of ROI in NF-kappaB-mediated HIV gene expression as well as the efficacy of LA as a therapeutic regimen for HIV infection and acquired immunodeficiency syndrome (AIDS). Moreover, this study validates the applicability of our present assay system which we primarily designed for the screening of candidate drugs against HIV-1 gene expression.

The Leaderless Bacteriocin Enterocin K1 Is Highly Potent against Enterococcus faecium: A Study on Structure, Target Spectrum and Receptor.

PubMed

Ovchinnikov, Kirill V; Kristiansen, Per Eugen; Straume, Daniel; Jensen, Marianne S; Aleksandrzak-Piekarczyk, Tamara; Nes, Ingolf F; Diep, Dzung B

2017-01-01

Enterocin K1 (EntK1), enterocin EJ97 (EntEJ97), and LsbB are three sequence related leaderless bacteriocins. Yet LsbB kills only lactococci while EntK1 and EntEJ97 target wider spectra with EntK1 being particularly active against Enterococcus faecium , including nosocomial multidrug resistant isolates. NMR study of EntK1 showed that it had a structure very similar to LsbB - both having an amphiphilic N-terminal α-helix and an unstructured C-terminus. The α-helix in EntK1 is, however, about 3-4 residues longer than that of LsbB. Enterococcal mutants highly resistant to EntEJ97 and EntK1 were found to have mutations within rseP , a gene encoding a stress response membrane-bound Zn-dependent protease. Heterologous expression of the enterococcal rseP rendered resistant cells of Streptococcus pneumoniae sensitive to EntK1 and EntEJ97, suggesting that RseP likely serves as the receptor for EntK1 and EntEJ97. It was also shown that the conserved proteolytic active site in E. faecalis RseP is partly required for EntK1 and EntEJ97 activity, since alanine substitutions of its conserved residues (HExxH) reduced the sensitivity of the clones to the bacteriocins. RseP is known to be involved in bacterial stress response. As expected, the growth of resistant mutants with mutations within rseP was severely affected when they were exposed to higher (stressing) growth temperatures, e.g., at 45°C, at which wild type cells still grew well. These findings allow us to design a hurdle strategy with a combination of the bacteriocin(s) and higher temperature that effectively kills bacteriocin sensitive bacteria and prevents the development of resistant cells.

Ambient Air Pollution Exaggerates Adipose Inflammation and Insulin Resistance in a Mouse Model of Diet-Induced Obesity

PubMed Central

Sun, Qinghua; Yue, Peibin; Deiuliis, Jeffrey A.; Lumeng, Carey N.; Kampfrath, Thomas; Mikolaj, Michael B.; Cai, Ying; Ostrowski, Michael C.; Lu, Bo; Parthasarathy, Sampath; Brook, Robert D.; Moffatt-Bruce, Susan D.; Chen, Lung Chi; Rajagopalan, Sanjay

2009-01-01

Background There is a strong link between urbanization and type 2 diabetes mellitus. Although a multitude of mechanisms have been proposed, there are no studies evaluating the impact of ambient air pollutants and the propensity to develop type 2 diabetes mellitus. We hypothesized that exposure to ambient fine particulate matter (<2.5 μm; PM2.5) exaggerates diet-induced insulin resistance, adipose inflammation, and visceral adiposity. Methods and Results Male C57BL/6 mice were fed high-fat chow for 10 weeks and randomly assigned to concentrated ambient PM2.5 or filtered air (n=14 per group) for 24 weeks. PM2.5-exposed C57BL/6 mice exhibited marked whole-body insulin resistance, systemic inflammation, and an increase in visceral adiposity. PM2.5 exposure induced signaling abnormalities characteristic of insulin resistance, including decreased Akt and endothelial nitric oxide synthase phosphorylation in the endothelium and increased protein kinase C expression. These abnormalilties were associated with abnormalities in vascular relaxation to insulin and acetylcholine. PM2.5 increased adipose tissue macrophages (F4/80+ cells) in visceral fat expressing higher levels of tumor necrosis factor-α/interleukin-6 and lower interleukin-10/N-acetyl-galactosamine specific lectin 1. To test the impact of PM2.5 in eliciting direct monocyte infiltration into fat, we rendered FVBN mice expressing yellow fluorescent protein (YFP) under control of a monocyte-specific promoter (c-fms, c-fmsYFP) diabetic over 10 weeks and then exposed these mice to PM2.5 or saline intratracheally. PM2.5 induced YFP cell accumulation in visceral fat and potentiated YFP cell adhesion in the microcirculation. Conclusion PM2.5 exposure exaggerates insulin resistance and visceral inflammation/adiposity. These findings provide a new link between air pollution and type 2 diabetes mellitus. PMID:19153269

Internalization of Ineffective Platinum Complex in Nanocapsules Renders It Cytotoxic.

PubMed

Vrana, Oldrich; Novohradsky, Vojtech; Medrikova, Zdenka; Burdikova, Jana; Stuchlikova, Olga; Kasparkova, Jana; Brabec, Viktor

2016-02-18

Anticancer therapy by platinum complexes, based on nanocarrier-based delivery, may offer a new approach to improve the efficacy and tolerability of the platinum family of anticancer drugs. The original rules for the design of new anticancer platinum drugs were affected by the fact that, although cisplatin (cis-[PtCl2 (NH3)2) was an anticancer drug, its isomer transplatin was not cytotoxic. For the first time, it is demonstrated that simple encapsulation of an inactive platinum compound in phospholipid bilayers transforms it into an efficient cytotoxic agent. Notably, the encapsulation of transplatin makes it possible to overcome the resistance mechanisms operating in cancer cells treated with cisplatin and prevents inactivation of transplatin in the extracellular environment. It is also shown that transplatin delivered to the cells in nanocapsules, in contrast to free (nonencapsulated) complex, forms cytotoxic cross-links on DNA. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High throughput microscopy identifies bisphenol AP, a bisphenol A analog, as a novel AR down-regulator.

PubMed

Stossi, Fabio; Dandekar, Radhika D; Bolt, Michael J; Newberg, Justin Y; Mancini, Maureen G; Kaushik, Akash K; Putluri, Vasanta; Sreekumar, Arun; Mancini, Michael A

2016-03-29

Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.

Chronic Myeloid Leukemia in the Era of Tyrosine Kinase Inhibitors: An Evolving Paradigm of Molecularly Targeted Therapy.

PubMed

Ali, Mohamed A M

2016-08-01

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the unrestrained expansion of pluripotent hematopoietic stem cells. CML was the first malignancy in which a unique chromosomal abnormality was identified and a pathophysiologic association was suggested. The hallmark of CML is a reciprocal chromosomal translocation between the long arms of chromosomes 9 and 22, t(9; 22)(q34; q11), creating a derivative 9q+ and a shortened 22q-. The latter, known as the Philadelphia (Ph) chromosome, harbors the breakpoint cluster region-abelson (BCR-ABL) fusion gene, encoding the constitutively active BCR-ABL tyrosine kinase that is necessary and sufficient for initiating CML. The successful implementation of tyrosine kinase inhibitors (TKIs) for the treatment of CML remains a flagship for molecularly targeted therapy in cancer. TKIs have changed the clinical course of CML; however, some patients nonetheless demonstrate primary or secondary resistance to such therapy and require an alternative therapeutic strategy. Therefore, the assessment of early response to treatment with TKIs has become an important tool in the clinical monitoring of CML patients. Although mutations in the BCR-ABL have proven to be the most prominent mechanism of resistance to TKIs, other mechanisms-either rendering the leukemic cells still dependent on BCR-ABL activity or supporting oncogenic properties of the leukemic cells independent of BCR-ABL signaling-have been identified. This article provides an overview of the current understanding of CML pathogenesis; recommendations for diagnostic tools, treatment strategies, and management guidelines; and highlights the BCR-ABL-dependent and -independent mechanisms that contribute to the development of resistance to TKIs.

Subcellular localization patterns and their relationship to photodynamic activity of pyropheophorbide-a derivatives.

PubMed

MacDonald, I J; Morgan, J; Bellnier, D A; Paszkiewicz, G M; Whitaker, J E; Litchfield, D J; Dougherty, T J

1999-11-01

To determine if subcellular localization is important to photodynamic therapy (PDT) efficacy, an in vitro fluorescence microscopy study was conducted with a congeneric series of pyropheophorbide-a derivatives in human pharyngeal squamous cell carcinoma (FaDu) cells and murine radiation-induced fibrosarcoma (RIF) mutant cells. In the FaDu cells the octyl, decyl and dodecyl ether derivatives localized to the lysosomes at extracellular concentrations less than needed to produce a 50% cell kill (LD50). At extracellular concentrations equal or greater than the LD50 the compounds localized mainly to mitochondria. The propyl, pentyl, hexyl and heptyl ether derivatives localized mainly to the mitochondria at all concentrations studied. This suggested that mitochondria are a sensitive PDT target for these derivatives. Similar experiments were performed with two Photofrin-PDT resistant RIF cell lines, one of which was found to be resistant to hexyl ether derivative (C6) mediated-PDT and the other sensitive to C6-PDT relative to the parent line. At extracellular concentrations of C6 below the LD50 of each cell line, the mutants exhibited lysosomal localization. At concentrations above these values the patterns shifted to a mainly mitochondrial pattern. In these cell lines mitochondrial localization also correlated with PDT sensitivity. Localization to mitochondria or lysosomes appeared to be affected by the aggregation state of the congeners, all of which are highly aggregated in aqueous medium. Monomers apparently were the active fraction of these compounds because equalizing the extracellular monomer concentrations produced equivalent intracellular concentrations, photoxicity and localization patterns. Compounds that were mainly aggregates localized to the lysosomes where they were rendered less active. Mitochondria appear to be a sensitive target for pyropheophorbide-a-mediated photodamage, and the degree of aggregation seems to be a determinant of the localization site.

Bacteriocin-like inhibitory activities of seven Lactobacillus delbrueckii subsp. bulgaricus strains against antibiotic susceptible and resistant Helicobacter pylori strains.

PubMed

Boyanova, L; Gergova, G; Markovska, R; Yordanov, D; Mitov, I

2017-12-01

The aim of the study was to detect anti-Helicobacter pylori activity of seven Lactobacillus delbrueckii subsp. bulgaricus (GLB) strains by four cell-free supernatant (CFS) types. Activity of non-neutralized and non-heat-treated (CFSs1), non-neutralized and heat-treated (CFSs2), pH neutralized, catalase-treated and non-heat-treated (CFSs3), or neutralized, catalase- and heat-treated (CFSs4) CFSs against 18 H. pylori strains (11 of which with antibiotic resistance) was evaluated. All GLB strains produced bacteriocin-like inhibitory substances (BLISs), the neutralized CFSs of two GLB strains inhibited >81% of test strains and those of four GLB strains were active against >71% of antibiotic resistant strains. Two H. pylori strains were BLIS resistant. The heating did not reduce the CFS activity. Briefly, all GLB strains evaluated produced heat-stable BLISs, although GLB and H. pylori strain susceptibility patterns exhibited differences. Bacteriocin-like inhibitory substance activity can be an advantage for the probiotic choice for H. pylori infection control. In this study, anti-Helicobacter pylori activity of seven Lactobacillus delbrueckii subsp. bulgaricus (GLB) strains was evaluated by four cell-free supernatant (CFS) types. The GLB strains produced heat-stable bacteriocin-like inhibitory substances (BLISs) with a strong anti-H. pylori activity and some neutralized, catalase- and heat-treated CFSs inhibited >83% of the test strains. Bacteriocin-like inhibitory substance production of GLB strains can render them valuable probiotics in the control of H. pylori infection. © 2017 The Society for Applied Microbiology.

Titanium dioxide antireflection coating for silicon solar cells by spray deposition

NASA Technical Reports Server (NTRS)

Kern, W.; Tracy, E.

1980-01-01

A high-speed production process is described for depositing a single-layer, quarter-wavelength thick antireflection coating of titanium dioxide on metal-patterned single-crystal silicon solar cells for terrestrial applications. Controlled atomization spraying of an organotitanium solution was selected as the most cost-effective method of film deposition using commercial automated equipment. The optimal composition consists of titanium isopropoxide as the titanium source, n-butyl acetate as the diluent solvent, sec-butanol as the leveling agent, and 2-ethyl-1-hexanol to render the material uniformly depositable. Application of the process to the coating of circular, large-diameter solar cells with either screen-printed silver metallization or with vacuum-evaporated Ti/Pd/Ag metallization showed increases of over 40% in the electrical conversion efficiency. Optical characteristics, corrosion resistance, and several other important properties of the spray-deposited film are reported. Experimental evidence indicates a wide tolerance in the coating thickness upon the overall efficiency of the cell. Considerations pertaining to the optimization of AR coatings in general are discussed, and a comprehensive critical survey of the literature is presented.

A biomimetic functionalization approach to integration of carbon nanoutbes into biological systems

NASA Astrophysics Data System (ADS)

Chen, Xing; Tam, Un Chong; Bertozzi, Carolyn; Zettl, Alex

2006-03-01

Due to their remarkable structural, electrical, and mechanical properties, carbon nanotubes (CNTs) have potential applications in biology ranging from imaging and tissue engineering. To realize these applications, however, new strategies for controlling the interaction between CNTs and biological systems such as proteins and cells are required. Here we describe a biomimetic approach to functionalize CNTs and therefore render them biocompatibility in order to facilitate their integration into biological systems. CNTs were coated with synthetic gycopolymers that mimic cell surface mucin gycoproteins. The functionalized CNTs were soluble in water, resisted non-specific protein binding and bound specifically to biomolecules. The coated CNTs could then be integrated onto mammalian cell surface by virtue of glycan-receptor interactions. Furthermore, the functionalized CNTs are non-toxic to cells. This strategy offers new opportunities for development of biosensor to probe biological processes. References: 1. X. Chen, G. S. Lee, A. Zettl, C. R. Bertozzi, Angewandte Chemie-International Edition 43, 6111 (2004). 2. X. Chen, U. C. Tam, J. L. Czlapanski, G. S. Lee, D. Rabuka, A. Zettl, C. R. Bertozzi, submitted.

Circadian and Melatonin Disruption by Exposure to Light at Night Drives Intrinsic Resistance to Tamoxifen Therapy in Breast Cancer

PubMed Central

Dauchy, Robert T.; Xiang, Shulin; Mao, Lulu; Brimer, Samantha; Wren, Melissa A.; Yuan, Lin; Anbalagan, Muralidharan; Hauch, Adam; Frasch, Tripp; Rowan, Brian G.; Blask, David E.; Hill, Steven M.

2014-01-01

Resistance to endocrine therapy is a major impediment to successful treatment of breast cancer. Preclinical and clinical evidence links resistance to anti-estrogen drugs in breast cancer cells with the overexpression and/or activation of various pro-oncogenic tyrosine kinases. Disruption of circadian rhythms by night shift work or disturbed sleep-wake cycles may lead to an increased risk of breast cancer and other diseases. Moreover, light exposure at night (LEN) suppresses the nocturnal production of melatonin that inhibits breast cancer growth. In this study, we used a rat model of ERα+ MCF-7 tumor xenografts to demonstrate how altering light/dark cycles with dim LEN (dLEN) speeds the development of breast tumors, increasing their metabolism and growth and conferring an intrinsic resistance to tamoxifen therapy. These characters were not produced in animals where circadian rhythms were not disrupted, or in animals subjected to dLEN if they received nocturnal melatonin replacement. Strikingly, our results also showed that melatonin acted both as a tumor metabolic inhibitor and a circadian-regulated kinase inhibitor to re-establish the sensitivity of breast tumors to tamoxifen and tumor regression. Together, our findings show how dLEN-mediated disturbances in nocturnal melatonin production can render tumors insensitive to tamoxifen. PMID:25062775

Involvement of the Chemokine RANTES (CCL5) in Resistance to Experimental Infection with Leishmania major

PubMed Central

da Costa Santiago, Helton; Oliveira, Carolina Ferreira; Santiago, Luciana; Ferraz, Fernanda Oliveira; da Glória de Souza, Daniele; Rodrigues De-Freitas, Luiz Antônio; Crocco Afonso, Luís Carlos; Teixeira, Mauro Martins; Gazzinelli, Ricardo Tostes; Vieira, Leda Quercia

2004-01-01

The expression and putative role of chemokines during infection with Leishmania major in mice were investigated. CCL5 expression correlates with resistance, and blockade of CCL5 rendered mice more susceptible to infection. CCL5 is part of the cascade of events leading to efficient parasite control in L. major infection. PMID:15271961

Tumor Cells Surviving Exposure to Proton or Photon Radiation Share a Common Immunogenic Modulation Signature, Rendering Them More Sensitive to T Cell–Mediated Killing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gameiro, Sofia R.; Malamas, Anthony S.; Bernstein, Michael B.

Purpose: To provide the foundation for combining immunotherapy to induce tumor antigen–specific T cells with proton radiation therapy to exploit the activity of those T cells. Methods and Materials: Using cell lines of tumors frequently treated with proton radiation, such as prostate, breast, lung, and chordoma, we examined the effect of proton radiation on the viability and induction of immunogenic modulation in tumor cells by flow cytometric and immunofluorescent analysis of surface phenotype and the functional immune consequences. Results: These studies show for the first time that (1) proton and photon radiation induced comparable up-regulation of surface molecules involved in immune recognition (histocompatibilitymore » leukocyte antigen, intercellular adhesion molecule 1, and the tumor-associated antigens carcinoembryonic antigen and mucin 1); (2) proton radiation mediated calreticulin cell-surface expression, increasing sensitivity to cytotoxic T-lymphocyte killing of tumor cells; and (3) cancer stem cells, which are resistant to the direct cytolytic activity of proton radiation, nonetheless up-regulated calreticulin after radiation in a manner similar to non-cancer stem cells. Conclusions: These findings offer a rationale for the use of proton radiation in combination with immunotherapy, including for patients who have failed radiation therapy alone or have limited treatment options.« less

PD-1 expression on dendritic cells suppresses CD8+ T cell function and antitumor immunity.

PubMed

Lim, Tong Seng; Chew, Valerie; Sieow, Je Lin; Goh, Siting; Yeong, Joe Poh-Sheng; Soon, Ai Ling; Ricciardi-Castagnoli, Paola

2016-03-01

Programmed death one (PD-1) is a well-established co-inhibitory regulator that suppresses proliferation and cytokine production of T cells. Despite remarkable progress in delineating the functional roles of PD-1 on T lymphocytes, little is known about the regulatory role of PD-1 expressed on myeloid cells such as dendritic cells (DCs). Here, we show that CD8 + T cells can be more potently activated to secrete IL-2 and IFNγ by PD-1-deficient DCs compared to wild-type DCs. Adoptive transfer of PD-1-deficient DCs demonstrated their superior capabilities in inducing antigen-specific CD8 + T cell proliferation in vivo . In addition, we provide first evidence demonstrating the existence of peripheral blood DCs and CD11c + tumor-infiltrating myeloid cells that co-express PD-1 in patients with hepatocellular carcinoma (HCC). The existence of PD-1-expressing HCC-infiltrating DCs (HIDCs) was further supported in a mouse model of HCC. Intratumoral transfer of PD-1-deficient DCs rendered recipient mice resistant to the growth of HCC by promoting tumor-infiltrating CD8 + effector T cells to secrete perforin and granzyme B. This novel finding provides a deeper understanding of the role of PD-1 in immune regulation and has significant implications for cancer immunotherapies targeting PD-1.

PD-1 expression on dendritic cells suppresses CD8+ T cell function and antitumor immunity

PubMed Central

Lim, Tong Seng; Chew, Valerie; Sieow, Je Lin; Goh, Siting; Yeong, Joe Poh-Sheng; Soon, Ai Ling; Ricciardi-Castagnoli, Paola

2016-01-01

ABSTRACT Programmed death one (PD-1) is a well-established co-inhibitory regulator that suppresses proliferation and cytokine production of T cells. Despite remarkable progress in delineating the functional roles of PD-1 on T lymphocytes, little is known about the regulatory role of PD-1 expressed on myeloid cells such as dendritic cells (DCs). Here, we show that CD8+ T cells can be more potently activated to secrete IL-2 and IFNγ by PD-1-deficient DCs compared to wild-type DCs. Adoptive transfer of PD-1-deficient DCs demonstrated their superior capabilities in inducing antigen-specific CD8+ T cell proliferation in vivo. In addition, we provide first evidence demonstrating the existence of peripheral blood DCs and CD11c+ tumor-infiltrating myeloid cells that co-express PD-1 in patients with hepatocellular carcinoma (HCC). The existence of PD-1-expressing HCC-infiltrating DCs (HIDCs) was further supported in a mouse model of HCC. Intratumoral transfer of PD-1-deficient DCs rendered recipient mice resistant to the growth of HCC by promoting tumor-infiltrating CD8+ effector T cells to secrete perforin and granzyme B. This novel finding provides a deeper understanding of the role of PD-1 in immune regulation and has significant implications for cancer immunotherapies targeting PD-1. PMID:27141339

SMAD4 gene mutation renders pancreatic cancer resistance to radiotherapy through promotion of autophagy.

PubMed

Wang, Feng; Xia, Xiaojun; Yang, Chunying; Shen, Jianliang; Mai, Junhua; Kim, Han-Cheon; Kirui, Dickson; Kang, Ya'an; Fleming, Jason B; Koay, Eugene J; Mitra, Sankar; Ferrari, Mauro; Shen, Haifa

2018-03-30

Understanding the mechanism of radioresistance could help develop strategies to improve therapeutic response of patients with PDAC. The SMAD4 gene is frequently mutated in pancreatic cancer. In this study, we investigated the role of SMAD4 deficiency in pancreatic cancer cells' response to radiotherapy. We downregulated SMAD4 expression with SMAD4 siRNA or SMAD4 shRNA and overexpressed SMAD4 in SMAD4 mutant pancreatic cancer cells followed by clonogenic survival assay to evaluate their effects on cell radioresistance. To study the mechanism of radioresistance, the effects of SMAD4 loss on reactive oxygen species (ROS) and autophagy were determined by Flow Cytometry and immunoblot analysis, respectively. Furthermore, we measured radioresistance by clonogenic survival assay after treatment with autophagy inhibitor (Chloroquine) and ROS inhibitor (N-acetyl-L-cysteine) in SMAD4 -depleted pancreatic cancer cells. Finally, the effects of SMAD4 on radioresistance were also confirmed in an orthotopic tumor model derived from SMAD4 -depleted Panc-1 cells. SMAD4 -depleted pancreatic cancer cells were more resistant to radiotherapy based on clonogenic survival assay. Overexpression of wild type SMAD4 in SMAD4 -mutant cells rescued their radiosensitivity. Radioresistance mediated by SMAD4 depletion was associated with persistently higher levels of ROS and radiation-induced autophagy. Finally, SMAD4 depletion induced in vivo radioresistance in Panc-1-derived orthotopic tumor model ( P = 0.038). More interestingly, we observed that the protein level of SMAD4 is inversely correlated with autophagy in orthotopic tumor tissue samples. Our results demonstrate that defective SMAD4 is responsible for radioresistance in pancreatic cancer through induction of ROS and increased level of radiation-induced autophagy. Copyright ©2018, American Association for Cancer Research.

Microbicidal Effects of Stored Aqueous Ozone Solution Generated by Nano-bubble Technology.

PubMed

Seki, Mineaki; Ishikawa, Tatsuya; Terada, Hiroshi; Nashimoto, Masayuki

2017-01-01

Clinically used disinfectants are often irritating and cause skin problems. Ozone water is unique among disinfectants. It does not damage skin cells and readily decomposes to oxygen without generating harmful residues. On the other hand, it rapidly loses its sanitizing activity. Recently developed nano-bubble ozone water (NBOW) can keep its sanitizing activity much longer. This study aimed to examine the microbicidal effects of NBOW after long-term storage. The concentration of ozone in NBOW was examined by measuring the NBOW redox potential. Microbicidal activity was evaluated by colony formation assays, after incubating bacteria with NBOW for set time periods. NBOW lost its microbicidal activity after 1 year of storage at 4°C. Stocked frozen, NBOW retained appreciable microbicidal activity after 1 year of storage. Mycobacterium smegmatis, one of the most disinfectant-resistant bacteria, was killed within 15 min. NBOW was resistant to freeze-thawing. NBOW that had been stored frozen possessed sufficient microbicidal activity to kill bacteria even after 1 year of storage. Moreover, it was shown that NBOW is freeze-thaw resistant. NBOW possesses desirable features rendering it an attractive alternative disinfectant. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Microbial colonization of biopolymeric thin films containing natural compounds and antibiotics fabricated by MAPLE

NASA Astrophysics Data System (ADS)

Cristescu, R.; Surdu, A. V.; Grumezescu, A. M.; Oprea, A. E.; Trusca, R.; Vasile, O.; Dorcioman, G.; Visan, A.; Socol, G.; Mihailescu, I. N.; Mihaiescu, D.; Enculescu, M.; Chifiriuc, M. C.; Boehm, R. D.; Narayan, R. J.; Chrisey, D. B.

2015-05-01

Although a great number of antibiotics are currently available, they are often rendered ineffective by the ability of microbial strains to develop genetic resistance and to grow in biofilms. Since many antimicrobial agents poorly penetrate biofilms, biofilm-associated infections often require high concentrations of antimicrobial agents for effective treatment. Among the various strategies that may be used to inhibit microbial biofilms, one strategy that has generated significant interest involves the use of bioactive surfaces that are resistant to microbial colonization. In this respect, we used matrix assisted pulsed laser evaporation (MAPLE) involving a pulsed KrF* excimer laser source (λ = 248 nm, τ = 25 ns, ν = 10 Hz) to obtain thin composite biopolymeric films containing natural (flavonoid) or synthetic (antibiotic) compounds as bioactive substances. Chemical composition and film structures were investigated by Fourier transform infrared spectroscopy and X-ray diffraction. Films morphology was studied by scanning electron microscopy and transmission electron microscopy. The antimicrobial assay of the microbial biofilms formed on these films was assessed by the viable cell counts method. The flavonoid-containing thin films showed increased resistance to microbial colonization, highlighting their potential to be used for the design of anti-biofilm surfaces.

Stem-like tumor-initiating cells isolated from IL13Rα2 expressing gliomas are targeted and killed by IL13-zetakine-redirected T Cells.

PubMed

Brown, Christine E; Starr, Renate; Aguilar, Brenda; Shami, Andrew F; Martinez, Catalina; D'Apuzzo, Massimo; Barish, Michael E; Forman, Stephen J; Jensen, Michael C

2012-04-15

To evaluate IL13Rα2 as an immunotherapeutic target for eliminating glioma stem-like cancer initiating cells (GSC) of high-grade gliomas, with particular focus on the potential of genetically engineered IL13Rα2-specific primary human CD8(+) CTLs (IL13-zetakine(+) CTL) to target this therapeutically resistant glioma subpopulation. A panel of low-passage GSC tumor sphere (TS) and serum-differentiated glioma lines were expanded from patient glioblastoma specimens. These glioblastoma lines were evaluated for expression of IL13Rα2 and for susceptibility to IL13-zetakine(+) CTL-mediated killing in vitro and in vivo. We observed that although glioma IL13Rα2 expression varies between patients, for IL13Rα2(pos) cases this antigen was detected on both GSCs and more differentiated tumor cell populations. IL13-zetakine(+) CTL were capable of efficient recognition and killing of both IL13Rα2(pos) GSCs and IL13Rα2(pos) differentiated cells in vitro, as well as eliminating glioma-initiating activity in an orthotopic mouse tumor model. Furthermore, intracranial administration of IL13-zetakine(+) CTL displayed robust antitumor activity against established IL13Rα2(pos) GSC TS-initiated orthotopic tumors in mice. Within IL13Rα2 expressing high-grade gliomas, this receptor is expressed by GSCs and differentiated tumor populations, rendering both targetable by IL13-zetakine(+) CTLs. Thus, our results support the potential usefullness of IL13Rα2-directed immunotherapeutic approaches for eradicating therapeutically resistant GSC populations. ©2012 AACR.

Up-regulation of Survivin during Immortalization of Human Myofibroblasts Is Linked to Repression of Tumor Suppressor p16INK4a Protein and Confers Resistance to Oxidative Stress*

PubMed Central

Kan, Chin-Yi; Petti, Carlotta; Bracken, Lauryn; Maritz, Michelle; Xu, Ning; O'Brien, Rosemary; Yang, Chen; Liu, Tao; Yuan, Jun; Lock, Richard B.; MacKenzie, Karen L.

2013-01-01

Survivin is an essential component of the chromosomal passenger complex and a member of the inhibitor of apoptosis family. It is expressed at high levels in a large variety of malignancies, where it has been implicated in drug resistance. It was also shown previously that survivin is up-regulated during telomerase-mediated immortalization, which occurs at a relatively early stage during carcinogenesis. This study shows that up-regulation of survivin during immortalization of human myofibroblasts is an indirect consequence of the repression of p16INK4a. Survivin and p16INK4a were functionally linked by assays that showed that either the up-regulation of survivin or repression of p16INK4a rendered telomerase-transduced MRC-5 myofibroblasts resistant to oxidative stress. Conversely, siRNA-mediated down-regulation of survivin activated caspases and enhanced the sensitivity of immortal MRC-5 cells to oxidative stress. The E2F1 transcription factor, which is negatively regulated by the pRB/p16INK4a tumor suppressor pathway, was implicated in the up-regulation of survivin. Using the ChIP assay, it was shown that E2F1 directly interacted with the survivin gene (BIRC5) promoter in cells that spontaneously silenced p16INK4a during telomerase-mediated immortalization. E2F1 binding to the BIRC5 was also enhanced in telomerase-transduced cells subjected to shRNA-mediated repression of p16INK4a. Together, these data show that repression of p16INK4a contributes to the up-regulation of survivin and thereby provides a survival advantage to cells exposed to oxidative stress during immortalization. The up-regulation of survivin during immortalization likely contributes to the vulnerability of immortal cells to transformation by oncogenes that alter intracellular redox state. PMID:23449974

Metformin combined with sodium dichloroacetate promotes B leukemic cell death by suppressing anti-apoptotic protein Mcl-1

PubMed Central

Melloni, Elisabetta; Gilli, Paola; Bertolasi, Valerio; Casciano, Fabio; Rigolin, Gian Matteo; Zauli, Giorgio; Secchiero, Paola

2016-01-01

Metformin and the mitochondrial targeting dichloroacetate (DCA) have recently received attention due to their ability to inhibit anaerobic glycolysis, which renders most cancer cells resistant to apoptosis induction. We observed that Metformin alone exhibited a dose-dependent anti-leukemic activity in both B leukemic cell lines and primary B-chronic lymphocytic leukemia (B-CLL) patients' cells and its anti-leukemic activity was enhanced when used in combination with DCA. In order to overcome the problems of poor bioavailability and cellular uptake, which limit DCA efficacy, we have designed and synthetized cocrystals consisting of Metformin and DCA (Met-DCA) at different stoichiometric ratios. Of note, the MetH2++•2DCA− cocrystal exhibited enhanced in vitro anti-leukemic activity, with respect to the treatment with the mix consisting of Metformin plus DCA. In particular, the treatment with the cocrystal MetH2++•2DCA− induced a synergistic apoptotic cell death coupled to a marked down-modulation of the anti-apoptotic Mcl-1 protein. Taken together, our data emphasize that innovative compounds based on Metformin-DCA combination merit to be further evaluated as chemotherapeutic agents for the treatment of B-CLL. PMID:26959881

Novel electrospun gas diffusion layers for polymer electrolyte membrane fuel cells: Part I. Fabrication, morphological characterization, and in situ performance

NASA Astrophysics Data System (ADS)

Chevalier, S.; Lavielle, N.; Hatton, B. D.; Bazylak, A.

2017-06-01

In this first of a series of two papers, we report an in-depth analysis of the impact of the gas diffusion layer (GDL) structure on the polymer electrolyte membrane (PEM) fuel cell performance through the use of custom GDLs fabricated in-house. Hydrophobic electrospun nanofibrous gas diffusion layers (eGDLs) are fabricated with controlled fibre diameter and alignment. The eGDLs are rendered hydrophobic through direct surface functionalization, and this molecular grafting is achieved in the absence of structural alteration. The fibre diameter, chemical composition, and electrical conductivity of the eGDL are characterized, and the impact of eGDL structure on fuel cell performance is analysed. We observe that the eGDL facilitates higher fuel cell power densities compared to a commercial GDL (Toray TGP-H-60) at highly humidified operating conditions. The ohmic resistance of the fuel cell is found to significantly increase with increasing inter-fiber distance. It is also observed that the addition of a hydrophobic treatment enhances membrane hydration, and fibres perpendicularly aligned to the channel direction may enhance the contact area between the catalyst layer and the GDL.

Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance.

PubMed

Kohli, Manish; Ho, Yeung; Hillman, David W; Van Etten, Jamie L; Henzler, Christine; Yang, Rendong; Sperger, Jamie M; Li, Yingming; Tseng, Elizabeth; Hon, Ting; Clark, Tyson; Tan, Winston; Carlson, Rachel E; Wang, Liguo; Sicotte, Hugues; Thai, Ho; Jimenez, Rafael; Huang, Haojie; Vedell, Peter T; Eckloff, Bruce W; Quevedo, Jorge F; Pitot, Henry C; Costello, Brian A; Jen, Jin; Wieben, Eric D; Silverstein, Kevin A T; Lang, Joshua M; Wang, Liewei; Dehm, Scott M

2017-08-15

Purpose: Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be coexpressed with AR-V7 and promote resistance to AR-targeted therapies. Experimental Design: We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Coexpression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate. Results: AR-V9 was frequently coexpressed with AR-V7. Both AR variant species were found to share a common 3' terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0; 95% confidence interval, 1.31-12.2; P = 0.02). Conclusions: AR-V9 may be an important component of therapeutic resistance in CRPC. Clin Cancer Res; 23(16); 4704-15. ©2017 AACR . ©2017 American Association for Cancer Research.

Lung adenocarcinoma originates from retrovirus infection of proliferating type 2 pneumocytes during pulmonary post-natal development or tissue repair.

PubMed

Murgia, Claudio; Caporale, Marco; Ceesay, Ousman; Di Francesco, Gabriella; Ferri, Nicola; Varasano, Vincenzo; de las Heras, Marcelo; Palmarini, Massimo

2011-03-01

Jaagsiekte sheep retrovirus (JSRV) is a unique oncogenic virus with distinctive biological properties. JSRV is the only virus causing a naturally occurring lung cancer (ovine pulmonary adenocarcinoma, OPA) and possessing a major structural protein that functions as a dominant oncoprotein. Lung cancer is the major cause of death among cancer patients. OPA can be an extremely useful animal model in order to identify the cells originating lung adenocarcinoma and to study the early events of pulmonary carcinogenesis. In this study, we demonstrated that lung adenocarcinoma in sheep originates from infection and transformation of proliferating type 2 pneumocytes (termed here lung alveolar proliferating cells, LAPCs). We excluded that OPA originates from a bronchioalveolar stem cell, or from mature post-mitotic type 2 pneumocytes or from either proliferating or non-proliferating Clara cells. We show that young animals possess abundant LAPCs and are highly susceptible to JSRV infection and transformation. On the contrary, healthy adult sheep, which are normally resistant to experimental OPA induction, exhibit a relatively low number of LAPCs and are resistant to JSRV infection of the respiratory epithelium. Importantly, induction of lung injury increased dramatically the number of LAPCs in adult sheep and rendered these animals fully susceptible to JSRV infection and transformation. Furthermore, we show that JSRV preferentially infects actively dividing cell in vitro. Overall, our study provides unique insights into pulmonary biology and carcinogenesis and suggests that JSRV and its host have reached an evolutionary equilibrium in which productive infection (and transformation) can occur only in cells that are scarce for most of the lifespan of the sheep. Our data also indicate that, at least in this model, inflammation can predispose to retroviral infection and cancer.

A nude mouse model of obesity to study the mechanisms of resistance to aromatase inhibitors.

PubMed

Schech, Amanda; Yu, Stephen; Goloubeva, Olga; McLenithan, John; Sabnis, Gauri

2015-08-01

Obesity is a risk factor for breast cancer progression. Breast cancer patients who are overweight or obese or have excess abdominal fat have an increased risk of local or distant recurrence and cancer-related death. Hormone depletion therapies can also cause weight gain, exacerbating the risk for these patients. To understand the effect of obesity on hormone-dependent human breast cancer tumors, we fed ovariectomized athymic nude mice a diet containing 45% kcal fat and 17% kcal sucrose (high fat sucrose diet (HFSD)), 10% kcal fat (low fat diet (LFD)), or a standard chow diet (chow). The mice fed the HFSD developed metabolic abnormalities consistent with the development of obesity such as weight gain, high fasting blood glucose, and impaired glucose tolerance. These mice also developed hyperinsulinemia and insulin resistance. The obese mice also had a higher tumor growth rate compared to the lean mice. Furthermore, the obese mice showed a significantly reduced responsiveness to letrozole. To understand the role of obesity in this reduced responsiveness, we examined the effect of insulin on the growth of MCF-7Ca cells in response to estrogen or letrozole. The presence of insulin rendered MCF-7Ca cells less responsive to estrogen and letrozole. Exogenous insulin treatment of MCF-7Ca cells also resulted in increased p-Akt as well as ligand-independent phosphorylation of ERα. These findings suggest that diet-induced obesity may result in reduced responsiveness of tumors to letrozole due to the development of hyperinsulinemia. We conclude that obesity influences the response and resistance of breast cancer tumors to aromatase inhibitor treatment. © 2015 Society for Endocrinology.

Saporin-S6: A Useful Tool in Cancer Therapy

PubMed Central

Polito, Letizia; Bortolotti, Massimo; Mercatelli, Daniele; Battelli, Maria Giulia; Bolognesi, Andrea

2013-01-01

Thirty years ago, the type 1 ribosome-inactivating protein (RIP) saporin-S6 (also known as saporin) was isolated from Saponaria officinalis L. seeds. Since then, the properties and mechanisms of action of saporin-S6 have been well characterized, and it has been widely employed in the construction of conjugates and immunotoxins for different purposes. These immunotoxins have shown many interesting results when used in cancer therapy, particularly in hematological tumors. The high enzymatic activity, stability and resistance to conjugation procedures and blood proteases make saporin-S6 a very useful tool in cancer therapy. High efficacy has been reported in clinical trials with saporin-S6-containing immunotoxins, at dosages that induced only mild and transient side effects, which were mainly fever, myalgias, hepatotoxicity, thrombocytopenia and vascular leak syndrome. Moreover, saporin-S6 triggers multiple cell death pathways, rendering impossible the selection of RIP-resistant mutants. In this review, some aspects of saporin-S6, such as the chemico-physical characteristics, the structural properties, its endocytosis, its intracellular routing and the pathogenetic mechanisms of the cell damage, are reported. In addition, the recent progress and developments of saporin-S6-containing immunotoxins in cancer immunotherapy are summarized, including in vitro and in vivo pre-clinical studies and clinical trials. PMID:24105401

Targeted nanoparticles for enhanced X-ray radiation killing of multidrug-resistant bacteria.

PubMed

Luo, Yang; Hossain, Mainul; Wang, Chaoming; Qiao, Yong; An, Jincui; Ma, Liyuan; Su, Ming

2013-01-21

This paper describes a nanoparticle enhanced X-ray irradiation based strategy that can be used to kill multidrug resistant (MDR) bacteria. In the proof-of-concept experiment using MDR Pseudomonas aeruginosa (P. aeruginosa) as an example, polyclonal antibody modified bismuth nanoparticles are introduced into bacterial culture to specifically target P. aeruginosa. After washing off uncombined bismuth nanoparticles, the bacteria are irradiated with X-rays, using a setup that mimics a deeply buried wound in humans. Results show that up to 90% of MDR P. aeruginosa are killed in the presence of 200 μg ml(-1) bismuth nanoparticles, whereas only ∼6% are killed in the absence of bismuth nanoparticles when exposed to 40 kVp X-rays for 10 min. The 200 μg ml(-1) bismuth nanoparticles enhance localized X-ray dose by 35 times higher than the control with no nanoparticles. In addition, no significant harmful effects on human cells (HeLa and MG-63 cells) have been observed with 200 μg ml(-1) bismuth nanoparticles and 10 min 40 kVp X-ray irradiation exposures, rendering the potential for future clinical use. Since X-rays can easily penetrate human tissues, this bactericidal strategy has the potential to be used in effectively killing deeply buried MDR bacteria in vivo.

Cyclin D1 Downregulation Contributes to Anti-Cancer Effect of Isorhapontigenin (ISO) on Human Bladder Cancer Cells

PubMed Central

Fang, Yong; Cao, Zipeng; Hou, Qi; Ma, Chen; Yao, Chunsuo; Li, Jingxia; Wu, Xue-Ru; Huang, Chuanshu

2013-01-01

Isorhapontigenin (ISO) is a new derivative of stilbene compound that was isolated from the Chinese herb Gnetum Cleistostachyum, and has been used for treatment of bladder cancers for centuries. In our current studies, we have explored the potential inhibitory effect and molecular mechanisms underlying ISO anti-cancer effects on anchorage-independent growth of human bladder cancer cell lines. We found that ISO showed a significant inhibitory effect on human bladder cancer cell growth and was accompanied with related cell cycle G0/G1 arrest as well as downregulation of Cyclin D1 expression at the transcriptional level in UMUC3 and RT112 cells. Further studies identified that ISO down-regulated Cyclin D1 gene transcription via inhibition of SP1 transactivation. Moreover, ectopic expression of GFP-Cyclin D1 rendered UMUC3 cells resistant to induction of cell cycle G0/G1 arrest and inhibition of cancer cell anchorage-independent growth by ISO treatment. Together, our studies demonstrate that ISO is an active compound that mediates for Gnetum Cleistostachyum’s induction of cell cycle G0/G1 arrest and inhibition of cancer cell anchorage-independent growth through down-regulating SP1/Cyclin D1 axis in bladder cancer cells. Our studies provide a novel insight into understanding the anti-cancer activity of the Chinese herb Gnetum Cleistostachyum and its isolate ISO. PMID:23723126

Technological characterization and survival of the exopolysaccharide-producing strain Lactobacillus delbrueckii subsp. lactis 193 and its bile-resistant derivative 193+ in simulated gastric and intestinal juices.

PubMed

Burns, Patricia; Vinderola, Gabriel; Reinheimer, Jorge; Cuesta, Isabel; de Los Reyes-Gavilán, Clara G; Ruas-Madiedo, Patricia

2011-08-01

The capacity of lactic acid bacteria to produce exopolysaccharides (EPS) conferring microorganisms a ropy phenotype could be an interesting feature from a technological point of view. Progressive adaptation to bile salts might render some lactobacilli able to overcome physiological gut barriers but could also modify functional properties of the strain, including the production of EPS. In this work some technological properties and the survival ability in simulated gastrointestinal conditions of Lactobacillus delbrueckii subsp. lactis 193, and Lb. delbrueckii subsp. lactis 193+, a strain with stable bile-resistant phenotype derived thereof, were characterized in milk in order to know whether the acquisition of resistance to bile could modify some characteristics of the microorganism. Both strains were able to grow and acidify milk similarly; however the production of ethanol increased at the expense of the aroma compound acetaldehyde in milk fermented by the strain 193+, with respect to milk fermented by the strain 193. Both microorganisms produced a heteropolysaccharide composed of glucose and galactose, and were able to increase the viscosity of fermented milks. In spite of the higher production yield of EPS by the bile-resistant strain 193+, it displayed a lower ability to increase viscosity than Lb. delbrueckii subsp. lactis 193. Milk increased survival in simulated gastric juice; the presence of bile improved adhesion to the intestinal cell line HT29-MTX in both strains. However, the acquisition of a stable resistance phenotype did not improve survival in simulated gastric and intestinal conditions or the adhesion to the intestinal cell line HT29-MTX. Thus, Lb. delbrueckii subsp. lactis 193 presents suitable technological properties for the manufacture of fermented dairy products; the acquisition of a stable bile-resistant phenotype modified some properties of the microorganism. This suggests that the possible use of bile-resistant derivative strains should be carefully evaluated in each specific application considering the influence that the acquisition of a stable bile-resistant phenotype could have in survival ability in gastric and intestinal conditions and in technological properties.

Genotoxic Effects of Low- and High-LET Radiation on Human Epithelial Cells Grown in 2-D Versus 3-D Culture

NASA Technical Reports Server (NTRS)

Patel, Z. S.; Cucinotta, F. A.; Huff, J. L.

2011-01-01

Risk estimation for radiation-induced cancer relies heavily on human epidemiology data obtained from terrestrial irradiation incidents from sources such as medical and occupational exposures as well as from the atomic bomb survivors. No such data exists for exposures to the types and doses of high-LET radiation that will be encountered during space travel; therefore, risk assessment for space radiation requires the use of data derived from cell culture and animal models. The use of experimental models that most accurately replicate the response of human tissues is critical for precision in risk projections. This work compares the genotoxic effects of radiation on normal human epithelial cells grown in standard 2-D monolayer culture compared to 3-D organotypic co-culture conditions. These 3-D organotypic models mimic the morphological features, differentiation markers, and growth characteristics of fully-differentiated normal human tissue and are reproducible using defined components. Cultures were irradiated with 2 Gy low-LET gamma rays or varying doses of high-LET particle radiation and genotoxic damage was measured using a modified cytokinesis block micronucleus assay. Our results revealed a 2-fold increase in residual damage in 2 Gy gamma irradiated cells grown under organotypic culture conditions compared to monolayer culture. Irradiation with high-LET particle radiation gave similar results, while background levels of damage were comparable under both scenarios. These observations may be related to the phenomenon of "multicellular resistance" where cancer cells grown as 3-D spheroids or in vivo exhibit an increased resistance to killing by chemotherapeutic agents compared to the same cells grown in 2-D culture. A variety of factors are likely involved in mediating this process, including increased cell-cell communication, microenvironment influences, and changes in cell cycle kinetics that may promote survival of damaged cells in 3-D culture that would otherwise die or be rendered reproductively inactive in 2-D culture.

Tanshinone IIA Pretreatment Renders Free Flaps against Hypoxic Injury through Activating Wnt Signaling and Upregulating Stem Cell-Related Biomarkers

PubMed Central

Xu, Zihan; Zhang, Zhenxin; Wu, Lijun; Sun, Yaowen; Guo, Yadong; Qin, Gaoping; Mu, Shengzhi; Fan, Ronghui; Wang, Benfeng; Gao, Wenjie

2014-01-01

Partial or total flap necrosis after flap transplantation is sometimes clinically encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. In this study, we determine whether tanshinone IIA (TSA) pretreatment can protect flap tissue against hypoxic injury and improve its viability. Primary epithelial cells isolated from the dorsal skin of mice were pretreated with TSA for two weeks. Cell counting kit-8 and Trypan Blue assays were carried out to examine the proliferation of TSA-pretreated cells after exposure to cobalt chloride. Then, Polymerase chain reaction and Western blot analysis were used to determine the expression of β-catenin, GSK-3β, SOX2, and OCT4 in TSA-treated cells. In vivo, after mice were pretreated with TSA for two weeks, a reproducible ischemic flap model was implemented, and the area of surviving tissue in the transplanted flaps was measured. Immunohistochemistry was also conducted to examine the related biomarkers mentioned above. Results show that epidermal cells, pretreated with TSA, showed enhanced resistance to hypoxia. Activation of the Wnt signaling pathway in TSA-pretreated cells was characterized by the upregulation of β-catenin and the downregulation of GSK-3β. The expression of SOX2 and OCT4 controlled by Wnt signaling were also found higher in TSA pretreated epithelial cells. In the reproducible ischaemic flap model, pretreatment with TSA enhanced resistance to hypoxia and increased the area of surviving tissue in transplanted flaps. The expression of Wnt signaling pathway components, stem-cell related biomarkers, and CD34, which are involved in the regeneration of blood vessels, was also upregulated in TSA-pretreated flap tissue. The results show that TSA pretreatment protects free flaps against hypoxic injury and increases the area of surviving tissue by activating Wnt signaling and upregulating stem cell-related biomarkers. PMID:25302618

Colony-Stimulating Factor 1 Receptor Antagonists Sensitize Human Immunodeficiency Virus Type 1-Infected Macrophages to TRAIL-Mediated Killing

PubMed Central

Cunyat, Francesc; Rainho, Jennifer N.; West, Brian; Swainson, Louise; McCune, Joseph M.

2016-01-01

ABSTRACT Strategies aimed at eliminating persistent viral reservoirs from HIV-1-infected individuals have focused on CD4+ T-cell reservoirs. However, very little attention has been given to approaches that could promote elimination of tissue macrophage reservoirs. HIV-1 infection of macrophages induces phosphorylation of colony-stimulating factor 1 receptor (CSF-1R), which confers resistance to apoptotic pathways driven by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), thereby promoting viral persistence. In this study, we assessed whether CSF-1R antagonists (PLX647, PLX3397, and PLX5622) restored apoptotic sensitivity of HIV-1-infected macrophages in vitro. PLX647, PLX3397, and PLX5622 at clinically relevant concentrations blocked the activation of CSF-1R and reduced the viability of infected macrophages, as well as the extent of viral replication. Our data show that strategies targeting monocyte colony-stimulating factor (MCSF) signaling could be used to promote elimination of HIV-1-infected myeloid cells and to contribute to the elimination of persistent viral reservoirs. IMPORTANCE As the HIV/AIDS research field explores approaches to eliminate HIV-1 in individuals on suppressive antiviral therapy, those approaches will need to eliminate both CD4+ T-cell and myeloid cell reservoirs. Most of the attention has focused on CD4+ T-cell reservoirs, and scant attention has been paid to myeloid cell reservoirs. The distinct nature of the infection in myeloid cells versus CD4+ T cells will likely dictate different approaches in order to achieve their elimination. For CD4+ T cells, most strategies focus on promoting virus reactivation to promote immune-mediated clearance and/or elimination by viral cytopathicity. Macrophages resist viral cytopathic effects and CD8+ T-cell killing. Therefore, we have explored clearance strategies that render macrophages sensitive to viral cytopathicity. This research helps inform the design of strategies to promote clearance of the macrophage reservoir in infected individuals on suppressive antiviral therapy. PMID:27122585

Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II.

PubMed

Dong, L-F; Low, P; Dyason, J C; Wang, X-F; Prochazka, L; Witting, P K; Freeman, R; Swettenham, E; Valis, K; Liu, J; Zobalova, R; Turanek, J; Spitz, D R; Domann, F E; Scheffler, I E; Ralph, S J; Neuzil, J

2008-07-17

Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

α-Tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II

PubMed Central

Dong, Lan-Feng; Low, Pauline; Dyason, Jeffrey C.; Wang, Xiu-Fang; Prochazka, Lubomir; Witting, Paul K.; Freeman, Ruth; Swettenham, Emma; Valis, Karel; Liu, Ji; Zobalova, Renata; Turanek, Jaroslav; Spitz, Doug R.; Domann, Frederick E.; Scheffler, Immo E.; Ralph, Stephen J.; Neuzil, Jiri

2009-01-01

α-Tocopheryl succinate (α-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of α-TOS has not been identified. Here we show that α-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ) binding site (QP and QD, respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of α-TOS compared to that of UbQ for the QP and QD sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and undergo apoptosis in the presence of α-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to α-TOS. We propose that α-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy. PMID:18372923

The Glutaminase-1 Inhibitor 968 Enhances Dihydroartemisinin-Mediated Antitumor Efficacy in Hepatocellular Carcinoma Cells

PubMed Central

Zheng, Meihong; Zhang, Yonghui; Chen, Aiping; Wu, Junhua; Wei, Jiwu

2016-01-01

Reprogrammed metabolism and redox homeostasis are potential targets of cancer therapy. Our previous study demonstrated that the kidney form of glutaminase (GLS1) is highly expressed in hepatocellular carcinoma (HCC) cells and can be used as a target for effective anticancer therapy. Dihydroartemisinin (DHA) increases intracellular reactive oxygen species (ROS) levels leading to cytotoxicity in cancer cells. However, the heterogeneity of cancer cells often leads to differing responses to oxidative lesions. For instance, cancer cells with high ratio of GSH/GSSG, a critical ROS scavenger, are resistant to ROS-induced cytotoxicity. We postulate that a combinatorial strategy firstly disrupting redox homeostasis followed by DHA might yield a profound antitumor efficacy. In this study, when HCC cells were treated with a GLS1 inhibitor 968, the ROS elimination capacity was significantly reduced in HCC cells, which rendered HCC cells but not normal endothelial cells more sensitive to DHA-mediated cytotoxicity. We further confirmed that this synergistic antitumor efficacy was mediated by excessive ROS generation in HCC cells. NAC, a ROS inhibitor, partly rescued the combinatorial cytotoxic effect of 968 and DHA. Given that GLS1 is a potential antitumor target and DHA has been safely used in clinic, our findings provide new insight into liver cancer therapy targeting glutamine metabolism combined with the ROS generator DHA, which can be readily translated into cancer clinical trials. PMID:27835669

Development of flame resistant treatment for nomex fibrous structures

NASA Technical Reports Server (NTRS)

Toy, M. S.

1978-01-01

Technology which renders aramid fibrous structures flame resistant through chemical modification was developed. The project scaled up flame resistant treatment from laboratory fabric swatches of a few inches to efficiently producing ten yards of commercial width (41 inches) aromatic polyamide. The radiation intensity problem of the processor was resolved. Further improvement of the processor cooling system was recommended for two reasons: (1) To advance current technology of flame proofing Nomex fabric to higher oxygen enriched atmospheres; and (2) To adapt the processor for direct applicability to low cost commercial fabrics.

