Cocamidopropyl betaine.

PubMed

Jacob, Sharon E; Amini, Sadegh

2008-01-01

Cocamidopropyl betaine (CAPB) is an amphoteric synthetic detergent that has been increasingly used in cosmetics and personal hygiene products (eg, shampoos, contact lens solutions, toothpaste detergents, makeup removers, bath gels, skin care products, cleansers, liquid soaps, antiseptics, and gynecologic and anal hygiene products) because it induces relatively mild skin irritation. Delayed T-cell-mediated type IV hypersensitivity reactions to CAPB have been reported, and contact sensitization prevalence is estimated at between 3.0 and 7.2%. The increasing rates of sensitization led to CAPB's being named Allergen of the Year in 2004. Related impurities rendered during the manufacturing process (such as amidoamine and dimethylaminopropylamine) are thought to play a role in sensitization.

GENETICS OF HUMAN CELL LINES

PubMed Central

Djordjevic, B.; Szybalski, Waclaw

1960-01-01

The human cell line D98S can be cultivated indefinitely in the presence of up to 3 x 10–5 M 5-bromodeoxyuridine (BUDR), without loss of cell viability. During this time, BUDR is incorporated into both strands of the DNA molecules, replacing up to 45 per cent of the thymidine and thereby rendering the cells highly sensitive to UV light and to x-rays. Cells grown for a limited period of time in the presence of 5-iododeoxyuridine (IUDR) become UV-sensitized, while prolonged cultivation with IUDR results in the loss of cell viability. The properties of the BUDR label permitted the demonstration that: (a) human DNA replicates in a "semiconservative" manner; (b) the degree of radiosensitization of BUDR-treated cells depends on whether the DNA has been substituted in one strand only ("unifilarly") or in both strands ("bifilarly"); (c) functional human DNA is produced during partial inhibition of protein synthesis. The potential applicability of this new rational principle of radiosensitization to the radiotherapy of neoplastic diseases is discussed. PMID:13723177

pH-Triggered Echogenicity and Contents Release from Liposomes

PubMed Central

2015-01-01

Liposomes are representative lipid nanoparticles widely used for delivering anticancer drugs, DNA fragments, or siRNA to cancer cells. Upon targeting, various internal and external triggers have been used to increase the rate for contents release from the liposomes. Among the internal triggers, decreased pH within the cellular lysosomes has been successfully used to enhance the rate for releasing contents. However, imparting pH sensitivity to liposomes requires the synthesis of specialized lipids with structures that are substantially modified at a reduced pH. Herein, we report an alternative strategy to render liposomes pH sensitive by encapsulating a precursor which generates gas bubbles in situ in response to acidic pH. The disturbance created by the escaping gas bubbles leads to the rapid release of the encapsulated contents from the liposomes. Atomic force microscopic studies indicate that the liposomal structure is destroyed at a reduced pH. The gas bubbles also render the liposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeted doxorubicin-encapsulated liposomes to the pancreatic ductal carcinoma cells that overexpress the folate receptor on the surface. In response to the decreased pH in the lysosomes, the encapsulated anticancer drug is efficiently released. Contents released from these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantially reduced viability for the pancreatic cancer cells (14%). PMID:25271780

pH-triggered echogenicity and contents release from liposomes.

PubMed

Nahire, Rahul; Hossain, Rayat; Patel, Rupa; Paul, Shirshendu; Meghnani, Varsha; Ambre, Avinash H; Gange, Kara N; Katti, Kalpana S; Leclerc, Estelle; Srivastava, D K; Sarkar, Kausik; Mallik, Sanku

2014-11-03

Liposomes are representative lipid nanoparticles widely used for delivering anticancer drugs, DNA fragments, or siRNA to cancer cells. Upon targeting, various internal and external triggers have been used to increase the rate for contents release from the liposomes. Among the internal triggers, decreased pH within the cellular lysosomes has been successfully used to enhance the rate for releasing contents. However, imparting pH sensitivity to liposomes requires the synthesis of specialized lipids with structures that are substantially modified at a reduced pH. Herein, we report an alternative strategy to render liposomes pH sensitive by encapsulating a precursor which generates gas bubbles in situ in response to acidic pH. The disturbance created by the escaping gas bubbles leads to the rapid release of the encapsulated contents from the liposomes. Atomic force microscopic studies indicate that the liposomal structure is destroyed at a reduced pH. The gas bubbles also render the liposomes echogenic, allowing ultrasound imaging. To demonstrate the applicability of this strategy, we have successfully targeted doxorubicin-encapsulated liposomes to the pancreatic ductal carcinoma cells that overexpress the folate receptor on the surface. In response to the decreased pH in the lysosomes, the encapsulated anticancer drug is efficiently released. Contents released from these liposomes are further enhanced by the application of continuous wave ultrasound (1 MHz), resulting in substantially reduced viability for the pancreatic cancer cells (14%).

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

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.

mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling

PubMed Central

Hämäläinen, Riikka H.; Ahlqvist, Kati J.; Ellonen, Pekka; Lepistö, Maija; Logan, Angela; Otonkoski, Timo; Murphy, Michael P.; Suomalainen, Anu

2015-01-01

Summary mtDNA mutagenesis in somatic stem cells leads to their dysfunction and to progeria in mouse. The mechanism was proposed to involve modification of reactive oxygen species (ROS)/redox signaling. We studied the effect of mtDNA mutagenesis on reprogramming and stemness of pluripotent stem cells (PSCs) and show that PSCs select against specific mtDNA mutations, mimicking germline and promoting mtDNA integrity despite their glycolytic metabolism. Furthermore, mtDNA mutagenesis is associated with an increase in mitochondrial H2O2, reduced PSC reprogramming efficiency, and self-renewal. Mitochondria-targeted ubiquinone, MitoQ, and N-acetyl-L-cysteine efficiently rescued these defects, indicating that both reprogramming efficiency and stemness are modified by mitochondrial ROS. The redox sensitivity, however, rendered PSCs and especially neural stem cells sensitive to MitoQ toxicity. Our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function. PMID:26027936

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

Tolerance of Erythrocytes in Poultry: Induction and Specificity

PubMed Central

Mitchison, N. A.

1962-01-01

Measurement of the rate of elimination of 51Cr-labelled erythrocytes provides a reliable test of immunity in fowls. Chickens can be rendered tolerant of homologous and turkey erythrocytes, as judged by this test, by receiving a series of transfusions of irradiated blood. The series were arranged so that foreign cells remained present in the circulation from the time of hatching. Tolerance induced by this treatment is generally incomplete, but can last indefinitely. In some chickens the manifestation of tolerance of turkey erythrocytes is delayed, probably because of passive transmission of antibody from the dam. Chickens old enough to react against small transfusions of homologous blood can still be rendered tolerant by massive transfusions. Tolerance of the erythrocytes from an individual donor extends only slightly to those from other donors. Tolerance acquired in this way, through transfusion of irradiated blood, stands in contrast to the more stable and complete tolerance that can be acquired through administration of viable cells. Viable cells, on the other hand, provide a less sensitive test, for birds which tolerate skin homografts often eliminate rapidly erythrocytes from the same donor. PMID:14474652

Efficacy of ATR inhibitors as single agents in Ewing sarcoma

PubMed Central

Lecona, Emilio; Murga, Matilde; Callen, Elsa; Azorin, Daniel; Alonso, Javier; Lopez, Andres J.; Nussenzweig, Andre; Fernandez-Capetillo, Oscar

2016-01-01

Ewing sarcomas (ES) are pediatric bone tumors that arise from a driver translocation, most frequently EWS/FLI1. Current ES treatment involves DNA damaging agents, yet the basis for the sensitivity to these therapies remains unknown. Oncogene-induced replication stress (RS) is a known source of endogenous DNA damage in cancer, which is suppressed by ATR and CHK1 kinases. We here show that ES suffer from high endogenous levels of RS, rendering them particularly dependent on the ATR pathway. Accordingly, two independent ATR inhibitors show in vitro toxicity in ES cell lines as well as in vivo efficacy in ES xenografts as single agents. Expression of EWS/FLI1 or EWS/ERG oncogenic translocations sensitizes non-ES cells to ATR inhibitors. Our data shed light onto the sensitivity of ES to genotoxic agents, and identify ATR inhibitors as a potential therapy for Ewing Sarcomas. PMID:27577084

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

Mavrakis, K. J.; McDonald, E. R.; Schlabach, M. R.

5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA–mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in anmore » MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP–deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.« less

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

Fast and accurate detection of cancer cell using a versatile three-channel plasmonic sensor

NASA Astrophysics Data System (ADS)

Hoseinian, M.; Ahmadi, A. R.; Bolorizadeh, M. A.

2016-09-01

Surface Plasmon Resonance (SPR) optical fiber sensors can be used as cost-effective small sized biosensors that are relatively simple to operate. Additionally, these instruments are label-free, hence rendering them highly sensitive to biological measurements. In this study, a three-channel microstructure optical fiber plasmonic-based portable biosensor is designed and analyzed using Finite Element Method. The proposed system is capable of determining changes in sample's refractive index with precision of order one thousandth. The biosensor measures three absorption resonance wavelengths of the analytes simultaneously. This property is one of the main advantages of the proposed biosensor since it reduces the error in the measured wavelength and enhances the accuracy of the results up to 10-5 m/RIU by reducing noise. In this paper, Jurkat cell, an indicator cell for leukemia cancer, is considered as the analyte; and its absorption resonance wavelengths as well as sensitivity in each channel are determined.

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

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.

Inhibition of MicroRNA-23 Contributes to the Isoflurane-Mediated Cardioprotection Against Oxidative Stress.

PubMed

Liu, Hai-Jian; Liu, Bin

2018-04-07

Isoflurane is one of the most frequently used volatile anesthetics in clinical practice for inhalational anesthesia. It is widely studied that isoflurane mediates cardioprotection during multiple pathological processes. However, the precise mechanisms have not been fully elucidated. Neonatal cardiomyocytes were isolated and cultured, followed by treatments with isoflurane at 0, 50, 100 or 200 µM. Rat cardiomyoblast cell line, H9c2, was treated with H 2 O 2 . Expression of miR-23 was measured by qRT-PCR. The cell survival rate of H9c2 in response to H 2 O 2 treatments was evaluated by MTT assay. The ROS and GSH/GSSG levels were measured using Superoxide Detection Kit and GSH/GSSG Ratio Detection Assay Kit. In this study, we report an isoflurane-miR-23-antioxidant axis in cardiomyocyte. We observed that miR-23 was suppressed by isoflurane treatments at 50, 100 or 200 µM. Moreover, cardiomyocyte with isoflurane exposure was insensitive to H 2 O 2 treatment in vitro. Inhibition of miR-23 protected cardiomyocyte against oxidative stress induced by H 2 O 2 treatments at 30, 60, 90 or 120 µM. In addition, overexpression of miR-23 induced ROS generation over twofolds and rendered cardiomyocyte sensitive to H 2 O 2 treatments. We demonstrate that miR-23 inhibited intracellular GSH, an antioxidant against oxidative stress. Our results reveal that with isoflurane exposure, overexpression of miR-23 rendered cardiomyocyte sensitive to H 2 O 2 treatments at 20, 30, 40, 50 µM. Pretreatments with GSH in miR-23 overexpressing cells rescued the cell death under oxidative stress. In summary, our results illustrate that the isoflurane-mediated protection of cardiomyocytes under oxidative stress is through inhibition of miR-23. This study provides an aspect for the miRNAs-modulated cardiomyocyte sensitivity to oxidative stress, contributing to the development of therapeutic agents.

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.

Deoxycytidine Deaminase-Deficient Escherichia coli Strains Display Acute Sensitivity to Cytidine, Adenosine, and Guanosine and Increased Sensitivity to a Range of Antibiotics, Including Vancomycin

PubMed Central

Kang, Tina Manzhu; Yuan, Jessica; Zhou, Alice; Beppler, Casey

2014-01-01

We show here that deoxycytidine deaminase (DCD)-deficient mutants of Escherichia coli are hypersensitive to killing by exogenous cytidine, adenosine, or guanosine, whereas wild-type cells are not. This hypersensitivity is reversed by exogenous thymidine. The mechanism likely involves the allosteric regulation of ribonucleotide reductase and severe limitations of the dTTP pools, resulting in thymineless death, the phenomenon of cell death due to thymidine starvation. We also report here that DCD-deficient mutants of E. coli are more sensitive to a series of different antibiotics, including vancomycin, and we show synergistic killing with the combination of vancomycin and cytidine. One possibility is that a very low, subinhibitory concentration of vancomycin enters Gram-negative cells and that this concentration is potentiated by chromosomal lesions resulting from the thymineless state. A second possibility is that the metabolic imbalance resulting from DCD deficiency affects the assembly of the outer membrane, which normally presents a barrier to drugs such as vancomycin. We consider these findings with regard to ideas of rendering Gram-negative bacteria sensitive to drugs such as vancomycin. PMID:24633874

Epstein-Barr virus infection and nasopharyngeal carcinoma: the other side of the coin.

PubMed

Perri, Francesco; Della Vittoria Scarpati, Giuseppina; Giuliano, Mario; D'Aniello, Carmine; Gnoni, Antonio; Cavaliere, Carla; Licchetta, Antonella; Pisconti, Salvatore

2015-11-01

Oncogenic viruses may have a significant impact on the therapeutic management of several malignancies besides their well-known role in tumor pathogenesis. Epstein-Barr virus (EBV) induces neoplastic transformation of epithelial cells of the nasopharynx by various molecular mechanisms mostly involving activation of oncogenes and inactivation of tumor-suppressor genes. EBV infection can also induce the expression of several immunogenic peptides on the plasma membrane of the infected cells. Importantly, these virus-related antigens may be used as targets for antitumor immunotherapy-based treatment strategies. Two different immunotherapy strategies, namely adoptive and active immunotherapy, have been developed and strongly improved in the recent years. Furthermore, EBV infection may influence the use of targeted therapies for nasopharyngeal carcinoma (NPC) considering that the presence of EBV can induce important modifications in cell signaling. As an example, latent membrane protein type 1 is a viral transmembrane protein mainly involved in the cancerogenesis process, which can also mediate overexpression of the epidermal growth factor receptor (EGFR) in NPC cells, rendering them more sensitive to anti-EGFR therapy. Finally, EBV may induce epigenetic changes in the infected cells, such as DNA hypermethylation and histone deacetylation, that can sustain tumor growth and can thus be considered potential targets for novel therapies. In conclusion, EBV infection can modify important biological features of NPC cells, rendering them more vulnerable to both immunotherapy and targeted therapy.

Connexin 43 inhibition sensitizes chemoresistant glioblastoma cells to temozolomide

PubMed Central

Murphy, Susan F; Varghese, Robin T; Lamouille, Samy; Guo, Sujuan; Pridham, Kevin J; Kanabur, Pratik; Osimani, Alyssa M; Sharma, Shaan; Jourdan, Jane; Rodgers, Cara M; Simonds, Gary R; Gourdie, Robert G; Sheng, Zhi

2015-01-01

Resistance of glioblastoma (GBM) to the front-line chemotherapeutic agent temozolomide (TMZ) continues to challenge GBM treatment efforts. The repair of TMZ-induced DNA damage by O-6-methylguanine-DNA methyltransferase (MGMT) confers one mechanism of TMZ resistance. Paradoxically, MGMT-deficient GBM patients survive longer despite still developing resistance to TMZ. Recent studies indicate that the gap junction protein connexin 43 (Cx43) renders GBM cells resistant to TMZ through its carboxyl terminus (CT). In this study, we report insights into how Cx43 promotes TMZ resistance. Cx43 levels were inversely correlated with TMZ sensitivity of GBM cells, including GBM stem cells. Moreover, Cx43 levels inversely correlated with patient survival, including as observed in MGMT-deficient GBM patients. Addition of the C-terminal peptide mimetic αCT1, a selective inhibitor of Cx43 channels, sensitized human MGMT-deficient and TMZ-resistant GBM cells to TMZ treatment. Moreover, combining αCT1 with TMZ blocked AKT/mTOR signaling, induced autophagy and apoptosis in TMZ-resistant GBM cells. Our findings suggest that Cx43 may offer a biomarker to predict the survival of patients with MGMT-independent TMZ resistance, and that combining a Cx43 inhibitor with TMZ could enhance therapeutic responses in GBM and perhaps other TMZ-resistant cancers. PMID:26542214

Non-canonical Wnt signalling modulates the endothelial shear stress flow sensor in vascular remodelling

PubMed Central

Franco, Claudio A; Jones, Martin L; Bernabeu, Miguel O; Vion, Anne-Clemence; Barbacena, Pedro; Fan, Jieqing; Mathivet, Thomas; Fonseca, Catarina G; Ragab, Anan; Yamaguchi, Terry P; Coveney, Peter V; Lang, Richard A; Gerhardt, Holger

2016-01-01

Endothelial cells respond to molecular and physical forces in development and vascular homeostasis. Deregulation of endothelial responses to flow-induced shear is believed to contribute to many aspects of cardiovascular diseases including atherosclerosis. However, how molecular signals and shear-mediated physical forces integrate to regulate vascular patterning is poorly understood. Here we show that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/Wnt11 renders endothelial cells more sensitive to shear, resulting in axial polarization and migration against flow at lower shear levels. Integration of flow modelling and polarity analysis in entire vascular networks demonstrates that polarization against flow is achieved differentially in artery, vein, capillaries and the primitive sprouting front. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus. DOI: http://dx.doi.org/10.7554/eLife.07727.001 PMID:26845523

Plantaricin A, a cationic peptide produced by Lactobacillus plantarum, permeabilizes eukaryotic cell membranes by a mechanism dependent on negative surface charge linked to glycosylated membrane proteins.

PubMed

Sand, Sverre L; Nissen-Meyer, Jon; Sand, Olav; Haug, Trude M

2013-02-01

Lactobacillus plantarum C11 releases plantaricin A (PlnA), a cationic peptide pheromone that has a membrane-permeabilizing, antimicrobial effect. We have previously shown that PlnA may also permeabilize eukaryotic cells, with a potency that differs between cell types. It is generally assumed that cationic antimicrobial peptides exert their effects through electrostatic attraction to negatively charged phospholipids in the membrane. The aim of the present study was to investigate if removal of the negative charge linked to glycosylated proteins at the cell surface reduces the permeabilizing potency of PlnA. The effects of PlnA were tested on clonal rat anterior pituitary cells (GH(4) cells) using patch clamp and microfluorometric techniques. In physiological extracellular solution, GH(4) cells are highly sensitive to PlnA, but the sensitivity was dramatically reduced in solutions that partly neutralize the negative surface charge of the cells, in agreement with the notion that electrostatic interactions are probably important for the PlnA effects. Trypsination of cells prior to PlnA exposure also rendered the cells less sensitive to the peptide, suggesting that negative charges linked to membrane proteins are involved in the permeabilizing action. Finally, pre-exposure of cells to a mixture of enzymes that split carbohydrate residues from the backbone of glycosylated proteins also impeded the PlnA-induced membrane permeabilization. We conclude that electrostatic attraction between PlnA and glycosylated membrane proteins is probably an essential first step before PlnA can interact with membrane phospholipids. Deviating glycosylation patterns may contribute to the variation in PlnA sensitivity of different cell types, including cancerous cells and their normal counterparts. Copyright © 2012 Elsevier B.V. All rights reserved.

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

Cell Culture on MEMS Platforms: A Review

PubMed Central

Ni, Ming; Tong, Wen Hao; Choudhury, Deepak; Rahim, Nur Aida Abdul; Iliescu, Ciprian; Yu, Hanry

2009-01-01

Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bio-incompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bio-incompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented. PMID:20054478

In vivo flow speed measurement of capillaries by photoacoustic correlation spectroscopy.

PubMed

Chen, Sung-Liang; Xie, Zhixing; Carson, Paul L; Wang, Xueding; Guo, L Jay

2011-10-15

We recently proposed photoacoustic correlation spectroscopy (PACS) and demonstrated a proof-of-concept experiment. Here we use the technique for in vivo flow speed measurement in capillaries in a chick embryo model. The photoacoustic microscopy system is used to render high spatial resolution and high sensitivity, enabling sufficient signals from single red blood cells. The probe beam size is calibrated by a blood-mimicking phantom. The results indicate the feasibility of using PACS to study flow speeds in capillaries.

Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine.

PubMed

Muta, Yu; Fujita, Yoshihisa; Sumiyama, Kenta; Sakurai, Atsuro; Taketo, M Mark; Chiba, Tsutomu; Seno, Hiroshi; Aoki, Kazuhiro; Matsuda, Michiyuki; Imajo, Masamichi

2018-06-05

Acting downstream of many growth factors, extracellular signal-regulated kinase (ERK) plays a pivotal role in regulating cell proliferation and tumorigenesis, where its spatiotemporal dynamics, as well as its strength, determine cellular responses. Here, we uncover the ERK activity dynamics in intestinal epithelial cells (IECs) and their association with tumour characteristics. Intravital imaging identifies two distinct modes of ERK activity, sustained and pulse-like activity, in IECs. The sustained and pulse-like activities depend on ErbB2 and EGFR, respectively. Notably, activation of Wnt signalling, the earliest event in intestinal tumorigenesis, augments EGFR signalling and increases the frequency of ERK activity pulses through controlling the expression of EGFR and its regulators, rendering IECs sensitive to EGFR inhibition. Furthermore, the increased pulse frequency is correlated with increased cell proliferation. Thus, ERK activity dynamics are defined by composite inputs from EGFR and ErbB2 signalling in IECs and their alterations might underlie tumour-specific sensitivity to pharmacological EGFR inhibition.

P2X receptors, sensory neurons and pain.

PubMed

Bele, Tanja; Fabbretti, Elsa

2015-01-01

Pain represents a very large social and clinical problem since the current treatment provides insufficient pain relief. Plasticity of pain receptors together with sensitisation of sensory neurons, and the role of soluble mediators released from non-neuronal cells render difficult to understand the spatial and temporal scale of pain development, neuronal responses and disease progression. In pathological conditions, ATP is one of the most powerful mediators that activates P2X receptors that behave as sensitive ATP-detectors, such as neuronal P2X3 receptor subtypes and P2X4 and P2X7 receptors expressed on non-neuronal cells. Dissecting the molecular mechanisms occurring in sensory neurons and in accessory cells allows to design appropriate tissue- and cell- targeted approaches to treat chronic pain.

(E,Z)-3-(3',5'-Dimethoxy-4'-hydroxy-benzylidene)-2-indolinone blocks mast cell degranulation.

PubMed

Kiefer, S; Mertz, A C; Koryakina, A; Hamburger, M; Küenzi, P

2010-05-12

(E,Z)-3-(3',5'-Dimethoxy-4'-hydroxy-benzylidene)-2-indolinone (indolinone) is an alkaloid that has been identified as a pharmacologically active compound in extracts of the traditional anti-inflammatory herb Isatis tinctoria. Indolinone has been shown to inhibit compound 48/80-induced mast cell degranulation in vitro. Application of indolinone to bone marrow derived mast cells showed that it was uniformly distributed in the cytoplasm and that cellular uptake was terminated within minutes. Pre-treatment of IgE-sensitized mast cells with 100nM indolinone rendered them insensitive against FcvarepsilonRI-receptor dependent degranulation. However, upstream signalling induced by antigen such as activation of PI3-K and MAPK remained unaffected. We conclude that indolinone blocks mast cell degranulation at the level of granule exocitosis with an IC(50) of 54nm.

MYCN drives glutaminolysis in neuroblastoma and confers sensitivity to an ROS augmenting agent.

PubMed

Wang, Tingting; Liu, Lingling; Chen, Xuyong; Shen, Yuqing; Lian, Gaojian; Shah, Nilay; Davidoff, Andrew M; Yang, Jun; Wang, Ruoning

2018-02-14

Heightened aerobic glycolysis and glutaminolysis are characteristic metabolic phenotypes in cancer cells. Neuroblastoma (NBL), a devastating pediatric cancer, is featured by frequent genomic amplification of MYCN, a member of the Myc oncogene family that is primarily expressed in the early stage of embryonic development and required for neural crest development. Here we report that an enriched glutaminolysis gene signature is associated with MYCN amplification in children with NBL. The partial knockdown of MYCN suppresses glutaminolysis in NBL cells. Conversely, forced overexpression of MYCN in neural crest progenitor cells enhances glutaminolysis. Importantly, glutaminolysis induces oxidative stress by producing reactive oxygen species (ROS), rendering NBL cells sensitive to ROS augmentation. Through a small-scale metabolic-modulator screening, we have found that dimethyl fumarate (DMF), a Food and Drug Administration-approved drug for multiple sclerosis, suppresses NBL cell proliferation in vitro and tumor growth in vivo. DMF suppresses NBL cell proliferation through inducing ROS and subsequently suppressing MYCN expression, which is rescued by an ROS scavenger. Our findings suggest that the metabolic modulation and ROS augmentation could be used as novel strategies in treating NBL and other MYC-driven cancers.

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

Superoxide and Nitric Oxide Mechanisms in Traumatic Brain Injury and Hemorrhagic Hypotension.

DTIC Science & Technology

1999-12-01

DISTRIBUTION CODE 13. ABSTRACT (Maximum 200 Words) Traumatic brain injury (TBI) renders the brain vulnerable to secondary ischemia and poor outcome...cerebral blood flow (CBF) and renders the brain vulnerable to secondary ischemia. There is clinical evidence that hypotension contributes to poor...without TBI. These data indicate that even moderate TBI renders the brain sensitive to ischemic injury during relative mild levels of hypotension that

Human SNM1B is required for normal cellular response to both DNA interstrand crosslink-inducing agents and ionizing radiation.

PubMed

Demuth, Ilja; Digweed, Martin; Concannon, Patrick

2004-11-11

DNA interstrand crosslinks (ICLs) are critical lesions for the mammalian cell since they affect both DNA strands and block transcription and replication. The repair of ICLs in the mammalian cell involves components of different repair pathways such as nucleotide-excision repair and the double-strand break/homologous recombination repair pathways. However, the mechanistic details of mammalian ICL repair have not been fully delineated. We describe here the complete coding sequence and the genomic organization of hSNM1B, one of at least three human homologs of the Saccharomyces cerevisiae PSO2 gene. Depletion of hSNM1B by RNA interference rendered cells hypersensitive to ICL-inducing agents. This requirement for hSNM1B in the cellular response to ICL has been hypothesized before but never experimentally verified. In addition, siRNA knockdown of hSNM1B rendered cells sensitive to ionizing radiation, suggesting the possibility of hSNM1B involvement in homologous recombination repair of double-strand breaks arising as intermediates of ICL repair. Monoubiquitination of FANCD2, a key step in the FANC/BRCA pathway, is not affected in hSNM1B-depleted HeLa cells, indicating that hSNM1B is probably not a part of the Fanconi anemia core complex. Nonetheless, similarities in the phenotype of hSNM1B-depleted cells and cultured cells from patients suffering from Fanconi anemia make hSNM1B a candidate for one of the as yet unidentified Fanconi anemia genes not involved in monoubiquitination of FANCD2.

Suppression of Reserve MCM Complexes Chemosensitizes to Gemcitabine and 5-Fluorouracil

PubMed Central

Bryant, Victoria L.; Elias, Roy M.; McCarthy, Susan M.; Yeatman, Timothy J.; Alexandrow, Mark G.

2015-01-01

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is very difficult to treat with conventional chemotherapeutic regimens. Gemcitabine and 5-fluorouracil (5-FU) are used in the management of PDAC and act by indirectly blocking replicative forks. However, these drugs are not highly effective at suppressing disease progression, indicating a need for the development of innovative therapeutic approaches. Recent studies indicate that suppression of the MCM helicase may provide a novel means to sensitize cancer cells to chemotherapeutic agents that inhibit replicative fork progression. Mammalian cells assemble more MCM complexes on DNA than are required to start S-phase. The excess MCM complexes function as back-up initiation sites under conditions of replicative stress. The current study provides definitive evidence that co-suppression of the excess/back-up MCM complexes sensitizes PDAC tumor lines to both gemcitabine and 5-FU, leading to increased loss of proliferative capacity compared to drugs alone. This occurs because reduced MCM levels prevent efficient recovery of DNA replication in tumor cells exposed to drug. PDAC tumor cells are more sensitive to MCM loss in the presence of gemcitabine than are non-tumor, immortalized epithelial cells. Similarly, colon tumor cells are rendered less viable when co-suppression of MCM complexes occurs during exposure to the crosslinking agent oxaliplatin or topoisomerase inhibitor etoposide. Implications These studies demonstrate that suppressing the back-up complement of MCM complexes provides an effective sensitizing approach with the potential to increase the therapeutic index of drugs used in the clinical management of PDAC and other cancers. PMID:26063742

Suppression of Reserve MCM Complexes Chemosensitizes to Gemcitabine and 5-Fluorouracil.

PubMed

Bryant, Victoria L; Elias, Roy M; McCarthy, Susan M; Yeatman, Timothy J; Alexandrow, Mark G

2015-09-01

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is very difficult to treat with conventional chemotherapeutic regimens. Gemcitabine and 5-fluorouracil are used in the management of PDAC and act by indirectly blocking replicative forks. However, these drugs are not highly effective at suppressing disease progression, indicating a need for the development of innovative therapeutic approaches. Recent studies indicate that suppression of the MCM helicase may provide a novel means to sensitize cancer cells to chemotherapeutic agents that inhibit replicative fork progression. Mammalian cells assemble more MCM complexes on DNA than are required to start S-phase. The excess MCM complexes function as backup initiation sites under conditions of replicative stress. The current study provides definitive evidence that cosuppression of the excess/backup MCM complexes sensitizes PDAC tumor lines to both gemcitabine and 5-FU, leading to increased loss of proliferative capacity compared with drugs alone. This occurs because reduced MCM levels prevent efficient recovery of DNA replication in tumor cells exposed to drug. PDAC tumor cells are more sensitive to MCM loss in the presence of gemcitabine than are nontumor, immortalized epithelial cells. Similarly, colon tumor cells are rendered less viable when cosuppression of MCM complexes occurs during exposure to the crosslinking agent oxaliplatin or topoisomerase inhibitor etoposide. These studies demonstrate that suppressing the backup complement of MCM complexes provides an effective sensitizing approach with the potential to increase the therapeutic index of drugs used in the clinical management of PDAC and other cancers. ©2015 American Association for Cancer Research.

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.

Downregulation of transcription factor GATA4 sensitizes human hepatoblastoma cells to doxorubicin-induced apoptosis.

PubMed

Soini, Tea; Pihlajoki, Marjut; Kyrönlahti, Antti; Andersson, Leif C; Wilson, David B; Heikinheimo, Markku

2017-03-01

Hepatoblastoma, the most common type of pediatric liver cancer, is treated with a combination of surgery and chemotherapy. An essential drug in the treatment of hepatoblastoma is doxorubicin, which in high doses is cardiotoxic. This adverse effect is due to downregulation of cardiac expression of transcription factor GATA4, leading in turn to diminished levels of anti-apoptotic BCL2 (B-cell lymphoma 2) protein family members. GATA4 is also expressed in early fetal liver, but absent from normal postnatal hepatocytes. However, GATA4 is highly expressed in hepatoblastoma tissue. In this study, we assessed the role of GATA4 in doxorubicin-induced apoptosis of hepatoblastoma cells. Herein, we demonstrate that doxorubicin decreases GATA4 expression and alters the expression pattern of BCL2 family members, most profoundly that of BCL2 and BAK, in the HUH6 hepatoblastoma cell line. Silencing of GATA4 by siRNA prior to doxorubicin treatment sensitizes HUH6 cells to the apoptotic effect of this drug by further shifting the balance of BCL2 family members to the pro-apoptotic direction. Specifically, expression levels of anti-apoptotic BCL2 were decreased and pro-apoptotic BID were increased after GATA4 silencing. On the whole, our results indicate that since high endogenous levels of transcription factor GATA4 likely protect hepatoblastoma cells from doxorubicin-induced apoptosis, these cells can be rendered more sensitive to the drug by downregulation of GATA4.

Thiazolides inhibit growth and induce glutathione-S-transferase Pi (GSTP1)-dependent cell death in human colon cancer cells.

PubMed

Müller, Joachim; Sidler, Daniel; Nachbur, Ueli; Wastling, Jonathan; Brunner, Thomas; Hemphill, Andrew

2008-10-15

Thiazolides are a novel class of broad-spectrum anti-infective drugs with promising in vitro and in vivo activities against intracellular and extracellular protozoan parasites. The nitrothiazole-analogue nitazoxanide (NTZ; 2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide) represents the thiazolide parent compound, and a number of bromo- and carboxy-derivatives with differing activities have been synthesized. Here we report that NTZ and the bromo-thiazolide RM4819, but not the carboxy-thiazolide RM4825, inhibited proliferation of the colon cancer cell line Caco2 and nontransformed human foreskin fibroblasts (HFF) at or below concentrations the compounds normally exhibit anti-parasitic activity. Thiazolides induced typical signs of apoptosis, such as nuclear condensation, DNA fragmentation and phosphatidylserine exposure. Interestingly, the apoptosis-inducing effect of thiazolides appeared to be cell cycle-dependent and induction of cell cycle arrest substantially inhibited the cell death-inducing activity of these compounds. Using affinity chromatography and mass spectrometry glutathione-S-transferase P1 (GSTP1) from the GST class Pi was identified as a major thiazolide-binding protein. GSTP1 expression was more than 10 times higher in the thiazolide-sensitive Caco2 cells than in the less sensitive HFF cells. The enzymatic activity of recombinant GSTP1 was strongly inhibited by thiazolides. Silencing of GSTP1 using siRNA rendered cells insensitive to RM4819, while overexpression of GSTP1 increased sensitivity to RM4819-induced cell death. Thiazolides may thus represent an interesting novel class of future cancer therapeutics.

What you can't feel won't hurt you: Evaluating haptic hardware using a haptic contrast sensitivity function.

PubMed

Salisbury, C M; Gillespie, R B; Tan, H Z; Barbagli, F; Salisbury, J K

2011-01-01

In this paper, we extend the concept of the contrast sensitivity function - used to evaluate video projectors - to the evaluation of haptic devices. We propose using human observers to determine if vibrations rendered using a given haptic device are accompanied by artifacts detectable to humans. This determination produces a performance measure that carries particular relevance to applications involving texture rendering. For cases in which a device produces detectable artifacts, we have developed a protocol that localizes deficiencies in device design and/or hardware implementation. In this paper, we present results from human vibration detection experiments carried out using three commercial haptic devices and one high performance voice coil motor. We found that all three commercial devices produced perceptible artifacts when rendering vibrations near human detection thresholds. Our protocol allowed us to pinpoint the deficiencies, however, and we were able to show that minor modifications to the haptic hardware were sufficient to make these devices well suited for rendering vibrations, and by extension, the vibratory components of textures. We generalize our findings to provide quantitative design guidelines that ensure the ability of haptic devices to proficiently render the vibratory components of textures.

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.

Inhibitors of Rho kinase (ROCK) signaling revert the malignant phenotype of breast cancer cells in 3D context.

PubMed

Matsubara, Masahiro; Bissell, Mina J

2016-05-31

Loss of polarity and quiescence along with increased cellular invasiveness are associated with breast tumor progression. ROCK plays a central role in actin-cytoskeletal rearrangement. We used physiologically relevant 3D cultures of nonmalignant and cancer cells in gels made of laminin-rich extracellular matrix, to investigate ROCK function. Whereas expression levels of ROCK1 and ROCK2 were elevated in cancer cells compared to nonmalignant cells, this was not observed in 2D cultures. Malignant cells showed increased phosphorylation of MLC, corresponding to disorganized F-actin. Inhibition of ROCK signaling restored polarity, decreased disorganization of F-actin, and led to reduction of proliferation. Inhibition of ROCK also decreased EGFR and Integrinβ1 levels, and consequently suppressed activation of Akt, MAPK and FAK as well as GLUT3 and LDHA levels. Again, ROCK inhibition did not inhibit these molecules in 2D. A triple negative breast cancer cell line, which lacks E-cadherin, had high levels of ROCK but was less sensitive to ROCK inhibitors. Exogenous overexpression of E-cadherin, however, rendered these cells strikingly sensitive to ROCK inhibition. Our results add to the growing literature that demonstrate the importance of context and tissue architecture in determining not only regulation of normal and malignant phenotypes but also drug response.