Geographical Distribution of MDR1 Expression in Leishmania Isolates, from Greece and Cyprus, Measured by the Rhodamine-123 Efflux Potential of the Isolates, Using Flow Cytometry

PubMed Central

Tsirigotakis, Nikolaos; Christodoulou, Vasiliki; Ntais, Pantelis; Mazeris, Apostolos; Koutala, Eleni; Messaritakis, Ippokratis; Antoniou, Maria

2016-01-01

Leishmaniasis, a neglected vector-borne disease caused by the protozoan parasite Leishmania, is encountered in 98 countries causing serious concerns to public health. The most alarming is the development of parasite drug resistance, a phenomenon increasingly encountered in the field rendering chemotherapy ineffective. Although resistance to drugs is a complex phenomenon, the rate of efflux of the fluorescent dye Rhodamine-123 from the parasite body, using flow cytometry, is an indication of the isolate's ability to efflux the drug, thus avoiding death. The rate of efflux measured 275 Leishmania strains, isolated from patients and dogs from Greece and Cyprus, was measured and mapped to study the geographical distribution of the multidrug resistance (MDR) gene expression as an indication of the drug resistance of the parasite. The map showed that out of the seven prefectures, where dogs presented high efflux rates, five also had patients with high efflux rates. In one, out of the 59 prefectures studied, the highest number of isolates with efflux slope α > 1, in both human and dog isolates, was found; a fact which may suggest that spread of drug resistance is taking place. The virulence of the Leishmania strains, assessed after infecting human macrophages of the THP-1 cell line, fluctuated from 1% to 59.3% with only 2.5% of the isolates showing infectivity > 50%. The most virulent strains were isolated from Attica and Crete. PMID:27001764

FAS system deregulation in T-cell lymphoblastic lymphoma

PubMed Central

Villa-Morales, M; Cobos, M A; González-Gugel, E; Álvarez-Iglesias, V; Martínez, B; Piris, M A; Carracedo, A; Benítez, J; Fernández-Piqueras, J

2014-01-01

The acquisition of resistance towards FAS-mediated apoptosis may be required for tumor formation. Tumors from various histological origins exhibit FAS mutations, the most frequent being hematological malignancies. However, data regarding FAS mutations or FAS signaling alterations are still lacking in precursor T-cell lymphoblastic lymphomas (T-LBLs). The available data on acute lymphoblastic leukemia, of precursor origin as well, indicate a low frequency of FAS mutations but often report a serious reduction in FAS-mediated apoptosis as well as chemoresistance, thus suggesting the occurrence of mechanisms able to deregulate the FAS signaling pathway, different from FAS mutation. Our aim at this study was to determine whether FAS-mediated apoptotic signaling is compromised in human T-LBL samples and the mechanisms involved. This study on 26 T-LBL samples confirms that the FAS system is impaired to a wide extent in these tumors, with 57.7% of the cases presenting any alteration of the pathway. A variety of mechanisms seems to be involved in such alteration, in order of frequency the downregulation of FAS, the deregulation of other members of the pathway and the occurrence of mutations at FAS. Considering these results together, it seems plausible to think of a cumulative effect of several alterations in each T-LBL, which in turn may result in FAS/FASLG system deregulation. Since defective FAS signaling may render the T-LBL tumor cells resistant to apoptotic cell death, the correct prognosis, diagnosis and thus the success of anticancer therapy may require such an in-depth knowledge of the complete scenario of FAS-signaling alterations. PMID:24603338

Circadian and melatonin disruption by exposure to light at night drives intrinsic resistance to tamoxifen therapy in breast cancer.

PubMed

Dauchy, Robert T; Xiang, Shulin; Mao, Lulu; Brimer, Samantha; Wren, Melissa A; Yuan, Lin; Anbalagan, Muralidharan; Hauch, Adam; Frasch, Tripp; Rowan, Brian G; Blask, David E; Hill, Steven M

2014-08-01

Resistance to endocrine therapy is a major impediment to successful treatment of breast cancer. Preclinical and clinical evidence links resistance to antiestrogen drugs in breast cancer cells with the overexpression and/or activation of various pro-oncogenic tyrosine kinases. Disruption of circadian rhythms by night shift work or disturbed sleep-wake cycles may lead to an increased risk of breast cancer and other diseases. Moreover, light exposure at night (LEN) suppresses the nocturnal production of melatonin that inhibits breast cancer growth. In this study, we used a rat model of estrogen receptor (ERα(+)) MCF-7 tumor xenografts to demonstrate how altering light/dark cycles with dim LEN (dLEN) speed the development of breast tumors, increasing their metabolism and growth and conferring an intrinsic resistance to tamoxifen therapy. These characteristics were not observed in animals in which the circadian melatonin rhythm was not disrupted, or in animals subjected to dLEN if they received nocturnal melatonin replacement. Strikingly, our results also showed that melatonin acted both as a tumor metabolic inhibitor and a circadian-regulated kinase inhibitor to reestablish the sensitivity of breast tumors to tamoxifen and tumor regression. Together, our findings show how dLEN-mediated disturbances in nocturnal melatonin production can render tumors insensitive to tamoxifen. ©2014 American Association for Cancer Research.

Medulloblastoma Genotype Dictates Blood Brain Barrier Phenotype.

PubMed

Phoenix, Timothy N; Patmore, Deanna M; Boop, Scott; Boulos, Nidal; Jacus, Megan O; Patel, Yogesh T; Roussel, Martine F; Finkelstein, David; Goumnerova, Liliana; Perreault, Sebastien; Wadhwa, Elizabeth; Cho, Yoon-Jae; Stewart, Clinton F; Gilbertson, Richard J

2016-04-11

The childhood brain tumor, medulloblastoma, includes four subtypes with very different prognoses. Here, we show that paracrine signals driven by mutant β-catenin in WNT-medulloblastoma, an essentially curable form of the disease, induce an aberrant fenestrated vasculature that permits the accumulation of high levels of intra-tumoral chemotherapy and a robust therapeutic response. In contrast, SHH-medulloblastoma, a less curable disease subtype, contains an intact blood brain barrier, rendering this tumor impermeable and resistant to chemotherapy. The medulloblastoma-endothelial cell paracrine axis can be manipulated in vivo, altering chemotherapy permeability and clinical response. Thus, medulloblastoma genotype dictates tumor vessel phenotype, explaining in part the disparate prognoses among medulloblastoma subtypes and suggesting an approach to enhance the chemoresponsiveness of other brain tumors. Copyright © 2016 Elsevier Inc. All rights reserved.

Medulloblastoma genotype dictates blood brain barrier phenotype

PubMed Central

Phoenix, Timothy N.; Patmore, Deanna M.; Boop, Scott; Boulos, Nidal; Jacus, Megan O.; Patel, Yogesh T.; Roussel, Martine F; Finkelstein, David; Goumnerova, Lilian; Perreault, Sebastien; Wadhwa, Elizabeth; Cho, Yoon-Jae; Stewart, Clinton F.; Gilbertson, Richard J.

2016-01-01

SUMMARY The childhood brain tumour medulloblastoma includes four subtypes with very different prognoses. Here, we show that paracrine signals driven by mutant Beta-Catenin in WNT-medulloblastoma – an essentially curable form of the disease – induce an aberrant fenestrated vasculature that permits the accumulation of high levels of intra-tumoural chemotherapy and a robust therapeutic response. In contrast, SHH-medulloblastoma – a less curable disease subtype – contains an intact blood brain barrier, rendering this tumour impermeable and resistant to chemotherapy. The medulloblastoma-endothelial cell paracrine axis can be manipulated in vivo, altering chemotherapy permeability and clinical response. Thus, medulloblastoma genotype dictates tumour vessel phenotype, explaining in part the disparate prognoses among medulloblastoma subtypes and suggesting an approach to enhance the chemoresponsiveness of other brain tumours. PMID:27050100

Evolution subverting essentiality: Dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus

PubMed Central

Martínez, Miguel A.; Verdaguer, Nuria; Mateu, Mauricio G.; Domingo, Esteban

1997-01-01

Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet—which is also a widespread motif involved in cell-to-cell adhesion—can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA viruses in nature. PMID:9192645

Unequal homologous recombination between tandemly arranged sequences stably incorporated into cultured rat cells.

PubMed Central

Stringer, J R; Kuhn, R M; Newman, J L; Meade, J C

1985-01-01

Cultured rat cells deficient in endogenous thymidine kinase activity (tk) were stably transformed with a recombination-indicator DNA substrate constructed in vitro by rearrangement of the herpes simplex virus tk gene sequences into a partially redundant permutation of the functional gene. The recombination-indicator DNA did not express tk, but was designed to allow formation of a functional tk gene via homologous recombination. A clonal cell line (519) was isolated that harbored several permuted herpes simplex virus tk genes. 519 cells spontaneously produced progeny that survived in medium containing hypoxanthine, aminopterin, and thymidine. Acquisition of resistance to hypoxanthine, aminopterin, and thymidine was accompanied by the rearrangement of the defective tk gene to functional configuration. The rearrangement apparently occurred by unequal exchange between one permuted tk gene and a replicated copy of itself. Recombination was between 500-base-pair tracts of DNA sequence homology that were separated by 3.4 kilobases. Exchanges occurred spontaneously at a frequency of approximately 5 X 10(-6) events per cell per generation. Recombination also mediated reversion to the tk- phenotype; however, the predominant mechanism by which cells escaped death in the presence of drugs rendered toxic by thymidine kinase was not recombination, but rather inactivation of the intact tk gene. Images PMID:3016511

Perhexiline maleate enhances antitumor efficacy of cisplatin in neuroblastoma by inducing over-expression of NDM29 ncRNA

PubMed Central

Vella, Serena; Penna, Ilaria; Longo, Luca; Pioggia, Giulia; Garbati, Patrizia; Florio, Tullio; Rossi, Fabio; Pagano, Aldo

2015-01-01

High Risk Neuroblastoma (HR-NB) is a pediatric cancer characterized by high malignancy and remarkable cell heterogeneity within the tumour nodules. In a recent study, we demonstrated that in vitro and in vivo over-expression of the non-coding RNA NDM29 (neuroblastoma differentiation marker 29) induces NB cell differentiation, dramatically reducing their malignancy. Among gene expression changes, differentiated phenotype induced by NDM29 is characterized by decrease of the expression of ABC transporters responsible for anticancer drug resistance. Thus, the pharmacological induction of NDM29, in principle, might represent a possible novel strategy to increase cytotoxic drug responses. In this work, we identify a small molecule able to induce the expression of NDM29 in NB cells, conferring to malignant cells increased susceptibility to cisplatin cytotoxic effects. We demonstrate that the pharmacological induction of NDM29 expression in vivo enhances the antitumoral effects of chemotherapy specifically on tumour initiating/cancer stem cells sub-population, usually refractory to therapies and responsible for tumour relapse. In summary, we suggest a novel therapeutical approach possibly useful to treat very aggressive NB cases with poor prognosis. This novel pharmacological strategy aims to promote differentiation of “stem-like” cells to render them more susceptible to the killing action of cytotoxic anticancer drugs. PMID:26674674

Perhexiline maleate enhances antitumor efficacy of cisplatin in neuroblastoma by inducing over-expression of NDM29 ncRNA.

PubMed

Vella, Serena; Penna, Ilaria; Longo, Luca; Pioggia, Giulia; Garbati, Patrizia; Florio, Tullio; Rossi, Fabio; Pagano, Aldo

2015-12-17

High Risk Neuroblastoma (HR-NB) is a pediatric cancer characterized by high malignancy and remarkable cell heterogeneity within the tumour nodules. In a recent study, we demonstrated that in vitro and in vivo over-expression of the non-coding RNA NDM29 (neuroblastoma differentiation marker 29) induces NB cell differentiation, dramatically reducing their malignancy. Among gene expression changes, differentiated phenotype induced by NDM29 is characterized by decrease of the expression of ABC transporters responsible for anticancer drug resistance. Thus, the pharmacological induction of NDM29, in principle, might represent a possible novel strategy to increase cytotoxic drug responses. In this work, we identify a small molecule able to induce the expression of NDM29 in NB cells, conferring to malignant cells increased susceptibility to cisplatin cytotoxic effects. We demonstrate that the pharmacological induction of NDM29 expression in vivo enhances the antitumoral effects of chemotherapy specifically on tumour initiating/cancer stem cells sub-population, usually refractory to therapies and responsible for tumour relapse. In summary, we suggest a novel therapeutical approach possibly useful to treat very aggressive NB cases with poor prognosis. This novel pharmacological strategy aims to promote differentiation of "stem-like" cells to render them more susceptible to the killing action of cytotoxic anticancer drugs.

Telomerase reverse transcriptase (TERT) is a therapeutic target of oleanane triterpenoid CDDO-Me in prostate cancer.

PubMed

Liu, Yongbo; Gao, Xiaohua; Deeb, Dorrah; Arbab, Ali S; Gautam, Subhash C

2012-12-11

Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) is an synthetic oleanane triterpenoid with strong antiprolifertive and proapoptotic activities in cancer cells. However, the effect of CDDO-Me on human telomerase reverse transcriptase (hTERT) and its telomerase activity in prostate cancer cells has not been studied. We investigated the role of hTERT in mediating the anticancer activity of CDDO-Me in prostate cancer cells in vitro and in vivo. The inhibition of cell proliferation and induction of apoptosis by CDDO-Me in LNCaP and PC-3 prostate cancer cell lines was associated with the inhibition of hTERT gene expression, hTERT telomerase activity and a number of proteins that regulate hTERT transcriptionally and post-translationally. Furthermore, ablation of hTERT protein increased the sensitivity of cancer cells to CDDO-Me, whereas its overexpression rendered them resistant to CDDO-Me. In addition, inhibition of progression of preneoplastic lesions (i.e., low and high-grade prostate intraepithelial neoplasms, PINs) to adenocarcinoma of the prostate by CDDO-Me in TRAMP mice was associated with significant decrease in TERT and its regulatory proteins in the prostate gland. These data provide evidence that telomerase is a potential target of CDDO-Me for the prevention and treatment of prostate cancer.

Transactivation of a cellular promoter by the NS1 protein of the parvovirus minute virus of mice through a putative hormone-responsive element.

PubMed Central

Vanacker, J M; Corbau, R; Adelmant, G; Perros, M; Laudet, V; Rommelaere, J

1996-01-01

The promoter of the thyroid hormone receptor alpha gene (c-erbA-1) is activated by the nonstructural protein 1 (NS1) of parvovirus minute virus of mice (prototype strain [MVMp]) in ras-transformed FREJ4 cells that are permissive for lytic MVMp replication. This stimulation may be related to the sensitivity of host cells to MVMp, as it does not take place in parental FR3T3 cells, which are resistant to the parvovirus killing effect. The analysis of a series of deletion and point mutants of the c-erbA-1 promoter led to the identification of an upstream region that is necessary for NS1-driven transactivation. This sequence harbors a putative hormone-responsive element and is sufficient to render a minimal promoter NS1 inducible in FREJ4 but not in FR3T3 cells, and it is involved in distinct interactions with proteins from the respective cell lines. The NS1-responsive element of the c-erbA-1 promoter bears no homology with sequences that were previously reported to be necessary for NS1 DNA binding and transactivation. Altogether, our data point to a novel, cell-specific mechanism of promoter activation by NS1. PMID:8642664

B lymphoma Moloney murine leukemia virus insertion region 1: An oncogenic mediator in prostate cancer.

PubMed

Liu, Qipeng; Li, Qiaqia; Zhu, Sen; Yi, Yang; Cao, Qi

2018-06-01

B lymphoma Moloney murine leukemia virus insertion region 1 (BMI1), a core member of polycomb repressive complex 1 (PRC1), has been intensely investigated in the field of cancer epigenetics for decades. Widely known as a critical regulator in cellular physiology, BMI1 is essential in self-renewal and differentiation in different lineages of stem cells. BMI1 also plays a significant role in cancer etiology for its involvement in pathological progress such as epithelial-mesenchymal transition (EMT) and cancer stem cell maintenance, propagation, and differentiation. Importantly, overexpression of BMI1 is predictive for drug resistance, tumor recurrence, and eventual therapy failure of various cancer subtypes, which renders the pharmacological targeting at BMI1 as a novel and promising therapeutic approach. The study on prostate cancer, a prevalent hormone-related cancer among men, has promoted enormous research advancements in cancer genetics and epigenetics. This review summarizes the role of BMI1 as an oncogenic and epigenetic regulator in tumor initiation, progression, and relapse of prostate cancer.

Suppression of NF-κB Survival Signaling by Nitrosylcobalamin Sensitizes Neoplasms to the Anti-tumor Effects of Apo2L/TRAIL*

PubMed Central

Chawla-Sarkar, Mamta; Bauer, Joseph A.; Lupica, Joseph A.; Morrison, Bei H.; Tang, Zhuo; Oates, Rhonda K.; Almasan, Alex; DiDonato, Joseph A.; Borden, Ernest C.; Lindner, Daniel J.

2007-01-01

We have previously demonstrated the anti-tumor activity of nitrosylcobalamin (NO-Cbl), an analog of vitamin B12 that delivers nitric oxide (NO) and increases the expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. The specific aim of this study was to examine whether NO-Cbl could sensitize drug-resistant melanomas to Apo2L/TRAIL. Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibro-blast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by TUNEL and confirmed by examining levels and activity of key mediators of apoptosis. The activation status of NF-κB was established by assaying DNA binding, luciferase reporter activity, the phosphorylation status of IκBα, and in vitro IKK activity. NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. Treatment with NO-Cbl followed by Apo2L/TRAIL induced apoptosis in Apo2L/TRAIL-resistant tumor cells, characterized by cleavage of caspase-3, caspase-8, and PARP. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of IκBα and inhibition of NF-κB DNA binding activity. NO-Cbl suppressed Apo2L/TRAIL- and TNF-α-mediated activation of a transfected NF-κB-driven luciferase reporter. XIAP, an inhibitor of apoptosis, was inactivated by NO-Cbl. NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of NO-Cbl and Apo2L/TRAIL capitalizes on the tumor-specific properties of both agents and represents a promising anti-cancer combination. PMID:12881518

Insights into the mechanism of drug resistance. X-ray structure analysis of multi-drug resistant HIV-1 protease ritonavir complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Zhigang; Yedidi, Ravikiran S.; Wang, Yong

2013-01-08

Ritonavir (RTV) is a first generation HIV-1 protease inhibitor with rapidly emerging drug resistance. Mutations at residues 46, 54, 82 and 84 render the HIV-1 protease drug resistant against RTV. We report the crystal structure of multi-drug resistant (MDR) 769 HIV-1 protease (carrying resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84 and 90) complexed with RTV and the in vitro enzymatic IC50 of RTV against MDR HIV-1 protease. The structural and functional studies demonstrate significant drug resistance of MDR HIV-1 protease against RTV, arising from reduced hydrogen bonds and Van der Waals interactions between RTVmore » and MDR HIV-1 protease.« less

Engineering hematopoietic stem cells toward a functional cure of human immunodeficiency virus infection.

PubMed

Wang, Jianbin; Holmes, Michael C

2016-11-01

The battle with human immunodeficiency virus (HIV) has been ongoing for more than 30 years, and although progress has been made, there are still significant challenges remaining. A few unique features render HIV to be one of the toughest viruses to conquer in the modern medicine era, such as the ability to target the host immune system, persist by integrating into the host genome and adapt to a hostile environment such as a single anti-HIV medication by continuously evolving. The finding of combination anti-retroviral therapy (cART) about 2 decades ago has transformed the treatment options for HIV-infected patients and significantly improved patient outcomes. However, finding an HIV cure has proven to be extremely challenging with the only known exception being the so-called "Berlin patient," whose immune system was replaced by stem cell transplants from a donor missing one of HIV's key co-receptors (CCR5). The broad application of this approach is limited by the requirement of an HLA-matched donor who is also homozygous for the rare CCR5 delta32 deletion. On the other hand, the Berlin patient provided the proof of concept of a potential cure for HIV using HIV-resistant hematopoietic stem cells (HSCs), revitalizing the hope to find an HIV cure that is broadly applicable. Here we will review strategies and recent attempts to engineer HIV-resistant HSCs as a path to an HIV cure. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

The genomes of Mycosphaerella graminicola and M. fijiensis

USDA-ARS?s Scientific Manuscript database

Mycosphaerella graminicola and M. fijiensis are two of the most important pathogens of wheat and banana, respectively. Both pathogens cause economically significant diseases that can be controlled with fungicides, but have rapidly developed resistance rendering chemical control less than satisfactor...

Biochar

USDA-ARS?s Scientific Manuscript database

Biochar, a carbonaceous material produced by pyrolysis, can be used as soil amendment to improve soil properties. As some of the carbon is converted into a recalcitrant form rendering it more resistant to biodegradation, land application of biochar is promoted as a beneficial mean for carbon sequest...

Human Keratinocytes Are Vanilloid Resistant

PubMed Central

Pecze, László; Szabó, Kornélia; Széll, Márta; Jósvay, Katalin; Kaszás, Krisztián; Kúsz, Erzsébet; Letoha, Tamás; Prorok, János; Koncz, István; Tóth, András; Kemény, Lajos; Vizler, Csaba; Oláh, Zoltán

2008-01-01

Background Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. Methods To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. Results Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 µM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. Conclusion TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials. PMID:18852901

Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall*

PubMed Central

Chatterjee, Subhasish; Prados-Rosales, Rafael; Itin, Boris; Casadevall, Arturo; Stark, Ruth E.

2015-01-01

Melanin pigments protect against both ionizing radiation and free radicals and have potential soil remediation capabilities. Eumelanins produced by pathogenic Cryptococcus neoformans fungi are virulence factors that render the fungal cells resistant to host defenses and certain antifungal drugs. Because of their insoluble and amorphous characteristics, neither the pigment bonding framework nor the cellular interactions underlying melanization of C. neoformans have yielded to comprehensive molecular-scale investigation. This study used the C. neoformans requirement of exogenous obligatory catecholamine precursors for melanization to produce isotopically enriched pigment “ghosts” and applied 2D 13C-13C correlation solid-state NMR to reveal the carbon-based architecture of intact natural eumelanin assemblies in fungal cells. We demonstrated that the aliphatic moieties of solid C. neoformans melanin ghosts include cell-wall components derived from polysaccharides and/or chitin that are associated proximally with lipid membrane constituents. Prior to development of the mature aromatic fungal pigment, these aliphatic moieties form a chemically resistant framework that could serve as the scaffold for melanin synthesis. The indole-based core aromatic moieties show interconnections that are consistent with proposed melanin structures consisting of stacked planar assemblies, which are associated spatially with the aliphatic scaffold. The pyrrole aromatic carbons of the pigments bind covalently to the aliphatic framework via glycoside or glyceride functional groups. These findings establish that the structure of the pigment assembly changes with time and provide the first biophysical information on the mechanism by which melanin is assembled in the fungal cell wall, offering vital insights that can advance the design of bioinspired conductive nanomaterials and novel therapeutics. PMID:25825492

Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall.

PubMed

Chatterjee, Subhasish; Prados-Rosales, Rafael; Itin, Boris; Casadevall, Arturo; Stark, Ruth E

2015-05-29

Melanin pigments protect against both ionizing radiation and free radicals and have potential soil remediation capabilities. Eumelanins produced by pathogenic Cryptococcus neoformans fungi are virulence factors that render the fungal cells resistant to host defenses and certain antifungal drugs. Because of their insoluble and amorphous characteristics, neither the pigment bonding framework nor the cellular interactions underlying melanization of C. neoformans have yielded to comprehensive molecular-scale investigation. This study used the C. neoformans requirement of exogenous obligatory catecholamine precursors for melanization to produce isotopically enriched pigment "ghosts" and applied 2D (13)C-(13)C correlation solid-state NMR to reveal the carbon-based architecture of intact natural eumelanin assemblies in fungal cells. We demonstrated that the aliphatic moieties of solid C. neoformans melanin ghosts include cell-wall components derived from polysaccharides and/or chitin that are associated proximally with lipid membrane constituents. Prior to development of the mature aromatic fungal pigment, these aliphatic moieties form a chemically resistant framework that could serve as the scaffold for melanin synthesis. The indole-based core aromatic moieties show interconnections that are consistent with proposed melanin structures consisting of stacked planar assemblies, which are associated spatially with the aliphatic scaffold. The pyrrole aromatic carbons of the pigments bind covalently to the aliphatic framework via glycoside or glyceride functional groups. These findings establish that the structure of the pigment assembly changes with time and provide the first biophysical information on the mechanism by which melanin is assembled in the fungal cell wall, offering vital insights that can advance the design of bioinspired conductive nanomaterials and novel therapeutics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms of Normal and Abnormal Endometrial Bleeding

PubMed Central

Lockwood, Charles J.

2011-01-01

Expression of tissue factor (TF), the primary initiator of coagulation, is enhanced in decidualized human endometrial stromal cells (HESC) during the progesterone-dominated luteal phase. Progesterone also augments a second HESC hemostatic factor, plasminogen activator inhibitor-1 (PAI-1). In contrast, progestins inhibit HESC matrix metalloproteinase (MMP)-1, 3 and 9 expression to stabilize endometrial stromal and vascular extracellular matrix. Through these mechanisms decidualized endometrium is rendered both hemostatic and resistant to excess trophoblast invasion in the mid-luteal phase and throughout gestation to prevent hemorrhage and accreta. In non-fertile cycles, progesterone withdrawal results in decreased HESC TF and PAI-expression and increased MMP activity and inflammatory cytokine production promoting the controlled hemorrhage of menstruation and related tissue sloughing. In contrast to these well ordered biochemical processes, unpredictable endometrial bleeding associated with anovulation reflects absence of progestational effects on TF, PAI-1 and MMP activity as well as unrestrained angiogenesis rendering the endometrium non-hemostatic, proteolytic and highly vascular. Abnormal bleeding associated with long-term progestin-only contraceptives results not from impaired hemostasis but from unrestrained angiogenesis leading to large fragile endometrial vessels. This abnormal angiogenesis reflects progestational inhibition of endometrial blood flow promoting local hypoxia and generation of reactive oxygen species that increase production of angiogenic factors such as vascular endothelial growth factor (VEGF) in HESCs and Angiopoietin-2 (Ang-2) in endometrial endothelial cells while decreasing HESC expression of angiostatic, Ang-1. The resulting vessel fragility promotes bleeding. Aberrant angiogenesis also underlies abnormal bleeding associated with myomas and endometrial polyps however there are gaps in our understanding of this pathology. PMID:21499503

DOE Office of Scientific and Technical Information (OSTI.GOV)

Small, Leo J.; Brumbach, Michael T.; Clem, Paul G.

A new multi-step, solution-phase method for the spontaneous deposition of tungsten from a room temperature ethereal solution is reported. This immersion process relies on the deposition of a sacrificial zinc coating which is galvanically displaced by the ether-mediated reduction of oxophilic WCl 6. Subsequent thermal treatment renders a crystalline, metallic tungsten film. The chemical evolution of the surface and formation of a complex intermediate tungsten species is characterized by X-ray diffraction, infrared spectroscopy, and X-ray photoelectron spectroscopy. Efficient metallic tungsten deposition is first characterized on a graphite substrate and then demonstrated on a functional carbon foam electrode. The resulting electrochemicalmore » performance of the modified electrode is interrogated with the canonical aqueous ferricyanide system. A tungsten-coated carbon foam electrode showed that both electrode resistance and overall electrochemical cell resistance were reduced by 50%, resulting in a concomitant decrease in redox peak separation from 1.902 V to 0.783 V. Furthermore, this process promises voltage efficiency gains in electrodes for energy storage technologies and demonstrates the viability of a new route to tungsten coating for technologies and industries where high conductivity and chemical stability are paramount.« less

Biomedical applications of nanodiamond (Review)

NASA Astrophysics Data System (ADS)

Turcheniuk, K.; Mochalin, Vadym N.

2017-06-01

The interest in nanodiamond applications in biology and medicine is on the rise over recent years. This is due to the unique combination of properties that nanodiamond provides. Small size (∼5 nm), low cost, scalable production, negligible toxicity, chemical inertness of diamond core and rich chemistry of nanodiamond surface, as well as bright and robust fluorescence resistant to photobleaching are the distinct parameters that render nanodiamond superior to any other nanomaterial when it comes to biomedical applications. The most exciting recent results have been related to the use of nanodiamonds for drug delivery and diagnostics—two components of a quickly growing area of biomedical research dubbed theranostics. However, nanodiamond offers much more in addition: it can be used to produce biodegradable bone surgery devices, tissue engineering scaffolds, kill drug resistant microbes, help us to fight viruses, and deliver genetic material into cell nucleus. All these exciting opportunities require an in-depth understanding of nanodiamond. This review covers the recent progress as well as general trends in biomedical applications of nanodiamond, and underlines the importance of purification, characterization, and rational modification of this nanomaterial when designing nanodiamond based theranostic platforms.

Effect of quantity of food residues on resistance to desiccation of food-related pathogens adhered to a stainless steel surface.

PubMed

Kuda, Takashi; Shibata, Gensui; Takahashi, Hajime; Kimura, Bon

2015-04-01

In order to study the effect of food residues on the survival of food-borne pathogens, Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes were subjected to drying conditions in the presence of small amounts of food such as carrot juice, aqueous solution of nori, milk, and soy-milk. After drying for 2 h at room temperature in the absence of food residue, cell counts of S. Typhimurium, S. aureus, and L. monocytogenes decreased from 8 to 3, 6, and 5 log cfu/dish, respectively. Five milligrams of fresh carrot, 0.05 mg dried nori, and 100 nL milk or soy milk per 10 mm φ surface were sufficient to demonstrate a protective effect on the adhered pathogens, as confirmed by atomic force microscopy. Results from this study suggest that small sediments of food, not only protein rich but also carbohydrate rich, increase the resistance of surface-adherent bacteria to desiccation, rendering sanitization processes ineffective and encouraging cross contamination. Copyright © 2014 Elsevier Ltd. All rights reserved.

Methylation of 23S rRNA Nucleotide G748 by RlmAII Methyltransferase Renders Streptococcus pneumoniae Telithromycin Susceptible

PubMed Central

Sato, Yoshiharu; Shoji, Tatsuma; Yamamoto, Tomoko

2013-01-01

Several posttranscriptional modifications of bacterial rRNAs are important in determining antibiotic resistance or sensitivity. In all Gram-positive bacteria, dimethylation of nucleotide A2058, located in domain V of 23S rRNA, by the dimethyltransferase Erm(B) results in low susceptibility and resistance to telithromycin (TEL). However, this is insufficient to produce high-level resistance to TEL in Streptococcus pneumoniae. Inactivation of the methyltransferase RlmAII, which methylates the N-1 position of nucleotide G748, located in hairpin 35 of domain II of 23S rRNA, results in increased resistance to TEL in erm(B)-carrying S. pneumoniae. Sixteen TEL-resistant mutants (MICs, 16 to 32 μg/ml) were obtained from a clinically isolated S. pneumoniae strain showing low TEL susceptibility (MIC, 2 μg/ml), with mutation resulting in constitutive dimethylation of A2058 because of nucleotide differences in the regulatory region of erm(B) mRNA. Primer extension analysis showed that the degree of methylation at G748 in all TEL-resistant mutants was significantly reduced by a mutation in the gene encoding RlmAII to create a stop codon or change an amino acid residue. Furthermore, RNA footprinting with dimethyl sulfate and a molecular modeling study suggested that methylation of G748 may contribute to the stable interaction of TEL with domain II of 23S rRNA, even after dimethylation of A2058 by Erm(B). This novel finding shows that methylation of G748 by RlmAII renders S. pneumoniae TEL susceptible. PMID:23716046

Methylation of 23S rRNA nucleotide G748 by RlmAII methyltransferase renders Streptococcus pneumoniae telithromycin susceptible.

PubMed

Takaya, Akiko; Sato, Yoshiharu; Shoji, Tatsuma; Yamamoto, Tomoko

2013-08-01

Several posttranscriptional modifications of bacterial rRNAs are important in determining antibiotic resistance or sensitivity. In all Gram-positive bacteria, dimethylation of nucleotide A2058, located in domain V of 23S rRNA, by the dimethyltransferase Erm(B) results in low susceptibility and resistance to telithromycin (TEL). However, this is insufficient to produce high-level resistance to TEL in Streptococcus pneumoniae. Inactivation of the methyltransferase RlmA(II), which methylates the N-1 position of nucleotide G748, located in hairpin 35 of domain II of 23S rRNA, results in increased resistance to TEL in erm(B)-carrying S. pneumoniae. Sixteen TEL-resistant mutants (MICs, 16 to 32 μg/ml) were obtained from a clinically isolated S. pneumoniae strain showing low TEL susceptibility (MIC, 2 μg/ml), with mutation resulting in constitutive dimethylation of A2058 because of nucleotide differences in the regulatory region of erm(B) mRNA. Primer extension analysis showed that the degree of methylation at G748 in all TEL-resistant mutants was significantly reduced by a mutation in the gene encoding RlmA(II) to create a stop codon or change an amino acid residue. Furthermore, RNA footprinting with dimethyl sulfate and a molecular modeling study suggested that methylation of G748 may contribute to the stable interaction of TEL with domain II of 23S rRNA, even after dimethylation of A2058 by Erm(B). This novel finding shows that methylation of G748 by RlmA(II) renders S. pneumoniae TEL susceptible.

HIV-1 protease-substrate coevolution in nelfinavir resistance.

PubMed

Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

2014-07-01

Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Preclinical Assessment of the Treatment of Second-Stage African Trypanosomiasis with Cordycepin and Deoxycoformycin

PubMed Central

Vodnala, Suman K.; Ferella, Marcela; Lundén-Miguel, Hilda; Betha, Evans; van Reet, Nick; Amin, Daniel Ndem; Öberg, Bo; Andersson, Björn; Kristensson, Krister; Wigzell, Hans; Rottenberg, Martin E.

2009-01-01

Background There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with Trypanosoma brucei (T.b.). Methodology/Principal Findings Cordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug. Conclusions/Significance Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT. PMID:19652702

Mechanistic Assessment of DNA Ligase as an Antibacterial Target in Staphylococcus aureus

PubMed Central

Podos, Steven D.; Thanassi, Jane A.

2012-01-01

We report the use of a known pyridochromanone inhibitor with antibacterial activity to assess the validity of NAD+-dependent DNA ligase (LigA) as an antibacterial target in Staphylococcus aureus. Potent inhibition of purified LigA was demonstrated in a DNA ligation assay (inhibition constant [Ki] = 4.0 nM) and in a DNA-independent enzyme adenylation assay using full-length LigA (50% inhibitory concentration [IC50] = 28 nM) or its isolated adenylation domain (IC50 = 36 nM). Antistaphylococcal activity was confirmed against methicillin-susceptible and -resistant S. aureus (MSSA and MRSA) strains (MIC = 1.0 μg/ml). Analysis of spontaneous resistance potential revealed a high frequency of emergence (4 × 10−7) of high-level resistant mutants (MIC > 64) with associated ligA lesions. There were no observable effects on growth rate in these mutants. Of 22 sequenced clones, 3 encoded point substitutions within the catalytic adenylation domain and 19 in the downstream oligonucleotide-binding (OB) fold and helix-hairpin-helix (HhH) domains. In vitro characterization of the enzymatic properties of four selected mutants revealed distinct signatures underlying their resistance to inhibition. The infrequent adenylation domain mutations altered the kinetics of adenylation and probably elicited resistance directly. In contrast, the highly represented OB fold domain mutations demonstrated a generalized resistance mechanism in which covalent LigA activation proceeds normally and yet the parameters of downstream ligation steps are altered. A resulting decrease in substrate Km and a consequent increase in substrate occupancy render LigA resistant to competitive inhibition. We conclude that the observed tolerance of staphylococcal cells to such hypomorphic mutations probably invalidates LigA as a viable target for antistaphylococcal chemotherapy. PMID:22585221

PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes.

PubMed

Soto-Feliciano, Yadira M; Bartlebaugh, Jordan M E; Liu, Yunpeng; Sánchez-Rivera, Francisco J; Bhutkar, Arjun; Weintraub, Abraham S; Buenrostro, Jason D; Cheng, Christine S; Regev, Aviv; Jacks, Tyler E; Young, Richard A; Hemann, Michael T

2017-05-15

Developmental and lineage plasticity have been observed in numerous malignancies and have been correlated with tumor progression and drug resistance. However, little is known about the molecular mechanisms that enable such plasticity to occur. Here, we describe the function of the plant homeodomain finger protein 6 (PHF6) in leukemia and define its role in regulating chromatin accessibility to lineage-specific transcription factors. We show that loss of Phf6 in B-cell leukemia results in systematic changes in gene expression via alteration of the chromatin landscape at the transcriptional start sites of B-cell- and T-cell-specific factors. Additionally, Phf6 KO cells show significant down-regulation of genes involved in the development and function of normal B cells, show up-regulation of genes involved in T-cell signaling, and give rise to mixed-lineage lymphoma in vivo. Engagement of divergent transcriptional programs results in phenotypic plasticity that leads to altered disease presentation in vivo, tolerance of aberrant oncogenic signaling, and differential sensitivity to frontline and targeted therapies. These findings suggest that active maintenance of a precise chromatin landscape is essential for sustaining proper leukemia cell identity and that loss of a single factor (PHF6) can cause focal changes in chromatin accessibility and nucleosome positioning that render cells susceptible to lineage transition. © 2017 Soto-Feliciano et al.; Published by Cold Spring Harbor Laboratory Press.

Both Leukotoxin and Poly-N-Acetylglucosamine Surface Polysaccharide Protect Aggregatibacter actinomycetemcomitans Cells from Macrophage Killing

PubMed Central

Venketaraman, Vishwanath; Lin, Albert K.; Le, Amy; Kachlany, Scott C.; Connell, Nancy D.; Kaplan, Jeffrey B.

2008-01-01

Two virulence factors produced by the periodontopathogen Aggregatibacter actinomycetemcomitans are leukotoxin, a secreted lipoprotein that kills human polymorphonuclear leukocytes and macrophages, and poly-N-acetylglucosamine (PGA), a surface polysaccharide that mediates intercellular adhesion, biofilm formation and detergent resistance. In this study we examined the roles of leukotoxin and PGA in protecting A. actinomycetemcomitans cells from killing by the human macrophage cell line THP-1. Monolayers of THP-1 cells were infected with single-cell suspensions of a wild-type A. actinomycetemcomitans strain, or of isogenic leukotoxin or PGA mutant strains. After 48 h, viable bacteria were enumerated by dilution plating, macrophage morphology was evaluated microscopically, and macrophage viability was measured by a Trypan blue dye exclusion assay. The number of A. actinomycetemcomitans CFUs increased approximately 2-fold in wells infected with the wild-type strain, but decreased by approximately 70–90% in wells infected with the leukotoxin and PGA mutant strains. Infection with the wild-type or leukotoxin mutant strain caused a significant decrease in THP-1 cell viability, whereas infection with the PGA mutant strain did not result in any detectable changes in THP-1 viability. Pre-treatment of wild-type A. actinomycetemcomitans cells with the PGA-hydrolyzing enzyme dispersin B rendered them sensitive to killing by THP-1 cells. We concluded that both leukotoxin and PGA are necessary for evasion of macrophage killing by A. actinomycetemcomitans. PMID:18573331

A protease-resistant Escherichia coli asparaginase with outstanding stability and enhanced anti-leukaemic activity in vitro.

PubMed

Maggi, Maristella; Mittelman, Steven D; Parmentier, Jean Hugues; Colombo, Giorgio; Meli, Massimiliano; Whitmire, Jeannette Marie; Merrell, D Scott; Whitelegge, Julian; Scotti, Claudia

2017-11-03

L-Asparaginases (ASNases) have been used as first line drugs for paediatric Acute Lymphoblastic Leukaemia (ALL) treatment for more than 40 years. Both the Escherichia coli (EcAII) and Erwinia chrysanthemi (ErAII) type II ASNases currently used in the clinics are characterized by high in vivo instability, short half-life and the requirement of several administrations to obtain a pharmacologically active concentration. Moreover, they are sensitive to proteases (cathepsin B and asparagine endopeptidase) that are over-expressed by resistant leukaemia lymphoblasts, thereby impairing drug activity and pharmacokinetics. Herein, we present the biochemical, structural and in vitro antiproliferative characterization of a new EcAII variant, N24S. The mutant shows completely preserved asparaginase and glutaminase activities, long-term storage stability, improved thermal parameters, and outstanding resistance to proteases derived from leukaemia cells. Structural analysis demonstrates a modification in the hydrogen bond network related to residue 24, while Normal Mode-based geometric Simulation and Molecular Dynamics predict a general rigidification of the monomer as compared to wild-type. These improved features render N24S a potential alternative treatment to reduce the number of drug administrations in vivo and to successfully address one of the major current challenges of ALL treatment: spontaneous, protease-dependent and immunological inactivation of ASNase.

4-(1-Ethyl-4-anisyl-imidazol-5-yl)-N-hydroxycinnamide – A new pleiotropic HDAC inhibitor targeting cancer cell signalling and cytoskeletal organisation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mahal, Katharina, E-mail: katharina.mahal@uni-bayreuth.de; Kahlen, Philip, E-mail: philip.kahlen@uni-bayreuth.de; Biersack, Bernhard, E-mail: bernhard.biersack@yahoo.com

2015-08-15

Histone deacetylases (HDAC) which play a crucial role in cancer cell proliferation are promising drug targets. However, HDAC inhibitors (HDACi) modelled on natural hydroxamic acids such as trichostatin A frequently lead to resistance or even an increased agressiveness of tumours. As a workaround we developed 4-(1-ethyl-4-anisyl-imidazol-5-yl)-N-hydroxycinnamide (etacrox), a hydroxamic acid that combines HDAC inhibition with synergistic effects of the 4,5-diarylimidazole residue. Etacrox proved highly cytotoxic against a panel of metastatic and resistant cancer cell lines while showing greater specificity for cancer over non-malignant cells when compared to the approved HDACi vorinostat. Like the latter, etacrox and the closely related imidazolesmore » bimacroxam and animacroxam acted as pan-HDACi yet showed some specificity for HDAC6. Akt signalling and interference with nuclear beta-catenin localisation were elicited by etacrox at lower concentrations when compared to vorinostat. Moreover, etacrox disrupted the microtubule and focal adhesion dynamics of cancer cells and inhibited the proteolytic activity of prometastatic and proangiogenic matrix metalloproteinases. As a consequence, etacrox acted strongly antimigratory and antiinvasive against various cancer cell lines in three-dimensional transwell invasion assays and also antiangiogenic in vivo with respect to blood vessel formation in the chorioallantoic membrane assay. These pleiotropic effects and its water-solubility and tolerance by mice render etacrox a promising new HDACi candidate. - Graphical abstract: A novel histone deacetylase inhibitor with pleiotropic anticancer effects. - Highlights: • Etacrox is a new HDACi with cytotoxic, antiangiogenic and antiinvasive activity. • Etacrox causes aberrant cancer cell signalling and cytoskeletal reorganisation. • Pro-metastatic and angiogenic matrix metalloproteinases are inhibited by etacrox. • Etacrox impairs blood vessel maturation in vivo and cancer cell invasion in vitro. • Etacrox is tolerated well by mice in doses as high as 150 mg/kg.« less

Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress.

PubMed

Kim, Kiyoon; Kim, Hunsung; Jeong, Kwon; Jung, Min Hyung; Hahn, Bum-Soo; Yoon, Kyung-Sik; Jin, Byung Kwan; Jahng, Geon-Ho; Kang, Insug; Ha, Joohun; Choe, Wonchae

2012-08-01

Cyclophilin, a cytosolic receptor for the immunosuppressive drug cyclosporin A, plays a role in diverse pathophysiologies along with its receptor, CD147. Although the interaction between cyclophilin A and CD147 is well established in inflammatory disease, that of cyclophilin B (CypB) with CD147 has not been fully explored, especially in cancer cell biology, and the exact molecular mechanism underlying such an association is poorly understood. In this study, we first identified high expression levels of CypB in 54 % of hepatocellular carcinoma patient tissues but in only 12.5 % of normal liver tissues. Then, we demonstrated that CypB overexpression protects human hepatoma cells against oxidative stress through its binding to CD147; this protective effect depends on the peptidyl prolyl isomerase activity of CypB. siRNA-mediated knockdown of CypB expression rendered hepatoma cells more vulnerable to ROS-mediated apoptosis. Furthermore, we also determined that a direct interaction between secreted CypB and CD147 regulates the extracellular signal-regulated kinase intracellular signaling pathway and is indispensible for the protective functions of CypB. For the first time, we demonstrated that CypB has an essential function in protecting hepatoma cells against oxidative stress through binding to CD147 and regulating the ERK pathway.

Minoxidil attenuates ischemia-induced apoptosis in cultured neonatal rat cardiomyocytes.

PubMed

Takatani, Tomoka; Takahashi, Kyoko; Jin, Chengshi; Matsuda, Takahisa; Cheng, Xinyao; Ito, Takashi; Azuma, Junichi

2004-06-01

The effects of minoxidil (a mitochondrial K+(ATP) channel opener) on ischemia-induced necrosis and apoptosis were examined using a cardiomyocyte model of simulated ischemia, since mitochondrial K+(ATP) channel openers have been suggested to be involved in the mechanisms of cardioprotective action against ischemia/reperfusion injury. In the absence of minoxidil, simulated ischemia led to cellular release of creatine phosphokinase (CPK), morphologic degeneration, and beating cessation within 24 to 72 hours. Based on the Hoechst 33258 staining pattern, a significant number of cells placed in sealed flasks underwent apoptosis. Myocytes treated with 5 microM of minoxidil failed to alter the degree of ischemia-induced CPK loss for 48 to 72 hours. However, minoxidil treatment prevented the loss of beating function in many of the ischemic cells, and attenuated the decline in intracellular ATP content after a 48-hour ischemic incubation. The number of nuclear fragmentation was significantly reduced in minoxidil-treated cells after a 72-hour ischemic insult compared with untreated ischemic cells. This effect was blocked by the mitochondrial K+(ATP) channel antagonist 5-HD. The data suggest that minoxidil renders the cell resistant to ischemia-induced necrosis and apoptosis. The beneficial effects of minoxidil appear to be related to the opening of mitochondrial K+(ATP) channels.

A subset of virus-specific CD161+ T cells selectively express the multidrug transporter MDR1 and are resistant to chemotherapy in AML

PubMed Central

Alsuliman, Abdullah; Muftuoglu, Muharrem; Khoder, Ahmad; Ahn, Yong-Oon; Basar, Rafet; Verneris, Michael R.; Muranski, Pawel; Barrett, A. John; Liu, Enli; Li, Li; Stringaris, Kate; Armstrong-James, Darius; Shaim, Hila; Kondo, Kayo; Imahashi, Nobuhiko; Andersson, Borje; Marin, David; Champlin, Richard E.; Shpall, Elizabeth J.

2017-01-01

The establishment of long-lived pathogen-specific T cells is a fundamental property of the adaptive immune response. However, the mechanisms underlying long-term persistence of antigen-specific CD4+ T cells are not well-defined. Here we identify a subset of memory CD4+ T cells capable of effluxing cellular toxins, including rhodamine (Rho), through the multidrug efflux protein MDR1 (also known as P-glycoprotein and ABCB1). Drug-effluxing CD4+ T cells were characterized as CD161+CD95+CD45RA−CD127hiCD28+CD25int cells with a distinct chemokine profile and a Th1-polarized pro-inflammatory phenotype. CD4+CD161+Rho-effluxing T cells proliferated vigorously in response to stimulation with anti-CD3/CD28 beads and gave rise to CD161− progeny in vitro. These cells were also capable of self-renewal and maintained their phenotypic and functional characteristics when cultured with homeostatic cytokines. Multidrug-effluxing CD4+CD161+ T cells were enriched within the viral-specific Th1 repertoire of healthy donors and patients with acute myeloid leukemia (AML) and survived exposure to daunorubicin chemotherapy in vitro. Multidrug-effluxing CD4+CD161+ T cells also resisted chemotherapy-induced cytotoxicity in vivo and underwent significant expansion in AML patients rendered lymphopenic after chemotherapy, contributing to the repopulation of anti-CMV immunity. Finally, after influenza vaccination, the proportion of influenza-specific CD4+ T cells coexpressing CD161 was significantly higher after 2 years compared with 4 weeks after immunization, suggesting CD161 is a marker for long-lived antigen-specific memory T cells. These findings suggest that CD4+CD161+ T cells with rapid efflux capacity contribute to the maintenance of viral-specific memory T cells. These data provide novel insights into mechanisms that preserve antiviral immunity in patients undergoing chemotherapy and have implications for the development of novel immunotherapeutic approaches. PMID:27821506

Differential effectiveness of Serratia plymuthica IC1270-induced systemic resistance against hemibiotrophic and necrotrophic leaf pathogens in rice

PubMed Central

De Vleesschauwer, David; Chernin, Leonid; Höfte, Monica M

2009-01-01

Background Induced resistance is a state of enhanced defensive capacity developed by a plant reacting to specific biotic or chemical stimuli. Over the years, several forms of induced resistance have been characterized, including systemic acquired resistance, which is induced upon localized infection by an avirulent necrotizing pathogen, and induced systemic resistance (ISR), which is elicited by selected strains of nonpathogenic rhizobacteria. However, contrary to the relative wealth of information on inducible defense responses in dicotyledoneous plants, our understanding of the molecular mechanisms underlying induced resistance phenomena in cereal crops is still in its infancy. Using a combined cytomolecular and pharmacological approach, we analyzed the host defense mechanisms associated with the establishment of ISR in rice by the rhizobacterium Serratia plymuthica IC1270. Results In a standardized soil-based assay, root treatment with IC1270 rendered foliar tissues more resistant to the hemibiotrophic pathogen Magnaporthe oryzae, causal agent of the devastating rice blast disease. Analysis of the cytological and biochemical alterations associated with restriction of fungal growth in IC1270-induced plants revealed that IC1270 primes rice for enhanced attacker-induced accumulation of reactive oxygen species (ROS) and autofluorescent phenolic compounds in and near epidermal cells displaying dense cytoplasmic granulation. Similar, yet more abundant, phenotypes of hypersensitively dying cells in the vicinity of fungal hyphae were evident in a gene-for-gene interaction with an avirulent M. oryzae strain, suggesting that IC1270-inducible ISR and R protein conditioned effector-triggered immunity (ETI) target similar defense mechanisms. Yet, this IC1270-inducible ISR response seems to act as a double-edged sword within the rice defense network as induced plants displayed an increased vulnerability to the necrotrophic pathogens Rhizoctonia solani and Cochliobolus miyabeanus. Artificial enhancement of ROS levels in inoculated leaves faithfully mimicked the opposite effects of IC1270 bacteria on aforementioned pathogens, suggesting a central role for oxidative events in the IC1270-induced resistance mechanism. Conclusion Besides identifying ROS as modulators of antagonistic defense mechanisms in rice, this work reveals the mechanistic similarities between S. plymuthica-mediated ISR and R protein-dictated ETI and underscores the importance of using appropriate innate defense mechanisms when breeding for broad-spectrum rice disease resistance. PMID:19161601

Differential effectiveness of Serratia plymuthica IC1270-induced systemic resistance against hemibiotrophic and necrotrophic leaf pathogens in rice.

PubMed

De Vleesschauwer, David; Chernin, Leonid; Höfte, Monica M

2009-01-22

Induced resistance is a state of enhanced defensive capacity developed by a plant reacting to specific biotic or chemical stimuli. Over the years, several forms of induced resistance have been characterized, including systemic acquired resistance, which is induced upon localized infection by an avirulent necrotizing pathogen, and induced systemic resistance (ISR), which is elicited by selected strains of nonpathogenic rhizobacteria. However, contrary to the relative wealth of information on inducible defense responses in dicotyledoneous plants, our understanding of the molecular mechanisms underlying induced resistance phenomena in cereal crops is still in its infancy. Using a combined cytomolecular and pharmacological approach, we analyzed the host defense mechanisms associated with the establishment of ISR in rice by the rhizobacterium Serratia plymuthica IC1270. In a standardized soil-based assay, root treatment with IC1270 rendered foliar tissues more resistant to the hemibiotrophic pathogen Magnaporthe oryzae, causal agent of the devastating rice blast disease. Analysis of the cytological and biochemical alterations associated with restriction of fungal growth in IC1270-induced plants revealed that IC1270 primes rice for enhanced attacker-induced accumulation of reactive oxygen species (ROS) and autofluorescent phenolic compounds in and near epidermal cells displaying dense cytoplasmic granulation. Similar, yet more abundant, phenotypes of hypersensitively dying cells in the vicinity of fungal hyphae were evident in a gene-for-gene interaction with an avirulent M. oryzae strain, suggesting that IC1270-inducible ISR and R protein conditioned effector-triggered immunity (ETI) target similar defense mechanisms. Yet, this IC1270-inducible ISR response seems to act as a double-edged sword within the rice defense network as induced plants displayed an increased vulnerability to the necrotrophic pathogens Rhizoctonia solani and Cochliobolus miyabeanus. Artificial enhancement of ROS levels in inoculated leaves faithfully mimicked the opposite effects of IC1270 bacteria on aforementioned pathogens, suggesting a central role for oxidative events in the IC1270-induced resistance mechanism. Besides identifying ROS as modulators of antagonistic defense mechanisms in rice, this work reveals the mechanistic similarities between S. plymuthica-mediated ISR and R protein-dictated ETI and underscores the importance of using appropriate innate defense mechanisms when breeding for broad-spectrum rice disease resistance.

Compositions containing amino acids, phosphate and manganese and their uses

DOEpatents

Daly, Michael J.; Gaidamakova, Elena K.

2016-01-12

The invention provides methods of producing vaccines directed against microorganisms, with the methods comprising culturing, harvesting and/or suspending the microorganism in the presence of a radiation-protective composition and irradiating the bacteria or viruses with a dose of radiation sufficient to render the microorganism replication-deficient and/or non-infective. The radiation-protective compositions used in the methods of the present invention comprise at least one nucleoside, at least one antioxidant and at least one small peptide. The invention also provides methods of rendering bacteria in culture resistant to ionizing radiation (IR), with these methods comprising culturing the bacteria in the presence of a radiation-protective composition.

Compositions containing amino acids, phosphate and manganese and their uses

DOEpatents

Daly, Michael J.; Gaidamakova, Elena K.

2017-09-12

The invention provides methods of producing vaccines directed against microorganisms, with the methods comprising culturing, harvesting and/or suspending the microorganism in the presence of a radiation-protective composition and irradiating the bacteria or viruses with a dose of radiation sufficient to render the microorganism replication-deficient and/or non-infective. The radiation-protective compositions used in the methods of the present invention comprise at least one nucleoside, at least one antioxidant and at least one small peptide. The invention also provides methods of rendering bacteria in culture resistant to ionizing radiation (IR), with these methods comprising culturing the bacteria in the presence of a radiation-protective composition.

Sequence- and structure-based computational analyses of Gram-negative tripartite efflux pumps in the context of bacterial membranes

DOE PAGES

Travers, Timothy; Wang, Katherine J.; Lopez, Cesar A.; ...