Inhibitors of Rho kinase (ROCK) signaling revert the malignant phenotype of breast cancer cells in 3D context

PubMed Central

Matsubara, Masahiro; Bissell, Mina J.

2016-01-01

Loss of polarity and quiescence along with increased cellular invasiveness are associated with breast tumor progression. ROCK plays a central role in actin-cytoskeletal rearrangement. We used physiologically relevant 3D cultures of nonmalignant and cancer cells in gels made of laminin-rich extracellular matrix, to investigate ROCK function. Whereas expression levels of ROCK1 and ROCK2 were elevated in cancer cells compared to nonmalignant cells, this was not observed in 2D cultures. Malignant cells showed increased phosphorylation of MLC, corresponding to disorganized F-actin. Inhibition of ROCK signaling restored polarity, decreased disorganization of F-actin, and led to reduction of proliferation. Inhibition of ROCK also decreased EGFR and Integrinβ1 levels, and consequently suppressed activation of Akt, MAPK and FAK as well as GLUT3 and LDHA levels. Again, ROCK inhibition did not inhibit these molecules in 2D. A triple negative breast cancer cell line, which lacks E-cadherin, had high levels of ROCK but was less sensitive to ROCK inhibitors. Exogenous overexpression of E-cadherin, however, rendered these cells strikingly sensitive to ROCK inhibition. Our results add to the growing literature that demonstrate the importance of context and tissue architecture in determining not only regulation of normal and malignant phenotypes but also drug response. PMID:27203208

Albendazole sensitizes cancer cells to ionizing radiation

PubMed Central

2011-01-01

Background Brain metastases afflict approximately half of patients with metastatic melanoma (MM) and small cell lung cancer (SCLC) and represent the direct cause of death in 60 to 70% of those affected. Standard of care remains ineffective in both types of cancer with the challenge of overcoming the blood brain barrier (BBB) exacerbating the clinical problem. Our purpose is to determine and characterize the potential of albendazole (ABZ) as a cytotoxic and radiosensitizing agent against MM and SCLC cells. Methods Here, ABZ's mechanism of action as a DNA damaging and microtubule disrupting agent is assessed through analysis of histone H2AX phosphorylation and cell cyle progression. The cytotoxicity of ABZ alone and in combination with radiation therapy is determined though clonogenic cell survival assays in a panel of MM and SCLC cell lines. We further establish ABZ's ability to act synergistically as a radio-sensitizer through combination index calculations and apoptotic measurements of poly (ADP-ribose) polymerase (PARP) cleavage. Results ABZ induces DNA damage as measured by increased H2AX phosphorylation. ABZ inhibits the growth of MM and SCLC at clinically achievable plasma concentrations. At these concentrations, ABZ arrests MM and SCLC cells in the G2/M phase of the cell cycle after 12 hours of treatment. Exploiting the notion that cells in the G2/M phase are the most sensitive to radiation therapy, we show that treatment of MM and SCLC cells treated with ABZ renders them more sensitive to radiation in a synergistic fashion. Additionally, MM and SCLC cells co-treated with ABZ and radiation exhibit increased apoptosis at 72 hours. Conclusions Our study suggests that the orally available antihelminthic ABZ acts as a potent radiosensitizer in MM and SCLC cell lines. Further evaluation of ABZ in combination with radiation as a potential treatment for MM and SCLC brain metastases is warranted. PMID:22094106

Water Transport Properties of Roots and Root Cortical Cells in Proton- and Al-Stressed Maize Varieties.

PubMed Central

Gunse, B.; Poschenrieder, C.; Barcelo, J.

1997-01-01

Root and root cell pressure-probe techniques were used to investigate the possible relationship between Al- or H+-induced alterations of the hydraulic conductivity of root cells (LPc) and whole-root water conductivity (LPr) in maize (Zea mays L.) plants. To distinguish between H+ and Al effects two varieties that differ in H+ and Al tolerance were assayed. Based on root elongation rates after 24 h in nutrient solution of pH 6.0, pH 4.5, or pH 4.5 plus 50 [mu]M Al, the variety Adour 250 was found to be H+-sensitive and Al-tolerant, whereas the variety BR 201 F was found to be H+-tolerant but Al-sensitive. No Al-induced decrease of root pressure and root cell turgor was observed in Al-sensitive BR 201 F, indicating that Al toxicity did not cause a general breakdown of membrane integrity and that ion pumping to the stele was maintained. Al reduced LPc more than LPr in Al-sensitive BR 201 F. Proton toxicity in Adour 250 affected LPr more than LPc. In this Al-tolerant variety LPc was increased by Al. Nevertheless, this positive effect on LPc did not render higher LPr values. In conclusion, there were no direct relationships between Al- or H+-induced decreases of LPr and the effects on LPc. To our knowledge, this is the first time that the influence of H+ and Al on root and root cell water relations has been directly measured by pressure-probe techniques. PMID:12223628

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.

Prostate cancer marker panel with single cell sensitivity in urine.

PubMed

Nickens, Kristen P; Ali, Amina; Scoggin, Tatiana; Tan, Shyh-Han; Ravindranath, Lakshmi; McLeod, David G; Dobi, Albert; Tacha, David; Sesterhenn, Isabell A; Srivastava, Shiv; Petrovics, Gyorgy

2015-06-15

Over one million men undergo prostate biopsies annually in the United States, a majority of whom due to elevated serum PSA. More than half of the biopsies turn out to be negative for prostate cancer (CaP). The limitations of both the PSA test and the biopsy procedure have led to the development for more precise CaP detection assays in urine (e.g., PCA3, TMPRSS2-ERG) or blood (e.g., PHI, 4K). Here, we describe the development and evaluation of the Urine CaP Marker Panel (UCMP) assay for sensitive and reproducible detection of CaP cells in post-digital rectal examination (post-DRE) urine. The cellular content of the post-DRE urine was captured on a translucent filter membrane, which is placed on Cytoclear slides for direct evaluation by microscopy and immuno-cytochemistry (ICC). Cells captured on the membrane were assayed for PSA and Prostein expression to identify prostate epithelial cells, and for ERG and AMACR to identify prostate tumor cells. Immunostained cells were analyzed for quantitative and qualitative features and correlated with biopsy positive and negative status for malignancy. The assay was optimized for single cell capture sensitivity and downstream evaluations by spiking a known number of cells from established CaP cell lines, LNCaP and VCaP, into pre-cleared control urine. The cells captured from the post-DRE urine of subjects, obtained prior to biopsy procedure, were co-stained for ERG, AMACR (CaP specific), and Prostein or PSA (prostate epithelium specific) rendering a whole cell based analysis and characterization. A feasibility cohort of 63 post-DRE urine specimens was assessed. Comparison of the UCMP results with blinded biopsy results showed an assay sensitivity of 64% (16 of 25) and a specificity of 68.8% (22 of 32) for CaP detection by biopsy. This pilot study assessing a minimally invasive CaP detection assay with single cell sensitivity cell-capture and characterization from the post-DRE urine holds promise for further development of this novel assay platform. Prostate 75: 969-975, 2015. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.

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.

The pancreatic niche inhibits the effectiveness of sunitinib treatment of pancreatic cancer

PubMed Central

Martínez-Bosch, Neus; Guerrero, Pedro Enrique; Moreno, Mireia; José, Anabel; Iglesias, Mar; Munné-Collado, Jessica; Anta, Héctor; Gibert, Joan; Orozco, Carlos Alberto; Vinaixa, Judith; Fillat, Cristina; Viñals, Francesc; Navarro, Pilar

2016-01-01

Current treatments for pancreatic ductal adenocarcinoma (PDA) are ineffective, making this the 4th leading cause of cancer deaths. Sunitinib is a broad-spectrum inhibitor of tyrosine kinase receptors mostly known for its anti-angiogenic effects. We tested the therapeutic effects of sunitinib in pancreatic cancer using the Ela-myc transgenic mouse model. We showed that Ela-myc pancreatic tumors express PDGFR and VEGFR in blood vessels and epithelial cells, rendering these tumors sensitive to sunitinib by more than only its anti-angiogenic activity. However, sunitinib treatment of Ela-myc mice with either early or advanced tumor progression had no impact on either survival or tumor burden. Further histopathological characterization of these tumors did not reveal differences in necrosis, cell differentiation, angiogenesis, apoptosis or proliferation. In stark contrast, in vitro sunitinib treatment of Ela-myc– derived cell lines showed high sensitivity to the drug, with increased apoptosis and reduced proliferation. Correspondingly, subcutaneous tumors generated from these cell lines completely regressed in vivo after sunitinib treatments. These data point at the pancreatic tumor microenvironment as the most likely barrier preventing sunitinib treatment efficiency in vivo. Combined treatments with drugs that disrupt tumor fibrosis may enhance sunitinib therapeutic effectiveness in pancreatic cancer treatment. PMID:27374084

Inhibition of the Electrostatic Interaction between β -amyloid Peptide and Membranes Prevents β -amyloid-induced Toxicity

NASA Astrophysics Data System (ADS)

Hertel, C.; Terzi, E.; Hauser, N.; Jakob-Rotne, R.; Seelig, J.; Kemp, J. A.

1997-08-01

The accumulation of β -amyloid peptides (Aβ ) into senile plaques is one of the hallmarks of Alzheimer disease. Aggregated Aβ is toxic to cells in culture and this has been considered to be the cause of neurodegeneration that occurs in the Alzheimer disease brain. The discovery of compounds that prevent Aβ toxicity may lead to a better understanding of the processes involved and ultimately to possible therapeutic drugs. Low nanomolar concentrations of Aβ 1-42 and the toxic fragment Aβ 25-35 have been demonstrated to render cells more sensitive to subsequent insults as manifested by an increased sensitivity to formazan crystals following MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction. Formation of the toxic β -sheet conformation by Aβ peptides is increased by negatively charged membranes. Here we demonstrate that phloretin and exifone, dipolar compounds that decrease the effective negative charge of membranes, prevent association of Aβ 1-40 and Aβ 25-35 to negatively charged lipid vesicles and Aβ induced cell toxicity. These results suggest that Aβ toxicity is mediated through a nonspecific physicochemical interaction with cell membranes.

Novel Connections Between DNA Replication, Telomere Homeostasis, and the DNA Damage Response Revealed by a Genome-Wide Screen for TEL1/ATM Interactions in Saccharomyces cerevisiae

PubMed Central

Piening, Brian D.; Huang, Dongqing; Paulovich, Amanda G.

2013-01-01

Tel1 is the budding yeast ortholog of the mammalian tumor suppressor and DNA damage response (DDR) kinase ATM. However, tel1-Δ cells, unlike ATM-deficient cells, do not exhibit sensitivity to DNA-damaging agents, but do display shortened (but stably maintained) telomere lengths. Neither the extent to which Tel1p functions in the DDR nor the mechanism by which Tel1 contributes to telomere metabolism is well understood. To address the first question, we present the results from a comprehensive genome-wide screen for genetic interactions with tel1-Δ that cause sensitivity to methyl methanesulfonate (MMS) and/or ionizing radiation, along with follow-up characterizations of the 13 interactions yielded by this screen. Surprisingly, many of the tel1-Δ interactions that confer DNA damage sensitivity also exacerbate the short telomere phenotype, suggesting a connection between these two phenomena. Restoration of normal telomere length in the tel1-Δ xxx-Δ mutants results in only minor suppression of the DNA damage sensitivity, demonstrating that the sensitivity of these mutants must also involve mechanisms independent of telomere length. In support of a model for increased replication stress in the tel1-Δ xxx-Δ mutants, we show that depletion of dNTP pools through pretreatment with hydroxyurea renders tel1-Δ cells (but not wild type) MMS-sensitive, demonstrating that, under certain conditions, Tel1p does indeed play a critical role in the DDR. PMID:23378069

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.

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.

The anti-androgen drug dutasteride renders triple negative breast cancer cells more sensitive to chemotherapy via inhibition of HIF-1α-/VEGF-signaling.

PubMed

von Wahlde, Marie-Kristin; Hülsewig, Carolin; Ruckert, Christian; Götte, Martin; Kiesel, Ludwig; Bernemann, Christof

2015-02-01

Triple negative breast cancer (TNBC) is characterized by lack of expression of both estrogen and progesterone receptor as well as lack of amplification of HER2. Patients with TNBC carry an unfavorable prognosis compared to other breast cancer subtypes given that endocrine or HER2 targeted therapies are not effective, rendering chemotherapy the sole effective treatment option to date. Therefore, there is a high demand for additional novel treatment options. We previously published a list of genes showing both higher gene expression rates in TNBC and, in addition, are known to encode targets of non-oncologic drugs. SRD5A1, which encodes the type-1 isoform of the steroid-5alpha-reductase, which is involved in androgen metabolism, was found to be one of these genes. Dutasteride is a dual blocker of both the type-1 and type-2 isoform of SRD5A1 and is indicated in the treatment of benign prostate hyperplasia. Treatment of TNBC cell lines with dutasteride was associated with a dose-dependent decrease in cell viability, altered protein expression of VEGF and HIF-1α and increased chemosensitivity. Our results demonstrate that the SRD5A1-corresponding anti-androgenic drug dutasteride might act as a combinatorial therapeutic option besides standard chemotherapy in highly aggressive TNBC.

Histone deacetylase inhibitor SAHA mediates mast cell death and epigenetic silencing of constitutively active D816V KIT in systemic mastocytosis.

PubMed

Lyberg, Katarina; Ali, Hani Abdulkadir; Grootens, Jennine; Kjellander, Matilda; Tirfing, Malin; Arock, Michel; Hägglund, Hans; Nilsson, Gunnar; Ungerstedt, Johanna

2017-02-07

Systemic mastocytosis (SM) is a clonal bone marrow disorder, where therapeutical options are limited. Over 90% of the patients carry the D816V point mutation in the KIT receptor that renders this receptor constitutively active. We assessed the sensitivity of primary mast cells (MC) and mast cell lines HMC1.2 (D816V mutated), ROSA (KIT WT) and ROSA (KIT D816V) cells to histone deacetylase inhibitor (HDACi) treatment. We found that of four HDACi, suberoyl anilide hydroxamic acid (SAHA) was the most effective in killing mutated MC. SAHA downregulated KIT, followed by major MC apoptosis. Primary SM patient MC cultured ex vivo were even more sensitive to SAHA than HMC1.2 cells, whereas primary MC from healthy subjects were less affected. There was a correlation between cell death and SM disease severity, where cell death was more pronounced in the case of aggressive SM, with almost 100% cell death among MC from the mast cell leukemia patient. Additionally, ROSA (KIT D816V) was more affected by HDACi than ROSA (KIT WT) cells. Using ChIP qPCR, we found that the level of active chromatin mark H3K18ac/H3 decreased significantly in the KIT region. This epigenetic silencing was seen only in the KIT region and not in control genes upstream and downstream of KIT, indicating that the downregulation of KIT is exerted by specific epigenetic silencing. In conclusion, KIT D816V mutation sensitized MC to HDACi mediated killing, and SAHA may be of value as specific treatment for SM, although the specific mechanism of action requires further investigation.

Histone deacetylase inhibitor SAHA mediates mast cell death and epigenetic silencing of constitutively active D816V KIT in systemic mastocytosis

PubMed Central

Lyberg, Katarina; Ali, Hani Abdulkadir; Grootens, Jennine; Kjellander, Matilda; Tirfing, Malin; Arock, Michel; Hägglund, Hans

2017-01-01

Systemic mastocytosis (SM) is a clonal bone marrow disorder, where therapeutical options are limited. Over 90% of the patients carry the D816V point mutation in the KIT receptor that renders this receptor constitutively active. We assessed the sensitivity of primary mast cells (MC) and mast cell lines HMC1.2 (D816V mutated), ROSA (KIT WT) and ROSA (KIT D816V) cells to histone deacetylase inhibitor (HDACi) treatment. We found that of four HDACi, suberoyl anilide hydroxamic acid (SAHA) was the most effective in killing mutated MC. SAHA downregulated KIT, followed by major MC apoptosis. Primary SM patient MC cultured ex vivo were even more sensitive to SAHA than HMC1.2 cells, whereas primary MC from healthy subjects were less affected. There was a correlation between cell death and SM disease severity, where cell death was more pronounced in the case of aggressive SM, with almost 100% cell death among MC from the mast cell leukemia patient. Additionally, ROSA (KIT D816V) was more affected by HDACi than ROSA (KIT WT) cells. Using ChIP qPCR, we found that the level of active chromatin mark H3K18ac/H3 decreased significantly in the KIT region. This epigenetic silencing was seen only in the KIT region and not in control genes upstream and downstream of KIT, indicating that the downregulation of KIT is exerted by specific epigenetic silencing. In conclusion, KIT D816V mutation sensitized MC to HDACi mediated killing, and SAHA may be of value as specific treatment for SM, although the specific mechanism of action requires further investigation. PMID:28038453

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.

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.

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.

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

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

GROWTH REGULATION IN RSV INFECTED CHECKEN EMBRYO FIBROBLASTS: THE ROLE OF THE src GENE

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

Parry, G.; Bartholomew, J.C.; Bissell, M.J.

1980-03-01

The relationship between growth regulation and cell transformation has been studied in many cultured cell lines transformed by a range of oncogenic agents. The main conclusion derived from these investigations is that the nature of the growth regulatory lesion in transformed cells is a function of the agent used to induce transformation. For example, when 3T3 fibroblasts are rendered stationary by serum deprivation, normal cells accumulate in G{sub 1} but SV40 transformed cells are arrested at all stages of the cell cycle. In contrast, 3T3 cells transformed with Rous sarcoma virus B77, accumulate in G{sub 1} upon serum deprivation. Thismore » is also true when mouse sarcoma virus (MSV) is used as the transforming agent. MSV-transformed cells accumulate in G{sub 1}, just as do normal cells. In this letter we report a detailed study of the mechanisms leading to loss of growth control in chicken embryo fibroblasts transformed by Rous sarcoma virus (RSV). We have been particularly concerned with the role of the src gene in the process, and have used RSV mutants temperature sensitive (ts) for transformation to investigate the nature of the growth regulatory lesion. Two principal findings have emerged: (a) the stationary phase of the cell cycle (G{sub 1}) in chick embryo fibroblasts has two distinct compartments, (for simplicity referred to as G{sub 1} and G{sub 0} states), (b) when rendered stationary at 41.5{sup o} by serum deprivation, normal cells enter a G{sub 0}-like state, but cells infected with the ts-mutant occupy a G{sub 1} state, even though a known src gene product, a kinase, should be inactive at this temperature. The possibility is discussed that viral factors other than the active src protein kinase influence growth control.« 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.

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

The MAP kinase-activated protein kinase Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen Candida albicans.

PubMed

Li, Xichuan; Du, Wei; Zhao, Jingwen; Zhang, Lilin; Zhu, Zhiyan; Jiang, Linghuo

2010-06-01

Rck2p is the Hog1p-MAP kinase-activated protein kinase required for the attenuation of protein synthesis in response to an osmotic challenge in Saccharomyces cerevisiae. Rck2p also regulates rapamycin sensitivity in both S. cerevisiae and Candida albicans. In this study, we demonstrate that the deletion of CaRCK2 renders C. albicans cells sensitive to, and CaRck2p translocates from the cytosol to the nucleus in response to, cell wall stresses caused by Congo red, Calcoflor White, elevated heat and zymolyase. However, the kinase activity of CaRck2p is not required for the cellular response to these cell wall stresses. Furthermore, transcripts of cell wall protein-encoding genes CaBGL2, CaHWP1 and CaXOG1 are reduced in C. albicans cells lacking CaRCK2. The deletion of CaRCK2 also reduces the in vitro filamentation of C. albicans and its virulence in a mouse model of systemic candidasis. The kinase activity of CaRck2p is required for the virulence, but not for the in vitro filamentation, in C. albicans. Therefore, Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen C. albicans.

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.

Dacarbazine-mediated upregulation of NKG2D ligands on tumor cells activates NK and CD8 T cells and restrains melanoma growth.

PubMed

Hervieu, Alice; Rébé, Cédric; Végran, Frédérique; Chalmin, Fanny; Bruchard, Mélanie; Vabres, Pierre; Apetoh, Lionel; Ghiringhelli, François; Mignot, Grégoire

2013-02-01

Dacarbazine (DTIC) is a cytotoxic drug widely used for melanoma treatment. However, the putative contribution of anticancer immune responses in the efficacy of DTIC has not been evaluated. By testing how DTIC affects host immune responses to cancer in a mouse model of melanoma, we unexpectedly found that both natural killer (NK) and CD8(+) T cells were indispensable for DTIC therapeutic effect. Although DTIC did not directly affect immune cells, it triggered the upregulation of NKG2D ligands on tumor cells, leading to NK cell activation and IFNγ secretion in mice and humans. NK cell-derived IFNγ subsequently favored upregulation of major histocompatibility complex class I molecules on tumor cells, rendering them sensitive to cytotoxic CD8(+) T cells. Accordingly, DTIC markedly enhanced cytotoxic T lymphocyte antigen 4 inhibition efficacy in vivo in an NK-dependent manner. These results underscore the immunogenic properties of DTIC and provide a rationale to combine DTIC with immunotherapeutic agents that relieve immunosuppression in vivo.

Cell Penetration Properties of a Highly Efficient Mini Maurocalcine Peptide

PubMed Central

Tisseyre, Céline; Bahembera, Eloi; Dardevet, Lucie; Sabatier, Jean-Marc; Ronjat, Michel; De Waard, Michel

2013-01-01

Maurocalcine is a highly potent cell-penetrating peptide isolated from the Tunisian scorpion Maurus palmatus. Many cell-penetrating peptide analogues have been derived from the full-length maurocalcine by internal cysteine substitutions and sequence truncation. Herein we have further characterized the cell-penetrating properties of one such peptide, MCaUF1-9, whose sequence matches that of the hydrophobic face of maurocalcine. This peptide shows very favorable cell-penetration efficacy compared to Tat, penetratin or polyarginine. The peptide appears so specialized in cell penetration that it seems hard to improve by site directed mutagenesis. A comparative analysis of the efficacies of similar peptides isolated from other toxin members of the same family leads to the identification of hadrucalcin’s hydrophobic face as an even better CPP. Protonation of the histidine residue at position 6 renders the cell penetration of MCaUF1-9 pH-sensitive. Greater cell penetration at acidic pH suggests that MCaUF1-9 can be used to specifically target cancer cells in vivo where tumor masses grow in more acidic environments. PMID:24276021

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.

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

FOXP3 renders activated human regulatory T cells resistant to restimulation-induced cell death by suppressing SAP expression.

PubMed

Katz, Gil; Voss, Kelsey; Yan, Toria F; Kim, Yong Chan; Kortum, Robert L; Scott, David W; Snow, Andrew L

2018-05-01

Restimulation-induced cell death (RICD) is an apoptotic program that regulates effector T cell expansion, triggered by repeated stimulation through the T cell receptor (TCR) in the presence of interleukin-2 (IL-2). Although CD4 + regulatory T cells (Tregs) consume IL-2 and experience frequent TCR stimulation, they are highly resistant to RICD. Resistance in Tregs is dependent on the forkhead box P3 (FOXP3) transcription factor, although the mechanism remains unclear. T cells from patients with X-linked lymphoproliferative disease (XLP-1), that lack the adaptor molecule SLAM-associated protein (SAP), are also resistant to RICD. Here we demonstrate that normal Tregs express very low levels of SAP compared to conventional T cells. FOXP3 reduces SAP expression by directly binding to and repressing the SH2D1A (SAP) promoter. Indeed, ectopic SAP expression restores RICD sensitivity in human FOXP3 + Tregs. Our findings illuminate the mechanism behind FOXP3-mediated RICD resistance in Tregs, providing new insight into their long-term persistence. Published by Elsevier Inc.

Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis.

PubMed

Adragna, Norma C; Ravilla, Nagendra B; Lauf, Peter K; Begum, Gulnaz; Khanna, Arjun R; Sun, Dandan; Kahle, Kristopher T

2015-01-01

The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K(+) and Cl(-) efflux via activation of K(+) channels, volume-regulated anion channels (VRACs), and the K(+)-Cl(-) cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter isoform 1 (NKCC1). This results in a rapid (<10 min) and significant (>90%) reduction in intracellular K(+) content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent [DCPIB (VRAC inhibitor)-sensitive] pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD.

Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis

PubMed Central

Adragna, Norma C.; Ravilla, Nagendra B.; Lauf, Peter K.; Begum, Gulnaz; Khanna, Arjun R.; Sun, Dandan; Kahle, Kristopher T.

2015-01-01

The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K+ and Cl− efflux via activation of K+ channels, volume-regulated anion channels (VRACs), and the K+-Cl− cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na+-K+-2Cl− cotransporter isoform 1 (NKCC1). This results in a rapid (<10 min) and significant (>90%) reduction in intracellular K+ content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent [DCPIB (VRAC inhibitor)-sensitive] pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD. PMID:26217182

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.

3D Microperiodic Hydrogel Scaffolds for Robust Neuronal Cultures

PubMed Central

Hanson Shepherd, Jennifer N.; Parker, Sara T.; Shepherd, Robert F.; Gillette, Martha U.; Lewis, Jennifer A.; Nuzzo, Ralph G.

2011-01-01

Three-dimensional (3D) microperiodic scaffolds of poly(2-hydroxyethyl methacrylate) (pHEMA) have been fabricated by direct-write assembly of a photopolymerizable hydrogel ink. The ink is initially composed of physically entangled pHEMA chains dissolved in a solution of HEMA monomer, comonomer, photoinitiator and water. Upon printing 3D scaffolds of varying architecture, the ink filaments are exposed to UV light, where they are transformed into an interpenetrating hydrogel network of chemically cross-linked and physically entangled pHEMA chains. These 3D microperiodic scaffolds are rendered growth compliant for primary rat hippocampal neurons by absorption of polylysine. Neuronal cells thrive on these scaffolds, forming differentiated, intricately branched networks. Confocal laser scanning microscopy reveals that both cell distribution and extent of neuronal process alignment depend upon scaffold architecture. This work provides an important step forward in the creation of suitable platforms for in vitro study of sensitive cell types. PMID:21709750

Signaling alkaline pH stress in the yeast Saccharomyces cerevisiae through the Wsc1 cell surface sensor and the Slt2 MAPK pathway.

PubMed

Serrano, Raquel; Martín, Humberto; Casamayor, Antonio; Ariño, Joaquín

2006-12-29

Alkalinization of the external environment represents a stress situation for Saccharomyces cerevisiae. Adaptation to this circumstance involves the activation of diverse response mechanisms, the components of which are still largely unknown. We show here that mutation of members of the cell integrity Pkc1/Slt2 MAPK module, as well as upstream and downstream elements of the system, confers sensitivity to alkali. Alkalinization resulted in fast and transient activation of the Slt2 MAPK, which depended on the integrity of the kinase module and was largely abolished by sorbitol. Lack of Wsc1, removal of specific extracellular and intracellular domains, or substitution of Tyr(303) in this putative membrane stress sensor rendered cells sensitive to alkali and considerably decreased alkali-induced Slt2 activation. In contrast, constitutive activation of Slt2 by the bck1-20 allele increased pH tolerance in the wsc1 mutant. DNA microarray analysis revealed that several genes encoding cell wall proteins, such as GSC2/FKS2, DFG5, SKT5, and CRH1, were induced, at least in part, by high pH in an Slt2-dependent manner. We observed that dfg5, skt5, and particularly dfg5 skt5 cells were alkali-sensitive. Therefore, our results show that an alkaline environment imposes a stress condition on the yeast cell wall. We propose that the Slt2-mediated MAPK pathway plays an important role in the adaptive response to this insult and that Wsc1 participates as an essential cell-surface pH sensor. Moreover, these results provide a new example of the complexity of the response of budding yeast to the alkalinization of the environment.

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

2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity.

PubMed

Sulkowski, Parker L; Corso, Christopher D; Robinson, Nathaniel D; Scanlon, Susan E; Purshouse, Karin R; Bai, Hanwen; Liu, Yanfeng; Sundaram, Ranjini K; Hegan, Denise C; Fons, Nathan R; Breuer, Gregory A; Song, Yuanbin; Mishra-Gorur, Ketu; De Feyter, Henk M; de Graaf, Robin A; Surovtseva, Yulia V; Kachman, Maureen; Halene, Stephanie; Günel, Murat; Glazer, Peter M; Bindra, Ranjit S

2017-02-01

2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability. Copyright © 2017, American Association for the Advancement of Science.

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.

Role of the Drug Transporter ABCC3 in Breast Cancer Chemoresistance

PubMed Central

Balaji, Sai A.; Udupa, Nayanabhirama; Chamallamudi, Mallikarjuna Rao; Gupta, Vaijayanti; Rangarajan, Annapoorni

2016-01-01

Increased expression of ABC-family of transporters is associated with chemotherapy failure. Although the drug transporters ABCG2, ABCB1 and ABCC1 have been majorly implicated in cancer drug resistance, recent studies have associated ABCC3 with multi drug resistance and poor clinical response. In this study, we have examined the expression of ABCC3 in breast cancers and studied its role in drug resistance and stemness of breast cancer cells in comparison with the more studied ABCC1. We observed that similar to ABCC1, the transcripts levels of ABCC3 was significantly high in breast cancers compared to adjacent normal tissue. Importantly, expression of both transporters was further increased in chemotherapy treated patient samples. Consistent with this, we observed that treatment of breast cancer cell lines with anti-cancer agents increased their mRNA levels of both ABCC1 and ABCC3. Further, similar to knockdown of ABCC1, knockdown of ABCC3 also significantly increased the retention of chemotherapeutic drugs in breast cancer cells and rendered them more chemo-sensitive. Interestingly, ABCC1 and ABCC3 knockdown cells also showed reduction in the expression of stemness genes, while ABCC3 knockdown additionally led to a reduction in the CD44high/CD24low breast cancer stem-like subpopulation. Consistent with this, their ability to form primary tumours was compromised. Importantly, down-modulation of ABCC3 rendered these cells increasingly susceptible to doxorubicin in xenograft mice models in vivo. Thus, our study highlights the importance of ABCC3 transporters in drug resistance to chemotherapy in the context of breast cancer. Further, these results suggest that combinatorial inhibition of these transporters together with standard chemotherapy can reduce therapy-induced resistance in breast cancer. PMID:27171227

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.

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.

"Spider"-shaped porphyrins with conjugated pyridyl anchoring groups as efficient sensitizers for dye-sensitized solar cells.

PubMed

Stangel, Christina; Bagaki, Anthi; Angaridis, Panagiotis A; Charalambidis, Georgios; Sharma, Ganesh D; Coutsolelos, Athanasios G

2014-11-17

Two novel "spider-shaped" porphyrins, meso-tetraaryl-substituted 1PV-Por and zinc-metalated 1PV-Zn-Por, bearing four oligo(p-phenylenevinylene) (oPPV) pyridyl groups with long dodecyloxy chains on the phenyl groups, have been synthesized. The presence of four pyridyl groups in both porphyrins, which allow them to act as anchoring groups upon coordination to various Lewis acid sites, the conjugated oPPV bridges, which offer the possibility of electronic communication between the porphyrin core and the pyridyl groups, and the dodecyloxy groups, which offer the advantage of high solubility in a variety of organic solvents of different polarities and could prevent porphyrin aggregation, renders porphyrins 1PV-Por and 1PV-Zn-Por very promising sensitizers for dye-sensitized solar cells (DSSCs). Photophysical measurements, together with electrochemistry experiments and density functional theory calculations, suggest that both porphyrins have frontier molecular orbital energy levels that favor electron injection and dye regeneration in DSSCs. Solar cells sensitized by 1PV-Por and 1PV-Zn-Por were fabricated, and it was found that they show power conversion efficiencies (PCEs) of 3.28 and 5.12%, respectively. Photovoltaic measurements (J-V curves) together with incident photon-to-electron conversion efficiency spectra of the two cells reveal that the higher PCE value of the DSSC based on 1PV-Zn-Por is ascribed to higher short-circuit current (Jsc), open-circuit voltage (Voc), and dye loading values. Emission spectra and electrochemistry experiments suggest a greater driving force for injection of the photogenerated electrons into the TiO2 conduction band for 1PV-Zn-Por rather than its free-base analogue. Furthermore, electrochemical impedance spectroscopy measurements prove that the utilization of 1PV-Zn-Por as a sensitizer offers a high charge recombination resistance and, therefore, leads to a longer electron lifetime.

Controlled drug-release system based on pH-sensitive chloride-triggerable liposomes.

PubMed

Wehunt, Mark P; Winschel, Christine A; Khan, Ali K; Guo, Tai L; Abdrakhmanova, Galya R; Sidorov, Vladimir

2013-03-01

New pH-sensitive lipids were synthesized and utilized in formulations of liposomes suitable for controlled drug release. These liposomes contain various amounts of NaCl in the internal aqueous compartments. The release of the drug model is triggered by an application of HCl cotransporter and exogenous physiologically relevant NaCl solution. HCl cotransporter allows an uptake of HCl by liposomes to the extent of their being proportional to the transmembrane Cl(-) gradient. Therefore, each set of liposomes undergoes internal acidification, which, ultimately, leads to the hydrolysis of the pH-sensitive lipids and content release at the desired time. The developed system releases the drug model in a stepwise fashion, with the release stages separated by periods of low activity. These liposomes were found to be insensitive to physiological concentrations of human serum albumin and to be nontoxic to cells at concentrations exceeding pharmacological relevance. These results render this new drug-release model potentially suitable for in vivo applications.

A Genome-Wide Knockout Screen to Identify Genes Involved in Acquired Carboplatin Resistance

DTIC Science & Technology

2016-07-01

library screen to identify genes that when knocked out render human ovarian cells > 2.5-fold resistant to CBDCA; 2) Validate the ability of...a GeCKOv2 library screen to identify genes that when knocked out render human ovarian cells > 2.5-fold resistant to CBDCA; 2) validate the ability of...resistance in either cell lines or clinical samples. The CRIPSR-cas9 technology now provides us with a major new tool to introduce knock out mutations

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.

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.

Epigenetic Reprogramming of the Type III Interferon Response Potentiates Antiviral Activity and Suppresses Tumor Growth

PubMed Central

Ding, Siyuan; Khoury-Hanold, William; Iwasaki, Akiko; Robek, Michael D.

2014-01-01

Type III interferon (IFN-λ) exhibits potent antiviral activity similar to IFN-α/β, but in contrast to the ubiquitous expression of the IFN-α/β receptor, the IFN-λ receptor is restricted to cells of epithelial origin. Despite the importance of IFN-λ in tissue-specific antiviral immunity, the molecular mechanisms responsible for this confined receptor expression remain elusive. Here, we demonstrate that the histone deacetylase (HDAC) repression machinery mediates transcriptional silencing of the unique IFN-λ receptor subunit (IFNLR1) in a cell-type-specific manner. Importantly, HDAC inhibitors elevate receptor expression and restore sensitivity to IFN-λ in previously nonresponsive cells, thereby enhancing protection against viral pathogens. In addition, blocking HDAC activity renders nonresponsive cell types susceptible to the pro-apoptotic activity of IFN-λ, revealing the combination of HDAC inhibitors and IFN-λ to be a potential antitumor strategy. These results demonstrate that the type III IFN response may be therapeutically harnessed by epigenetic rewiring of the IFN-λ receptor expression program. PMID:24409098

Notch3 overexpression enhances progression and chemoresistance of urothelial carcinoma.

PubMed

Zhang, Heng; Liu, Limei; Liu, Chungang; Pan, Jinhong; Lu, Gensheng; Zhou, Zhansong; Chen, Zhiwen; Qian, Cheng

2017-05-23

Abnormal activation of Notch signaling is involved in the etiology of various diseases, including cancer, but the association between Notch3 expression in urothelial cancer and clinical outcome remains unclear, and the molecular mechanisms underlying Notch3 signaling activation are not well defined. In this study we examined 59 urothelial cancer patients and found that Notch3 was more highly expressed in human urothelial cancer tissues than in non-tumorous bladder tissue samples, with Notch3 overexpression being associated with poor clinical outcome. Notch3 knockdown resulted in decreased proliferation of urothelial cancer cells in vitro and decreased xenograft tumor growth in vivo. In addition, Notch3 knockdown rendered urothelial cancer cells more sensitive to cisplatin. Furthermore, suberoylanilide hydroxamic acid (SAHA, a histone deacetylase [HDAC] inhibitor) induced acetylation of NOTCH3, downregulated Notch 3, prevented urothelial cancer cell proliferation, and induced cell cycle arrest. Taken together, these data suggested that Notch 3 overexpression promotes growth and chemoresistance in urothelial cancer.

Notch3 overexpression enhances progression and chemoresistance of urothelial carcinoma

PubMed Central

Zhang, Heng; Liu, Limei; Liu, Chungang; Pan, Jinhong; Lu, Gensheng; Zhou, Zhansong; Chen, Zhiwen; Qian, Cheng

2017-01-01

Abnormal activation of Notch signaling is involved in the etiology of various diseases, including cancer, but the association between Notch3 expression in urothelial cancer and clinical outcome remains unclear, and the molecular mechanisms underlying Notch3 signaling activation are not well defined. In this study we examined 59 urothelial cancer patients and found that Notch3 was more highly expressed in human urothelial cancer tissues than in non-tumorous bladder tissue samples, with Notch3 overexpression being associated with poor clinical outcome. Notch3 knockdown resulted in decreased proliferation of urothelial cancer cells in vitro and decreased xenograft tumor growth in vivo. In addition, Notch3 knockdown rendered urothelial cancer cells more sensitive to cisplatin. Furthermore, suberoylanilide hydroxamic acid (SAHA, a histone deacetylase [HDAC] inhibitor) induced acetylation of NOTCH3, downregulated Notch 3, prevented urothelial cancer cell proliferation, and induced cell cycle arrest. Taken together, these data suggested that Notch 3 overexpression promotes growth and chemoresistance in urothelial cancer. PMID:28416766

Light induced cytosolic drug delivery from liposomes with gold nanoparticles.

PubMed

Lajunen, Tatu; Viitala, Lauri; Kontturi, Leena-Stiina; Laaksonen, Timo; Liang, Huamin; Vuorimaa-Laukkanen, Elina; Viitala, Tapani; Le Guével, Xavier; Yliperttula, Marjo; Murtomäki, Lasse; Urtti, Arto

2015-04-10

Externally triggered drug release at defined targets allows site- and time-controlled drug treatment regimens. We have developed liposomal drug carriers with encapsulated gold nanoparticles for triggered drug release. Light energy is converted to heat in the gold nanoparticles and released to the lipid bilayers. Localized temperature increase renders liposomal bilayers to be leaky and triggers drug release. The aim of this study was to develop a drug releasing system capable of releasing its cargo to cell cytosol upon triggering with visible and near infrared light signals. The liposomes were formulated using either heat-sensitive or heat- and pH-sensitive lipid compositions with star or rod shaped gold nanoparticles. Encapsulated fluorescent probe, calcein, was released from the liposomes after exposure to the light. In addition, the pH-sensitive formulations showed a faster drug release in acidic conditions than in neutral conditions. The liposomes were internalized into human retinal pigment epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVECs) and did not show any cellular toxicity. The light induced cytosolic delivery of calcein from the gold nanoparticle containing liposomes was shown, whereas no cytosolic release was seen without light induction or without gold nanoparticles in the liposomes. The light activated liposome formulations showed a controlled content release to the cellular cytosol at a specific location and time. Triggering with visual and near infrared light allows good tissue penetration and safety, and the pH-sensitive liposomes may enable selective drug release in the intracellular acidic compartments (endosomes, lysosomes). Thus, light activated liposomes with gold nanoparticles are an attractive option for time- and site-specific drug delivery into the target cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Overexpression of isocitrate dehydrogenase mutant proteins renders glioma cells more sensitive to radiation.

PubMed

Li, Sichen; Chou, Arthur P; Chen, Weidong; Chen, Ruihuan; Deng, Yuzhong; Phillips, Heidi S; Selfridge, Julia; Zurayk, Mira; Lou, Jerry J; Everson, Richard G; Wu, Kuan-Chung; Faull, Kym F; Cloughesy, Timothy; Liau, Linda M; Lai, Albert

2013-01-01

Mutations in isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) are found in a subset of gliomas. Among the many phenotypic differences between mutant and wild-type IDH1/2 gliomas, the most salient is that IDH1/2 mutant glioma patients demonstrate markedly improved survival compared with IDH1/2 wild-type glioma patients. To address the mechanism underlying the superior clinical outcome of IDH1/2 mutant glioma patients, we investigated whether overexpression of the IDH1(R132H) protein could affect response to therapy in the context of an isogenic glioma cell background. Stable clonal U87MG and U373MG cell lines overexpressing IDH1(WT) and IDH1(R132H) were generated, as well as U87MG cell lines overexpressing IDH2(WT) and IDH2(R172K). In vitro experiments were conducted to characterize baseline growth and migration and response to radiation and temozolomide. In addition, reactive oxygen species (ROS) levels were measured under various conditions. U87MG-IDH1(R132H) cells, U373MG-IDH1(R132H) cells, and U87MG-IDH2(R172K) cells demonstrated increased sensitivity to radiation but not to temozolomide. Radiosensitization of U87MG-IDH1(R132H) cells was accompanied by increased apoptosis and accentuated ROS generation, and this effect was abrogated by the presence of the ROS scavenger N-acetyl-cysteine. Interestingly, U87MG-IDH1(R132H) cells also displayed decreased growth at higher cell density and in soft agar, as well as decreased migration. Overexpression of IDH1(R132H) and IDH2(R172K) mutant protein in glioblastoma cells resulted in increased radiation sensitivity and altered ROS metabolism and suppression of growth and migration in vitro. These findings provide insight into possible mechanisms contributing to the improved outcomes observed in patients with IDH1/2 mutant gliomas.

Homeostasis of naive and memory CD4+ T cells: IL-2 and IL-7 differentially regulate the balance between proliferation and Fas-mediated apoptosis.

PubMed

Jaleco, Sara; Swainson, Louise; Dardalhon, Valérie; Burjanadze, Maryam; Kinet, Sandrina; Taylor, Naomi

2003-07-01

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo, the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4(+) T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE, whereas adult CD4(+) T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However, the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone, RTE, as well as mature naive and memory CD4(+) T cells, are rendered only minimally sensitive to Fas-mediated cell death. However, in the presence of the two cytokines, Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4(+) T cells. In contrast, equivalently treated memory CD4(+) T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4(+) T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4(+) T cells. Thus, IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets.

A mutated dph3 gene causes sensitivity of Schizosaccharomyces pombe cells to cytotoxic agents.

PubMed

Villahermosa, Desirée; Knapp, Karen; Fleck, Oliver

2017-12-01

Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3-ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress.

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

Roughness based perceptual analysis towards digital skin imaging system with haptic feedback.

PubMed

Kim, K

2016-08-01

To examine psoriasis or atopic eczema, analyzing skin roughness by palpation is essential to precisely diagnose skin diseases. However, optical sensor based skin imaging systems do not allow dermatologists to touch skin images. To solve the problem, a new haptic rendering technology that can accurately display skin roughness must be developed. In addition, the rendering algorithm must be able to filter spatial noises created during 2D to 3D image conversion without losing the original roughness on the skin image. In this study, a perceptual way to design a noise filter that will remove spatial noises and in the meantime recover maximized roughness is introduced by understanding human sensitivity on surface roughness. A visuohaptic rendering system that can provide a user with seeing and touching digital skin surface roughness has been developed including a geometric roughness estimation method from a meshed surface. In following, a psychophysical experiment was designed and conducted with 12 human subjects to measure human perception with the developed visual and haptic interfaces to examine surface roughness. From the psychophysical experiment, it was found that touch is more sensitive at lower surface roughness, and vice versa. Human perception with both senses, vision and touch, becomes less sensitive to surface distortions as roughness increases. When interact with both channels, visual and haptic interfaces, the performance to detect abnormalities on roughness is greatly improved by sensory integration with the developed visuohaptic rendering system. The result can be used as a guideline to design a noise filter that can perceptually remove spatial noises while recover maximized roughness values from a digital skin image obtained by optical sensors. In addition, the result also confirms that the developed visuohaptic rendering system can help dermatologists or skin care professionals examine skin conditions by using vision and touch at the same time. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

HLA-A11-mediated protection from NK cell-mediated lysis: role of HLA-A11-presented peptides.

PubMed

Gavioli, R; Zhang, Q J; Masucci, M G

1996-08-01

The capacity of MHC class I to protect target cells from NK is well established, but the mechanism by which these molecules influence NK recognition and the physical properties associated with this function remain poorly defined. We have examined this issue using as a model the HLA-A11 allele. HLA-A11 expression correlated with reduced susceptibility to NK and interferon-activated cytotoxicity in transfected sublines of the A11-defective Burkitt's lymphoma WW2-BL and the HLA class I A,B-null C1R cell line. Protection was also achieved by transfection of HLA-A11 in the peptide processing mutant T2 cells line (T2/A11), despite a very low expression of the transfected product at the cell surface. Induction of surface HLA-A11 by culture of T2/A11 cells at 26 degrees C or in the presence of beta 2m did not affect lysis, whereas NK sensitivity was restored by culture in the presence of HLA-All-binding synthetic peptides derived from viral or cellular proteins. Acid treatment rendered T2/A11 and C1R/A11 cells sensitive to lysis, but protection was restored after preincubation with peptide preparations derived from surface stripping of T2/A11 cells. Similar peptide preparations from T2 cells had no effect. The results suggest that NK protection is mediated by HLA-A11 molecules carrying a particular set of peptides that are translocated to the site of MHC class I assembly in the ER in a TAP-independent fashion.

Homeostatic enhancement of active mechanotransduction

NASA Astrophysics Data System (ADS)

Milewski, Andrew; O'Maoiléidigh, Dáibhid; Hudspeth, A. J.

2018-05-01

Our sense of hearing boasts exquisite sensitivity to periodic signals. Experiments and modeling imply, however, that the auditory system achieves this performance for only a narrow range of parameter values. As a result, small changes in these values could compromise the ability of the mechanosensory hair cells to detect stimuli. We propose that, rather than exerting tight control over parameters, the auditory system employs a homeostatic mechanism that ensures the robustness of its operation to variation in parameter values. Through analytical techniques and computer simulations we investigate whether a homeostatic mechanism renders the hair bundle's signal-detection ability more robust to alterations in experimentally accessible parameters. When homeostasis is enforced, the range of values for which the bundle's sensitivity exceeds a threshold can increase by more than an order of magnitude. The robustness of cochlear function based on somatic motility or hair bundle motility may be achieved by employing the approach we describe here.

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

PI3K inhibitor GDC-0941 enhances apoptotic effects of BH-3 mimetic ABT-737 in AML cells in the hypoxic bone marrow microenvironment

PubMed Central

Kojima, Kensuke; Shikami, Masato; Benito, Julina; Ruvolo, Vivian; Wang, Rui-Yu; McQueen, Teresa; Ciurea, Stefan O.; Miida, Takashi; Andreeff, Michael; Konopleva, Marina

2013-01-01

Both phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling and antiapoptotic Bcl-2 family members are critical for survival of acute myeloid leukemia (AML) cells. Here we demonstrate the antileukemic effects of simultaneous inhibition of PI3K by the selective class I PI3K inhibitor GDC-0941 and of Bcl-2 family members by the BH3 mimetic ABT-737 in the context of the bone marrow microenvironment, where hypoxia and interactions with bone marrow stromal cells promote AML cell survival and chemoresistance. The combination of GDC-0941 and ABT-737 profoundly downregulated antiapoptotic Mcl-1 expression levels, activated BAX, and induced mitochondrial apoptosis in AML cells co-cultured with bone marrow stromal cells under hypoxic conditions. Hypoxia caused degradation of Mcl-1 and rendered Mcl-1-overexpressing OCI-AML3 cells sensitive to ABT-737. Our findings suggest that pharmacologic PI3K inhibition by GDC-0941 enhances ABT-737–induced leukemia cell death even under the protective conditions afforded by the bone marrow microenvironment. PMID:23955073

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

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.

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

Using Dynamic Sensitivity Analysis to Assess Testability

NASA Technical Reports Server (NTRS)

Voas, Jeffrey; Morell, Larry; Miller, Keith

1990-01-01

This paper discusses sensitivity analysis and its relationship to random black box testing. Sensitivity analysis estimates the impact that a programming fault at a particular location would have on the program's input/output behavior. Locations that are relatively \\"insensitive" to faults can render random black box testing unlikely to uncover programming faults. Therefore, sensitivity analysis gives new insight when interpreting random black box testing results. Although sensitivity analysis is computationally intensive, it requires no oracle and no human intervention.

Photoelectrochemical cell having photoanode with thin boron phosphide coating as a corrosion resistant layer

DOEpatents

Baughman, Richard J.; Ginley, David S.

1984-01-01

A surface prone to corrosion in corrosive environments is rendered anticorrosive by CVD growing a thin continuous film, e.g., having no detectable pinholes, thereon, of boron phosphide. In one embodiment, the film is semiconductive. In another aspect, the invention is an improved photoanode, and/or photoelectrochemical cell with a photoanode having a thin film of boron phosphide thereon rendering it anitcorrosive, and providing it with unexpectedly improved photoresponsive properties.

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.

GAS PHOTOTUBE CIRCUIT

DOEpatents

Richardson, J.H.

1958-03-01

This patent pertains to electronic circuits for measuring the intensity of light and is especially concerned with measurement between preset light thresholds. Such a circuit has application in connection with devices for reading-out information stored on punch cards or tapes where the cards and tapes are translucent. By the novel arrangement of this invention thc sensitivity of a gas phototube is maintained at a low value when the light intensity is below a first threshold level. If the light level rises above the first threshold level, the tube is rendered highly sensitive and an output signal will vary in proportion to the light intensity change. When the light level decreases below a second threshold level, the gas phototube is automatically rendered highly insensitive. Each of these threshold points is adjustable.

Using the MCF10A/MCF10CA1a Breast Cancer Progression Cell Line Model to Investigate the Effect of Active, Mutant Forms of EGFR in Breast Cancer Development and Treatment Using Gefitinib

PubMed Central

Bessette, Darrell C.; Tilch, Erik; Seidens, Tatjana; Quinn, Michael C. J.; Wiegmans, Adrian P.; Shi, Wei; Cocciardi, Sibylle; McCart-Reed, Amy; Saunus, Jodi M.; Simpson, Peter T.; Grimmond, Sean M.; Lakhani, Sunil R.; Khanna, Kum Kum; Waddell, Nic; Al-Ejeh, Fares; Chenevix-Trench, Georgia

2015-01-01

Background Basal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of epidermal growth factor receptor (EGFR) occurs in ~50% of basal-like breast cancer, and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown. Materials and Methods Constructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated. Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed. Results Mutant EGFR increased MCF10A and MCF10CA1a proliferation and MCF10A gefitinib sensitivity. The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth. Compared to MCF10A cells, MCF10CA1a cells exhibited large regions of gain on chromosomes 3 and 9, deletion on chromosome 7, and mutations in many genes implicated in cancer. Conclusions Mutant EGFR enhances the oncogenic properties of MCF10A cell line, and increases sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased gefitinib sensitivity, perhaps due to additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer, accompanying mutations in PIK3CA may confer gefitinib resistance. PMID:25969993

Pathway-selective sensitization of Mycobacterium tuberculosis for target-based whole-cell screening

PubMed Central

Abrahams, Garth L.; Kumar, Anuradha; Savvi, Suzana; Hung, Alvin W.; Wen, Shijun; Abell, Chris; Barry, Clifton E.; Sherman, David R.; Boshoff, Helena I.M.; Mizrahi, Valerie

2012-01-01

SUMMARY Whole-cell screening of Mycobacterium tuberculosis (Mtb) remains a mainstay of drug discovery but subsequent target elucidation often proves difficult. Conditional mutants that under-express essential genes have been used to identify compounds with known mechanism of action by target-based whole-cell screening (TB-WCS). Here, the feasibility of TB-WCS in Mtb was assessed by generating mutants that conditionally express pantothenate synthetase (panC), diaminopimelate decarboxylase (lysA) and isocitrate lyase (icl1). The essentiality of panC and lysA, and conditional essentiality of icl1 for growth on fatty acids, was confirmed. Depletion of PanC and Icl1 rendered the mutants hypersensitive to target-specific inhibitors. Stable reporter strains were generated for use in high-throughput screening, and their utility demonstrated by identifying compounds that display greater potency against a PanC-depleted strain. These findings illustrate the power of TB-WCS as a tool for tuberculosis drug discovery. PMID:22840772

HIF-2α is essential for carotid body development and function

PubMed Central

Cowburn, Andrew S; Torres-Torrelo, Hortensia; Ortega-Sáenz, Patricia; López-Barneo, José

2018-01-01

Mammalian adaptation to oxygen flux occurs at many levels, from shifts in cellular metabolism to physiological adaptations facilitated by the sympathetic nervous system and carotid body (CB). Interactions between differing forms of adaptive response to hypoxia, including transcriptional responses orchestrated by the Hypoxia Inducible transcription Factors (HIFs), are complex and clearly synergistic. We show here that there is an absolute developmental requirement for HIF-2α, one of the HIF isoforms, for growth and survival of oxygen sensitive glomus cells of the carotid body. The loss of these cells renders mice incapable of ventilatory responses to hypoxia, and this has striking effects on processes as diverse as arterial pressure regulation, exercise performance, and glucose homeostasis. We show that the expansion of the glomus cells is correlated with mTORC1 activation, and is functionally inhibited by rapamycin treatment. These findings demonstrate the central role played by HIF-2α in carotid body development, growth and function. PMID:29671738

Anti-PD-L1/TGFβR2 (M7824) fusion protein induces immunogenic modulation of human urothelial carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis.

PubMed

Grenga, Italia; Donahue, Renee N; Gargulak, Morgan L; Lepone, Lauren M; Roselli, Mario; Bilusic, Marijo; Schlom, Jeffrey

2018-03-01

Avelumab has recently been approved by the Food and Drug Administration for the therapy of Merkel cell carcinoma and urothelial carcinoma. M7824 is a novel first-in-class bifunctional fusion protein comprising a monoclonal antibody against programmed death-ligand 1 (PD-L1, avelumab), fused to the extracellular domain of human transforming growth factor beta (TGFβ) receptor 2, which functions as a TGFβ "trap." Advanced urothelial tumors have been shown to express TGFβ, which possesses immunosuppressive properties that promote cancer progression and metastasis. The rationale for a combined molecule is to block the PD-1/PD-L1 interaction between tumor cells and immune cell infiltrate and simultaneously reduce or eliminate TGFβ from the tumor microenvironment. In this study, we explored the effect of M7824 on invasive urothelial carcinoma cell lines. Human urothelial (transitional cell) carcinoma cell lines HTB-4, HTB-1, and HTB-5 were treated with M7824, M7824mut (M7824 that is mutated in the anti-PD-L1 portion of the molecule and thus does not bind PD-L1), anti-PD-L1 (avelumab), or IgG1 isotype control monoclonal antibody, and were assessed for gene expression, cell-surface phenotype, and sensitivity to lysis by TRAIL, antigen-specific cytotoxic T lymphocytes and natural killer cells. M7824 retains the ability to mediate antibody-dependent cellular cytotoxicity of tumor cells, although in some cases to a lesser extent than anti-PD-L1. However, compared to anti-PD-L1, M7824 increases (A) gene expression of molecules involved in T-cell trafficking in the tumor (e.g., CXCL11), (B) TRAIL-mediated tumor cell lysis, and (C) antigen-specific CD8 + T-cell-mediated lysis of tumor cells. These studies demonstrate the immunomodulatory properties of M7824 on both tumor cell phenotype and immune-mediated lysis. Compared to anti-PD-L1 or M7824mut, M7824 induces immunogenic modulation of urothelial carcinoma cell lines, rendering them more susceptible to immune-mediated recognition and lysis. These findings show the relevance of the dual blockade of PD-L1 and TGFβ in urothelial carcinoma cell lines and thus support the rationale for future clinical studies of M7824 in patients with urothelial cancer. Published by Elsevier Inc.

Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells.

PubMed

Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

2015-11-26

As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations.

Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells

PubMed Central

Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

2015-01-01

As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations. PMID:26608589

Aminophthalocyanine-Mediated Photodynamic Inactivation of Leishmania tropica

PubMed Central

Al-Qahtani, Ahmed; Alkahtani, Saad; Kolli, Bala; Tripathi, Pankaj; Dutta, Sujoy; Al-Kahtane, Abdullah A.; Jiang, Xiong-Jie; Ng, Dennis K. P.

2016-01-01

Photodynamic inactivation of Leishmania spp. requires the cellular uptake of photosensitizers, e.g., endocytosis of silicon(IV)-phthalocyanines (PC) axially substituted with bulky ligands. We report here that when substituted with amino-containing ligands, the PCs (PC1 and PC2) were endocytosed and displayed improved potency against Leishmania tropica promastigotes and axenic amastigotes in vitro. The uptake of these PCs by both Leishmania stages followed saturation kinetics, as expected. Sensitive assays were developed for assessing the photodynamic inactivation of Leishmania spp. by rendering them fluorescent in two ways: transfecting promastigotes to express green fluorescent protein (GFP) and loading them with carboxyfluorescein succinimidyl ester (CFSE). PC-sensitized Leishmania tropica strains were seen microscopically to lose their motility, structural integrity, and GFP/CFSE fluorescence after exposure to red light (wavelength, ∼650 nm) at a fluence of 1 to 2 J cm−2. Quantitative fluorescence assays based on the loss of GFP/CFSE from live Leishmania tropica showed that PC1 and PC2 dose dependently sensitized both stages for photoinactivation, consistent with the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Leishmania tropica strains are >100 times more sensitive than their host cells or macrophages to PC1- and PC2-mediated photoinactivation, judging from the estimated 50% effective concentrations (EC50s) of these cells. Axial substitution of the PC with amino groups instead of other ligands appears to increase its leishmanial photolytic activity by up to 40-fold. PC1 and PC2 are thus potentially useful for photodynamic therapy of leishmaniasis and for oxidative photoinactivation of Leishmania spp. for use as vaccines or vaccine carriers. PMID:26824938

Transport of pyruvate into mitochondria is involved in methylmercury toxicity

PubMed Central

Lee, Jin-Yong; Ishida, Yosuke; Takahashi, Tsutomu; Naganuma, Akira; Hwang, Gi-Wook

2016-01-01

We have previously demonstrated that the overexpression of enzymes involved in the production of pyruvate, enolase 2 (Eno2) and D-lactate dehydrogenase (Dld3) renders yeast highly sensitive to methylmercury and that the promotion of intracellular pyruvate synthesis may be involved in intensifying the toxicity of methylmercury. In the present study, we showed that the addition of pyruvate to culture media in non-toxic concentrations significantly enhanced the sensitivity of yeast and human neuroblastoma cells to methylmercury. The results also suggested that methylmercury promoted the transport of pyruvate into mitochondria and that the increased pyruvate concentrations in mitochondria were involved in intensifying the toxicity of methylmercury without pyruvate being converted to acetyl-CoA. Furthermore, in human neuroblastoma cells, methylmercury treatment alone decreased the mitochondrial membrane potential, and the addition of pyruvate led to a further significant decrease. In addition, treatment with N-acetylcysteine (an antioxidant) significantly alleviated the toxicity of methylmercury and significantly inhibited the intensification of methylmercury toxicity by pyruvate. Based on these data, we hypothesize that methylmercury exerts its toxicity by raising the level of pyruvate in mitochondria and that mitochondrial dysfunction and increased levels of reactive oxygen species are involved in the action of pyruvate. PMID:26899208

Tuning of activation thresholds explains flexibility in the selection and development of T cells in the thymus

PubMed Central

Grossman, Zvi; Singer, Alfred

1996-01-01

Immature CD4+CD8+ thymocytes expressing T-cell antigen receptors (TCR) are selected by TCR-mediated recognition of peptides associated with major histocompatibility complex molecules on thymic stromal cells. Selection ensures reactivity of the mature cells to foreign antigens and tolerance to self. Although much has been learned about the factors that determine whether a thymocyte with a given specificity will be positively or negatively selected, selection as an aspect of the developmental process as a whole is less well-understood. Here we invoke a model in which thymocytes tune their response characteristics individually and dynamically in the course of development. Cellular development and selection are driven by receptor-mediated metabolic perturbations. Perturbation is a measure of the net intracellular change induced by external stimulation. It results from the integration of several signals and countersignals over time and therefore depends on the environment and the maturation stage of the cell. Individual cell adaptation limits the range of perturbations. Such adaptation renders thymocytes less sensitive to the level of stimulation per se, but responsive to environmental changes in that level. This formulation begins to explain the mechanisms that link developmental and selection events to each other. PMID:8962126

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

PubMed Central

Seif, R; Martin, R G

1979-01-01

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation. Images PMID:229274

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

PubMed

Seif, R; Martin, R G

1979-12-01

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.

Persistent response of Fanconi anemia haematopoietic stem and progenitor cells to oxidative stress.

PubMed

Li, Yibo; Amarachintha, Surya; Wilson, Andrew F; Li, Xue; Du, Wei

2017-06-18

Oxidative stress is considered as an important pathogenic factor in many human diseases including Fanconi anemia (FA), an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Members of the FA protein family are involved in DNA damage and other cellular stress responses. Loss of FA proteins renders cells hypersensitive to oxidative stress and cancer transformation. However, how FA cells respond to oxidative DNA damage remains unclear. By using an in vivo stress-response mouse strain expressing the Gadd45β-luciferase transgene, we show here that haematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc persistently responded to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. Furthermore, genetic correction of Fanca deficiency almost completely abolished the persistent oxidative stress-induced G 2 /M arrest and DNA damage response in vivo. Our study suggests that FA pathway is an integral part of a versatile cellular mechanism by which HSPCs respond to oxidative stress.

Persistent response of Fanconi anemia haematopoietic stem and progenitor cells to oxidative stress

PubMed Central

Wilson, Andrew F.; Li, Xue

2017-01-01

ABSTRACT Oxidative stress is considered as an important pathogenic factor in many human diseases including Fanconi anemia (FA), an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Members of the FA protein family are involved in DNA damage and other cellular stress responses. Loss of FA proteins renders cells hypersensitive to oxidative stress and cancer transformation. However, how FA cells respond to oxidative DNA damage remains unclear. By using an in vivo stress-response mouse strain expressing the Gadd45β-luciferase transgene, we show here that haematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc persistently responded to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. Furthermore, genetic correction of Fanca deficiency almost completely abolished the persistent oxidative stress-induced G2/M arrest and DNA damage response in vivo. Our study suggests that FA pathway is an integral part of a versatile cellular mechanism by which HSPCs respond to oxidative stress. PMID:28475398

Microstructured graphene arrays for highly sensitive flexible tactile sensors.

PubMed

Zhu, Bowen; Niu, Zhiqiang; Wang, Hong; Leow, Wan Ru; Wang, Hua; Li, Yuangang; Zheng, Liyan; Wei, Jun; Huo, Fengwei; Chen, Xiaodong

2014-09-24

A highly sensitive tactile sensor is devised by applying microstructured graphene arrays as sensitive layers. The combination of graphene and anisotropic microstructures endows this sensor with an ultra-high sensitivity of -5.53 kPa(-1) , an ultra-fast response time of only 0.2 ms, as well as good reliability, rendering it promising for the application of tactile sensing in artificial skin and human-machine interface. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Appropriate indices for color rendition and their recommended values for UHDTV production using white LED lighting.

PubMed

Hayashida, Tetsuya; Iwasaki, Hiroaki; Masaoka, Kenichiro; Shimizu, Masanori; Yamashita, Takayuki; Iwai, Wataru

2017-06-26

We selected appropriate indices for color rendition and determined their recommended values for ultra-high-definition television (UHDTV) production using white LED lighting. Since the spectral sensitivities of UHDTV cameras can be designed to approximate the ideal spectral sensitivities of UHDTV colorimetry, they have more accurate color reproduction than HDTV cameras, and thus the color-rendering properties of the lighting are critical. Comparing images taken under white LEDs with conventional color rendering indices (R a , R 9-14 ) and recently proposed methods for evaluating color rendition of CQS, TM-30, Q a , and SSI, we found the combination of R a and R 9 appropriate. For white LED lighting, R a ≥ 90 and R 9 ≥ 80 are recommended for UHDTV production.

Detection of compression vessels in trigeminal neuralgia by surface-rendering three-dimensional reconstruction of 1.5- and 3.0-T magnetic resonance imaging.

PubMed

Shimizu, Masahiro; Imai, Hideaki; Kagoshima, Kaiei; Umezawa, Eriko; Shimizu, Tsuneo; Yoshimoto, Yuhei

2013-01-01

Surface-rendered three-dimensional (3D) 1.5-T magnetic resonance (MR) imaging is useful for presurgical simulation of microvascular decompression. This study compared the sensitivity and specificity of 1.5- and 3.0-T surface-rendered 3D MR imaging for preoperative identification of the compression vessels of trigeminal neuralgia. One hundred consecutive patients underwent microvascular decompression for trigeminal neuralgia. Forty and 60 patients were evaluated by 1.5- and 3.0-T MR imaging, respectively. Three-dimensional MR images were constructed on the basis of MR imaging, angiography, and venography data and evaluated to determine the compression vessel before surgery. MR imaging findings were compared with the microsurgical findings to compare the sensitivity and specificity of 1.5- and 3.0-T MR imaging. The agreement between MR imaging and surgical findings depended on the compression vessels. For superior cerebellar artery, 1.5- and 3.0-T MR imaging had 84.4% and 82.7% sensitivity and 100% and 100% specificity, respectively. For anterior inferior cerebellar artery, 1.5- and 3.0-T MR imaging had 33.3% and 50% sensitivity and 92.9% and 95% specificity, respectively. For the petrosal vein, 1.5- and 3.0-T MR imaging had 75% and 64.3% sensitivity and 79.2% and 78.1% specificity, respectively. Complete pain relief was obtained in 36 of 40 and 55 of 60 patients undergoing 1.5- and 3.0-T MR imaging, respectively. The present study showed that both 1.5- and 3.0-T MR imaging provided high sensitivity and specificity for preoperative assessment of the compression vessels of trigeminal neuralgia. Preoperative 3D imaging provided very high quality presurgical simulation, resulting in excellent clinical outcomes. Copyright © 2013 Elsevier Inc. All rights reserved.

Development of a counterselectable seamless mutagenesis system in lactic acid bacteria.

PubMed

Xin, Yongping; Guo, Tingting; Mu, Yingli; Kong, Jian

2017-07-05

Lactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment. The pheS gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome. When mutant pheS gene (pheS*) under the control of the Lc. lactis NZ9000 L-lactate dehydrogenase promoter (P ldh ) was expressed from a plasmid, the resulted PheS* with an A312G substitution rendered cells sensitive to the phenylalanine analog p-chloro-phenylalanine (p-Cl-Phe). This result suggested pheS* was suitable to be used as a counterselectable marker in Lc. lactis. However, the expression level of pheS* from a chromosomal copy was too low to confer p-Cl-Phe sensitivity. Therefore, a strategy of cascading promoters was attempted for strengthening the expression level of pheS*. Expectedly, a cassette 5Pldh-pheS* with five tandem repetitive promoters P ldh resulted in a sensitivity to 15 mM p-Cl-Phe. Subsequently, a counterselectable seamless mutagenesis system PheS*/pG + host9 based on a temperature-sensitive plasmid pG + host9 harboring a 5Pldh-pheS* cassette was developed in Lc. lactis. We also demonstrated the possibility of applying pheS* to be a counterselectable marker in Lactobacillus casei BL23. As reported in E. coli, pheS* as a counterselectable marker has been demonstrated to be functional in targeted gene(s) deletion in Lc. lactis as well as in L. casei. Moreover, the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG + host9 could be used in the wild-type host cells without pretreatment.

Comparison of three different methods for the detection of circulating tumor cells in mice with lung metastasis.

PubMed

Xu, Weifeng; Wu, Bing; Fu, Lengxi; Chen, Junying; Wang, Zeng; Huang, Fei; Chen, Jinrong; Zhang, Mei; Zhang, Zhenhuan; Lin, Jingan; Lan, Ruilong; Chen, Ruiqing; Chen, Wei; Chen, Long; Hong, Jinsheng; Zhang, Weijian; Ding, Yuxiong; Okunieff, Paul; Lin, Jianhua; Zhang, Lurong

2017-06-01

Circulating tumor cells (CTCs) represent the key step of cancer cell dissemination. The alteration of CTCs correlates with the treatment outcome and prognosis. To enrich and identify CTCs from billions of blood cells renders a very challenging task, which triggers development of several methods, including lysis of RBC plus negative or positive enrichment using antibodies, and filter membrane or spiral microfluidics to capture CTCs. To compare the advantages of different enrichment methods for CTCs, we utilized the 4T1 breast cancer cells transfected with both green fluorescent protein (GFP) and luciferase to trace CTCs in the experimental lung metastasis model. Three methods were used to detect CTCs at the same time: bioluminescence assay, smearing method, and membrane filter method. The in vivo alive mouse imaging was used to dynamically monitor the growth of lung metastases. The sensitivity and accuracy of three detection methods were compared side-by-side. Our results showed that 1) the sensitivity of bioluminescence assay was the highest, but there was no information of CTC morphology; 2) the smearing method and membrane filter method could observe the detail of CTC morphology, such as in single or in cluster, while their sensitivity was lower than bioluminescence assay; 3) A dynamic observation at a 7-day intervals, the lung metastatic cancer grew at a log speed, while CTCs were increased at a low speed. This might be due to the activated immune cells eliminating the CTCs at a speed much faster than CTCs were generated. This comparison of three CTC detection methods in mouse model suggests that bioluminescence assay could be used in quantitative study of the effect of certain agent on the suppression of CTCs, while GFP-based morphological assays could be used to study the dissemination mechanism of CTCs. The combination of both bioluminescence assay and GFP-based assay would generate more information for quantity and quality of CTCs.

A computational framework for testing arrhythmia marker sensitivities to model parameters in functionally calibrated populations of atrial cells

NASA Astrophysics Data System (ADS)

Vagos, Márcia R.; Arevalo, Hermenegild; de Oliveira, Bernardo Lino; Sundnes, Joakim; Maleckar, Mary M.

2017-09-01

Models of cardiac cell electrophysiology are complex non-linear systems which can be used to gain insight into mechanisms of cardiac dynamics in both healthy and pathological conditions. However, the complexity of cardiac models can make mechanistic insight difficult. Moreover, these are typically fitted to averaged experimental data which do not incorporate the variability in observations. Recently, building populations of models to incorporate inter- and intra-subject variability in simulations has been combined with sensitivity analysis (SA) to uncover novel ionic mechanisms and potentially clarify arrhythmogenic behaviors. We used the Koivumäki human atrial cell model to create two populations, representing normal Sinus Rhythm (nSR) and chronic Atrial Fibrillation (cAF), by varying 22 key model parameters. In each population, 14 biomarkers related to the action potential and dynamic restitution were extracted. Populations were calibrated based on distributions of biomarkers to obtain reasonable physiological behavior, and subjected to SA to quantify correlations between model parameters and pro-arrhythmia markers. The two populations showed distinct behaviors under steady state and dynamic pacing. The nSR population revealed greater variability, and more unstable dynamic restitution, as compared to the cAF population, suggesting that simulated cAF remodeling rendered cells more stable to parameter variation and rate adaptation. SA revealed that the biomarkers depended mainly on five ionic currents, with noted differences in sensitivities to these between nSR and cAF. Also, parameters could be selected to produce a model variant with no alternans and unaltered action potential morphology, highlighting that unstable dynamical behavior may be driven by specific cell parameter settings. These results ultimately suggest that arrhythmia maintenance in cAF may not be due to instability in cell membrane excitability, but rather due to tissue-level effects which promote initiation and maintenance of reentrant arrhythmia.

Novel T lymphocyte proliferation assessment using whole mouse cryo-imaging

NASA Astrophysics Data System (ADS)

Wuttisarnwattana, Patiwet; Raza, Syed A.; Eid, Saada; Cooke, Kenneth R.; Wilson, David L.