2018-02-09

Gram-negative multidrug resistance currently presents a serious threat to public health with infections effectively rendered untreatable. Multiple molecular mechanisms exist that cause antibiotic resistance and in addition, the last three decades have seen slowing rates of new drug development. In this paper, we summarize the use of various computational techniques for investigating the mechanisms of multidrug resistance mediated by Gram-negative tripartite efflux pumps and membranes. Recent work in our lab combines data-driven sequence and structure analyses to study the interactions and dynamics of these bacterial components. Computational studies can complement experimental methodologies for gaining crucial insights into combatting multidrug resistance.

Sequence- and structure-based computational analyses of Gram-negative tripartite efflux pumps in the context of bacterial membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Travers, Timothy; Wang, Katherine J.; Lopez, Cesar A.

Gram-negative multidrug resistance currently presents a serious threat to public health with infections effectively rendered untreatable. Multiple molecular mechanisms exist that cause antibiotic resistance and in addition, the last three decades have seen slowing rates of new drug development. In this paper, we summarize the use of various computational techniques for investigating the mechanisms of multidrug resistance mediated by Gram-negative tripartite efflux pumps and membranes. Recent work in our lab combines data-driven sequence and structure analyses to study the interactions and dynamics of these bacterial components. Computational studies can complement experimental methodologies for gaining crucial insights into combatting multidrug resistance.

First-line treatment of advanced ALK-positive non-small-cell lung cancer

PubMed Central

Gandhi, Shipra; Chen, Hongbin; Zhao, Yujie; Dy, Grace K

2015-01-01

Non-small-cell lung cancer (NSCLC) is one of the leading causes of cancer deaths, both within the US and worldwide. There have been major treatment advances in NSCLC over the past decade with the discovery of molecular drivers of NSCLC, which has ushered in an era of personalized medicine. There are several actionable genetic aberrations in NSCLC, such as epidermal growth factor receptor and anaplastic lymphoma kinase (ALK). In 3%–7% of NSCLC, a chromosomal inversion event in chromosome 2 leads to fusion of a portion of the ALK gene with the echinoderm microtubule–associated protein-like 4 (EML4) gene. The constitutive activation of the ALK fusion oncogene renders it vulnerable to therapeutic intervention. This review focuses on the first-line treatment of advanced ALK-positive NSCLC using ALK inhibitors. Crizotinib was the first agent proven to be efficacious as first-line treatment for ALK-positive NSCLC. However, acquired resistance inevitably develops. The central nervous system is a sanctuary site that represents a common site for disease progression as well. Hence, more potent, selective next-generation ALK inhibitors that are able to cross the blood–brain barrier have been developed for treatment against crizotinib-resistant ALK-positive NSCLC and are also currently being evaluated for first-line therapy as well. In this review, we provide summary of the clinical experience with these drugs in the treatment of ALK-positive NSCLC. PMID:28210152

Global Analysis of the Evolution and Mechanism of Echinocandin Resistance in Candida glabrata

PubMed Central

Singh-Babak, Sheena D.; Babak, Tomas; Diezmann, Stephanie; Hill, Jessica A.; Xie, Jinglin Lucy; Chen, Ying-Lien; Poutanen, Susan M.; Rennie, Robert P.; Heitman, Joseph; Cowen, Leah E.

2012-01-01

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential. PMID:22615574

Mixed biofilm formation by Shiga toxin-producing Escherichia coli and Salmonella enterica serovar Typhimurium enhanced bacterial resistance to sanitization due to extracellular polymeric substances.

PubMed

Wang, Rong; Kalchayanand, Norasak; Schmidt, John W; Harhay, Dayna M

2013-09-01

Shiga toxin-producing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium are important foodborne pathogens capable of forming single-species biofilms or coexisting in multispecies biofilm communities. Bacterial biofilm cells are usually more resistant to sanitization than their planktonic counterparts, so these foodborne pathogens in biofilms pose a serious food safety concern. We investigated how the coexistence of E. coli O157:H7 and Salmonella Typhimurium strains would affect bacterial planktonic growth competition and mixed biofilm composition. Furthermore, we also investigated how mixed biofilm formation would affect bacterial resistance to common sanitizers. Salmonella Typhimurium strains were able to outcompete E. coli strains in the planktonic growth phase; however, mixed biofilm development was highly dependent upon companion strain properties in terms of the expression of bacterial extracellular polymeric substances (EPS), including curli fimbriae and exopolysaccharide cellulose. The EPS-producing strains with higher biofilm-forming abilities were able to establish themselves in mixed biofilms more efficiently. In comparison to single-strain biofilms, Salmonella or E. coli strains with negative EPS expression obtained significantly enhanced resistance to sanitization by forming mixed biofilms with an EPS-producing companion strain of the other species. These observations indicate that the bacterial EPS components not only enhance the sanitizer resistance of the EPS-producing strains but also render protections to their companion strains, regardless of species, in mixed biofilms. Our study highlights the potential risk of cross-contamination by multispecies biofilms in food safety and the need for increased attention to proper sanitization practices in food processing facilities.

High-density antimicrobial peptide coating with broad activity and low cytotoxicity against human cells.

PubMed

Rai, Akhilesh; Pinto, Sandra; Evangelista, Marta B; Gil, Helena; Kallip, Silvar; Ferreira, Mario G S; Ferreira, Lino

2016-03-01

Medical device-associated infections are a multi-billion dollar burden for the worldwide healthcare systems. The modification of medical devices with non-leaching coatings capable of killing microorganisms on contact is one of the strategies being investigated to prevent microorganism colonization. Here we developed a robust antimicrobial coating based on the chemical immobilization of the antimicrobial peptide (AMP), cecropin-melittin (CM), on gold nanoparticles coated surfaces. The concentration of AMP immobilized (110 μg/cm(2)) was higher than most of the studies reported so far (<10 μg/cm(2)). This translated onto a coating with high antimicrobial activity against Gram positive and negative bacteria sp., as well as multi-drug resistant bacteria. Studies with E. coli reporter bacteria showed that these coatings induced the permeability of the outer membrane of bacteria in less than 5 min and the inner membrane in approximately 20 min. Importantly, the antimicrobial properties of the coating are maintained in the presence of 20% (v/v) human serum, and have low probability to induce bacteria resistance. We further show that coatings have low toxicity against human endothelial and fibroblast cells and is hemocompatible since it does not induce platelet and complement activation. The antimicrobial coating described here may be promising to prevent medical device-associated infections. In recent years, antimicrobial peptides (AMPs) have been chemically immobilized on surfaces of medical devices to render them with antimicrobial properties. Surfaces having immobilized cationic peptides are susceptible to be adsorbed by plasma proteins with the subsequent loss of antimicrobial activity. Furthermore, with the exception of very few studies that have determined the cytotoxicity of surfaces in mammalian cells, the effect of the immobilized AMP on human cells is relatively unknown. Here we report a coating based on cecropin-melittin peptide (CM) that maintains its antimicrobial activity against Gram-positive and negative bacteria including multi-drugs resistance bacteria in the presence of serum and has relatively low cytotoxicity against human cells. The reported coatings may be translated on to variety of substrates (glass and titanium) and medical devices to prevent device-associated microbial infection. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanyan; Hu, Yahui; Feng, Yidong

2014-01-15

Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity andmore » multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein. - Highlights: • The cytotoxicity and reversing effect of HZ08 was measured in Caco-2 cell line. • HZ08 inhibited the transport of Rhodamine123 across Caco-2 cell monolayer. • The efflux ratio of HZ08 was dropped when combined with P-glycoprotein inhibitors. • The accumulation of HZ08 increased via gene interference targeting P-glycoprotein. • HZ08 competitively bound to P-glycoprotein under the presence of verapamil.« less

Histone 3.3 Participates in a Self-Sustaining Cascade of Apoptosis That Contributes to the Progression of Chronic Obstructive Pulmonary Disease

PubMed Central

Barrero, Carlos A.; Perez-Leal, Oscar; Aksoy, Mark; Moncada, Camilo; Ji, Rong; Lopez, Yolanda; Mallilankaraman, Karthik; Madesh, Muniswamy; Criner, Gerard J.; Kelsen, Steven G.

2013-01-01

Rationale: Shifts in the gene expression of nuclear protein in chronic obstructive pulmonary disease (COPD), a progressive disease that is characterized by extensive lung inflammation and apoptosis, are common; however, the extent of the elevation of the core histones, which are the major components of nuclear proteins and their consequences in COPD, has not been characterized, which is important because extracellular histones are cytotoxic to endothelial and airway epithelial cells. Objectives: To investigate the role of extracellular histones in COPD disease progression. Methods: We analyzed the nuclear lung proteomes of ex-smokers with and without the disease. Further studies on the consequences of H3.3 were also performed. Measurements and Main Results: A striking finding was a COPD-specific eightfold increase of hyperacetylated histone H3.3. The hyperacetylation renders H3.3 resistant to proteasomal degradation despite ubiquitination; when combined with the reduction in proteasome activity that is known for COPD, this resistance helps account for the increased levels of H3.3. Using anti-H3 antibodies, we found H3.3 in the airway lumen, alveolar fluid, and plasma of COPD samples. H3.3 was cytotoxic to lung structural cells via a mechanism that involves the perturbation of Ca2+ homeostasis and mitochondrial toxicity. We used the primary human airway epithelial cells and found that the antibodies to either the C or N terminus of H3 could partially reverse H3.3 toxicity. Conclusions: Our data indicate that there is an uncontrolled positive feedback loop in which the damaged cells release acetylated H3.3, which causes more damage, adds H3.3 release, and contributes toward the disease progression. PMID:23924319

Unraveling Fungal Radiation Resistance Regulatory Networks through the Genome-Wide Transcriptome and Genetic Analyses of Cryptococcus neoformans

PubMed Central

Jung, Kwang-Woo; Yang, Dong-Hoon; Kim, Min-Kyu; Seo, Ho Seong

2016-01-01

ABSTRACT The basidiomycetous fungus Cryptococcus neoformans has been known to be highly radiation resistant and has been found in fatal radioactive environments such as the damaged nuclear reactor at Chernobyl. To elucidate the mechanisms underlying the radiation resistance phenotype of C. neoformans, we identified genes affected by gamma radiation through genome-wide transcriptome analysis and characterized their functions. We found that genes involved in DNA damage repair systems were upregulated in response to gamma radiation. Particularly, deletion of recombinase RAD51 and two DNA-dependent ATPase genes, RAD54 and RDH54, increased cellular susceptibility to both gamma radiation and DNA-damaging agents. A variety of oxidative stress response genes were also upregulated. Among them, sulfiredoxin contributed to gamma radiation resistance in a peroxiredoxin/thioredoxin-independent manner. Furthermore, we found that genes involved in molecular chaperone expression, ubiquitination systems, and autophagy were induced, whereas genes involved in the biosynthesis of proteins and fatty acids/sterols were downregulated. Most importantly, we discovered a number of novel C. neoformans genes, the expression of which was modulated by gamma radiation exposure, and their deletion rendered cells susceptible to gamma radiation exposure, as well as DNA damage insults. Among these genes, we found that a unique transcription factor containing the basic leucine zipper domain, named Bdr1, served as a regulator of the gamma radiation resistance of C. neoformans by controlling expression of DNA repair genes, and its expression was regulated by the evolutionarily conserved DNA damage response protein kinase Rad53. Taken together, the current transcriptome and functional analyses contribute to the understanding of the unique molecular mechanism of the radiation-resistant fungus C. neoformans. PMID:27899501

Ad-Hoc Sensor Networks for Maritime Interdiction Operations and Regional Security

DTIC Science & Technology

2012-09-01

as resistant to rough sea conditions as the SHARC, since its maximum operation limit is sea state 3. Its maximum speed approaches three knots and...which renders it corrosion resistant and lightweight. Its length is 3.2 meters with a rotor diameter at 3.3 meters. It flies at speeds of 50 knots...NMIOTC main building and to a moored training ship (see Figure 50), (2) GSM/GPRS was networked with swimmers , (3) security patrol and target vessels

The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance

PubMed Central

Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L.; Abbas, Tarek; Larner, James M.

2017-01-01

Lung cancer resists radiation therapy, making it one of the deadliest forms of cancer. Here we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor p21 protein is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene (CDKN1A) in CHIP knockdown cells. Conversely, over-expression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone heat shock protein 70 (HSP70). These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity; thus, suggesting a novel strategy for the treatment of lung cancer. Implications The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. PMID:28232384

The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance.

PubMed

Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L; Abbas, Tarek; Larner, James M

2017-06-01

Lung cancer resists radiotherapy, making it one of the deadliest forms of cancer. Here, we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically, ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor, p21 protein, is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene ( CDKN1A) in CHIP knockdown cells. Conversely, overexpression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone HSP70. These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity, thus suggesting a novel strategy for the treatment of lung cancer. Implications: The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. Mol Cancer Res; 15(6); 651-9. ©2017 AACR . ©2017 American Association for Cancer Research.

Glucose Addiction in Cancer Therapy: Advances and Drawbacks.

PubMed

Granja, Sara; Pinheiro, Céline; Reis, Rui Manuel; Martinho, Olga; Baltazar, Fátima

2015-01-01

While normal differentiated cells primarily use mitochondrial respiration to generate the required energy for cellular processes, most cancer cells rely on glycolysis, even in sufficient oxygen conditions. This phenomenon is known as the "Warburg effect" or aerobic glycolysis and the metabolic reprogramming of cancer cells towards this altered energy metabolism is currently recognized as one of the "hallmarks of cancer". Aerobic glycolysis underlies the rapid growth of tumor cells, with high rates of glucose consumption and lactic acid production, leading to cellular acidosis. Metabolic reprogramming renders cancer cells dependent on specific metabolic enzymes or pathways that could be exploited in cancer therapy. The development of treatments that target tumor glucose metabolism is receiving renewed attention, with several drugs targeting metabolic pathways currently in clinical trials. The search for suitable targets, however, is limited by the high plasticity of the metabolic network that can induce compensatory routes. Deregulated glucose metabolism is a prominent feature associated with resistance to classical chemotherapy or oncogene-targeted therapies, strengthening the clinical potential of combining these therapies with glycolysis inhibitors. The aim of this review is to compare the advances of different therapeutic strategies targeting the glucose "addiction" of tumor cells, highlighting their potential as effective weapons against cancer. We further discuss recent evidence for the involvement of glucose metabolism as a compensatory response to the use of drugs that target different signaling pathways, where the combination with glycolysis inhibitors could prove extraordinarily useful.

Methods of generating synthetic acoustic logs from resistivity logs for gas-hydrate-bearing sediments

USGS Publications Warehouse

Lee, Myung W.

1999-01-01

Methods of predicting acoustic logs from resistivity logs for hydrate-bearing sediments are presented. Modified time average equations derived from the weighted equation provide a means of relating the velocity of the sediment to the resistivity of the sediment. These methods can be used to transform resistivity logs into acoustic logs with or without using the gas hydrate concentration in the pore space. All the parameters except the unconsolidation constants, necessary for the prediction of acoustic log from resistivity log, can be estimated from a cross plot of resistivity versus porosity values. Unconsolidation constants in equations may be assumed without rendering significant errors in the prediction. These methods were applied to the acoustic and resistivity logs acquired at the Mallik 2L-38 gas hydrate research well drilled at the Mackenzie Delta, northern Canada. The results indicate that the proposed method is simple and accurate.

Ultra-high aspect ratio copper nanowires as transparent conductive electrodes for dye sensitized solar cells

NASA Astrophysics Data System (ADS)

Zhu, Zhaozhao; Mankowski, Trent; Shikoh, Ali Sehpar; Touati, Farid; Benammar, Mohieddine A.; Mansuripur, Masud; Falco, Charles M.

2016-09-01

We report the synthesis of ultra-high aspect ratio copper nanowires (CuNW) and fabrication of CuNW-based transparent conductive electrodes (TCE) with high optical transmittance (>80%) and excellent sheet resistance (Rs <30 Ω/sq). These CuNW TCEs are subsequently hybridized with aluminum-doped zinc oxide (AZO) thin-film coatings, or platinum thin film coatings, or nickel thin-film coatings. Our hybrid transparent electrodes can replace indium tin oxide (ITO) films in dye-sensitized solar cells (DSSCs) as either anodes or cathodes. We highlight the challenges of integrating bare CuNWs into DSSCs, and demonstrate that hybridization renders the solar cell integrations feasible. The CuNW/AZO-based DSSCs have reasonably good open-circuit voltage (Voc = 720 mV) and short-circuit current-density (Jsc = 0.96 mA/cm2), which are comparable to what is obtained with an ITO-based DSSC fabricated with a similar process. Our CuNW-Ni based DSSCs exhibit a good open-circuit voltage (Voc = 782 mV) and a decent short-circuit current (Jsc = 3.96 mA/cm2), with roughly 1.5% optical-to-electrical conversion efficiency.

Transcriptional Changes during Naturally Acquired Zika Virus Infection Render Dendritic Cells Highly Conducive to Viral Replication.

PubMed

Sun, Xiaoming; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Einkauf, Kevin; Tse, Samantha; Ard, Kevin; Ciaranello, Andrea; Yawetz, Sigal; Sax, Paul; Rosenberg, Eric S; Lichterfeld, Mathias; Yu, Xu G

2017-12-19

Although dendritic cells are among the human cell population best equipped for cell-intrinsic antiviral immune defense, they seem highly susceptible to infection with the Zika virus (ZIKV). Using highly purified myeloid dendritic cells isolated from individuals with naturally acquired acute infection, we here show that ZIKV induces profound perturbations of transcriptional signatures relative to healthy donors. Interestingly, we noted a remarkable downregulation of antiviral interferon-stimulated genes and innate immune sensors, suggesting that ZIKV can actively suppress interferon-dependent immune responses. In contrast, several host factors known to support ZIKV infection were strongly upregulated during natural ZIKV infection; these transcripts included AXL, the main entry receptor for ZIKV; SOCS3, a negative regulator of ISG expression; and IDO-1, a recognized inducer of regulatory T cell responses. Thus, during in vivo infection, ZIKV can transform the transcriptome of dendritic cells in favor of the virus to render these cells highly conducive to ZIKV infection. Published by Elsevier Inc.

THE MECHANISM OF THE INHIBITION OF HEMOLYSIS

PubMed Central

Ponder, Eric

1946-01-01

This paper is concerned with hemolytic systems containing sodium taurocholate, sodium glycocholate, or saponin, and either human red cells or the ghosts (stromata) of human red cells. The lysins are allowed to react with the cells for a short time (1 minute or less), and with the ghosts for a long time (4 hours), and the quantity of lysin remaining in the bulk phase, after the removal of the cells or of the ghosts, is found by (a) colorimetric methods, and (b) methods which measure its hemolytic activity. In the experiments in which the lysins react with the cells for a time so short that none of them is hemolyzed, it is found: (1) that the suspension medium of a cell suspension contains inhibitory substances which render a small amount of the lysin non-lytic, (2) that on the addition of the lysin to the cell suspension, a further and much larger amount of lysin is rendered non-lytic, and (3) that the quantity of chromogenic material in the bulk phase, after the lysin has been in contact with the cells and the latter have been removed, is substantially the same as that initially present. No appreciable quantity of lysin, accordingly, accumulates in increased concentration at the cell surfaces. The results of the colorimetric determinations show that the apparent disappearance of lysin from the bulk phase, once thought to be due to an accumulation of lysin at the cell surfaces, is the result either of an inhibitory process or of a sudden utilization of lysin unrecognized by existing theory. In the experiments in which the lysins react with stromata for 4 hours, it is found: (1) that the suspension medium of a stroma suspension contains inhibitory substances which render some of the lysin non-lytic, (2) that when the lysin reacts with the stromata over a period of time, a much greater quantity of lysin is rendered non-lytic, and (3) that the concentration of chromogenic material in the bulk phase of the lysin-stroma system, after 4 hours of reaction, is substantially the same as it was initially. The observations can be accounted for by supposing that the lysin molecules are as chromogenic after reacting with the cell components as before, and by their not being bound to the cell or to the ghost, but diffusing back, combined with the components with which they have reacted, into the bulk phase. Such a process would have similarities to the process of penetration and breakdown of mixed protein-lipoid films. Because it is not possible at present to decide how much of the lysin is rendered inert because of utilization in a reaction with cell components, and how much because of the effect of inhibitory substances, difficulties and uncertainties arise in connection with the expressions which have been used to describe the kinetics of hemolysis. PMID:19873457

49 CFR 173.35 - Hazardous materials in IBCs.

Code of Federal Regulations, 2013 CFR

2013-10-01

..., cracks, cuts, or other damage which would render it unable to pass the prescribed design type test to... metal, rigid plastic, or composite IBC that is appropriately resistant to an increase in internal pressure likely to develop during transportation. (1) A rigid plastic or composite IBC may only be filled...

Discovery of novel phosphonate natural products and their biosynthetic pathways by large-scale genome mining

USDA-ARS?s Scientific Manuscript database

Genome mining has revolutionized the field of natural products, providing hope that new antibiotics can be discovered in time before all remainders are rendered useless against multidrug resistant pathogens. While this approach has been successful in academic settings focused on small collections or...

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

PubMed Central

Geoghegan, Ivey A.

2017-01-01

Summary Chitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear. PMID:28371146

Temozolomide-modulated glioma proteome: role of interleukin-1 receptor-associated kinase-4 (IRAK4) in chemosensitivity.

PubMed

Kumar, Durairaj M; Patil, Vikas; Ramachandran, Bini; Nila, Murugesan V; Dharmalingam, Kuppamuthu; Somasundaram, Kumaravel

2013-07-01

The current treatment for glioblastoma includes temozolomide (TMZ) chemotherapy, yet the mechanism of action of TMZ is not thoroughly understood. Here, we investigated the TMZ-induced changes in the proteome of the glioma-derived cell line (U251) by 2D DIGE. We found 95 protein spots to be significantly altered in their expression after TMZ treatment. MS identified four upregulated spots: aspartyl tRNA synthetase glutathione synthetase, interleukin-1 receptor-associated kinase-4 (IRAK4), and breast carcinoma amplified sequence-1 and one downregulated spot: optineurin. TMZ-induced regulation of these five genes was validated by RT-qPCR and Western blot analysis. RNAi-mediated knockdown of IRAK4, an important mediator of Toll-like receptors signaling and chemoresistance, rendered the glioma cells resistant to TMZ. High levels of IRAK4 induced upon TMZ treatment resulted in IRAK1 downregulation and inhibition of NFkB pathway. Endogenous IRAK4 protein, but not transcript levels in glioma cell lines, correlated with TMZ sensitivity. Thus, we have identified several TMZ-modulated proteins and discovered an important novel role for IRAK4 in determining TMZ sensitivity of glioma cells through its ability to inhibit Toll-like receptor signaling and NFkB pathway. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modulation of caspases and their non-apoptotic functions by Legionella pneumophila.

PubMed

Amer, Amal O

2010-02-01

Legionella pneumophila has become a model system to decipher the non-apoptotic functions of caspases and their role in immunity. In permissive cells, the L. pneumophila-containing vacuole evades endosomal traffic and is remodelled by the endoplasmic reticulum. Evasion of the endosomes is mediated by the Dot/Icm type IV secretion system. Upon L. pneumophila infection of genetically restrictive cells such as wild-type (WT) C57Bl/6J murine macrophages, flagellin is sensed by the NOD-like receptor Nlrc4 leading to caspase-1 activation by the inflammasome complex. Then, caspase-7 is activated downstream of the Nlrc4 inflammasome, promoting non-apoptotic functions such as L. pneumophila-containing phagosome maturation and bacterial degradation. Interestingly, caspase-3 is activated in permissive cells during early stages of infection. However, caspase-3 activation does not lead to apoptosis until late stages of infection because it is associated with potent Dot/Icm-mediated anti-apoptotic stimuli that render the infected cells resistant to external apoptotic inducers. Therefore, the role of caspase-1 and non-apoptotic functions of executioner caspases are temporally and spatially modulated during infection by L. pneumophila, which determine permissiveness to intracellular bacterial proliferation. This review will examine the novel activation pathways of caspases by L. pneumophila and discuss their role in genetic restriction and permissiveness to infection.

BRAF-mutated cells activate GCN2-mediated integrated stress response as a cytoprotective mechanism in response to vemurafenib.

PubMed

Nagasawa, Ikuko; Kunimasa, Kazuhiro; Tsukahara, Satomi; Tomida, Akihiro

2017-01-22

In BRAF-mutated melanoma cells, the BRAF inhibitor, vemurafenib, induces phosphorylation of eukaryotic initiation factor 2α (eIF2α) and subsequent induction of activating transcription factor 4 (ATF4), the central regulation node of the integrated stress response (ISR). While the ISR supports cellular adaptation to various stresses, the role of vemurafenib-triggered ISR has not been fully characterized. Here, we showed that in response to vemurafenib, BRAF-mutated melanoma and colorectal cancer cells rapidly induced the ISR as a cytoprotective mechanism through activation of general control nonderepressible 2 (GCN2), an eIF2α kinase sensing amino acid levels. The vemurafenib-triggered ISR, an event independent of downstream MEK inhibition, was specifically prevented by silencing GCN2, but not other eIF2α kinases, including protein kinase-like endoplasmic reticulum kinase, which transmits endoplasmic reticulum (ER) stress. Consistently, the ER stress gatekeeper, GRP78, was not induced by vemurafenib. Interestingly, ATF4 silencing by siRNA rendered BRAF-mutated melanoma cells sensitive to vemurafenib. Thus, the GCN2-mediated ISR can promote cellular adaptation to vemurafenib-induced stress, providing an insight into the development of drug resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

Resistive-pulse and rectification sensing with glass and carbon nanopipettes

PubMed Central

Wang, Yixian; Wang, Dengchao

2017-01-01

Along with more prevalent solid-state nanopores, glass or quartz nanopipettes have found applications in resistive-pulse and rectification sensing. Their advantages include the ease of fabrication, small physical size and needle-like geometry, rendering them useful for local measurements in small spaces and delivery of nanoparticles/biomolecules. Carbon nanopipettes fabricated by depositing a thin carbon layer on the inner wall of a quartz pipette provide additional means for detecting electroactive species and fine-tuning the current rectification properties. In this paper, we discuss the fundamentals of resistive-pulse sensing with nanopipettes and our recent studies of current rectification in carbon pipettes. PMID:28413354

Resistive-pulse and rectification sensing with glass and carbon nanopipettes.

PubMed

Wang, Yixian; Wang, Dengchao; Mirkin, Michael V

2017-03-01

Along with more prevalent solid-state nanopores, glass or quartz nanopipettes have found applications in resistive-pulse and rectification sensing. Their advantages include the ease of fabrication, small physical size and needle-like geometry, rendering them useful for local measurements in small spaces and delivery of nanoparticles/biomolecules. Carbon nanopipettes fabricated by depositing a thin carbon layer on the inner wall of a quartz pipette provide additional means for detecting electroactive species and fine-tuning the current rectification properties. In this paper, we discuss the fundamentals of resistive-pulse sensing with nanopipettes and our recent studies of current rectification in carbon pipettes.

Inhibition of apoptosis by Escherichia coli K1 is accompanied by increased expression of BclXL and blockade of mitochondrial cytochrome c release in macrophages.

PubMed

Sukumaran, Sunil K; Selvaraj, Suresh K; Prasadarao, Nemani V

2004-10-01

Escherichia coli K1 survival in the blood is a critical step for the onset of meningitis in neonates. Therefore, the circulating bacteria are impelled to avoid host defense mechanisms by finding a niche to survive and multiply. Our recent studies have shown that E. coli K1 enters and survives in both monocytes and macrophages in the newborn rat model of meningitis as well as in macrophage cell lines. Here we demonstrate that E. coli K1 not only extends the survival of human and murine infected macrophage cell lines but also renders them resistant to apoptosis induced by staurosporine. Macrophages infected with wild-type E. coli expressing outer membrane protein A (OmpA), but not with OmpA- E. coli, are resistant to DNA fragmentation and phosphatidylserine exposure induced by staurosporine. Infection with OmpA+ E. coli induces the expression of Bcl(XL), an antiapoptotic protein, both at the mRNA level as assessed by gene array analysis and at the protein level as evaluated by immunoblotting. OmpA- E. coli infection of macrophages induced the release of cytochrome c from mitochondria into the cytosol and the activation of caspases 3, 6, and 9, events that were significantly blocked in OmpA+ E. coli-infected macrophages. In addition, OmpA+ E. coli-infected cells were resistant to a decrease in the transmembrane potential of mitochondria induced by staurosporine as measured by the MitoCapture fluorescence technique. Complementation of OmpA- E. coli with a plasmid containing the ompA gene restored the ability of OmpA- E. coli to inhibit the apoptosis of infected macrophages, further demonstrating that E. coli OmpA expression is critical for inducing macrophage survival and thereby finding a safe haven for its growth.

Self-assembled arginine-rich peptides as effective antimicrobial agents.

PubMed

Mi, Gujie; Shi, Di; Herchek, Whitney; Webster, Thomas J

2017-04-01

Bacteria can adapt to their ever-changing environment to develop a resistance to commonly used antibiotics. This escalating evolution of bacteria coupled with a diminished number of effective antibiotics has caused a global healthcare crisis. New antimicrobials and novel approaches to tackle this problem are urgently needed. Antimicrobial peptides are of particular interest in this endeavor due to their broad spectrum antimicrobial properties as well as ability to combat multi-drug resistant bacteria. Most peptides have both hydrophobic and hydrophilic regions that enable them to be soluble in an aqueous solution, yet can insert into and subsequently disintegrate lipid rich membranes through diverse mechanisms. In this study, a novel class of cationic nanoparticles (formed by the self-assembly of an amphiphilic peptide) were shown to have strong antimicrobial properties against gram-positive bacteria, specifically Staphylococcus aureus, Staphylococcus epidermidis, and methicillin-resistant Staphylococcus aureus (MRSA) with minimal toxicity to human dermal fibroblasts. The particular self-assembled structure tested here included an arginine rich nanoparticle (C 17 H 35 GR7RGDS or amphiphilic peptide nanoparticles, APNPs) which incorporated seven arginine residues (imparting a positive charge to improve membrane interactions), a hydrophobic block which drove the self-assembly process, and the presence of an amino acid quadruplet arginine-glycine-aspartic acid-serine (RGDS) which may render these nanoparticles capable of attracting healthy cells while competing bacterial adherence to fibronectin, an adhesive protein found on cell surfaces. As such, this in vitro study demonstrated that the presently formulated APNPs should be further studied for a wide range of antibacterial applications where antibiotics are no longer useful. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1046-1054, 2017. © 2017 Wiley Periodicals, Inc.

The CopRS Two-Component System Is Responsible for Resistance to Copper in the Cyanobacterium Synechocystis sp. PCC 68031[C][W][OA

PubMed Central

Giner-Lamia, Joaquín; López-Maury, Luis; Reyes, José C.; Florencio, Francisco J.

2012-01-01

Photosynthetic organisms need copper for cytochrome oxidase and for plastocyanin in the fundamental processes of respiration and photosynthesis. However, excess of free copper is detrimental inside the cells and therefore organisms have developed homeostatic mechanisms to tightly regulate its acquisition, sequestration, and efflux. Herein we show that the CopRS two-component system (also known as Hik31-Rre34) is essential for copper resistance in Synechocystis sp. PCC 6803. It regulates expression of a putative heavy-metal efflux-resistance nodulation and division type copper efflux system (encoded by copBAC) as well as its own expression (in the copMRS operon) in response to the presence of copper in the media. Mutants in this two-component system or the efflux system render cells more sensitive to the presence of copper in the media and accumulate more intracellular copper than the wild type. Furthermore, CopS periplasmic domain is able to bind copper, suggesting that CopS could be able to detect copper directly. Both operons (copMRS and copBAC) are also induced by the photosynthetic inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone but this induction requires the presence of copper in the media. The reduced response of two mutant strains to copper, one lacking plastocyanin and a second one impaired in copper transport to the thylakoid, due to the absence of the PI-type ATPases PacS and CtaA, suggests that CopS can detect intracellular copper. In addition, a tagged version of CopS with a triple HA epitope localizes to both the plasma and the thylakoid membranes, suggesting that CopS could be involved in copper detection in both the periplasm and the thylakoid lumen. PMID:22715108

The CopRS two-component system is responsible for resistance to copper in the cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Giner-Lamia, Joaquín; López-Maury, Luis; Reyes, José C; Florencio, Francisco J

2012-08-01

Photosynthetic organisms need copper for cytochrome oxidase and for plastocyanin in the fundamental processes of respiration and photosynthesis. However, excess of free copper is detrimental inside the cells and therefore organisms have developed homeostatic mechanisms to tightly regulate its acquisition, sequestration, and efflux. Herein we show that the CopRS two-component system (also known as Hik31-Rre34) is essential for copper resistance in Synechocystis sp. PCC 6803. It regulates expression of a putative heavy-metal efflux-resistance nodulation and division type copper efflux system (encoded by copBAC) as well as its own expression (in the copMRS operon) in response to the presence of copper in the media. Mutants in this two-component system or the efflux system render cells more sensitive to the presence of copper in the media and accumulate more intracellular copper than the wild type. Furthermore, CopS periplasmic domain is able to bind copper, suggesting that CopS could be able to detect copper directly. Both operons (copMRS and copBAC) are also induced by the photosynthetic inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone but this induction requires the presence of copper in the media. The reduced response of two mutant strains to copper, one lacking plastocyanin and a second one impaired in copper transport to the thylakoid, due to the absence of the P(I)-type ATPases PacS and CtaA, suggests that CopS can detect intracellular copper. In addition, a tagged version of CopS with a triple HA epitope localizes to both the plasma and the thylakoid membranes, suggesting that CopS could be involved in copper detection in both the periplasm and the thylakoid lumen.

Challenging Oneself Intermittently to Improve Health

PubMed Central

Mattson, Mark P.

2014-01-01

Humans and their predecessors evolved in environments where they were challenged intermittently with: 1) food scarcity; 2) the need for aerobic fitness to catch/kill prey and avoid or repel attackers; and 3) exposure to biological toxins present in foodstuffs. Accordingly, cells and organ systems acquired and retained molecular signaling and metabolic pathways through which the environmental challenges enhanced the functionality and resilience of the cells and organisms. Within the past 60 years there has been a precipitous diminution of such challenges in modern societies because of the development of technologies that provide a continuous supply of energy-dense processed foods and that largely eliminate the need for physical exertion. As a consequence of the modern ‘couch potato’ lifestyle, signaling pathways that mediate beneficial effects of environmental challenges on health and disease resistance are disengaged, thereby rendering people vulnerable to obesity, diabetes, cardiovascular disease, cancers and neurodegenerative disorders. Reversal of the epidemic of diseases caused by unchallenging lifestyles will require a society-wide effort to re-introduce intermittent fasting, exercise and consumption of plants containing hormetic phytochemicals into daily and weekly routines. PMID:25552960

The Ski oncoprotein interacts with the Smad proteins to repress TGFbeta signaling.

PubMed

Luo, K; Stroschein, S L; Wang, W; Chen, D; Martens, E; Zhou, S; Zhou, Q

1999-09-01

Smad proteins are critical signal transducers downstream of the receptors of the transforming growth factor-beta (TGFbeta) superfamily. On phosphorylation and activation by the active TGFbeta receptor complex, Smad2 and Smad3 form hetero-oligomers with Smad4 and translocate into the nucleus, where they interact with different cellular partners, bind to DNA, regulate transcription of various downstream response genes, and cross-talk with other signaling pathways. Here we show that a nuclear oncoprotein, Ski, can interact directly with Smad2, Smad3, and Smad4 on a TGFbeta-responsive promoter element and repress their abilities to activate transcription through recruitment of the nuclear transcriptional corepressor N-CoR and possibly its associated histone deacetylase complex. Overexpression of Ski in a TGFbeta-responsive cell line renders it resistant to TGFbeta-induced growth inhibition and defective in activation of JunB expression. This ability to overcome TGFbeta-induced growth arrest may be responsible for the transforming activity of Ski in human and avian cancer cells. Our studies suggest a new paradigm for inactivation of the Smad proteins by an oncoprotein through transcriptional repression.

The Ski oncoprotein interacts with the Smad proteins to repress TGFβ signaling

PubMed Central

Luo, Kunxin; Stroschein, Shannon L.; Wang, Wei; Chen, Dan; Martens, Eric; Zhou, Sharleen; Zhou, Qiang

1999-01-01

Smad proteins are critical signal transducers downstream of the receptors of the transforming growth factor-β (TGFβ) superfamily. On phosphorylation and activation by the active TGFβ receptor complex, Smad2 and Smad3 form hetero-oligomers with Smad4 and translocate into the nucleus, where they interact with different cellular partners, bind to DNA, regulate transcription of various downstream response genes, and cross-talk with other signaling pathways. Here we show that a nuclear oncoprotein, Ski, can interact directly with Smad2, Smad3, and Smad4 on a TGFβ-responsive promoter element and repress their abilities to activate transcription through recruitment of the nuclear transcriptional corepressor N-CoR and possibly its associated histone deacetylase complex. Overexpression of Ski in a TGFβ-responsive cell line renders it resistant to TGFβ-induced growth inhibition and defective in activation of JunB expression. This ability to overcome TGFβ-induced growth arrest may be responsible for the transforming activity of Ski in human and avian cancer cells. Our studies suggest a new paradigm for inactivation of the Smad proteins by an oncoprotein through transcriptional repression. PMID:10485843

Treatment of Pneumocystis jirovecii pneumonia in HIV-infected patients: a review.

PubMed

Huang, Yu-Shan; Yang, Jen-Jia; Lee, Nan-Yao; Chen, Guan-Jhou; Ko, Wen-Chien; Sun, Hsin-Yun; Hung, Chien-Ching

2017-09-01

Pneumocystis pneumonia is a potentially life-threatening pulmonary infection that occurs in immunocompromised individuals and HIV-infected patients with a low CD4 cell count. Trimethoprim-sulfamethoxazole has been used as the first-line agent for treatment, but mutations within dihydropteroate synthase gene render potential resistance to sulfamide. Despite advances of combination antiretroviral therapy (cART), Pneumocystis pneumonia continues to occur in HIV-infected patients with late presentation for cART or virological and immunological failure after receiving cART. Areas covered: This review summarizes the diagnosis and first-line and alternative treatment and prophylaxis for Pneumocystis pneumonia in HIV-infected patients. Articles for this review were identified through searching PubMed. Search terms included: 'Pneumocystis pneumonia', 'Pneumocystis jirovecii pneumonia', 'Pneumocystis carinii pneumonia', 'trimethoprim-sulfamethoxazole', 'primaquine', 'trimetrexate', 'dapsone', 'pentamidine', 'atovaquone', 'echinocandins', 'human immunodeficiency virus infection', 'acquired immunodeficiency syndrome', 'resistance to sulfamide' and combinations of these terms. We limited the search to English language papers that were published between 1981 and March 2017. We screened all identified articles and cross-referenced studies from retrieved articles. Expert commentary: Trimethoprim-sulfamethoxazole will continue to be the first-line agent for Pneumocystis pneumonia given its cost, availability of both oral and parenteral formulations, and effectiveness or efficacy in both treatment and prophylaxis. Whether resistance due to mutations within dihydropteroate synthase gene compromises treatment effectiveness remains controversial. Continued search for effective alternatives with better safety profiles for Pneumocystis pneumonia is warranted.

Reversal of the multidrug resistance by drug combination using multifunctional liposomes

NASA Astrophysics Data System (ADS)

Patel, Niravkumar R.

One of the major obstacles to the success of cancer chemotherapy is the multi-drug resistance (MDR) that results due mainly to the over-expression of drug efflux transporter pumps such as P-glycoprotein (P-gp). Highly efficacious third generation P-gp inhibitors, like tariquidar, have shown promising results against MDR. However, P-gp is also expressed in normal tissues like the blood-brain barrier, gastrointestinal tract, liver and kidney. It is therefore important to limit the exposure of P-gp inhibitors to normal tissues and increase their co-localization with anticancer agents in tumor tissues to maximize the efficacy of a P-gp inhibitor. To minimize non-specific binding and increase its delivery to tumor tissues, liposomes, self-assembling phospholipid vesicles, were chosen as a drug delivery vehicle. The liposome has been identified as a system capable of carrying molecules with diverse physicochemical properties. It can also alter the pharmacokinetic profile of loaded molecules which is a concern with both tariquidar and paclitaxel. Liposomes can easily be surface-modified rendering them cell-specific as well as organelle-specific. The main objective of present study was to develop an efficient liposomal delivery system which would deliver therapeutic molecules of interest to tumor tissues and avoid interaction with normal tissues. In this study, the co-delivery of tariquidar and paclitaxel into tumor cells to reverse the MDR using long-circulating cationic liposomes was investigated. SKOV-3TR, the resistant variant of SKOV-3 and MCF-7/ADR, the resistant variant of MCF-7 were used as model cell lines. Uniform liposomal formulations were generated with high incorporation efficiency and no apparent decrease in tariquidar potency towards P-gp. Tariquidar- and paclitaxel- co-loaded long-circulating liposomes showed significant re-sensitization of SKOV-3TR and MCF-7/ADR for paclitaxel in vitro. Further modification of these liposomes with antitumor 2C5 resulted in increased cell association with these cancer cells. The 2C5-modified immunoliposomes, along with unmodified liposomes co-loaded with tariquidar and paclitaxel were tested for their antitumor effects in vivo. Significant tumor growth inhibition occurred with combination therapy in resistant as well as sensitive cell lines. However, immunoliposomes failed to increase antitumor effect in vivo as spontaneous accumulation of liposomes at added dose may have saturated tumor accumulation. We were also interested in evaluating physiological factors responsible for the MDR. Spheroids grown in vitro provided platform to demonstrate many characteristics of tumor tissues such as cell-cell interaction, a hypoxic core, low pH environment at core and a relevant genetic profile. In this study, spheroids were utilized to evaluate paclitaxel cytotoxity and to evaluate effects of 2C5 modification on cellular uptake. Lack of cytotoxicity was observed in spheroids treated with paclitaxel alone as well as in combination with tariquidar. Likely explanations could be the presence of cells in diverse cell cycle stages and limited penetration. Also, increased uptake was observed in spheroids when treated with 2C5-modified Rh-labeled liposomes compared to UPC10-modified Rh-labeled liposomes. Such results have clearly demonstrated the importance of using this novel research model in cancer research.

NRAM: a disruptive carbon-nanotube resistance-change memory.

PubMed

Gilmer, D C; Rueckes, T; Cleveland, L

2018-04-03

Advanced memory technology based on carbon nanotubes (CNTs) (NRAM) possesses desired properties for implementation in a host of integrated systems due to demonstrated advantages of its operation including high speed (nanotubes can switch state in picoseconds), high endurance (over a trillion), and low power (with essential zero standby power). The applicable integrated systems for NRAM have markets that will see compound annual growth rates (CAGR) of over 62% between 2018 and 2023, with an embedded systems CAGR of 115% in 2018-2023 (http://bccresearch.com/pressroom/smc/bcc-research-predicts:-nram-(finally)-to-revolutionize-computer-memory). These opportunities are helping drive the realization of a shift from silicon-based to carbon-based (NRAM) memories. NRAM is a memory cell made up of an interlocking matrix of CNTs, either touching or slightly separated, leading to low or higher resistance states respectively. The small movement of atoms, as opposed to moving electrons for traditional silicon-based memories, renders NRAM with a more robust endurance and high temperature retention/operation which, along with high speed/low power, is expected to blossom in this memory technology to be a disruptive replacement for the current status quo of DRAM (dynamic RAM), SRAM (static RAM), and NAND flash memories.

NRAM: a disruptive carbon-nanotube resistance-change memory

NASA Astrophysics Data System (ADS)

Gilmer, D. C.; Rueckes, T.; Cleveland, L.

2018-04-01

Advanced memory technology based on carbon nanotubes (CNTs) (NRAM) possesses desired properties for implementation in a host of integrated systems due to demonstrated advantages of its operation including high speed (nanotubes can switch state in picoseconds), high endurance (over a trillion), and low power (with essential zero standby power). The applicable integrated systems for NRAM have markets that will see compound annual growth rates (CAGR) of over 62% between 2018 and 2023, with an embedded systems CAGR of 115% in 2018-2023 (http://bccresearch.com/pressroom/smc/bcc-research-predicts:-nram-(finally)-to-revolutionize-computer-memory). These opportunities are helping drive the realization of a shift from silicon-based to carbon-based (NRAM) memories. NRAM is a memory cell made up of an interlocking matrix of CNTs, either touching or slightly separated, leading to low or higher resistance states respectively. The small movement of atoms, as opposed to moving electrons for traditional silicon-based memories, renders NRAM with a more robust endurance and high temperature retention/operation which, along with high speed/low power, is expected to blossom in this memory technology to be a disruptive replacement for the current status quo of DRAM (dynamic RAM), SRAM (static RAM), and NAND flash memories.

Volatile anesthetics affect nutrient availability in yeast.

PubMed Central

Palmer, Laura K; Wolfe, Darren; Keeley, Jessica L; Keil, Ralph L

2002-01-01

Volatile anesthetics affect all cells and tissues tested, but their mechanisms and sites of action remain unknown. To gain insight into the cellular activities of anesthetics, we have isolated genes that, when overexpressed, render Saccharomyces cerevisiae resistant to the volatile anesthetic isoflurane. One of these genes, WAK3/TAT1, encodes a permease that transports amino acids including leucine and tryptophan, for which our wild-type strain is auxotrophic. This suggests that availability of amino acids may play a key role in anesthetic response. Multiple lines of evidence support this proposal: (i) Deletion or overexpression of permeases that transport leucine and/or tryptophan alters anesthetic response; (ii) prototrophic strains are anesthetic resistant; (iii) altered concentrations of leucine and tryptophan in the medium affect anesthetic response; and (iv) uptake of leucine and tryptophan is inhibited during anesthetic exposure. Not all amino acids are critical for this response since we find that overexpression of the lysine permease does not affect anesthetic sensitivity. These findings are consistent with models in which anesthetics have a physiologically important effect on availability of specific amino acids by altering function of their permeases. In addition, we show that there is a relationship between nutrient availability and ubiquitin metabolism in this response. PMID:12072454

Deposition of tungsten metal by an immersion process

DOE PAGES

Small, Leo J.; Brumbach, Michael T.; Clem, Paul G.; ...

2017-03-23

A new multi-step, solution-phase method for the spontaneous deposition of tungsten from a room temperature ethereal solution is reported. This immersion process relies on the deposition of a sacrificial zinc coating which is galvanically displaced by the ether-mediated reduction of oxophilic WCl 6. Subsequent thermal treatment renders a crystalline, metallic tungsten film. The chemical evolution of the surface and formation of a complex intermediate tungsten species is characterized by X-ray diffraction, infrared spectroscopy, and X-ray photoelectron spectroscopy. Efficient metallic tungsten deposition is first characterized on a graphite substrate and then demonstrated on a functional carbon foam electrode. The resulting electrochemicalmore » performance of the modified electrode is interrogated with the canonical aqueous ferricyanide system. A tungsten-coated carbon foam electrode showed that both electrode resistance and overall electrochemical cell resistance were reduced by 50%, resulting in a concomitant decrease in redox peak separation from 1.902 V to 0.783 V. Furthermore, this process promises voltage efficiency gains in electrodes for energy storage technologies and demonstrates the viability of a new route to tungsten coating for technologies and industries where high conductivity and chemical stability are paramount.« less

How "resistant" is artemisinin resistant malaria? - The risks of ambiguity using the term "resistant" malaria.

PubMed

Hanscheid, Thomas; Hardisty, David W

2018-04-30

In scientific discourse, few would consider the widely used term resistance as ambiguous. The definition and usage of the term antimicrobial resistance revolves around the concept that microorganisms change in ways that render antimicrobial medications clinically ineffective. Because artemisinins have become the cornerstone for antimalarial therapy, the widely used term artemisinin resistance in scientific literature is highly alarming. Naturally, many people will assume that artemisinin resistance must essentially be the same as antimicrobial resistance, which means it is clinically ineffective. However, this is incorrect, and the WHO defines artemisinin resistance differently to antimicrobial resistance as "partial/relative resistance". This means that parasite clearance times are increased but does not automatically mean that artemisinins have become clinically inefficacious. Is the ambiguous use of the term resistance justified and appropriate, although it might be misleading biomedical researchers, the media, policy makers and possibly attending physicians? Science is also about clear and unambiguous use of terminology, so that a message is accurately communicated and understood. Ambiguity can lead to misunderstandings, and misunderstandings can cause wrong actions; unnecessarily so. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mechanism of Resistance in Mungbean [Vigna radiata (L.) R. Wilczek var. radiata] to bruchids, Callosobruchus spp. (Coleoptera: Bruchidae).

PubMed

War, Abdul R; Murugesan, Surya; Boddepalli, Venkata N; Srinivasan, Ramasamy; Nair, Ramakrishnan M

2017-01-01

Mungbean [ Vigna radiata (L.) R. Wilczek var. radiata ] is an important pulse crop in Asia, and is consumed as dry seeds and as bean sprouts. It is an excellent source of digestible protein. Bruchids [ Callosobruchus chinensis (L.) and Callosobruchus maculatus (F.)] are the important pests of mungbean and cause damage in the field and in storage. Bruchid infestation reduces the nutritional and market value of the grain and renders seeds unfit for human consumption, agricultural and commercial uses. These pests are controlled mainly by fumigation with highly toxic chemicals such as carbon disulfide, phosphene, and methyl bromide, or by dusting with several other insecticides, which leave residues on the grain, thus, threatening food safety. Some plant-based extracts have been found useful in controlling bruchids, but are not fully successful due to their short-term activity, rapid degradability, and potentially negative effect on seed germination. Although some wild sources of bruchid resistance in mungbean have been reported, which have been used to develop bruchid- resistant lines, undesirable genetic linkages threaten the proper exploitation of genetic diversity from wild germplasm into commercial cultivars. Further, biotype variation in bruchids has rendered some mungbean lines susceptible that otherwise would have been resistant to the pest. Host plant resistance is a cost-effective and a safe alternative to control bruchids in mungbean and is associated with morphological, biochemical, and molecular traits. These traits affect insect growth and development, thereby, reduce the yield losses by the pests. Understanding the defense mechanisms against insect pests could be utilized in exploiting these traits in crop breeding. This review discusses different traits in mungbean involved in defense against bruchids and their utility in pest management. We also highlight the breeding constraints for developing bruchid-resistant mungbean and how can these constraints be minimized. We further highlight the importance of supporting conventional breeding techniques by molecular techniques such as molecular markers linked to bruchid resistance.

Mechanism of Resistance in Mungbean [Vigna radiata (L.) R. Wilczek var. radiata] to bruchids, Callosobruchus spp. (Coleoptera: Bruchidae)

PubMed Central

War, Abdul R.; Murugesan, Surya; Boddepalli, Venkata N.; Srinivasan, Ramasamy; Nair, Ramakrishnan M.

2017-01-01

Mungbean [Vigna radiata (L.) R. Wilczek var. radiata] is an important pulse crop in Asia, and is consumed as dry seeds and as bean sprouts. It is an excellent source of digestible protein. Bruchids [Callosobruchus chinensis (L.) and Callosobruchus maculatus (F.)] are the important pests of mungbean and cause damage in the field and in storage. Bruchid infestation reduces the nutritional and market value of the grain and renders seeds unfit for human consumption, agricultural and commercial uses. These pests are controlled mainly by fumigation with highly toxic chemicals such as carbon disulfide, phosphene, and methyl bromide, or by dusting with several other insecticides, which leave residues on the grain, thus, threatening food safety. Some plant-based extracts have been found useful in controlling bruchids, but are not fully successful due to their short-term activity, rapid degradability, and potentially negative effect on seed germination. Although some wild sources of bruchid resistance in mungbean have been reported, which have been used to develop bruchid- resistant lines, undesirable genetic linkages threaten the proper exploitation of genetic diversity from wild germplasm into commercial cultivars. Further, biotype variation in bruchids has rendered some mungbean lines susceptible that otherwise would have been resistant to the pest. Host plant resistance is a cost-effective and a safe alternative to control bruchids in mungbean and is associated with morphological, biochemical, and molecular traits. These traits affect insect growth and development, thereby, reduce the yield losses by the pests. Understanding the defense mechanisms against insect pests could be utilized in exploiting these traits in crop breeding. This review discusses different traits in mungbean involved in defense against bruchids and their utility in pest management. We also highlight the breeding constraints for developing bruchid-resistant mungbean and how can these constraints be minimized. We further highlight the importance of supporting conventional breeding techniques by molecular techniques such as molecular markers linked to bruchid resistance. PMID:28676807

A Critical Role for the Anti-apoptotic Protein ARC (Apoptosis Repressor with CARD) in Hypoxia-Induced Pulmonary Hypertension

PubMed Central

Zaiman, Ari L; Damico, Rachel; Thoms-Chesley, Alan; Files, D Clark; Kesari, Priya; Johnston, Laura; Swaim, Mara; Mozammel, Shehzin; Myers, Alan C; Halushka, Marc; El-Haddad, Hasim; Shimoda, Larissa A; Peng, Chang-Fu; Hassoun, Paul M; Champion, Hunter C; Kitsis, Richard N; Crow, Michael T

2015-01-01

Background Pulmonary hypertension (PH) is a lethal syndrome associated with the pathogenic remodeling of the pulmonary vasculature and the emergence of apoptosis-resistant cells. ARC (Apoptosis Repressor with Caspase Recruitment Domain) is an inhibitor of multiple forms of cell death known to be abundantly expressed in striated muscle. We show for the first time that ARC is expressed in arterial smooth muscle cells of the pulmonary vasculature and is markedly up-regulated in several experimental models of PH. In this study, we test the hypothesis that ARC expression is essential for the development of chronic hypoxia-induced PH. Methods and Results Experiments in which cells or mice were rendered ARC-deficient revealed that ARC not only protected pulmonary arterial smooth muscle cells from hypoxia-induced death, but also facilitated growth factor-induced proliferation and hypertrophy and hypoxia-induced down-regulation of selective voltage-gated potassium channels, the latter a hallmark of the syndrome in humans. Moreover, ARC-deficient mice exhibited diminished vascular remodeling, increased apoptosis, and decreased proliferation in response to chronic hypoxia, resulting in marked protection from PH in vivo. Patients with PH have significantly increased ARC expression not only in remodeled vessels but also in the lumen-occluding lesions associated with severe disease. Conclusions These data show that ARC, previously unlinked to pulmonary hypertension, is a critical determinant of vascular remodeling in this syndrome. PMID:22082675

Genetic and Biochemical Analysis of High Iron Toxicity in Yeast

PubMed Central

Lin, Huilan; Li, Liangtao; Jia, Xuan; Ward, Diane McVey; Kaplan, Jerry

2011-01-01

Iron storage in yeast requires the activity of the vacuolar iron transporter Ccc1. Yeast with an intact CCC1 are resistant to iron toxicity, but deletion of CCC1 renders yeast susceptible to iron toxicity. We used genetic and biochemical analysis to identify suppressors of high iron toxicity in Δccc1 cells to probe the mechanism of high iron toxicity. All genes identified as suppressors of high iron toxicity in aerobically grown Δccc1 cells encode organelle iron transporters including mitochondrial iron transporters MRS3, MRS4, and RIM2. Overexpression of MRS3 suppressed high iron toxicity by decreasing cytosolic iron through mitochondrial iron accumulation. Under anaerobic conditions, Δccc1 cells were still sensitive to high iron toxicity, but overexpression of MRS3 did not suppress iron toxicity and did not result in mitochondrial iron accumulation. We conclude that Mrs3/Mrs4 can sequester iron within mitochondria under aerobic conditions but not anaerobic conditions. We show that iron toxicity in Δccc1 cells occurred under both aerobic and anaerobic conditions. Microarray analysis showed no evidence of oxidative damage under anaerobic conditions, suggesting that iron toxicity may not be solely due to oxidative damage. Deletion of TSA1, which encodes a peroxiredoxin, exacerbated iron toxicity in Δccc1 cells under both aerobic and anaerobic conditions, suggesting a unique role for Tsa1 in iron toxicity. PMID:21115478

Hybrid- and complex-type N-glycans are not essential for Newcastle disease virus infection and fusion of host cells.