2014-03-01

New imaging technologies enable one to assess T-cell proliferation, an important feature of the immunological response. However, none of the traditional imaging modalities allow one to examine quantiatively T-cell function with microscopic resolution and single cell sensitivity over an entire mouse. To address this need, we established T-cells proliferation assays using 3D microscopic cryo-imaging. Assays include: (1) biodistribution of T-cells, (2) secondary lymphoid organ (SLO) volume measurement, (3) carboxyfluorescein succinimidyl ester (CFSE) dilution per cell as cells divide. To demonstrate the application, a graft-versus-host-disease (GVHD) model was used. 3D visualization show that T-cells specifically homed to the SLOs (spleen and lymph nodes) as well as GVHD target organs (such as GI-tract, liver, skin and thymus).The spleen was chosen as representative of the SLOs. For spleen size analysis, volumes of red and white pulp were measured. Spleen volumes of the allogeneic mice (with GVHD) were significantly larger than those of the syngeneic mice (without GVHD) at 72 to 120 hours post-transplant. For CFSE dilution approach, we employed color-coded volume rendering and probability density function (PDF) of single cell intensity to assess T-cell proliferation in the spleen. As compared to syngeneic T-cells, the allogeneic T-cells quickly aggregated in the spleen as indicated by increasing of CFSE signal over the first 48 hours. Then they rapidly proliferated as evidenced by reduced CFSE intensity (at 48-96 hours). Results suggest that assays can be used to study GVHD treatments using T-cell proliferation and biodistibution as assays. In summary, this is the first time that we are able to track and visualize T-cells in whole mouse with single cell sensitivity. We believe that our technique can be an alternative choice to traditional in vitro immunological proliferation assays by providing assessment of proliferation in an in vivo model.

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.

A Synthetic Interaction Screen Identifies Factors Selectively Required for Proliferation and TERT Transcription in p53-Deficient Human Cancer Cells

PubMed Central

Park, Sung Mi; Zhu, Lihua J.; Debily, Marie-anne; Kittler, Ellen L. W.; Zapp, Maria L.; Lapointe, David; Gobeil, Stephane; Virbasius, Ching-Man; Green, Michael R.

2012-01-01

Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi)–based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53−) human cancer cells. We find that compared to p53-competent (p53+) human cancer cell lines, diverse p53− human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53− cells, RNAi–mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53− but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53− cancer cells. PMID:23284306

Gastric Helicobacter Infection Inhibits Development of Oral Tolerance to Food Antigens in Mice

PubMed Central

Matysiak-Budnik, Tamara; van Niel, Guillaume; Mégraud, Francis; Mayo, Kathryn; Bevilacqua, Claudia; Gaboriau-Routhiau, Valérie; Moreau, Marie-Christiane; Heyman, Martine

2003-01-01

The increase in the transcellular passage of intact antigens across the digestive epithelium infected with Helicobacter pylori may interfere with the regulation of mucosal immune responses. The aim of this work was to study the capacity of Helicobacter infection to inhibit the development of oral tolerance or to promote allergic sensitization and the capacity of a gastro-protective agent, rebamipide, to interfere with these processes in mice. Oral tolerance to ovalbumin (OVA) was studied in 48 C3H/He 4-week-old mice divided into four groups: (i) OVA-sensitized mice; (ii) OVA-“tolerized” mice (that is, mice that were rendered immunologically tolerant); (iii) H. felis-infected, OVA-tolerized mice; (iv) and H. felis-infected, OVA-tolerized, rebamipide-treated mice. Oral sensitization to hen egg lysozyme (HEL) was studied in 48 mice divided into four groups: (i) controls; (ii) HEL-sensitized mice; (iii) H. felis-infected, HEL-sensitized mice; and (iv) H. felis-infected, HEL-sensitized, rebamipide-treated mice. Specific anti-OVA or anti-HEL immunoglobulin E (IgE) and IgG1/IgG2a serum titers were measured by enzyme-linked immunosorbent assay. Additionally, the capacity of rebamipide to interfere with antigen presentation and T-cell activation in vitro, as well as absorption of rebamipide across the epithelial monolayer, was tested. H. felis infection led to the inhibition of oral tolerance to OVA, but rebamipide prevented this inhibitive effect of H. felis. H. felis infection did not enhance the sensitization to HEL, but rebamipide inhibited the development of this sensitization. Moreover, rebamipide inhibited in a dose-dependent manner antigen presentation and T-cell activation in vitro and was shown to be able to cross the epithelium at a concentration capable of inducing this inhibitory effect. We conclude that H. felis can inhibit the development of oral tolerance to OVA in mice and that this inhibition is prevented by rebamipide. PMID:12933867

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

Malonyl-coenzyme-A is a potential mediator of cytotoxicity induced by fatty-acid synthase inhibition in human breast cancer cells and xenografts.

PubMed

Pizer, E S; Thupari, J; Han, W F; Pinn, M L; Chrest, F J; Frehywot, G L; Townsend, C A; Kuhajda, F P

2000-01-15

A biologically aggressive subset of human breast cancers and other malignancies is characterized by elevated fatty-acid synthase (FAS) enzyme expression, elevated fatty acid (FA) synthesis, and selective sensitivity to pharmacological inhibition of FAS activity by cerulenin or the novel compound C75. In this study, inhibition of FA synthesis at the physiologically regulated step of carboxylation of acetyl-CoA to malonyl-CoA by 5-(tetradecyloxy)-2-furoic acid (TOFA) was not cytotoxic to breast cancer cells in clonogenic assays. FAS inhibitors induced a rapid increase in intracellular malonyl-CoA to several fold above control levels, whereas TOFA reduced intracellular malonyl-CoA by 60%. Simultaneous exposure of breast cancer cells to TOFA and an FAS inhibitor resulted in significantly reduced cytotoxicity and apoptosis. Subcutaneous xenografts of MCF7 breast cancer cells in nude mice treated with C75 showed FA synthesis inhibition, apoptosis, and inhibition of tumor growth to less than 1/8 of control volumes, without comparable toxicity in normal tissues. The data suggest that differences in intermediary metabolism render tumor cells susceptible to toxic fluxes in malonyl-CoA, both in vitro and in vivo.

GROWTH REGULATION IN ROUS SARCOMA VIRUS INFECTED CHICKEN EMBRYO FIBROBLASTS: THE ROLE OF THE src GENE

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

Parry, G.; Bartholomew, J.A.; Blssell, M.J.

1980-07-01

We report here a study of the mechanisms leading to loss of growth control in chicken embryo fibroblasts transformed by Rous sarcoma virus (RSV). We have been particularly concerned with the role of the src gene in this process, and have used RSV mutants temperature sensitive (ts) for transformation to investigate the nature of the growth regulatory lesion. The two principal findings were (1) the stationary phase of the cell cycle (G{sub 1}) in chick embryo fibroblasts seems to have two distinct regulatory compartments (using the terminology of Brooks et al. we refer to these as 'Q' and 'A' states).more » When rendered stationary at 41.5 C by serum deprivation, normal cells enter a Q state, but cells infected with the ts-mutant occupy an A state. (2) Whereas normal cells can occupy either state depending on culture conditions, the ts-infected cells, at 41.5 C, do not seem to enter Q even though a known src gene product, a kinase, is reported to be inactive at this temperature. We discuss the possibility that viral factors other than the active src protein kinase influence growth control in infected cultures.« less

Catalase, a remarkable enzyme: targeting the oldest antioxidant enzyme to find a new cancer treatment approach.

PubMed

Glorieux, Christophe; Calderon, Pedro Buc

2017-09-26

This review is centered on the antioxidant enzyme catalase and will present different aspects of this particular protein. Among them: historical discovery, biological functions, types of catalases and recent data with regard to molecular mechanisms regulating its expression. The main goal is to understand the biological consequences of chronic exposure of cells to hydrogen peroxide leading to cellular adaptation. Such issues are of the utmost importance with potential therapeutic extrapolation for various pathologies. Catalase is a key enzyme in the metabolism of H2O2 and reactive nitrogen species, and its expression and localization is markedly altered in tumors. The molecular mechanisms regulating the expression of catalase, the oldest known and first discovered antioxidant enzyme, are not completely elucidated. As cancer cells are characterized by an increased production of reactive oxygen species (ROS) and a rather altered expression of antioxidant enzymes, these characteristics represent an advantage in terms of cell proliferation. Meanwhile, they render cancer cells particularly sensitive to an oxidant insult. In this context, targeting the redox status of cancer cells by modulating catalase expression is emerging as a novel approach to potentiate chemotherapy.

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

Jenei, Veronika; Andersson, Tommy; Jakus, Judit

E3B1, a human homologue of the mouse gene product Abi-1, has been implicated in growth-factor-mediated regulation of the small GTPases p21{sup Ras} and Rac. E3b1 is a regulator of Rac because it can form a complex with Sos-1 and eps8, and such a Sos-1-e3B1-eps8 complex serves as a guanine nucleotide exchange factor for Rac. In the present study, we found that overexpression of e3B1 in NIH3T3/EGFR cells sensitized EGF-induced activation of Rac1, whereas it had no impact on EGF-induced activation of p21{sup Ras}. Remarkably, we found that EGF-induced activation of the p21{sup Ras}-related GTPase Rap1 was also sensitized in NIH3T3/EGFR-e3B1more » cells. Thus, in NIH3T3/EGFR-e3B1 cells, maximal EGF-induced activation of Rap1 occurs with a dose of EGF much lower than in NIH3T3/EGFR cells. We also report that overexpression of e3B1 in NIH3T3/EGFR cells renders EGF-induced activation of Rap1 completely dependent on Src tyrosine kinases but not on c-Abl. However, EGF-induced tyrosine phosphorylation of the Rap GEF C3G occurred regardless of whether e3B1 was overexpressed or not, and this did not involve Src tyrosine kinases. Accordingly, we propose that overexpression of e3B1 in NIH3T3/EGFR cells leads to mobilization of Src tyrosine kinases that participate in EGF-induced activation of Rap1 and inhibition of cell proliferation.« less

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.

Ultrafast charge separation dynamics in opaque, operational dye-sensitized solar cells revealed by femtosecond diffuse reflectance spectroscopy

PubMed Central

Ghadiri, Elham; Zakeeruddin, Shaik M.; Hagfeldt, Anders; Grätzel, Michael; Moser, Jacques-E.

2016-01-01

Efficient dye-sensitized solar cells are based on highly diffusive mesoscopic layers that render these devices opaque and unsuitable for ultrafast transient absorption spectroscopy measurements in transmission mode. We developed a novel sub-200 femtosecond time-resolved diffuse reflectance spectroscopy scheme combined with potentiostatic control to study various solar cells in fully operational condition. We studied performance optimized devices based on liquid redox electrolytes and opaque TiO2 films, as well as other morphologies, such as TiO2 fibers and nanotubes. Charge injection from the Z907 dye in all TiO2 morphologies was observed to take place in the sub-200 fs time scale. The kinetics of electron-hole back recombination has features in the picosecond to nanosecond time scale. This observation is significantly different from what was reported in the literature where the electron-hole back recombination for transparent films of small particles is generally accepted to occur on a longer time scale of microseconds. The kinetics of the ultrafast electron injection remained unchanged for voltages between +500 mV and –690 mV, where the injection yield eventually drops steeply. The primary charge separation in Y123 organic dye based devices was clearly slower occurring in two picoseconds and no kinetic component on the shorter femtosecond time scale was recorded. PMID:27095505

Human cytomegalovirus renders cells non-permissive for replication of herpes simplex viruses

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

Cockley, K.D.

1988-01-01

The herpes simplex virus (HSV) genome during production infection in vitro may be subject to negative regulation which results in modification of the cascade of expression of herpes virus macromolecular synthesis leading to establishment of HSV latency. In the present study, human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of HSV type-1 (HSV-1). A delay in HSV replication of 15 hr as well as a consistent, almost 1000-fold inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 hr after superinfection were observed compared with controls infected with HSV alone. HSV type-2 (HSV-2)more » replication was similarly inhibited in HCMV-infected HEL cells. Prior ultraviolet-irradiation (UV) of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HCMV deoxyribonucleic acid (DNA) negative temperature-sensitive (ts) mutants inhibited HSV replications as efficiently as wild-type (wt) HCMV at the non-permissive temperature. Evidence for penetration and replication of superinfecting HSV into HCMV-infected cells was provided by blot hybridization of HSV DNA synthesized in HSV-superinfected cell cultures and by cesium chloride density gradient analysis of ({sup 3}H)-labeled HSV-1-superinfected cells.« less

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

Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii

PubMed Central

Morales-Menchén, Ana; Navarro-García, Federico; Guirao-Abad, José P.; Román, Elvira; Prieto, Daniel; Coman, Ioana V.; Pla, Jesús; Alonso-Monge, Rebeca

2018-01-01

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains. PMID:29774204

The putative hydrolase YycJ (WalJ) affects the coordination of cell division with DNA replication in Bacillus subtilis and may play a conserved role in cell wall metabolism.

PubMed

Biller, Steven J; Wayne, Kyle J; Winkler, Malcolm E; Burkholder, William F

2011-02-01

Bacteria must accurately replicate and segregate their genetic information to ensure the production of viable daughter cells. The high fidelity of chromosome partitioning is achieved through mechanisms that coordinate cell division with DNA replication. We report that YycJ (WalJ), a predicted member of the metallo-β-lactamase superfamily found in most low-G+C Gram-positive bacteria, contributes to the fidelity of cell division in Bacillus subtilis. B. subtilis ΔwalJ (ΔwalJ(Bsu)) mutants divide over unsegregated chromosomes more frequently than wild-type cells, and this phenotype is exacerbated when DNA replication is inhibited. Two lines of evidence suggest that WalJ(Bsu) and its ortholog in the Gram-positive pathogen Streptococcus pneumoniae, WalJ(Spn) (VicX), play a role in cell wall metabolism: (i) strains of B. subtilis and S. pneumoniae lacking walJ exhibit increased sensitivity to a narrow spectrum of cephalosporin antibiotics, and (ii) reducing the expression of a two-component system that regulates genes involved in cell wall metabolism, WalRK (YycFG), renders walJ essential for growth in B. subtilis, as observed previously with S. pneumoniae. Together, these results suggest that the enzymatic activity of WalJ directly or indirectly affects cell wall metabolism and is required for accurate coordination of cell division with DNA replication.

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

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.

Preclinical Evaluation of Sequential Combination of Oncolytic Adenovirus Delta-24-RGD and Phosphatidylserine-Targeting Antibody in Pancreatic Ductal Adenocarcinoma.

PubMed

Dai, Bingbing; Roife, David; Kang, Ya'an; Gumin, Joy; Rios Perez, Mayrim V; Li, Xinqun; Pratt, Michael; Brekken, Rolf A; Fueyo-Margareto, Juan; Lang, Frederick F; Fleming, Jason B

2017-04-01

Delta-24-RGD (DNX-2401) is a conditional replication-competent oncolytic virus engineered to preferentially replicate in and lyse tumor cells with abnormality of p16/RB/E2F pathway. In a phase I clinical trial, Delta-24-RGD has shown favorable safety profile and promising clinical efficacy in brain tumor, which prompted us to evaluate its anticancer activity in pancreatic ductal adenocarcinoma (PDAC), which also has high frequency of homozygous deletion and promoter methylation of CDKN2A encoding the p16 protein. Our results demonstrate that Delta-24-RGD can induce dramatic cytotoxicity in a subset of PDAC cell lines with high cyclin D1 expression. Induction of autophagy and apoptosis by Delta-24-RGD in sensitive PDAC cells was confirmed with LC3B-GFP autophagy reporter and acridine orange staining as well as Western blotting analysis of LC3B-II expression. Notably, we found that Delta-24-RGD induced phosphatidylserine exposure in infected cells independent of cells' sensitivity to Delta-24-RGD, which renders a rationale for combination of Delta-24-RGD viral therapy and phosphatidylserine targeting antibody for PDAC. In a mouse PDAC model derived from a liver metastatic pancreatic cancer cell line, Delta-24-RGD significantly inhibited tumor growth compared with control ( P < 0.001), and combination of phosphatidylserine targeting antibody 1N11 further enhanced its anticancer activity ( P < 0.01) possibly through inducing synergistic anticancer immune responses. Given that these 2 agents are currently in clinical evaluation, our study warrants further clinical evaluation of this novel combination strategy in pancreatic cancer therapy. Mol Cancer Ther; 16(4); 662-70. ©2016 AACR . ©2017 American Association for Cancer Research.

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.

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

Ecotoxicological Assessment of Immersion Samples from Façade Render Containing Free or Encapsulated Biocides.

PubMed

Vermeirssen, Etiënne L M; Campiche, Sophie; Dietschweiler, Conrad; Werner, Inge; Burkhardt, Michael

2018-05-22

To protect house façades from fouling by microorganisms, biocides can be added to a render or paint before it is applied. During driving rain events, these biocides gradually leach out and have the potential to pollute soil or aquatic ecosystems. We studied the leaching behaviour of biocides and toxicity of leachates from renders with either free or encapsulated biocides. Both render types contained equal amounts of terbutryn, 2-octyl-3(2H)-isothiazolinone (OIT) and 4,5-dichloro-2-n-octyl-4-isothiazolino-3-one (DCOIT). Leachate samples were generated over nine immersion cycles according to a European standard and biocides were quantified. The first and ninth samples were tested using bioassays with algae, bacteria and water flea, the first sample with earthworms and springtails. Encapsulation reduced leaching of terbutryn, OIT and DCOIT four-, 17-, and 27-fold. For aquatic organisms, the toxicity of water from render containing encapsulated biocides was always lower than that of render with free biocides. Furthermore, toxicity decreased four- to five-fold over the nine immersion cycles. Inhibition of photosynthesis was the most sensitive endpoint, followed by algal growth rate, bacterial bioluminescence and water flea reproduction. Toxicity to algae was explained by terbutryn and toxicity to bacteria by OIT. None of the samples affected soil organisms. Results demonstrate that combining standardised leaching tests with standardised bioassays is a promising approach to evaluate the ecotoxicity of biocides that leach from façade renders. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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

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

Display modes for CT colonography. Part II. Blinded comparison of axial CT and virtual endoscopic and panoramic endoscopic volume-rendered studies.

PubMed

Beaulieu, C F; Jeffrey, R B; Karadi, C; Paik, D S; Napel, S

1999-07-01

To determine the sensitivity of radiologist observers for detecting colonic polyps by using three different data review (display) modes for computed tomographic (CT) colonography, or "virtual colonoscopy." CT colonographic data in a patient with a normal colon were used as base data for insertion of digitally synthesized polyps. Forty such polyps (3.5, 5, 7, and 10 mm in diameter) were randomly inserted in four copies of the base data. Axial CT studies, volume-rendered virtual endoscopic movies, and studies from a three-dimensional mode termed "panoramic endoscopy" were reviewed blindly and independently by two radiologists. Detection improved with increasing polyp size. Trends in sensitivity were dependent on whether all inserted lesions or only visible lesions were considered, because modes differed in how completely the colonic surface was depicted. For both reviewers and all polyps 7 mm or larger, panoramic endoscopy resulted in significantly greater sensitivity (90%) than did virtual endoscopy (68%, P = .014). For visible lesions only, the sensitivities were 85%, 81%, and 60% for one reader and 65%, 62%, and 28% for the other for virtual endoscopy, panoramic endoscopy, and axial CT, respectively. Three-dimensional displays were more sensitive than two-dimensional displays (P < .05). The sensitivity of panoramic endoscopy is higher than that of virtual endoscopy, because the former displays more of the colonic surface. Higher sensitivities for three-dimensional displays may justify the additional computation and review time.

Detoxification of mercury, cadmium, and lead in Klebsiella aerogenes NCTC 418 growing in continuous culture

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

Aiking, H.; Govers, H.; van 'T Riet, J.

1985-11-01

Klebsiella aerogenes NCTC 418 growing in the presence of cadmium under glucose-, sulfate-, or phosphate-limited conditions in continuous culture exhibited sulfide formation and P/sub i/ accumulation as the only demonstrable detoxification mechanisms. In the presence of mercury under similar conditions only HgS formation could be confirmed, by an increased sensitivity to mercury under sulfate-limited conditions, among others. The fact that the cells were most sensitive to cadmium under conditions of phosphate limitation and most sensitive to mercury under conditions of sulfate limitation led to the hypothesis that these inorganic detoxification mechanisms generally depended on a kind of facilitated precipitation. Themore » process was coined thus because heavy metals were probably accumulated and precipitated near the cell perimeter due to the relatively high local concentrations of sulfide and phosphate there. Depending on the growth-limiting nutrient, mercury proved to be 25-fold (phosphate limitation), 75-fold (glycerol limitation), or 150-fold (sulfate limitation) more toxic than cadmium to this organism. In the presence of lead, PbS formation was suggested. since no other detoxification mechanisms were detected, for example, rendering heavy metal ions innocuous as metallo-organic compounds, it was concluded that formation of heavy metal precipitates is crucially important to this organism. In addition, it was observed that several components of a defined mineral medium were able to reduce mercuric ions to elemental mercury. This abiotic mercury volatilization was studied in detail, and its general and environmental implications are discussed.« less

Review. The incentive sensitization theory of addiction: some current issues.

PubMed

Robinson, Terry E; Berridge, Kent C

2008-10-12

We present a brief overview of the incentive sensitization theory of addiction. This posits that addiction is caused primarily by drug-induced sensitization in the brain mesocorticolimbic systems that attribute incentive salience to reward-associated stimuli. If rendered hypersensitive, these systems cause pathological incentive motivation ('wanting') for drugs. We address some current questions including: what is the role of learning in incentive sensitization and addiction? Does incentive sensitization occur in human addicts? Is the development of addiction-like behaviour in animals associated with sensitization? What is the best way to model addiction symptoms using animal models? And, finally, what are the roles of affective pleasure or withdrawal in addiction?

The Spindle Assembly Checkpoint Is Not Essential for Viability of Human Cells with Genetically Lowered APC/C Activity.

PubMed

Wild, Thomas; Larsen, Marie Sofie Yoo; Narita, Takeo; Schou, Julie; Nilsson, Jakob; Choudhary, Chunaram

2016-03-01

The anaphase-promoting complex/cyclosome (APC/C) and the spindle assembly checkpoint (SAC), which inhibits the APC/C, are essential determinants of mitotic timing and faithful division of genetic material. Activation of the APC/C is known to depend on two APC/C-interacting E2 ubiquitin-conjugating enzymes-UBE2C and UBE2S. We show that APC/C activity in human cells is tuned by the combinatorial use of three E2s, namely UBE2C, UBE2S, and UBE2D. Genetic deletion of UBE2C and UBE2S, individually or in combination, leads to discriminative reduction in APC/C function and sensitizes cells to UBE2D depletion. Reduction of APC/C activity results in loss of switch-like metaphase-to-anaphase transition and, strikingly, renders cells insensitive to chemical inhibition of MPS1 and genetic ablation of MAD2, both of which are essential for the SAC. These results provide insights into the regulation of APC/C activity and demonstrate that the essentiality of the SAC is imposed by the strength of the APC/C. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Solution-gated graphene transistors for chemical and biological sensors.

PubMed

Yan, Feng; Zhang, Meng; Li, Jinhua

2014-03-01

Graphene has attracted much attention in biomedical applications for its fascinating properties. Because of the well-known 2D structure, every atom of graphene is exposed to the environment, so the electronic properties of graphene are very sensitive to charged analytes (ions, DNA, cells, etc.) or an electric field around it, which renders graphene an ideal material for high-performance sensors. Solution-gated graphene transistors (SGGTs) can operate in electrolytes and are thus excellent candidates for chemical and biological sensors, which have been extensively studied in the recent 5 years. Here, the device physics, the sensing mechanisms, and the performance of the recently developed SGGT-based chemical and biological sensors, including pH, ion, cell, bacterial, DNA, protein, glucose sensors, etc., are introduced. Their advantages and shortcomings, in comparison with some conventional techniques, are discussed. Conclusions and challenges for the future development of the field are addressed in the end. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ssaD1, a suppressor of secA51(Ts) that renders growth of Escherichia coli cold sensitive, is an early amber mutation in the transcription factor gene nusB.

PubMed Central

Rajapandi, T.; Oliver, D.

1994-01-01

Complementation analysis of the ssaD1 mutation, isolated as a suppressor of the secA51(Ts) mutation that renders growth of Escherichia coli cold sensitive, was used to show that ssaD corresponds to nusB, a gene known to be important in transcription antitermination. DNA sequence analysis of the ssaD1 allele showed that it creates an amber mutation in the 15th codon of nusB. Analysis of the effect of different levels of NusB protein on secA transcription and translation suggested that NusB plays little or no role in the control of secA expression. Accordingly, mechanisms by which nusB inactivation can lead to suppression of secA51(Ts) and secY24(Ts) mutations without affecting secA expression need to be considered. PMID:8021230

Thermal effects on bacterial bioaerosols in continuous air flow.

PubMed

Jung, Jae Hee; Lee, Jung Eun; Kim, Sang Soo

2009-08-01

Exposure to bacterial bioaerosols can have adverse effects on health, such as infectious diseases, acute toxic effects, and allergies. The search for ways of preventing and curing the harmful effects of bacterial bioaerosols has created a strong demand for the study and development of an efficient method of controlling bioaerosols. We investigated the thermal effects on bacterial bioaerosols of Escherichia coli and Bacillus subtilis by using a thermal electric heating system in continuous air flow. The bacterial bioaerosols were exposed to a surrounding temperature that ranged from 20 degrees C to 700 degrees C for about 0.3 s. Both E. coli and B. subtilis vegetative cells were rendered more than 99.9% inactive at 160 degrees C and 350 degrees C of wall temperature of the quartz tube, respectively. Although the data on bacterial injury showed that the bacteria tended to sustain greater damage as the surrounding temperature increased, Gram-negative E. coli was highly sensitive to structural injury but Gram-positive B. subtilis was slightly more sensitive to metabolic injury. In addition, the inactivation of E. coli endotoxins was found to range from 9.2% (at 200 degrees C) to 82.0% (at 700 degrees C). However, the particle size distribution and morphology of both bacterial bioaerosols were maintained, despite exposure to a surrounding temperature of 700 degrees C. Our results show that thermal heating in a continuous air flow can be used with short exposure time to control bacterial bioaerosols by rendering the bacteria and endotoxins to a large extent inactive. This result could also be useful for developing more effective thermal treatment strategies for use in air purification or sterilization systems to control bioaerosols.

PI3K inhibitor GDC-0941 enhances apoptotic effects of BH-3 mimetic ABT-737 in AML cells in the hypoxic bone marrow microenvironment.

PubMed

Jin, Linhua; Tabe, Yoko; Kojima, Kensuke; Shikami, Masato; Benito, Julina; Ruvolo, Vivian; Wang, Rui-Yu; McQueen, Teresa; Ciurea, Stefan O; Miida, Takashi; Andreeff, Michael; Konopleva, Marina

2013-12-01

Both phosphatidylinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling and antiapoptotic Bcl-2 family members are critical for survival of acute myeloid leukemia (AML) cells. Here, we demonstrate the antileukemic effects of simultaneous inhibition of PI3K by the selective class I PI3K inhibitor GDC-0941 and of Bcl-2 family members by the BH3 mimetic ABT-737 in the context of the bone marrow microenvironment, where hypoxia and interactions with bone marrow stromal cells promote AML cell survival and chemoresistance. The combination of GDC-0941 and ABT-737 profoundly downregulated antiapoptotic Mcl-1 expression levels, activated BAX, and induced mitochondrial apoptosis in AML cells co-cultured with bone marrow stromal cells under hypoxic conditions. Hypoxia caused degradation of Mcl-1 and rendered Mcl-1-overexpressing OCI-AML3 cells sensitive to ABT-737. Our findings suggest that pharmacologic PI3K inhibition by GDC-0941 enhances ABT-737-induced leukemia cell death even under the protective conditions afforded by the bone marrow microenvironment. Combined blockade of PI3K and Bcl-2 pathways down-regulates anti-apoptotic Mcl-1 expression PI3K and Bcl-2 induced Mcl-1 down-regulation activates BAX PI3K and Bcl-2 blockage induces apoptosis in AML under hypoxic BM microenvironment.

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.

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.

Effects of Pulsed Electromagnetic Fields on Breast Cancer Cell Line MCF 7 Using Absorption Spectroscopy.

PubMed

Alcantara, Dominic Z; Soliman, Ian Jerry S; Pobre, Romeric F; Naguib, Raouf N G

2017-07-01

We present an analysis of the effects of pulsed electromagnetic fields (PEMF) with 3.3 MHz carrier frequency and modulated by audio resonant frequencies on the MCF-7 breast cancer cell line in vitro using absorption spectroscopy. This involves a fluorescence dye called PrestoBlue™ Cell Viability Reagent and a spectrophotometry to test the viability of MCF-7 breast cancer cells under different PEMF treatment conditions in terms of the cell absorption values. The DNA molecule of the MCF-7 breast cancer cells has an electric dipole property that renders it sensitive and reactive to applied electromagnetic fields. Resonant frequencies derived from four genes mutated in MCF-7 breast cancer cells [rapamycin-insensitive companion of mammalian target of rapamycin (RICTOR), peroxisome proliferator-activated receptor (PPARG), Nijmegen breakage syndrome 1 (NBN) and checkpoint kinase 2 (CHEK2)] were applied in generating square pulsed electromagnetic waves. Effects were monitored through measurement of absorption of the samples with PrestoBlue™, and the significance of the treatment was determined using the t-test. There was a significant effect on MCF-7 cells after treatment with PEMF at the resonant frequencies of the following genes for specific durations of exposure: RICTOR for 10 min, PPARG for 10 min, NBN for 15 min, and CHEK2 for 5 min. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

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

Electrocatalytic Zinc Composites as the Efficient Counter Electrodes of Dye-Sensitized Solar Cells: Study on the Electrochemical Performances and Density Functional Theory Calculations.

PubMed

Li, Chun-Ting; Chang, Hung-Yu; Li, Yu-Yan; Huang, Yi-June; Tsai, Yu-Lin; Vittal, R; Sheng, Yu-Jane; Ho, Kuo-Chuan

2015-12-30

Highly efficient zinc compounds (Zn3N2, ZnO, ZnS, and ZnSe) have been investigated as low-cost electrocatalysts for the counter electrodes (CE) of dye-sensitized solar cells (DSSCs). Among them, Zn3N2 and ZnSe are introduced for the first time in DSSCs. The zinc compounds were separately mixed with a conducting binder, poly(3,4-ethylene-dioxythiophene):poly(styrenesulfonate) ( PSS), and thereby four composite films of Zn3N2/PEDOT:PSS, ZnO/PEDOT:PSS, ZnS/PEDOT:PSS, and ZnSe/ PSS were coated on the tin-doped indium oxide (ITO) substrates through a simple drop-coating process. In the composite film, nanoparticles of the zinc compound form active sites for the electrocatalytic reduction of triiodide ions, and PSS provides a continuous conductive matrix for fast electron transfer. By varying the weight percentage (5-20 wt %) of a zinc compound with respect to the weight of the PSS, the optimized concentration of a zinc compound was found to be 10 wt % in all four cases, based on the photovoltaic performances of the corresponding DSSCs. At this concentration (10 wt %), the composites films with Zn3N2 (Zn3N2-10), ZnO (ZnO-10), ZnS (ZnS-10), and ZnSe (ZnSe-10) rendered, for their DSSCs, power conversion efficiencies (η) of 8.73%, 7.54%, 7.40%, and 8.13%, respectively. The difference in the power conversion efficiency is explained based on the electrocatalytic abilities of those composite films as determined by cyclic voltammetry (CV), Tafel polarization plots, and electrochemical impedance spectroscopy (EIS) techniques. The energy band gaps of the zinc compounds, obtained by density functional theory (DFT) calculations, were used to explain the electrocatalytic behaviors of the compounds. Among all the zinc-based composites, the one with Zn3N2-10 showed the best electrocatalytic ability and thereby rendered for its DSSC the highest η of 8.73%, which is even higher than that of the cell with the traditional Pt CE (8.50%). Therefore, Zn3N2 can be considered as a promising inexpensive electrocatalyst to replace the rare and expensive Pt.

The role of the inhibitors of interleukin-6 signal transduction SHP2 and SOCS3 for desensitization of interleukin-6 signalling.

PubMed Central

Fischer, Patrick; Lehmann, Ute; Sobota, Radoslaw M; Schmitz, Jochen; Niemand, Claudia; Linnemann, Sonja; Haan, Serge; Behrmann, Iris; Yoshimura, Akihiko; Johnston, James A; Müller-Newen, Gerhard; Heinrich, Peter C; Schaper, Fred

2004-01-01

The immediate early response of cells treated with IL-6 (interleukin-6) is the activation of the signal transducer and activator of transcription (STAT)3. The Src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP2 and the feedback inhibitor SOCS3 (suppressor of cytokine signalling) are potent inhibitors of IL-6 signal transduction. Impaired function of SOCS3 or SHP2 leads to enhanced and prolonged IL-6 signalling. The inhibitory function of both proteins depends on their recruitment to the tyrosine motif 759 within glycoprotein gp130. In contrast to inactivation, desensitization of signal transduction is regarded as impaired responsiveness due to prestimulation. Usually, after activation the sensing receptor becomes inactivated by modifications such as phosphorylation, internalization or degradation. We designed an experimental approach which allows discrimination between desensitization and inactivation of IL-6 signal transduction. We observed that pre-stimulation with IL-6 renders cells less sensitive to further stimulation with IL-6. After several hours, the cells become sensitive again. We show that not only signal transduction through previously activated receptors is affected by desensitization but signalling through receptors which were not targeted by the first stimulation was also attenuated ( trans -desensitization). Interestingly, in contrast to inhibition, desensitization does not depend on the presence of functional SHP2. Furthermore, cells lacking SOCS3 show constitutive STAT3 activation which is not affected by pre-stimulation with IL-6. All these observations suggest that desensitization and inhibition of signalling are mechanistically distinct. PMID:14611646

ΔNp63 activates the Fanconi anemia DNA repair pathway and limits the efficacy of cisplatin treatment in squamous cell carcinoma.

PubMed

Bretz, Anne Catherine; Gittler, Miriam P; Charles, Joël P; Gremke, Niklas; Eckhardt, Ines; Mernberger, Marco; Mandic, Robert; Thomale, Jürgen; Nist, Andrea; Wanzel, Michael; Stiewe, Thorsten

2016-04-20

TP63, a member of the p53 gene family gene, encodes the ΔNp63 protein and is one of the most frequently amplified genes in squamous cell carcinomas (SCC) of the head and neck (HNSCC) and lungs (LUSC). Using an epiallelic series of siRNAs with intrinsically different knockdown abilities, we show that the complete loss of ΔNp63 strongly impaired cell proliferation, whereas partial ΔNp63 depletion rendered cells hypersensitive to cisplatin accompanied by an accumulation of DNA damage. Expression profiling revealed wide-spread transcriptional regulation of DNA repair genes and in particular Fanconi anemia (FA) pathway components such as FANCD2 and RAD18 - known to be crucial for the repair of cisplatin-induced interstrand crosslinks. In SCC patients ΔNp63 levels significantly correlate with FANCD2 and RAD18 expression confirming ΔNp63 as a key activator of the FA pathway in vivo Mechanistically, ΔNp63 bound an upstream enhancer of FANCD2 inactive in primary keratinocytes but aberrantly activated by ΔNp63 in SCC. Consistently, depletion of FANCD2 sensitized to cisplatin similar to depletion of ΔNp63. Together, our results demonstrate that ΔNp63 directly activates the FA pathway in SCC and limits the efficacy of cisplatin treatment. Targeting ΔNp63 therefore would not only inhibit SCC proliferation but also sensitize tumors to chemotherapy. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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.

Endogenous S-sulfhydration of PTEN helps protect against modification by nitric oxide

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

Ohno, Kazuki; Okuda, Kosaku; Uehara, Takashi, E-mail: uehara@pharm.okayama-u.ac.jp

2015-01-02

Highlights: • PTEN is S-sulfhydrated endogenously in SH-SY5Y human neuroblastoma cells. • Preventing this modification by knocking down CBS renders PTEN sensitive to NO. • pAkt levels are increased significantly in CBS siRNA-transfected cells. • H{sub 2}S functions as an endogenous regulator of PTEN in neuronal cells. - Abstract: Hydrogen sulfide (H{sub 2}S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H{sub 2}S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and themore » oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H{sub 2}S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine β-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H{sub 2}S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H{sub 2}S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions.« 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

DNA damage, metabolism and aging in pro-inflammatory T cells: Rheumatoid arthritis as a model system.