PubMed

Sun, Qing; Zhao, Lixiang; Song, Qingqing; Wang, Zheng; Qiu, Xusheng; Zhang, Wenjun; Zhao, Mingjun; Zhao, Guo; Liu, Wenbo; Liu, Haiyan; Li, Yunsen; Liu, Xiufan

2012-03-01

N-linked glycans are composed of three major types: high-mannose (Man), hybrid or complex. The functional role of hybrid- and complex-type N-glycans in Newcastle disease virus (NDV) infection and fusion was examined in N-acetylglucosaminyltransferase I (GnT I)-deficient Lec1 cells, a mutant Chinese hamster ovary (CHO) cell incapable of synthesizing hybrid- and complex-type N-glycans. We used recombinant NDV expressing green fluorescence protein or red fluorescence protein to monitor NDV infection, syncytium formation and viral yield. Flow cytometry showed that CHO-K1 and Lec1 cells had essentially the same degree of NDV infection. In contrast, Lec2 cells were found to be resistant to NDV infection. Compared with CHO-K1 cells, Lec1 cells were shown to more sensitive to fusion induced by NDV. Viral attachment was found to be comparable in both lines. We found that there were no significant differences in the yield of progeny virus produced by both CHO-K1 and Lec1 cells. Quantitative analysis revealed that NDV infection and fusion in Lec1 cells were also inhibited by treatment with sialidase. Pretreatment of Lec1 cells with Galanthus nivalis agglutinin specific for terminal α1-3-linked Man prior to inoculation with NDV rendered Lec1 cells less sensitive to cell-to-cell fusion compared with mock-treated Lec1 cells. Treatment of CHO-K1 and Lec1 cells with tunicamycin, an inhibitor of N-glycosylation, significantly blocked fusion and infection. In conclusion, our results suggest that hybrid- and complex-type N-glycans are not required for NDV infection and fusion. We propose that high-Man-type N-glycans could play an important role in the cell-to-cell fusion induced by NDV.

Synergistic antimicrobial therapy using nanoparticles and antibiotics for the treatment of multidrug-resistant bacterial infection

NASA Astrophysics Data System (ADS)

Gupta, Akash; Saleh, Neveen M.; Das, Riddha; Landis, Ryan F.; Bigdeli, Arafeh; Motamedchaboki, Khatereh; Rosa Campos, Alexandre; Pomeroy, Kenneth; Mahmoudi, Morteza; Rotello, Vincent M.

2017-06-01

Infections caused by multidrug-resistant (MDR) bacteria pose a serious global burden of mortality, causing thousands of deaths each year. Antibiotic treatment of resistant infections further contributes to the rapidly increasing number of antibiotic-resistant species and strains. Synthetic macromolecules such as nanoparticles (NPs) exhibit broad-spectrum activity against MDR species, however lack of specificity towards bacteria relative to their mammalian hosts limits their widespread therapeutic application. Here, we demonstrate synergistic antimicrobial therapy using hydrophobically functionalized NPs and fluoroquinolone antibiotics for treatment of MDR bacterial strains. An 8-16-fold decrease in antibiotic dosage is achieved in presence of engineered NPs to combat MDR strains. This strategy demonstrates the potential of using NPs to ‘revive’ antibiotics that have been rendered ineffective due to the development of resistance by pathogenic bacteria.

Accelerating bacterial growth detection and antimicrobial susceptibility assessment in integrated picoliter droplet platform.

PubMed

Kaushik, Aniruddha M; Hsieh, Kuangwen; Chen, Liben; Shin, Dong Jin; Liao, Joseph C; Wang, Tza-Huei

2017-11-15

There remains an urgent need for rapid diagnostic methods that can evaluate antibiotic resistance for pathogenic bacteria in order to deliver targeted antibiotic treatments. Toward this end, we present a rapid and integrated single-cell biosensing platform, termed dropFAST, for bacterial growth detection and antimicrobial susceptibility assessment. DropFAST utilizes a rapid resazurin-based fluorescent growth assay coupled with stochastic confinement of bacteria in 20 pL droplets to detect signal from growing bacteria after 1h incubation, equivalent to 2-3 bacterial replications. Full integration of droplet generation, incubation, and detection into a single, uninterrupted stream also renders this platform uniquely suitable for in-line bacterial phenotypic growth assessment. To illustrate the concept of rapid digital antimicrobial susceptibility assessment, we employ the dropFAST platform to evaluate the antibacterial effect of gentamicin on E. coli growth. Copyright © 2017 Elsevier B.V. All rights reserved.

Inhibition of human papillomavirus expression using DNAzymes.

PubMed

Benítez-Hess, María Luisa; Reyes-Gutiérrez, Pablo; Alvarez-Salas, Luis Marat

2011-01-01

Deoxyribozymes (DXZs) are catalytic oligodeoxynucleotides capable of performing diverse functions including the specific cleavage of a target RNA. These molecules represent a new type of therapeutic oligonucleotides combining the efficiency of ribozymes and the intracellular endurance and simplicity of modified antisense oligonucleotides. Commonly used DXZs include the 8-17 and 10-23 motifs, which have been engineered to destroy disease-associated genes with remarkable efficiency. Targeting DXZs to disease-associated transcripts requires extensive biochemical testing to establish target RNA accessibility, catalytic efficiency, and nuclease sensibility. The usage of modified nucleotides to render nuclease-resistance DXZs must be counterweighted against deleterious consequences on catalytic activity. Further intracellular testing is required to establish the effect of microenvironmental conditions on DXZ activity and off-target issues. Application of modified DXZs to cervical cancer results in specific growth inhibition, cell death, and apoptosis. Thus, DXZs represent a highly effective antisense moiety with minimal secondary effects.

Evaluation of a new toxic house fly scatter bait

USDA-ARS?s Scientific Manuscript database

Toxic scatter baits have been a popular tool for house fly control for many years. In fact, Starbar’s Golden Malrin has been sold since 1966. Because of long-term use, resistance to methomyl, the active ingredient in Golden Malrin, has rendered the bait useless in many parts of the US. Increased res...

Effects of pre- and post-harvest application of selenium on inducing disease resistance and selenium accumulation in fruits

USDA-ARS?s Scientific Manuscript database

Botrytis cinerea, a ubiquitous fungal pathogen, causes severe damage (gray mold rot) on a large number of economically important fruits, vegetables, and ornamental crops at both pre- and post-harvest, which renders fruits unmarketable. Penicillium expansum is a widely spread fungal pathogen that cau...

The Principal and "The Prince": Machiavelli and School Leadership.

ERIC Educational Resources Information Center

English, Fenwick W.

1992-01-01

Because most people will resist law and reason (government), power must be rendered in someone else's name to make ruling palatable. Executives, including principals, represent the law. To enforce a given law, executives must often exceed their base of authority to act decisively--despite recent rhetoric about staff empowerment and site-based…

Influence of pumice and plant roots on substrate physical properties over time

USDA-ARS?s Scientific Manuscript database

An experiment was conducted to test the hypothesis that either pumice or plant roots maintain air space and porosity over time, or renders substrates more resistant to shrinkage. Treatment design was a 3×2 factorial with three substrate types and either presence or absence of a plant. The three su...

Technological Difficulties: A Theoretical Frame for Understanding the Non-Relativistic Permanence of Traditional Print Literacy in Elementary Education

ERIC Educational Resources Information Center

Hassett, Dawnene D.

2006-01-01

Currently, definitions of "science", "reading", and "literacy" in the US lend a seemingly nonrelativistic permanence to these terms, and render them resistant to critique. This paper offers a theoretical frame for critiquing this permanence, analysing why early-literacy instruction is tightly tied to traditional forms…

Cultural Capital as Whiteness? Examining Logics of Ethno-Racial Representation and Resistance

ERIC Educational Resources Information Center

Wallace, Derron

2018-01-01

There is a significant, longstanding tradition in British sociological research that renders cultural capital synonymous with whiteness. This article suggests that one substantive factor that contributes to the enduring relationship between whiteness and cultural capital is the paucity of research on the Black and ethnic minority middle classes.…

The Coordinated Actions of TIM-3 on Cancer and Myeloid Cells in the Regulation of Tumorigenicity and Clinical Prognosis in Clear Cell Renal Cell Carcinomas.

PubMed

Komohara, Yoshihiro; Morita, Tomoko; Annan, Dorcas A; Horlad, Hasita; Ohnishi, Koji; Yamada, Sohsuke; Nakayama, Toshiyuki; Kitada, Shohei; Suzu, Shinya; Kinoshita, Ichiro; Dosaka-Akita, Hirotoshi; Akashi, Koichi; Takeya, Motohiro; Jinushi, Masahisa

2015-09-01

Clear cell renal cell carcinoma (ccRCC) is one of most common cancers in urogenital organs. Although recent experimental and clinical studies have shown the immunogenic properties of ccRCC as illustrated by the clinical sensitivities to various immunotherapies, the detailed immunoregulatory machineries governing the tumorigenicity of human ccRCC remain largely obscure. In this study, we demonstrated the clinical significance and functional relevance of T-cell immunoglobulin and mucin domain-containing molecule-3 (TIM-3) expressed on tumor cells and myeloid cells in patients with ccRCC. TIM-3 expression was detected on cancer cells and CD204(+) tumor-associated macrophages (TAM), and higher expression level of TIM-3 was positively correlated with shorter progression-free survival (PFS) in patients with ccRCC. We found that TIM-3 expression was detected on a large number of tumors, and there was significant correlation between an increased number of TAMs and high expression level of TIM-3 in patients with ccRCC. Furthermore, TIM-3 rendered RCC cells with the ability to induce resistance to sunitinib and mTOR inhibitors, the standard regimen for patients with ccRCC, as well as stem cell activities. TIM-3 expression was induced on CD14(+) monocytes upon long-term stimulation with RCC cells, and TIM-3-expressing myeloid cells play a critical role in augmenting tumorigenic activities of TIM-3-negative RCC cells. More importantly, treatment with anti-TIM-3 mAb suppressed its tumorigenic effects in in vitro and in vivo settings. These findings indicate the coordinated action of TIM-3 in cancer cells and in myeloid cells regulates the tumorigenicity of human RCC. ©2015 American Association for Cancer Research.

AMPK activators inhibit the proliferation of human melanomas bearing the activated MAPK pathway.

PubMed

Petti, Carlotta; Vegetti, Claudia; Molla, Alessandra; Bersani, Ilaria; Cleris, Loredana; Mustard, Kirsty J; Formelli, Franca; Hardie, Grahame D; Sensi, Marialuisa; Anichini, Andrea

2012-10-01

Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker β-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.

ATP-binding cassette transporter 1 participates in LDL oxidation by artery wall cells.

PubMed

Reddy, Srinivasa T; Hama, Susan; Ng, Carey; Grijalva, Victor; Navab, Mohamad; Fogelman, Alan M

2002-11-01

We have previously reported that products of the lipoxygenase pathway, hydroperoxyoctadecadienoic acid and hydroperoxyeicosatetraenoic acid, as well as cholesterol linoleate hydroperoxides, collectively termed seeding molecules, are removed by apolipoprotein A-I (apoA-I) from the artery wall cells and render low density lipoprotein (LDL) resistant to oxidation by human artery wall cells. The mechanisms by which oxidized lipids are transported and/or transferred to lipoproteins and the pathways by which apoA-I facilitates their removal remain unclear. ATP-binding cassette transporter 1 (ABCA1) is known to facilitate the release of cellular phospholipids and cholesterol from the plasma membrane to apoA-I and high density lipoprotein. Therefore, we evaluated whether ABCA1 participates in LDL oxidation. In this report, we show that (1) chemical inhibitors of ABCA1 function, glyburide and DIDS, block artery wall cell-mediated oxidative modification of LDL, (2) inhibition of ABCA1 with the use of antisense (but not sense) oligonucleotides prevents LDL-induced lipid hydroperoxide formation and LDL-induced monocyte chemotactic activity by the artery wall cells, and (3) oxysterols that induce ABCA1 expression, such as 22(R)hydroxycholesterol, enhance cell-mediated LDL oxidation. Furthermore, we also show that 22(R)hydroxycholesterol induces the production of reactive oxygen species in the artery wall cells, which can be removed by incubating the artery wall cells with apoA-I. Our data suggest that ABCA1 plays an important role in artery wall cell-mediated modification/oxidation of LDL by modulating the release of reactive oxygen species from artery wall cells that are necessary for LDL oxidation.

Enhanced Transcription of the Arabidopsis Disease Resistance Genes RPW8.1 and RPW8.2 via a Salicylic Acid–Dependent Amplification Circuit Is Required for Hypersensitive Cell Death

PubMed Central

Xiao, Shunyuan; Brown, Samantha; Patrick, Elaine; Brearley, Charles; Turner, John G.

2003-01-01

The Arabidopsis disease resistance (R) genes RPW8.1 and RPW8.2 couple the recognition of powdery mildew pathogens of this plant with the subsequent induction of a localized necrosis, or hypersensitive response (HR). The HR restricts the spread of the infection and renders the plant resistant. One-third of Arabidopsis plants transformed with a genomic fragment containing RPW8.1 and RPW8.2 developed spontaneous HR-like lesions (SHL) in the absence of pathogens. We demonstrate that SHL occurs in transgenic lines that contain multiple copies of the transgene and express RPW8.1 and RPW8.2 at high levels. SHL is associated with salicylic acid (SA) accumulation, and at the site of the lesion, there is increased expression of RPW8.1, increased production of H2O2, and increased expression of pathogenesis-related genes. These lesions are physiologically similar to the pathogen-induced HR mediated by RPW8.1 and RPW8.2. Significantly, environmental conditions that suppress SHL suppress the transcription of RPW8.1 and RPW8.2 and also suppress resistance to powdery mildews, even in transgenic lines containing RPW8.1 and RPW8.2 that normally do not express SHL. Furthermore, treatment with SA increases the transcription of RPW8.1 and RPW8.2, induces SHL, and enhances resistance to powdery mildews. We conclude that HR requires the transcription of RPW8.1 and RPW8.2, which is regulated independently of the pathogen by SA-dependent feedback amplification. PMID:12509520

Induction of steatohepatitis (NASH) with insulin resistance in wildtype B6 mice by a western-type diet containing soybean oil and cholesterol.

PubMed

Henkel, Janin; Coleman, Charles Dominic; Schraplau, Anne; Jӧhrens, Korinna; Weber, Daniela; Castro, José Pedro; Hugo, Martin; Schulz, Tim Julius; Krämer, Stephanie; Schürmann, Annette; Püschel, Gerhard Paul

2017-03-21

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are hepatic manifestations of the metabolic syndrome. Many currently used animal models of NAFLD/NASH lack clinical features of either NASH or metabolic syndrome such as hepatic inflammation and fibrosis (e.g. high-fat diets) or overweight and insulin resistance (e.g. methionine-choline-deficient diets) or they are based on monogenetic defects (e.g. ob/ob mice). In the current study, a western-type diet containing soybean oil with high n 6-PUFA and 0.75% cholesterol (SOD+Cho) induced steatosis, inflammation and fibrosis accompanied by hepatic lipid peroxidation and oxidative stress in livers of C57BL/6-mice which in addition showed increased weight gain and insulin resistance, thus displaying a phenotype closely resembling all clinical features of NASH in patients with metabolic syndrome. In striking contrast a soybean oil-containing western-type diet without cholesterol (SOD) induced only mild steatosis but neither hepatic inflammation nor fibrosis, weight gain or insulin resistance. Another high-fat diet mainly consisting of lard and supplemented with fructose in drinking water (LAD+Fru) resulted in more prominent weight gain, insulin resistance and hepatic steatosis than SOD+Cho but livers were devoid of inflammation and fibrosis. Although both LAD+Fru- and SOD+Cho-fed animals had high plasma cholesterol, liver cholesterol was elevated only in SOD+Cho animals. Cholesterol induced expression of chemotactic and inflammatory cytokines in cultured Kupffer cells and rendered hepatocytes more susceptible to apoptosis. Summarizing, dietary cholesterol in SOD+Cho diet may trigger hepatic inflammation and fibrosis. SOD+Cho-fed animals may be a useful disease model displaying many clinical features of patients with the metabolic syndrome and NASH.

High temperature solid electrolyte fuel cell with ceramic electrodes

DOEpatents

Marchant, David D.; Bates, J. Lambert

1984-01-01

A solid oxide electrolyte fuel cell is described having a central electrolyte comprised of a HfO.sub.2 or ZrO.sub.2 ceramic stabilized and rendered ionically conductive by the addition of Ca, Mg, Y, La, Nd, Sm, Gd, Dy Er, or Yb. The electrolyte is sandwiched between porous electrodes of a HfO.sub.2 or ZrO.sub.2 ceramic stabilized by the addition of a rare earth and rendered electronically conductive by the addition of In.sub.2 O.sub.3. Alternatively, the anode electrode may be made of a metal such as Co, Ni, Ir Pt, or Pd.

High temperature solid electrolyte fuel cell with ceramic electrodes

DOEpatents

Bates, J.L.; Marchant, D.D.

A solid oxide electrolyte fuel cell is described having a central electrolyte comprised of a HfO/sub 2/ or ZrO/sub 2/ ceramic stabilized and rendered ionically conductive by the addition of Ca, Mg, Y, La, Nd, Sm, Gd, Dy Er, or Yb. The electrolyte is sandwiched between porous electrodes of a HfO/sub 2/ or ZrO/sub 2/ ceramic stabilized by the addition of a rare earth and rendered electronically conductive by the addition of In/sub 2/O/sub 3/. Alternatively, the anode electrode may be made of a metal such as Co, Ni, Ir Pt, or Pd.

Antitumor activity of fermented noni exudates and its fractions

PubMed Central

LI, JINHUA; CHANG, LENG-CHEE; WALL, MARISA; WONG, D.K.W.; YU, XIANZHONG; WEI, YANZHANG

2013-01-01

Noni has been extensively used in folk medicine by Polynesians for over 2000 year. Recent studies have shown that noni has a wide spectrum of therapeutic activities including inhibition of angiogenesis, anti-inflammatory effects and anti-cancer activities. Intraperitoneal (i.p.) injection of fermented noni exudates (fNE) were previously found to induce significant tumor rejection in a S180 mouse sarcoma tumor model, while natural killer (NK) cells were demonstrated to be markedly involved in fNE-induced antitumor activity. In this study, fNE was partitioned into three fractions and their antitumor effects were examined using i.p. injection or as water supplement. The in vivo animal study results showed that when delivered by i.p. injection, n-butanol fraction of fNE (BuOH) effectively rejected (100%) tumor challenge and eradicated existing tumors (75%). When delivered as a water supplement, 62.5% of the mice receiving the n-butanol or ethyl acetate fractions resisted tumor cells. The tumor-resistant mice effectively rejected more and higher doses of tumor challenge, indicating that the immune system was activated. The findings confirm those of an earlier study showing fNE to have anti-tumor activity and demonstrating that the n-butanol fraction of fNE contains active antitumor components, to be further identified. More importantly, the antitumor effect of fNE and its fractions as water supplements renders a significant potential for identifying novel and powerful new dietary products for cancer prevention. PMID:24649140

Antitumor activity of fermented noni exudates and its fractions.

PubMed

Li, Jinhua; Chang, Leng-Chee; Wall, Marisa; Wong, D K W; Yu, Xianzhong; Wei, Yanzhang

2013-01-01

Noni has been extensively used in folk medicine by Polynesians for over 2000 year. Recent studies have shown that noni has a wide spectrum of therapeutic activities including inhibition of angiogenesis, anti-inflammatory effects and anti-cancer activities. Intraperitoneal (i.p.) injection of fermented noni exudates (fNE) were previously found to induce significant tumor rejection in a S180 mouse sarcoma tumor model, while natural killer (NK) cells were demonstrated to be markedly involved in fNE-induced antitumor activity. In this study, fNE was partitioned into three fractions and their antitumor effects were examined using i.p. injection or as water supplement. The in vivo animal study results showed that when delivered by i.p. injection, n-butanol fraction of fNE (BuOH) effectively rejected (100%) tumor challenge and eradicated existing tumors (75%). When delivered as a water supplement, 62.5% of the mice receiving the n-butanol or ethyl acetate fractions resisted tumor cells. The tumor-resistant mice effectively rejected more and higher doses of tumor challenge, indicating that the immune system was activated. The findings confirm those of an earlier study showing fNE to have anti-tumor activity and demonstrating that the n-butanol fraction of fNE contains active antitumor components, to be further identified. More importantly, the antitumor effect of fNE and its fractions as water supplements renders a significant potential for identifying novel and powerful new dietary products for cancer prevention.

Cavitation Resistance in Seedless Vascular Plants: The Structure and Function of Interconduit Pit Membranes1[W][OPEN

PubMed Central

Brodersen, Craig; Jansen, Steven; Choat, Brendan; Rico, Christopher; Pittermann, Jarmila

2014-01-01

Plant water transport occurs through interconnected xylem conduits that are separated by partially digested regions in the cell wall known as pit membranes. These structures have a dual function. Their porous construction facilitates water movement between conduits while limiting the spread of air that may enter the conduits and render them dysfunctional during a drought. Pit membranes have been well studied in woody plants, but very little is known about their function in more ancient lineages such as seedless vascular plants. Here, we examine the relationships between conduit air seeding, pit hydraulic resistance, and pit anatomy in 10 species of ferns (pteridophytes) and two lycophytes. Air seeding pressures ranged from 0.8 ± 0.15 MPa (mean ± sd) in the hydric fern Athyrium filix-femina to 4.9 ± 0.94 MPa in Psilotum nudum, an epiphytic species. Notably, a positive correlation was found between conduit pit area and vulnerability to air seeding, suggesting that the rare-pit hypothesis explains air seeding in early-diverging lineages much as it does in many angiosperms. Pit area resistance was variable but averaged 54.6 MPa s m−1 across all surveyed pteridophytes. End walls contributed 52% to the overall transport resistance, similar to the 56% in angiosperm vessels and 64% in conifer tracheids. Taken together, our data imply that, irrespective of phylogenetic placement, selection acted on transport efficiency in seedless vascular plants and woody plants in equal measure by compensating for shorter conduits in tracheid-bearing plants with more permeable pit membranes. PMID:24777347

Identification and characterization of ALK kinase splicing isoforms in non-small-cell lung cancer

PubMed Central

de Figueiredo-Pontes, Lorena Lobo; Wong, Daisy Wing-Sze; Tin, Vick Pui-Chi; Chung, Lap-Ping; Yasuda, Hiroyuki; Yamaguchi, Norihiro; Nakayama, Sohei; Jänne, Pasi Antero; Wong, Maria Pik; Kobayashi, Susumu Soeda; Costa, Daniel Botelho

2014-01-01

Purpose: Anaplastic lymphoma kinase (ALK) rearrangements are present in an important subset of non-small-cell lung cancer (NSCLC) and predict for response to the tyrosine kinase inhibitor crizotinib. In this study, we evaluated the yet unknown frequency and functional role of ALK splicing isoforms in NSCLC. Experimental Design: We analyzed 270 cases of NSCLC for ALK kinase domain splicing aberrations, and in addition generated constructs with full length EML4-ALK (E13;A20) and a splicing isoform. Results: Splicing isoforms of the kinase domain of ALK - including complete skipping of exon 23 (ALKdel23, ALK p.I1171fs*42) and exon 27 (ALKdel27, ALK p.T1312fs*0) - were identified in 11.1% (30/270 cases) of NSCLC, and these changes co-existed with ALK rearrangements, KRAS mutations and EGFR mutations. ALK splicing isoforms were observed with full length EML4-ALK in crizotinib-naïve and treated NSCLCs. ALK T1312fs*0 was unable to render cells solely dependent on ALK signaling. Unlike EML4-ALK and EML4-ALK p.L1196M, EML4-ALK T1312fs*0 did not autophosphorylate ALK or other phospho-tyrosine sites. Co-expression of equal amounts of EML4-ALK T1312fs*0 and EML4-ALK did not result in resistance to crizotinib, while co-expression of EML4-ALK L1196M with EML4-ALK resulted in resistance to inhibition of ALK by crizotinib. Conclusions: ALK kinase splicing isoforms were present in NSCLC and even if translated seemed to be non-functional variants of ALK. PMID:24419423

Towards rhombohedral SiGe epitaxy on 150mm c-plane sapphire substrates

NASA Astrophysics Data System (ADS)

Duzik, Adam J.; Park, Yeonjoon; Choi, Sang H.

2015-04-01

Previous work demonstrated for the first time the ability to epitaxially grow uniform single crystal diamond cubic SiGe (111) films on trigonal sapphire (0001) substrates. While SiGe (111) forms two possible crystallographic twins on sapphire (0001), films consisting primarily of one twin were produced on up to 99.95% of the total wafer area. This permits new bandgap engineering possibilities and improved group IV based devices that can exploit the higher carrier mobility in Ge compared to Si. Models are proposed on the epitaxy of such dissimilar crystal structures based on the energetic favorability of crystallographic twins and surface reconstructions. This new method permits Ge (111) on sapphire (0001) epitaxy, rendering Ge an economically feasible replacement for Si in some applications, including higher efficiency Si/Ge/Si quantum well solar cells. Epitaxial SiGe films on sapphire showed a 280% increase in electron mobility and a 500% increase in hole mobility over single crystal Si. Moreover, Ge possesses a wider bandgap for solar spectrum conversion than Si, while the transparent sapphire substrate permits an inverted device structure, increasing the total efficiency to an estimated 30-40%, much higher than traditional Si solar cells. Hall Effect mobility measurements of the Ge layer in the Si/Ge/Si quantum well structure were performed to demonstrate the advantage in carrier mobility over a pure Si solar cell. Another application comes in the use of microelectromechanical devices technology, where high-resistivity Si is currently used as a substrate. Sapphire is a more resistive substrate and offers better performance via lower parasitic capacitance and higher film carrier mobility over the current Si-based technology.

A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin

DOE PAGES

Liu, Yao; Zhu, Wenhan; Tan, Yunhao; ...

2017-01-27

Legionella pneumophila, the etiological agent of Legionnaires' disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H 95E XXH 99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cellmore » rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Furthermore, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.« less

Platelet and Erythrocyte Sources of S1P Are Redundant for Vascular Development and Homeostasis, but Both Rendered Essential After Plasma S1P Depletion in Anaphylactic Shock.

PubMed

Gazit, Salomé L; Mariko, Boubacar; Thérond, Patrice; Decouture, Benoit; Xiong, Yuquan; Couty, Ludovic; Bonnin, Philippe; Baudrie, Véronique; Le Gall, Sylvain M; Dizier, Blandine; Zoghdani, Nesrine; Ransinan, Jessica; Hamilton, Justin R; Gaussem, Pascale; Tharaux, Pierre-Louis; Chun, Jerold; Coughlin, Shaun R; Bachelot-Loza, Christilla; Hla, Timothy; Ho-Tin-Noé, Benoit; Camerer, Eric

2016-09-30

Sphingosine-1-phosphate (S1P) signaling is essential for vascular development and postnatal vascular homeostasis. The relative importance of S1P sources sustaining these processes remains unclear. To address the level of redundancy in bioactive S1P provision to the developing and mature vasculature. S1P production was selectively impaired in mouse platelets, erythrocytes, endothelium, or smooth muscle cells by targeted deletion of genes encoding sphingosine kinases -1 and -2. S1P deficiency impaired aggregation and spreading of washed platelets and profoundly reduced their capacity to promote endothelial barrier function ex vivo. However, and in contrast to recent reports, neither platelets nor any other source of S1P was essential for vascular development, vascular integrity, or hemostasis/thrombosis. Yet rapid and profound depletion of plasma S1P during systemic anaphylaxis rendered both platelet- and erythrocyte-derived S1P essential for survival, with a contribution from blood endothelium observed only in the absence of circulating sources. Recovery was sensitive to aspirin in mice with but not without platelet S1P, suggesting that platelet activation and stimulus-response coupling is needed. S1P deficiency aggravated vasoplegia in this model, arguing a vital role for S1P in maintaining vascular resistance during recovery from circulatory shock. Accordingly, the S1P2 receptor mediated most of the survival benefit of S1P, whereas the endothelial S1P1 receptor was dispensable for survival despite its importance for maintaining vascular integrity. Although source redundancy normally secures essential S1P signaling in developing and mature blood vessels, profound depletion of plasma S1P renders both erythrocyte and platelet S1P pools necessary for recovery and high basal plasma S1P levels protective during anaphylactic shock. © 2016 American Heart Association, Inc.

Analysis of Tomato spotted wilt virus NSs protein indicates the importance of the N-terminal domain for avirulence and RNA silencing suppression.

PubMed

de Ronde, Dryas; Pasquier, Adrien; Ying, Su; Butterbach, Patrick; Lohuis, Dick; Kormelink, Richard

2014-02-01

Recently, Tomato spotted wilt virus (TSWV) nonstructural protein NSs has been identified unambiguously as an avirulence (Avr) determinant for Tomato spotted wilt (Tsw)-based resistance. The observation that NSs from two natural resistance-breaking isolates had lost RNA silencing suppressor (RSS) activity and Avr suggested a link between the two functions. To test this, a large set of NSs mutants was generated by alanine substitutions in NSs from resistance-inducing wild-type strains (NSs(RI) ), amino acid reversions in NSs from resistance-breaking strains (NSs(RB)), domain deletions and swapping. Testing these mutants for their ability to suppress green fluorescent protein (GFP) silencing and to trigger a Tsw-mediated hypersensitive response (HR) revealed that the two functions can be separated. Changes in the N-terminal domain were found to be detrimental for both activities and indicated the importance of this domain, additionally supported by domain swapping between NSs(RI) and NSs(RB). Swapping domains between the closely related Tospovirus Groundnut ringspot virus (GRSV) NSs and TSWV NSs(RI) showed that Avr functionality could not simply be transferred between species. Although deletion of the C-terminal domain rendered NSs completely dysfunctional, only a few single-amino-acid mutations in the C-terminus affected both functions. Mutation of a GW/WG motif (position 17/18) rendered NSs completely dysfunctional for RSS and Avr activity, and indicated a putative interaction between NSs and Argonaute 1 (AGO1), and its importance in TSWV virulence and viral counter defence against RNA interference. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Application of Nanoparticle Technologies in the Combat against Anti-Microbial Resistance.

PubMed

Kumar, Mayur; Curtis, Anthony; Hoskins, Clare

2018-01-14

Anti-microbial resistance is a growing problem that has impacted the world and brought about the beginning of the end for the old generation of antibiotics. Increasingly, more antibiotics are being prescribed unnecessarily and this reckless practice has resulted in increased resistance towards these drugs, rendering them useless against infection. Nanotechnology presents a potential answer to anti-microbial resistance, which could stimulate innovation and create a new generation of antibiotic treatments for future medicines. Preserving existing antibiotic activity through novel formulation into or onto nanotechnologies can increase clinical longevity of action against infection. Additionally, the unique physiochemical properties of nanoparticles can provide new anti-bacterial modes of action which can also be explored. Simply concentrating on antibiotic prescribing habits will not resolve the issue but rather mitigate it. Thus, new scientific approaches through the development of novel antibiotics and formulations is required in order to employ a new generation of therapies to combat anti-microbial resistance.

cellVIEW: a Tool for Illustrative and Multi-Scale Rendering of Large Biomolecular Datasets

PubMed Central

Le Muzic, Mathieu; Autin, Ludovic; Parulek, Julius; Viola, Ivan

2017-01-01

In this article we introduce cellVIEW, a new system to interactively visualize large biomolecular datasets on the atomic level. Our tool is unique and has been specifically designed to match the ambitions of our domain experts to model and interactively visualize structures comprised of several billions atom. The cellVIEW system integrates acceleration techniques to allow for real-time graphics performance of 60 Hz display rate on datasets representing large viruses and bacterial organisms. Inspired by the work of scientific illustrators, we propose a level-of-detail scheme which purpose is two-fold: accelerating the rendering and reducing visual clutter. The main part of our datasets is made out of macromolecules, but it also comprises nucleic acids strands which are stored as sets of control points. For that specific case, we extend our rendering method to support the dynamic generation of DNA strands directly on the GPU. It is noteworthy that our tool has been directly implemented inside a game engine. We chose to rely on a third party engine to reduce software development work-load and to make bleeding-edge graphics techniques more accessible to the end-users. To our knowledge cellVIEW is the only suitable solution for interactive visualization of large bimolecular landscapes on the atomic level and is freely available to use and extend. PMID:29291131

Teen Girls, Working-Class Femininity and Resistance: Retheorising Fantasy and Desire in Educational Contexts of Heterosexualised Violence

ERIC Educational Resources Information Center

Ringrose, Jessica; Renold, Emma

2012-01-01

This paper challenges post-feminist discourses and recuperative masculinity politics in education that have evoked mythical constructions of the successful "achieving" girl in ways that flatten out social and cultural difference and render invisible ongoing gendered and sexualised inequalities and violence in the social worlds of schools…

'The First Step Is the Two-Step': Hegemonic Masculinity and Dancing in a Country-Western Gay Bar

ERIC Educational Resources Information Center

Johnson, Corey W.

2005-01-01

Gay men are often rendered feminized males according to masculine, heterosexual ideologies. This research demonstrates that gay men are actually more creative and active agents in their gender performance, simultaneously resisting and reinforcing dominant ideologies around gender and sexuality. One context where the creative agency of gay men is…

PLUTONIUM-URANIUM-TITANIUM ALLOYS

DOEpatents

Coffinberry, A.S.

1959-07-28

A plutonium-uranium alloy suitable for use as the fuel element in a fast breeder reactor is described. The alloy contains from 15 to 60 at.% titanium with the remainder uranium and plutonium in a specific ratio, thereby limiting the undesirable zeta phase and rendering the alloy relatively resistant to corrosion and giving it the essential characteristic of good mechanical workability.

Constructing the Hydrogeological Model of the Choushuichi Fan-delta in Central Taiwan with the Electrical Resistivity Measurements

NASA Astrophysics Data System (ADS)

Chang, P.; Chang, L.; Chen, W.; Chiang, C.

2012-12-01

In the study we used the resistivity measurements across the Choushuichi Fan-delta to establish a three-dimensional hydrogeological model. The resistivity measurements includes the half-Schlumberger surveys conducted during the year of 1990-2000 across the entire fan-delta area, and the two-dimensional resistivity data collected recently for the purpose of characterizing the recharge zone boundaries between the upper-fan gravels and the lower-fan clayey sediments. Core records from the monitoring wells in the area were used for the training data to help determining the resistivity ranges of the gavel, sand, and muddy sediments in the fan-delta. The resistivity measurements were inverted and converted into 1-D data form and interpolated for rendering a three dimensional resistivity volume that represents the general resistivity distribution in the Choushuichi fan-delta. We categorize the hydrogeological materials into gravels, sands, and clayey sediments with the resistivity ranges from the previous statistical analysis. Hence we are able to quickly construct a three-dimensional hydrogeological model with simple three materials.

Intracellular Serine Protease Inhibitor SERPINB4 Inhibits Granzyme M-Induced Cell Death

PubMed Central

de Koning, Pieter J. A.; Kummer, J. Alain; de Poot, Stefanie A. H.; Quadir, Razi; Broekhuizen, Roel; McGettrick, Anne F.; Higgins, Wayne J.; Devreese, Bart; Worrall, D. Margaret; Bovenschen, Niels

2011-01-01

Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1′ triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×104 M−1s−1. SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death. PMID:21857942

Nrf2 protects human bladder urothelial cells from arsenite and monomethylarsonous acid toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Xiaojun; Sun Zheng; Chen Weimin

2007-12-01

Arsenic is widely spread in our living environment and imposes a big challenge on human health worldwide. Arsenic damages biological systems through multiple mechanisms including the generation of reactive oxygen species. The transcription factor Nrf2 regulates the cellular antioxidant response that protects cells from various insults. In this study, the protective role of Nrf2 in arsenic toxicity was investigated in a human bladder urothelial cell line, UROtsa. Using a UROtsa cell line stably infected with Nrf2-siRNA, we clearly demonstrate that compromised Nrf2 expression sensitized the cells to As(III)- and MMA(III)-induced toxicity. On the other hand, the activation of the Nrf2more » pathway by tert-butylhydroquinone (tBHQ) and sulforaphane (SF), the known Nrf2-inducers, rendered UROtsa cells more resistant to As(III) and MMA(III). Furthermore, the wild-type mouse embryo fibroblast (WT-MEF) cells were protected from As(III)- and MMA(III)-induced toxicity following Nrf2 activation by tBHQ or SF, whereas neither tBHQ nor SF conferred protection in the Nrf2{sup -/-}MEF cells, demonstrating that tBHQ- or SF-mediated protection against As(III)- and MMA(III)-induced toxicity depends on Nrf2 activation. These results, obtained by both loss of function and gain of function analyses, clearly demonstrate the protective role of Nrf2 in arsenic-induced toxicity. The current work lays the groundwork for using Nrf2 activators for therapeutic and dietary interventions against adverse effects of arsenic.« less

In-depth analysis of switchable glycerol based polymeric coatings for cell sheet engineering.

PubMed

Becherer, Tobias; Heinen, Silke; Wei, Qiang; Haag, Rainer; Weinhart, Marie

2015-10-01

Scaffold-free cell sheet engineering using thermoresponsive substrates provides a promising alternative to conventional tissue engineering which in general employs biodegradable scaffold materials. We have previously developed a thermoresponsive coating with glycerol based linear copolymers that enables gentle harvesting of entire cell sheets. In this article we present an in-depth analysis of these thermoresponsive linear polyglycidyl ethers and their performance as coating for substrates in cell culture in comparison with commercially available poly(N-isopropylacrylamide) (PNIPAM) coated culture dishes. A series of copolymers of glycidyl methyl ether (GME) and glycidyl ethyl ether (EGE) was prepared in order to study their thermoresponsive properties in solution and on the surface with respect to the comonomer ratio. In both cases, when grafted to planar surfaces or spherical nanoparticles, the applied thermoresponsive polyglycerol coatings render the respective surfaces switchable. Protein adsorption experiments on copolymer coated planar surfaces with surface plasmon resonance (SPR) spectroscopy reveal the ability of the tested thermoresponsive coatings to be switched between highly protein resistant and adsorptive states. Cell culture experiments demonstrate that these thermoresponsive coatings allow for adhesion and proliferation of NIH 3T3 fibroblasts comparable to TCPS and faster than on PNIPAM substrates. Temperature triggered detachment of complete cell sheets from copolymer coated substrates was accomplished within minutes while maintaining high viability of the harvested cells. Thus such glycerol based copolymers present a promising alternative to PNIPAM as a thermoresponsive coating of cell culture substrates. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

DNA modification and functional delivery into human cells using Escherichia coli DH10B

PubMed Central

Narayanan, Kumaran; Warburton, Peter E.

2003-01-01

The availability of almost the complete human genome as cloned BAC libraries represents a valuable resource for functional genomic analysis, which, however, has been somewhat limited by the ability to modify and transfer this DNA into mammalian cells intact. Here we report a novel comprehensive Escherichia coli-based vector system for the modification, propagation and delivery of large human genomic BAC clones into mammalian cells. The GET recombination inducible homologous recombination system was used in the BAC host strain E.coli DH10B to precisely insert an EGFPneo cassette into the vector portion of a ∼200 kb human BAC clone, providing a relatively simple method to directly convert available BAC clones into suitable vectors for mammalian cells. GET recombination was also used for the targeted deletion of the asd gene from the E.coli chromosome, resulting in defective cell wall synthesis and diaminopimelic acid auxotrophy. Transfer of the Yersinia pseudotuberculosis invasin gene into E.coli DH10B asd– rendered it competent to invade HeLa cells and deliver DNA, as judged by transient expression of green fluorescent protein and stable neomycin-resistant colonies. The efficiency of DNA transfer and survival of HeLa cells has been optimized for incubation time and multiplicity of infection of invasive E.coli with HeLa cells. This combination of E.coli-based homologous recombination and invasion technologies using BAC host strain E.coli DH10B will greatly improve the utility of the available BAC libraries from the human and other genomes for gene expression and functional genomic studies. PMID:12711696

Thymosin From Bombyx mori Is Down-Regulated in Expression by BmNPV Exhibiting Antiviral Activity.

PubMed

Zhang, Chen; Wang, Yongdi; Fang, Qiang; Xu, Minlin; Lv, Mengyuan; Liao, Jinxu; Li, Si; Nie, Zuoming; Zhang, Wenping

2016-01-01

Thymosins have been highly conserved during evolution. These hormones exist in many animal species and play an essential role in many biological events. However, little is known regarding the physiological function of silkworm Bombyx mori thymosin (BmTHY). In this study, we investigated the expression pattern of BmTHY in a Bombyx mori larval ovarian cell line (BmN) challenged with Bombyx mori nuclear polyhydrosis virus (BmNPV) and the antiviral effect of recombinant BmTHY (rBmTHY) for Bombyx mori against BmNPV. Western-blot assay and qRT-PCR analysis revealed that the level of BmTHY protein expression and transcription decreased over time when BmN cells were infected by BmNPV. Treatment with endotoxin-free rBmTHY led to a significant reduction in viral titer in the supernatant of BmN cells challenged with BmNPV. The results from antiviral tests performed in vitro and in vivo showed that endotoxin-free rBmTHY improved the survival rate of Bombyx mori infected with BmNPV. These findings suggest that BmTHY exerts immunomodulatory effects on Bombyx mori, rendering them resistant to viral infection. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America.

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations.

PubMed

Saito, Kohta; Warrier, Thulasi; Somersan-Karakaya, Selin; Kaminski, Lina; Mi, Jianjie; Jiang, Xiuju; Park, Suna; Shigyo, Kristi; Gold, Ben; Roberts, Julia; Weber, Elaina; Jacobs, William R; Nathan, Carl F

2017-06-13

Mycobacterium tuberculosis (Mtb) encounters stresses during the pathogenesis and treatment of tuberculosis (TB) that can suppress replication of the bacteria and render them phenotypically tolerant to most available drugs. Where studied, the majority of Mtb in the sputum of most untreated subjects with active TB have been found to be nonreplicating by the criterion that they do not grow as colony-forming units (cfus) when plated on agar. However, these cells are viable because they grow when diluted in liquid media. A method for generating such "differentially detectable" (DD) Mtb in vitro would aid studies of the biology and drug susceptibility of this population, but lack of independent confirmation of reported methods has contributed to skepticism about their existence. Here, we identified confounding artifacts that, when avoided, allowed development of a reliable method of producing cultures of ≥90% DD Mtb in starved cells. We then characterized several drugs according to whether they contribute to the generation of DD Mtb or kill them. Of the agents tested, rifamycins led to DD Mtb generation, an effect lacking in a rifampin-resistant strain with a mutation in rpoB , which encodes the canonical rifampin target, the β subunit of RNA polymerase. In contrast, thioridazine did not generate DD Mtb from starved cells but killed those generated by rifampin.

Isolation and characterization of Escherichia coli K-12 mutants unable to induce the adaptive response to simple alkylating agents.

PubMed Central

Jeggo, P

1979-01-01

When Esherichia coli cells are exposed to a low level of simple alkylating agents, they induce the adaptive response which renders them more resistant to the killing and the mutagenic effects of the same or other alkylating agents. This paper describes the isolation of one strain that was deficient in mutagenic adaptation and five that were deficient in both mutagenic and killing adaptation, confirming previous suggestions that killing and mutagenic adaptation are, at least to some extent, separable. These six strains have been called Ada mutants. They were more sensitive to the killing and mutagenic effects of N-methy-N'-nitro-N-nitrosoguanidine (MNNG) than the unadapted Ada+ parent. Thus, the adaptation pathway is responsible for circumventing some alkylation-induced damage even in cells that are preinduced. The increase in mutation frequency seen in Ada cells treated with MNNG was the same whether the cells were lexA+ or lexA, showing that the extra mutations found in Ada- strains do not depend upon the SOS pathway. Ada strains accumulated more O6-methyl guanine lesions than the Ada+ parent on prolonged exposure to MNNG, and this supports the idea that O6-methyl guanine is the most important lesion for MNNG-induced mutagenesis. The ada mutations have been shown to map in the 47 to 53-min region of the E. coli chromosome. PMID:383692

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae.

PubMed

Geoghegan, Ivey A; Gurr, Sarah J

2017-09-01

Chitin deacetylation results in the formation of chitosan, a polymer of β1,4-linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth-CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear. © 2017 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

Induction of homologous recombination in Saccharomyces cerevisiae.

PubMed

Simon, J R; Moore, P D

1988-09-01

We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, or the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.

Dual inhibition of ATR and ATM potentiates the activity of trabectedin and lurbinectedin by perturbing the DNA damage response and homologous recombination repair.

PubMed

Lima, Michelle; Bouzid, Hana; Soares, Daniele G; Selle, Frédéric; Morel, Claire; Galmarini, Carlos M; Henriques, João A P; Larsen, Annette K; Escargueil, Alexandre E

2016-05-03

Trabectedin (Yondelis®, ecteinascidin-743, ET-743) is a marine-derived natural product approved for treatment of advanced soft tissue sarcoma and relapsed platinum-sensitive ovarian cancer. Lurbinectedin is a novel anticancer agent structurally related to trabectedin. Both ecteinascidins generate DNA double-strand breaks that are processed through homologous recombination repair (HRR), thereby rendering HRR-deficient cells particularly sensitive. We here characterize the DNA damage response (DDR) to trabectedin and lurbinectedin in HeLa cells. Our results show that both compounds activate the ATM/Chk2 (ataxia-telangiectasia mutated/checkpoint kinase 2) and ATR/Chk1 (ATM and RAD3-related/checkpoint kinase 1) pathways. Interestingly, pharmacological inhibition of Chk1/2, ATR or ATM is not accompanied by any significant improvement of the cytotoxic activity of the ecteinascidins while dual inhibition of ATM and ATR strongly potentiates it. Accordingly, concomitant inhibition of both ATR and ATM is an absolute requirement to efficiently block the formation of γ-H2AX, MDC1, BRCA1 and Rad51 foci following exposure to the ecteinascidins. These results are not restricted to HeLa cells, but are shared by cisplatin-sensitive and -resistant ovarian carcinoma cells. Together, our data identify ATR and ATM as central coordinators of the DDR to ecteinascidins and provide a mechanistic rationale for combining these compounds with ATR and ATM inhibitors.

Shikonin protects dopaminergic cell line PC12 against 6-hydroxydopamine-mediated neurotoxicity via both glutathione-dependent and independent pathways and by inhibiting apoptosis.

PubMed

Esmaeilzadeh, Emran; Gardaneh, Mossa; Gharib, Ehsan; Sabouni, Farzaneh

2013-08-01

We have investigated the mechanism of shikonin function on protection of dopaminergic neurons against 6-OHDA-induced neurotoxicity. Treatment of rat pheochromocytoma cell line PC12 by serial dilutions of shikonin determined 10 μM of the compound as its optimum concentration for protection saving nearly 70 % of the cells against toxicity. Reverse transcription-PCR analysis of shikonin-treated cells showed threefold increase in mRNA levels of glutathione peroxidase-1 (GPX-1) as a representative component of the intracellular anti-oxidant defense system. To elucidate shikonin-GPX1 relationships and maximize protection, we transduced PC12 cells using recombinant lentivirus vectors that harbored GPX-1 coding sequence. This change upregulated GPX-1 expression, increased peroxidase activity and made neuronal cells resistant to 6-OHDA-mediated toxicity. More importantly, addition of shikonin to GPX1-overexpressing PC12 cells augmented GPX-1 protein content by eightfold leading to fivefold increase of enzymatic activity, 91 % cell survival against neurotoxicity and concomitant increases in intracellular glutathione (GSH) levels. Depletion of intracellular GSH rendered all cell groups highly susceptible to toxicity; however, shikonin was capable of partially saving them. Subsequently, GSH-independent superoxide dismutase mRNA was found upregulated by shikonin. As signs of apoptosis inhibition, the compound upregulated Bcl-2, downregulated Bax, and prevented cell nuclei from undergoing morphological changes typical of apoptosis. Also, a co-staining method demonstrated GPX-1 overexpression significantly increases the percent of live cells that is maximized by shikonin treatment. Our data indicate that shikonin as an antioxidant compound protects dopaminergic neurons against 6-OHDA toxicity and enhances their survival via both glutathione-dependent and direct anti-apoptotic pathways.

Inhibition of oncogenic Pim-3 kinase modulates transformed growth and chemosensitizes pancreatic cancer cells to gemcitabine

PubMed Central

Xu, Dapeng; Cobb, Michael G.; Gavilano, Lily; Witherspoon, Sam M.; Williams, Daniel; White, Catherine D.; Taverna, Pietro; Bednarski, Brian K.; Kim, Hong Jin; Baldwin, Albert S.; Baines, Antonio T.

2013-01-01

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a 5-year survival rate of only 6%. Although the cytosine analog gemcitabine is the drug commonly used to treat PDAC, chemoresistance unfortunately renders the drug ineffective. Thus, strategies that can decrease this resistance will be essential for improving the dismal outcome of patients suffering from this disease. We previously observed that oncogenic Pim-1 kinase was aberrantly expressed in PDAC tissues and cell lines and was responsible for radioresistance. Furthermore, members of the Pim family have been shown to reduce the efficacy of chemotherapeutic drugs in cancer. Therefore, we attempted to evaluate the role of Pim-3 in chemoresistance of PDAC cells. We were able to confirm upregulation of the Pim-3 oncogene in PDAC tissues and cell lines vs. normal samples. Biological consequences of inhibiting Pim-3 expression with shRNA-mediated suppression included decreases in anchorage-dependent growth, invasion through Matrigel and chemoresistance to gemcitabine as measured by caspase-3 activity. Additionally, we were able to demonstrate that Pim-1 and Pim-3 play overlapping but non-identical roles as it relates to gemcitabine sensitivity of pancreatic cancer cells. To further support the role of Pim-3 suppression in sensitizing PDAC cells to gemcitabine, we used the pharmacological Pim kinase inhibitor SGI-1776. Treatment of PDAC cells with SGI-1776 resulted in decreased phosphorylation of the proapoptotic protein Bad and cell cycle changes. When SGI-1776 was combined with gemcitabine, there was a greater decrease in cell viability in the PDAC cells vs. cells treated with either of the drugs separately. These results suggest combining drug therapies that inhibit Pim kinases, such as Pim-3, with chemotherapeutic agents, to aid in decreasing chemoresistance in pancreatic cancer. PMID:23760491

Synthesis and in vitro antibacterial activity of oxazolidine LBM-415 analogs as peptide deformylase inhibitors.

PubMed

Yu, Linliang; Zhou, Weicheng; Wang, Zhenyu

2011-03-01

The drug resistant bacteria pose a severe threat to human health. The increasing resistance of those pathogens to traditional antibacterial therapy renders the identification of new antibacterial agents with novel antibacterial mechanisms an urgent need. In this study, a series of (2S)-N-substituted-1-[(formyhydroxyamino)methyl]-1-oxohexyl]-2-oxazolidinecarboxamides were designed, synthesized and evaluated for in vitro antibacterial activity. Most of these compounds displayed good activities against Gram-positive organisms comparable to reference agent LBM-415. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

Green Synthesis of Oxovanadium(IV)/chitosan Nanocomposites and Its Ameliorative Effect on Hyperglycemia, Insulin Resistance, and Oxidative Stress.

PubMed

Liu, Yanjun; Jie, Xu; Guo, Yongli; Zhang, Xin; Wang, Jingfeng; Xue, Changhu

2016-02-01

In this paper, the preparation, characterization, and ameliorative effect on high-fat high-sucrose diet-induced hyperglycemia, insulin resistance, oxidative stress in mice of novel oxovanadium(IV)/chitosan (OV/CS) nanocomposites were investigated. The nanobiocomposite was produced by chemical reduction by chitosan and L-ascorbic acid using microwave heating, under environment-friendly conditions, using aqueous solutions, and notably, by using both mediators as reducing and stabilizing agents. In addition, OV/CS nanocomposites were characterized by transmission electron microscopy, energy dispersive spectroscopy, particle size, and zeta potential measurements. In vivo experiments were designed to examine whether the OV/CS nanocomposites would provide additional benefits on oxidative stress, hyperglycemia, and insulin resistance in mice with type 2 diabetes. The results rendered insulin resistant by treating with OV/CS nanocomposites alleviate insulin resistance and improve oxidative stress. Such nanocomposite seem to be a valuable therapy to achieve and/or maintain glycemic control and therapeutic agents in the treatment arsenal for insulin resistance and type 2 diabetes.