PubMed

Li, Yinyin; Goronzy, Jörg J; Weyand, Cornelia M

2018-05-01

The aging process is the major driver of morbidity and mortality, steeply increasing the risk to succumb to cancer, cardiovascular disease, infection and neurodegeneration. Inflammation is a common denominator in age-related pathologies, identifying the immune system as a gatekeeper in aging overall. Among immune cells, T cells are long-lived and exposed to intense replication pressure, making them sensitive to aging-related abnormalities. In successful T cell aging, numbers of naïve cells, repertoire diversity and activation thresholds are preserved as long as possible; in maladaptive T cell aging, protective T cell functions decline and pro-inflammatory effector cells are enriched. Here, we review in the model system of rheumatoid arthritis (RA) how maladaptive T cell aging renders the host susceptible to chronic, tissue-damaging inflammation. In T cells from RA patients, known to be about 20years pre-aged, three interconnected functional domains are altered: DNA damage repair, metabolic activity generating energy and biosynthetic precursor molecules, and shaping of plasma membranes to promote T cell motility. In each of these domains, key molecules and pathways have now been identified, including the glycolytic enzymes PFKFB3 and G6PD; the DNA repair molecules ATM, DNA-PKcs and MRE11A; and the podosome marker protein TKS5. Some of these molecules may help in defining targetable pathways to slow the T cell aging process. Copyright © 2017 Elsevier Inc. All rights reserved.

A non-neuronal cholinergic system regulates cellular ATP levels to maintain cell viability.

PubMed

Oikawa, Shino; Iketani, Mitsue; Kakinuma, Yoshihiko

2014-01-01

We previously suggested that a non-neuronal cholinergic system modulates energy metabolism through the mitochondria. However, the mechanisms responsible for making this system crucial remained undetermined. In this study, we developed a fusion protein expression vector containing a luciferase gene fused to the folic acid receptor-α gene. This protein of the vector was confirmed to target the plasma membrane of transfected HEK293 cells, and vector-derived luciferase activities and ATP levels in viable cells were positively correlated (r = 0.599). Using this luciferase vector, choline acetyltransferase (ChAT)-expressing cells (i.e., cells with an activated non-neuronal cholinergic system) had increased cellular ATP levels. ChAT-expressing cells also had upregulated IGF-1R and Glut-1 protein expressions as well as increased glucose uptake. This activated non-neuronal cholinergic system with efficient glucose metabolism rendered cells resistant to serum depletion-induced cell death. Our results indicate that a non-neuronal cholinergic system is involved in sustaining ATP levels to render cells resistant to a nutrient-deficient environment. © 2014 S. Karger AG, Basel.

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

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

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.

Multifunctional Iodide-Free Polymeric Ionic Liquid for Quasi-Solid-State Dye-Sensitized Solar Cells with a High Open-Circuit Voltage.

PubMed

Lin, Yi-Feng; Li, Chun-Ting; Lee, Chuan-Pei; Leu, Yow-An; Ezhumalai, Yamuna; Vittal, R; Chen, Ming-Chou; Lin, Jiang-Jen; Ho, Kuo-Chuan

2016-06-22

A polymeric ionic liquid, poly(oxyethylene)-imide-imidazolium selenocyanate (POEI-IS), was newly synthesized and used for a multifunctional gel electrolyte in a quasi-solid-state dye-sensitized solar cell (QSS-DSSC). POEI-IS has several functions: (a) acts as a gelling agent for the electrolyte of the DSSC, (b) possesses a redox mediator of SeCN(-), which is aimed to form a SeCN(-)/(SeCN)3(-) redox couple with a more positive redox potential than that of traditional I(-)/I3(-), (c) chelates the potassium cations through the lone pair electrons of the oxygen atoms of its poly(oxyethylene)-imide-imidazolium (POEI-I) segments, and (d) obstructs the recombination of photoinjected electrons with (SeCN)3(-) ions in the electrolyte through its POEI-I segments. Thus, the POEI-IS renders a high open-circuit voltage (VOC) to the QSS-DSSC due to its functions of b-d and prolongs the stability of the cell due to its function of a. The QSS-DSSC with the gel electrolyte containing 30 wt % of the POEI-IS in liquid selenocyanate electrolyte exhibited a high VOC of 825.50 ± 3.51 mV and a high power conversion efficiency (η) of 8.18 ± 0.02%. The QSS-DSSC with 30 wt % POEI-IS retained up to 95% of its initial η after an at-rest stability test with the period of more than 1,000 h.

Immune activation of the host cell induces drug tolerance in Mycobacterium tuberculosis both in vitro and in vivo

PubMed Central

Liu, Yancheng; Tan, Shumin; Huang, Lu; Abramovitch, Robert B.; Rohde, Kyle H.; Zimmerman, Matthew D.; Chen, Chao; Dartois, Véronique; VanderVen, Brian C.

2016-01-01

Successful chemotherapy against Mycobacterium tuberculosis (Mtb) must eradicate the bacterium within the context of its host cell. However, our understanding of the impact of this environment on antimycobacterial drug action remains incomplete. Intriguingly, we find that Mtb in myeloid cells isolated from the lungs of experimentally infected mice exhibit tolerance to both isoniazid and rifampin to a degree proportional to the activation status of the host cells. These data are confirmed by in vitro infections of resting versus activated macrophages where cytokine-mediated activation renders Mtb tolerant to four frontline drugs. Transcriptional analysis of intracellular Mtb exposed to drugs identified a set of genes common to all four drugs. The data imply a causal linkage between a loss of fitness caused by drug action and Mtb’s sensitivity to host-derived stresses. Interestingly, the environmental context exerts a more dominant impact on Mtb gene expression than the pressure on the drugs’ primary targets. Mtb’s stress responses to drugs resemble those mobilized after cytokine activation of the host cell. Although host-derived stresses are antimicrobial in nature, they negatively affect drug efficacy. Together, our findings demonstrate that the macrophage environment dominates Mtb’s response to drug pressure and suggest novel routes for future drug discovery programs. PMID:27114608

Inactivation of the Fanconi anemia/BRCA pathway in lung and oral cancers: implications for treatment and survival.

PubMed

Marsit, Carmen J; Liu, Mei; Nelson, Heather H; Posner, Marshall; Suzuki, Makoto; Kelsey, Karl T

2004-01-29

Inactivation of the FANC-BRCA pathway via promoter methylation of the FANCF gene renders cells sensitive to DNA crosslinking agents, and has been identified in ovarian cancer cell lines and sporadic primary tumor tissues. We investigated epigenetic alterations in the FANC-BRCA pathway in head and neck squamous cell carcinomas (HNSCC) and non-small-cell lung cancers (NSCLC) using methylation-specific PCR. Promoter methylation of FANCF occurred in 15% (13/89) of HNSCCs and 14% (22/158) of NSCLCs. Methylation of BRCA1 occurred only in 6/158 NSCLC, and was limited to adenocarcinomas and large-cell carcinomas of the lung. No methylation of BRCA2 was detected. FANCF methylation was associated with a shorter duration of tobacco use (P=0.03) and a younger age of starting smoking (P=0.06) in NSCLC, and with a greater number of years of alcohol drinking (P=0.02) in HNSCC. In adenocarcinomas of the lung, FANCF promoter methylation was a significant predictor of poor survival with a hazard ratio of 3.1 (95% CI 1.2-7.9). This study demonstrates that inactivation of the FANC-BRCA pathway is relatively common in solid tumors and may be related to tobacco and alcohol exposure and survival of these patients.

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.

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.

Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing

PubMed Central

Gameiro, Sofia R.; Jammed, Momodou L.; Wattenberg, Max M.; Tsang, Kwong Y.; Ferrone, Soldano; Hodge, James W.

2014-01-01

Radiation therapy (RT) is used for local tumor control through direct killing of tumor cells. Radiation-induced cell death can trigger tumor antigen-specific immune responses, but these are often noncurative. Radiation has been demonstrated to induce immunogenic modulation (IM) in various tumor types by altering the biology of surviving cells to render them more susceptible to T cell-mediated killing. Little is known about the mechanism(s) underlying IM elicited by sub-lethal radiation dosing. We have examined the molecular and immunogenic consequences of radiation exposure in breast, lung, and prostate human carcinoma cells. Radiation induced secretion of ATP and HMGB1 in both dying and surviving tumor cells. In vitro and in vivo tumor irradiation induced significant upregulation of multiple components of the antigen-processing machinery and calreticulin cell-surface expression. Augmented CTL lysis specific for several tumor-associated antigens was largely dictated by the presence of calreticulin on the surface of tumor cells and constituted an adaptive response to endoplasmic reticulum stress, mediated by activation of the unfolded protein response. This study provides evidence that radiation induces a continuum of immunogenic alterations in tumor biology, from immunogenic modulation to immunogenic cell death. We also expand the concept of immunogenic modulation, where surviving tumor cells recovering from radiation-induced endoplasmic reticulum stress become more sensitive to CTL killing. These observations offer a rationale for the combined use of radiation with immunotherapy, including for patients failing RT alone. PMID:24480782

Thin boron phosphide coating as a corrosion-resistant layer

DOEpatents

Not Available

1982-08-25

A surface prone to corrosion in corrosive environments is rendered anticorrosive by CVD growing a thin continuous film, e.g., having no detectable pinholes, thereon, of boron phosphide. In one embodiment, the film is semiconductive. In another aspect, the invention is an improved photoanode, and/or photoelectrochemical cell with a photoanode having a thin film of boron phosphide thereon rendering it anticorrosive, and providing it with unexpectedly improved photoresponsive properties.

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.

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

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

The Expansion of Explosives Safety Education for the 21st Century

DTIC Science & Technology

2010-07-01

shape charges, explosive welding, thermite reaction – Sensitivity testing: drop hammer, electrospark discharge, friction – Physics of explosives, history... ATF ) • Phytoremediation workers use plants to remove explosives from soil and render the explosives harmless • Sales of explosives detection

Imaging circulating tumor cells in freely moving awake small animals using a miniaturized intravital microscope.

PubMed

Sasportas, Laura Sarah; Gambhir, Sanjiv Sam

2014-01-01

Metastasis, the cause for 90% of cancer mortality, is a complex and poorly understood process involving the invasion of circulating tumor cells (CTCs) into blood vessels. These cells have potential prognostic value as biomarkers for early metastatic risk. But their rarity and the lack of specificity and sensitivity in measuring them render their interrogation by current techniques very challenging. How and when these cells are circulating in the blood, on their way to potentially give rise to metastasis, is a question that remains largely unanswered. In order to provide an insight into this "black box" using non-invasive imaging, we developed a novel miniature intravital microscopy (mIVM) strategy capable of real-time long-term monitoring of CTCs in awake small animals. We established an experimental 4T1-GL mouse model of metastatic breast cancer, in which tumor cells express both fluorescent and bioluminescent reporter genes to enable both single cell and whole body tumor imaging. Using mIVM, we monitored blood vessels of different diameters in awake mice in an experimental model of metastasis. Using an in-house software algorithm we developed, we demonstrated in vivo CTC enumeration and computation of CTC trajectory and speed. These data represent the first reported use we know of for a miniature mountable intravital microscopy setup for in vivo imaging of CTCs in awake animals.

Imaging Circulating Tumor Cells in Freely Moving Awake Small Animals Using a Miniaturized Intravital Microscope

PubMed Central

Sasportas, Laura Sarah; Gambhir, Sanjiv Sam

2014-01-01

Metastasis, the cause for 90% of cancer mortality, is a complex and poorly understood process involving the invasion of circulating tumor cells (CTCs) into blood vessels. These cells have potential prognostic value as biomarkers for early metastatic risk. But their rarity and the lack of specificity and sensitivity in measuring them render their interrogation by current techniques very challenging. How and when these cells are circulating in the blood, on their way to potentially give rise to metastasis, is a question that remains largely unanswered. In order to provide an insight into this "black box" using non-invasive imaging, we developed a novel miniature intravital microscopy (mIVM) strategy capable of real-time long-term monitoring of CTCs in awake small animals. We established an experimental 4T1-GL mouse model of metastatic breast cancer, in which tumor cells express both fluorescent and bioluminescent reporter genes to enable both single cell and whole body tumor imaging. Using mIVM, we monitored blood vessels of different diameters in awake mice in an experimental model of metastasis. Using an in-house software algorithm we developed, we demonstrated in vivo CTC enumeration and computation of CTC trajectory and speed. These data represent the first reported use we know of for a miniature mountable intravital microscopy setup for in vivo imaging of CTCs in awake animals. PMID:24497977

β-Glucuronidase-coupled assays of glucuronoyl esterases.

PubMed

Fraňová, Lucia; Puchart, Vladimír; Biely, Peter

2016-10-01

Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl β-D-glucuronides for qualitative and quantitative GE assay coupled with β-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries. Copyright © 2016 Elsevier Inc. All rights reserved.

Implication of Highly Cytotoxic Natural Killer Cells for Esophageal Squamous Cell Carcinoma Treatment.

PubMed

Lim, Kee Siang; Mimura, Kosaku; Kua, Ley-Fang; Shiraishi, Kensuke; Kono, Koji

2018-04-20

Esophageal squamous cell carcinoma (ESCC) is an aggressive upper gastrointestinal cancer and effective treatments are limited. Previous studies reported that natural killer (NK) cells expanded by coculturing with K562-mb15-41BBL feeder cells, a genetically modified K562 leukemia cell line that expresses membrane-bound interleukin (IL)-15 and 41BBL ligand, were highly proliferative and highly cytotoxic. Here, we investigated the potential of expanded NK cells for ESCC treatment. We analyzed both genetic and surface expression levels of NKG2D ligands (NKG2DLs) in ESCC using publicly available microarray data sets and ESCC cell lines. The cytotoxicity of resting and of IL-2-activated NK cells against ESCC cell lines was compared with that of expanded NK cells. We then also investigated the effect of epithelial mesenchymal transition (EMT) inducers, GSK3β inhibitor and epidermal growth factor, on NKG2DLs expressions. As a result, MICA and MICB were significantly overexpressed in ESCC compared with adjacent normal tissues and surface NKG2DLs were expressed in ESCC cell lines. Expanded NK cells were much potent than IL-2-activated and resting NK cells against ESCC cell lines. Blocking of NKG2D with anti-NKG2D monoclonal antibody dampened expanded NK cell cytotoxicity, suggesting that the NKG2DLs-NKG2D interaction is crucial for NK cells to eliminate ESCC cells. EMT inducers concurrently induced EMT and NKG2DLs expression in ESCC cell lines rendering transitioned cells more sensitive to expanded NK cells. In conclusion, expanded NK cells were highly cytotoxic against NKG2DLs-expressing ESCC cells, particularly the EMT phenotype. These results provide a strong rationale for clinical use of these NK cells in ESCC patients.

Targeted disruption of FANCC and FANCG in human cancer provides a preclinical model for specific therapeutic options.

PubMed

Gallmeier, Eike; Calhoun, Eric S; Rago, Carlo; Brody, Jonathan R; Cunningham, Steven C; Hucl, Tomas; Gorospe, Myriam; Kohli, Manu; Lengauer, Christoph; Kern, Scott E

2006-06-01

How specifically to treat pancreatic and other cancers harboring Fanconi anemia gene mutations has raised great interest recently, yet preclinical studies have been hampered by the lack of well-controlled human cancer models. We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance. FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function. On treatment with DNA interstrand-cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation. The extent of hypersensitivity varied among agents, with ratios of inhibitory concentration 50% ranging from 2-fold for oxaliplatin to 14-fold for melphalan, a drug infrequently used in solid tumors. No hypersensitivity was observed on gemcitabine, etoposide, 3-aminobenzamide, NU1025, or hydrogen peroxide. FANCC and FANCG disruption also resulted in increased clastogenic damage on irradiation, but only FANCG disruption caused a subsequent decrease in relative survival. Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer. Our human cancer cell model provides optimal controls to elucidate fundamental biologic features of individual Fanconi anemia gene defects and facilitates preclinical studies of therapeutic options. The impact of Fanconi gene defects on drug and irradiation sensitivity renders these genes promising targets for a specific, genotype-based therapy for individual cancer patients, providing a strong rationale for clinical trials.

Inhibition of autophagy stimulate molecular iodine-induced apoptosis in hormone independent breast tumors

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

Singh, Preeti; Godbole, Madan, E-mail: madangodbole@yahoo.co.in; Rao, Geeta

2011-11-11

Highlights: Black-Right-Pointing-Pointer Molecular iodine (I{sub 2}) causes non-apoptotic cell death in MDA-MB231 breast tumor cells. Black-Right-Pointing-Pointer Autophagy is activated as a survival mechanism in response to I{sub 2} in MDA-MB231. Black-Right-Pointing-Pointer Autophagy inhibition sensitizes tumor cells to I{sub 2}-induced apoptotic cell death. Black-Right-Pointing-Pointer Autophagy inhibitor potentiates apoptosis and tumor regressive effects of I{sub 2} in mice. -- Abstract: Estrogen receptor negative (ER{sup -ve}) and p53 mutant breast tumors are highly aggressive and have fewer treatment options. Previously, we showed that molecular Iodine (I{sub 2}) induces apoptosis in hormone responsive MCF-7 breast cancer cells, and non-apoptotic cell death in ER{sup -ve}-p53more » mutant MDA-MB231 cells (Shrivastava, 2006). Here we show that I{sub 2} (3 {mu}M) treatment enhanced the features of autophagy in MDA-MB231 cells. Since autophagy is a cell survival response to most anti-cancer therapies, we used both in vitro and in vivo systems to determine whether ER{sup -ve} mammary tumors could be sensitized to I{sub 2}-induced apoptosis by inhibiting autophagy. Autophagy inhibition with chloroquine (CQ) and inhibitors for PI3K (3MA, LY294002) and H+/ATPase (baflomycin) resulted in enhanced cell death in I{sub 2} treated MDA-MB231 cells. Further, CQ (20 {mu}M) in combination with I{sub 2}, showed apoptotic features such as increased sub-G1 fraction ({approx}5-fold), expression of cleaved caspase-9 and -3 compared to I{sub 2} treatment alone. Flowcytometry of I{sub 2} and CQ co-treated cells revealed increase in mitochondrial membrane permeability (p < 0.01) and translocation of cathepsin D activity to cytosol relative to I{sub 2} treatment. For in vivo studies ICRC mice were transplanted subcutaneously with MMTV-induced mammary tumors. A significant reduction in tumor volumes, as measured by MRI, was found in I{sub 2} and CQ co-treated mice relative to I{sub 2} or vehicle treated mice. These data indicate that inhibition of autophagy renders ER{sup -ve} breast tumor cells more sensitive to I{sub 2} induced apoptosis. Thus, I{sub 2} together with autophagy inhibitor could have a potential tumorostatic role in ER{sup -ve} aggressive breast tumors that may be evaluated in future studies.« less

Photoacoustic microscopy of human teeth

NASA Astrophysics Data System (ADS)

Rao, Bin; Cai, Xin; Favazza, Christopher; Yao, Junjie; Li, Li; Duong, Steven; Liaw, Lih-Huei; Holtzman, Jennifer; Wilder-Smith, Petra; Wang, Lihong V.

2011-03-01

Photoacoustic microscopy (PAM) utilizes short laser pulses to deposit energy into light absorbers and sensitively detects the ultrasonic waves the absorbers generate in response. PAM directly renders a three-dimensional spatial distribution of sub-surface optical absorbers. Unlike other optical imaging technologies, PAM features label-free optical absorption contrast and excellent imaging depths. Standard dental imaging instruments are limited to X-ray and CCD cameras. Subsurface optical dental imaging is difficult due to the highly-scattering enamel and dentin tissue. Thus, very few imaging methods can detect dental decay or diagnose dental pulp, which is the innermost part of the tooth, containing the nerves, blood vessels, and other cells. Here, we conducted a feasibility study on imaging dental decay and dental pulp with PAM. Our results showed that PAM is sensitive to the color change associated with dental decay. Although the relative PA signal distribution may be affected by surface contours and subsurface reflections from deeper dental tissue, monitoring changes in the PA signals (at the same site) over time is necessary to identify the progress of dental decay. Our results also showed that deep-imaging, near-infrared (NIR) PAM can sensitively image blood in the dental pulp of an in vitro tooth. In conclusion, PAM is a promising tool for imaging both dental decay and dental pulp.

The promise of solution-processed Fe 2GeS 4 thin films in iron chalcogenide photovoltaics

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

Liu, Mimi; Berg, Dominik M.; Hwang, Po-Yu

The olivine Fe 2GeS 4, featuring non-toxic elements, cost-effective synthesis, and suitable optoelectronic properties, recently emerged as a promising light-absorbing candidate. Fe 2GeS 4 precursor powders obtained via a simple solution-based process were converted to highly crystalline Fe 2GeS 4 powders upon a thermal treatment in controlled atmosphere. Thin films fabricated by dip coating in the Fe 2GeS 4 precursor dispersion and subjected to the same thermal treatment render high-purity Fe 2GeS 4 thin films with a band gap of 1.4 eV, measured by room-temperature photoluminescence. Using Fe 2GeS 4 thin films as the sole absorber in a solution-based solarmore » cell, open-circuit voltages of 361 mV are observed, while the use of the Fe 2GeS 4 films as counter electrodes in dye-sensitized solar cell constructs enhances the overall power conversion efficiency of the cell by a factor of five. Finally, this is the first report of a photovoltaic device based onFe 2GeS 4.« less

Low-cost microcontroller platform for studying lymphatic biomechanics in vitro

PubMed Central

Kornuta, Jeffrey A.; Nipper, Matthew E.; Dixon, J. Brandon

2012-01-01

The pumping innate to collecting lymphatic vessels routinely exposes the endothelium to oscillatory wall shear stress and other dynamic forces. However, studying the mechanical sensitivity of the lymphatic endothelium remains a difficult task due to limitations of commercial or custom systems to apply a variety of time-varying stresses in vitro. Current biomechanical in vitro testing devices are very expensive, limited in capability, or highly complex; rendering them largely inaccessible to the endothelial cell biology community. To address these short-comings, the authors propose a reliable, low-cost platform for augmenting the capabilities of commercially available pumps to produce a wide variety of flow rate waveforms. In particular, the Arduino Uno, a microcontroller development board, is used to provide open-loop control of a digital peristaltic pump using precisely-timed serial commands. In addition, the flexibility of this platform is further demonstrated through its support of a custom-built cell-straining device capable of producing oscillatory strains with varying amplitudes and frequencies. Hence, this microcontroller development board is shown to be an inexpensive, precise, and easy-to-use tool for supplementing in vitro assays to quantify the effects of biomechanical forces on lymphatic endothelial cells. PMID:23178036

The promise of solution-processed Fe 2GeS 4 thin films in iron chalcogenide photovoltaics

DOE PAGES

Liu, Mimi; Berg, Dominik M.; Hwang, Po-Yu; ...

2018-02-06

The olivine Fe 2GeS 4, featuring non-toxic elements, cost-effective synthesis, and suitable optoelectronic properties, recently emerged as a promising light-absorbing candidate. Fe 2GeS 4 precursor powders obtained via a simple solution-based process were converted to highly crystalline Fe 2GeS 4 powders upon a thermal treatment in controlled atmosphere. Thin films fabricated by dip coating in the Fe 2GeS 4 precursor dispersion and subjected to the same thermal treatment render high-purity Fe 2GeS 4 thin films with a band gap of 1.4 eV, measured by room-temperature photoluminescence. Using Fe 2GeS 4 thin films as the sole absorber in a solution-based solarmore » cell, open-circuit voltages of 361 mV are observed, while the use of the Fe 2GeS 4 films as counter electrodes in dye-sensitized solar cell constructs enhances the overall power conversion efficiency of the cell by a factor of five. Finally, this is the first report of a photovoltaic device based onFe 2GeS 4.« less

Low-cost microcontroller platform for studying lymphatic biomechanics in vitro.

PubMed

Kornuta, Jeffrey A; Nipper, Matthew E; Dixon, J Brandon

2013-01-04

The pumping innate to collecting lymphatic vessels routinely exposes the endothelium to oscillatory wall shear stress and other dynamic forces. However, studying the mechanical sensitivity of the lymphatic endothelium remains a difficult task due to limitations of commercial or custom systems to apply a variety of time-varying stresses in vitro. Current biomechanical in vitro testing devices are very expensive, limited in capability, or highly complex; rendering them largely inaccessible to the endothelial cell biology community. To address these shortcomings, the authors propose a reliable, low-cost platform for augmenting the capabilities of commercially available pumps to produce a wide variety of flow rate waveforms. In particular, the Arduino Uno, a microcontroller development board, is used to provide open-loop control of a digital peristaltic pump using precisely timed serial commands. In addition, the flexibility of this platform is further demonstrated through its support of a custom-built cell-straining device capable of producing oscillatory strains with varying amplitudes and frequencies. Hence, this microcontroller development board is shown to be an inexpensive, precise, and easy-to-use tool for supplementing in vitro assays to quantify the effects of biomechanical forces on lymphatic endothelial cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Iron addiction: a novel therapeutic target in ovarian cancer

DOE PAGES

Basuli, D.; Tesfay, L.; Deng, Z.; ...

2017-03-20

Ovarian cancer is a lethal malignancy that has not seen a major therapeutic advance in over 30 years. We demonstrate that ovarian cancer exhibits a targetable alteration in iron metabolism. Ferroportin (FPN), the iron efflux pump, is decreased, and transferrin receptor (TFR1), the iron importer, is increased in tumor tissue from patients with high grade but not low grade serous ovarian cancer. A similar profile of decreased FPN and increased TFR1 is observed in a genetic model of ovarian cancer tumor-initiating cells (TICs). The net result of these changes is an accumulation of excess intracellular iron and an augmented dependencemore » on iron for proliferation. A forced reduction in intracellular iron reduces the proliferation of ovarian cancer TICs in vitro, and inhibits both tumor growth and intraperitoneal dissemination of tumor cells in vivo. Some mechanistic studies demonstrate that iron increases metastatic spread by facilitating invasion through expression of matrix metalloproteases and synthesis of interleukin 6 (IL-6). Here, we show that the iron dependence of ovarian cancer TICs renders them exquisitely sensitive in vivo to agents that induce iron-dependent cell death (ferroptosis) as well as iron chelators, and thus creates a metabolic vulnerability that can be exploited therapeutically.« less

Iron addiction: a novel therapeutic target in ovarian cancer

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

Basuli, D.; Tesfay, L.; Deng, Z.

Ovarian cancer is a lethal malignancy that has not seen a major therapeutic advance in over 30 years. We demonstrate that ovarian cancer exhibits a targetable alteration in iron metabolism. Ferroportin (FPN), the iron efflux pump, is decreased, and transferrin receptor (TFR1), the iron importer, is increased in tumor tissue from patients with high grade but not low grade serous ovarian cancer. A similar profile of decreased FPN and increased TFR1 is observed in a genetic model of ovarian cancer tumor-initiating cells (TICs). The net result of these changes is an accumulation of excess intracellular iron and an augmented dependencemore » on iron for proliferation. A forced reduction in intracellular iron reduces the proliferation of ovarian cancer TICs in vitro, and inhibits both tumor growth and intraperitoneal dissemination of tumor cells in vivo. Some mechanistic studies demonstrate that iron increases metastatic spread by facilitating invasion through expression of matrix metalloproteases and synthesis of interleukin 6 (IL-6). Here, we show that the iron dependence of ovarian cancer TICs renders them exquisitely sensitive in vivo to agents that induce iron-dependent cell death (ferroptosis) as well as iron chelators, and thus creates a metabolic vulnerability that can be exploited therapeutically.« less

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

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.

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.

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.

ALLOZYME ANALYSIS OF THE RELATIONSHIP AMONG CONTAMINANT-TOLERANT AND CONTAMINANT SENSITIVE POPULATIONS OF FUNDULUS HETEROCLITUS

EPA Science Inventory

Genetic diversity is essential for adaptation to environmental change. Exposure to toxic chemicals causes directional selection that can reduce genetic diversity, rendering exposed populations less able to adapt to environmental change. Populations of the estuarine killifish Fund...

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...

Inhibition of BRCA2 and Thymidylate Synthase Creates Multidrug Sensitive Tumor Cells via the Induction of Combined "Complementary Lethality".

PubMed

Rytelewski, Mateusz; Ferguson, Peter J; Maleki Vareki, Saman; Figueredo, Rene; Vincent, Mark; Koropatnick, James

2013-03-12

A high mutation rate leading to tumor cell heterogeneity is a driver of malignancy in human cancers. Paradoxically, however, genomic instability can also render tumors vulnerable to therapeutic attack. Thus, targeting DNA repair may induce an intolerable level of DNA damage in tumor cells. BRCA2 mediates homologous recombination repair, and BRCA2 polymorphisms increase cancer risk. However, tumors with BRCA2 mutations respond better to chemotherapy and are associated with improved patient prognosis. Thymidylate synthase (TS) is also involved in DNA maintenance and generates cellular thymidylate. We determined that antisense downregulation of BRCA2 synergistically potentiated drugs with mechanisms of action related to BRCA2 function (cisplatin, melphalan), a phenomenon we named "complementary lethality." TS knockdown induced complementary lethality to TS-targeting drugs (5-FUdR and pemetrexed) but not DNA cross-linking agents. Combined targeting of BRCA2 and TS induced complementary lethality to both DNA-damaging and TS-targeting agents, thus creating multidrug sensitive tumors. In addition, we demonstrated for the first time that simultaneous downregulation of both targets induced combined complementary lethality to multiple mechanistically different drugs in the same cell population. In this study, we propose and define the concept of "complementary lethality" and show that actively targeting BRCA2 and TS is of potential therapeutic benefit in multidrug treatment of human tumors. This work has contributed to the development of a BRCA2-targeting antisense oligdeoxynucleotide (ASO) "BR-1" which we will test in vivo in combination with our TS-targeting ASO "SARI 83" and attempt early clinical trials in the future.Molecular Therapy - Nucleic Acids (2013) 2, e78; doi:10.1038/mtna.2013.7 published online 12 March 2013.

Cells with dysfunctional telomeres are susceptible to reactive oxygen species hydrogen peroxide via generation of multichromosomal fusions and chromosomal fragments bearing telomeres

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

Woo, Seon Rang; Department of Biochemistry, College of Medicine, Korea University, Seoul 136-705; Park, Jeong-Eun

2012-01-06

Highlights: Black-Right-Pointing-Pointer Under conditions of telomere erosion, cells become extremely sensitive to H{sub 2}O{sub 2}. Black-Right-Pointing-Pointer Chromosomal regions adjacent to telomeres are cleaved by H{sub 2}O{sub 2} under such conditions. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} thus causes multichromosomal fusions and generation of small chromosomal fragments. Black-Right-Pointing-Pointer N-acetylcysteine prevents H{sub 2}O{sub 2}-induced chromosomal aberrations. -- Abstract: During genotoxic stress, reactive oxygen species hydrogen peroxide (H{sub 2}O{sub 2}) is a prime mediator of the DNA damage response. Telomeres function both to assist in DNA damage repair and to inhibit chromosomal end-to-end fusion. Here, we show that telomere dysfunction renders cells susceptible to H{submore » 2}O{sub 2}, via generation of multichromosomal fusion and chromosomal fragments. H{sub 2}O{sub 2} caused formation of multichromosomal end-to-end fusions involving more than three chromosomes, preferentially when telomeres were erosive. Interestingly, extensive chromosomal fragmentation (yielding small-sized fragments) occurred only in cells exhibiting such multichromosomal fusions. Telomeres were absent from fusion points, being rather present in the small fragments, indicating that H{sub 2}O{sub 2} cleaves chromosomal regions adjacent to telomeres. Restoration of telomere function or addition of the antioxidant N-acetylcysteine prevented development of chromosomal aberrations and rescued the observed hypersensitivity to H{sub 2}O{sub 2}. Thus, chromosomal regions adjacent to telomeres become sensitive to reactive oxygen species hydrogen peroxide when telomeres are dysfunctional, and are cleaved to produce multichromosomal fusions and small chromosomal fragments bearing the telomeres.« less

Repression of mammosphere formation of human breast cancer cells by soy isoflavone genistein and blueberry polyphenolic acids suggests diet-mediated targeting of cancer stem-like/progenitor cells

USDA-ARS?s Scientific Manuscript database

Mammary stem cells are undifferentiated epithelial cells which initiate mammary tumors and render them resistant to anticancer therapies, when deregulated. Diets rich in fruits and vegetables are implicated in breast cancer risk reduction, yet underlying mechanisms are poorly understood. Here, we ad...

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.

A novel, sensitive and label-free loop-mediated isothermal amplification detection method for nucleic acids using luminophore dyes.

PubMed

Roy, Sharmili; Wei, Sim Xiao; Ying, Jean Liew Zhi; Safavieh, Mohammadali; Ahmed, Minhaz Uddin

2016-12-15

Electrochemiluminescence (ECL) has been widely rendered for nucleic acid testing. Here, we integrate loop-mediated isothermal amplification (LAMP) with ECL technique for DNA detection and quantification. The target LAMP DNA bound electrostatically with [Ru(bpy)3](+2) on the carbon electrode surface, and an ECL reaction was triggered by tripropylamine (TPrA) to yield luminescence. We illustrated this method as a new and highly sensitive strategy for the detection of sequence-specific DNA from different meat species at picogram levels. The proposed strategy renders the signal amplification capacities of TPrA and combines LAMP with inherently high sensitivity of the ECL technique, to facilitate the detection of low quantities of DNA. By leveraging this technique, target DNA of Sus scrofa (pork) meat was detected as low as 1pg/µL (3.43×10(-1)copies/µL). In addition, the proposed technique was applied for detection of Bacillus subtilis DNA samples and detection limit of 10pg/µL (2.2×10(3)copies/µL) was achieved. The advantages of being isothermal, sensitive and robust with ability for multiplex detection of bio-analytes makes this method a facile and appealing sensing modality in hand-held devices to be used at the point-of-care (POC). Copyright © 2016 Elsevier B.V. All rights reserved.

Multi-Laboratory Study of Five Methods for the Determination of Brevetoxins in Shellfish Tissue Extracts.

PubMed

Dickey, Robert W; Plakas, Steven M; Jester, Edward L E; El Said, Kathleen R; Johannessen, Jan N; Flewelling, Leanne J; Scott, Paula; Hammond, Dan G; Van Dolah, Frances M; Leighfield, Tod A; Bottein Dachraoui, Marie-Yasmine; Ramsdell, John S; Pierce, Richard H; Henry, Mike S; Poli, Mark A; Walker, Calvin; Kurtz, Jan; Naar, Jerome; Baden, Daniel G; Musser, Steve M; White, Kevin D; Truman, Penelope; Miller, Aaron; Hawryluk, Timothy P; Wekell, Marleen M; Stirling, David; Quilliam, Michael A; Lee, Jung K

A thirteen-laboratory comparative study tested the performance of four methods as alternatives to mouse bioassay for the determination of brevetoxins in shellfish. The methods were N2a neuroblastoma cell assay, two variations of the sodium channel receptor binding assay, competitive ELISA, and LC/MS. Three to five laboratories independently performed each method using centrally prepared spiked and naturally incurred test samples. Competitive ELISA and receptor binding (96-well format) compared most favorably with mouse bioassay. Between-laboratory relative standard deviations (RSDR) ranged from 10 to 20% for ELISA and 14 to 31% for receptor binding. Within-laboratory (RSDr) ranged from 6 to 15% for ELISA, and 5 to 31% for receptor binding. Cell assay was extremely sensitive but data variation rendered it unsuitable for statistical treatment. LC/MS performed as well as ELISA on spiked test samples but was inordinately affected by lack of toxin-metabolite standards, uniform instrumental parameters, or both, on incurred test samples. The ELISA and receptor binding assay are good alternatives to mouse bioassay for the determination of brevetoxins in shellfish.

Construction and Evaluation of an Ultra Low Latency Frameless Renderer for VR.