Accumulation of MxB/Mx2-resistant HIV-1 Capsid Variants During Expansion of the HIV-1 Epidemic in Human Populations.

PubMed

Wei, Wei; Guo, Haoran; Ma, Min; Markham, Richard; Yu, Xiao-Fang

2016-06-01

Recent studies have identified human myxovirus resistance protein 2 (MxB or Mx2) as an interferon induced inhibitor of HIV-1 replication. However, whether HIV-1 can overcome MxB restriction without compromise of viral fitness has been undefined. Here, we have discovered that naturally occurring capsid (CA) variants can render HIV-1 resistant to the activity of MxB without losing viral infectivity or the ability to escape from interferon induction. Moreover, these MxB resistant HIV-1 variants do not lose MxB recognition. Surprisingly, MxB resistant CA variants are most commonly found in the Clade C HIV-1 that is the most rapidly expanding Clade throughout the world. Accumulation of MxB resistant mutations is also observed during HIV-1 spreading in human populations. These findings support a potential role for MxB as a selective force during HIV-1 transmission and evolution. Copyright © 2016. Published by Elsevier B.V.

Neuropil threads occur in dendrites of tangle-bearing nerve cells.

PubMed

Braak, H; Braak, E

1988-01-01

Transparent Golgi preparations counterstained for Alzheimer's neurofibrillary changes rendered possible the demonstration of neuropil threads in defined cellular processes. Only dendrites of tangle-bearing cortical nerve cells were found to contain neuropil threads. Processes of glial cells as well as axons present in the material were devoid of neuropil threads.

Effects of prolonged chlorine exposures upon PCR detection of Helicobacter pylori DNA.

EPA Science Inventory

The effect of low doses of free chlorine on the detection by qPCR of Helicobacter pylori (H. pylori) cells by qPCR in tap water was monitored. H. pylori target sequences (within suspended, intact cells at densities of 102 to 103 cells /ml) were rendered undetectable by qPCR an...

Embedded top-coat for reducing the effect out of band radiation in EUV lithography

NASA Astrophysics Data System (ADS)

Du, Ke; Siauw, Meiliana; Valade, David; Jasieniak, Marek; Voelcker, Nico; Trefonas, Peter; Thackeray, Jim; Blakey, Idriss; Whittaker, Andrew

2017-03-01

Out of band (OOB) radiation from the EUV source has significant implications for the performance of EUVL photoresists. Here we introduce a surface-active polymer additive, capable of partitioning to the top of the resist film during casting and annealing, to protect the underlying photoresist from OOB radiation. Copolymers were prepared using reversible addition-fragmentation chain transfer (RAFT) polymerization, and rendered surface active by chain extension with a block of fluoro-monomer. Films were prepared from the EUV resist with added surface-active Embedded Barrier Layer (EBL), and characterized using measurements of contact angles and spectroscopic ellipsometry. Finally, the lithographic performance of the resist containing the EBL was evaluated using Electron Beam Lithography exposure

Eosinophil Activities Modulate the Immune/Inflammatory Character of Allergic Respiratory Responses in Mice

PubMed Central

Jacobsen, Elizabeth A.; LeSuer, William E.; Willetts, Lian; Zellner, Katie R.; Mazzolini, Kirea; Antonios, Nathalie; Beck, Brandon; Protheroe, Cheryl; Ochkur, Sergei I.; Colbert, Dana; Lacy, Paige; Moqbel, Redwan; Appleton, Judith; Lee, Nancy A.; Lee, James J.

2014-01-01

Background The importance and specific role(s) of eosinophils in modulating the immune/inflammatory phenotype of allergic pulmonary disease remain to be defined. Established animals models assessing the role(s) of eosinophils as contributors and/or causative agents of disease have relied on congenitally deficient mice where the developmental consequences of eosinophil depletion are unknown. Methods We developed a novel conditional eosinophil-deficient strain of mice (iPHIL) through a gene knock-in strategy inserting the human diphtheria toxin (DT) receptor (DTR) into the endogenous eosinophil peroxidase genomic locus. Results Expression of DTR rendered resistant mouse eosinophil progenitors sensitive to DT without affecting any other cell types. The presence of eosinophils was shown to be unnecessary during the sensitization phase of either ovalbumin (OVA) or house dust mite (HDM) acute asthma models. However, eosinophil ablation during airway challenge led to a predominantly neutrophilic phenotype (>15% neutrophils) accompanied by allergen-induced histopathologies and airway hyperresponsiveness in response to methacholine indistinguishable from eosinophilic wild type mice. Moreover, the iPHIL neutrophilic airway phenotype was shown to be a steroid-resistant allergic respiratory variant that was reversible upon restoration of peripheral eosinophils. Conclusions Eosinophil contributions to allergic immune/inflammatory responses appear to be limited to the airway challenge and not the sensitization phase of allergen provocation models. The reversible steroid-resistant character of the iPHIL neutrophilic airway variant suggests underappreciated mechanisms by which eosinophils shape the character of allergic respiratory responses. PMID:24266710

Differential Metabolic Rearrangements after Cold Storage Are Correlated with Chilling Injury Resistance of Peach Fruits

PubMed Central

Bustamante, Claudia A.; Monti, Laura L.; Gabilondo, Julieta; Scossa, Federico; Valentini, Gabriel; Budde, Claudio O.; Lara, María V.; Fernie, Alisdair R.; Drincovich, María F.

2016-01-01

Reconfiguration of the metabolome is a key component involved in the acclimation to cold in plants; however, few studies have been devoted to the analysis of the overall metabolite changes after cold storage of fruits prior to consumption. Here, metabolite profiling of six peach varieties with differential susceptibility to develop mealiness, a chilling-injury (CI) symptom, was performed. According to metabolic content at harvest; after cold treatment; and after ripening, either following cold treatment or not; peach fruits clustered in distinct groups, depending on harvest-time, cold treatment, and ripening state. Both common and distinct metabolic responses among the six varieties were found; common changes including dramatic galactinol and raffinose rise; GABA, Asp, and Phe increase; and 2-oxo-glutarate and succinate decrease. Raffinose content after long cold treatment quantitatively correlated to the degree of mealiness resistance of the different peach varieties; and thus, raffinose emerges as a candidate biomarker of this CI disorder. Xylose increase after cold treatment was found only in the susceptible genotypes, indicating a particular cell wall reconfiguration of these varieties while being cold-stored. Overall, results indicate that peach fruit differential metabolic rearrangements due to cold treatment, rather than differential metabolic priming before cold, are better related with CI resistance. The plasticity of peach fruit metabolism renders it possible to induce a diverse metabolite array after cold, which is successful, in some genotypes, to avoid CI. PMID:27746802

Ovarian tumor-initiating cells display a flexible metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, Angela S.; Roberts, Paul C.; Frisard, Madlyn I.

2014-10-15

An altered metabolism during ovarian cancer progression allows for increased macromolecular synthesis and unrestrained growth. However, the metabolic phenotype of cancer stem or tumor-initiating cells, small tumor cell populations that are able to recapitulate the original tumor, has not been well characterized. In the present study, we compared the metabolic phenotype of the stem cell enriched cell variant, MOSE-L{sub FFLv} (TIC), derived from mouse ovarian surface epithelial (MOSE) cells, to their parental (MOSE-L) and benign precursor (MOSE-E) cells. TICs exhibit a decrease in glucose and fatty acid oxidation with a concomitant increase in lactate secretion. In contrast to MOSE-L cells,more » TICs can increase their rate of glycolysis to overcome the inhibition of ATP synthase by oligomycin and can increase their oxygen consumption rate to maintain proton motive force when uncoupled, similar to the benign MOSE-E cells. TICs have an increased survival rate under limiting conditions as well as an increased survival rate when treated with AICAR, but exhibit a higher sensitivity to metformin than MOSE-E and MOSE-L cells. Together, our data show that TICs have a distinct metabolic profile that may render them flexible to adapt to the specific conditions of their microenvironment. By better understanding their metabolic phenotype and external environmental conditions that support their survival, treatment interventions can be designed to extend current therapy regimens to eradicate TICs. - Highlights: • Ovarian cancer TICs exhibit a decreased glucose and fatty acid oxidation. • TICs are more glycolytic and have highly active mitochondria. • TICs are more resistant to AICAR but not metformin. • A flexible metabolism allows TICs to adapt to their microenvironment. • This flexibility requires development of specific drugs targeting TIC-specific changes to prevent recurrent TIC outgrowth.« less

Inhibition of Mycobacterial Infection by the Tumor Suppressor PTEN*

PubMed Central

Huang, Guochang; Redelman-Sidi, Gil; Rosen, Neal; Glickman, Michael S.; Jiang, Xuejun

2012-01-01

The tumor suppressor PTEN is a lipid phosphatase that is frequently mutated in various human cancers. PTEN suppresses tumor cell proliferation, survival, and growth mainly by inhibiting the PI3K-Akt signaling pathway through dephosphorylation of phosphatidylinositol 3,4,5-triphosphate. In addition to it role in tumor suppression, the PTEN-PI3K pathway controls many cellular functions, some of which may be important for cellular resistance to infection. Currently, the intersection between tumorigenic signaling pathways and cellular susceptibility to infection is not well defined. In this study we report that PTEN signaling regulates infection of both noncancerous and cancerous cells by multiple intracellular mycobacterial pathogens and that pharmacological modulation of PTEN signaling can affect mycobacterial infection. We found that PTEN deficiency renders multiple types of cells hyper-susceptible to infection by Mycoplasma and Mycobacterium bovis Bacillus Calmette-Guérin (BCG). The lipid phosphatase activity of PTEN is required for attenuating infection. Furthermore, we found mycobacterial infection activates host cell Akt phosphorylation, and pharmacological inhibition of Akt or PI3K activity reduced levels of intracellular infection. Intriguingly, inhibition of mTOR, one of the downstream components of the Akt signaling and a promising cancer therapeutic target, also lowered intracellular Bacillus Calmette-Guérin levels in mammary epithelial cancer MCF-7 cells. These findings demonstrate a critical role of PTEN-regulated pathways in pathogen infection. The relationship of PTEN-PI3K-Akt mTOR status and susceptibility to mycobacterial infection suggests that the interaction of mycobacterial pathogens with cancer cells may be influenced by genetic alterations in the tumor cells. PMID:22613768

Promyelocytic extracellular chromatin exacerbates coagulation and fibrinolysis in acute promyelocytic leukemia

PubMed Central

Cao, Muhua; Li, Tao; He, Zhangxiu; Wang, Lixiu; Yang, Xiaoyan; Kou, Yan; Zou, Lili; Dong, Xue; Novakovic, Valerie A.; Bi, Yayan; Kou, Junjie; Yu, Bo; Fang, Shaohong; Wang, Jinghua; Zhou, Jin

2017-01-01

Despite routine treatment of unselected acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA), early death because of hemorrhage remains unacceptably common, and the mechanism underlying this complication remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin. However, the role of promyelocytic extracellular chromatin in APL-associated coagulation remains unclear. Our objectives were to identify the novel role of ATRA-promoted extracellular chromatin in inducing a hypercoagulable and hyperfibrinolytic state in APL and to evaluate its interaction with fibrin and endothelial cells (ECs). Results from a series of coagulation assays have shown that promyelocytic extracellular chromatin increases thrombin and plasmin generation, causes a shortening of plasma clotting time of APL cells, and increases fibrin formation. DNase I but not anti-tissue factor antibody could inhibit these effects. Immunofluorescence staining showed that promyelocytic extracellular chromatin and phosphatidylserine on APL cells provide platforms for fibrin deposition and render clots more resistant to fibrinolysis. Additionally, coincubation assays revealed that promyelocytic extracellular chromatin is cytotoxic to ECs, converting them to a procoagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C by 31%. Our current results thus delineate the pathogenic role of promyelocytic extracellular chromatin in APL coagulopathy. Furthermore, the remaining coagulation disturbance in high-risk APL patients after ATRA administration may be treatable by intrinsic pathway inhibition via accelerating extracellular chromatin degradation. PMID:28053193

75 FR 18952 - Graco Children's Products Inc., Receipt of Petition for Decision of Inconsequential Noncompliance

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-13

... other fabric, including other warning labels for the MyRide seat, are flame resistant. The small size of... of all other fabric and label components of the MyRide seat, render the Label's noncompliance... yards of fabric that is used to make the pad and the ``soft goods'' for the MyRide seat. Proportionally...

Three-dimensional confocal microscopy of the living cornea and ocular lens

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1991-07-01

The three-dimensional reconstruction of the optic zone of the cornea and the ocular crystalline lens has been accomplished using confocal microscopy and volume rendering computer techniques. A laser scanning confocal microscope was used in the reflected light mode to obtain the two-dimensional images from the cornea and the ocular lens of a freshly enucleated rabbit eye. The light source was an argon ion laser with a 488 nm wavelength. The microscope objective was a Leitz X25, NA 0.6 water immersion lens. The 400 micron thick cornea was optically sectioned into 133 three micron sections. The semi-transparent cornea and the in-situ ocular lens was visualized as high resolution, high contrast two-dimensional images. The structures observed in the cornea include: superficial epithelial cells and their nuclei, basal epithelial cells and their 'beaded' cell borders, basal lamina, nerve plexus, nerve fibers, nuclei of stromal keratocytes, and endothelial cells. The structures observed in the in- situ ocular lens include: lens capsule, lens epithelial cells, and individual lens fibers. The three-dimensional data sets of the cornea and the ocular lens were reconstructed in the computer using volume rendering techniques. Stereo pairs were also created of the two- dimensional ocular images for visualization. The stack of two-dimensional images was reconstructed into a three-dimensional object using volume rendering techniques. This demonstration of the three-dimensional visualization of the intact, enucleated eye provides an important step toward quantitative three-dimensional morphometry of the eye. The important aspects of three-dimensional reconstruction are discussed.

Mitochondrial Superoxide Dismutase and Yap1p Act as a Signaling Module Contributing to Ethanol Tolerance of the Yeast Saccharomyces cerevisiae.

PubMed

Zyrina, Anna N; Smirnova, Ekaterina A; Markova, Olga V; Severin, Fedor F; Knorre, Dmitry A

2017-02-01

There are two superoxide dismutases in the yeast Saccharomyces cerevisiae-cytoplasmic and mitochondrial enzymes. Inactivation of the cytoplasmic enzyme, Sod1p, renders the cells sensitive to a variety of stresses, while inactivation of the mitochondrial isoform, Sod2p, typically has a weaker effect. One exception is ethanol-induced stress. Here we studied the role of Sod2p in ethanol tolerance of yeast. First, we found that repression of SOD2 prevents ethanol-induced relocalization of yeast hydrogen peroxide-sensing transcription factor Yap1p, one of the key stress resistance proteins. In agreement with this, the levels of Trx2p and Gsh1p, proteins encoded by Yap1 target genes, were decreased in the absence of Sod2p. Analysis of the ethanol sensitivities of the cells lacking Sod2p, Yap1p, or both indicated that the two proteins act in the same pathway. Moreover, preconditioning with hydrogen peroxide restored the ethanol resistance of yeast cells with repressed SOD2 Interestingly, we found that mitochondrion-to-nucleus signaling by Rtg proteins antagonizes Yap1p activation. Together, our data suggest that hydrogen peroxide produced by Sod2p activates Yap1p and thus plays a signaling role in ethanol tolerance. Baker's yeast harbors multiple systems that ensure tolerance to high concentrations of ethanol. Still, the role of mitochondria under severe ethanol stress in yeast is not completely clear. Our study revealed a signaling function of mitochondria which contributes significantly to the ethanol tolerance of yeast cells. We found that mitochondrial superoxide dismutase Sod2p and cytoplasmic hydrogen peroxide sensor Yap1p act together as a module of the mitochondrion-to-nucleus signaling pathway. We also report cross talk between this pathway and the conventional retrograde signaling cascade activated by dysfunctional mitochondria. Copyright © 2017 American Society for Microbiology.

Mitochondrial Superoxide Dismutase and Yap1p Act as a Signaling Module Contributing to Ethanol Tolerance of the Yeast Saccharomyces cerevisiae

PubMed Central

Zyrina, Anna N.; Smirnova, Ekaterina A.; Markova, Olga V.; Severin, Fedor F.

2016-01-01

ABSTRACT There are two superoxide dismutases in the yeast Saccharomyces cerevisiae—cytoplasmic and mitochondrial enzymes. Inactivation of the cytoplasmic enzyme, Sod1p, renders the cells sensitive to a variety of stresses, while inactivation of the mitochondrial isoform, Sod2p, typically has a weaker effect. One exception is ethanol-induced stress. Here we studied the role of Sod2p in ethanol tolerance of yeast. First, we found that repression of SOD2 prevents ethanol-induced relocalization of yeast hydrogen peroxide-sensing transcription factor Yap1p, one of the key stress resistance proteins. In agreement with this, the levels of Trx2p and Gsh1p, proteins encoded by Yap1 target genes, were decreased in the absence of Sod2p. Analysis of the ethanol sensitivities of the cells lacking Sod2p, Yap1p, or both indicated that the two proteins act in the same pathway. Moreover, preconditioning with hydrogen peroxide restored the ethanol resistance of yeast cells with repressed SOD2. Interestingly, we found that mitochondrion-to-nucleus signaling by Rtg proteins antagonizes Yap1p activation. Together, our data suggest that hydrogen peroxide produced by Sod2p activates Yap1p and thus plays a signaling role in ethanol tolerance. IMPORTANCE Baker's yeast harbors multiple systems that ensure tolerance to high concentrations of ethanol. Still, the role of mitochondria under severe ethanol stress in yeast is not completely clear. Our study revealed a signaling function of mitochondria which contributes significantly to the ethanol tolerance of yeast cells. We found that mitochondrial superoxide dismutase Sod2p and cytoplasmic hydrogen peroxide sensor Yap1p act together as a module of the mitochondrion-to-nucleus signaling pathway. We also report cross talk between this pathway and the conventional retrograde signaling cascade activated by dysfunctional mitochondria. PMID:27864171

Ras-Related Small GTPases RalA and RalB Regulate Cellular Survival After Ionizing Radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kidd, Ambrose R.; Snider, Jared L.; Martin, Timothy D.

2010-09-01

Purpose: Oncogenic activation of Ras renders cancer cells resistant to ionizing radiation (IR), but the mechanisms have not been fully characterized. The Ras-like small GTPases RalA and RalB are downstream effectors of Ras function and are critical for both tumor growth and survival. The Ral effector RalBP1/RLIP76 mediates survival of mice after whole-body irradiation, but the role of the Ral GTPases themselves in response to IR is unknown. We have investigated the role of RalA and RalB in cellular responses to IR. Methods and Materials: RalA, RalB, and their major effectors RalBP1 and Sec5 were knocked down by stable expressionmore » of short hairpin RNAs in the K-Ras-dependent pancreatic cancer-derived cell line MIA PaCa-2. Radiation responses were measured by standard clonogenic survival assays for reproductive survival, {gamma}H2AX expression for double-strand DNA breaks (DSBs), and poly(ADP-ribose)polymerase (PARP) cleavage for apoptosis. Results: Knockdown of K-Ras, RalA, or RalB reduced colony-forming ability post-IR, and knockdown of either Ral isoform decreased the rate of DSB repair post-IR. However, knockdown of RalB, but not RalA, increased cell death. Surprisingly, neither RalBP1 nor Sec5 suppression affected colony formation post-IR. Conclusions: Both RalA and RalB contribute to K-Ras-dependent IR resistance of MIA PaCa-2 cells. Sensitization due to suppressed Ral expression is likely due in part to decreased efficiency of DNA repair (RalA and RalB) and increased susceptibility to apoptosis (RalB). Ral-mediated radioresistance does not depend on either the RalBP1 or the exocyst complex, the two best-characterized Ral effectors, and instead may utilize an atypical or novel effector.« less

Resistance to excitotoxin-induced seizures and neuronal death in mice lacking the preprotachykinin A gene.

PubMed

Liu, H; Cao, Y; Basbaum, A I; Mazarati, A M; Sankar, R; Wasterlain, C G

1999-10-12

Epileptic seizures are associated with increases in hippocampal excitability, but the mechanisms that render the hippocampus hyperexcitable chronically (in epilepsy) or acutely (in status epilepticus) are poorly understood. Recent evidence suggests that substance P (SP), a peptide that has been implicated in cardiovascular function, inflammatory responses, and nociception, also contributes to hippocampal excitability and status epilepticus, in part by enhancing glutamate release. Here we report that mice with disruption of the preprotachykinin A gene, which encodes SP and neurokinin A, are resistant to kainate excitoxicity. The mice show a reduction in the duration and severity of seizures induced by kainate or pentylenetetrazole, and both necrosis and apoptosis of hippocampal neurons are prevented. Although kainate induced the expression of bax and caspase 3 in the hippocampus of wild-type mice, these critical intracellular mediators of cell death pathways were not altered by kainate injection in the mutant mice. These results indicate that the reduction of seizure activity and the neuroprotection observed in preprotachykinin A null mice are caused by the extinction of a SP/neurokinin A-mediated signaling pathway that is activated by seizures. They suggest that these neurokinins are critical to the control of hippocampal excitability, hippocampal seizures, and hippocampal vulnerability.

Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection

PubMed Central

Bohn, Yu-Sing Tammy; Brandes, Gudrun; Rakhimova, Elza; Horatzek, Sonja; Salunkhe, Prabhakar; Munder, Antje; van Barneveld, Andrea; Jordan, Doris; Bredenbruch, Florian; Häußler, Susanne; Riedel, Kathrin; Eberl, Leo; Jensen, Peter Østrup; Bjarnsholt, Thomas; Moser, Claus; Hoiby, Niels; Tümmler, Burkhard; Wiehlmann, Lutz

2008-01-01

Polymorphonuclear neutrophils are the most important mammalian host defence cells against infections with Pseudomonas aeruginosa. Screening of a signature tagged mutagenesis library of the non-piliated P. aeruginosa strain TBCF10839 uncovered that transposon inactivation of its pilY1 gene rendered the bacterium more resistant against killing by neutrophils than the wild type and any other of the more than 3000 tested mutants. Inactivation of pilY1 led to the loss of twitching motility in twitching-proficient wild-type PA14 and PAO1 strains, predisposed to autolysis and impaired the secretion of quinolones and pyocyanin, but on the other hand promoted growth in stationary phase and bacterial survival in murine airway infection models. The PilY1 population consisted of a major full-length and a minor shorter PilY1* isoform. PilY1* was detectable in small extracellular quinolone-positive aggregates, but not in the pilus. P. aeruginosa PilY1 is not an adhesin on the pilus tip, but assists in pilus biogenesis, twitching motility, secretion of secondary metabolites and in the control of cell density in the bacterial population. PMID:19054330

INDUCTION OF IMMUNOLOGIC TOLERANCE IN OLDER NEW ZEALAND MICE REPOPULATED WITH YOUNG SPLEEN, BONE MARROW, OR THYMUS

PubMed Central

Staples, Parker J.; Steinberg, Alfred D.; Talal, Norman

1970-01-01

Newborn, 7–9 day, and 16–18 day old NZB and B/W mice were, unlike older New Zealand mice, rendered tolerant to single doses of 8–10 mg of soluble BGG. After challenge, this tolerance was of short duration and escape occurred rapidly. Age-matched and similarly treated C3H, Balb/c and C57Bl mice did not escape from tolerance. Partial tolerance could be maintained by repeated injections of BGG. Biofiltration ruled out hyperphagocytosis as an explanation for this resistance to tolerance. Tolerance could be induced in older B/W mice if they were thymectomized, irradiated, and repopulated with young (12–15 day), but not old (2–3 month), spleen or bone marrow cells. Old bone marrow cells gave a non-tolerant response even when combined with young thymic grafts. Young bone marrow gave a tolerant response which was followed by the expected rapid escape only if a young thymus graft was also present. Escape was retarded if old thymus, or old irradiated thymus, was combined with young bone marrow. These results are best explained by abnormalities of both lymphoid precursors and thymic regulation. PMID:4192570

Highly dynamic biodegradable micelles capable of lysing Gram-positive and Gram-negative bacterial membrane.

PubMed

Qiao, Yuan; Yang, Chuan; Coady, Daniel J; Ong, Zhan Yuin; Hedrick, James L; Yang, Yi-Yan

2012-02-01

The development of biodegradable antimicrobial polymers adds to the toolbox of attractive antimicrobial agents against antibiotic-resistant microbes. To this end, the potential of polycarbonate polymers as such materials were explored. A series of random polycarbonate polymers consisting of monomers MTC-OEt and MTC-CH(2)CH(3)Cl were designed and synthesized using metal-free organocatalytic ring-opening polymerization. Random polycarbonate polymers self-assembled in solution but appeared highly dynamic; such behaviors are desirable as ready disassembly of polymers at the microbial membrane facilitates membrane disruption. Their activities against clinically relevant Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (E.coli and Pseudomonas aeruginosa) revealed that the hydrophobic-hydrophilic composition balance in polymers are important to render antimicrobial potency. Scanning electron microscopy (SEM) studies indicated microbial cell surface damage after treatment with polymers, and confocal microscopy studies also showed entry of FITC-dextran dye in Escherichia coli as a result of membrane disruption. On the other hand, the polymers exhibited minimal toxicity against red blood cells in hemolysis tests. Therefore, these random polycarbonate polymers are promising antimicrobial agents against both Gram-positive and Gram-negative bacteria for various biomedical applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

Acidosis Promotes Bcl-2 Family-mediated Evasion of Apoptosis

PubMed Central

Ryder, Christopher; McColl, Karen; Zhong, Fei; Distelhorst, Clark W.

2012-01-01

Acidosis arises in solid and lymphoid malignancies secondary to altered nutrient supply and utilization. Tumor acidosis correlates with therapeutic resistance, although the mechanism behind this effect is not fully understood. Here we show that incubation of lymphoma cell lines in acidic conditions (pH 6.5) blocks apoptosis induced by multiple cytotoxic metabolic stresses, including deprivation of glucose or glutamine and treatment with dexamethasone. We sought to examine the role of the Bcl-2 family of apoptosis regulators in this process. Interestingly, we found that acidic culture causes elevation of both Bcl-2 and Bcl-xL, while also attenuating glutamine starvation-induced elevation of p53-up-regulated modulator of apoptosis (PUMA) and Bim. We confirmed with knockdown studies that these shifts direct survival decisions during starvation and acidosis. Importantly, the promotion of a high anti- to pro-apoptotic Bcl-2 family member ratio by acidosis renders cells exquisitely sensitive to the Bcl-2/Bcl-xL antagonist ABT-737, suggesting that acidosis causes Bcl-2 family dependence. This dependence appears to be mediated, in part, by the acid-sensing G protein-coupled receptor, GPR65, via a MEK/ERK pathway. PMID:22685289

Antimicrobial activities of amphiphilic peptides covalently bonded to a water-insoluble resin.

PubMed Central

Haynie, S L; Crum, G A; Doele, B A

1995-01-01

A series of polymer-bound antimicrobial peptides was prepared, and the peptides were tested for their antimicrobial activities. The immobilized peptides were prepared by a strategy that used solid-phase peptide synthesis that linked the carboxy-terminal amino acid with an ethylenediamine-modified polyamide resin (PepsynK). The acid-stable, permanent amide bond between the support and the nascent peptide renders the peptide resistant to cleavage from the support during the final acid-catalyzed deprotection step in the synthesis. Select immobilized peptides containing amino acid sequences that ranged from the naturally occurring magainin to simpler synthetic sequences with idealized secondary structures were excellent antimicrobial agents against several organisms. The immobilized peptides typically reduced the number of viable cells by > or = 5 log units. We show that the reduction in cell numbers cannot be explained by the action of a soluble component. We observed no leached or hydrolyzed peptide from the resin, nor did we observe any antimicrobial activity in soluble extracts from the immobilized peptide. The immobilized peptides were washed and reused for repeated microbial contact and killing. These results suggest that the surface actions by magainins and structurally related antimicrobial peptides are sufficient for their lethal activities. PMID:7726486

Human IgG1 antibodies antagonizing activating receptor NKG2D on natural killer cells

PubMed Central

Steigerwald, Jutta; Raum, Tobias; Pflanz, Stefan; Cierpka, Ronny; Mangold, Susanne; Rau, Doris; Hoffmann, Patrick; Kvesic, Majk; Zube, Christina; Linnerbauer, Stefanie; Lumsden, John; Sriskandarajah, Mirnaalini; Kufer, Peter; Baeuerle, Patrick A

2009-01-01

NKG2D is a surface receptor expressed on NK cells but also on CD8+ T cells, γδ T cells, and auto-reactive CD4+/CD28− T cells of patients with rheumatoid arthritis. Various studies suggested that NKG2D plays a critical role in autoimmune diseases, e.g., in diabetes, celiac disease and rheumatoid arthritis (RA), rendering the activating receptor a potential target for antibody-based therapies. Here, we describe the generation and characteristics of a panel of human, high-affinity anti-NKG2D IgG1 monoclonal antibodies (mAbs) derived by phage display. The lead molecule mAb E4 bound with an affinity (KD) of 2.7 ± 1.4 × 10−11 M to soluble and membrane-bound human NKG2D, and cross-reacted with NKG2D from cynomolgus macaque, indicating potential suitability for studies in a relevant primate model. MAb E4 potently antagonized the cytolytic activity of NKL cells against BaF/3-MICA cells expressing NKG2D ligand, and blocked the NKG2D ligand-induced secretion of TNFα, IFNγ and GM-CSF, as well as surface expression of CRTAM by NK cells cultured on immobilized MICA or ULBP-1 ligands. The antibody did not show a detectable loss of binding to NKG2D after seven days in human serum at 37°C, and resisted thermal inactivation up to 70°C. Based on these results, anti-human NKG2D mAb E4 provides an ideal candidate for development of a novel therapeutic agent antagonizing a key receptor of NK and cytotoxic T cells with implications in autoimmune diseases. PMID:20061825

T cell-derived IL-10 determines leishmaniasis disease outcome and is suppressed by a dendritic cell based vaccine.

PubMed

Schwarz, Tobias; Remer, Katharina A; Nahrendorf, Wiebke; Masic, Anita; Siewe, Lisa; Müller, Werner; Roers, Axel; Moll, Heidrun

2013-01-01

In the murine model of Leishmania major infection, resistance or susceptibility to the parasite has been associated with the development of a Th1 or Th2 type of immune response. Recently, however, the immunosuppressive effects of IL-10 have been ascribed a crucial role in the development of the different clinical correlates of Leishmania infection in humans. Since T cells and professional APC are important cellular sources of IL-10, we compared leishmaniasis disease progression in T cell-specific, macrophage/neutrophil-specific and complete IL-10-deficient C57BL/6 as well as T cell-specific and complete IL-10-deficient BALB/c mice. As early as two weeks after infection of these mice with L. major, T cell-specific and complete IL-10-deficient animals showed significantly increased lesion development accompanied by a markedly elevated secretion of IFN-γ or IFN-γ and IL-4 in the lymph nodes draining the lesions of the C57BL/6 or BALB/c mutants, respectively. In contrast, macrophage/neutrophil-specific IL-10-deficient C57BL/6 mice did not show any altered phenotype. During the further course of disease, the T cell-specific as well as the complete IL-10-deficient BALB/c mice were able to control the infection. Furthermore, a dendritic cell-based vaccination against leishmaniasis efficiently suppresses the early secretion of IL-10, thus contributing to the control of parasite spread. Taken together, IL-10 secretion by T cells has an influence on immune activation early after infection and is sufficient to render BALB/c mice susceptible to an uncontrolled Leishmania major infection.

The drug efflux pump Pgp1 in pro-inflammatory lymphocytes is a target for novel treatment strategies in COPD.

PubMed

Hodge, Greg; Holmes, Mark; Jersmann, Hubertus; Reynolds, Paul N; Hodge, Sandra

2013-06-03

Pro-inflammatory/cytotoxic T cells (IFNγ, TNFα, granzyme B+) are increased in the peripheral circulation in COPD. NKT-like and NK cells are effector lymphocytes that we have also shown to be major sources of pro-inflammatory cytokines and granzymes. P-glycoprotein 1 (Pgp1) is a transmembrane efflux pump well characterised in drug resistant cancer cells. We hypothesized that Pgp1 would be increased in peripheral blood T, NKT-like and NK cells in patients with COPD, and that this would be accompanied by increased expression of IFNγ, TNFα and granzyme B. We further hypothesized that treatment with cyclosporine A, a Pgp1 inhibitor, would render cells more sensitive to treatment with corticosteroids. Pgp1, granzyme B, IFNγ and TNFα expression were measured in peripheral blood T, NK and NKT-like cells from COPD patients and control subjects (± cyclosporine A and prednisolone) following in vitro stimulation and results correlated with uptake of efflux dye Calcein-AM using flow cytometry. There was increased Pgp1 expression by peripheral blood T, NKT-like and NK cells co-expressing IFNγ, TNFα and granzyme B in COPD patients compared with controls (e.g. %IFNγ/Pgp1 T, NKT-like, NK for COPD (Control): 25(6), 54(27), 39(23)). There was an inverse correlation between Pgp1 expression and Calcein-AM uptake. Treatment with 2.5 ng/ml cylosporin A and10-6 M prednisolone resulted in synergistic inhibition of pro-inflammatory cytokines in Pgp1 + cells (p < 0.05 for all). Treatment strategies that target Pgp1 in T, NKT-like and NK cells may reduce systemic inflammatory mediators in COPD and improve patient morbidity.

Bone microenvironment-mediated resistance of cancer cells to bisphosphonates and impact on bone osteocytes/stem cells.

PubMed

Alasmari, Abeer; Lin, Shih-Chun; Dibart, Serge; Salih, Erdjan

2016-08-01

Anti-resorptive bisphosphonates (BPs) have been clinically used to prevent cancer-bone metastasis and cancer-induced bone pathologies despite the fact that the phenotypic response of the cancer-bone interactions to BP exposure is "uncharted territory". This study offers unique insights into the interplay between cancer stem cells and osteocytes/osteoblasts and mesenchymal stem cells using a three-dimensional (3D) live cancer-bone interactive model. We provide extraordinary cryptic details of the biological events that occur as a result of alendronate (ALN) treatment using 3D live cancer-bone model systems under specific bone remodeling stages. While cancer cells are susceptible to BP treatment in the absence of bone, they are totally unaffected in the presence of bone. Cancer cells colonize live bone irrespective of whether the bone is committed to bone resorption or formation and hence, cancer-bone metastasis/interactions are though to be "independent of bone remodeling stages". In our 3D live bone model systems, ALN inhibited bone resorption at the osteoclast differentiation level through effects of mineral-bound ALN on osteocytes and osteoblasts. The mineral-bound ALN rendered bone incapable of osteoblast differentiation, while cancer cells colonize the bone with striking morphological adaptations which led to a conclusion that a direct anti-cancer effect of BPs in a "live or in vivo" bone microenvironment is implausible. The above studies were complemented with mass spectrometric analysis of the media from cancer-bone organ cultures in the absence and presence of ALN. The mineral-bound ALN impacts the bone organs by limiting transformation of mesenchymal stem cells to osteoblasts and leads to diminished endosteal cell population and degenerated osteocytes within the mineralized bone matrix.

Memory Applications Using Resonant Tunneling Diodes

NASA Astrophysics Data System (ADS)

Shieh, Ming-Huei

Resonant tunneling diodes (RTDs) producing unique folding current-voltage (I-V) characteristics have attracted considerable research attention due to their promising application in signal processing and multi-valued logic. The negative differential resistance of RTDs renders the operating points self-latching and stable. We have proposed a multiple -dimensional multiple-state RTD-based static random-access memory (SRAM) cell in which the number of stable states can significantly be increased to (N + 1)^ m or more for m number of N-peak RTDs connected in series. The proposed cells take advantage of the hysteresis and folding I-V characteristics of RTD. Several cell designs are presented and evaluated. A two-dimensional nine-state memory cell has been implemented and demonstrated by a breadboard circuit using two 2-peak RTDs. The hysteresis phenomenon in a series of RTDs is also further analyzed. The switch model provided in SPICE 3 can be utilized to simulate the hysteretic I-V characteristics of RTDs. A simple macro-circuit is described to model the hysteretic I-V characteristic of RTD for circuit simulation. A new scheme for storing word-wide multiple-bit information very efficiently in a single memory cell using RTDs is proposed. An efficient and inexpensive periphery circuit to read from and write into the cell is also described. Simulation results on the design of a 3-bit memory cell scheme using one-peak RTDs are also presented. Finally, a binary transistor-less memory cell which is only composed of a pair of RTDs and an ordinary rectifier diode is presented and investigated. A simple means for reading and writing information from or into the memory cell is also discussed.

Prevalence and Characterization of Salmonella in Animal Meals Collected from Rendering Operations.

PubMed

Jiang, Xiuping

2016-06-01

As part of the Salmonella Education Reduction Program, the Animal Protein Producers Industry initiated a yearlong microbiological survey of animal meals from 1 January to 31 December 2010. The types of animal meals included poultry meal, pork and beef crax, meat meal, meat and bone meal, feather meal, blood meal, and fish meal from a variety of rendering operations (n = 65). Salmonella was positive in 731 (8.3%) of 8,783 analyzed samples, with contamination rates as 1.0, 33.2, and 21.3% from samples collected right after press, being loaded out, or unidentified, respectively. The randomly selected positive Salmonella samples (n = 100) representing 1.1% of the total samples tested were enumerated by the most-probable-number (MPN) method. The Salmonella contamination level ranged from <0.03 (below the detection limit) to 240 MPN/g with a median MPN per gram of 0.036. Among 102 Salmonella isolates from those 100 positive samples, a total of 42 Salmonella serotypes or groups were identified with Montevideo (13%), Senftenberg (11%), Mbandaka (7%), Orion (7%), Livingstone (6%), Tennessee (4%), Infantis (4%), Cerro (4%), and group C1 (4%) as the most predominant ones. Those Salmonella isolates were further analyzed for antimicrobial resistance to the 15 most common antibiotics by using the National Antimicrobial Resistance Monitoring System gram-negative plate. Most Salmonella isolates (n = 94) were sensitive to all antibiotics tested, with seven isolates resistant to one antibiotic and one resistant to seven antibiotics. Clearly, the prevalence of Salmonella in animal meals declined compared with previous surveys, and none of the Salmonella serotypes concerning target animal health were isolated. In addition, most Salmonella isolates remained susceptible to the majority of the 15 most commonly used antibiotics.

Estradiol Protects Proopiomelanocortin Neurons Against Insulin Resistance.

PubMed

Qiu, Jian; Bosch, Martha A; Meza, Cecilia; Navarro, Uyen-Vy; Nestor, Casey C; Wagner, Edward J; Rønnekleiv, Oline K; Kelly, Martin J

2018-02-01

Insulin resistance is at the core of the metabolic syndrome, and men exhibit a higher incidence of metabolic syndrome than women in early adult life, but this sex advantage diminishes sharply when women reach the postmenopausal state. Because 17β-estradiol (E2) augments the excitability of the anorexigenic proopiomelanocortin (POMC) neurons, we investigated the neuroprotective effects of E2 against insulin resistance in POMC neurons from diet-induced obese (DIO) female and male mice. The efficacy of insulin to activate canonical transient receptor potential 5 (TRPC5) channels and depolarize POMC neurons was significantly reduced in DIO male mice but not in DIO female mice. However, the insulin response in POMC neurons was abrogated in ovariectomized DIO females but restored with E2 replacement. E2 increased T-type calcium channel Cav3.1 messenger RNA (mRNA) expression and whole-cell currents but downregulated stromal-interaction molecule 1 mRNA, which rendered POMC neurons more excitable and responsive to insulin-mediated TRPC5 channel activation. Moreover, E2 prevented the increase in suppressor of cytokine signaling-3 mRNA expression with DIO as seen in DIO males. As proof of principle, insulin [intracerebroventricular injection into the third ventricle (ICV)] decreased food intake and increased metabolism in female but not male guinea pigs fed a high-fat diet. The uncoupling of the insulin receptor from its downstream effector system was corroborated by the reduced expression of phosphorylated protein kinase B in the arcuate nucleus of male but not female guinea pigs following insulin. Therefore, E2 protects female POMC neurons from insulin resistance by enhancing POMC neuronal excitability and the coupling of insulin receptor to TRPC5 channel activation. Copyright © 2018 Endocrine Society.

Functional sites of the Ada regulatory protein of Escherichia coli. Analysis by amino acid substitutions.

PubMed

Takano, K; Nakabeppu, Y; Sekiguchi, M

1988-05-20

Specific cysteine residues at possible methyl acceptor sites of the Ada protein of Escherichia coli were converted to other amino acids by site-directed mutagenesis of the cloned ada gene of E. coli. Ada protein with the cysteine residue at 321 replaced by alanine was capable of accepting the methyl group from the methylphosphotriester but not from O6-methylguanine or O4-methylthymine of alkylated DNA, whereas the protein with alanine at position 69 accepted the methyl group from the methylated bases but not from the methylphosphotriester. These two mutants were used to elucidate the biological significance of repair of the two types of alkylation lesions. Introduction of the ada gene with the Ala69 mutation into an ada- cell rendered the cell more resistant to alkylating agents with respect to both killing and induction of mutations, but the gene with the Ala321 mutation exhibited no such activity. Replacement of the cysteine residue at position 69, but not at position 321, abolished the ability of Ada protein to promote transcription of both ada and alkA genes in vitro. These results are compatible with the idea that methylation of the cysteine residue at position 69 renders Ada protein active as a transcriptional regulator, whilst the cysteine residue at position 321 is responsible for repair of pre-mutagenic and lethal lesions in DNA. The actions of mutant Ada proteins on the ada and alkA promoters in vivo were investigated using an artificially composed gene expression system. When the ada gene with the Ala69 mutation was introduced into the cell, there was little induction of expression of either the ada or the alkA genes, even after treatment with an alkylating agent, in agreement with the data obtained from studies in vitro. With the Ala321 mutation, however, a considerable degree of ada gene expression occurred without adaptive treatment. The latter finding suggests that the cysteine residue at position 321, which is located near the C terminus of the Ada protein, is involved in regulating activity, as the transcriptional activator.

Dysregulated mTORC1 renders cells critically dependent on desaturated lipids for survival under tumor-like stress

PubMed Central

Young, Regina M.; Ackerman, Daniel; Quinn, Zachary L.; Mancuso, Anthony; Gruber, Michaela; Liu, Liping; Giannoukos, Dionysios N.; Bobrovnikova-Marjon, Ekaterina; Diehl, J. Alan; Keith, Brian; Simon, M. Celeste

2013-01-01

Solid tumors exhibit heterogeneous microenvironments, often characterized by limiting concentrations of oxygen (O2), glucose, and other nutrients. How oncogenic mutations alter stress response pathways, metabolism, and cell survival in the face of these challenges is incompletely understood. Here we report that constitutive mammalian target of rapamycin complex 1 (mTORC1) activity renders hypoxic cells dependent on exogenous desaturated lipids, as levels of de novo synthesized unsaturated fatty acids are reduced under low O2. Specifically, we demonstrate that hypoxic Tsc2−/− (tuberous sclerosis complex 2−/−) cells deprived of serum lipids exhibit a magnified unfolded protein response (UPR) but fail to appropriately expand their endoplasmic reticulum (ER), leading to inositol-requiring protein-1 (IRE1)-dependent cell death that can be reversed by the addition of unsaturated lipids. UPR activation and apoptosis were also detected in Tsc2-deficient kidney tumors. Importantly, we observed this phenotype in multiple human cancer cell lines and suggest that cells committed to unregulated growth within ischemic tumor microenvironments are unable to balance lipid and protein synthesis due to a critical limitation in desaturated lipids. PMID:23699409

The microRNA miR393 re-directs secondary metabolite biosynthesis away from camalexin and towards glucosinolates.

PubMed

Robert-Seilaniantz, Alexandre; MacLean, Dan; Jikumaru, Yusuke; Hill, Lionel; Yamaguchi, Shinjiro; Kamiya, Yuji; Jones, Jonathan D G

2011-07-01

flg22 treatment increases levels of miR393, a microRNA that targets auxin receptors. Over-expression of miR393 renders plants more resistant to biotroph pathogens and more susceptible to necrotroph pathogens. In contrast, over-expression of AFB1, an auxin receptor whose mRNA is partially resistant to miR393 degradation, renders the plant more susceptible to biotroph pathogens. Here we investigate the mechanism by which auxin signalling and miR393 influence plant defence. We show that auxin signalling represses SA levels and signalling. We also show that miR393 represses auxin signalling, preventing it from antagonizing SA signalling. In addition, over-expression of miR393 increases glucosinolate levels and decreases the levels of camalexin. Further studies on pathogen interactions in auxin signalling mutants revealed that ARF1 and ARF9 negatively regulate glucosinolate accumulation, and that ARF9 positively regulates camalexin accumulation. We propose that the action of miR393 on auxin signalling triggers two complementary responses. First, it prevents suppression of SA levels by auxin. Second, it stabilizes ARF1 and ARF9 in inactive complexes. As a result, the plant is able to mount a full SA response and to re-direct metabolic flow toward the most effective anti-microbial compounds for biotroph resistance. We propose that miR393 levels can fine-tune plant defences and prioritize resources. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Activity of Norspermidine on Bacterial Biofilms of Multidrug-Resistant Clinical Isolates Associated with Persistent Extremity Wound Infections.

PubMed

Cardile, Anthony P; Woodbury, Ronald L; Sanchez, Carlos J; Becerra, Sandra C; Garcia, Rebecca A; Mende, Katrin; Wenke, Joseph C; Akers, Kevin S

2017-01-01

Biofilm formation is a major virulence factor for numerous pathogenic bacteria and is cited as a central event in the pathogenesis of chronic human infections, which is in large part due to excessive extracellular matrix secretion and metabolic changes that occur within the biofilm rendering them highly tolerant to antimicrobial treatments. Polyamines, including norspermidine, play central roles in bacterial biofilm development, but have also recently been shown to inhibit biofilm formation in select strains of various pathogenic bacteria. The aim of this study was to evaluate in vitro the biofilm dispersive and inhibitory activities of norspermidine against multidrug-resistant clinical isolates of Acinetobacter baumannii(n = 4), Klebsiella pneumoniae (n = 3), Pseudomonas aeruginosa (n = 5) and Staphylococcus aureus (n = 4) associated with chronic extremity wound infections using the semi-quantitative 96-well plate method and confocal laser microscopy. In addition to the antibiofilm activity, biocompatibility of norspermidine was also evaluated by measuring toxicity in vitro to human cell lines and whole porcine tissue explants using MTT viability assay and histological analysis. Norspermidine (5-20 mM) had variable dispersive and inhibitory activity on biofilms which was dependent on both the strain and species. Of the clinical bacterial species evaluated herein, A. baumannii isolates were the most sensitive to the effect of norspermidine, which was in part due to the inhibitory effects of norspermidine on bacterial motility and expression of genes involved in the production of homoserine lactones and quorum sensing molecules both essential for biofilm formation. Importantly, exposure of cell lines and whole tissues to norspermidine for prolonged periods of time (≥24 h) was observed to reduce viability and alter tissue histology in a time and concentration dependent manner, with 20 mM exposure having the greatest negative effects on both tissues and individual cell lines. Collectively our findings demonstrate that, similar to other polyamines, norspermidine displays both inhibitory and dispersive activities on biofilms of clinical multidrug-resistant bacterial isolates, in particular for strains of A. baumannii. Additionally our findings suggest that direct application may be considered on tissues, albeit for limited exposure times.

Dissecting and Targeting Latent Metastasis

DTIC Science & Technology

2015-09-01

distinct class of stem-like cancer cells , which primed to enter quiescence and evade innate immunity after infiltrating distant organs. LCC cells express...state and actively silencing WNT signaling, LCC cells can enter quiescence and evade innate immunity to remain latent for extended periods. These...mutation in Foxn1 renders the mice athymic, severely blunting the maturation of effector T cells but preserving innate immunity components including

Temporal variations in patterns of Escherichia coli strain diversity and antimicrobial resistance in the migrant Egyptian vulture

PubMed Central

Maherchandani, Sunil; Shringi, B. N.; Kashyap, Sudhir Kumar

2018-01-01

ABSTRACT Aims: Multiple antimicrobial resistance in Escherichia coli of wild vertebrates is a global concern with scarce assessments on the subject from developing countries that have high human-wild species interactions. We studied the ecology of E. coli in a wintering population of Egyptian Vultures in India to understand temporal changes in both E. coli strains and patterns of antimicrobial resistance. Methods and Results: We ribotyped E. coli strains and assessed antimicrobial resistance from wintering vultures at a highly synanthropic carcass dump in north-west India. Both E. coli occurence (90.32%) and resistance to multiple antimicrobials (71.43%) were very high. Clear temporal patterns were apparent. Diversity of strains changed and homogenized at the end of the Vultures’ wintering period, while the resistance pattern showed significantly difference inter-annually, as well as between arrival and departing individuals within a wintering cycle. Significance of study: The carcass dump environment altered both E. coli strains and multiple antimicrobial resistance in migratory Egyptian Vultures within a season. Long-distance migratory species could therefore disseminate resistant E. coli strains across broad geographical scales rendering regional mitigation strategies to control multiple antimicrobial resistance in bacteria ineffective. PMID:29755700

Vortex-dislodged cells from bone marrow trephine biopsy yield satisfactory results for flow cytometric immunophenotyping.

PubMed

Bommannan, K; Sachdeva, M U S; Gupta, M; Bose, P; Kumar, N; Sharma, P; Naseem, S; Ahluwalia, J; Das, R; Varma, N

2016-10-01

A good bone marrow (BM) sample is essential in evaluating many hematologic disorders. An unsuccessful BM aspiration (BMA) procedure precludes a successful flow cytometric immunophenotyping (FCI) in most hematologic malignancies. Apart from FCI, most ancillary diagnostic techniques in hematology are less informative. We describe the feasibility of FCI in vortex-dislodged cell preparation obtained from unfixed trephine biopsy (TB) specimens. In pancytopenic patients and dry tap cases, routine diagnostic BMA and TB samples were complemented by additional trephine biopsies. These supplementary cores were immediately transferred into sterile tubes filled with phosphate-buffered saline, vortexed, and centrifuged. The cell pellet obtained was used for flow cytometric immunophenotyping. Of 7955 BMAs performed in 42 months, 34 dry tap cases were eligible for the study. Vortexing rendered a cell pellet in 94% of the cases (32 of 34), and FCI rendered a rapid diagnosis in 100% of the cases (32 of 32) where cell pellets were available. We describe an efficient procedure which could be effectively utilized in resource-limited centers and reduce the frequency of repeat BMA procedures. © 2016 John Wiley & Sons Ltd.

Alteration of Escherichia coli topoisomerase IV to novobiocin resistance.

PubMed

Hardy, Christine D; Cozzarelli, Nicholas R

2003-03-01

DNA gyrase and topoisomerase IV (topo IV) are the two essential type II topoisomerases of Escherichia coli. Gyrase is responsible for maintaining negative supercoiling of the bacterial chromosome, whereas topo IV's primary role is in disentangling daughter chromosomes following DNA replication. Coumarins, such as novobiocin, are wide-spectrum antimicrobial agents that primarily interfere with DNA gyrase. In this work we designed an alteration in the ParE subunit of topo IV at a site homologous to that which confers coumarin resistance in gyrase. This parE mutation renders the encoded topo IV approximately 40-fold resistant to inhibition by novobiocin in vitro and imparts a similar resistance to inhibition of topo IV-mediated relaxation of supercoiled DNA in vivo. We conclude that topo IV is a secondary target of novobiocin and that it is very likely to be inhibited by the same mechanism as DNA gyrase.

Engineering Vaccines to Reprogram Immunity against Head and Neck Cancer.

PubMed

Tan, Y S; Sansanaphongpricha, K; Prince, M E P; Sun, D; Wolf, G T; Lei, Y L

2018-06-01

The recent Food and Drug Administration's approval of monoclonal antibodies targeting immune checkpoint receptors (ICRs) for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) offers exciting promise to improve patient outcome and reduce morbidities. A favorable response to ICR blockade relies on an extensive collection of preexisting tumor-specific T cells in the tumor microenvironment (TME). ICR blockade reinvigorates exhausted CD8 + T cells and enhances immune killing. However, resistance to ICR blockade is observed in about 85% of patients with HNSCC, therefore highlighting the importance of characterizing the mechanisms underlying HNSCC immune escape and exploring combinatorial strategies to sensitize hypoimmunogenic cold HNSCC to ICR inhibition. Cancer vaccines are designed to bypass the cold TME and directly deliver cancer antigens to antigen-presenting cells (APCs); these vaccines epitomize a priming strategy to synergize with ICR inhibitors. Cancer cells are ineffective antigen presenters, and poor APC infiltration as well as the M2-like polarization in the TME further dampens antigen uptake and processing, both of which render ineffective innate and adaptive immune detection. Cancer vaccines directly activate APC and expand the tumor-specific T-cell repertoire. In addition, cancer vaccines often contain an adjuvant, which further improves APC function, promotes epitope spreading, and augments host intrinsic antitumor immunity. Thus, the vaccine-induced immune priming generates a pool of effectors whose function can be enhanced by ICR inhibitors. In this review, we summarize the major HNSCC immune evasion strategies, the ongoing effort toward improving HNSCC vaccines, and the current challenges limiting the efficacy of cancer vaccines.

Potential usage of proteasome inhibitor bortezomib (Velcade, PS-341) in the treatment of metastatic melanoma: basic and clinical aspects

PubMed Central

Shahshahan, Mohammad A; Beckley, Maureen N; Jazirehi, Ali R

2011-01-01

Protein degradation by proteasome is essential to the regulation of important cellular functions including cell cycle progression, proliferation, differentiation and apoptosis. Abnormal proteasomal degradation of key regulatory proteins perturbs the normal dynamics of these cellular processes culminating in uncontrolled cell cycle progression and decreased apoptosis leading to the characteristic cancer cell phenotype. Proteasome inhibitors are a novel group of therapeutic agents designed to oppose the increased proteasomal degradation observed in various cancers while restoring key cellular functions such as apoptosis, cell cycle progression, and the inhibition of angiogenesis. Several proteasome inhibitors have been evaluated in pre- and clinical studies for their potential usage in clinical oncology. Bortezomib (Velcade, PS-341) is the first Food and Drug Administration-approved proteasome inhibitor for the treatment of multiple myeloma and mantle cell lymphoma. Bortezomib's ability to preferentially induce toxicity and cell death in tumor cells while rendering healthy cells unaffected makes it a powerful therapeutic agent and has extended its use in other types of malignancies. The ability of bortezomib and other proteasome inhibitors to synergize with conventional therapies in killing tumors in various in vitro and in vivo models makes this class of drugs a powerful tool in overcoming acquired and inherent resistance observed in many cancers. This is achieved through modulation of aberrant cellular survival signal transduction pathways and their downstream anti-apoptotic gene products. This review will discuss the anti-neoplastic effects of various proteasome inhibitors in a variety of cancers with a special emphasis on bortezomib, its mechanism of action and role in cancer therapy. We further discuss the potential use of bortezomib in the treatment of metastatic melanoma. PMID:22016836

Inhibition of JNK Sensitizes Hypoxic Colon Cancer Cells to DNA Damaging Agents

PubMed Central

Vasilevskaya, Irina A.; Selvakumaran, Muthu; Hierro, Lucia Cabal; Goldstein, Sara R.; Winkler, Jeffrey D.; O'Dwyer, Peter J.

2015-01-01

Purpose We showed previously that in HT29 colon cancer cells, modulation of hypoxia-induced stress signaling affects oxaliplatin cytotoxicity. To further study the significance of hypoxia-induced signaling through JNK, we set out to investigate how modulation of kinase activities influences cellular responses of hypoxic colon cancer cells to cytotoxic drugs. Experimental design In a panel of cell lines we investigated effects of pharmacological and molecular inhibition of JNK on sensitivity to oxaliplatin, SN-38 and 5-FU. Combination studies for the drugs and JNK inhibitor CC-401 were carried out in vitro and in vivo. Results Hypoxia-induced JNK activation was associated with resistance to oxaliplatin. CC-401 in combination with chemotherapy demonstrates synergism in colon cancer cell lines, though synergy is not always hypoxia-specific. A more detailed analysis focused on HT29 and SW620 (responsive), and HCT116 (non-responsive) lines. In HT29 and SW620 cells CC-401 treatment results in greater DNA damage in the sensitive cells. In vivo, potentiation of bevacizumab, oxaliplatin, and the combination by JNK inhibition was confirmed in HT29-derived mouse xenografts, where tumor growth delay was greater in the presence of CC-401. Finally, stable introduction of a dominant negative JNK1, but not JNK2, construct into HT29 cells rendered them more sensitive to oxaliplatin under hypoxia, suggesting differing input of JNK isoforms in cellular responses to chemotherapy. Conclusions These findings demonstrate that signaling through JNK is a determinant of response to therapy in colon cancer models, and support the testing of JNK inhibition to sensitize colon tumors in the clinic. PMID:26023085

Suppression of SRC Signaling Is Effective in Reducing Synergy between Glioblastoma and Stromal Cells.

PubMed

Calgani, Alessia; Vignaroli, Giulia; Zamperini, Claudio; Coniglio, Federica; Festuccia, Claudio; Di Cesare, Ernesto; Gravina, Giovanni Luca; Mattei, Claudia; Vitale, Flora; Schenone, Silvia; Botta, Maurizio; Angelucci, Adriano

2016-07-01

Glioblastoma cells efficiently interact with and infiltrate the surrounding normal tissue, rendering surgical resection and adjuvant chemo/radiotherapy ineffective. New therapeutic targets, able to interfere with glioblastoma's capacity to synergize with normal brain tissue, are currently under investigation. The compound Si306, a pyrazolo[3,4-d]pyrimidine derivative, selected for its favorable activity against SRC, was tested in vitro and in vivo on glioblastoma cell lines. In vivo, combination treatment with Si306 and radiotherapy was strongly active in reducing U-87 xenograft growth with respect to control and single treatments. The histology revealed a significant difference in the stromal compartment of tumoral tissue derived from control or radiotherapy-treated samples with respect to Si306-treated samples, showing in the latter a reduced presence of collagen and α-SMA-positive cells. This effect was paralleled in vitro by the capacity of Si306 to interfere with myofibroblastic differentiation of normal fibroblasts induced by U-87 cells. In the presence of Si306, TGF-β released by U-87 cells, mainly in hypoxia, was ineffective in upregulating α-SMA and β-PDGFR in fibroblasts. Si306 efficiently reached the brain and significantly prolonged the survival of mice orthotopically injected with U-87 cells. Drugs that target SRC could represent an effective therapeutic strategy in glioblastoma, able to block positive paracrine loop with stromal cells based on the β-PDGFR axis and the formation of a tumor-promoting microenvironment. This approach could be important in combination with conventional treatments in the effort to reduce tumor resistance to therapy. Mol Cancer Ther; 15(7); 1535-44. ©2016 AACR. ©2016 American Association for Cancer Research.

A toxin-antitoxin module as a target for antimicrobial development.

PubMed

Lioy, Virginia S; Rey, Oscar; Balsa, Dolors; Pellicer, Teresa; Alonso, Juan C

2010-01-01

The emergence and spread of pathogenic bacteria that have become resistant to multiple antibiotics through lateral gene transfer have created the need of novel antimicrobials. Toxin-antitoxin (TA) modules, which have been implicated in plasmid maintenance and stress management, are ubiquitous among plasmids from vancomycin or methicillin resistant bacteria. In the Streptococcus pyogenes pSM19035-encoded TA loci, the labile epsilon antitoxin binds to free zeta toxin and neutralizes it. When the zeta toxin is freed from the epsilon antitoxin, it induces a reversible state of growth arrest with a drastic reduction on the rate of replication, transcription and translation. However, upon prolonged zeta toxin action, the cells can no longer be rescued from their stasis state. A compound that disrupts the epsilon.zeta interaction can be considered as an attractive antimicrobial agent. Gene epsilon was fused to luc (Luc-epsilon antitoxin) and zeta to the gfp gene (zeta-GFP). Luc-epsilon or epsilon antitoxin neutralizes the toxic effect of the zeta or zeta-GFP toxin. In the absence of the antitoxin, free zeta or zeta-GFP triggers a reversible loss of cell proliferation, but the zetaK46A-GFP variant fails to block growth. Bioluminescence resonance energy transfer (BRET) assay was developed for high-throughput screening (HTS). To develop the proper controls, molecular dynamics studies were used to predict that the Asp18 and/or Glu22 residues might be relevant for epsilon.zeta interaction. Luc-epsilon efficiently transfers the excited energy to the fluorescent acceptor molecule (zeta-GFP or zetaK46A-GFP) and rendered high bioluminescence BRET signals. The exchange of Asp18 to Ala from zeta (D18A) affects Luc-epsilon.zetaD18A K46A-GFP interaction. In this study, we validate the hypothesis that it is possible to disrupt a TA module and offer a novel and unexploited targets to fight against antibiotic-resistant strains. Copyright 2009 Elsevier Inc. All rights reserved.

Prevalence and antimicrobial resistance of Listeria, Salmonella, and Yersinia species isolates in ducks and geese.

PubMed

Jamali, Hossein; Radmehr, Behrad; Ismail, Salmah

2014-04-01

The aims of this study were to determine the prevalence and antimicrobial resistance of Listeria, Salmonella, and Yersinia spp. isolated from duck and goose intestinal contents. A total of 471 samples, including 291 duck and 180 goose intestinal contents, were purchased from wet markets between November 2008 and July 2010. Listeria, Salmonella, and Yersinia spp. were isolated from 58 (12.3%), 107 (22.7%), and 80 (17%) of the samples, respectively. It was concluded that Listeria ivanovii, Salmonella Thompson, and Yersinia enterocolitica were the predominant serovars among Listeria, Salmonella, and Yersinia spp., respectively. Moreover, resistance to tetracycline was common in Listeria (48.3%) and Salmonella spp. (63.6%), whereas 51.3% of the Yersinia spp. isolates were resistant to cephalothin. Therefore, continued surveillance of the prevalence of the pathogens and also of emerging antibiotic resistance is needed to render possible the recognition of foods that may represent risks and also ensure the effective treatment of listeriosis, salmonellosis, and yersiniosis.

Metabolic QTL Analysis Links Chloroquine Resistance in Plasmodium falciparum to Impaired Hemoglobin Catabolism

PubMed Central

Olszewski, Kellen L.; Cobbold, Simon A.; Baska, Katelynn S.; Tan, Asako; Ferdig, Michael T.; Llinás, Manuel

2014-01-01

Drug resistant strains of the malaria parasite, Plasmodium falciparum, have rendered chloroquine ineffective throughout much of the world. In parts of Africa and Asia, the coordinated shift from chloroquine to other drugs has resulted in the near disappearance of chloroquine-resistant (CQR) parasites from the population. Currently, there is no molecular explanation for this phenomenon. Herein, we employ metabolic quantitative trait locus mapping (mQTL) to analyze progeny from a genetic cross between chloroquine-susceptible (CQS) and CQR parasites. We identify a family of hemoglobin-derived peptides that are elevated in CQR parasites and show that peptide accumulation, drug resistance, and reduced parasite fitness are all linked in vitro to CQR alleles of the P. falciparum chloroquine resistance transporter (pfcrt). These findings suggest that CQR parasites are less fit because mutations in pfcrt interfere with hemoglobin digestion by the parasite. Moreover, our findings may provide a molecular explanation for the reemergence of CQS parasites in wild populations. PMID:24391526

Oxidative Stress Response Tips the Balance in Aspergillus terreus Amphotericin B Resistance

PubMed Central

Blatzer, Michael; Posch, Wilfried; Steger, Marion; Binder, Ulrike; Lass-Flörl, Cornelia

2017-01-01

ABSTRACT In this study, we characterize the impact of antioxidative enzymes in amphotericin B (AmB)-resistant (ATR) and rare AmB-susceptible (ATS) clinical Aspergillus terreus isolates. We elucidate expression profiles of superoxide dismutase (SOD)- and catalase (CAT)-encoding genes, enzymatic activities of SODs, and superoxide anion production and signaling pathways involved in the oxidative stress response (OSR) in ATS and ATR strains under AmB treatment conditions. We show that ATR strains possess almost doubled basal SOD activity compared to that of ATS strains and that ATR strains exhibit an enhanced OSR, with significantly higher sod2 mRNA levels and significantly increased cat transcripts in ATR strains upon AmB treatment. In particular, inhibition of SOD and CAT proteins renders resistant isolates considerably susceptible to the drug in vitro. In conclusion, this study shows that SODs and CATs are crucial for AmB resistance in A. terreus and that targeting the OSR might offer new treatment perspectives for resistant species. PMID:28739793

Application of Nanoparticle Technologies in the Combat against Anti-Microbial Resistance

PubMed Central

Kumar, Mayur; Curtis, Anthony

2018-01-01

Anti-microbial resistance is a growing problem that has impacted the world and brought about the beginning of the end for the old generation of antibiotics. Increasingly, more antibiotics are being prescribed unnecessarily and this reckless practice has resulted in increased resistance towards these drugs, rendering them useless against infection. Nanotechnology presents a potential answer to anti-microbial resistance, which could stimulate innovation and create a new generation of antibiotic treatments for future medicines. Preserving existing antibiotic activity through novel formulation into or onto nanotechnologies can increase clinical longevity of action against infection. Additionally, the unique physiochemical properties of nanoparticles can provide new anti-bacterial modes of action which can also be explored. Simply concentrating on antibiotic prescribing habits will not resolve the issue but rather mitigate it. Thus, new scientific approaches through the development of novel antibiotics and formulations is required in order to employ a new generation of therapies to combat anti-microbial resistance. PMID:29342903

Non-volatile, solid state bistable electrical switch

NASA Technical Reports Server (NTRS)

Williams, Roger M. (Inventor)

1994-01-01

A bistable switching element is made of a material whose electrical resistance reversibly decreases in response to intercalation by positive ions. Flow of positive ions between the bistable switching element and a positive ion source is controlled by means of an electrical potential applied across a thermal switching element. The material of the thermal switching element generates heat in response to electrical current flow therethrough, which in turn causes the material to undergo a thermal phase transition from a high electrical resistance state to a low electrical resistance state as the temperature increases above a predetermined value. Application of the electrical potential in one direction renders the thermal switching element conductive to pass electron current out of the ion source. This causes positive ions to flow from the source into the bistable switching element and intercalate the same to produce a non-volatile, low resistance logic state. Application of the electrical potential in the opposite direction causes reverse current flow which de-intercalates the bistable logic switching element and produces a high resistance logic state.

Lack of formylated methionyl-tRNA has pleiotropic effects on Bacillus subtilis.

PubMed

Cai, Yanfei; Chandrangsu, Pete; Gaballa, Ahmed; Helmann, John D

2017-02-01

Bacteria initiate translation using a modified amino acid, N-formylmethionine (fMet), adapted specifically for this function. Most proteins are processed co-translationally by peptide deformylase (PDF) to remove this modification. Although PDF activity is essential in WT cells and is the target of the antibiotic actinonin, bypass mutations in the fmt gene that eliminate the formylation of Met-tRNAMet render PDF dispensable. The extent to which the emergence of fmt bypass mutations might compromise the therapeutic utility of actinonin is determined, in part, by the effects of these bypass mutations on fitness. Here, we characterize the phenotypic consequences of an fmt null mutation in the model organism Bacillus subtilis. An fmt null mutant is defective for several post-exponential phase adaptive programmes including antibiotic resistance, biofilm formation, swarming and swimming motility and sporulation. In addition, a survey of well-characterized stress responses reveals an increased sensitivity to metal ion excess and oxidative stress. These diverse phenotypes presumably reflect altered synthesis or stability of key proteins involved in these processes.

Sb7Te3/Ge multilayer films for low power and high speed phase-change memory

NASA Astrophysics Data System (ADS)

Chen, Shiyu; Wu, Weihua; Zhai, Jiwei; Song, Sannian; Song, Zhitang

2017-06-01

Phase-change memory has attracted enormous attention for its excellent properties as compared to flash memories due to their high speed, high density, better date retention and low power consumption. Here we present Sb7Te3/Ge multilayer films by using a magnetron sputtering method. The 10 years’ data retention temperature is significantly increased compared with pure Sb7Te3. When the annealing temperature is above 250 °C, the Sb7Te3/Ge multilayer thin films have better interface properties, which renders faster crystallization speed and high thermal stability. The decrease in density of ST/Ge multilayer films is only around 5%, which is very suitable for phase change materials. Moreover, the low RESET power benefits from high resistivity and better thermal stability in the PCM cells. This work demonstrates that the multilayer configuration thin films with tailored properties are beneficial for improving the stability and speed in phase change memory applications.

Microprojectile Bombardment Transformation of Date Palm Using the Insecticidal Cholesterol Oxidase (ChoA) Gene.

PubMed

Allam, Mai A; Saker, Mahmoud M

2017-01-01

The overall objective of this work is to optimize the transformation system for date palm as a first step toward production of date palm clones resistant to noxious pests. A construct harboring the cholesterol oxidase (ChoA) gene, which renders plant resistance against insect attack, is introduced into embryogenic date palm callus using the PDS-1000/He particle bombardment system. The process involves the establishment of embryogenic callus cultures as well as immature embryo-derived microcalli that are used as target tissues for shooting and optimization of transformation conditions. This chapter in addition explains molecular and histochemical assays conducted to confirm gene integration and expression.

Validating continuous digital light processing (cDLP) additive manufacturing accuracy and tissue engineering utility of a dye-initiator package.

PubMed

Wallace, Jonathan; Wang, Martha O; Thompson, Paul; Busso, Mallory; Belle, Vaijayantee; Mammoser, Nicole; Kim, Kyobum; Fisher, John P; Siblani, Ali; Xu, Yueshuo; Welter, Jean F; Lennon, Donald P; Sun, Jiayang; Caplan, Arnold I; Dean, David

2014-03-01

This study tested the accuracy of tissue engineering scaffold rendering via the continuous digital light processing (cDLP) light-based additive manufacturing technology. High accuracy (i.e., <50 µm) allows the designed performance of features relevant to three scale spaces: cell-scaffold, scaffold-tissue, and tissue-organ interactions. The biodegradable polymer poly (propylene fumarate) was used to render highly accurate scaffolds through the use of a dye-initiator package, TiO2 and bis (2,4,6-trimethylbenzoyl)phenylphosphine oxide. This dye-initiator package facilitates high accuracy in the Z dimension. Linear, round, and right-angle features were measured to gauge accuracy. Most features showed accuracies between 5.4-15% of the design. However, one feature, an 800 µm diameter circular pore, exhibited a 35.7% average reduction of patency. Light scattered in the x, y directions by the dye may have reduced this feature's accuracy. Our new fine-grained understanding of accuracy could be used to make further improvements by including corrections in the scaffold design software. Successful cell attachment occurred with both canine and human mesenchymal stem cells (MSCs). Highly accurate cDLP scaffold rendering is critical to the design of scaffolds that both guide bone regeneration and that fully resorb. Scaffold resorption must occur for regenerated bone to be remodeled and, thereby, achieve optimal strength.

Automated 3-D cell counting method for grading uveitis of rodent eye in vivo with optical coherence tomograph.

PubMed

Choi, Woo June; Pepple, Kathryn L; Wang, Ruikang K

2018-05-24

In preclinical vision research, cell grading in small animal models is essential for the quantitative evaluation of intraocular inflammation. Here, we present a new and practical optical coherence tomography (OCT) image analysis method for the automated detection and counting of aqueous cells in the anterior chamber (AC) of a rodent model of uveitis. Anterior segment OCT (AS-OCT) images are acquired with a 100kHz swept-source OCT (SS-OCT) system. The proposed method consists of two steps. In the first step, we first despeckle and binarize each OCT image. After removing AS structures in the binary image, we then apply area thresholding to isolate cell-like objects. Potential cell candidates are selected based on their best fit to roundness. The second step performs the cell counting within the whole AC, in which additional cell tracking analysis is conducted on the successive OCT images to eliminate redundancy in cell counting. Finally, 3-D cell grading using the proposed method is demonstrated in longitudinal OCT imaging of a mouse model of anterior uveitis in vivo. Rendering of anterior segment (orange) of mouse eye and automatically counted anterior chamber cells (green). Inset is a top view of the rendering, showing the cell distribution across the anterior chamber. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Cross-resistance, mode of inheritance and stability of resistance to emamectin in Spodoptera litura (Lepidoptera: Noctuidae).

PubMed

Shad, Sarfraz Ali; Sayyed, Ali H; Saleem, Mushtaq A

2010-08-01

Spodoptera litura (F.) is a cosmopolitan pest that has developed resistance to several insecticides. The aim of the present study was to establish whether an emamectin-selected (Ema-SEL) population could render cross-resistance to other insecticides, and to investigate the genetics of resistance. Bioassays at G(1) gave resistance ratios (RRs) of 80-, 2980-, 3050- and 2800-fold for emamectin, abamectin, indoxacarb and acetamiprid, respectively, compared with a laboratory susceptible population Lab-PK. After three rounds of selection, resistance to emamectin in Ema-SEL increased significantly, with RRs of 730-fold and 13-fold compared with the Lab-PK and unselected (UNSEL) population respectively. Further studies revealed that three generations were required for a tenfold increase in resistance to emamectin. Resistance to abamectin, indoxacarb, acetamiprid and emamectin in UNSEL declined significantly compared with the field population at G(1). Furthermore, selection with emamectin reduced resistance to abamectin, indoxacarb and acetamiprid on a par with UNSEL. Crosses between Ema-SEL and Lab-PK indicated autosomal and incomplete dominance of resistance. A direct test of a monogenic model and Land's method suggested that resistance to emamectin was controlled by more than one locus. Instability of resistance and lack of cross-resistance to other insecticides suggest that insecticides with different modes of action should be recommended to reduce emamectin selection pressure. Copyright (c) 2010 Society of Chemical Industry.

RNA splicing, cell signaling, and response to therapies.

PubMed

Abou Faycal, Cherine; Gazzeri, Sylvie; Eymin, Beatrice

2016-01-01

PremRNA alternative splicing is more a rule than an exception as it affects more than 90% of multiexons genes and plays a key role in proteome diversity. Here, we discuss some recent studies published in the extensively growing field linking RNA splicing and cancer. These last years, the development of high-throughput studies together with appropriate bioinformatic tools have led to the identification of new cancer-specific splicing patterns that allow to distinguish various cancer types, and provide new prognosis biomarkers. In addition, the functional consequences of hot spot mutations affecting various components of the spliceosome machinery in cancers have been described. As an example, missplicing of the enhancer of zeste homolog 2 histone methyltransferase premRNA in response to hot spot mutation of the splicing factor SRSF2 was found to participate to the pathogenesis of myelodysplastic syndrome. Moreover, proofs of principle that targeting the RNA splicing machinery can be used to correct aberrant missplicing, kill oncogene-driven cancer cells, or reverse resistance of tumor cells to targeted therapies have been done. As another example, the core spliceosomal function was recently found to be critical for the survival of Myc-driven breast cancer cells, rendering them hypersensitive to spliceosome inhibitors. Dysregulation of premRNA alternative splicing appears to be one of the hallmarks of cancer. The characterization of novel splicing signatures in cancer as well as the identification of original signaling networks involving RNA splicing regulators should allow to decipher novel oncogenic mechanisms and to develop new therapeutic strategies.

Engineering Cartilage

MedlinePlus

... Research Matters NIH Research Matters March 3, 2014 Engineering Cartilage Artistic rendering of human stem cells on ... situations has been a major goal in tissue engineering. Cartilage contains water, collagen, proteoglycans, and chondrocytes. Collagens ...

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations

PubMed Central

Saito, Kohta; Warrier, Thulasi; Somersan-Karakaya, Selin; Kaminski, Lina; Mi, Jianjie; Jiang, Xiuju; Park, Suna; Shigyo, Kristi; Gold, Ben; Roberts, Julia; Weber, Elaina; Jacobs, William R.; Nathan, Carl F.

2017-01-01

Mycobacterium tuberculosis (Mtb) encounters stresses during the pathogenesis and treatment of tuberculosis (TB) that can suppress replication of the bacteria and render them phenotypically tolerant to most available drugs. Where studied, the majority of Mtb in the sputum of most untreated subjects with active TB have been found to be nonreplicating by the criterion that they do not grow as colony-forming units (cfus) when plated on agar. However, these cells are viable because they grow when diluted in liquid media. A method for generating such “differentially detectable” (DD) Mtb in vitro would aid studies of the biology and drug susceptibility of this population, but lack of independent confirmation of reported methods has contributed to skepticism about their existence. Here, we identified confounding artifacts that, when avoided, allowed development of a reliable method of producing cultures of ≥90% DD Mtb in starved cells. We then characterized several drugs according to whether they contribute to the generation of DD Mtb or kill them. Of the agents tested, rifamycins led to DD Mtb generation, an effect lacking in a rifampin-resistant strain with a mutation in rpoB, which encodes the canonical rifampin target, the β subunit of RNA polymerase. In contrast, thioridazine did not generate DD Mtb from starved cells but killed those generated by rifampin. PMID:28559332

Effects of grain refinement on the biocorrosion and in vitro bioactivity of magnesium.

PubMed

Saha, Partha; Roy, Mangal; Datta, Moni Kanchan; Lee, Boeun; Kumta, Prashant N

2015-12-01

Magnesium is a new class of biodegradable metals potentially suitable for bone fracture fixation due to its suitable mechanical properties, high degradability and biocompatibility. However, rapid corrosion and loss in mechanical strength under physiological conditions render it unsuitable for load-bearing applications. In the present study, grain refinement was implemented to control bio-corrosion demonstrating improved in vitro bioactivity of magnesium. Pure commercial magnesium was grain refined using different amounts of zirconium (0.25 and 1.0 wt.%). Corrosion behavior was studied by potentiodynamic polarization (PDP) and mass loss immersion tests demonstrating corrosion rate decrease with grain size reduction. In vitro biocompatibility tests conducted by MC3T3-E1 pre-osteoblast cells and measured by DNA quantification demonstrate significant increase in cell proliferation for Mg-1 wt.% Zr at day 5. Similarly, alkaline phosphatase (ALP) activity was higher for grain refined Mg. Alloys were also tested for ability to support osteoclast differentiation using RAW264.7 monocytes with receptor activator of nuclear factor kappa-β ligand (RANKL) supplemented cell culture. Osteoclast differentiation process was observed to be severely restricted for smaller grained Mg. Overall, the results indicate grain refinement to be useful not only for improving corrosion resistance of Mg implants for bone fixation devices but also potentially modulate bone regeneration around the implant. Copyright © 2015 Elsevier B.V. All rights reserved.

Oxidative stress and myocardial injury in the diabetic heart

PubMed Central

Ansley, David M.; Wang, Baohua

2013-01-01

Reactive oxygen or nitrogen species play an integral role in both myocardial injury and repair. This dichotomy is differentiated at the level of species type, amount, duration of free radical generated. Homeostatic mechanisms designed to prevent free radical generation in the first instance, scavenge, or enzymatically convert them to less toxic forms and water, play crucial roles in maintenance of cellular structure and function. The outcome between functional recovery and dysfunction is dependent upon the inherent ability of these homeostatic antioxidant defenses to withstand acute free radical generation, in the order of seconds to minutes. Alternatively, pre-existent antioxidant capacity (from intracellular and extracellular sources) may regulate the degree of free radical generation. This converts reactive oxygen and nitrogen species to the role of second messenger involved in cell signalling. The adaptive capacity of the cell is altered by the balance between death or survival signal converging at the level of the mitochondria, with distinct pathophysiologic consequences that extends the period of injury from hours to days and weeks. Hyperglycemia, hyperlipidemia, and insulin resistance enhance oxidative stress in diabetic myocardium that cannot adapt to ischemia reperfusion. Altered glucose flux, mitochondrial derangements and nitric oxide synthase uncoupling in the presence of decreased antioxidant defense and impaired prosurvival cell signalling may render the diabetic myocardium more vulnerable to injury, remodelling and heart failure. PMID:23011912

Nuclear Calcium Signaling Controls Expression of a Large Gene Pool: Identification of a Gene Program for Acquired Neuroprotection Induced by Synaptic Activity

PubMed Central

Zhang, Sheng-Jia; Zou, Ming; Lu, Li; Lau, David; Ditzel, Désirée A. W.; Delucinge-Vivier, Celine; Aso, Yoshinori; Descombes, Patrick; Bading, Hilmar

2009-01-01

Synaptic activity can boost neuroprotection through a mechanism that requires synapse-to-nucleus communication and calcium signals in the cell nucleus. Here we show that in hippocampal neurons nuclear calcium is one of the most potent signals in neuronal gene expression. The induction or repression of 185 neuronal activity-regulated genes is dependent upon nuclear calcium signaling. The nuclear calcium-regulated gene pool contains a genomic program that mediates synaptic activity-induced, acquired neuroprotection. The core set of neuroprotective genes consists of 9 principal components, termed Activity-regulated Inhibitor of Death (AID) genes, and includes Atf3, Btg2, GADD45β, GADD45γ, Inhibin β-A, Interferon activated gene 202B, Npas4, Nr4a1, and Serpinb2, which strongly promote survival of cultured hippocampal neurons. Several AID genes provide neuroprotection through a common process that renders mitochondria more resistant to cellular stress and toxic insults. Stereotaxic delivery of AID gene-expressing recombinant adeno-associated viruses to the hippocampus confers protection in vivo against seizure-induced brain damage. Thus, treatments that enhance nuclear calcium signaling or supplement AID genes represent novel therapies to combat neurodegenerative conditions and neuronal cell loss caused by synaptic dysfunction, which may be accompanied by a deregulation of calcium signal initiation and/or propagation to the cell nucleus. PMID:19680447

Inactivation of the inhA-Encoded Fatty Acid Synthase II (FASII) Enoyl-Acyl Carrier Protein Reductase Induces Accumulation of the FASI End Products and Cell Lysis of Mycobacterium smegmatis

PubMed Central

Vilchèze, Catherine; Morbidoni, Hector R.; Weisbrod, Torin R.; Iwamoto, Hiroyuki; Kuo, Mack; Sacchettini, James C.; Jacobs, William R.

2000-01-01

The mechanism of action of isoniazid (INH), a first-line antituberculosis drug, is complex, as mutations in at least five different genes (katG, inhA, ahpC, kasA, and ndh) have been found to correlate with isoniazid resistance. Despite this complexity, a preponderance of evidence implicates inhA, which codes for an enoyl-acyl carrier protein reductase of the fatty acid synthase II (FASII), as the primary target of INH. However, INH treatment of Mycobacterium tuberculosis causes the accumulation of hexacosanoic acid (C26:0), a result unexpected for the blocking of an enoyl-reductase. To test whether inactivation of InhA is identical to INH treatment of mycobacteria, we isolated a temperature-sensitive mutation in the inhA gene of Mycobacterium smegmatis that rendered InhA inactive at 42°C. Thermal inactivation of InhA in M. smegmatis resulted in the inhibition of mycolic acid biosynthesis, a decrease in hexadecanoic acid (C16:0) and a concomitant increase of tetracosanoic acid (C24:0) in a manner equivalent to that seen in INH-treated cells. Similarly, INH treatment of Mycobacterium bovis BCG caused an inhibition of mycolic acid biosynthesis, a decrease in C16:0, and a concomitant accumulation of C26:0. Moreover, the InhA-inactivated cells, like INH-treated cells, underwent a drastic morphological change, leading to cell lysis. These data show that InhA inactivation, alone, is sufficient to induce the accumulation of saturated fatty acids, cell wall alterations, and cell lysis and are consistent with InhA being a primary target of INH. PMID:10869086

Frequent Nuclear/Cytoplasmic Localization of β-Catenin without Exon 3 Mutations in Malignant Melanoma

PubMed Central

Rimm, David L.; Caca, Karel; Hu, Gang; Harrison, Frank B.; Fearon, Eric R.

1999-01-01

β-Catenin has a critical role in E-cadherin-mediated cell-cell adhesion, and it also functions as a downstream signaling molecule in the wnt pathway. Mutations in the putative glycogen synthase kinase 3β phosphorylation sites near the β-catenin amino terminus have been found in some cancers and cancer cell lines. The mutations render β-catenin resistant to regulation by a complex containing the glycogen synthase kinase 3β, adenomatous polyposis coli, and axin proteins. As a result, β-catenin accumulates in the cytosol and nucleus and activates T-cell factor/lymphoid enhancing factor transcription factors. Previously, 6 of 27 melanoma cell lines were found to have β-catenin exon 3 mutations affecting the N-terminal phosphorylation sites (Rubinfeld B, Robbins P, Elgamil M, Albert I, Porfiri E, Polakis P: Stabilization of beta-catenin by genetic defects in melanoma cell lines. Science 1997, 275:1790–1792). To assess the role of β-catenin defects in primary melanomas, we undertook immunohistochemical and DNA sequencing studies in 65 melanoma specimens. Nuclear and/or cytoplasmic localization of β-catenin, a potential indicator of wnt pathway activation, was seen focally within roughly one third of the tumors, though a clonal somatic mutation in β-catenin was found in only one case (codon 45 Ser→Pro). Our findings demonstrate that β-catenin mutations are rare in primary melanoma, in contrast to the situation in melanoma cell lines. Nonetheless, activation of β-catenin, as indicated by its nuclear and/or cytoplasmic localization, appears to be frequent in melanoma, and in some cases, it may reflect focal and transient activation of the wnt pathway within the tumor. PMID:10027390

Up-regulated transcriptional repressors SnoN and Ski bind Smad proteins to antagonize transforming growth factor-beta signals during liver regeneration.

PubMed

Macias-Silva, Marina; Li, Wei; Leu, Julia I; Crissey, Mary Ann S; Taub, Rebecca

2002-08-09

Transforming growth factor-beta (TGF-beta) functions as an antiproliferative factor for hepatocytes. However, for unexplained reasons, hepatocytes become resistant to TGF-beta signals and can proliferate despite the presence of TGF-beta during liver regeneration. TGF-beta is up-regulated during liver regeneration, although it is not known whether it is active or latent. TGF-beta activity may be examined by assessing Smad activation, a downstream signaling pathway. Smad pathway activation during liver regeneration induced by partial hepatectomy or CC4 injury was examined by assessing the levels of phospho-Smad2 and Smad2-Smad4 complexes. We found that Smad proteins were slightly activated in quiescent liver, but that their activation was further enhanced in regenerating liver. Interestingly, TGF-beta/Smad pathway inhibitors (SnoN and Ski) were up-regulated during regeneration, and notably, SnoN was induced mainly in hepatocytes. SnoN and Ski are transcriptional repressors that may render some cells resistant to TGF-beta via binding Smad proteins. Complexes between SnoN, Ski, and the activated Smad proteins were detected from 2 to 120 h during the major proliferative phase in regenerating liver. Inhibitory complexes decreased after liver mass restitution (5-15 days), suggesting that persistently activated Smad proteins might participate in returning the liver to a quiescent state. Our data show that active TGF-beta/Smad signals are present during regeneration and suggest that SnoN/Ski induction might explain hepatocyte resistance to TGF-beta during the proliferative phase.

Diagnosis of Compartment Syndrome Based on Tissue Oxygenation

DTIC Science & Technology

2015-06-01

distal ends at the midpoint between the anterior and posterior ends. Hydroxyethyl starch colloid fluid, chosen because of convenient storage and same...below). It is not felt that the lower number of animals affected the findings of the study.  Hydroxyethyl starch (Hespan) was used for infusion...renders readings unreliable. It is essential that the probe not be advanced farther than the length of the catheter. If resistance is encountered during

Use of triphenyl phosphate as risk mitigant for metal amide hydrogen storage materials

DOEpatents

Cortes-Concepcion, Jose A.; Anton, Donald L.

2016-04-26

A process in a resulting product of the process in which a hydrogen storage metal amide is modified by a ball milling process using an additive of TPP. The resulting product provides for a hydrogen storage metal amide having a coating that renders the hydrogen storage metal amide resistant to air, ambient moisture, and liquid water while improving useful hydrogen storage and release kinetics.

Low susceptibility of NC/Nga mice to the lipopolysaccharide-mediated lethality with D-galactosamine sensitization and the involvement of fewer natural killer T cells.

PubMed

Koide, Naoki; Morikawa, Akiko; Odkhuu, Erdenezaya; Haque, Abedul; Badamtseren, Battuvshin; Naiki, Yoshikazu; Komatsu, Takayuki; Yoshida, Tomoaki; Yokochi, Takashi

2012-02-01

The LPS-mediated lethality of NC/Nga mice, having fewer NKT cells, was examined by using d-galactosamine (d-GalN)-sensitization. The NC/Nga mice were not killed by a simultaneous administration of d-GalN and LPS whereas all C57BL/6 (B6) control mice were killed. The injection of d-GalN and LPS failed to elevate the levels of serum alanine aminotransferase and caspase 3 in the liver tissues of NC/Nga mice. Further, the nitric oxide (NO) level of the d-GalN- and LPS-injected NC/Nga mice was much lower than those of the B6 mice. The expression of an inducible NO synthase (iNOS) was significantly reduced in the livers of NC/Nga mice. However, there was no significant difference in LPS-induced TNF-α production between B6 mice and NC/Nga mice. The NC/Nga mice had an impaired expression of IFN-γ protein and mRNA in response to d-GalN and LPS. The pretreatment with α-galactosylceramide (α-GalCer), which activates Vα14(+) NKT cells and induces the production of IFN-γ, rendered NC/Nga mice more susceptible to the LPS-mediated lethality. The livers of NC/Nga mice had fewer NKT cells compared to B6 mice. Taken together, it is suggested that the resistance of NC/Nga mice to the LPS-mediated lethality with d-GalN sensitization depended on the impaired IFN-γ production caused by fewer NKT cells and reduced NO production that followed.

Bardoxolone methyl (CDDO-Me or RTA402) induces cell cycle arrest, apoptosis and autophagy via PI3K/Akt/mTOR and p38 MAPK/Erk1/2 signaling pathways in K562 cells.

PubMed

Wang, Xin-Yu; Zhang, Xue-Hong; Peng, Li; Liu, Zheng; Yang, Yin-Xue; He, Zhi-Xu; Dang, Hong-Wan; Zhou, Shu-Feng

2017-01-01

Chronic myeloid leukemia (CML) treatment remains a challenge due to drug resistance and severe side effect, rendering the need on the development of novel therapeutics. CDDO-Me (Bardoxolone methyl), a potent Nrf2 activator and NF-κB inhibitor, is a promising candidate for cancer treatment including leukemia. However, the underlying mechanism for CDDO-Me in CML treatment is unclear. This study aimed to evaluate the molecular interactome of CDDO-Me in K562 cells using the quantitative proteomics approach stable-isotope labeling by amino acids in cell culture (SILAC) and explore the underlying mechanisms using cell-based functional assays. A total of 1,555 proteins responded to CDDO-Me exposure, including FANCI, SRPK2, XPO5, HP1BP3, NELFCD, Na + ,K + -ATPase 1, etc. in K562 cells. A total of 246 signaling pathways and 25 networks regulating cell survival and death, cellular function and maintenance, energy production, protein synthesis, response to oxidative stress, and nucleic acid metabolism were involved. Our verification experiments confirmed that CDDO-Me down-regulated Na + ,K + -ATPase α1 in K562 cells, and significantly arrested cells in G 2 /M and S phases, accompanied by remarkable alterations in the expression of key cell cycle regulators. CDDO-Me caused mitochondria-, death receptor-dependent and ER stress-mediated apoptosis in K562 cells, also induced autophagy with the suppression of PI3K/Akt/mTOR signaling pathway. p38 MAPK/Erk1/2 signaling pathways contributed to both apoptosis- and autophagy-inducing effects of CDDO-Me in K562 cells. Taken together, these data demonstrate that CDDO-Me is a potential anti-cancer agent that targets cell cycle, apoptosis, and autophagy in the treatment of CML.

Bardoxolone methyl (CDDO-Me or RTA402) induces cell cycle arrest, apoptosis and autophagy via PI3K/Akt/mTOR and p38 MAPK/Erk1/2 signaling pathways in K562 cells

PubMed Central

Wang, Xin-Yu; Zhang, Xue-Hong; Peng, Li; Liu, Zheng; Yang, Yin-Xue; He, Zhi-Xu; Dang, Hong-Wan; Zhou, Shu-Feng

2017-01-01

Chronic myeloid leukemia (CML) treatment remains a challenge due to drug resistance and severe side effect, rendering the need on the development of novel therapeutics. CDDO-Me (Bardoxolone methyl), a potent Nrf2 activator and NF-κB inhibitor, is a promising candidate for cancer treatment including leukemia. However, the underlying mechanism for CDDO-Me in CML treatment is unclear. This study aimed to evaluate the molecular interactome of CDDO-Me in K562 cells using the quantitative proteomics approach stable-isotope labeling by amino acids in cell culture (SILAC) and explore the underlying mechanisms using cell-based functional assays. A total of 1,555 proteins responded to CDDO-Me exposure, including FANCI, SRPK2, XPO5, HP1BP3, NELFCD, Na+,K+-ATPase 1, etc. in K562 cells. A total of 246 signaling pathways and 25 networks regulating cell survival and death, cellular function and maintenance, energy production, protein synthesis, response to oxidative stress, and nucleic acid metabolism were involved. Our verification experiments confirmed that CDDO-Me down-regulated Na+,K+-ATPase α1 in K562 cells, and significantly arrested cells in G2/M and S phases, accompanied by remarkable alterations in the expression of key cell cycle regulators. CDDO-Me caused mitochondria-, death receptor-dependent and ER stress-mediated apoptosis in K562 cells, also induced autophagy with the suppression of PI3K/Akt/mTOR signaling pathway. p38 MAPK/Erk1/2 signaling pathways contributed to both apoptosis- and autophagy-inducing effects of CDDO-Me in K562 cells. Taken together, these data demonstrate that CDDO-Me is a potential anti-cancer agent that targets cell cycle, apoptosis, and autophagy in the treatment of CML. PMID:29118925

Genome Structure and Reproductive Behaviour Influence the Evolutionary Potential of a Fungal Phytopathogen

PubMed Central

Daverdin, Guillaume; Rouxel, Thierry; Gout, Lilian; Aubertot, Jean-Noël; Fudal, Isabelle; Meyer, Michel; Parlange, Francis; Carpezat, Julien; Balesdent, Marie-Hélène

2012-01-01

Modern agriculture favours the selection and spread of novel plant diseases. Furthermore, crop genetic resistance against pathogens is often rendered ineffective within a few years of its commercial deployment. Leptosphaeria maculans, the cause of phoma stem canker of oilseed rape, develops gene-for-gene interactions with its host plant, and has a high evolutionary potential to render ineffective novel sources of resistance in crops. Here, we established a four-year field experiment to monitor the evolution of populations confronted with the newly released Rlm7 resistance and to investigate the nature of the mutations responsible for virulence against Rlm7. A total of 2551 fungal isolates were collected from experimental crops of a Rlm7 cultivar or a cultivar without Rlm7. All isolates were phenotyped for virulence and a subset was genotyped with neutral genetic markers. Virulent isolates were investigated for molecular events at the AvrLm4-7 locus. Whilst virulent isolates were not found in neighbouring crops, their frequency had reached 36% in the experimental field after four years. An extreme diversity of independent molecular events leading to virulence was identified in populations, with large-scale Repeat Induced Point mutations or complete deletion of AvrLm4-7 being the most frequent. Our data suggest that increased mutability of fungal genes involved in the interactions with plants is directly related to their genomic environment and reproductive system. Thus, rapid allelic diversification of avirulence genes can be generated in L. maculans populations in a single field provided that large population sizes and sexual reproduction are favoured by agricultural practices. PMID:23144620

Ceftaroline--An Anti-MRSA Cephalosporin and Its Implications for Singapore.

PubMed

Chen, Hui Hiong; Hon, Pei Yun; Hsu, Li Yang

2014-03-01

Ceftaroline is a fifth-generation cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA) that was recently launched in Singapore. It received approval from the United States (US) Food Drug Administration (FDA) and European Commission for the treatment of adult patients with community-acquired pneumonia (CAP) and complicated skin and soft tissue infections (cSSTI). This study aimed to review current published data and determine its clinical role, particularly in the local setting. A literature review on published articles in English on ceftaroline, focusing in particular on clinical trials and other clinical reports. Susceptibility testing was also performed on a limited sample of local MRSA and Streptococcus pneumoniae isolates. Ceftaroline has an extensive spectrum of activity, including coverage of MRSA and multidrug-resistant S. pneumoniae. However, it has limited activity against non-fermenting Gram-negative bacteria and is susceptible to hydrolysis by extended spectrum beta-lactamases. It is only available for intravenous delivery, with a reconstituted stability of just 6 hours, rendering it unavailable for use for outpatient antibiotic therapy. Clinical trials demonstrate non-inferiority compared to first-line comparators in the treatment of CAP and cSSTI. Published case reports/series suggest a potential greater role in the treatment of MRSA bacteremia and endocarditis. No resistance was found among local archived MRSA and S. pneumoniae isolates. We believe ceftaroline will occupy primarily niche roles for culture-directed treatment of various infections--in particular those caused by MRSA--until further clinical trial data become available. A variety of factors render it less useful or appealing for empirical treatment of CAP or healthcare-associated infections.

CD8+ T Cells Induce Fatal Brainstem Pathology during Cerebral Malaria via Luminal Antigen-Specific Engagement of Brain Vasculature

PubMed Central

Swanson, Phillip A.; Hart, Geoffrey T.; Russo, Matthew V.; Nayak, Debasis; Yazew, Takele; Peña, Mirna; Khan, Shahid M.; Pierce, Susan K.; McGavern, Dorian B.

2016-01-01

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. The in vivo mechanisms underlying this fatal condition are not entirely understood. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the pathogenesis of CM. Fatal disease was associated with alterations in tight junction proteins, vascular breakdown in the meninges / parenchyma, edema, and ultimately neuronal cell death in the brainstem, which is consistent with cerebral herniation as a cause of death. At the peak of ECM, we revealed using intravital two-photon microscopy that myelomonocytic cells and parasite-specific CD8+ T cells associated primarily with the luminal surface of CNS blood vessels. Myelomonocytic cells participated in the removal of parasitized red blood cells (pRBCs) from cerebral blood vessels, but were not required for the disease. Interestingly, the majority of disease-inducing parasite-specific CD8+ T cells interacted with the lumen of brain vascular endothelial cells (ECs), where they were observed surveying, dividing, and arresting in a cognate peptide-MHC I dependent manner. These activities were critically dependent on IFN-γ, which was responsible for activating cerebrovascular ECs to upregulate adhesion and antigen-presenting molecules. Importantly, parasite-specific CD8+ T cell interactions with cerebral vessels were impaired in chimeric mice rendered unable to present EC antigens on MHC I, and these mice were in turn resistant to fatal brainstem pathology. Moreover, anti-adhesion molecule (LFA-1 / VLA-4) therapy prevented fatal disease by rapidly displacing luminal CD8+ T cells from cerebrovascular ECs without affecting extravascular T cells. These in vivo data demonstrate that parasite-specific CD8+ T cell-induced fatal vascular breakdown and subsequent neuronal death during ECM is associated with luminal, antigen-dependent interactions with cerebrovasculature. PMID:27907215

Forespore engulfment mediated by a ratchet-like mechanism.

PubMed

Broder, Dan H; Pogliano, Kit

2006-09-08

A key step in bacterial endospore formation is engulfment, during which one bacterial cell engulfs another in a phagocytosis-like process that normally requires SpoIID, SpoIIM, and SpoIIP (DMP). We here describe a second mechanism involving the zipper-like interaction between the forespore protein SpoIIQ and its mother cell ligand SpoIIIAH, which are essential for engulfment when DMP activity is reduced or SpoIIB is absent. They are also required for the rapid engulfment observed during the enzymatic removal of peptidoglycan, a process that does not require DMP. These results suggest the existence of two separate engulfment machineries that compensate for one another in intact cells, thereby rendering engulfment robust. Photobleaching analysis demonstrates that SpoIIQ assembles a stationary structure, suggesting that SpoIIQ and SpoIIIAH function as a ratchet that renders forward membrane movement irreversible. We suggest that ratchet-mediated engulfment minimizes the utilization of chemical energy during this dramatic cellular reorganization, which occurs during starvation.

Fcγ receptor I alpha chain (CD64) expression in macrophages is critical for the onset of meningitis by Escherichia coli K1.

PubMed

Mittal, Rahul; Sukumaran, Sunil K; Selvaraj, Suresh K; Wooster, David G; Babu, M Madan; Schreiber, Alan D; Verbeek, J Sjef; Prasadarao, Nemani V

2010-11-18

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa(-/-) mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa(-/-) macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa(-/-) mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1.

The MerR-like regulator BrlR confers biofilm tolerance by activating multidrug efflux pumps in Pseudomonas aeruginosa biofilms.

PubMed

Liao, Julie; Schurr, Michael J; Sauer, Karin

2013-08-01

A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. In Pseudomonas aeruginosa, biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated that brlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR, with DNA binding assays confirming BrlR binding to the promoter regions of the mexAB-oprM and mexEF-oprN operons. Quantitative reverse transcriptase PCR (qRT-PCR) analysis further indicated BrlR to be an activator of mexAB-oprM and mexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressing brlR. Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by bactericidal agents. Overexpression of either efflux pump in a ΔbrlR strain partly restored tolerance of ΔbrlR biofilms to antibiotics. Expression of brlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild-type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance to P. aeruginosa biofilms and to resist the action of antimicrobial agents.

Differential sensitivity of TREK–1, TREK–2 and TRAAK background potassium channels to the polycationic dye ruthenium red

PubMed Central

Braun, G; Lengyel, M; Enyedi, P; Czirják, G

2015-01-01

Background and Purpose Pharmacological separation of the background potassium currents of closely related K2P channels is a challenging problem. We previously demonstrated that ruthenium red (RR) inhibits TASK-3 (K2P9.1), but not TASK-1 (K2P3.1) channels. RR has been extensively used to distinguish between TASK currents in native cells. In the present study, we systematically investigate the RR sensitivity of a more comprehensive set of K2P channels. Experimental Approach K+ currents were measured by two-electrode voltage clamp in Xenopus oocytes and by whole-cell patch clamp in mouse dorsal root ganglion (DRG) neurons. Key Results RR differentiates between two closely related members of the TREK subfamily. TREK-2 (K2P10.1) proved to be highly sensitive to RR (IC50 = 0.2 μM), whereas TREK-1 (K2P2.1) was not affected by the compound. We identified aspartate 135 (D135) as the target of the inhibitor in mouse TREK-2c. D135 lines the wall of the extracellular ion pathway (EIP), a tunnel structure through the extracellular cap characteristic for K2P channels. TREK-1 contains isoleucine in the corresponding position. The mutation of this isoleucine (I110D) rendered TREK-1 sensitive to RR. The third member of the TREK subfamily, TRAAK (K2P4.1) was more potently inhibited by ruthenium violet, a contaminant in some RR preparations, than by RR. DRG neurons predominantly express TREK-2 and RR-resistant TREK-1 and TRESK (K2P18.1) background K+ channels. We detected the RR-sensitive leak K+ current component in DRG neurons. Conclusions and Implications We propose that RR may be useful for distinguishing TREK-2 (K2P10.1) from TREK-1 (K2P2.1) and other RR-resistant K2P channels in native cells. PMID:25409575

Dietary sugars, serum and the biocide chlorhexidine digluconate modify the population and structural dynamics of mixed Candida albicans and Escherichia coli biofilms.

PubMed

Thein, Z M; Smaranayake, Y H; Smaranayake, L P

2007-11-01

Despite the increasing recognition of the role played by mixed species biofilms in health and disease, the behavior and factors modulating these biofilms remain elusive. We therefore compared the effect of serum, two dietary sugars (sucrose and galactose) and a biocide, chlorhexidine digluconate, on a dual species biofilm (DSB) of Candida albicans and Escherichia coli and, their single species biofilm (SSB) counterparts. Both modes of biofilm growth on polystyrene plastic surfaces were quantified using a viable cell count method and visualized using confocal scanning laser microscopy (CSLM). Present data indicate that co-culture of C. albicans with varying initial concentrations of E. coli leads to a significant inhibition of yeast growth (r=-0.964; p<0.001). Parallel ultrastructural studies using CSLM and a Live/Dead stain confirmed that E. coli growth rendered blastospores and hyphal yeasts non-viable in DSB. SSB of C. albicans showed pronounced growth when its growth surface was pretreated with serum and by sugar supplements in the incubating medium (p<0.05). Intriguingly, C. albicans in DSB was more resistant to the antiseptic effect of chlorhexidine digluconate. Taken together, the current data elucidate some features of the colonization resistance offered by bacteria in mixed bacterial/fungal habitats and how such phenomena may contribute to the development of fungal superinfection during antimicrobial therapy.

Stochastic switching of TiO2-based memristive devices with identical initial memory states

PubMed Central

2014-01-01

In this work, we show that identical TiO2-based memristive devices that possess the same initial resistive states are only phenomenologically similar as their internal structures may vary significantly, which could render quite dissimilar switching dynamics. We experimentally demonstrated that the resistive switching of practical devices with similar initial states could occur at different programming stimuli cycles. We argue that similar memory states can be transcribed via numerous distinct active core states through the dissimilar reduced TiO2-x filamentary distributions. Our hypothesis was finally verified via simulated results of the memory state evolution, by taking into account dissimilar initial filamentary distribution. PMID:24994953

The history and background of Augmentin.

PubMed

Rolinson, G N

1982-07-28

Bacterial resistance to the beta-lactam group of antibiotics is frequently due to the production of beta-lactamase which brings about the inactivation of the antibiotic. Clavulanic acid is a naturally occurring inhibitor of beta-lactamase which is capable of rendering penicillin- and cephalosporin-resistant organisms sensitive. The compound is obtained by fermentation from Streptomyces clavuligerus. Clavulanic acid shows some structural similarity to the penicillins and cephalosporins and functions as a progressive inhibitor of a wide range of beta-lactamases including those found in Escherichia coli, Klebsiella aerogenes, Proteus species, Bacteroides fragilis, Haemophilus influenzae, Neisseria gonorrhoeae and Staphylococcus aureus. Clavulanic acid is well absorbed when given by mouth and a formulation with amoxycillin (Augmentin; Beechams) is now available for clinical use.

Fine line structures of ceramic films formed by patterning of metalorganic precursors using photolithography and ion beams

NASA Astrophysics Data System (ADS)

Hung, L. S.; Zheng, L. R.

1992-05-01

Fine line structures of ceramic thin films were fabricated by patterning of metalorganic precursors using photolithography and ion beams. A trilevel structure was developed with an outer resist layer to transfer patterns, a silver delineated layer as an implantation mask, and a planar resist layer protecting the precursor film from chemical attacking and sputtering. Ion irradiation through the Ag stencil rendered metal carboxylates insoluble in 2-ethylhexanoic acid, permitting patterning of the precursor film with patterning features on micron scales. The potential of this technique was demonstrated in patterning of Bi2Sr2CaCu2O(8+x) and Pb(Zr(0.53)Ti(0.47) thin films.