PubMed

Friston, Sebastian; Steed, Anthony; Tilbury, Simon; Gaydadjiev, Georgi

2016-04-01

Latency - the delay between a user's action and the response to this action - is known to be detrimental to virtual reality. Latency is typically considered to be a discrete value characterising a delay, constant in time and space - but this characterisation is incomplete. Latency changes across the display during scan-out, and how it does so is dependent on the rendering approach used. In this study, we present an ultra-low latency real-time ray-casting renderer for virtual reality, implemented on an FPGA. Our renderer has a latency of ~1 ms from 'tracker to pixel'. Its frameless nature means that the region of the display with the lowest latency immediately follows the scan-beam. This is in contrast to frame-based systems such as those using typical GPUs, for which the latency increases as scan-out proceeds. Using a series of high and low speed videos of our system in use, we confirm its latency of ~1 ms. We examine how the renderer performs when driving a traditional sequential scan-out display on a readily available HMO, the Oculus Rift OK2. We contrast this with an equivalent apparatus built using a GPU. Using captured human head motion and a set of image quality measures, we assess the ability of these systems to faithfully recreate the stimuli of an ideal virtual reality system - one with a zero latency tracker, renderer and display running at 1 kHz. Finally, we examine the results of these quality measures, and how each rendering approach is affected by velocity of movement and display persistence. We find that our system, with a lower average latency, can more faithfully draw what the ideal virtual reality system would. Further, we find that with low display persistence, the sensitivity to velocity of both systems is lowered, but that it is much lower for ours.

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

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

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.

Flavanols from evening primrose (Oenothera paradoxa) defatted seeds inhibit prostate cells invasiveness and cause changes in Bcl-2/Bax mRNA ratio.

PubMed

Lewandowska, Urszula; Szewczyk, Karolina; Owczarek, Katarzyna; Hrabec, Zbigniew; Podsędek, Anna; Koziołkiewicz, Maria; Hrabec, Elżbieta

2013-03-27

In this study, we assessed the influence of an evening primrose flavanol preparation (EPFP) on proliferation and invasiveness of human prostate cancer cells (DU 145) and immortalized prostate epithelial cells (PNT1A). We report for the first time that EPFP reduces DU 145 cell proliferation (IC50 = 97 μM GAE for 72 h incubation) and invasiveness (by 24% versus control at 75 μM GAE). EPFP strongly inhibited PNT1A invasiveness in a concentration-dependent manner (by 67% versus control at 75 μM GAE) and did not cause a reduction in their proliferation. Furthermore, EPFP inhibited the activities of MMP-2 and MMP-9 secreted to culture medium by PNT1A cells by 84% and 34% versus control at 100 μM GAE, respectively. In the case of DU 145, MMP-9 activity at 100 μM GAE was reduced by 37% versus control. Moreover, the evening primrose seed flavanols suppressed the expression of selected genes (MMP-1, MMP-9, MMP-14, c-Fos, c-Jun, and VEGF) and also caused favorable changes in Bcl-2/Bax mRNA ratio which render DU 145 cells more sensitive to apoptosis-triggering agents. An additional confirmation of the proapoptotic activity of EPFP toward DU 145 was visualization of characteristic apoptotic bodies by DAPI staining. In conclusion, this study suggests that EPFP may increase apoptosis and reduce angiogenesis of prostate cancer cells.

Reactive oxygen species as a signal in glucose-stimulated insulin secretion.

PubMed

Pi, Jingbo; Bai, Yushi; Zhang, Qiang; Wong, Victoria; Floering, Lisa M; Daniel, Kiefer; Reece, Jeffrey M; Deeney, Jude T; Andersen, Melvin E; Corkey, Barbara E; Collins, Sheila

2007-07-01

One of the unique features of beta-cells is their relatively low expression of many antioxidant enzymes. This could render beta-cells susceptible to oxidative damage but may also provide a system that is sensitive to reactive oxygen species as signals. In isolated mouse islets and INS-1(832/13) cells, glucose increases intracellular accumulation of H2O2. In both models, insulin secretion could be stimulated by provision of either exogenous H2O2 or diethyl maleate, which raises intracellular H2O2 levels. Provision of exogenous H2O2 scavengers, including cell permeable catalase and N-acetyl-L-cysteine, inhibited glucose-stimulated H2O2 accumulation and insulin secretion (GSIS). In contrast, cell permeable superoxide dismutase, which metabolizes superoxide into H2O2, had no effect on GSIS. Because oxidative stress is an important risk factor for beta-cell dysfunction in diabetes, the relationship between glucose-induced H2O2 generation and GSIS was investigated under various oxidative stress conditions. Acute exposure of isolated mouse islets or INS-1(832/13) cells to oxidative stressors, including arsenite, 4-hydroxynonenal, and methylglyoxal, led to decreased GSIS. This impaired GSIS was associated with increases in a battery of endogenous antioxidant enzymes. Taken together, these findings suggest that H2O2 derived from glucose metabolism is one of the metabolic signals for insulin secretion, whereas oxidative stress may disturb its signaling function.

Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells.

PubMed

Zamporlini, Federica; Ruggieri, Silverio; Mazzola, Francesca; Amici, Adolfo; Orsomando, Giuseppe; Raffaelli, Nadia

2014-11-01

The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma. © 2014 FEBS.

Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads

PubMed Central

Wang, Lin; Acosta, Miguel A.; Leach, Jennie B.; Carrier, Rebecca L.

2013-01-01

Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems. PMID:23443975

Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads.

PubMed

Wang, Lin; Acosta, Miguel A; Leach, Jennie B; Carrier, Rebecca L

2013-04-21

Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems.

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

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

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.

32 CFR 154.60 - Evaluating continued security eligibility.

Code of Federal Regulations, 2011 CFR

2011-07-01

... in sensitive positions who are experiencing problems in their personal lives with respect to such... personal problem which may have a bearing upon the individual's continued eligibility for access. (1) In... potential problem areas at an early stage so that any assistance rendered by the employing activity will...

32 CFR 154.60 - Evaluating continued security eligibility.

Code of Federal Regulations, 2010 CFR

2010-07-01

... in sensitive positions who are experiencing problems in their personal lives with respect to such... personal problem which may have a bearing upon the individual's continued eligibility for access. (1) In... potential problem areas at an early stage so that any assistance rendered by the employing activity will...

Immobilized lysozyme for the continuous lysis of lactic bacteria in wine: Bench-scale fluidized-bed reactor study.

PubMed

Cappannella, Elena; Benucci, Ilaria; Lombardelli, Claudio; Liburdi, Katia; Bavaro, Teodora; Esti, Marco

2016-11-01

Lysozyme from hen egg white (HEWL) was covalently immobilized on spherical supports based on microbial chitosan in order to develop a system for the continuous, efficient and food-grade enzymatic lysis of lactic bacteria (Oenococcus oeni) in white and red wine. The objective is to limit the sulfur dioxide dosage required to control malolactic fermentation, via a cell concentration typical during this process. The immobilization procedure was optimized in batch mode, evaluating the enzyme loading, the specific activity, and the kinetic parameters in model wine. Subsequently, a bench-scale fluidized-bed reactor was developed, applying the optimized process conditions. HEWL appeared more effective in the immobilized form than in the free one, when the reactor was applied in real white and red wine. This preliminary study suggests that covalent immobilization renders the enzyme less sensitive to the inhibitory effect of wine flavans. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of correlated parameters and uncertainty in electronic-structure-based chemical kinetic modelling

NASA Astrophysics Data System (ADS)

Sutton, Jonathan E.; Guo, Wei; Katsoulakis, Markos A.; Vlachos, Dionisios G.

2016-04-01

Kinetic models based on first principles are becoming common place in heterogeneous catalysis because of their ability to interpret experimental data, identify the rate-controlling step, guide experiments and predict novel materials. To overcome the tremendous computational cost of estimating parameters of complex networks on metal catalysts, approximate quantum mechanical calculations are employed that render models potentially inaccurate. Here, by introducing correlative global sensitivity analysis and uncertainty quantification, we show that neglecting correlations in the energies of species and reactions can lead to an incorrect identification of influential parameters and key reaction intermediates and reactions. We rationalize why models often underpredict reaction rates and show that, despite the uncertainty being large, the method can, in conjunction with experimental data, identify influential missing reaction pathways and provide insights into the catalyst active site and the kinetic reliability of a model. The method is demonstrated in ethanol steam reforming for hydrogen production for fuel cells.

High pressure processing and its application to the challenge of virus-contaminated foods.

PubMed

Kingsley, David H

2013-03-01

High pressure processing (HPP) is an increasingly popular non-thermal food processing technology. Study of HPP's potential to inactivate foodborne viruses has defined general pressure levels required to inactivate hepatitis A virus, norovirus surrogates, and human norovirus itself within foods such as shellfish and produce. The sensitivity of a number of different picornaviruses to HPP is variable. Experiments suggest that HPP inactivates viruses via denaturation of capsid proteins which render the virus incapable of binding to its receptor on the surface of its host cell. Beyond the primary consideration of treatment pressure level, the effects of extending treatment times, temperature of initial pressure application, and matrix composition have been identified as critical parameters for designing HPP inactivation strategies. Research described here can serve as a preliminary guide to whether a current commercial process could be effective against HuNoV or HAV.

[Mutants of the yeast Saccharomyces cerevisiae characterized by enhanced induced mutagenesis. III. Effect of the him mutation on the effectiveness and specificity of UF-induced mutagenesis].

PubMed

Ivanov, E L; Koval'tsova, S V; Korolev, V G

1987-09-01

We have studied the influence of him1-1, him2-1, him3-1 and himX mutations on induction frequency and specificity of UV-induced adenine-dependent mutations in the yeast Saccharomyces cerevisiae. Him mutations do not render haploid cells more sensitive to the lethal action of UV-light; however, in him strains adenine-dependent mutations (ade1, ade2) were induced more frequently (1.5--2-fold), as compared to the HIM strain. An analysis of the molecular nature of ade2 mutants revealed that him1-1, him2-1 and himX mutations increase specifically the yield of transitions (AT----GC and GC----AT), whereas in the him3-1 strain the yield of transversions was enhanced as well. We suggest him mutations analysed to affect specific repair pathway for mismatch correction.

Charge collection and pore filling in solid-state dye-sensitized solar cells.

PubMed

Snaith, Henry J; Humphry-Baker, Robin; Chen, Peter; Cesar, Ilkay; Zakeeruddin, Shaik M; Grätzel, Michael

2008-10-22

The solar to electrical power conversion efficiency for dye-sensitized solar cells (DSCs) incorporating a solid-state organic hole-transporter can be over 5%. However, this is for devices significantly thinner than the optical depth of the active composites and by comparison to the liquid electrolyte based DSCs, which exhibit efficiencies in excess of 10%, more than doubling of this efficiency is clearly attainable if all the steps in the photovoltaic process can be optimized. Two issues are currently being addressed by the field. The first aims at enhancing the electron diffusion length by either reducing the charge recombination or enhancing the charge transport rates. This should enable a larger fraction of photogenerated charges to be collected. The second, though less actively investigated, aims to improve the physical composite formation, which in this instance is the infiltration of mesoporous TiO(2) with the organic hole-transporter 2,2',7,7'-tetrakis(N,N-di-p-methoxypheny-amine)-9,9'-spirobifluorene (spiro-MeOTAD). Here, we perform a broad experimental study to elucidate the limiting factors to the solar cell performance. We first investigate the charge transport and recombination in the solid-state dye-sensitized solar cell under realistic working conditions via small perturbation photovoltage and photocurrent decay measurements. From these measurements we deduce that the electron diffusion length near short-circuit is as long as 20 µm. However, at applied biases approaching open-circuit potential under realistic solar conditions, the diffusion length becomes comparable with the film thickness, ∼2 µm, illustrating that real losses to open-circuit voltage, fill factor and hence efficiency are occurring due to ineffective charge collection. The long diffusion length near short-circuit, on the other hand, illustrates that another process, separate from ineffective charge collection, is rendering the solar cell less than ideal. We investigate the process of TiO(2) mesopore infiltration with spiro-MeOTAD by examining the cross-sectional images of and performing photo-induced absorption spectroscopy on devices with a range of thickness, infiltrated with spiro-MeOTAD with a range of concentrations. We present our interpretation of the mechanism for material infiltration, and by improving the casting conditions demonstrate efficient charge collection through devices of over 7 µm in thickness. This investigation represents an improvement in our understanding of the limiting factors to the dye-sensitized solar cell. However, much work, focused on composite formation and improved kinetic competition, is required to realize the true potential of this concept.

Photoinitiator-Free Synthesis of Endothelial Cell Adhesive and Enzymatically Degradable Hydrogels

PubMed Central

Jones, Derek R.; Marchant, Roger E.; von Recum, Horst; Gupta, Anirban Sen; Kottke-Marchant, Kandice

2015-01-01

We report on a photoinitiator-free synthetic method of incorporating bioactivity into poly(ethylene glycol) (PEG) hydrogels in order to control physical properties, enzymatic biodegradability and cell-specific adhesiveness of the polymer network, while eliminating the need for UV-mediated photopolymerization. To accomplish this, hydrogel networks were polymerized using Michael addition with four-arm PEG acrylate (10 kDa), using a collagenase sensitive peptide (CSP) as a crosslinker, and introducing an endothelial cell adhesive peptide either terminally (RGD) or attached to the crosslinking peptide sequence (CSP-RGD). The efficiency of the Michael addition reactions were determined by NMR and Ellman’s assay. Successful decoupling of cell adhesivity and physical properties was demonstrated by quantifying and comparing the swelling ratios and Young’s Moduli of various hydrogel formulations. Degradation profiles were established by incubating functionalized hydrogels in collagenase solutions (0.0 – 1.0 µg/mL), demonstrating that functionalized hydrogels degraded at a rate dependent upon collagenase concentration. Moreover, it was shown that the degradation rate was independent of CSP-RGD concentration. Cell attachment and proliferation on functionalized hydrogels were compared for various RGD concentrations, providing evidence that cell attachment and proliferation were directly related to relative amounts of the CSP-RGD combination peptide. An increase in cell viability was achieved using Michael addition techniques when compared to UV-polymerization, and was assessed by a LIVE/DEAD fluorescence assay. This photoinitiator-free method shows promise in creating hydrogel-based tissue engineering scaffolds allow for decoupled cell adhesivity and physical properties and that render greater cell viability. PMID:25462848

Visualization and Enumeration of Marine Planktonic Archaea and Bacteria by Using Polyribonucleotide Probes and Fluorescent In Situ Hybridization

PubMed Central

DeLong, Edward F.; Taylor, Lance Trent; Marsh, Terence L.; Preston, Christina M.

1999-01-01

Fluorescent in situ hybridization (FISH) using rRNA-specific oligonucleotide probes has emerged as a popular technique for identifying individual microbial cells. In natural samples, however, the signal derived from fluor-labeled oligonucleotide probes often is undetectable above background fluorescence in many cells. To circumvent this difficulty, we applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine planktonic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identification and enumeration of planktonic cells to ocean depths of 3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total 4′,6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study (R. Massana, A. E. Murray, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50–56, 1997), group I and II marine archaea predominate in different zones in the water column, with maximal cell densities of 105/ml. The high cell densities of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, rendering them easily detectable with polyribonucleotide probes. These results imply that the majority of planktonic cells visualized by DAPI do not represent lysed cells or “ghosts,” as was suggested in a previous report. PMID:10584017

Green-Light-Sensitive BODIPY Photoprotecting Groups for Amines

PubMed Central

2018-01-01

We describe a series of easily accessible, visible-light-sensitive (λ > 500 nm) BODIPY (boron-dipyrromethene)-based photoprotecting groups (PPGs) for primary and secondary amines, based on a carbamate linker. The caged compounds are stable under aqueous conditions for 24 h and can be efficiently uncaged in vitro with visible light (λ = 530 nm). These properties allow efficient photodeprotection of amines, rendering these novel PPGs potentially suitable for various applications, including the delivery of caged drugs and their remote activation. PMID:29369628

Poly(styrene-co-maleic acid)-based pH-sensitive liposomes mediate cytosolic delivery of drugs for enhanced cancer chemotherapy.

PubMed

Banerjee, Shubhadeep; Sen, Kacoli; Pal, Tapan K; Guha, Sujoy K

2012-10-15

pH-responsive polymers render liposomes pH-sensitive and facilitate the intracellular release of encapsulated payload by fusing with endovascular membranes under mildly acidic conditions found inside cellular endosomes. The present study reports the use of high-molecular weight poly(styrene-co-maleic acid) (SMA), which exhibits conformational transition from a charged extended structure to an uncharged globule below its pK(1) value, to confer pH-sensitive property to liposomes. The changes in the co-polymer chain conformation resulted in destabilization of the liposomes at mildly acidic pH due to vesicle fusion and/or channel formation within the membrane bilayer, and ultimately led to the release of the encapsulated cargo. The vesicles preserved their pH-sensitivity and stability in serum unlike other polymer-based liposomes and exhibited no hemolytic activity at physiological pH. The lysis of RBCs at endosomal pH due to SMA-based liposome-induced alterations in the bilayer organization leading to spherocyte formation indicated the potential of these vesicles to mediate cytosolic delivery of bio-active molecules through endosome destabilization. The SMA-loaded liposomes exhibiting excellent cytocompatibility, efficiently delivered chemotherapeutic agent 5-Fluorouracil (5-FU) within colon cancer cells HT-29 in comparison to neat liposomes. This caused increased cellular-availability of the drug, which resulted in enhanced apoptosis and highlighted the clinical potential of SMA-based vesicles. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

Trichostatin A Sensitizes Hepatocellular Carcinoma Cells to Enhanced NK Cell-mediated Killing by Regulating Immune-related Genes.

PubMed

Shin, Sangsu; Kim, Miok; Lee, Seon-Jin; Park, Kang-Seo; Lee, Chang Hoon

2017-01-01

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. The ability of HCC to avoid immune detection is considered one of the main factors making it difficult to cure. Abnormal histone deacetylation is thought to be one of the mechanisms for HCC immune escape, making histone deacetylases (HDACs) attractive targets for HCC treatment. Here, we investigated the effect of trichostatin A (TSA), a highly potent HDAC inhibitor, on HCC (HepG2) gene expression and function. A genome wide-transcriptional microarray was used to identify genes regulated by TSA in HepG2 cells. Gene Ontology was used to identify pathways regulated by TSA, and these changes were confirmed by qPCR. The effect of TSA on natural killer (NK) cell-mediated killing of HCC cell lines were analyzed by both flow cytometry and LDH cytotoxicity assay. A study was also conducted in a Balb/c nude mice xenograft model to assess the anti-tumor activity of TSA. TSA regulated the transcription of numerous innate immunity & tumor antigen recognition-associated genes, such as ULBP1 and RAET1G, in HCC cells. In vivo, TSA reduced tumor cell growth in an NK cell-dependent manner. In vitro, TSA treatment of HepG2 cells rendered them more susceptible to NK cell-mediated killing while increasing the expression of NKGD2 ligands, including ULBP1/2/3 and MICA/B. TSA also induced direct killing of HCC cells by stimulating apoptosis. TSA likely increases killing of HCC cells indirectly by increasing NK cell-directed killing and directly by increasing apoptosis. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Trichostatin A Sensitizes Hepatocellular Carcinoma Cells to Enhanced NK Cell-mediated Killing by Regulating Immune-related Genes

PubMed Central

SHIN, SANGSU; KIM, MIOK; LEE, SEON-JIN; PARK, KANG-SEO

2017-01-01

Background/Aim: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. The ability of HCC to avoid immune detection is considered one of the main factors making it difficult to cure. Abnormal histone deacetylation is thought to be one of the mechanisms for HCC immune escape, making histone deacetylases (HDACs) attractive targets for HCC treatment. Here, we investigated the effect of trichostatin A (TSA), a highly potent HDAC inhibitor, on HCC (HepG2) gene expression and function. Materials and Methods: A genome wide-transcriptional microarray was used to identify genes regulated by TSA in HepG2 cells. Gene Ontology was used to identify pathways regulated by TSA, and these changes were confirmed by qPCR. The effect of TSA on natural killer (NK) cell-mediated killing of HCC cell lines were analyzed by both flow cytometry and LDH cytotoxicity assay. A study was also conducted in a Balb/c nude mice xenograft model to assess the anti-tumor activity of TSA. Results: TSA regulated the transcription of numerous innate immunity & tumor antigen recognition-associated genes, such as ULBP1 and RAET1G, in HCC cells. In vivo, TSA reduced tumor cell growth in an NK cell-dependent manner. In vitro, TSA treatment of HepG2 cells rendered them more susceptible to NK cell-mediated killing while increasing the expression of NKGD2 ligands, including ULBP1/2/3 and MICA/B. TSA also induced direct killing of HCC cells by stimulating apoptosis. Conclusion: TSA likely increases killing of HCC cells indirectly by increasing NK cell-directed killing and directly by increasing apoptosis. PMID:28871002

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.

Oxidovanadium(IV/V) complexes as new redox mediators in dye-sensitized solar cells: a combined experimental and theoretical study.

PubMed

Apostolopoulou, Andigoni; Vlasiou, Manolis; Tziouris, Petros A; Tsiafoulis, Constantinos; Tsipis, Athanassios C; Rehder, Dieter; Kabanos, Themistoklis A; Keramidas, Anastasios D; Stathatos, Elias

2015-04-20

Corrosiveness is one of the main drawbacks of using the iodide/triiodide redox couple in dye-sensitized solar cells (DSSCs). Alternative redox couples including transition metal complexes have been investigated where surprisingly high efficiencies for the conversion of solar to electrical energy have been achieved. In this paper, we examined the development of a DSSC using an electrolyte based on square pyramidal oxidovanadium(IV/V) complexes. The oxidovanadium(IV) complex (Ph4P)2[V(IV)O(hybeb)] was combined with its oxidized analogue (Ph4P)[V(V)O(hybeb)] {where hybeb(4-) is the tetradentate diamidodiphenolate ligand [1-(2-hydroxybenzamido)-2-(2-pyridinecarboxamido)benzenato}and applied as a redox couple in the electrolyte of DSSCs. The complexes exhibit large electron exchange and transfer rates, which are evident from electron paramagnetic resonance spectroscopy and electrochemistry, rendering the oxidovanadium(IV/V) compounds suitable for redox mediators in DSSCs. The very large self-exchange rate constant offered an insight into the mechanism of the exchange reaction most likely mediated through an outer-sphere exchange mechanism. The [V(IV)O(hybeb)](2-)/[V(V)O(hybeb)](-) redox potential and the energy of highest occupied molecular orbital (HOMO) of the sensitizing dye N719 and the HOMO of [V(IV)O(hybeb)](2-) were calculated by means of density functional theory electronic structure calculation methods. The complexes were applied as a new redox mediator in DSSCs, while the cell performance was studied in terms of the concentration of the reduced and oxidized form of the complexes. These studies were performed with the commercial Ru-based sensitizer N719 absorbed on a TiO2 semiconducting film in the DSSC. Maximum energy conversion efficiencies of 2% at simulated solar light (AM 1.5; 1000 W m(-2)) with an open circuit voltage of 660 mV, a short-circuit current of 5.2 mA cm(-2), and a fill factor of 0.58 were recorded without the presence of any additives in the electrolyte.

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.

Polymer-coated nanoparticles: Carrier platforms for hydrophobic water- and air-sensitive metallo-organic compounds.

PubMed

Valdeperez, Daniel; Wang, Tianqiang; Eußner, Jens P; Weinert, Bastian; Hao, Jianyuan; Parak, Wolfgang J; Dehnen, Stefanie; Pelaz, Beatriz

2017-03-01

Many of the relevant compounds for anticancer therapy are metal-based compounds (metallodrugs), being platinum-based drugs such as cisplatin, carboplatin (Paraplatin ® ), and oxaliplatin (Eloxatin ® ) the most widely used. Despite this, their application is limited by issues such as cell-acquired platinum resistance and manifold side effects following systemic delivery. Thus, the development of new metal-based compounds is highly needed. The catalytic properties of a variety of metal-based compounds are nowadays very well known, which opens new opportunities to take advantage of them inside living cells or organisms. However, many of these compounds are hydrophobic and thus not soluble in aqueous solution, as they lack stability against water or oxygen presence. Thus, versatile platforms capable of enhancing the features of these compounds in aqueous solutions are of importance in the development of new drugs. Surface engineered nanoparticles may render metallodrugs with good colloidal stability in water and in complex media containing high salt concentration and/or proteins. Herein, polymer coated nanoparticles are proposed as a platform to link insoluble and water/oxygen sensitive drugs. The linkage of insoluble and oxygen sensitive tin clusters to nanoparticles is presented, aiming to enhance both, the solubility and the stability of these compounds in water, which may be an alternative approach in the development of metal-based drugs. The formation of the cluster-nanoparticle system was confirmed via inductively coupled plasma mass spectrometry experiments. The catalytic activity and the stability of the cluster in water were studied through the reduction of methylene blue. Results demonstrate that in fact the tin clusters could be transferred into aqueous solution and retained their catalytic activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Harnessing cancer cell metabolism for theranostic applications using metabolic glycoengineering of sialic acid in breast cancer as a pioneering example

PubMed Central

Badr, Haitham A.; AlSadek, Dina M.M.; El-Houseini, Motawa E.; Saeui, Christopher T.; Mathew, Mohit P.; Yarema, Kevin J.; Ahmed, Hafiz

2016-01-01

Abnormal cell surface display of sialic acids – a family of unusual 9-carbon sugars - is widely recognized as distinguishing feature of many types of cancer. Sialoglycans, however, typically cannot be identified with sufficiently high reproducibility and sensitivity to serve as clinically accepted biomarkers and similarly, almost all efforts to exploit cancer-specific differences in sialylation signatures for therapy remain in early stage development. In this report we provide an overview of important facets of glycosylation that contribute to cancer in general with a focus on breast cancer as an example of malignant disease characterized by aberrant sialylation. We then describe how cancer cells experience nutrient deprivation during oncogenesis and discuss how the resulting metabolic reprogramming, which endows breast cancer cells with the ability to obtain nutrients during scarcity, constitutes an “Achilles’ heel” that we believe can be exploited by metabolic glycoengineering (MGE) strategies to develop new diagnostic methods and therapeutic approaches. In particular, we hypothesize that adaptations made by breast cancer cells that allow them to efficiently scavenge sialic acid during times of nutrient deprivation renders them vulnerable to MGE, which refers to the use of exogenously-supplied, non-natural monosaccharide analogues to modulate targeted aspects of glycosylation in living cells and animals. In specific, once non-natural sialosides are incorporated into the cancer “sialome” they can be exploited as epitopes for immunotherapy or as chemical tags for targeted delivery of imaging or therapeutic agents selectively to tumors. PMID:27926828

A Cytosolic Amphiphilic α-Helix Controls the Activity of the Bile Acid-sensitive Ion Channel (BASIC)*

PubMed Central

Schmidt, Axel; Löhrer, Daniel; Alsop, Richard J.; Lenzig, Pia; Oslender-Bujotzek, Adrienne; Wirtz, Monika; Rheinstädter, Maikel C.; Gründer, Stefan; Wiemuth, Dominik

2016-01-01

The bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na+ channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic α-helical structure within its N-terminal domain. This α-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction. PMID:27679529

A Cytosolic Amphiphilic α-Helix Controls the Activity of the Bile Acid-sensitive Ion Channel (BASIC).

PubMed

Schmidt, Axel; Löhrer, Daniel; Alsop, Richard J; Lenzig, Pia; Oslender-Bujotzek, Adrienne; Wirtz, Monika; Rheinstädter, Maikel C; Gründer, Stefan; Wiemuth, Dominik

2016-11-18

The bile acid-sensitive ion channel (BASIC) is a member of the degenerin/epithelial Na + channel (Deg/ENaC) family of ion channels. It is mainly found in bile duct epithelial cells, the intestinal tract, and the cerebellum and is activated by alterations of its membrane environment. Bile acids, one class of putative physiological activators, exert their effect by changing membrane properties, leading to an opening of the channel. The physiological function of BASIC, however, is unknown. Deg/ENaC channels are characterized by a trimeric subunit composition. Each subunit is composed of two transmembrane segments, which are linked by a large extracellular domain. The termini of the channels protrude into the cytosol. Many Deg/ENaC channels contain regulatory domains and sequence motifs within their cytosolic domains. In this study, we show that BASIC contains an amphiphilic α-helical structure within its N-terminal domain. This α-helix binds to the cytosolic face of the plasma membrane and stabilizes a closed state. Truncation of this domain renders the channel hyperactive. Collectively, we identify a cytoplasmic domain, unique to BASIC, that controls channel activity via membrane interaction. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay

PubMed Central

Ni, Julie Z.; Grate, Leslie; Donohue, John Paul; Preston, Christine; Nobida, Naomi; O’Brien, Georgeann; Shiue, Lily; Clark, Tyson A.; Blume, John E.; Ares, Manuel

2007-01-01

Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified >50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called “ultraconserved” elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression. We suggest that the extreme genomic conservation surrounding these regulatory splicing events within splicing factor genes demonstrates the evolutionary importance of maintaining tightly tuned homeostasis of RNA-binding protein levels in the vertebrate cell. PMID:17369403

Aging and the genetic road towards the positivity effect in memory.

PubMed

Mammarella, Nicola; Di Domenico, Alberto; Fairfield, Beth

2016-09-01

Better memory for positive information compared to negative and neutral information has been repeatedly associated with successful aging. The main psychological explanations for this so-called "positivity effect" in memory principally rely on emotional, motivational, and cognitive mechanisms that make older adults' cognition highly sensitive to positive information according to ultimate goals of well-being. However, emerging evidence also delineates a genetic profile for positivity effects in memory, which may render some older adults more prone than others to encoding and remembering positive memories. First, we present a brief overview of behavioral and neuroimaging studies about the positivity effect in aging. Subsequently, we report studies on candidate genes associated with positive memories. In particular, we review work to date on several candidate genes that are sensitive to stimulus valence such as ADRA2B, COMT, and 5HTTLPR. Finally, we propose that the future approach to the study of genetic correlates of positivity effects in memory should also include mitochondrial functioning (TOMM40). Altogether, the study of genetics and cell biology of positivity effects in memory can help us to reveal the underlying bottom-up pathways to positive affect in healthy aging. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

[131I]FIAU labeling of genetically transduced, tumor-reactive lymphocytes: cell-level dosimetry and dose-dependent toxicity.

PubMed

Zanzonico, Pat; Koehne, Guenther; Gallardo, Humilidad F; Doubrovin, Mikhail; Doubrovina, Ekaterina; Finn, Ronald; Blasberg, Ronald G; Riviere, Isabelle; O'Reilly, Richard J; Sadelain, Michel; Larson, Steven M

2006-09-01

Donor T cells have been shown to be reactive against and effective in adoptive immunotherapy of Epstein-Barr virus (EBV) lymphomas which develop in some leukemia patients post marrow transplantation. These T cells may be genetically modified by incorporation of a replication-incompetent viral vector (NIT) encoding both an inactive mutant nerve growth factor receptor (LNGFR), as an immunoselectable surface marker, and a herpes simplex virus thymidine kinase (HSV-TK), rendering the cells sensitive to ganciclovir. The current studies are based on the selective HSV-TK-catalyzed trapping (phosphorylation) of the thymidine analog [(131)I]-2'-fluoro-2'-deoxy-1-beta-D-arabinofuransyl-5-iodo-uracil (FIAU) as a means of stably labeling such T cells for in vivo trafficking (including tumor targeting) studies. Because of the radiosensitivity of lymphocytes and the potentially high absorbed dose to the nucleus from intracellular (131)I (even at tracer levels), the nucleus absorbed dose (D ( n )) and dose-dependent immune functionality were evaluated for NIT(+) T cells labeled ex vivo in [(131)I]FIAU-containing medium. Based on in vitro kinetic studies of [(131)I]FIAU uptake by NIT(+) T cells, D ( n ) was calculated using an adaptation of the MIRD formalism and the recently published MIRD cellular S factors. Immune cytotoxicity of [(131)I]FIAU-labeled cells was assayed against (51)Cr-labeled target cells [B-lymphoblastoid cells (BLCLs)] in a standard 4-h release assay. At median nuclear absorbed doses up to 830 cGy, a (51)Cr-release assay against BLCLs showed no loss of immune cytotoxicity, thus demonstrating the functional integrity of genetically transduced, tumor-reactive T cells labeled at this dose level for in vivo cell trafficking and tumor targeting studies.

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.

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.

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

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 ...

The Enemy within: Innate Surveillance-Mediated Cell Death, the Common Mechanism of Neurodegenerative Disease

PubMed Central

Richards, Robert I.; Robertson, Sarah A.; O'Keefe, Louise V.; Fornarino, Dani; Scott, Andrew; Lardelli, Michael; Baune, Bernhard T.

2016-01-01

Neurodegenerative diseases comprise an array of progressive neurological disorders all characterized by the selective death of neurons in the central nervous system. Although, rare (familial) and common (sporadic) forms can occur for the same disease, it is unclear whether this reflects several distinct pathogenic pathways or the convergence of different causes into a common form of nerve cell death. Remarkably, neurodegenerative diseases are increasingly found to be accompanied by activation of the innate immune surveillance system normally associated with pathogen recognition and response. Innate surveillance is the cell's quality control system for the purpose of detecting such danger signals and responding in an appropriate manner. Innate surveillance is an “intelligent system,” in that the manner of response is relevant to the magnitude and duration of the threat. If possible, the threat is dealt with within the cell in which it is detected, by degrading the danger signal(s) and restoring homeostasis. If this is not successful then an inflammatory response is instigated that is aimed at restricting the spread of the threat by elevating degradative pathways, sensitizing neighboring cells, and recruiting specialized cell types to the site. If the danger signal persists, then the ultimate response can include not only the programmed cell death of the original cell, but the contents of this dead cell can also bring about the death of adjacent sensitized cells. These responses are clearly aimed at destroying the ability of the detected pathogen to propagate and spread. Innate surveillance comprises intracellular, extracellular, non-cell autonomous and systemic processes. Recent studies have revealed how multiple steps in these processes involve proteins that, through their mutation, have been linked to many familial forms of neurodegenerative disease. This suggests that individuals harboring these mutations may have an amplified response to innate-mediated damage in neural tissues, and renders innate surveillance mediated cell death a plausible common pathogenic pathway responsible for neurodegenerative diseases, in both familial and sporadic forms. Here we have assembled evidence in favor of the hypothesis that neurodegenerative disease is the cumulative result of chronic activation of the innate surveillance pathway, triggered by endogenous or environmental danger or damage associated molecular patterns in a progressively expanding cascade of inflammation, tissue damage and cell death. PMID:27242399

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

Differential KrasV12 protein levels control a switch regulating lung cancer cell morphology and motility

PubMed Central

Schäfer, C.; Mohan, A.; Burford, W.; Driscoll, M. K.; Ludlow, A. T.; Wright, W. E.; Shay, J. W.; Danuser, G.

2016-01-01

Introduction Oncogenic Kras mutations are important drivers of lung cancer development and metastasis. They are known to activate numerous cellular signaling pathways implicated in enhanced proliferation, survival, tumorigenicity and motility during malignant progression. Objectives Most previous studies of Kras in cancer have focused on the comparison of cell states in the absence or presence of oncogenic Kras mutations. Here we show that differential expression of the constitutively active mutation KrasV12 has profound effects on cell morphology and motility that drive metastatic processes. Methods The study relies on lung cancer cell transformation models, patient-derived lung cancer cell lines, and human lung tumor sections combined with molecular biology techniques, live-cell imaging and staining methods. Results Our analysis shows two cell functional states driven by KrasV12 protein levels: a non-motile state associated with high KrasV12 levels and tumorigenicity, and a motile state associated with low KrasV12 levels and cell dissemination. Conversion between the states is conferred by differential activation of a mechano-sensitive double-negative feedback between KrasV12/ERK/Myosin II and matrix-adhesion signaling. KrasV12 expression levels change upon cues such as hypoxia and integrin-mediated cell-matrix adhesion, rendering KrasV12 levels an integrator of micro-environmental signals that translate into cellular function. By live cell imaging of tumor models we observe shedding of mixed high and low KrasV12 expressers forming multi-functional collectives with potentially optimal metastatic properties composed of a highly mobile and a highly tumorigenic unit. Discussion Together these data highlight previously unappreciated roles for the quantitative effects of expression level variation of oncogenic signaling molecules in conferring fundamental alterations in cell function regulation required for cancer progression. PMID:29057096

DNA Clutch Probes for Circulating Tumor DNA Analysis.