How Listeria monocytogenes organizes its surface for virulence

PubMed Central

Carvalho, Filipe; Sousa, Sandra; Cabanes, Didier

2014-01-01

Listeria monocytogenes is a Gram-positive pathogen responsible for the manifestation of human listeriosis, an opportunistic foodborne disease with an associated high mortality rate. The key to the pathogenesis of listeriosis is the capacity of this bacterium to trigger its internalization by non-phagocytic cells and to survive and even replicate within phagocytes. The arsenal of virulence proteins deployed by L. monocytogenes to successfully promote the invasion and infection of host cells has been progressively unveiled over the past decades. A large majority of them is located at the cell envelope, which provides an interface for the establishment of close interactions between these bacterial factors and their host targets. Along the multistep pathways carrying these virulence proteins from the inner side of the cytoplasmic membrane to their cell envelope destination, a multiplicity of auxiliary proteins must act on the immature polypeptides to ensure that they not only maturate into fully functional effectors but also are placed or guided to their correct position in the bacterial surface. As the major scaffold for surface proteins, the cell wall and its metabolism are critical elements in listerial virulence. Conversely, the crucial physical support and protection provided by this structure make it an ideal target for the host immune system. Therefore, mechanisms involving fine modifications of cell envelope components are activated by L. monocytogenes to render it less recognizable by the innate immunity sensors or more resistant to the activity of antimicrobial effectors. This review provides a state-of-the-art compilation of the mechanisms used by L. monocytogenes to organize its surface for virulence, with special focus on those proteins that work “behind the frontline”, either supporting virulence effectors or ensuring the survival of the bacterium within its host. PMID:24809022

Predictive modelling of a novel anti-adhesion therapy to combat bacterial colonisation of burn wounds

PubMed Central

Huebinger, Ryan M.; Keen, Emma

2018-01-01

As the development of new classes of antibiotics slows, bacterial resistance to existing antibiotics is becoming an increasing problem. A potential solution is to develop treatment strategies with an alternative mode of action. We consider one such strategy: anti-adhesion therapy. Whereas antibiotics act directly upon bacteria, either killing them or inhibiting their growth, anti-adhesion therapy impedes the binding of bacteria to host cells. This prevents bacteria from deploying their arsenal of virulence mechanisms, while simultaneously rendering them more susceptible to natural and artificial clearance. In this paper, we consider a particular form of anti-adhesion therapy, involving biomimetic multivalent adhesion molecule 7 coupled polystyrene microbeads, which competitively inhibit the binding of bacteria to host cells. We develop a mathematical model, formulated as a system of ordinary differential equations, to describe inhibitor treatment of a Pseudomonas aeruginosa burn wound infection in the rat. Benchmarking our model against in vivo data from an ongoing experimental programme, we use the model to explain bacteria population dynamics and to predict the efficacy of a range of treatment strategies, with the aim of improving treatment outcome. The model consists of two physical compartments: the host cells and the exudate. It is found that, when effective in reducing the bacterial burden, inhibitor treatment operates both by preventing bacteria from binding to the host cells and by reducing the flux of daughter cells from the host cells into the exudate. Our model predicts that inhibitor treatment cannot eliminate the bacterial burden when used in isolation; however, when combined with regular or continuous debridement of the exudate, elimination is theoretically possible. Lastly, we present ways to improve therapeutic efficacy, as predicted by our mathematical model. PMID:29723210

Measuring The Contact Resistances Of Photovoltaic Cells

NASA Technical Reports Server (NTRS)

Burger, D. R.

1985-01-01

Simple method devised to measure contact resistances of photovoltaic solar cells. Method uses readily available equipment and applicable at any time during life of cell. Enables evaluation of cell contact resistance, contact-end resistance, contact resistivity, sheet resistivity, and sheet resistivity under contact.

XIAP Limits Autophagic Degradation of Sox2 and Is A Therapeutic Target in Nasopharyngeal Carcinoma Stem Cells

PubMed Central

Ji, Jiao; Yu, Yan; Li, Zhi-Ling; Chen, Ming-Yuan; Deng, Rong; Huang, Xiang; Wang, Guang-Feng; Zhang, Meng-Xia; Yang, Qi; Ravichandran, Senthilkumar; Feng, Gong-Kan; Xu, Xue-Lian; Yang, Chen-Lu; Qiu, Miao-Zhen; Jiao, Lin; Yang, Dajun; Zhu, Xiao-Feng

2018-01-01

Rationale: Nasopharyngeal carcinoma (NPC) is the most frequent head and neck tumor in South China. The presence of cancer stem cells (CSCs) in NPC contributes to tumor maintenance and therapeutic resistance, while the ability of CSCs to escape from the apoptosis pathway may render them the resistant property to the therapies. Inhibitor of apoptosis proteins family proteins (IAPs), which are overexpressed in nasopharyngeal carcinoma stem cells, may play an important role in maintaining nasopharyngeal cancer stem cell properties. Here, we develop a novel CSC-targeting strategy to treat NPC through inhibiting IAPs. Methods: Human NPC S-18 and S-26 cell lines were used as the model system in vitro and in vivo. Fluorescence activated cell sorting (FACS) assay was used to detect nasopharyngeal SP cells and CD44+ cells. The characteristics of CSCs were defined by sphere suspension culture, colony formation assay and cell migration. The role of XIAP on the regulation of Sox2 protein stability and ERK1-mediated phosphorylation of Sox2 signaling pathway were analyzed using immunoblotting, immunoprecipitation, immunofluorescence, phosphorylation mass spectrometry, siRNA silencing and plasmid overexpression. The correlation between XIAP and Sox2 in NPC biopsies and their role in prognosis was performed by immunohistochemistry. APG-1387 or chemotherapies-induced cell death and apoptosis in S-18 and S-26 were determined by WST, immunoblotting and flow cytometry assay. Results: IAPs, especially X chromosome-linked IAP (XIAP), were elevated in CSCs of NPC, and these proteins were critically involved in the maintenance of CSCs properties by enhancing the stability of Sox2. Mechanistically, ERK1 kinase promoted autophagic degradation of Sox2 via phosphorylation of Sox2 at Ser251 and further SUMOylation of Sox2 at Lys245 in non-CSCs. However, XIAP blocked autophagic degradation of Sox2 by inhibiting ERK1 activation in CSCs. Additionally, XIAP was positively correlated with Sox2 expression in NPC tissues, which were associated with NPC progression. Finally, we discovered that a novel antagonist of IAPs, APG-1387, exerted antitumor effect on CSCs. Also, the combination of APG-1387 with CDDP /5-FU has a synergistic effect on NPC. Conclusion: Our study highlights the importance of IAPs in the maintenance of CSCs in NPC. Thus, XIAP is a promising therapeutic target in CSCs and suggests that NPC patients may benefit from a combination treatment of APG-1387 with conventional chemotherapy. PMID:29556337

From Human Mesenchymal Stem Cells to Insulin-Producing Cells: Comparison between Bone Marrow- and Adipose Tissue-Derived Cells.

PubMed

Gabr, Mahmoud M; Zakaria, Mahmoud M; Refaie, Ayman F; Abdel-Rahman, Engy A; Reda, Asmaa M; Ali, Sameh S; Khater, Sherry M; Ashamallah, Sylvia A; Ismail, Amani M; Ismail, Hossam El-Din A; El-Badri, Nagwa; Ghoneim, Mohamed A

2017-01-01

The aim of this study is to compare human bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs), for their differentiation potentials to form insulin-producing cells. BM-MSCs were obtained during elective orthotopic surgery and AT-MSCs from fatty aspirates during elective cosmetics procedures. Following their expansion, cells were characterized by phenotyping, trilineage differentiation ability, and basal gene expression of pluripotency genes and for their metabolic characteristics. Cells were differentiated according to a Trichostatin-A based protocol. The differentiated cells were evaluated by immunocytochemistry staining for insulin and c-peptide. In addition the expression of relevant pancreatic endocrine genes was determined. The release of insulin and c-peptide in response to a glucose challenge was also quantitated. There were some differences in basal gene expression and metabolic characteristics. After differentiation the proportion of the resulting insulin-producing cells (IPCs), was comparable among both cell sources. Again, there were no differences neither in the levels of gene expression nor in the amounts of insulin and c-peptide release as a function of glucose challenge. The properties, availability, and abundance of AT-MSCs render them well-suited for applications in regenerative medicine. Conclusion . BM-MSCs and AT-MSCs are comparable regarding their differential potential to form IPCs. The availability and properties of AT-MSCs render them well-suited for applications in regenerative medicine.

From Human Mesenchymal Stem Cells to Insulin-Producing Cells: Comparison between Bone Marrow- and Adipose Tissue-Derived Cells

PubMed Central

Abdel-Rahman, Engy A.; Reda, Asmaa M.; Ashamallah, Sylvia A.; Ismail, Amani M.; Ismail, Hossam El-Din A.; El-Badri, Nagwa

2017-01-01

The aim of this study is to compare human bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs), for their differentiation potentials to form insulin-producing cells. BM-MSCs were obtained during elective orthotopic surgery and AT-MSCs from fatty aspirates during elective cosmetics procedures. Following their expansion, cells were characterized by phenotyping, trilineage differentiation ability, and basal gene expression of pluripotency genes and for their metabolic characteristics. Cells were differentiated according to a Trichostatin-A based protocol. The differentiated cells were evaluated by immunocytochemistry staining for insulin and c-peptide. In addition the expression of relevant pancreatic endocrine genes was determined. The release of insulin and c-peptide in response to a glucose challenge was also quantitated. There were some differences in basal gene expression and metabolic characteristics. After differentiation the proportion of the resulting insulin-producing cells (IPCs), was comparable among both cell sources. Again, there were no differences neither in the levels of gene expression nor in the amounts of insulin and c-peptide release as a function of glucose challenge. The properties, availability, and abundance of AT-MSCs render them well-suited for applications in regenerative medicine. Conclusion. BM-MSCs and AT-MSCs are comparable regarding their differential potential to form IPCs. The availability and properties of AT-MSCs render them well-suited for applications in regenerative medicine. PMID:28584815

Breast Cancer Resistance Protein and P-glycoprotein in Brain Cancer: Two Gatekeepers Team Up

PubMed Central

Agarwal, Sagar; Hartz, Anika M.S.; Elmquist, William F.; Bauer, Björn

2012-01-01

Brain cancer is a devastating disease. Despite extensive research, treatment of brain tumors has been largely ineffective and the diagnosis of brain cancer remains uniformly fatal. Failure of brain cancer treatment may be in part due to limitations in drug delivery, influenced by the ABC drug efflux transporters P-gp and BCRP at the blood-brain and blood-tumor barriers, in brain tumor cells, as well as in brain tumor stem-like cells. P-gp and BCRP limit various anti-cancer drugs from entering the brain and tumor tissues, thus rendering chemotherapy ineffective. To overcome this obstacle, two strategies – targeting transporter regulation and direct transporter inhibition – have been proposed. In this review, we focus on these strategies. We first introduce the latest findings on signaling pathways that could potentially be targeted to down-regulate P-gp and BCRP expression and/or transport activity. We then highlight in detail the new paradigm of P-gp and BCRP working as a “cooperative team of gatekeepers” at the blood-brain barrier, discuss its ramifications for brain cancer therapy, and summarize the latest findings on dual P-gp/BCRP inhibitors. Finally, we provide a brief summary with conclusions and outline the perspectives for future research endeavors in this field. PMID:21827403

The human tyrosine kinase Kit and its gatekeeper mutant T670I, show different kinetic properties: Implications for drug design.

PubMed

Kissova, Miroslava; Maga, Giovanni; Crespan, Emmanuele

2016-10-01

The tyrosine kinase Kit, a receptor for Stem Cell Factor, is involved, among others, in processes associated to cell survival, proliferation and migration. Upon physiological conditions, the activity of Kit is tightly regulated. However, primary mutations that lead to its constitutive activation are the causal oncogenic driver of gastrointestinal stromal tumours (GISTs). GISTs are known to be refractory to conventional therapies but the introduction of Imatinib, a selective inhibitor of tyrosine kinases Abl and Kit, significantly ameliorated the treatment options of GISTs patients. However, the acquisition of secondary mutations renders Kit resistant towards all available drugs. Mutation involving gatekeeper residues (such as V654a and T670I) influence both the structure and the catalytic activity of the enzyme. Therefore, detailed knowledge of the enzymatic properties of the mutant forms, in comparison with the wild type enzyme, is an important pre-requisite for the rational development of specific inhibitors. In this paper we report a thorough kinetic analysis of the reaction catalyzed by the Kit kinase and its gatekeeper mutated form T670I. Our results revealed the different mechanisms of action of these two enzymes and may open a new avenue for the future design of specific Kit inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibition of intracellular bacterial replication in fibroblasts is dependent on the perforin-like protein (Perforin-2) encoded by macrophage expressed gene 1

PubMed Central

McCormack, Ryan; de Armas, Lesley R.; Shiratsuchi, Motoaki; Ramos, Jay; Podack, Eckhard R.

2013-01-01

Fibroblasts are known to eliminate intracellular bacteria, but the lethal hit of the bactericidal mechanism has not been defined. We show that primary embryonic and established fibroblasts can be induced by interferons or by intracellular bacterial infection to express a perforin-like mRNA previously described as macrophage expressed gene 1 (mpeg1). The presence and level of the perforin-like mRNA correlate with the ability of primary mouse embryonic fibroblasts (MEF) to eliminate intracellular bacteria. In addition, siRNA knock-down of the perforin-like molecule abolishes bactericidal activity and allows intracellular bacterial replication. Complementation of MEF in which the endogenous perforin-like molecule has been knocked down with an RFP-tagged version restores bactericidal activity. The perforin-like molecule has broad bactericidal specificity for pathogenic and non-pathogenic bacteria including Gram positive, Gram negative and acid fast bacteria. The perforin-like molecule renders previously lysozyme-resistant bacteria sensitive to lysis by lysozyme suggesting physical damage of the outer cell wall by the perforin-like protein. MEFs damage cell walls of intracellular bacteria by insertion, polymerization and pore-formation of the perforin-like protein, analogous to pore-formers of complement and Perforin-1 of cytolytic lymphocytes. We propose the name Perforin-2. PMID:23257510

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae.

PubMed

Hu, Kejin; Guo, Shuguang; Yan, Gonghong; Yuan, Wenjie; Zheng, Yin; Jiang, Yu

2016-04-01

In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth-related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12-rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2(W2041R) ) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non-covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization. © 2016 John Wiley & Sons Ltd.

Pathogenesis of Dengue Vaccine Viruses in Mosquitoes.

DTIC Science & Technology

1985-07-01

small percentage of engorging mosquitoes became infected. To determine if Fc receptors might be a determinate of virus infection of midgut cells, blood...somehow alter glycoprotein conformation rendering the virus less capable of interacting with midgut cell receptors , 2) virus in cells might be protected...virus preparations are known to be much less efficient than a viresic host in mediating midgut infection. The artificial meal must be several logs

Hydroxypropyl cellulose methacrylate as a photo-patternable and biodegradable hybrid paper substrate for cell culture and other bioapplications.

PubMed

Qi, Aisha; Hoo, Siew Pei; Friend, James; Yeo, Leslie; Yue, Zhilian; Chan, Peggy P Y

2014-04-01

In addition to the choice of appropriate material properties of the tissue construct to be used, such as its biocompatibility, biodegradability, cytocompatibility, and mechanical rigidity, the ability to incorporate microarchitectural patterns in the construct to mimic that found in the cellular microenvironment is an important consideration in tissue engineering and regenerative medicine. Both these issues are addressed by demonstrating a method for preparing biodegradable and photo-patternable constructs, where modified cellulose is cross-linked to form an insoluble structure in an aqueous environment. Specifically, hydroxypropyl cellulose (HPC) is rendered photocrosslinkable by grafting with methylacrylic anhydride, whose linkages also render the cross-linked construct hydrolytically degradable. The HPC is then cross-linked via a photolithography-based fabrication process. The feasibility of functionalizing these HPC structures with biochemical cues is verified post-fabrication, and shown to facilitate the adhesion of mesenchymal progenitor cells. The HPC constructs are shown to be biocompatible and hydrolytically degradable, thus enabling cell proliferation and cell migration, and therefore constituting an ideal candidate for long-term cell culture and implantable tissue scaffold applications. In addition, the potential of the HPC structure is demonstrated as an alternative substrate to paper microfluidic diagnostic devices for protein and cell assays. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plasticized phenolphthalein polycarbonate

NASA Technical Reports Server (NTRS)

Harrison, E. S.

1976-01-01

Phenolphthalein polycarbonate was successfully plasticized with polychlorinated biphenyls (e.g., Aroclor 1231) or tricresyl phosphate and cast from tetrahydrofuran to give clear films without loss of fire resistance. At loadings of 20 to 30 percent plasticizer the Tg was lowered to approximately 100 C which would render phenolphthalein polycarbonate easily moldable. Although these materials had some mechanical integrity as shown by their film forming ability, the room temperature toughness of the plasticized polymer was not significantly improved over unmodified polymer.

The Growing Threat of Multidrug-Resistant Gram-Negative Infections in Patients with Hematologic Malignancies

PubMed Central

Baker, Thomas M.; Satlin, Michael J.

2016-01-01

Prolonged neutropenia and chemotherapy-induced mucositis render patients with hematologic malignancies highly vulnerable to Gram-negative bacteremia. Unfortunately, multidrug-resistant (MDR) Gram-negative bacteria are increasingly encountered globally, and current guidelines for empirical antibiotic coverage in these patients may not adequately treat these bacteria. This expansion of resistance, coupled with traditional culturing techniques requiring 2-4 days for bacterial identification and antimicrobial susceptibility results, have grave implications for these immunocompromised hosts. This review characterizes the epidemiology, risk factors, resistance mechanisms, recommended treatments, and outcomes of the MDR Gram-negative bacteria that commonly cause infections in patients with hematologic malignancies. We also examine infection prevention strategies in hematology patients, such as infection control practices, antimicrobial stewardship, and targeted decolonization. Finally, we assess strategies to improve outcomes of infected patients, including gastrointestinal screening to guide empirical antibiotic therapy, new rapid diagnostic tools for expeditious identification of MDR pathogens, and use of two new antimicrobial agents, ceftolozane/tazobactam and ceftazidime/avibactam. PMID:27339405

In vitro Development of Chemotherapy and Targeted Therapy Drug-Resistant Cancer Cell Lines: A Practical Guide with Case Studies

PubMed Central

McDermott, Martina; Eustace, Alex J.; Busschots, Steven; Breen, Laura; Crown, John; Clynes, Martin; O’Donovan, Norma; Stordal, Britta

2014-01-01

The development of a drug-resistant cell line can take from 3 to 18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval, and optimizing the dose of drug for the parent cell line. Clinically relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between two- and eight-fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299 and H460) and temozolomide-resistant melanoma (Malme-3M and HT144) cell lines. Continuous selection produced a lapatinib-resistant breast cancer cell line (HCC1954). Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy, or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug resistance. PMID:24639951

Simulation and training of lumbar punctures using haptic volume rendering and a 6DOF haptic device

NASA Astrophysics Data System (ADS)

Färber, Matthias; Heller, Julika; Handels, Heinz

2007-03-01

The lumbar puncture is performed by inserting a needle into the spinal chord of the patient to inject medicaments or to extract liquor. The training of this procedure is usually done on the patient guided by experienced supervisors. A virtual reality lumbar puncture simulator has been developed in order to minimize the training costs and the patient's risk. We use a haptic device with six degrees of freedom (6DOF) to feedback forces that resist needle insertion and rotation. An improved haptic volume rendering approach is used to calculate the forces. This approach makes use of label data of relevant structures like skin, bone, muscles or fat and original CT data that contributes information about image structures that can not be segmented. A real-time 3D visualization with optional stereo view shows the punctured region. 2D visualizations of orthogonal slices enable a detailed impression of the anatomical context. The input data consisting of CT and label data and surface models of relevant structures is defined in an XML file together with haptic rendering and visualization parameters. In a first evaluation the visible human male data has been used to generate a virtual training body. Several users with different medical experience tested the lumbar puncture trainer. The simulator gives a good haptic and visual impression of the needle insertion and the haptic volume rendering technique enables the feeling of unsegmented structures. Especially, the restriction of transversal needle movement together with rotation constraints enabled by the 6DOF device facilitate a realistic puncture simulation.

Map-based cloning and characterization of a brown planthopper resistance gene BPH26 from Oryza sativa L. ssp. indica cultivar ADR52.

PubMed

Tamura, Yasumori; Hattori, Makoto; Yoshioka, Hirofumi; Yoshioka, Miki; Takahashi, Akira; Wu, Jianzhong; Sentoku, Naoki; Yasui, Hideshi

2014-07-29

The brown planthopper (BPH) is the most serious insect pest of rice in Asia. The indica rice cultivar ADR52 carries two BPH resistance genes, BPH26 (brown planthopper resistance 26) and BPH25. Map-based cloning of BPH26 revealed that BPH26 encodes a coiled-coil-nucleotide-binding-site-leucine-rich repeat (CC-NBS-LRR) protein. BPH26 mediated sucking inhibition in the phloem sieve element. BPH26 was identical to BPH2 on the basis of DNA sequence analysis and feeding ability of the BPH2-virulent biotype of BPH. BPH2 was widely incorporated in elite rice cultivars and was well-cultivated in many Asian countries as a favorable gene resource in rice breeding against BPH. However, BPH2 was rendered ineffective by a virulent biotype of BPH in rice fields in Asia. In this study, we suggest that BPH2 can be reused by combining with other BPH resistance genes, such as BPH25, to ensure durable resistance to BPH.

Map-based Cloning and Characterization of a Brown Planthopper Resistance Gene BPH26 from Oryza sativa L. ssp. indica Cultivar ADR52

PubMed Central

Tamura, Yasumori; Hattori, Makoto; Yoshioka, Hirofumi; Yoshioka, Miki; Takahashi, Akira; Wu, Jianzhong; Sentoku, Naoki; Yasui, Hideshi

2014-01-01

The brown planthopper (BPH) is the most serious insect pest of rice in Asia. The indica rice cultivar ADR52 carries two BPH resistance genes, BPH26 (BROWN PLANTHOPPER RESISTANCE 26) and BPH25. Map-based cloning of BPH26 revealed that BPH26 encodes a coiled-coil-nucleotide-binding-site–leucine-rich repeat (CC–NBS–LRR) protein. BPH26 mediated sucking inhibition in the phloem sieve element. BPH26 was identical to BPH2 on the basis of DNA sequence analysis and feeding ability of the BPH2-virulent biotype of BPH. BPH2 was widely incorporated in elite rice cultivars and was well-cultivated in many Asian countries as a favorable gene resource in rice breeding against BPH. However, BPH2 was rendered ineffective by a virulent biotype of BPH in rice fields in Asia. In this study, we suggest that BPH2 can be reused by combining with other BPH resistance genes, such as BPH25, to ensure durable resistance to BPH. PMID:25076167

Mucin-like protein, a saliva component involved in brown planthopper virulence and host adaptation.

PubMed

Huang, Hai-Jian; Liu, Cheng-Wen; Xu, Hai-Jun; Bao, Yan-Yuan; Zhang, Chuan-Xi

2017-04-01

The rice brown planthopper (BPH), Nilaparvata lugens, can rapidly adapt to new resistant rice varieties within several generations, rendering its management burdensome. However, the molecular mechanism underlying its adaptability remains unclear. In this study, we investigated the potential role of mucin-like protein (NlMul) in N. lugens virulence and adaptation to host resistance. NlMul is an important glycoprotein that constitutes both gelling and watery saliva, and specifically expressed in the salivary glands at all developmental stages except the egg period. Knocking down the expression of NlMul resulted in the secretion of short and single-branched salivary sheaths. NlMul might help BPH deal with plant resistance, and altered gene expression was observed when BPHs were transferred from a susceptible rice variety to a resistant one. The NlMul-deficient BPHs showed disordered developmental duration and a portion of these insects reared on resistant rice exhibited lethal effects. Our results uncover a saliva-mediated interaction between insect and host plant, and provide useful information in rice breeding and planthopper management. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differential Resistance Mechanisms to Glyphosate Result in Fitness Cost for Lolium perenne and L. multiflorum

PubMed Central

Fernández-Moreno, Pablo T.; Alcántara-de la Cruz, Ricardo; Smeda, Reid J.; De Prado, Rafael

2017-01-01

Multiple mechanisms of resistance to glyphosate are exhibited by populations of Lolium spp. worldwide. Association of resistance with growth and reproductive fitness is an important predictor for long-term success of glyphosate-resistant (R) versus glyphosate-susceptible (S) biotypes. Numerous studies were conducted on R- and S-biotypes of Italian ryegrass (Lolium multiflorum) and perennial ryegrass (L. perenne) to characterize the underlying mechanism(s) of glyphosate resistance and associate this with growth and reproductive fitness. L. perenne expressed both altered uptake and translocation as well as a genetic change at 106-Pro to –Ser, This pattern for two resistance mechanisms is unique. L. multiflorum also exhibited altered uptake and translocation as well as duplication of EPSPS gene copies. Reduced plant biomass and height for R-versus S-biotypes of both species was evident over two growing seasons. This resulted in S- versus R- L. multiflorum producing up to 47 and 38% more seeds in 2014 and 2015, respectively. S- L. perenne produced up to 20 and 30% more seeds in 2014 and 2015, respectively. Both non-target site and target-site mechanisms of glyphosate resistance can render Lolium spp. at a competitive disadvantage. This has long-term implications for the success of glyphosate-resistant plants in the absence of selection pressure. PMID:29089958

Extended spectrum β-lactamases, carbapenemases and mobile genetic elements responsible for antibiotics resistance in Gram-negative bacteria.

PubMed

El Salabi, Allaaeddin; Walsh, Timothey R; Chouchani, Chedly

2013-05-01

Infectious diseases due to Gram-negative bacteria are a leading cause of morbidity and mortality worldwide. Antimicrobial agents represent one major therapeutic tools implicated to treat these infections. The misuse of antimicrobial agents has resulted in the emergence of resistant strains of Gram-negatives in particular Enterobacteriaceae and non-fermenters; they have an effect not only on a human but on the public health when bacteria use the resistance mechanisms to spread in the hospital environment and to the community outside the hospitals by means of mobile genetic elements. Gram-negative bacteria have become increasingly resistant to antimicrobial agents. They have developed several mechanisms by which they can withstand to antimicrobials, these mechanisms include the production of Extended-spectrum β-lactamases (ESBLs) and carbapenemases, furthermore, Gram-negative bacteria are now capable of spreading such resistance between members of the family Enterobacteriaceae and non-fermenters using mobile genetic elements as vehicles for such resistance mechanisms rendering antibiotics useless. Therefore, addressing the issue of mechanisms of antimicrobial resistance is considered one of most urgent priorities. This review will help to illustrate different resistance mechanisms; ESBLs, carbapenemases encoded by genes carried by mobile genetic elements, which are used by Gram-negative bacteria to escape antimicrobial effect.

A reassessment of the risk of rust fungi developing resistance to fungicides.

PubMed

Oliver, Richard P

2014-11-01

Rust fungi are major pathogens of many annual and perennial crops. Crop protection is largely based on genetic and chemical control. Fungicide resistance is a significant issue that has affected many crop pathogens. Some pathogens have rapidly developed resistance and hence are regarded as high-risk species. Rust fungi have been classified as being low risk, in spite of sharing many relevant features with high-risk pathogens. An examination of the evidence suggests that rust fungi may be wrongly classified as low risk. Of the nine classes of fungicide to which resistance has developed, six are inactive against rusts. The three remaining classes are quinone outside inhibitors (QoIs), demethylation inhibitors (DMIs) and succinate dehydrogenase inhibitors (SDHIs). QoIs have been protected by a recently discovered intron that renders resistant mutants unviable. Low levels of resistance have developed to DMIs, but with limited field significance. Older SDHI fungicides were inactive against rusts. Some of the SDHIs introduced since 2003 are active against rusts, so it may be that insufficient time has elapsed for resistance to develop, especially as SDHIs are generally sold in mixtures with other actives. It would therefore seem prudent to increase the level of vigilance for possible cases of resistance to established and new fungicides in rusts. © 2014 Society of Chemical Industry.

FabH Mutations Confer Resistance to FabF-Directed Antibiotics in Staphylococcus aureus

PubMed Central

Parsons, Joshua B.; Yao, Jiangwei; Frank, Matthew W.

2014-01-01

Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were isolated. All mutants selected against one antibiotic were cross-resistant to the other two antibiotics. Mutations were not detected in fabF, but the resistant strains harbored missense mutations in fabH. The altered amino acids clustered in and around the FabH active-site tunnel. The mutant FabH proteins were catalytically compromised based on the low activities of the purified enzymes, a fatty acid-dependent growth phenotype, and elevated expression of the fabHF operon in the mutant strains. Independent manipulation of fabF and fabH expression levels showed that the FabH/FabF activity ratio was a major determinant of antibiotic sensitivity. Missense mutations that reduce FabH activity are sufficient to confer resistance to multiple antibiotics that bind to the FabF acyl-enzyme intermediate in S. aureus. PMID:25403676

Hepatitis C Virus Cell-Cell Transmission and Resistance to Direct-Acting Antiviral Agents

PubMed Central

Heydmann, Laura; Barth, Heidi; Soulier, Eric; Habersetzer, François; Doffoël, Michel; Bukh, Jens; Patel, Arvind H.; Zeisel, Mirjam B.; Baumert, Thomas F.

2014-01-01

Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs. PMID:24830295

Radiation induction of drug resistance in RIF-1 tumors and tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hopwood, L.E.; Moulder, J.E.

1989-11-01

The RIF-1 tumor cell line contains a small number of cells (1-20 per 10(6) cells) that are resistant to various single antineoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), and adriamycin (ADR). For 5FU the frequency of drug resistance is lower for tumor-derived cells than for cells from cell culture; for MTX the reverse is true, and for ADR there is no difference. In vitro irradiation at 5 Gy significantly increased the frequency of drug-resistant cells for 5FU, MTX, and ADR. In vivo irradiation at 3 Gy significantly increased the frequency of drug-resistant cells for 5FU and MTX, but not formore » ADR. The absolute risk for in vitro induction of MTX, 5FU, and ADR resistance, and for in vivo induction of 5FU resistance, was 1-3 per 10(6) cells per Gy; but the absolute risk for in vivo induction of MTX resistance was 54 per 10(6) cells per Gy. The frequency of drug-resistant cells among individual untreated tumors was highly variable; among individual irradiated tumors the frequency of drug-resistant cells was significantly less variable. These studies provide supporting data for models of the development of tumor drug resistance, and imply that some of the drug resistance seen when chemotherapy follows radiotherapy may be due to radiation-induced drug resistance.« less

Combining R gene and quantitative resistance increases effectiveness of cultivar resistance against Leptosphaeria maculans in Brassica napus in different environments

PubMed Central

Mitrousia, Georgia K.; Sidique, Siti Nordahliawate M.; Qi, Aiming; Fitt, Bruce D. L.

2018-01-01

Using cultivar resistance against pathogens is one of the most economical and environmentally friendly methods for control of crop diseases. However, cultivar resistance can be easily rendered ineffective due to changes in pathogen populations or environments. To test the hypothesis that combining R gene-mediated resistance and quantitative resistance (QR) in one cultivar can provide more effective resistance than use of either type of resistance on its own, effectiveness of resistance in eight oilseed rape (Brassica napus) cultivars with different R genes and/or QR against Leptosphaeria maculans (phoma stem canker) was investigated in 13 different environments/sites over three growing seasons (2010/2011, 2011/2012 and 2012/2013). Cultivar Drakkar with no R genes and no QR was used as susceptible control and for sampling L. maculans populations. Isolates of L. maculans were obtained from the 13 sites in 2010/2011 to assess frequencies of avirulent alleles of different effector genes (AvrLm1, AvrLm4 or AvrLm7) corresponding to the resistance genes (Rlm1, Rlm4 or Rlm7) used in the field experiments. Results of field experiments showed that cultivars DK Cabernet (Rlm1 + QR) and Adriana (Rlm4 + QR) had significantly less severe phoma stem canker than cultivars Capitol (Rlm1) and Bilbao (Rlm4), respectively. Results of controlled environment experiments confirmed the presence of Rlm genes and/or QR in these four cultivars. Analysis of L. maculans populations from different sites showed that the mean frequencies of AvrLm1 (10%) and AvrLm4 (41%) were less than that of AvrLm7 (100%), suggesting that Rlm1 and Rlm4 gene-mediated resistances were partially rendered ineffective while Rlm7 resistance was still effective. Cultivar Excel (Rlm7 + QR) had less severe canker than cultivar Roxet (Rlm7), but the difference between them was not significant due to influence of the effective resistance gene Rlm7. For the two cultivars with only QR, Es-Astrid (QR) had less severe stem canker than NK Grandia (QR). Analysis of the relationship between severity of stem canker and weather data among the 13 sites in the three growing seasons showed that increased severity of stem canker was associated with increased rainfall during the phoma leaf spot development stage and increased temperature during the stem canker development stage. Further analysis of cultivar response to environmental factors showed that cultivars with both an Rlm gene and QR (e.g. DK Cabernet, Adriana and Excel) were less sensitive to a change in environment than cultivars with only Rlm genes (e.g. Capitol, Bilbao) or only QR (e.g. DK Grandia). These results suggest that combining R gene and QR can provide effective, stable control of phoma stem canker in different environments. PMID:29791484

Education Catching Up with Science: Preparing Students for Three-Dimensional Literacy in Cell Biology

PubMed Central

Kramer, IJsbrand M.; Dahmani, Hassen-Reda; Delouche, Pamina; Bidabe, Marissa; Schneeberger, Patricia

2012-01-01

The large number of experimentally determined molecular structures has led to the development of a new semiotic system in the life sciences, with increasing use of accurate molecular representations. To determine how this change impacts students’ learning, we incorporated image tests into our introductory cell biology course. Groups of students used a single text dealing with signal transduction, which was supplemented with images made in one of three iconographic styles. Typically, we employed realistic renderings, using computer-generated Protein Data Bank (PDB) structures; realistic-schematic renderings, using shapes inspired by PDB structures; or schematic renderings, using simple geometric shapes to represent cellular components. The control group received a list of keywords. When students were asked to draw and describe the process in their own style and to reply to multiple-choice questions, the three iconographic approaches equally improved the overall outcome of the tests (relative to keywords). Students found the three approaches equally useful but, when asked to select a preferred style, they largely favored a realistic-schematic style. When students were asked to annotate “raw” realistic images, both keywords and schematic representations failed to prepare them for this task. We conclude that supplementary images facilitate the comprehension process and despite their visual clutter, realistic representations do not hinder learning in an introductory course. PMID:23222839

Education catching up with science: preparing students for three-dimensional literacy in cell biology.

PubMed

Kramer, Ijsbrand M; Dahmani, Hassen-Reda; Delouche, Pamina; Bidabe, Marissa; Schneeberger, Patricia

2012-01-01

The large number of experimentally determined molecular structures has led to the development of a new semiotic system in the life sciences, with increasing use of accurate molecular representations. To determine how this change impacts students' learning, we incorporated image tests into our introductory cell biology course. Groups of students used a single text dealing with signal transduction, which was supplemented with images made in one of three iconographic styles. Typically, we employed realistic renderings, using computer-generated Protein Data Bank (PDB) structures; realistic-schematic renderings, using shapes inspired by PDB structures; or schematic renderings, using simple geometric shapes to represent cellular components. The control group received a list of keywords. When students were asked to draw and describe the process in their own style and to reply to multiple-choice questions, the three iconographic approaches equally improved the overall outcome of the tests (relative to keywords). Students found the three approaches equally useful but, when asked to select a preferred style, they largely favored a realistic-schematic style. When students were asked to annotate "raw" realistic images, both keywords and schematic representations failed to prepare them for this task. We conclude that supplementary images facilitate the comprehension process and despite their visual clutter, realistic representations do not hinder learning in an introductory course.

Exosomes promote cetuximab resistance via the PTEN/Akt pathway in colon cancer cells.

PubMed

Zhang, S; Zhang, Y; Qu, J; Che, X; Fan, Y; Hou, K; Guo, T; Deng, G; Song, N; Li, C; Wan, X; Qu, X; Liu, Y

2017-11-13

Cetuximab is widely used in patients with metastatic colon cancer expressing wildtype KRAS. However, acquired drug resistance limits its clinical efficacy. Exosomes are nanosized vesicles secreted by various cell types. Tumor cell-derived exosomes participate in many biological processes, including tumor invasion, metastasis, and drug resistance. In this study, exosomes derived from cetuximab-resistant RKO colon cancer cells induced cetuximab resistance in cetuximab-sensitive Caco-2 cells. Meanwhile, exosomes from RKO and Caco-2 cells showed different levels of phosphatase and tensin homolog (PTEN) and phosphor-Akt. Furthermore, reduced PTEN and increased phosphorylated Akt levels were found in Caco-2 cells after exposure to RKO cell-derived exosomes. Moreover, an Akt inhibitor prevented RKO cell-derived exosome-induced drug resistance in Caco-2 cells. These findings provide novel evidence that exosomes derived from cetuximab-resistant cells could induce cetuximab resistance in cetuximab-sensitive cells, by downregulating PTEN and increasing phosphorylated Akt levels.

Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110

PubMed Central

Deisting, Wibke; Raum, Tobias; Kufer, Peter; Baeuerle, Patrick A.; Münz, Markus

2015-01-01

Background Bispecific T cell engager (BiTE®) are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a polyclonal cytotoxic T cell response that is not restricted by T cell receptor (TCR) specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3-bispecific BiTE® antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl-2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-β), interleukin-10 (IL-10) and adenosine in culture medium can impact redirected lysis by AMG 110-engaged T cells. Methods The seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO) target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co-culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE®-induced proliferation of previously resting human peripheral T cells. Findings An inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overexpression of serpin PI-9, Bcl-2, TGF-βand PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE®-induced lysis, and even various combinations could not. Conclusions Our data suggest that diverse mechanisms employed by cancer cells to fend off T cells cannot inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE® antibody construct. PMID:26510188

6-DoF Haptic Rendering Using Continuous Collision Detection between Points and Signed Distance Fields.

PubMed

Hongyi Xu; Barbic, Jernej

2017-01-01

We present an algorithm for fast continuous collision detection between points and signed distance fields, and demonstrate how to robustly use it for 6-DoF haptic rendering of contact between objects with complex geometry. Continuous collision detection is often needed in computer animation, haptics, and virtual reality applications, but has so far only been investigated for polygon (triangular) geometry representations. We demonstrate how to robustly and continuously detect intersections between points and level sets of the signed distance field. We suggest using an octree subdivision of the distance field for fast traversal of distance field cells. We also give a method to resolve continuous collisions between point clouds organized into a tree hierarchy and a signed distance field, enabling rendering of contact between rigid objects with complex geometry. We investigate and compare two 6-DoF haptic rendering methods now applicable to point-versus-distance field contact for the first time: continuous integration of penalty forces, and a constraint-based method. An experimental comparison to discrete collision detection demonstrates that the continuous method is more robust and can correctly resolve collisions even under high velocities and during complex contact.

Effects of host resistance and injury on the susceptibility of Aedes taeniorhynchus to mosquito iridescent virus.

PubMed

Undeen, A H; Fukuda, T

1994-03-01

An iridescent virus is found at low prevalence in population of Aedes taenirhynchus. Attempts at experimental transmission produced low levels of infection, regardless of the dosage applied. In a test for genetic resistance in colonized Ae. taeniorhynchus, the mean infection rates +/- SD for groups of randomly selected and sibling larvae were compared. The standard deviation of the sibling groups was not higher than the random groups (4.0 +/- 3.1% and 3.0 +/- 2.1%), rendering genetic resistance unlikely. Injury to the larvae by feeding silicon carbide fibers consistently caused higher infection rates (4.8 +/- 2.0% by virus alone and 17.5 +/- 5.3% by virus and fibers concurrently). Similar results were obtained for vertical transmission. These results support the hypothesis that this virus has no active means of penetration, invading only through random breaks in the cuticle or peritrophic membrane.

Treatment of Gram-negative bacterial infections by potentiation of antibiotics.

PubMed

Zabawa, Thomas P; Pucci, Michael J; Parr, Thomas R; Lister, Troy

2016-10-01

Infections caused by antibiotic-resistant pathogens, particularly Gram-negative bacteria, represent significant treatment challenges for physicians resulting in high rates of morbidity and mortality. The outer membrane of Gram-negative bacteria acts as a permeability barrier to many compounds that would otherwise be effective antibacterial agents, including those effective against Gram-positive pathogens. Potentiator molecules disrupt this barrier allowing entry of otherwise impermeant molecules, thus providing a strategy to render multi-drug resistant pathogens susceptible to a broader range of antibiotics. Potentiator molecules are cationic and the mechanism of disruption involves interaction with the negatively charged outer membrane. This physical attribute, along with an often high degree of lipophilicity typically endears these molecules with unacceptable toxicity. Presented herein are examples of advanced potentiator molecules being evaluated for use in combination therapy for the treatment of resistant Gram-negative infections. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enhancing caries resistance with a short-pulsed CO2 9.3-μm laser: a laboratory study (Conference Presentation)

NASA Astrophysics Data System (ADS)

Rechmann, Peter; Rechmann, Beate M.; Groves, William H.; Le, Charles; Rapozo-Hilo, Marcia L.; Featherstone, John D. B.

2016-02-01

The objective of this laboratory study was to test whether irradiation with a new 9.3µm microsecond short-pulsed CO2-laser enhances enamel caries resistance with and without additional fluoride applications. 101 human enamel samples were divided into 7 groups. Each group was treated with different laser parameters (Carbon-dioxide laser, wavelength 9.3µm, 43Hz pulse-repetition rate, pulse duration between 3μs to 7μs (1.5mJ/pulse to 2.9mJ/pulse). Using a pH-cycling model and cross-sectional microhardness testing determined the mean relative mineral loss delta Z (∆Z) for each group. The pH-cycling was performed with or without additional fluoride. The CO2 9.3μm short-pulsed laser energy rendered enamel caries resistant with and without additional fluoride use.

Isoliquiritigenin, a strong nod gene- and glyceollin resistance-inducing flavonoid from soybean root exudate.

PubMed

Kape, R; Parniske, M; Brandt, S; Werner, D

1992-05-01

Isoflavonoid signal molecules from soybean (Glycine max (L.) Merr.) seed and root exudate induce the transcription of nodulation (nod) genes in Bradyrhizobium japonicum. In this study, a new compound with symbiotic activity was isolated from soybean root exudate. The isolated 2',4',4-trihydroxychalcone (isoliquiritigenin) is characterized by its strong inducing activity for the nod genes of B. japonicum. These genes are already induced at concentrations 1 order of magnitude below those required of the previously described isoflavonoid inducers genistein and daidzein. Isoliquiritigenin is also a potent inducer of glyceollin resistance in B. japonicum, which renders this bacterium insensitive to potentially bactericidal concentrations of glyceollin, the phytoalexin of G. max. No chemotactic effect of isoliquiritigenin was observed. The highly efficient induction of nod genes and glyceollin resistance by isoliquiritigenin suggests the ecological significance of this compound, although it is not a major flavonoid constituent of the soybean root exudate in quantitative terms.

Effects of sample treatments on genome recovery via single-cell genomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clingenpeel, Scott; Schwientek, Patrick; Hugenholtz, Philip

2014-06-13

It is known that single-cell genomics is a powerful tool for accessing genetic information from uncultivated microorganisms. Methods of handling samples before single-cell genomic amplification may affect the quality of the genomes obtained. Using three bacterial strains we demonstrate that, compared to cryopreservation, lower-quality single-cell genomes are recovered when the sample is preserved in ethanol or if the sample undergoes fluorescence in situ hybridization, while sample preservation in paraformaldehyde renders it completely unsuitable for sequencing.

Evaluation of Aminoglycoside and Carbapenem Resistance in a Collection of Drug-Resistant Pseudomonas aeruginosa Clinical Isolates.

PubMed

Holbrook, Selina Y L; Garneau-Tsodikova, Sylvie

2017-12-20

Pseudomonas aeruginosa, a Gram-negative bacterium, is a member of the ESKAPE pathogens and one of the leading causes of healthcare-associated infections worldwide. Aminoglycosides (AGs) are recognized for their efficacy against P. aeruginosa. The most common resistance mechanism against AGs is the acquisition of AG-modifying enzymes (AMEs) by the bacteria, including AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases (ANTs). In this study, we obtained 122 multidrug-resistant P. aeruginosa clinical isolates and evaluated the antibacterial effects of six AGs and two carbapenems alone against all clinical isolates, and in combination against eight selected strains. We further probed for four representatives of the most common AME genes [aac(6')-Ib, aac(3)-IV, ant(2")-Ia, and aph(3')-Ia] by polymerase chain reaction (PCR) and compared the AME patterns of these 122 clinical isolates to their antibiotic resistance profile. Among the diverse antibiotics resistance profile displayed by these clinical isolates, we found correlations between the resistance to various AGs as well as between the resistance to one AG and the resistance to carbapenems. PCR results revealed that the presence of aac(6')-Ib renders these isolates more resistant to a variety of antibiotics. The correlation between resistance to various AGs and carbapenems partially reflects the complex resistance strategies adapted in these pathogens and encourages the development of strategic treatment for each P. aeruginosa infection by considering the genetic information of each isolated bacteria.

Downregulation of PMCA2 increases the vulnerability of midbrain neurons to mitochondrial complex I inhibition.

PubMed

Brendel, Alexander; Renziehausen, Jana; Behl, Christian; Hajieva, Parvana

2014-01-01

Parkinson's disease is an age-associated disorder characterized by selective degeneration of dopaminergic neurons. The molecular mechanisms underlying the selective vulnerability of this subset of neurons are, however, not fully understood. Employing SH-SY5Y neuroblastoma cells and primary mesencephalic neurons, we here demonstrate a significant increase in cytosolic calcium after inhibition of mitochondrial complex I by means of MPP(+), which is a well-established environmental toxin-based in vitro model of Parkinson's disease. This increase in calcium is correlated with a downregulation of the neuron-specific plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2). Interestingly, two other important mediators of calcium efflux, sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), and Na(+)-Ca(2+)-exchanger (NCX), remained unaltered, indicating a specific role of PMCA2 in maintaining calcium homeostasis in neurons. The observed PMCA2 downregulation was accompanied by reduced levels of phosphorylated CREB protein, an intracellular signaling molecule and transcriptional regulator. In order to investigate the potential influence of PMCA2 on neuronal vulnerability, experimental downregulation of PMCA2 by means of siRNA was performed. The results demonstrate a significant impairment of cell survival under conditions of PMCA2 suppression. Hence, in our cell models increased cytosolic calcium levels as a consequence of insufficient calcium efflux lead to an increased vulnerability of neuronal cells. Moreover, overexpression of PMCA2 rendered the neurons significantly resistant to complex I inhibition. Our findings point toward a dysregulation of calcium homeostasis in Parkinson's disease and suggest a potential molecular mechanism of neurodegeneration via PMCA2. Copyright © 2013 Elsevier Inc. All rights reserved.

Activated STAT5 promotes long-lived cytotoxic CD8+ T cells that induce regression of autochthonous melanoma.

PubMed

Grange, Magali; Buferne, Michel; Verdeil, Grégory; Leserman, Lee; Schmitt-Verhulst, Anne-Marie; Auphan-Anezin, Nathalie

2012-01-01

Immunotherapy based on adoptive transfer of tumor antigen-specific CD8(+) T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8(+) effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytoma- or melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti-IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8(+) TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs. ©2011 AACR.

p75 Neurotrophin Receptor in the Skin: Beyond Its Neurotrophic Function.

PubMed

Pincelli, Carlo

2017-01-01

p75 neurotrophin receptor (p75 NTR ), also known as CD271, is the low-affinity receptor that, together with the tyrosine kinase receptor tropomyosin-receptor kinase (Trk), mediate neurotrophin (NT) functions. Beside their classic role in skin innervation, NT and their receptors constitute a complex cutaneous network associated with a number of autocrine and paracrine activities. In this context, the role of p75 NTR is becoming more and more important. This review will focus on the intriguing functions of p75 NTR in healthy and diseased skin. First, p75 NTR counterbalances the proliferative and survival activities of its cognate receptor Trk by inducing keratinocyte apoptosis. In addition, p75 NTR identifies an early transit-amplifying (TA) keratinocyte population and plays a critical role in keratinocyte stem cell transition to its progeny as well as in epidermal differentiation. p75 NTR is absent in psoriatic TA cells, thus rendering these cells resistant to apoptosis. On the other hand, p75 NTR infection restores NT-induced apoptosis in psoriatic keratinocytes. Taken together, these results provide evidence for a critical role of p75 NTR in epidermal homeostasis, while its lack may account for the TA defect in psoriasis. While the issue of p75 NTR as a marker of melanoma initiating cells is still to be solved, there is strong evidence that downregulation of this receptor is a precondition to melanoma invasion and metastasis in vitro and in vivo . All in all, this review points to p75 NTR as a major actor in both physiologic and pathologic conditions at the skin level.

Longitudinal Multiplexed Measurement of Quantitative Proteomic Signatures in Mouse Lymphoma Models Using Magneto-Nanosensors

PubMed Central

Lee, Jung-Rok; Appelmann, Iris; Miething, Cornelius; Shultz, Tyler O.; Ruderman, Daniel; Kim, Dokyoon; Mallick, Parag; Lowe, Scott W.; Wang, Shan X.

2018-01-01

Cancer proteomics is the manifestation of relevant biological processes in cancer development. Thus, it reflects the activities of tumor cells, host-tumor interactions, and systemic responses to cancer therapy. To understand the causal effects of tumorigenesis or therapeutic intervention, longitudinal studies are greatly needed. However, most of the conventional mouse experiments are unlikely to accommodate frequent collection of serum samples with a large enough volume for multiple protein assays towards single-object analysis. Here, we present a technique based on magneto-nanosensors to longitudinally monitor the protein profiles in individual mice of lymphoma models using a small volume of a sample for multiplex assays. Methods: Drug-sensitive and -resistant cancer cell lines were used to develop the mouse models that render different outcomes upon the drug treatment. Two groups of mice were inoculated with each cell line, and treated with either cyclophosphamide or vehicle solution. Serum samples taken longitudinally from each mouse in the groups were measured with 6-plex magneto-nanosensor cytokine assays. To find the origin of IL-6, experiments were performed using IL-6 knock-out mice. Results: The differences in serum IL-6 and GCSF levels between the drug-treated and untreated groups were revealed by the magneto-nanosensor measurement on individual mice. Using the multiplex assays and mouse models, we found that IL-6 is secreted by the host in the presence of tumor cells upon the drug treatment. Conclusion: The multiplex magneto-nanosensor assays enable longitudinal proteomic studies on mouse tumor models to understand tumor development and therapy mechanisms more precisely within a single biological object. PMID:29507628

Longitudinal Multiplexed Measurement of Quantitative Proteomic Signatures in Mouse Lymphoma Models Using Magneto-Nanosensors.

PubMed

Lee, Jung-Rok; Appelmann, Iris; Miething, Cornelius; Shultz, Tyler O; Ruderman, Daniel; Kim, Dokyoon; Mallick, Parag; Lowe, Scott W; Wang, Shan X

2018-01-01

Cancer proteomics is the manifestation of relevant biological processes in cancer development. Thus, it reflects the activities of tumor cells, host-tumor interactions, and systemic responses to cancer therapy. To understand the causal effects of tumorigenesis or therapeutic intervention, longitudinal studies are greatly needed. However, most of the conventional mouse experiments are unlikely to accommodate frequent collection of serum samples with a large enough volume for multiple protein assays towards single-object analysis. Here, we present a technique based on magneto-nanosensors to longitudinally monitor the protein profiles in individual mice of lymphoma models using a small volume of a sample for multiplex assays. Methods: Drug-sensitive and -resistant cancer cell lines were used to develop the mouse models that render different outcomes upon the drug treatment. Two groups of mice were inoculated with each cell line, and treated with either cyclophosphamide or vehicle solution. Serum samples taken longitudinally from each mouse in the groups were measured with 6-plex magneto-nanosensor cytokine assays. To find the origin of IL-6, experiments were performed using IL-6 knock-out mice. Results: The differences in serum IL-6 and GCSF levels between the drug-treated and untreated groups were revealed by the magneto-nanosensor measurement on individual mice. Using the multiplex assays and mouse models, we found that IL-6 is secreted by the host in the presence of tumor cells upon the drug treatment. Conclusion: The multiplex magneto-nanosensor assays enable longitudinal proteomic studies on mouse tumor models to understand tumor development and therapy mechanisms more precisely within a single biological object.

The Function of Neuroendocrine Cells in Prostate Cancer

DTIC Science & Technology

2012-04-01

high profile article describing the technology developed by our group (Goldstein et al. Nat Protoc. 2011; 6:656-67). We explored the utility of...UGSM cells and transplanted in vivo into immune-deficient mice. We utilize an activated form of AKT (myristoylated rendering it membrane-bound) which... utilized for research can vary greatly. The second possibility is that the SV40 T-antigen is toxic to naïve benign primary human prostate cells when

Intracellular Targeting Specificity of Novel Phthalocyanines Assessed in a Host-Parasite Model for Developing Potential Photodynamic Medicine

PubMed Central

Dutta, Sujoy; Ongarora, Benson G.; Li, Hairong; Vicente, Maria da Graca H.; Kolli, Bala K.; Chang, Kwang Poo

2011-01-01

Photodynamic therapy, unlikely to elicit drug-resistance, deserves attention as a strategy to counter this outstanding problem common to the chemotherapy of all diseases. Previously, we have broadened the applicability of this modality to photodynamic vaccination by exploiting the unusual properties of the trypanosomatid protozoa, Leishmania, i.e., their innate ability of homing to the phagolysosomes of the antigen-presenting cells and their selective photolysis therein, using transgenic mutants endogenously inducible for porphyrin accumulation. Here, we extended the utility of this host-parasite model for in vitro photodynamic therapy and vaccination by exploring exogenously supplied photosensitizers. Seventeen novel phthalocyanines (Pcs) were screened in vitro for their photolytic activity against cultured Leishmania. Pcs rendered cationic and soluble (csPcs) for cellular uptake were phototoxic to both parasite and host cells, i.e., macrophages and dendritic cells. The csPcs that targeted to mitochondria were more photolytic than those restricted to the endocytic compartments. Treatment of infected cells with endocytic csPcs resulted in their accumulation in Leishmania-containing phagolysosomes, indicative of reaching their target for photodynamic therapy, although their parasite versus host specificity is limited to a narrow range of csPc concentrations. In contrast, Leishmania pre-loaded with csPc were selectively photolyzed intracellularly, leaving host cells viable. Pre-illumination of such csPc-loaded Leishmania did not hinder their infectivity, but ensured their intracellular lysis. Ovalbumin (OVA) so delivered by photo-inactivated OVA transfectants to mouse macrophages and dendritic cells were co-presented with MHC Class I molecules by these antigen presenting cells to activate OVA epitope-specific CD8+T cells. The in vitro evidence presented here demonstrates for the first time not only the potential of endocytic csPcs for effective photodynamic therapy against Leishmania but also their utility in photo-inactivation of Leishmania to produce a safe carrier to express and deliver a defined antigen with enhanced cell-mediated immunity. PMID:21673971

The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas

Research highlights: {yields} Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; {yields} Kaempferol causes cytoplasmic mislocalization of HIF-1{alpha} by impairing the MAPK pathway. {yields} Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1{alpha} subunit, an important target of anti-cancer therapy, is observed in many cancers includingmore » HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1{alpha} as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC{sub 50} = 5.16 {mu}M). The mechanism of this inhibition did not involve suppression of HIF-1{alpha} protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC{sub 50} = 4.75 {mu}M). Exposure of Huh7 cells to 10 {mu}{Mu} kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 {mu}M) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.« less

Deficiency of Huntingtin Has Pleiotropic Effects in the Social Amoeba Dictyostelium discoideum

PubMed Central

Myre, Michael A.; Lumsden, Amanda L.; Thompson, Morgan N.; Wasco, Wilma; MacDonald, Marcy E.; Gusella, James F.