PubMed

Das, Jagotamoy; Ivanov, Ivaylo; Sargent, Edward H; Kelley, Shana O

2016-08-31

Progress toward the development of minimally invasive liquid biopsies of disease is being bolstered by breakthroughs in the analysis of circulating tumor DNA (ctDNA): DNA released from cancer cells into the bloodstream. However, robust, sensitive, and specific methods of detecting this emerging analyte are lacking. ctDNA analysis has unique challenges, since it is imperative to distinguish circulating DNA from normal cells vs mutation-bearing sequences originating from tumors. Here we report the electrochemical detection of mutated ctDNA in samples collected from cancer patients. By developing a strategy relying on the use of DNA clutch probes (DCPs) that render specific sequences of ctDNA accessible, we were able to readout the presence of mutated ctDNA. DCPs prevent reassociation of denatured DNA strands: they make one of the two strands of a dsDNA accessible for hybridization to a probe, and they also deactivate other closely related sequences in solution. DCPs ensure thereby that only mutated sequences associate with chip-based sensors detecting hybridization events. The assay exhibits excellent sensitivity and specificity in the detection of mutated ctDNA: it detects 1 fg/μL of a target mutation in the presence of 100 pg/μL of wild-type DNA, corresponding to detecting mutations at a level of 0.01% relative to wild type. This approach allows accurate analysis of samples collected from lung cancer and melanoma patients. This work represents the first detection of ctDNA without enzymatic amplification.

Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research.

PubMed

Loroch, Stefan; Schommartz, Tim; Brune, Wolfram; Zahedi, René Peiman; Sickmann, Albert

2015-05-01

Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research. Copyright © 2015. Published by Elsevier B.V.

Development of a Sensitive and Specific Polyclonal Antibody for Serological Detection of Clavibacter michiganensis subsp. sepedonicus

PubMed Central

Przewodowska, Agnieszka

2017-01-01

The quarantine bacterium Clavibacter michiganensis subsp. sepedonicus (Cms) causes bacterial ring rot (BRR) in potato but is difficult to detect, hampering the diagnosis of this disease. ELISA immunoassays have not been widely used to detect Cms because commercially available anti-Cms antibodies detect mainly EPS-producing bacteria and can fail to detect strains that do not produce EPS. In the current study, we developed a new type of polyclonal antibody that specifically detects Clavibacter michiganensis subsp. sepedonicus bacteria irrespective of their EPS level. We first found that the presence of bacterial EPS precluded quantitative measurement of bacteria by currently available immunoenzymatic methods, but that washing Cms cells with acidic and basic buffers to remove EPS before analysis successfully standardized ELISA results. We used a mix of three strains of Cms with diverse EPS levels to generate antigen for production of antibodies recognizing Cms cells with and without an EPS layer (IgG-EPS and IgG-N-EPS, respectively). The resulting IgG-N-EPS recognized almost all Cms strains tested in this work regardless of their mucoidal level. The availability of this new antibody renders immunological diagnostics of Cms more sensitive and reliable, as our newly developed antibodies can be used in many type of immunoassays. This work represents an important step forward in efforts to diagnose and prevent the spread of BRR, and the methods and solutions developed in this work are covered by six Polish, one European and one US patents. PMID:28068400

Aversive Memory Reactivation Engages in the Amygdala Only Some Neurotransmitters Involved in Consolidation

ERIC Educational Resources Information Center

Bucherelli, Corrado; Baldi, Elisabetta; Mariottini, Chiara; Passani, Maria Beatrice; Blandina, Patrizio

2006-01-01

Consolidation refers to item stabilization in long-term memory. Retrieval renders a consolidated memory sensitive, and a "reconsolidation" process has been hypothesized to keep the original memory persistent. Some authors could not detect this phenomenon. Here we show that retrieved contextual fear memory is vulnerable to amnesic treatments and…

EEG synchronization and migraine

NASA Astrophysics Data System (ADS)

Stramaglia, Sebastiano; Angelini, Leonardo; Pellicoro, Mario; Hu, Kun; Ivanov, Plamen Ch.

2004-03-01

We investigate phase synchronization in EEG recordings from migraine patients. We use the analytic signal technique, based on the Hilbert transform, and find that migraine brains are characterized by enhanced alpha band phase synchronization in presence of visual stimuli. Our findings show that migraine patients have an overactive regulatory mechanism that renders them more sensitive to external stimuli.

Sampling methods for bats.

Treesearch

D.W. Thomas; S.D. West

1989-01-01

Bats represent the second most diverse group of mammals inhabiting the western slopes of the Cascade Range in southern Washington and the Oregon Coast Range. Bat populations may well be sensitive to changes in forest age, structure, or distribution, but their nocturnal habits and high mobility render the study of the habitat requirements of bats problematical. Unlike...

Ability of various materials to detect explosive vapors by fluorescent technologies: a comparative study.

PubMed

Bouhadid, Myriam; Caron, Thomas; Veignal, Florian; Pasquinet, Eric; Ratsimihety, Amédée; Ganachaud, François; Montméat, Pierre

2012-10-15

For the development of fluorescent sensors, one of the key points is choosing the sensitive material. In this article, we aim at evaluating, under strictly identical experimental conditions, the performance of three materials for the detection of dinitrotoluene (a volatile marker of trinitrotoluene) through different parameters: response time, fluorescence intensity, sensitivity, reversibility, reaction after successive exposures and long-term stability. The results are discussed according to the nature of the sensitive materials. This first study rendered it possible to select a conjugated molecule as the best sensitive material for the development of a lab-made prototype. In a second part, the selectivity of this particular sensitive material was studied and its ability to detect TNT could be demonstrated. Copyright © 2012. Published by Elsevier B.V.

User Authentication in Smartphones for Telehealth

PubMed Central

SMITH, KATHERINE A.; ZHOU, LEMING; WATZLAF, VALERIE J. M.

2017-01-01

Many functions previously conducted on desktop computers are now performed on smartphones. Smartphones provide convenience, portability, and connectivity. When smartphones are used in the conduct of telehealth, sensitive data is invariably accessed, rendering the devices in need of user authentication to ensure data protection. User authentication of smartphones can help mitigate potential Health Insurance Portability and Accountability Act (HIPAA) breaches and keep sensitive patient information protected, while also facilitating the convenience of smartphones within everyday life and healthcare. This paper presents and examines several types of authentication methods available to smartphone users to help ensure security of sensitive data from attackers. The applications of these authentication methods in telehealth are discussed. PMID:29238444

User Authentication in Smartphones for Telehealth.

PubMed

Smith, Katherine A; Zhou, Leming; Watzlaf, Valerie J M

2017-01-01

Many functions previously conducted on desktop computers are now performed on smartphones. Smartphones provide convenience, portability, and connectivity. When smartphones are used in the conduct of telehealth, sensitive data is invariably accessed, rendering the devices in need of user authentication to ensure data protection. User authentication of smartphones can help mitigate potential Health Insurance Portability and Accountability Act (HIPAA) breaches and keep sensitive patient information protected, while also facilitating the convenience of smartphones within everyday life and healthcare. This paper presents and examines several types of authentication methods available to smartphone users to help ensure security of sensitive data from attackers. The applications of these authentication methods in telehealth are discussed.

Polarization: A Key Difference between Man-made and Natural Electromagnetic Fields, in regard to Biological Activity.

PubMed

Panagopoulos, Dimitris J; Johansson, Olle; Carlo, George L

2015-10-12

In the present study we analyze the role of polarization in the biological activity of Electromagnetic Fields (EMFs)/Electromagnetic Radiation (EMR). All types of man-made EMFs/EMR - in contrast to natural EMFs/EMR - are polarized. Polarized EMFs/EMR can have increased biological activity, due to: 1) Ability to produce constructive interference effects and amplify their intensities at many locations. 2) Ability to force all charged/polar molecules and especially free ions within and around all living cells to oscillate on parallel planes and in phase with the applied polarized field. Such ionic forced-oscillations exert additive electrostatic forces on the sensors of cell membrane electro-sensitive ion channels, resulting in their irregular gating and consequent disruption of the cell's electrochemical balance. These features render man-made EMFs/EMR more bioactive than natural non-ionizing EMFs/EMR. This explains the increasing number of biological effects discovered during the past few decades to be induced by man-made EMFs, in contrast to natural EMFs in the terrestrial environment which have always been present throughout evolution, although human exposure to the latter ones is normally of significantly higher intensities/energy and longer durations. Thus, polarization seems to be a trigger that significantly increases the probability for the initiation of biological/health effects.

Anti-tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC-dependent vulnerability.

PubMed

Iwai, Kenichi; Yaguchi, Masahiro; Nishimura, Kazuho; Yamamoto, Yukiko; Tamura, Toshiya; Nakata, Daisuke; Dairiki, Ryo; Kawakita, Yoichi; Mizojiri, Ryo; Ito, Yoshiteru; Asano, Moriteru; Maezaki, Hironobu; Nakayama, Yusuke; Kaishima, Misato; Hayashi, Kozo; Teratani, Mika; Miyakawa, Shuichi; Iwatani, Misa; Miyamoto, Maki; Klein, Michael G; Lane, Wes; Snell, Gyorgy; Tjhen, Richard; He, Xingyue; Pulukuri, Sai; Nomura, Toshiyuki

2018-06-01

The modulation of pre-mRNA splicing is proposed as an attractive anti-neoplastic strategy, especially for the cancers that exhibit aberrant pre-mRNA splicing. Here, we discovered that T-025 functions as an orally available and potent inhibitor of Cdc2-like kinases (CLKs), evolutionally conserved kinases that facilitate exon recognition in the splicing machinery. Treatment with T-025 reduced CLK-dependent phosphorylation, resulting in the induction of skipped exons, cell death, and growth suppression in vitro and in vivo Further, through growth inhibitory characterization, we identified high CLK2 expression or MYC amplification as a sensitive-associated biomarker of T-025. Mechanistically, the level of CLK2 expression correlated with the magnitude of global skipped exons in response to T-025 treatment. MYC activation, which altered pre-mRNA splicing without the transcriptional regulation of CLKs, rendered cancer cells vulnerable to CLK inhibitors with synergistic cell death. Finally, we demonstrated in vivo anti-tumor efficacy of T-025 in an allograft model of spontaneous, MYC-driven breast cancer, at well-tolerated dosage. Collectively, our results suggest that the novel CLK inhibitor could have therapeutic benefits, especially for MYC-driven cancer patients. © 2018 Takeda Pharmaceutical Company Published under the terms of the CC BY 4.0 license.

Telomere attrition and genomic instability in xeroderma pigmentosum type-b deficient fibroblasts under oxidative stress

PubMed Central

Ting, Aloysius Poh Leong; Low, Grace Kah Mun; Gopalakrishnan, Kalpana; Hande, M Prakash

2010-01-01

Abstract Xeroderma pigmentosum B (XPB/ERCC3/p89) is an ATP-dependent 3′→5′ directed DNA helicase involved in basal RNA transcription and the nucleotide excision repair (NER) pathway. While the role of NER in alleviating oxidative DNA damage has been acknowledged it remains poorly understood. To study the involvement of XPB in repair of oxidative DNA damage, we utilized primary fibroblasts from a patient suffering from XP with Cockayne syndrome and hydrogen peroxide (H2O2) to induce oxidative stress. Mutant cells retained higher viability and cell cycle dysfunction after H2O2 exposure. Cytokinesis blocked micronucleus assay revealed increased genome instability induced by H2O2. Single cell gel electrophoresis (comet) assay showed that the missense mutation caused a reduced repair capacity for oxidative DNA damage. Mutant fibroblasts also displayed decreased population doubling rate, increased telomere attrition rate and early emergence of senescent characteristics under chronic low dose exposure to H2O2. Fibroblasts from a heterozygous individual displayed intermediate traits in some assays and normal traits in others, indicating possible copy number dependence. The results show that a deficiency in functional XPB paradoxically renders cells more sensitive to the genotoxic effects of oxidative stress while reducing the cytotoxic effects. These findings have implications in the mechanisms of DNA repair, mutagenesis and carcinogenesis and ageing in normal physiological systems. PMID:19840190

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

Trace amines depress D2-autoreceptor-mediated responses on midbrain dopaminergic cells

PubMed Central

Ledonne, Ada; Federici, Mauro; Giustizieri, Michela; Pessia, Mauro; Imbrici, Paola; Millan, Mark J; Bernardi, Giorgio; Mercuri, Nicola B

2010-01-01

Background and purpose: Although trace amines (TAs) are historically considered ‘false neurotransmitters’ on the basis of their ability to induce catecholamine release, there is evidence that they directly affect neuronal activity via TA receptors, ligand-gated receptor channels and/or σ receptors. Here, we have investigated the effects of two TAs, tyramine (TYR) and β-phenylethylamine (β-PEA), on electrophysiological responses of substantia nigra pars compacta (SNpc) dopaminergic cells to the D2 receptor agonist, quinpirole. Experimental approach: Electrophysiological recordings of D2 receptor-activated G-protein-gated inward rectifier K+ channel (GIRK) currents were performed on dopaminergic cells from midbrain slices of mice and on Xenopus oocytes expressing D2 receptors and GIRK channels. Key results: TYR and β-PEA reversibly reduced D2 receptor-activated GIRK currents in a concentration-dependent manner on SNpc neurones. The inhibitory effect of TAs was still present in transgenic mice with genetically deleted TA1 receptors and they could not be reproduced by the selective TA1 agonist, o-phenyl-3-iodotyramine (O-PIT). Pretreatment with antagonists of σ1 and σ2 receptors did not block TA-induced effects. In GTPγS-loaded neurones, the irreversibly-activated GIRK-current was still reversibly reduced by β-PEA. Moreover, β-PEA did not affect basal or dopamine-evoked GIRK-currents in Xenopus oocytes. Conclusions and implications: TAs reduced dopamine-induced responses on SNpc neurones by acting at sites different from TA1, σ-receptors, D2 receptors or GIRK channels. Although their precise mechanism of action remains to be identified, TAs, by antagonizing the inhibitory effects of dopamine, may render dopaminergic neurones less sensitive to autoreceptor feedback inhibition and hence enhance their sensitivity to stimulation. PMID:20590640

Tuning the colors of c-Si solar cells by exploiting plasmonic effects

NASA Astrophysics Data System (ADS)

Peharz, G.; Grosschädl, B.; Prietl, C.; Waldhauser, W.; Wenzl, F. P.

2016-09-01

The color of a crystalline silicon (c-Si) solar cell is mainly determined by its anti-reflective coating. This is a lambda/4 coating made from a transparent dielectric material. The thickness of the anti-reflective coating is optimized for maximal photocurrent generation, resulting in the typical blue or black colors of c-Si solar cells. However, for building-integrated photovoltaic (BiPV) applications the color of the solar cells is demanded to be tunable - ideally by a cheap and flexible coating process on standard (low cost) c-Si solar cells. Such a coating can be realized by applying plasmonic coloring which is a rapidly growing technology for high-quality color filtering and rendering for different fields of application (displays, imaging,…). In this contribution, we present results of an approach for tuning the color of standard industrial c-Si solar cells that is based on coating them with metallic nano-particles. In particular, thin films (< 20 nm) of a metal (e.g., silver) were sputtered onto c-Si solar cells and thermally annealed subsequently. The sizes and the shapes of the nano-particles (characterized by SEM) were found to depend on the thickness of the deposited films and the surface roughness of the substrates/solar cells. With such an approach it is possible to tune the color of the standard c-Si cells from blue to green and brownish/red. The position of the resonance peak in the reflection spectrum was found to be almost independent from the angle of incidence. This low angular sensitivity is a clear advantage compared to alternative color tuning methods, for which additional dielectric thin films are deposited on c-Si solar cells.

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

Caspase inhibition augmented oridonin-induced cell death in murine fibrosarcoma l929 by enhancing reactive oxygen species generation.

PubMed

Wu, Jin-Nan; Huang, Jian; Yang, Jia; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2008-09-01

Oridonin, a diterpenoid isolated from Rabdosia rubescences, has been reported to have antitumor effects. In this study, the growth-inhibitory activity of oridonin for L929 cells was exerted in a time-and dose-dependent manner. After treatment with oridonin for 24 h, L929 cells underwent both apoptosis and necrosis as measured by an lactate dehydrogenase (LDH) activity-based assay. A rapid generation of reactive oxygen species (ROS) was triggered by oridonin, and subsequently up-regulation of phospho-p53 (ser 15) expression and an increased expression ratio of Bax/Bcl-2 was observed. Furthermore, there was a significant fall in mitochondrial membrane potential (MMP) and increase in caspase-3 activity after exposure to oridonin for 24 h. Surprisingly, the pan-caspase inhibitor z-VAD-fmk and caspase3 inhibitor z-DEVD-fmk rendered L929 cells more sensitive to oridonin, rather than preventing oridonin-induced cell death. Oridonin and z-VAD-fmk co-treatment not only resulted in an even higher ROS production, but also made a more significant reduction in the MMP. Pretreatment of ROS scavenger N-acetylcysteine (NAC) led to a complete inhibition of oridonin-induced cell death, intracellular ROS generation, and MMP collapse. NAC treatment also reversed the potentiation of cell death by the pan-caspase inhibitor z-VAD-fmk. Taken together, these observations showed that oridonin-induced cell death in L929 cells involved intracellular ROS generation, activation of phospho-p53 (ser 15), and up-regulation of the Bax/Bcl-2 ratio; and the augmented cell death by z-VAD-fmk was dependent on an increased ROS production.

Stress and strain in the contractile and cytoskeletal filaments of airway smooth muscle.

PubMed

Deng, Linhong; Bosse, Ynuk; Brown, Nathan; Chin, Leslie Y M; Connolly, Sarah C; Fairbank, Nigel J; King, Greg G; Maksym, Geoffrey N; Paré, Peter D; Seow, Chun Y; Stephen, Newman L

2009-10-01

Stress and strain are omnipresent in the lung due to constant lung volume fluctuation associated with respiration, and they modulate the phenotype and function of all cells residing in the airways including the airway smooth muscle (ASM) cell. There is ample evidence that the ASM cell is very sensitive to its physical environment, and can alter its structure and/or function accordingly, resulting in either desired or undesired consequences. The forces that are either conferred to the ASM cell due to external stretching or generated inside the cell must be borne and transmitted inside the cytoskeleton (CSK). Thus, maintaining appropriate levels of stress and strain within the CSK is essential for maintaining normal function. Despite the importance, the mechanisms regulating/dysregulating ASM cytoskeletal filaments in response to stress and strain remained poorly understood until only recently. For example, it is now understood that ASM length and force are dynamically regulated, and both can adapt over a wide range of length, rendering ASM one of the most malleable living tissues. The malleability reflects the CSK's dynamic mechanical properties and plasticity, both of which strongly interact with the loading on the CSK, and all together ultimately determines airway narrowing in pathology. Here we review the latest advances in our understanding of stress and strain in ASM cells, including the organization of contractile and cytoskeletal filaments, range and adaptation of functional length, structural and functional changes of the cell in response to mechanical perturbation, ASM tone as a mediator of strain-induced responses, and the novel glassy dynamic behaviors of the CSK in relation to asthma pathophysiology.

Lytic agents, cell permeability, and monolayer penetrability.

PubMed

Salton, M R

1968-07-01

Cell lysis induced by lytic agents is the terminal phase of a series of events leading to membrane disorganization and breadkdown with the release of cellular macromolecules. Permeability changes following exposure to lytic systems may range from selective effects on ion fluxes to gross membrane damage and cell leakage. Lysis can be conceived as an interfacial phenomenon, and the action of surface-active agents on erythrocytes has provided a model in which to investigate relationships between hemolysis and chemical structure, ionic charge, surface tension lowering, and ability to penetrate monolayers of membrane lipid components. Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid. Similarities of chemical composition of membranes from various cell types could account for lytic responses observed on interaction with surface-active agents. Cell membranes usually contain about 20-30 % lipid and 50-75 % protein. One or two major phospholipids are present in all cell membranes, but sterols are not detectable in bacterial membranes other than those of the Mycoplasma group. The rigid cell wall in bacteria has an important bearing on their response to treatment with lytic agents. Removal of the wall renders the protoplast membrane sensitive to rapid lysis with surfactants. Isolated membranes of erythrocytes and bacteria are rapidly dissociated by surface-active agents. Products of dissociation of bacterial membranes have uniform behavior in the ultracentrifuge (sedimentation coefficients 2-3S). Dissociation of membrane proteins from lipids and the isolation and characterization of these proteins will provide a basis for investigating the specificity of interaction of lytic agents with biomembranes.

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.

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 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.

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

Educational Leadership and Context: A Rendering of an Inseparable Relationship

ERIC Educational Resources Information Center

Clarke, Simon; O'Donoghue, Tom

2017-01-01

The amount of empirical research on leadership of educational organisations, and especially of schools, which has stressed the importance of being sensitive to context, is not great. This paper seeks to highlight the challenge presented by this situation. First, context is defined. Second, attention is drawn to what can be learned in the area of…

Two Distinct Origins of Long-Term Learning Effects in Verbal Short-Term Memory

ERIC Educational Resources Information Center

Majerus, Steve; Perez, Trecy Martinez; Oberauer, Klaus

2012-01-01

Verbal short-term memory (STM) is highly sensitive to learning effects: digit sequences or nonword sequences which have been rendered more familiar via repeated exposure are recalled more accurately. In this study we show that sublist-level, incidental learning of item co-occurrence regularities affects immediate serial recall of words and…

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.

Opposite rheological properties of neuronal microcompartments predict axonal vulnerability in brain injury.

PubMed

Grevesse, Thomas; Dabiri, Borna E; Parker, Kevin Kit; Gabriele, Sylvain

2015-03-30

Although pathological changes in axonal morphology have emerged as important features of traumatic brain injury (TBI), the mechanical vulnerability of the axonal microcompartment relative to the cell body is not well understood. We hypothesized that soma and neurite microcompartments exhibit distinct mechanical behaviors, rendering axons more sensitive to a mechanical injury. In order to test this assumption, we combined protein micropatterns with magnetic tweezer rheology to probe the viscoelastic properties of neuronal microcompartments. Creep experiments revealed two opposite rheological behaviors within cortical neurons: the cell body was soft and characterized by a solid-like response, whereas the neurite compartment was stiffer and viscous-like. By using pharmacological agents, we demonstrated that the nucleus is responsible for the solid-like behavior and the stress-stiffening response of the soma, whereas neurofilaments have a predominant contribution in the viscous behavior of the neurite. Furthermore, we found that the neurite is a mechanosensitive compartment that becomes softer and adopts a pronounced viscous state on soft matrices. Together, these findings highlight the importance of the regionalization of mechanical and rigidity-sensing properties within neuron microcompartments in the preferential damage of axons during traumatic brain injury and into potential mechanisms of axonal outgrowth after injury.

Hormonal stimulation of mitochondrial pyruvate carboxylation in filipin-treated hepatocytes.

PubMed Central

Allan, E H; Chisholm, A B; Titheradge, M A

1983-01-01

A method is described for measuring rates of mitochondrial pyruvate carboxylation in hepatocytes treated with the polyene antibiotic, filipin, to render the plasma membrane permeable to substrates. With this approach it was possible to demonstrate that treatment of cells with glucagon or catecholamines results in a stimulation of mitochondrial CO2 fixation measured in situ comparable with that observed in the isolated mitochondria, in terms of time of onset of the response, hormone selectivity and sensitivity. In addition, angiotensin II and vasopressin were shown to enhance the activity of pyruvate carboxylase in both the intact mitochondria and filipin-treated cells, thus strengthening the postulate that this site is a major locus of hormone action in the control of gluconeogenesis. Addition of 3-mercaptopicolinic acid, to inhibit gluconeogenesis at the level of phosphoenolpyruvate carboxykinase, had no significant effect on the stimulation of pyruvate carboxylation by adrenaline, suggesting that the effect of the hormone at this site is independent of changes in activity of other enzymes further on in the pathway. The data presented preclude the possibility that acute effects of hormones on mitochondrial metabolism are solely artifacts of the preparation procedure. PMID:6411066

Opposite rheological properties of neuronal microcompartments predict axonal vulnerability in brain injury

NASA Astrophysics Data System (ADS)

Grevesse, Thomas; Dabiri, Borna E.; Parker, Kevin Kit; Gabriele, Sylvain

2015-03-01

Although pathological changes in axonal morphology have emerged as important features of traumatic brain injury (TBI), the mechanical vulnerability of the axonal microcompartment relative to the cell body is not well understood. We hypothesized that soma and neurite microcompartments exhibit distinct mechanical behaviors, rendering axons more sensitive to a mechanical injury. In order to test this assumption, we combined protein micropatterns with magnetic tweezer rheology to probe the viscoelastic properties of neuronal microcompartments. Creep experiments revealed two opposite rheological behaviors within cortical neurons: the cell body was soft and characterized by a solid-like response, whereas the neurite compartment was stiffer and viscous-like. By using pharmacological agents, we demonstrated that the nucleus is responsible for the solid-like behavior and the stress-stiffening response of the soma, whereas neurofilaments have a predominant contribution in the viscous behavior of the neurite. Furthermore, we found that the neurite is a mechanosensitive compartment that becomes softer and adopts a pronounced viscous state on soft matrices. Together, these findings highlight the importance of the regionalization of mechanical and rigidity-sensing properties within neuron microcompartments in the preferential damage of axons during traumatic brain injury and into potential mechanisms of axonal outgrowth after injury.

Critical Role of Interface and Crystallinity on the Performance and Photostability of Perovskite Solar Cell on Nickel Oxide

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

Nie, Wanyi; Tsai, Hsinhan; Blancon, Jean -Christophe

Hybrid perovskites are on a trajectory toward realizing the most efficient single-junction, solution-processed photovoltaic devices. However, a critical issue is the limited understanding of the correlation between the degree of crystallinity and the emergent perovskite/hole (or electron) transport layer on device performance and photostability. Here, the controlled growth of hybrid perovskites on nickel oxide (NiO) is shown, resulting in the formation of thin films with enhanced crystallinity with characteristic peak width and splitting reminiscent of the tetragonal phase in single crystals. Photophysical and interface sensitive measurements reveal a reduced trap density at the perovskite/NiO interface in comparison with perovskites grownmore » on poly(3,4-ethylene dioxy thiophene) polystyrene sulfonate. Photovoltaic cells exhibit a high open circuit voltage (1.12 V), indicating a near-ideal energy band alignment. Moreover, photostability of photovoltaic devices up to 10-Suns is observed, which is a direct result of the superior crystallinity of perovskite thin films on NiO. Here, these results elucidate the critical role of the quality of the perovskite/hole transport layer interface in rendering high-performance and photostable optoelectronic devices.« less

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

Critical Role of Interface and Crystallinity on the Performance and Photostability of Perovskite Solar Cell on Nickel Oxide.

PubMed

Nie, Wanyi; Tsai, Hsinhan; Blancon, Jean-Christophe; Liu, Fangze; Stoumpos, Costas C; Traore, Boubacar; Kepenekian, Mikael; Durand, Olivier; Katan, Claudine; Tretiak, Sergei; Crochet, Jared; Ajayan, Pulickel M; Kanatzidis, MercouriG; Even, Jacky; Mohite, Aditya D

2018-02-01

Hybrid perovskites are on a trajectory toward realizing the most efficient single-junction, solution-processed photovoltaic devices. However, a critical issue is the limited understanding of the correlation between the degree of crystallinity and the emergent perovskite/hole (or electron) transport layer on device performance and photostability. Here, the controlled growth of hybrid perovskites on nickel oxide (NiO) is shown, resulting in the formation of thin films with enhanced crystallinity with characteristic peak width and splitting reminiscent of the tetragonal phase in single crystals. Photophysical and interface sensitive measurements reveal a reduced trap density at the perovskite/NiO interface in comparison with perovskites grown on poly(3,4-ethylene dioxy thiophene) polystyrene sulfonate. Photovoltaic cells exhibit a high open circuit voltage (1.12 V), indicating a near-ideal energy band alignment. Moreover, photostability of photovoltaic devices up to 10-Suns is observed, which is a direct result of the superior crystallinity of perovskite thin films on NiO. These results elucidate the critical role of the quality of the perovskite/hole transport layer interface in rendering high-performance and photostable optoelectronic devices. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Critical Role of Interface and Crystallinity on the Performance and Photostability of Perovskite Solar Cell on Nickel Oxide

DOE PAGES

Nie, Wanyi; Tsai, Hsinhan; Blancon, Jean -Christophe; ...

2017-12-11

Hybrid perovskites are on a trajectory toward realizing the most efficient single-junction, solution-processed photovoltaic devices. However, a critical issue is the limited understanding of the correlation between the degree of crystallinity and the emergent perovskite/hole (or electron) transport layer on device performance and photostability. Here, the controlled growth of hybrid perovskites on nickel oxide (NiO) is shown, resulting in the formation of thin films with enhanced crystallinity with characteristic peak width and splitting reminiscent of the tetragonal phase in single crystals. Photophysical and interface sensitive measurements reveal a reduced trap density at the perovskite/NiO interface in comparison with perovskites grownmore » on poly(3,4-ethylene dioxy thiophene) polystyrene sulfonate. Photovoltaic cells exhibit a high open circuit voltage (1.12 V), indicating a near-ideal energy band alignment. Moreover, photostability of photovoltaic devices up to 10-Suns is observed, which is a direct result of the superior crystallinity of perovskite thin films on NiO. Here, these results elucidate the critical role of the quality of the perovskite/hole transport layer interface in rendering high-performance and photostable optoelectronic devices.« less

Regioisomeric effects on the electronic features of indenothiophene-bridged D-π-A'-A DSSC sensitizers.

PubMed

Chou, Shu-Hua; Tsai, Chih-Hung; Wu, Chung-Chih; Kumar, Dhirendra; Wong, Ken-Tsung

2014-12-08

Two D-π-A'-A regioisomers (A-IDT-D and D-IDT-A) featuring 4,4'-di-p-tolyl-4 H-indeno[1,2-b]-thiophene as a π linker (π) between the diarylamino donor (D) and the pyrimidine-cyanoacrylic acid acceptor (A'-A) have been successfully synthesized and characterized as efficient sensitizers for the dye-sensitized solar cells (DSSCs). The different arrangements of the D and A'-A blocks on the unsymmetrical indenothiophene (IDT) core render the dipole of IDT being along (A-IDT-D) or opposite (D-IDT-A) to the direction of intramolecular (donor-to-acceptor) charge transfer, and thus induce variations in the physical properties. The experimental observations correlated well with the theoretical analyses, clearly revealing the trade-off between the molar extinction coefficient (ε) and the S0 →S1 transition energy. As a result, a superior ε value was observed for D-IDT-A, whereas a bathochromic shift in the absorption occurred in A-IDT-D. The larger ε value of D-IDT-A together with its more favorable energy level relative to TiO2 led to a higher power conversion efficiency of 7.41 % for the D-IDT-A-based DSSC, retaining approximately 95 % of the N719-based DSSC efficiency. This work manifests the clear structure-property relationship for the case of donor and acceptor components being connected by an unsymmetrical π linker and provides insights for molecular engineering of organic sensitizers. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nicotine Activation of α4* Receptors: Sufficient for Reward, Tolerance, and Sensitization

NASA Astrophysics Data System (ADS)

Tapper, Andrew R.; McKinney, Sheri L.; Nashmi, Raad; Schwarz, Johannes; Deshpande, Purnima; Labarca, Cesar; Whiteaker, Paul; Marks, Michael J.; Collins, Allan C.; Lester, Henry A.

2004-11-01

The identity of nicotinic receptor subtypes sufficient to elicit both the acute and chronic effects of nicotine dependence is unknown. We engineered mutant mice with α4 nicotinic subunits containing a single point mutation, Leu9' --> Ala9' in the pore-forming M2 domain, rendering α4* receptors hypersensitive to nicotine. Selective activation of α4* nicotinic acetylcholine receptors with low doses of agonist recapitulates nicotine effects thought to be important in dependence, including reinforcement in response to acute nicotine administration, as well as tolerance and sensitization elicited by chronic nicotine administration. These data indicate that activation of α4* receptors is sufficient for nicotine-induced reward, tolerance, and sensitization.

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.

Lack of the Sodium-Driven Chloride Bicarbonate Exchanger NCBE Impairs Visual Function in the Mouse Retina

PubMed Central

Hilgen, Gerrit; Huebner, Antje K.; Tanimoto, Naoyuki; Sothilingam, Vithiyanjali; Seide, Christina; Garrido, Marina Garcia; Schmidt, Karl-Friedrich; Seeliger, Mathias W.; Löwel, Siegrid; Weiler, Reto

2012-01-01

Regulation of ion and pH homeostasis is essential for normal neuronal function. The sodium-driven chloride bicarbonate exchanger NCBE (Slc4a10), a member of the SLC4 family of bicarbonate transporters, uses the transmembrane gradient of sodium to drive cellular net uptake of bicarbonate and to extrude chloride, thereby modulating both intracellular pH (pHi) and chloride concentration ([Cl−]i) in neurons. Here we show that NCBE is strongly expressed in the retina. As GABAA receptors conduct both chloride and bicarbonate, we hypothesized that NCBE may be relevant for GABAergic transmission in the retina. Importantly, we found a differential expression of NCBE in bipolar cells: whereas NCBE was expressed on ON and OFF bipolar cell axon terminals, it only localized to dendrites of OFF bipolar cells. On these compartments, NCBE colocalized with the main neuronal chloride extruder KCC2, which renders GABA hyperpolarizing. NCBE was also expressed in starburst amacrine cells, but was absent from neurons known to depolarize in response to GABA, like horizontal cells. Mice lacking NCBE showed decreased visual acuity and contrast sensitivity in behavioral experiments and smaller b-wave amplitudes and longer latencies in electroretinograms. Ganglion cells from NCBE-deficient mice also showed altered temporal response properties. In summary, our data suggest that NCBE may serve to maintain intracellular chloride and bicarbonate concentration in retinal neurons. Consequently, lack of NCBE in the retina may result in changes in pHi regulation and chloride-dependent inhibition, leading to altered signal transmission and impaired visual function. PMID:23056253

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.

Elevated YKL40 is associated with advanced prostate cancer (PCa) and positively regulates invasion and migration of PCa cells

PubMed Central

Jeet, Varinder; Tevz, Gregor; Lehman, Melanie; Hollier, Brett; Nelson, Colleen

2014-01-01

Chitinase 3-like 1 (CHI3L1 or YKL40) is a secreted glycoprotein highly expressed in tumours from patients with advanced stage cancers, including prostate cancer (PCa). The exact function of YKL40 is poorly understood, but it has been shown to play an important role in promoting tumour angiogenesis and metastasis. The therapeutic value and biological function of YKL40 are unknown in PCa. The objective of this study was to examine the expression and function of YKL40 in PCa. Gene expression analysis demonstrated that YKL40 was highly expressed in metastatic PCa cells when compared with less invasive and normal prostate epithelial cell lines. In addition, the expression was primarily limited to androgen receptor-positive cell lines. Evaluation of YKL40 tissue expression in PCa patients showed a progressive increase in patients with aggressive disease when compared with those with less aggressive cancers and normal controls. Treatment of LNCaP and C4-2B cells with androgens increased YKL40 expression, whereas treatment with an anti-androgen agent decreased the gene expression of YKL40 in androgen-sensitive LNCaP cells. Furthermore, knockdown of YKL40 significantly decreased invasion and migration of PCa cells, whereas overexpression rendered them more invasive and migratory, which was commensurate with an enhancement in the anchorage-independent growth of cells. To our knowledge, this study characterises the role of YKL40 for the first time in PCa. Together, these results suggest that YKL40 plays an important role in PCa progression and thus inhibition of YKL40 may be a potential therapeutic strategy for the treatment of PCa. PMID:24981110

Elevated YKL40 is associated with advanced prostate cancer (PCa) and positively regulates invasion and migration of PCa cells.