2011-01-01

Huntingtin is a large HEAT repeat protein first identified in humans, where a polyglutamine tract expansion near the amino terminus causes a gain-of-function mechanism that leads to selective neuronal loss in Huntington's disease (HD). Genetic evidence in humans and knock-in mouse models suggests that this gain-of-function involves an increase or deregulation of some aspect of huntingtin's normal function(s), which remains poorly understood. As huntingtin shows evolutionary conservation, a powerful approach to discovering its normal biochemical role(s) is to study the effects caused by its deficiency in a model organism with a short life-cycle that comprises both cellular and multicellular developmental stages. To facilitate studies aimed at detailed knowledge of huntingtin's normal function(s), we generated a null mutant of hd, the HD ortholog in Dictyostelium discoideum. Dictyostelium cells lacking endogenous huntingtin were viable but during development did not exhibit the typical polarized morphology of Dictyostelium cells, streamed poorly to form aggregates by accretion rather than chemotaxis, showed disorganized F-actin staining, exhibited extreme sensitivity to hypoosmotic stress, and failed to form EDTA-resistant cell–cell contacts. Surprisingly, chemotactic streaming could be rescued in the presence of the bivalent cations Ca2+ or Mg2+ but not pulses of cAMP. Although hd − cells completed development, it was delayed and proceeded asynchronously, producing small fruiting bodies with round, defective spores that germinated spontaneously within a glassy sorus. When developed as chimeras with wild-type cells, hd − cells failed to populate the pre-spore region of the slug. In Dictyostelium, huntingtin deficiency is compatible with survival of the organism but renders cells sensitive to low osmolarity, which produces pleiotropic cell autonomous defects that affect cAMP signaling and as a consequence development. Thus, Dictyostelium provides a novel haploid organism model for genetic, cell biological, and biochemical studies to delineate the functions of the HD protein. PMID:21552328

Comparative Expression Profiling of Distinct T Cell Subsets Undergoing Oxidative Stress

PubMed Central

Lichtenfels, Rudolf; Mougiakakos, Dimitrios; Johansson, C. Christian; Dressler, Sven P.; Recktenwald, Christian V.; Kiessling, Rolf; Seliger, Barbara

2012-01-01

The clinical outcome of adoptive T cell transfer-based immunotherapies is often limited due to different escape mechanisms established by tumors in order to evade the hosts' immune system. The establishment of an immunosuppressive micromilieu by tumor cells along with distinct subsets of tumor-infiltrating lymphocytes is often associated with oxidative stress that can affect antigen-specific memory/effector cytotoxic T cells thereby substantially reducing their frequency and functional activation. Therefore, protection of tumor-reactive cytotoxic T lymphocytes from oxidative stress may enhance the anti-tumor-directed immune response. In order to better define the key pathways/proteins involved in the response to oxidative stress a comparative 2-DE-based proteome analysis of naïve CD45RA+ and their memory/effector CD45RO+ T cell counterparts in the presence and absence of low dose hydrogen peroxide (H2O2) was performed in this pilot study. Based on the profiling data of these T cell subpopulations under the various conditions, a series of differentially expressed spots were defined, members thereof identified by mass spectrometry and subsequently classified according to their cellular function and localization. Representative targets responding to oxidative stress including proteins involved in signaling pathways, in regulating the cellular redox status as well as in shaping/maintaining the structural cell integrity were independently verified at the transcript and protein level under the same conditions in both T cell subsets. In conclusion the resulting profiling data describe complex, oxidative stress-induced, but not strictly concordant changes within the respective expression profiles of CD45RA+ and CD45RO+ T cells. Some of the differentially expressed genes/proteins might be further exploited as potential targets toward modulating the redox capacity of the distinct lymphocyte subsets thereby providing the basis for further studies aiming at rendering them more resistant to tumor micromilieu-induced oxidative stress. PMID:22911781

Crystal structure of KPC-2

PubMed Central

Ke, Wei; Bethel, Christopher R.; Thomson, Jodi M.; Bonomo, Robert A.; van den Akker, Focco

2008-01-01

β-lactamases inactivate β-lactam antibiotics and are a major cause of antibiotic resistance. The recent outbreaks of Klebsiella pneumoniae carbapenem-resistant (KPC) infections mediated by KPC type β-lactamases are creating a serious threat to our “last resort” antibiotics, the carbapenems. KPC β-lactamases are thus carbapenemases and are a subclass of Class A β-lactamases that have evolved to efficiently hydrolyze carbapenems and cephamycins which contain substitutions at the α position proximal to the carbonyl group that normally render these β-lactams resistant to hydrolysis. To investigate the molecular basis of this carbapenemase activity, we have determined the structure of KPC-2 at 1.85Å resolution. The active site of KPC-2 reveals the presence of a bicine buffer molecule which interacts via its carboxyl group with conserved active site residues S130, K234, T235, and T237; this likely resembles the interactions the β-lactam carboxyl moiety makes in the Michaelis-Menten complex. Comparison of the KPC-2 structure with non-carbapenemases and previously determined NMC-A and SME-1 carbapenemase structures shows several active site alterations that are unique among carbapenemases. An outward shift of the catalytic S70 residue renders the active sites of the carbapenemases more shallow likely allowing easier access of the bulkier substrates. Further space for the α-substituents is likely provided by shifts in N132 and N170 in addition to concerted movements in the postulated carboxyl binding pocket that might allow the substrates to bind in a slightly different angle to accommodate these α-substituents. The structure of KPC-2 thus provides key insights into the carbapenemase activity of emerging Class A β-lactamases. PMID:17441734

Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid.

PubMed

Toh, Seok-Ming; Xiong, Liqun; Arias, Cesar A; Villegas, Maria V; Lolans, Karen; Quinn, John; Mankin, Alexander S

2007-06-01

Linezolid, which targets the ribosome, is a new synthetic antibiotic that is used for treatment of infections caused by Gram-positive pathogens. Clinical resistance to linezolid, so far, has been developing only slowly and has involved exclusively target site mutations. We have discovered that linezolid resistance in a methicillin-resistant Staphylococcus aureus hospital strain from Colombia is determined by the presence of the cfr gene whose product, Cfr methyltransferase, modifies adenosine at position 2503 in 23S rRNA in the large ribosomal subunit. The molecular model of the linezolid-ribosome complex reveals localization of A2503 within the drug binding site. The natural function of cfr likely involves protection against natural antibiotics whose site of action overlaps that of linezolid. In the chromosome of the clinical strain, cfr is linked to ermB, a gene responsible for dimethylation of A2058 in 23S rRNA. Coexpression of these two genes confers resistance to all the clinically relevant antibiotics that target the large ribosomal subunit. The association of the ermB/cfr operon with transposon and plasmid genetic elements indicates its possible mobile nature. This is the first example of clinical resistance to the synthetic drug linezolid which involves a natural resistance gene with the capability of disseminating among Gram-positive pathogenic strains.

Recognizing the real threat of biological terror.

PubMed

Wenzel, Richard P

2002-01-01

Weapons of mass destruction can be used to harm and terrorize populations. Such weapons include those with chemical, nuclear or biological properties. Obviously computer viruses can add additional barriers to a quick response. The most effective, least costly and greatest threats are biologicals. Biological terror is not new, and biological weapons have been used for centuries. However, as a result of modern technology, the risks are greater now and the outcomes more terrible. Today they include live pathogens, various toxins, and theoretically "bioregulators"--biochemicals affecting cell signaling. Altered cell signaling could be used to induce apoptosis-cell death, or a heightened outpouring of cytokines mimicking overwhelming sepsis, or even an intracellular, biochemical "strike" causing cellular paralysis. Biological weaponeers now have the frightening ability to alter the genetic makeup of pathogens, rendering them resistant not only to available antibiotic therapy but also to currently effective vaccines. In dark corners of some fringe groups, bioweaponeers are searching for the capability of designing pathogens that target specific races, by virtue of discriminating ligands (1). The resulting morbidity and mortality from use of any biological weapons will be accompanied by chaos, governmental and social instability, panic, an extraordinary utilization of available resources, and an ongoing epidemic of sleepless nights (2,3). Herein I will review some of the issues and some of the currently available biological weapons. The major goal is to highlight the clinical presentations of patients with infections that could be used as biological weapons.

Therapeutic potential of MEK inhibition in acute myelogenous leukemia: rationale for "vertical" and "lateral" combination strategies.

PubMed

Ricciardi, Maria Rosaria; Scerpa, Maria Cristina; Bergamo, Paola; Ciuffreda, Ludovica; Petrucci, Maria Teresa; Chiaretti, Sabina; Tavolaro, Simona; Mascolo, Maria Grazia; Abrams, Stephen L; Steelman, Linda S; Tsao, Twee; Marchetti, Antonio; Konopleva, Marina; Del Bufalo, Donatella; Cognetti, Francesco; Foà, Robin; Andreeff, Michael; McCubrey, James A; Tafuri, Agostino; Milella, Michele

2012-10-01

In hematological malignancies, constitutive activation of the RAF/MEK/ERK pathway is frequently observed, conveys a poor prognosis, and constitutes a promising target for therapeutic intervention. Here, we investigated the molecular and functional effects of pharmacological MEK inhibition in cell line models of acute myeloid leukemia (AML) and freshly isolated primary AML samples. The small-molecule, ATP-non-competitive, MEK inhibitor PD0325901 markedly inhibited ERK phosphorylation and growth of several AML cell lines and approximately 70 % of primary AML samples. Growth inhibition was due to G(1)-phase arrest and induction of apoptosis. Transformation by constitutively active upstream pathway elements (HRAS, RAF-1, and MEK) rendered FDC-P1 cells exquisitely prone to PD0325901-induced apoptosis. Gene and protein expression profiling revealed a selective effect of PD0325901 on ERK phosphorylation and compensatory upregulation of the RAF/MEK and AKT/p70( S6K ) kinase modules, potentially mediating resistance to drug-induced growth inhibition. Consequently, in appropriate cellular contexts, both "vertical" (i.e., inhibition of RAF and MEK along the MAPK pathway) and "lateral" (i.e., simultaneous inhibition of the MEK/ERK and mTOR pathways) combination strategies may result in synergistic anti-leukemic effects. Overall, MEK inhibition exerts potent growth inhibitory and proapoptotic activity in preclinical models of AML, particularly in combination with other pathway inhibitors. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective targeted strategies for the treatment of AML.

Chronic corticosterone affects brain weight, and mitochondrial, but not glial volume fraction in hippocampal area CA3.

PubMed

Coburn-Litvak, P S; Tata, D A; Gorby, H E; McCloskey, D P; Richardson, G; Anderson, B J

2004-01-01

Corticosterone (CORT), the predominant glucocorticoid in rodents, is known to damage hippocampal area CA3. Here we investigate how that damage is represented at the cellular and ultrastructural level of analyses. Rats were injected with CORT (26.8 mg/kg, s.c.) or vehicle for 56 days. Cell counts were estimated with the physical disector method. Glial and mitochondrial volume fractions were obtained from electron micrographs. The effectiveness of the CORT dose used was demonstrated in two ways. First, CORT significantly inhibited body weight gain relative to vehicles. Second, CORT significantly reduced adrenal gland, heart and gastrocnemius muscle weight. Both the adrenal and gastrocnemius muscle weight to body weight ratios were also significantly reduced. Although absolute brain weight was reduced, the brain to body weight ratio was higher in the CORT group relative to vehicles, suggesting that the brain is more resistant to the effects of CORT than many peripheral organs and muscles. Consistent with that interpretation, CORT did not alter CA3 cell density, cell layer volume, or apical dendritic neuropil volume. Likewise, CORT did not significantly alter glial volume fraction, but did reduce mitochondrial volume fraction. These findings highlight the need for ultrastructural analyses in addition to cellular level analyses before conclusions can be drawn about the damaging effects of prolonged CORT elevations. The relative reduction in mitochondria may indicate a reduction in bioenergetic capacity that, in turn, could render CA3 vulnerable to metabolic challenges.

Divide and conquer: The Pseudomonas aeruginosa two-component hybrid SagS enables biofilm formation and recalcitrance of biofilm cells to antimicrobial agents via distinct regulatory circuits

PubMed Central

Petrova, Olga E.; Gupta, Kajal; Liao, Julie; Goodwine, James S.; Sauer, Karin

2017-01-01

The opportunistic pathogen Pseudomonas aeruginosa forms antimicrobial resistant biofilms through sequential steps requiring several two-component regulatory systems. The sensor-regulator hybrid SagS plays a central role in biofilm development by enabling the switch from the planktonic to the biofilm mode of growth, and by facilitating the transition of biofilm cells to a highly tolerant state. However, the mechanism by which SagS accomplishes both functions is unknown. SagS harbors a periplasmic sensory HmsP, and phosphorelay HisKA and Rec domains. We used SagS domain constructs and site-directed mutagenesis to elucidate how SagS performs its dual functions. We demonstrate that HisKA-Rec and the phospho-signaling between SagS and BfiS contribute to the switch to the biofilm mode of growth, but not to the tolerant state. Instead, expression of SagS domain constructs harboring HmsP rendered ΔsagS biofilm cells as recalcitrant to antimicrobial agents as wild-type biofilms, likely by restoring BrlR production and cellular c-di-GMP levels to wild-type levels. Restoration of biofilm tolerance by HmsP was independent of biofilm biomass accumulation, RsmA, RsmYZ, HptB, and BfiSR-downstream targets. Our findings thus suggest that SagS likely makes use of a “divide-and-conquer” mechanism to regulate its dual switch function, by activating two distinct regulatory networks via its individual domains. PMID:28263038

The effect of aqueous speciation and cellular ligand binding on the biotransformation and bioavailability of methylmercury in mercury-resistant bacteria.

PubMed

Ndu, Udonna; Barkay, Tamar; Schartup, Amina Traore; Mason, Robert P; Reinfelder, John R

2016-02-01

Mercury resistant bacteria play a critical role in mercury biogeochemical cycling in that they convert methylmercury (MeHg) and inorganic mercury to elemental mercury, Hg(0). To date there are very few studies on the effects of speciation and bioavailability of MeHg in these organisms, and even fewer studies on the role that binding to cellular ligands plays on MeHg uptake. The objective of this study was to investigate the effects of thiol complexation on the uptake of MeHg by measuring the intracellular demethylation-reduction (transformation) of MeHg to Hg(0) in Hg-resistant bacteria. Short-term intracellular transformation of MeHg was quantified by monitoring the loss of volatile Hg(0) generated during incubations of bacteria containing the complete mer operon (including genes from putative mercury transporters) exposed to MeHg in minimal media compared to negative controls with non-mer or heat-killed cells. The results indicate that the complexes MeHgOH, MeHg-cysteine, and MeHg-glutathione are all bioavailable in these bacteria, and without the mer operon there is very little biological degradation of MeHg. In both Pseudomonas stutzeri and Escherichia coli, there was a pool of MeHg that was not transformed to elemental Hg(0), which was likely rendered unavailable to Mer enzymes by non-specific binding to cellular ligands. Since the rates of MeHg accumulation and transformation varied more between the two species of bacteria examined than among MeHg complexes, microbial bioavailability, and therefore microbial demethylation, of MeHg in aquatic systems likely depends more on the species of microorganism than on the types and relative concentrations of thiols or other MeHg ligands present.

Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)*

PubMed Central

van der Post, Sjoerd; Subramani, Durai B.; Bäckström, Malin; Johansson, Malin E. V.; Vester-Christensen, Malene B.; Mandel, Ulla; Bennett, Eric P.; Clausen, Henrik; Dahlén, Gunnar; Sroka, Aneta; Potempa, Jan; Hansson, Gunnar C.

2013-01-01

The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation. PMID:23546879

Effective and specific in planta RNAi in cyst nematodes: expression interference of four parasitism genes reduces parasitic success.

PubMed

Sindhu, Anoop S; Maier, Tom R; Mitchum, Melissa G; Hussey, Richard S; Davis, Eric L; Baum, Thomas J

2009-01-01

Cyst nematodes are highly evolved sedentary plant endoparasites that use parasitism proteins injected through the stylet into host tissues to successfully parasitize plants. These secretory proteins likely are essential for parasitism as they are involved in a variety of parasitic events leading to the establishment of specialized feeding cells required by the nematode to obtain nourishment. With the advent of RNA interference (RNAi) technology and the demonstration of host-induced gene silencing in parasites, a new strategy to control pests and pathogens has become available, particularly in root-knot nematodes. Plant host-induced silencing of cyst nematode genes so far has had only limited success but similarly should disrupt the parasitic cycle and render the host plant resistant. Additional in planta RNAi data for cyst nematodes are being provided by targeting four parasitism genes through host-induced RNAi gene silencing in transgenic Arabidopsis thaliana, which is a host for the sugar beet cyst nematode Heterodera schachtii. Here it is reported that mRNA abundances of targeted nematode genes were specifically reduced in nematodes feeding on plants expressing corresponding RNAi constructs. Furthermore, this host-induced RNAi of all four nematode parasitism genes led to a reduction in the number of mature nematode females. Although no complete resistance was observed, the reduction of developing females ranged from 23% to 64% in different RNAi lines. These observations demonstrate the relevance of the targeted parasitism genes during the nematode life cycle and, potentially more importantly, suggest that a viable level of resistance in crop plants may be accomplished in the future using this technology against cyst nematodes.

Intracellular Copper Does Not Catalyze the Formation of Oxidative DNA Damage in Escherichia coli▿

PubMed Central

Macomber, Lee; Rensing, Christopher; Imlay, James A.

2007-01-01

Because copper catalyzes the conversion of H2O2 to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity. Elimination of the copper export genes, copA, cueO, and cusCFBA, rendered Escherichia coli sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the copper-loaded mutants were actually less sensitive to killing by H2O2 than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative PCR analysis confirmed that copper decreased the rate at which H2O2 damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H2O2 resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl radical formation. However, copper EPR spectroscopy suggested that the majority of H2O2-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in this compartment and away from the DNA. Indeed, while E. coli responds to H2O2 stress by inducing iron sequestration proteins, H2O2-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore, copper toxicity must occur by a different mechanism. PMID:17189367

TNF-dependent regulation and activation of innate immune cells are essential for host protection against cerebral tuberculosis.

PubMed

Francisco, Ngiambudulu M; Hsu, Nai-Jen; Keeton, Roanne; Randall, Philippa; Sebesho, Boipelo; Allie, Nasiema; Govender, Dhirendra; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston; Jacobs, Muazzam

2015-06-26

Tuberculosis (TB) affects one third of the global population, and TB of the central nervous system (CNS-TB) is the most severe form of tuberculosis which often associates with high mortality. The pro-inflammatory cytokine tumour necrosis factor (TNF) plays a critical role in the initial and long-term host immune protection against Mycobacterium tuberculosis (M. tuberculosis) which involves the activation of innate immune cells and structure maintenance of granulomas. However, the contribution of TNF, in particular neuron-derived TNF, in the control of cerebral M. tuberculosis infection and its protective immune responses in the CNS were not clear. We generated neuron-specific TNF-deficient (NsTNF(-/-)) mice and compared outcomes of disease against TNF(f/f) control and global TNF(-/-) mice. Mycobacterial burden in brains, lungs and spleens were compared, and cerebral pathology and cellular contributions analysed by microscopy and flow cytometry after M. tuberculosis infection. Activation of innate immune cells was measured by flow cytometry and cell function assessed by cytokine and chemokine quantification using enzyme-linked immunosorbent assay (ELISA). Intracerebral M. tuberculosis infection of TNF(-/-) mice rendered animals highly susceptible, accompanied by uncontrolled bacilli replication and eventual mortality. In contrast, NsTNF(-/-) mice were resistant to infection and presented with a phenotype similar to that in TNF(f/f) control mice. Impaired immunity in TNF(-/-) mice was associated with altered cytokine and chemokine synthesis in the brain and characterised by a reduced number of activated innate immune cells. Brain pathology reflected enhanced inflammation dominated by neutrophil influx. Our data show that neuron-derived TNF has a limited role in immune responses, but overall TNF production is necessary for protective immunity against CNS-TB.

MYCN and HDAC5 transcriptionally repress CD9 to trigger invasion and metastasis in neuroblastoma

PubMed Central

Fabian, Johannes; Opitz, Desirée; Althoff, Kristina; Lodrini, Marco; Hero, Barbara; Volland, Ruth; Beckers, Anneleen; de Preter, Katleen; Decock, Anneleen; Patil, Nitin; Abba, Mohammed; Kopp-Schneider, Annette; Astrahantseff, Kathy; Wünschel, Jasmin; Pfeil, Sebastian; Ercu, Maria; Künkele, Annette; Hu, Jamie; Thole, Theresa; Schweizer, Leonille; Mechtersheimer, Gunhild; Carter, Daniel; Cheung, Belamy B.; Popanda, Odilia; von Deimling, Andreas; Koster, Jan; Versteeg, Rogier; Schwab, Manfred; Marshall, Glenn M.; Speleman, Frank; Erb, Ulrike; Zoeller, Margot; Allgayer, Heike; Simon, Thorsten; Fischer, Matthias; Kulozik, Andreas E.; Eggert, Angelika; Witt, Olaf; Schulte, Johannes H.; Deubzer, Hedwig E.

2016-01-01

The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma. PMID:27572323

MYCN and HDAC5 transcriptionally repress CD9 to trigger invasion and metastasis in neuroblastoma.

PubMed

Fabian, Johannes; Opitz, Desirée; Althoff, Kristina; Lodrini, Marco; Hero, Barbara; Volland, Ruth; Beckers, Anneleen; de Preter, Katleen; Decock, Anneleen; Patil, Nitin; Abba, Mohammed; Kopp-Schneider, Annette; Astrahantseff, Kathy; Wünschel, Jasmin; Pfeil, Sebastian; Ercu, Maria; Künkele, Annette; Hu, Jamie; Thole, Theresa; Schweizer, Leonille; Mechtersheimer, Gunhild; Carter, Daniel; Cheung, Belamy B; Popanda, Odilia; von Deimling, Andreas; Koster, Jan; Versteeg, Rogier; Schwab, Manfred; Marshall, Glenn M; Speleman, Frank; Erb, Ulrike; Zoeller, Margot; Allgayer, Heike; Simon, Thorsten; Fischer, Matthias; Kulozik, Andreas E; Eggert, Angelika; Witt, Olaf; Schulte, Johannes H; Deubzer, Hedwig E

2016-10-11

The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma.

Heat shock protein 27 as a prognostic and predictive biomarker in pancreatic ductal adenocarcinoma

PubMed Central

Schäfer, Claus; Seeliger, Hendrik; Bader, Dominik C; Assmann, Gerald; Buchner, Denise; Guo, Yang; Ziesch, Andreas; Palagyi, Andreas; Ochs, Stephanie; Laubender, Rüdiger P; Jung, Andreas; De Toni, Enrico N; Kirchner, Thomas; Göke, Burkhard; Bruns, Christiane; Gallmeier, Eike

2012-01-01

Abstract A role of heat shock protein 27 (HSP27) as a potential biomarker has been reported in various tumour entities, but comprehensive studies in pancreatic cancer are lacking. Applying tissue microarray (TMA) analysis, we correlated HSP27 protein expression status with clinicopathologic parameters in pancreatic ductal adenocarcinoma specimens from 86 patients. Complementary, we established HSP27 overexpression and RNA-interference models to assess the impact of HSP27 on chemo- and radiosensitivity directly in pancreatic cancer cells. In the TMA study, HSP27 expression was found in 49% of tumour samples. Applying univariate analyses, a significant correlation was found between HSP27 expression and survival. In the multivariate Cox-regression model, HSP27 expression emerged as an independent prognostic factor. HSP27 expression also correlated inversely with nuclear p53 accumulation, indicating either protein interactions between HSP27 and p53 or TP53 mutation-dependent HSP27-regulation in pancreatic cancer. In the sensitivity studies, HSP27 overexpression rendered HSP27 low-expressing PL5 pancreatic cancer cells more susceptible towards treatment with gemcitabine. Vice versa, HSP27 protein depletion in HSP27 high-expressing AsPC-1 cells caused increased gemcitabine resistance. Importantly, HSP27 expression was inducible in pancreatic cancer cell lines as well as primary cells. Taken together, our study suggests a role for HSP27 as a prognostic and predictive marker in pancreatic cancer. Assessment of HSP27 expression could thus facilitate the identification of specific patient subpopulations that might benefit from individualized treatment options. Additional studies need to clarify whether modulation of HSP27 expression could represent an attractive concept to support the incorporation of hyperthermia in clinical treatment protocols for pancreatic cancer. PMID:22004109

Species-specific disruption of STING-dependent antiviral cellular defenses by the Zika virus NS2B3 protease.

PubMed

Ding, Qiang; Gaska, Jenna M; Douam, Florian; Wei, Lei; Kim, David; Balev, Metodi; Heller, Brigitte; Ploss, Alexander

2018-06-18

The limited host tropism of numerous viruses causing disease in humans remains incompletely understood. One example is Zika virus (ZIKV), an RNA virus that has reemerged in recent years. Here, we demonstrate that ZIKV efficiently infects fibroblasts from humans, great apes, New and Old World monkeys, but not rodents. ZIKV infection in human-but not murine-cells impairs responses to agonists of the cGMP-AMP synthase/stimulator of IFN genes (cGAS/STING) signaling pathway, suggesting that viral mechanisms to evade antiviral defenses are less effective in rodent cells. Indeed, human, but not mouse, STING is subject to cleavage by proteases encoded by ZIKV, dengue virus, West Nile virus, and Japanese encephalitis virus, but not that of yellow fever virus. The protease cleavage site, located between positions 78/79 of human STING, is only partially conserved in nonhuman primates and rodents, rendering these orthologs resistant to degradation. Genetic disruption of STING increases the susceptibility of mouse-but not human-cells to ZIKV. Accordingly, expression of only mouse, not human, STING in murine STING knockout cells rescues the ZIKV suppression phenotype. STING-deficient mice, however, did not exhibit increased susceptibility, suggesting that other redundant antiviral pathways control ZIKV infection in vivo. Collectively, our data demonstrate that numerous RNA viruses evade cGAS/STING-dependent signaling and affirm the importance of this pathway in shaping the host range of ZIKV. Furthermore, our results explain-at least in part-the decreased permissivity of rodent cells to ZIKV, which could aid in the development of mice model with inheritable susceptibility to ZIKV and other flaviviruses.

Inhibition by stabilization: targeting the Plasmodium falciparum aldolase-TRAP complex.

PubMed

Nemetski, Sondra Maureen; Cardozo, Timothy J; Bosch, Gundula; Weltzer, Ryan; O'Malley, Kevin; Ejigiri, Ijeoma; Kumar, Kota Arun; Buscaglia, Carlos A; Nussenzweig, Victor; Sinnis, Photini; Levitskaya, Jelena; Bosch, Jürgen

2015-08-20

Emerging resistance of the malaria parasite Plasmodium to current therapies underscores the critical importance of exploring novel strategies for disease eradication. Plasmodium species are obligate intracellular protozoan parasites. They rely on an unusual form of substrate-dependent motility for their migration on and across host-cell membranes and for host cell invasion. This peculiar motility mechanism is driven by the 'glideosome', an actin-myosin associated, macromolecular complex anchored to the inner membrane complex of the parasite. Myosin A, actin, aldolase, and thrombospondin-related anonymous protein (TRAP) constitute the molecular core of the glideosome in the sporozoite, the mosquito stage that brings the infection into mammals. Virtual library screening of a large compound library against the PfAldolase-TRAP complex was used to identify candidate compounds that stabilize and prevent the disassembly of the glideosome. The mechanism of these compounds was confirmed by biochemical, biophysical and parasitological methods. A novel inhibitory effect on the parasite was achieved by stabilizing a protein-protein interaction within the glideosome components. Compound 24 disrupts the gliding and invasive capabilities of Plasmodium parasites in in vitro parasite assays. A high-resolution, ternary X-ray crystal structure of PfAldolase-TRAP in complex with compound 24 confirms the mode of interaction and serves as a platform for future ligand optimization. This proof-of-concept study presents a novel approach to anti-malarial drug discovery and design. By strengthening a protein-protein interaction within the parasite, an avenue towards inhibiting a previously "undruggable" target is revealed and the motility motor responsible for successful invasion of host cells is rendered inactive. This study provides new insights into the malaria parasite cell invasion machinery and convincingly demonstrates that liver cell invasion is dramatically reduced by 95 % in the presence of the small molecule stabilizer compound 24.

Silencing of six susceptibility genes results in potato late blight resistance.

PubMed

Sun, Kaile; Wolters, Anne-Marie A; Vossen, Jack H; Rouwet, Maarten E; Loonen, Annelies E H M; Jacobsen, Evert; Visser, Richard G F; Bai, Yuling

2016-10-01

Phytophthora infestans, the causal agent of late blight, is a major threat to commercial potato production worldwide. Significant costs are required for crop protection to secure yield. Many dominant genes for resistance (R-genes) to potato late blight have been identified, and some of these R-genes have been applied in potato breeding. However, the P. infestans population rapidly accumulates new virulent strains that render R-genes ineffective. Here we introduce a new class of resistance which is based on the loss-of-function of a susceptibility gene (S-gene) encoding a product exploited by pathogens during infection and colonization. Impaired S-genes primarily result in recessive resistance traits in contrast to recognition-based resistance that is governed by dominant R-genes. In Arabidopsis thaliana, many S-genes have been detected in screens of mutant populations. In the present study, we selected 11 A. thaliana S-genes and silenced orthologous genes in the potato cultivar Desiree, which is highly susceptible to late blight. The silencing of five genes resulted in complete resistance to the P. infestans isolate Pic99189, and the silencing of a sixth S-gene resulted in reduced susceptibility. The application of S-genes to potato breeding for resistance to late blight is further discussed.

A Review of the Antiviral Susceptibility of Human and Avian Influenza Viruses over the Last Decade

PubMed Central

Oh, Ding Yuan; Hurt, Aeron C.

2014-01-01

Antivirals play an important role in the prevention and treatment of influenza infections, particularly in high-risk or severely ill patients. Two classes of influenza antivirals have been available in many countries over the last decade (2004–2013), the adamantanes and the neuraminidase inhibitors (NAIs). During this period, widespread adamantane resistance has developed in circulating influenza viruses rendering these drugs useless, resulting in the reliance on the most widely available NAI, oseltamivir. However, the emergence of oseltamivir-resistant seasonal A(H1N1) viruses in 2008 demonstrated that NAI-resistant viruses could also emerge and spread globally in a similar manner to that seen for adamantane-resistant viruses. Previously, it was believed that NAI-resistant viruses had compromised replication and/or transmission. Fortunately, in 2013, the majority of circulating human influenza viruses remain sensitive to all of the NAIs, but significant work by our laboratory and others is now underway to understand what enables NAI-resistant viruses to retain the capacity to replicate and transmit. In this review, we describe how the susceptibility of circulating human and avian influenza viruses has changed over the last ten years and describe some research studies that aim to understand how NAI-resistant human and avian influenza viruses may emerge in the future. PMID:24800107

Unraveling Fungal Radiation Resistance Regulatory Networks through the Genome-Wide Transcriptome and Genetic Analyses of Cryptococcus neoformans.

PubMed

Jung, Kwang-Woo; Yang, Dong-Hoon; Kim, Min-Kyu; Seo, Ho Seong; Lim, Sangyong; Bahn, Yong-Sun

2016-11-29

The basidiomycetous fungus Cryptococcus neoformans has been known to be highly radiation resistant and has been found in fatal radioactive environments such as the damaged nuclear reactor at Chernobyl. To elucidate the mechanisms underlying the radiation resistance phenotype of C. neoformans, we identified genes affected by gamma radiation through genome-wide transcriptome analysis and characterized their functions. We found that genes involved in DNA damage repair systems were upregulated in response to gamma radiation. Particularly, deletion of recombinase RAD51 and two DNA-dependent ATPase genes, RAD54 and RDH54, increased cellular susceptibility to both gamma radiation and DNA-damaging agents. A variety of oxidative stress response genes were also upregulated. Among them, sulfiredoxin contributed to gamma radiation resistance in a peroxiredoxin/thioredoxin-independent manner. Furthermore, we found that genes involved in molecular chaperone expression, ubiquitination systems, and autophagy were induced, whereas genes involved in the biosynthesis of proteins and fatty acids/sterols were downregulated. Most importantly, we discovered a number of novel C. neoformans genes, the expression of which was modulated by gamma radiation exposure, and their deletion rendered cells susceptible to gamma radiation exposure, as well as DNA damage insults. Among these genes, we found that a unique transcription factor containing the basic leucine zipper domain, named Bdr1, served as a regulator of the gamma radiation resistance of C. neoformans by controlling expression of DNA repair genes, and its expression was regulated by the evolutionarily conserved DNA damage response protein kinase Rad53. Taken together, the current transcriptome and functional analyses contribute to the understanding of the unique molecular mechanism of the radiation-resistant fungus C. neoformans IMPORTANCE: Although there are no natural environments under intense radiation, some living organisms have been found to show high radiation resistance. Organisms harboring the ability of radiation resistance have unique regulatory networks to overcome this stress. Cryptococcus neoformans is one of the radiation-resistant fungi and is found in highly radioactive environments. However, it remains elusive how radiation-resistant eukaryotic microorganisms work differentially from radiation-sensitive ones. Here, we performed transcriptome analysis of C. neoformans to explore gene expression profiles after gamma radiation exposure and functionally characterized some of identified radiation resistance genes. Notably, we identified a novel regulator of radiation resistance, named Bdr1 (a bZIP TF for DNA damage response 1), which is a transcription factor (TF) that is not closely homologous to any known TF and is transcriptionally controlled by the Rad53 kinase. Therefore, our work could shed light on understanding not only the radiation response but also the radiation resistance mechanism of C. neoformans. Copyright © 2016 Jung et al.

Marine-Derived Biocatalysts: Importance, Accessing, and Application in Aromatic Pollutant Bioremediation

PubMed Central

Nikolaivits, Efstratios; Dimarogona, Maria; Fokialakis, Nikolas; Topakas, Evangelos

2017-01-01

The aim of the present review is to highlight the potential use of marine biocatalysts (whole cells or enzymes) as an alternative bioprocess for the degradation of aromatic pollutants. Firstly, information about the characteristics of the still underexplored marine environment and the available scientific tools used to access novel marine-derived biocatalysts is provided. Marine-derived enzymes, such as dioxygenases and dehalogenases, and the involved catalytic mechanisms for the degradation of aromatic and halogenated compounds, are presented, with the purpose of underpinning their potential use in bioremediation. Emphasis is given on persistent organic pollutants (POPs) that are organic compounds with significant impact on health and environment due to their resistance in degradation. POPs bioaccumulate mainly in the fatty tissue of living organisms, therefore current efforts are mostly focused on the restriction of their use and production, since their removal is still unclear. A brief description of the guidelines and criteria that render a pollutant POP is given, as well as their potential biodegradation by marine microorganisms by surveying recent developments in this rather unexplored field. PMID:28265269

Fatty acid conjugation enhances the activities of antimicrobial peptides.

PubMed

Li, Zhining; Yuan, Penghui; Xing, Meng; He, Zhumei; Dong, Chuanfu; Cao, Yongchang; Liu, Qiuyun

2013-04-01

Antimicrobial peptides are small molecules that play a crucial role in innate immunity in multi-cellular organisms, and usually expressed and secreted constantly at basal levels to prevent infection, but local production can be augmented upon an infection. The clock is ticking as rising antibiotic abuse has led to the emergence of many drug resistance bacteria. Due to their broad spectrum antibiotic and antifungal activities as well as anti-viral and anti-tumor activities, efforts are being made to develop antimicrobial peptides into future microbial agents. This article describes some of the recent patents on antimicrobial peptides with fatty acid conjugation. Potency and selectivity of antimicrobial peptide can be modulated with fatty acid tails of variable length. Interaction between membranes and antimicrobial peptides was affected by fatty acid conjugation. At concentrations above the critical miscelle concentration (CMC), propensity of solution selfassembly hampered binding of the peptide to cell membranes. Overall, fatty acid conjugation has enhanced the activities of antimicrobial peptides, and occasionally it rendered inactive antimicrobial peptides to be bioactive. Antimicrobial peptides can not only be used as medicine but also as food additives.

Functional bacterial amyloid increases Pseudomonas biofilm hydrophobicity and stiffness

PubMed Central

Zeng, Guanghong; Vad, Brian S.; Dueholm, Morten S.; Christiansen, Gunna; Nilsson, Martin; Tolker-Nielsen, Tim; Nielsen, Per H.; Meyer, Rikke L.; Otzen, Daniel E.

2015-01-01

The success of Pseudomonas species as opportunistic pathogens derives in great part from their ability to form stable biofilms that offer protection against chemical and mechanical attack. The extracellular matrix of biofilms contains numerous biomolecules, and it has recently been discovered that in Pseudomonas one of the components includes β-sheet rich amyloid fibrils (functional amyloid) produced by the fap operon. However, the role of the functional amyloid within the biofilm has not yet been investigated in detail. Here we investigate how the fap-based amyloid produced by Pseudomonas affects biofilm hydrophobicity and mechanical properties. Using atomic force microscopy imaging and force spectroscopy, we show that the amyloid renders individual cells more resistant to drying and alters their interactions with hydrophobic probes. Importantly, amyloid makes Pseudomonas more hydrophobic and increases biofilm stiffness 20-fold. Deletion of any one of the individual members of in the fap operon (except the putative chaperone FapA) abolishes this ability to increase biofilm stiffness and correlates with the loss of amyloid. We conclude that amyloid makes major contributions to biofilm mechanical robustness. PMID:26500638

Functional bacterial amyloid increases Pseudomonas biofilm hydrophobicity and stiffness.

PubMed

Zeng, Guanghong; Vad, Brian S; Dueholm, Morten S; Christiansen, Gunna; Nilsson, Martin; Tolker-Nielsen, Tim; Nielsen, Per H; Meyer, Rikke L; Otzen, Daniel E

2015-01-01

The success of Pseudomonas species as opportunistic pathogens derives in great part from their ability to form stable biofilms that offer protection against chemical and mechanical attack. The extracellular matrix of biofilms contains numerous biomolecules, and it has recently been discovered that in Pseudomonas one of the components includes β-sheet rich amyloid fibrils (functional amyloid) produced by the fap operon. However, the role of the functional amyloid within the biofilm has not yet been investigated in detail. Here we investigate how the fap-based amyloid produced by Pseudomonas affects biofilm hydrophobicity and mechanical properties. Using atomic force microscopy imaging and force spectroscopy, we show that the amyloid renders individual cells more resistant to drying and alters their interactions with hydrophobic probes. Importantly, amyloid makes Pseudomonas more hydrophobic and increases biofilm stiffness 20-fold. Deletion of any one of the individual members of in the fap operon (except the putative chaperone FapA) abolishes this ability to increase biofilm stiffness and correlates with the loss of amyloid. We conclude that amyloid makes major contributions to biofilm mechanical robustness.

Age-related changes in glial cells of dopamine midbrain subregions in rhesus monkeys.

PubMed

Kanaan, Nicholas M; Kordower, Jeffrey H; Collier, Timothy J

2010-06-01

Aging remains the strongest risk factor for developing Parkinson's disease (PD), and there is selective vulnerability in midbrain dopamine (DA) neuron degeneration in PD. By tracking normal aging-related changes with an emphasis on regional specificity, factors involved in selective vulnerability and resistance to degeneration can be studied. Towards this end, we sought to determine whether age-related changes in microglia and astrocytes in rhesus monkeys are region-specific, suggestive of involvement in regional differences in vulnerability to degeneration that may be relevant to PD pathogenesis. Gliosis in midbrain DA subregions was measured by estimating glia number using unbiased stereology, assessing fluorescence intensity for proteins upregulated during activation, and rating morphology. With normal aging, microglia exhibited increased staining intensity and a shift to more activated morphologies preferentially in the vulnerable substantia nigra-ventral tier (vtSN). Astrocytes did not exhibit age-related changes consistent with an involvement in regional vulnerability in any measure. Our results suggest advancing age is associated with chronic mild inflammation in the vtSN, which may render these DA neurons more vulnerable to degeneration. Copyright 2008 Elsevier Inc. All rights reserved.

Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection.

PubMed

Slack, Jeffrey M; Lawrence, Susan D; Krell, Peter J; Arif, Basil M

2008-10-01

Baculovirus occlusion-derived virions (ODVs) contain a number of infectivity factors essential for the initiation of infection in larval midgut cells. Deletion of any of these factors neutralizes infectivity by the per os route. We have observed that P74 of the group I alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is N-terminally cleaved when a soluble form of the protein was incubated with insect midgut tissues under alkaline conditions and that cleavage was prevented by soybean trypsin inhibitor (SBTI). Presently, biological assays were carried out that suggest SBTI inhibits and trypsin enhances baculovirus per os infectivity. We developed a method to rescue per os infectivity of a P74 null virus involving co-transfection of viral DNA with a plasmid that transiently expresses p74. We used this plasmid rescue method to functionally characterize P74. A series of site-directed mutants were generated at the N terminus to evaluate if trypsin cleavage sites were necessary for function. Mutagenesis of R195, R196 and R199 compromised per os infectivity and rendered P74 resistant to midgut trypsin.

Lack of formylated methionyl-tRNA has pleiotropic effects on Bacillus subtilis

PubMed Central

Cai, Yanfei; Chandrangsu, Pete; Gaballa, Ahmed; Helmann, John D

2017-01-01

Bacteria initiate translation using a modified amino acid, N-formylmethionine (fMet), adapted specifically for this function. Most proteins are processed co-translationally by peptide deformylase (PDF) to remove this modification. Although PDF activity is essential in WT cells and is the target of the antibiotic actinonin, bypass mutations in the fmt gene that eliminate the formylation of Met-tRNAMet render PDF dispensable. The extent to which the emergence of fmt bypass mutations might compromise the therapeutic utility of actinonin is determined, in part, by the effects of these bypass mutations on fitness. Here, we characterize the phenotypic consequences of an fmt null mutation in the model organism Bacillus subtilis. An fmt null mutant is defective for several post-exponential phase adaptive programmes including antibiotic resistance, biofilm formation, swarming and swimming motility and sporulation. In addition, a survey of well-characterized stress responses reveals an increased sensitivity to metal ion excess and oxidative stress. These diverse phenotypes presumably reflect altered synthesis or stability of key proteins involved in these processes. PMID:27983482

Resistive Switching Memory Phenomena in PEDOT PSS: Coexistence of Switchable Diode Effect and Write Once Read Many Memory

PubMed Central

Nguyen, Viet Cuong; Lee, Pooi See

2016-01-01

We study resistive switching memory phenomena in conducting polymer PEDOT PSS. In the same film, there are two types of memory behavior coexisting; namely, the switchable diode effect and write once read many memory. This is the first report on switchable diode phenomenon based on conducting organic materials. The effect was explained as charge trapping of PEDOT PSS film and movement of proton. The same PEDOT PSS device also exhibits write once read many memory (WORM) phenomenon which arises due to redox reaction that reduces PEDOT PSS and renders it non-conducting. The revelation of these two types of memory phenomena in PEDOT PSS highlights the remarkable versatility of this conducting conjugated polymer. PMID:26806868

The Weathering of Aluminum Alloy Sheet Materials Used in Aircraft

NASA Technical Reports Server (NTRS)

Mutchler, Willard

1935-01-01

This report presents the results of an investigation of the corrosion of aluminum alloy sheet materials used in aircraft. It has for its purpose to study the causes of corrosion embrittlement in duralumin-type alloys and the development of methods for its elimination. The report contains results, obtained in an extensive series of weather-exposure tests, which reveal the extent to which the resistance of the materials to corrosion was affected by variable factors in their heat treatment and by the application of various surface protective coatings. The results indicate that the sheet materials are to be regarded as thoroughly reliable, from the standpoint of their permanence in service, provided proper precautions are taken to render them corrosion-resistant.

[A novel chemo-resistant gene MSX2 discovered by establishment of two pancreatic cancer drug resistant cell lines JF305/CDDP and PANC-1/GEM].

PubMed

Yuan, W; Sui, C G; Ma, X; Ma, J

2018-05-23

Objective: To explore new multidrug resistant genes of pancreatic cancer by establishment and characterization of chemo-resistant cell lines. Methods: The cisplatin-resistant cell line JF305/CDDP and the gemcitabine-resistant cell line PANC-1/GEM were induced by high-dose intermittent treatment. CCK-8 assay was used to detect the 50% inhibiting concentration (IC(50)), drug resistance index (R), cross-resistance, and growth difference of different cells. The changes of cell cycle and migration ability of drug-resistant cells were determined by flow cytometry and transwell assay, respectively. And then real-time fluorescence quantitative PCR was used to detect the expression of multidrug resistance-related genes. Results: The drug resistance indexes of JF305/CDDP and PANC-1/GEM were 15.3 and 27.31, respectively, and there was cross-resistance. Compared with the parental cells, the proliferation rate of JF305/CDDP was decreased by 40% on the fourth day ( P <0.05); the proportion of S phase was decreased from (45±2)% to (30±2)% ( P <0.05), and the migration ability was enhanced from (32 ±1) cells per field to (158±5) cells per field ( P <0.01). The expression of multidrug resistance-related genes MRP2, MDR1, LRP and MSX2 was increased in JF305/CDDP cells ( P <0.05). Knockdown of MSX2 in JF305 cells reduced the expression of MRP2, whereas overexpression of MSX2 in PANC-1 cells upregulated MRP2 level ( P <0.05). Conclusions: Two stable multidrug resistant cell lines of pancreatic cancer, JF305/CDDP and PANC-1/GEM, were successfully established. MSX2 might be a new drug resistance related gene in pancreatic cancer cells by up-regulation of MRP2 expression.

Linezolid susceptibility in Helicobacter pylori, including strains with multidrug resistance.

PubMed

Boyanova, Lyudmila; Evstatiev, Ivailo; Gergova, Galina; Yaneva, Penka; Mitov, Ivan

2015-12-01

Only a few studies have evaluated Helicobacter pylori susceptibility to linezolid. The aim of the present study was to assess linezolid susceptibility in H. pylori, including strains with double/multidrug resistance. The susceptibility of 53 H. pylori strains was evaluated by Etest and a breakpoint susceptibility testing method. Helicobacter pylori resistance rates were as follows: amoxicillin, 1.9%; metronidazole, 37.7%; clarithromycin, 17.0%; tetracycline, 1.9%; levofloxacin, 24.5%; and linezolid (>4 mg/L), 39.6%. The linezolid MIC50 value was 31.2-fold higher than that of clarithromycin and 10.5-fold higher than that of levofloxacin; however, 4 of 11 strains with double/multidrug resistance were linezolid-susceptible. The MIC range of the oxazolidinone agent was larger (0.125-64 mg/L) compared with those in the previous two reports. The linezolid resistance rate was 2.2-fold higher in metronidazole-resistant strains and in strains resistant to at least one antibiotic compared with the remaining strains. Briefly, linezolid was less active against H. pylori compared with clarithromycin and levofloxacin, and linezolid resistance was linked to resistance to metronidazole as well as to resistance to at least one antibiotic. However, linezolid activity against some strains with double/multidrug resistance may render the agent appropriate to treat some associated H. pylori infections following in vitro susceptibility testing of the strains. Clinical trials are required to confirm this suggestion. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Identification of Natural Compound Inhibitors for Multidrug Efflux Pumps of Escherichia coli and Pseudomonas aeruginosa Using In Silico High-Throughput Virtual Screening and In Vitro Validation

PubMed Central

Aparna, Vasudevan; Dineshkumar, Kesavan; Mohanalakshmi, Narasumani; Velmurugan, Devadasan; Hopper, Waheeta

2014-01-01

Pseudomonas aeruginosa and Escherichia coli are resistant to wide range of antibiotics rendering the treatment of infections very difficult. A main mechanism attributed to the resistance is the function of efflux pumps. MexAB-OprM and AcrAB-TolC are the tripartite efflux pump assemblies, responsible for multidrug resistance in P. aeruginosa and E. coli respectively. Substrates that are more susceptible for efflux are predicted to have a common pharmacophore feature map. In this study, a new criterion of excluding compounds with efflux substrate-like features was used, thereby refining the selection process and enriching the inhibitor identification process. An in-house database of phytochemicals was created and screened using high-throughput virtual screening against AcrB and MexB proteins and filtered by matching with the common pharmacophore models (AADHR, ADHNR, AAHNR, AADHN, AADNR, AAADN, AAADR, AAANR, AAAHN, AAADD and AAADH) generated using known efflux substrates. Phytochemical hits that matched with any one or more of the efflux substrate models were excluded from the study. Hits that do not have features similar to the efflux substrate models were docked using XP docking against the AcrB and MexB proteins. The best hits of the XP docking were validated by checkerboard synergy assay and ethidium bromide accumulation assay for their efflux inhibition potency. Lanatoside C and diadzein were filtered based on the synergistic potential and validated for their efflux inhibition potency using ethidium bromide accumulation study. These compounds exhibited the ability to increase the accumulation of ethidium bromide inside the bacterial cell as evidenced by these increase in fluorescence in the presence of the compounds. With this good correlation between in silico screening and positive efflux inhibitory activity in vitro, the two compounds, lanatoside C and diadzein could be promising efflux pump inhibitors and effective to use in combination therapy against drug resistant strains of P. aeruginosa and E. coli. PMID:25025665

Identification of natural compound inhibitors for multidrug efflux pumps of Escherichia coli and Pseudomonas aeruginosa using in silico high-throughput virtual screening and in vitro validation.

PubMed

Aparna, Vasudevan; Dineshkumar, Kesavan; Mohanalakshmi, Narasumani; Velmurugan, Devadasan; Hopper, Waheeta

2014-01-01

Pseudomonas aeruginosa and Escherichia coli are resistant to wide range of antibiotics rendering the treatment of infections very difficult. A main mechanism attributed to the resistance is the function of efflux pumps. MexAB-OprM and AcrAB-TolC are the tripartite efflux pump assemblies, responsible for multidrug resistance in P. aeruginosa and E. coli respectively. Substrates that are more susceptible for efflux are predicted to have a common pharmacophore feature map. In this study, a new criterion of excluding compounds with efflux substrate-like features was used, thereby refining the selection process and enriching the inhibitor identification process. An in-house database of phytochemicals was created and screened using high-throughput virtual screening against AcrB and MexB proteins and filtered by matching with the common pharmacophore models (AADHR, ADHNR, AAHNR, AADHN, AADNR, AAADN, AAADR, AAANR, AAAHN, AAADD and AAADH) generated using known efflux substrates. Phytochemical hits that matched with any one or more of the efflux substrate models were excluded from the study. Hits that do not have features similar to the efflux substrate models were docked using XP docking against the AcrB and MexB proteins. The best hits of the XP docking were validated by checkerboard synergy assay and ethidium bromide accumulation assay for their efflux inhibition potency. Lanatoside C and diadzein were filtered based on the synergistic potential and validated for their efflux inhibition potency using ethidium bromide accumulation study. These compounds exhibited the ability to increase the accumulation of ethidium bromide inside the bacterial cell as evidenced by these increase in fluorescence in the presence of the compounds. With this good correlation between in silico screening and positive efflux inhibitory activity in vitro, the two compounds, lanatoside C and diadzein could be promising efflux pump inhibitors and effective to use in combination therapy against drug resistant strains of P. aeruginosa and E. coli.

Expression of a unique drug-resistant Hsp90 ortholog by the nematode Caenorhabditis elegans.

PubMed

David, Cynthia L; Smith, Harold E; Raynes, Deborah A; Pulcini, Elizabeth J; Whitesell, Luke

2003-01-01

In all species studied to date, the function of heat shock protein 90 (Hsp90), a ubiquitous and evolutionarily conserved molecular chaperone, is inhibited selectively by the natural product drugs geldanamycin (GA) and radicicol. Crystal structures of the N-terminal region of yeast and human Hsp90 have revealed that these compounds interact with the chaperone in a Bergerat-type adenine nucleotide-binding fold shared throughout the gyrase, Hsp90, histidine kinase mutL (GHKL) superfamily of adenosine triphosphatases. To better understand the consequences of disrupting Hsp90 function in a genetically tractable multicellular organism, we exposed the soil-dwelling nematode Caenorhabditis elegans to GA under a variety of conditions designed to optimize drug uptake. Mutations in the gene encoding C elegans Hsp90 affect larval viability, dauer development, fertility, and life span. However, exposure of worms to GA produced no discernable phenotypes, although the amino acid sequence of worm Hsp90 is 85% homologous to that of human Hsp90. Consistent with this observation, we found that solid phase-immobilized GA failed to bind worm Hsp90 from worm protein extracts or when translated in a rabbit reticulocyte lysate system. Further, affinity precipitation studies using chimeric worm-vertebrate fusion proteins or worm C-terminal truncations expressed in reticulocyte lysate revealed that the conserved nucleotide-binding fold of worm Hsp90 exhibits the novel ability to bind adenosine triphosphate but not GA. Despite its unusual GA resistance, worm Hsp90 appeared fully functional when expressed in a vertebrate background. It heterodimerized with its vertebrate counterpart and showed no evidence of compromising its essential cellular functions. Heterologous expression of worm Hsp90 in tumor cells, however, did not render them GA resistant. These findings provide new insights into the nature of unusual N-terminal nucleotide-binding fold of Hsp90 and suggest that target-related drug resistance is unlikely to emerge in patients receiving GA-like chemotherapeutic agents.

Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.

PubMed

Tebas, Pablo; Stein, David; Tang, Winson W; Frank, Ian; Wang, Shelley Q; Lee, Gary; Spratt, S Kaye; Surosky, Richard T; Giedlin, Martin A; Nichol, Geoff; Holmes, Michael C; Gregory, Philip D; Ando, Dale G; Kalos, Michael; Collman, Ronald G; Binder-Scholl, Gwendolyn; Plesa, Gabriela; Hwang, Wei-Ting; Levine, Bruce L; June, Carl H

2014-03-06

CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe. We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.).