PubMed

Jeet, Varinder; Tevz, Gregor; Lehman, Melanie; Hollier, Brett; Nelson, Colleen

2014-10-01

Chitinase 3-like 1 (CHI3L1 or YKL40) is a secreted glycoprotein highly expressed in tumours from patients with advanced stage cancers, including prostate cancer (PCa). The exact function of YKL40 is poorly understood, but it has been shown to play an important role in promoting tumour angiogenesis and metastasis. The therapeutic value and biological function of YKL40 are unknown in PCa. The objective of this study was to examine the expression and function of YKL40 in PCa. Gene expression analysis demonstrated that YKL40 was highly expressed in metastatic PCa cells when compared with less invasive and normal prostate epithelial cell lines. In addition, the expression was primarily limited to androgen receptor-positive cell lines. Evaluation of YKL40 tissue expression in PCa patients showed a progressive increase in patients with aggressive disease when compared with those with less aggressive cancers and normal controls. Treatment of LNCaP and C4-2B cells with androgens increased YKL40 expression, whereas treatment with an anti-androgen agent decreased the gene expression of YKL40 in androgen-sensitive LNCaP cells. Furthermore, knockdown of YKL40 significantly decreased invasion and migration of PCa cells, whereas overexpression rendered them more invasive and migratory, which was commensurate with an enhancement in the anchorage-independent growth of cells. To our knowledge, this study characterises the role of YKL40 for the first time in PCa. Together, these results suggest that YKL40 plays an important role in PCa progression and thus inhibition of YKL40 may be a potential therapeutic strategy for the treatment of PCa. © 2014 The authors.

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.

Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

PubMed Central

Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, Luísa

2012-01-01

Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements. PMID:22530027

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

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.

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

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

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.

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

Species-specific differences in peroxisome proliferation, catalase, and SOD2 upregulation as well as toxicity in human, mouse, and rat hepatoma cells induced by the explosive and environmental pollutant 2,4,6-trinitrotoluene.

PubMed

Naumenko, Ekaterina Anatolevna; Ahlemeyer, Barbara; Baumgart-Vogt, Eveline

2017-03-01

2,4,6-Trinitrotoluene (TNT) has been widely used as an explosive substance and its toxicity is still of interest as it persisted in polluted areas. TNT is metabolized in hepatocytes which are prone to its toxicity. Since analysis of the human liver or hepatocytes is restricted due to ethical reasons, we investigated the effects of TNT on cell viability, reactive oxygen species (ROS) production, peroxisome proliferation, and antioxidative enzymes in human (HepG2), mouse (Hepa 1-6), and rat (H4IIEC3) hepatoma cell lines. Under control conditions, hepatoma cells of all three species were highly comparable exhibiting identical proliferation rates and distribution of their cell cycle phases. However, we found strong differences in TNT toxicity with the lowest IC 50 values (highest cell death rate) for rat cells, whereas human and mouse cells were three to sevenfold less sensitive. Moreover, a strong decrease in cellular dehydrogenase activity (MTT assay) and increased ROS levels were noted. TNT caused peroxisome proliferation with rat hepatoma cells being most responsive followed by those from mouse and human. Under control conditions, rat cells contained fivefold higher peroxisomal catalase and mitochondrial SOD2 activities and a twofold higher capacity to reduce MTT than human and mouse cells. TNT treatment caused an increase in catalase and SOD2 mRNA and protein levels in human and mouse, but not in rat cells. Similarly, human and mouse cells upregulated SOD2 activity, whereas rat cells failed therein. We conclude that TNT induced oxidative stress, peroxisome proliferation and mitochondrial damage which are highest in rat cells rendering them most susceptible toward TNT. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 989-1006, 2017. © 2016 Wiley Periodicals, Inc.

Potential Cost Savings of Contrast-Enhanced Digital Mammography.

PubMed

Patel, Bhavika K; Gray, Richard J; Pockaj, Barbara A

2017-06-01

The purpose of this article is to discuss whether the sensitivity and specificity of contrast-enhanced digital mammography (CEDM) render it a viable diagnostic alternative to breast MRI. That CEDM couples low-energy images (comparable to the diagnostic quality of standard mammography) and subtracted contrast-enhanced mammograms make it a cost-effective modality and a realistic substitute for the more costly breast MRI.

Steady-State Visual Evoked Potentials and Phase Synchronization in Migraine Patients

NASA Astrophysics Data System (ADS)

Angelini, L.; Tommaso, M. De; Guido, M.; Hu, K.; Ivanov, P. Ch.; Marinazzo, D.; Nardulli, G.; Nitti, L.; Pellicoro, M.; Pierro, C.; Stramaglia, S.

2004-07-01

We investigate phase synchronization in EEG recordings from migraine patients. We use the analytic signal technique, based on the Hilbert transform, and find that migraine brains are characterized by enhanced alpha band phase synchronization in the presence of visual stimuli. Our findings show that migraine patients have an overactive regulatory mechanism that renders them more sensitive to external stimuli.

FOA Information from the Research Institute of Swedish National Defence on Electronic Warfare (FOA Orientar om Electronic Warfare),

DTIC Science & Technology

1967-01-01

probablity ot penetring to target in the preaence of atr do- fance . Curve P, =probebility of destro),ing target in the presence of air defence. Weapon...so rendering the lions. At high frequencies ( gen - quency range and sector of ter- traffic less sensitive to jamming. erally those tsed on radio links

Characterization of Squalene Epoxidase of Saccharomyces cerevisiae by Applying Terbinafine-Sensitive Variants▿

PubMed Central

Ruckenstuhl, Christoph; Lang, Silvia; Poschenel, Andrea; Eidenberger, Armin; Baral, Pravas Kumar; Kohút, Peter; Hapala, Ivan; Gruber, Karl; Turnowsky, Friederike

2007-01-01

Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1-knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G25S, D335X (W, F, P), and G210A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G30S and L37P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R269G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines. PMID:17043127

Methods for imaging individual cilia in living echinoid embryos.

PubMed

Morris, Robert L; Pope, Hans W; Sholi, Adam N; Williams, Leah M; Ettinger, Chelsea R; Beacham, Gwendolyn M; Shintaku, Tatsushi; Abbott, Zachary D; Doherty, Elyse M

2015-01-01

The embryos of echinoids (sea urchins and sand dollars) serve as excellent models for studying cilia differentiation and stages of the cilia life cycle including ciliogenic initiation, growth, maintenance, and retraction. Early in echinoid development, uniform motile cilia form on all cells simultaneously but then rapidly differentiate into multiple cilia types that differ in morphology, motility, and signaling sensitivity. Metal ion treatments that shift germ layer boundaries and thereby "animalize" or "vegetalize" embryos can be used to enrich for low-abundance cilia types rendering those specialized cilia and the differentiation processes they exhibit much easier to study. The experimental advantages of having robust cilia growth and differentiation is tempered by the challenge of restraining ciliated embryos well enough to view the process of ciliogenesis live. We have developed four observation chambers as modifications of the Kiehart chamber for long-term light microscopic imaging of ciliated echinoid embryos. One of these systems employs paramagnetic beads to render ciliated larvae magnetic so they can be gently and reversibly trapped directly under the objective lens. With this magnetic trapping system, the larva can be positioned and repositioned until they achieve the orientation with the clearest view of any cilia of interest. These methods of gentle embryo restraint allow normal embryo development and the normal ciliogenic cycle and ciliary differentiation processes to continue in direct view. Sequential image series can then be collected and analyzed to quantitatively study the wide spectrum of cilia behaviors and properties that arise in developing echinoid embryos. Copyright © 2015. Published by Elsevier Inc.

Subclinical Doses of ATP-Sensitive Potassium Channel Modulators Prevent Alterations in Memory and Synaptic Plasticity Induced by Amyloid-β.

PubMed

Salgado-Puga, Karla; Rodríguez-Colorado, Javier; Prado-Alcalá, Roberto A; Peña-Ortega, Fernando

2017-01-01

In addition to coupling cell metabolism and excitability, ATP-sensitive potassium channels (KATP) are involved in neural function and plasticity. Moreover, alterations in KATP activity and expression have been observed in Alzheimer's disease (AD) and during amyloid-β (Aβ)-induced pathology. Thus, we tested whether KATP modulators can influence Aβ-induced deleterious effects on memory, hippocampal network function, and plasticity. We found that treating animals with subclinical doses (those that did not change glycemia) of a KATP blocker (Tolbutamide) or a KATP opener (Diazoxide) differentially restrained Aβ-induced memory deficit, hippocampal network activity inhibition, and long-term synaptic plasticity unbalance (i.e., inhibition of LTP and promotion of LTD). We found that the protective effect of Tolbutamide against Aβ-induced memory deficit was strong and correlated with the reestablishment of synaptic plasticity balance, whereas Diazoxide treatment produced a mild protection against Aβ-induced memory deficit, which was not related to a complete reestablishment of synaptic plasticity balance. Interestingly, treatment with both KATP modulators renders the hippocampus resistant to Aβ-induced inhibition of hippocampal network activity. These findings indicate that KATP are involved in Aβ-induced pathology and they heighten the potential role of KATP modulation as a plausible therapeutic strategy against AD.

Dietary modulation and structure prediction of rat mucosal pentraxin (Mptx) protein and loss of function in humans

PubMed Central

van der Meer-van Kraaij, Cindy; Siezen, Roland; Kramer, Evelien; Reinders, Marjolein; Blokzijl, Hans; van der Meer, Roelof

2007-01-01

Mucosal pentraxin (Mptx), identified in rats, is a short pentraxin of unknown function. Other subfamily members are Serum amyloid P component (SAP), C-reactive protein (CRP) and Jeltraxin. Rat Mptx mRNA is predominantly expressed in colon and in vivo is strongly (30-fold) regulated by dietary heme and calcium, modulators of colon cancer risk. This renders Mptx a potential nutrient sensitive biomarker of gut health. To support a role as biomarker, we examined whether the pentraxin protein structure is conserved, whether Mptx protein is nutrient-sensitively expressed and whether Mptx is expressed in mouse and human. Sequence comparison and 3D modelling showed that rat Mptx is highly homologous to the other pentraxins. The calcium-binding site and subunit interaction sites are highly conserved, while a loop deletion and charged residues contribute to a distinctive “top” face of the pentamer. In accordance with mRNA expression, Mptx protein is strongly down-regulated in rat colon mucosa in response to high dietary heme intake. Mptx mRNA is expressed in rat and mouse colon, but not in human colon. A stop codon at the beginning of human exon two indicates loss of function, which may be related to differences in intestinal cell turnover between man and rodents. PMID:18850182

Photoinduced Oxidative DNA Damage Revealed by an Agarose Gel Nicking Assay: A Biophysical Chemistry Laboratory Experiment

NASA Astrophysics Data System (ADS)

Shafirovich, Vladimir; Singh, Carolyn; Geacintov, Nicholas E.

2003-11-01

Oxidative damage of DNA molecules associated with electron-transfer reactions is an important phenomenon in living cells, which can lead to mutations and contribute to carcinogenesis and the aging processes. This article describes the design of several simple experiments to explore DNA damage initiated by photoinduced electron-transfer reactions sensitized by the acridine derivative, proflavine (PF). A supercoiled DNA agarose gel nicking assay is employed as a sensitive probe of DNA strand cleavage. A low-cost experimental and computer-interfaced imaging apparatus is described allowing for the digital recording and analysis of the gel electrophoresis results. The first experiment describes the formation of direct strand breaks in double-stranded DNA induced by photoexcitation of the intercalated PF molecules. The second experiment demonstrates that the addition of the well-known electron acceptor, methylviologen, gives rise to a significant enhancement of the photochemical DNA strand cleavage effect. This occurs by an electron transfer step to methylviologen that renders the inital photoinduced charge separation between photoexcited PF and DNA irreversible. The third experiment demonstrates that the action spectrum of the DNA photocleavage matches the absorption spectrum of DNA-bound, intercalated PF molecules, which differs from that of free PF molecules. This result demonstrates that the photoinduced DNA strand cleavage is initiated by intercalated rather than free PF molecules.

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

Interferon response factor 3 is essential for house dust mite-induced airway allergy.

PubMed

Marichal, Thomas; Bedoret, Denis; Mesnil, Claire; Pichavant, Muriel; Goriely, Stanislas; Trottein, François; Cataldo, Didier; Goldman, Michel; Lekeux, Pierre; Bureau, Fabrice; Desmet, Christophe J

2010-10-01

Pattern-recognition receptors (PRRs) are critically involved in the pathophysiology of airway allergy, yet most of the signaling pathways downstream of PRRs implicated in allergic airway sensitization remain unknown. We sought to study the effects of genetic depletion of interferon response factor (IRF) 3 and IRF7, important transcription factors downstream of various PRRs, in a murine model of house dust mite (HDM)-induced allergic asthma. We compared HDM-induced allergic immune responses in IRF3-deficient (IRF3(-/-)), IRF7(-/-), and wild-type mice. Parameters of airway allergy caused by HDM exposure were strongly attenuated in IRF3(-/-), but not IRF7(-/-), mice compared with those in wild-type mice. Indeed, in HDM-exposed IRF3(-/-) mice HDM-specific T(H)2 cell responses did not develop. This correlated with impaired maturation and migration of IRF3(-/-) lung dendritic cells (DCs) on HDM treatment. Furthermore, adoptive transfer of HDM-loaded DCs indicated that IRF3(-/-) DCs had an intrinsic defect rendering them unable to migrate and to prime HDM-specific T(H)2 responses. Intriguingly, we also show that DC function and allergic airway sensitization in response to HDM were independent of signaling by type I interferons, the main target genes of IRF3. Through its role in DC function, IRF3, mainly known as a central activator of antiviral immunity, is essential for the development of T(H)2-type responses to airway allergens. Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

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

Effect of light intensity on the degree of ammonia toxicity on PSII activity of Arthrospira platensis and Chlorella vulgaris.

PubMed

Markou, Giorgos; Muylaert, Koenraad

2016-09-01

Herein the effect of increasing light intensity on the degree of ammonia toxicity and its impact on the photosynthetic performance of Arthrospira and Chlorella was investigated using Chl fluorescence as a technique to characterize their photosystem II (PSII) activity. The results revealed that the increase of light intensity amplifies the ammonia toxicity on PSII. Chl fluorescence transients shown that at a given free ammonia (FA) concentration (100mg-N/L), the photochemistry potential decreased by increasing light intensity. The inhibition of the PSII was not reversible either by re-incubating the cells under dark or under decreased FA concentration. Moreover, the decrease of photochemical and non-photochemical quenching (NPQ) of fluorescence suggest that ammonia toxicity decreases the open available PSII centers, as well the inability of PSII to transfer the generated electrons beyond QA. The collapse of NPQ suggests that ammonia toxicity inhibits the photoprotection mechanism(s) and hence renders PSII more sensitive to photoinhibition. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

An enhanced mangiferaindica for dye sensitized solar cell application

NASA Astrophysics Data System (ADS)

Uno, U. E.; Emetere, M. E.; Fadipe, L. A.; Oluranti, Jonathan

2016-02-01

Titanium dioxide (T1O2) is preferred to Zinc oxide as mesoporous oxide layer because it raised the efficiency of DSSCs from 1% to 7%. The chemistry of the process however seem rigorous to allow the light induced electron injection from the adsorbed dye into the nanocrystallites i.e. which renders the TiO2 conductive. The DSSC fabricated consist of 2.25 cm2 active area of titanium dioxide coated on FTO glass (fluorine tin oxide) immersed in ethanol solution of natural dye extracted as an anode (electrode) and counter electrode. These two electrodes were coupled together and the space between them was filled with the Iodolyte AN-50 as solid electrolyte or redox mediator. The photo electrochemical parameters of the dye extracted (Mango fruit Peel) from the results obtained are short circuit current (Isc)= 1.22×10-2, current density (Jsc)=4.07×10-2, open circuit voltage (voc) =0.53V, fill factor (FF) of 0.16 and the overall conversion efficiency (Eff) =0.345%.

Methylfolate Trap Promotes Bacterial Thymineless Death by Sulfa Drugs

PubMed Central

Pham, Thanh H.; Jakubowski, Hieronim; Wolff, Kerstin A.; Ogwang, Sam; Timpona, Joseph L.; Gogula, Soumya; Jacobs, Michael R.; Ruetz, Markus; Kräutler, Bernhard; Jacobsen, Donald W.; Zhang, Guo-Fang; Nguyen, Liem

2016-01-01

The methylfolate trap, a metabolic blockage associated with anemia, neural tube defects, Alzheimer’s dementia, cardiovascular diseases, and cancer, was discovered in the 1960s, linking the metabolism of folate, vitamin B12, methionine and homocysteine. However, the existence or physiological significance of this phenomenon has been unknown in bacteria, which synthesize folate de novo. Here we identify the methylfolate trap as a novel determinant of the bacterial intrinsic death by sulfonamides, antibiotics that block de novo folate synthesis. Genetic mutagenesis, chemical complementation, and metabolomic profiling revealed trap-mediated metabolic imbalances, which induced thymineless death, a phenomenon in which rapidly growing cells succumb to thymine starvation. Restriction of B12 bioavailability, required for preventing trap formation, using an “antivitamin B12” molecule, sensitized intracellular bacteria to sulfonamides. Since boosting the bactericidal activity of sulfonamides through methylfolate trap induction can be achieved in Gram-negative bacteria and mycobacteria, it represents a novel strategy to render these pathogens more susceptible to existing sulfonamides. PMID:27760199

Evaluation of a hyperspectral image database for demosaicking purposes

NASA Astrophysics Data System (ADS)

Larabi, Mohamed-Chaker; Süsstrunk, Sabine

2011-01-01

We present a study on the the applicability of hyperspectral images to evaluate color filter array (CFA) design and the performance of demosaicking algorithms. The aim is to simulate a typical digital still camera processing pipe-line and to compare two different scenarios: evaluate the performance of demosaicking algorithms applied to raw camera RGB values before color rendering to sRGB, and evaluate the performance of demosaicking algorithms applied on the final sRGB color rendered image. The second scenario is the most frequently used one in literature because CFA design and algorithms are usually tested on a set of existing images that are already rendered, such as the Kodak Photo CD set containing the well-known lighthouse image. We simulate the camera processing pipe-line with measured spectral sensitivity functions of a real camera. Modeling a Bayer CFA, we select three linear demosaicking techniques in order to perform the tests. The evaluation is done using CMSE, CPSNR, s-CIELAB and MSSIM metrics to compare demosaicking results. We find that the performance, and especially the difference between demosaicking algorithms, is indeed significant depending if the mosaicking/demosaicking is applied to camera raw values as opposed to already rendered sRGB images. We argue that evaluating the former gives a better indication how a CFA/demosaicking combination will work in practice, and that it is in the interest of the community to create a hyperspectral image dataset dedicated to that effect.

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

Finding the bottom and using it

PubMed Central

Sandoval, Ruben M.; Wang, Exing; Molitoris, Bruce A.

2014-01-01

Maximizing 2-photon parameters used in acquiring images for quantitative intravital microscopy, especially when high sensitivity is required, remains an open area of investigation. Here we present data on correctly setting the black level of the photomultiplier tube amplifier by adjusting the offset to allow for accurate quantitation of low intensity processes. When the black level is set too high some low intensity pixel values become zero and a nonlinear degradation in sensitivity occurs rendering otherwise quantifiable low intensity values virtually undetectable. Initial studies using a series of increasing offsets for a sequence of concentrations of fluorescent albumin in vitro revealed a loss of sensitivity for higher offsets at lower albumin concentrations. A similar decrease in sensitivity, and therefore the ability to correctly determine the glomerular permeability coefficient of albumin, occurred in vivo at higher offset. Finding the offset that yields accurate and linear data are essential for quantitative analysis when high sensitivity is required. PMID:25313346

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

High sensitive vectorial B-probe for low frequency plasma waves.

PubMed

Ullrich, Stefan; Grulke, Olaf; Klinger, Thomas; Rahbarnia, Kian

2013-11-01

A miniaturized multidimensional magnetic probe is developed for application in a low-temperature plasma environment. A very high sensitivity for low-frequency magnetic field fluctuations with constant phase run, a very good signal-to-noise ratio combined with an efficient electrostatic pickup rejection, renders the probe superior compared with any commercial solution. A two-step calibration allows for absolute measurement of amplitude and direction of magnetic field fluctuations. The excellent probe performance is demonstrated by measurements of the parallel current pattern of coherent electrostatic drift wave modes in the VINETA (versatile instrument for studies on nonlinearity, electromagnetism, turbulence, and applications) experiment.

Rationalizing the suitability of rhodamines as chromophores in dye-sensitized solar cells: A systematic molecular design study

DOE PAGES

Pepe, Giulio; Cole, Jacqueline M.; Waddell, Paul G.; ...

2016-09-30

Here, rhodamines are chromophores that are employed in many dye applications. Their strong optical absorption in the visible region of the electromagnetic spectrum renders them attractive dye candidates for dye-sensitized solar cells (DSCs). However, they have not yet been systematically tested in DSCs as single- or co-sensitizers. Recent advances in concerted experimental and computational workflows involving molecular design protocols can afford a better understanding of the molecular origins of the optoelectronic properties in these sensitizers. Herein, we examine the suitability of rhodamines R560 (1), R575 (2), R590 (3), R610 (4), R620 (5), R640 (6), and R3B (7) as chromophores inmore » co-sensitized DSCs. Our study follows a stepwise approach. Initially, structural and optical properties of the dyes are investigated by experimental and computational methods to reveal structure-property relationships and other useful features for DSC applications. Subsequently, 1-7 are investigated at the dye···TiO 2 interface, both by calculations of dye-adsorption onto the surface of a modeled (TiO 2) 9 cluster, and by experimental studies of dye-adsorption on TiO 2. For that purpose, a selection of rhodamine dyes are paired together (1 and 5) and (1 and 7) for co-sensitization, among which 5 is also co-adsorbed with a fluorescein dye in order to explore chemical compatibility factors. The best dye candidates are identified from the findings of these adsorption studies in terms of dye aggregation, anchoring modes, and panchromatic response. Despite their promising dye…TiO 2 adsorption and optical prospects, our results show that rhodamines lack a suitable intramolecular charge transfer pathway for dye-to-TiO 2 electron injection to occur, thus precluding their photovoltaic function as DSC dyes. Our results are then assessed against ostensibly disparate reports of rhodamines performing successfully in DSC devices; this comparison necessitated the internal reproduction of previously reported co-sensitization experiments on 2 with the industrial standard reference dye, N3. We achieve reconciliation between our results and those in the literature by reasoning that while rhodamines cannot deliver photovoltaic function in DSCs in their own right, they can either act to facilitate or deplete the photovoltaic output of a DSC indirectly by affecting the TiO 2 adsorption prospects of a photovoltaic-active dye with which a rhodamine is co-sensitized.« less

DDS, 4,4′-diaminodiphenylsulfone, extends organismic lifespan

PubMed Central

Keam, Bhumsuk; Choi, Jung Min; Cho, Yunje; Hyun, Soonsil; Park, Sang Chul; Lee, Junho

2010-01-01

DDS, 4,4′-diaminodiphenylsulfone, is the most common drug prescribed to treat Hansen disease patients. In addition to its antibacterial activity, DDS has been reported to be involved in other cellular processes that occur in eukaryotic cells. Because DDS treatment significantly enhances the antioxidant activity in humans, we examined its effect on lifespan extension. Here we show that DDS extends organismic lifespan using Caenorhabditis elegans as a model system. DDS treatment caused a delay in aging and decreased the levels of a mitochondrial complex. The oxygen consumption rate was also significantly lowered. Consistent with these data, paraquat treatment evoked less reactive oxygen species in DDS-treated worms, and these worms were less sensitive to paraquat. Interestingly enough, all of the molecular events caused by DDS treatment were consistently reproduced in mice treated with DDS for 3 mo and in the C2C12 muscle cell line. Structural prediction identified pyruvate kinase (PK) as a protein target of DDS. Indeed, DDS bound and inhibited PK in vitro and inhibited it in vivo, and a PK mutation conferred extended lifespan of C. elegans. Supplement of pyruvate to the media protected C2C12 cells from apoptosis caused by paraquat. Our findings establish the significance of DDS in lowering reactive oxygen species generation and extending the lifespan, which renders the rationale to examining the possible effect of DDS on human lifespan extension. PMID:20974969

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.

CHX14 is a plasma membrane K-efflux transporter that regulates K(+) redistribution in Arabidopsis thaliana.

PubMed

Zhao, Jian; Li, Penghui; Motes, Christy M; Park, Sunghun; Hirschi, Kendal D

2015-11-01

Potassium (K(+) ) is essential for plant growth and development, yet the molecular identity of many K(+) transporters remains elusive. Here we characterized cation/H(+) exchanger (CHX) 14 as a plasma membrane K(+) transporter. CHX14 expression was induced by elevated K(+) and histochemical analysis of CHX14 promoter::GUS transgenic plants indicated that CHX14 was expressed in xylem parenchyma of root and shoot vascular tissues of seedlings. CHX14 knockout (chx14) and CHX14 overexpression seedlings displayed different growth phenotypes during K(+) stress as compared with wild-type seedlings. Roots of mutant seedlings displayed higher K(+) uptake rates than wild-type roots. CHX14 expression in yeast cells deficient in K(+) uptake renders the mutant cells more sensitive to deficiencies of K(+) in the medium. CHX14 mediates K(+) efflux in yeast cells loaded with high K(+) . Uptake experiments using (86) Rb(+) as a tracer for K(+) with both yeast and plant mutants demonstrated that CHX14 expression in yeast and in planta mediated low-affinity K(+) efflux. Functional green fluorescent protein (GFP)-tagged versions of CHX14 were localized to both the yeast and plant plasma membranes. Taken together, we suggest that CHX14 is a plasma membrane K(+) efflux transporter involved in K(+) homeostasis and K(+) recirculation. © 2015 John Wiley & Sons Ltd.

Ganglioside GT1b protects human spermatozoa from hydrogen peroxide-induced DNA and membrane damage.

PubMed

Gavella, Mirjana; Garaj-Vrhovac, Verica; Lipovac, Vaskresenija; Antica, Mariastefania; Gajski, Goran; Car, Nikica

2010-06-01

We have reported previously that various gangliosides, the sialic acid containing glycosphingolipids, provide protection against sperm injury caused by reactive oxygen species (ROS). In this study, we investigated the effect of treatment of human spermatozoa with ganglioside GT1b on hydrogen peroxide (H(2)O(2))-induced DNA fragmentation and plasma membrane damage. Single-cell gel electrophoresis (Comet assay) used in the assessment of sperm DNA integrity showed that in vitro supplemented GT1b (100 microm) significantly reduced DNA damage induced by H(2)O(2) (200 microm) (p < 0.05). Measurements of Annexin V binding in combination with the propidium iodide vital dye labelling demonstrated that the spermatozoa pre-treated with GT1b exhibited a significant increase (p < 0.05) in the percentage of live cells with intact membrane and decreased phosphatidylserine translocation after exposure to H(2)O(2). Flow cytometry using the intracellular ROS-sensitive fluorescence dichlorodihydrofluorescein diacetate dye employed to investigate the transport of the extracellularly supplied H(2)O(2) into the cell interior revealed that ganglioside GT1b completely inhibited the passage of H(2)O(2) through the sperm membrane. These results suggest that ganglioside GT1b may protect human spermatozoa from H(2)O(2)-induced damage by rendering sperm membrane more hydrophobic, thus inhibiting the diffusion of H(2)O(2) across the membrane.

AN EXPERIMENTAL ANALYSIS OF BACTERIAL ALLERGY

PubMed Central

Zinsser, Hans; Tamiya, Takeo

1926-01-01

Our experiments have confirmed the fact that the so called bacterial allergies are dependent upon a mechanism which differs materially from that determining true protein anaphylaxis. Anaphylaxis to protein substances of the bacteria probably occurs but plays a relatively unimportant rôle in the phenomena of infection. The bacterial allergies, however, are of great importance since they develop rapidly and render the infected animal highly vulnerable to products of the bacterial growth which are relatively innocuous for the normal animal. Neither the type-specific carbohydrate "residue antigens" (the "soluble specific substances" of Avery and Heidelberger) nor the antibodies reacting with them play any part whatever in bacterial allergy, and since these type-specific substances represent the haptophore groups of the whole bacteria by which they react with the agglutinins, precipitins, sensitizers, etc., of immune serum, allergy, as previously determined by Mackenzie and Woo, is in no way related to that phase of resistance which is determined by these antibodies. This does not, however, preclude the possibility that allergic hypersusceptibility may not in some way be related to other factors of resistance more definitely associated with cellular rather than with intravascular reactions. Our previous studies with Jennings and Ward in tuberculosis point in this direction (20). Guinea pigs can be actively sensitized with all the bacteria with which we have worked when repeated injections of whole bacteria or of the protein (nucleoprotein) fraction are administered. Large amounts of the latter are necessary since these materials are indifferent antigens, possibly because of the severe manipulations necessary in their production. Sensitiveness develops usually within 10 days after the first dose and increases with continued treatment for 3 or 4 weeks. Sensitiveness is relatively specific, by which we mean that there is a definite specificity which, however, in highly sensitive animals is not absolute and shows considerable overlapping. Continued treatment with considerable quantities of the above substances leads to gradual desensitization in animals in which there are no chronic foci present, which, as in tuberculosis, tends to continue the sensitization. Attempts at passive sensitization have been irregular and inconclusive. When any degree of sensitiveness has developed after the injection of immune sera, it has appeared late and has been of doubtful specificity. Conversely we have failed in any case to neutralize the activity of the active allergic constituents of bacterial extracts by incubation with any type of immune serum. We have failed so far to show any increased fixation of tuberculin material on the part of tuberculous tissues or on that of living tuberculous animals. These failures, however, seem to us of relatively slight importance since quantitative experiments of this nature are extremely difficult in the case of a substance as delicately potent for the tuberculous animal. On the other hand we have obtained definite, though irregular evidence that the incubation of O.T. with fragments of tuberculous lung tissue (less clearly with other tissues) leads to the formation of a substance that produces allergy-like lesions in the skin of normal guinea pigs. With somewhat greater regularity, similar treatment of O.T. has enhanced the potency of the tuberculin for tuberculous animals. And, in these experiments there was evidence that the factor responsible for this action was not easily separable from the cells themselves. When these experimental data are analytically considered they appear in many respects confusing and contradictory. There has been so much work done on the tuberculin reaction, moreover, that, in the face of experimental inconsistencies it would seem foolhardy to formulate more than tentative suggestions to explain the mechanism of these reactions. Nevertheless there are a few outstanding and sufficiently reliable facts which compel a limited number of definite deductions. In the first place there is no question of the complete independence of the true allergic phenomena from the ordinary bacterial antigen-antibody reactions. We know, moreover, that the allergic substance is chemically separable from the carbohydrate "residue" or haptophore group of the bacteria (Mueller, Laidlaw and Dudley). Indeed it has been shown by Long and Seibert (21) that the active allergic substance is either a protein in itself, or at any rate closely associated with the bacterial protein. Furthermore, the distinct, though limited, specificity of the allergic sensitiveness compels the conclusion that we are dealing with an immunological process in which the tissue cells acquire an increased specific capacity to react with this nitrogenous material, a capacity which, in principle, is not far removed from the supposed "sessile receptor" apparatus which is conventionally held responsible for protein anaphylaxis; and this analogy is further amplified by the apparent desensitization which continued treatment produced in many of our own experiments as well as in those of Mackenzie and Woo. Here, however, the analogy with protein anaphylaxis ends. Passive sensitization with any form of immune serum or with the sera of highly sensitized animals is either feeble or entirely unsuccessful and indicates quite convincingly that, whatever the receptor apparatus of the cells may be, it is not easily given up to the blood stream as are ordinary antibodies. Further than this, our tissue-tuberculin experiments, irregular and occasional as they were, nevertheless convinced us that: 1. The contact with the tissues of tuberculous animals results in the production of a toxic factor, not unlike the autolytic toxic materials of some bacteria. 2. The active cell constituent by which this action is wrought, is not easily separated from the cells, even by energetic methods of extraction. This close association of the entire process with the cells themselves is particularly significant in view of the obvious cell injury in which these delayed allergic effects differ from the ordinary urticarial, evanescent reactions associated with protein anaphylaxis. The process of allergy, as far as we can approach it then, may be conceived as follows: A nitrogenous, probably protein, constituent of the bacterial growth or of its body substance stimulates a specific reaction in the tissue cell by which its specific capacity to establish contact with this constituent is enhanced. The cell is thereby enabled to exert a, probably, enzyme-like effect upon this material in consequence of which a toxic substance is liberated, largely upon or possibly within the cell itself. Both processes may be dependent upon one and the same reaction body. But it seems more likely that increased contact and the increased cell activity are separately developed, an assumption which is rendered probable by the association of the highest degrees of allergy with inflammatory cell reactions, and by the fact that moderate and less specific allergic sensitiveness follows 10 or more days after the administration of considerable amounts of indifferent protein substances to guinea pigs. We interpret this as signifying that such injections may non-speciffcally increase cellular activity, a change which many earlier workers have spoken of as "cell irritability." Both processes are closely associated with the altered cell itself and the factors by which the reaction is brought about are not easily given up to the blood stream as are the antibodies formed in response to injections of proteins or whole bacteria. We are confronted, therefore, with an immunological mechanism which has some close analogies to those others in which circulating antibodies are formed, but which differs from these mainly in the intimacy with which the entire reacting system is associated with the cells themselves. It is difficult to conceive that a functional cell alteration, as profound as this, should be entirely unrelated to the phenomena of susceptibility or resistance. PMID:19869221

Superresolution with the focused plenoptic camera

NASA Astrophysics Data System (ADS)

Georgiev, Todor; Chunev, Georgi; Lumsdaine, Andrew

2011-03-01

Digital images from a CCD or CMOS sensor with a color filter array must undergo a demosaicing process to combine the separate color samples into a single color image. This interpolation process can interfere with the subsequent superresolution process. Plenoptic superresolution, which relies on precise sub-pixel sampling across captured microimages, is particularly sensitive to such resampling of the raw data. In this paper we present an approach for superresolving plenoptic images that takes place at the time of demosaicing the raw color image data. Our approach exploits the interleaving provided by typical color filter arrays (e.g., Bayer filter) to further refine plenoptic sub-pixel sampling. Our rendering algorithm treats the color channels in a plenoptic image separately, which improves final superresolution by a factor of two. With appropriate plenoptic capture we show the theoretical possibility for rendering final images at full sensor resolution.

Mapping Ribonucleotides Incorporated into DNA by Hydrolytic End-Sequencing.

PubMed

Orebaugh, Clinton D; Lujan, Scott A; Burkholder, Adam B; Clausen, Anders R; Kunkel, Thomas A

2018-01-01

Ribonucleotides embedded within DNA render the DNA sensitive to the formation of single-stranded breaks under alkali conditions. Here, we describe a next-generation sequencing method called hydrolytic end sequencing (HydEn-seq) to map ribonucleotides inserted into the genome of Saccharomyce cerevisiae strains deficient in ribonucleotide excision repair. We use this method to map several genomic features in wild-type and replicase variant yeast strains.

The biodistribution of gold nanoparticles designed for renal clearance

NASA Astrophysics Data System (ADS)

Alric, Christophe; Miladi, Imen; Kryza, David; Taleb, Jacqueline; Lux, François; Bazzi, Rana; Billotey, Claire; Janier, Marc; Perriat, Pascal; Roux, Stéphane; Tillement, Olivier

2013-06-01

Owing to their tunable optical properties and their high absorption cross-section of X- and γ-ray, gold nanostructures appear as promising agents for remotely controlled therapy. Since the efficiency of cancer therapy is not limited to the eradication of the tumour but rests also on the sparing of healthy tissue, a biodistribution study is required in order to determine whether the behaviour of the nanoparticles after intravenous injection is safe (no accumulation in healthy tissue, no uptake by phagocytic cell-rich organs (liver, spleen) and renal clearance). The biodistribution of Au@DTDTPA nanoparticles which are composed of a gold core and a DTDTPA (dithiolated polyaminocarboxylate) shell can be established by X-ray imaging (owing to the X-ray absorption of the gold core) and by magnetic resonance imaging (MRI) since the DTDTPA shell was designed for the immobilization of paramagnetic gadolinium ions. However scintigraphy appears better suited for a biodistribution study owing to a great sensitivity. The successful immobilization of radioelements (99mTc, 111In) in the DTDTPA shell, instead of gadolinium ions, renders possible the follow up of Au@DTDTPA by scintigraphy which showed that Au@DTDTPA nanoparticles exhibit a safe behaviour after intravenous injection to healthy rats.Owing to their tunable optical properties and their high absorption cross-section of X- and γ-ray, gold nanostructures appear as promising agents for remotely controlled therapy. Since the efficiency of cancer therapy is not limited to the eradication of the tumour but rests also on the sparing of healthy tissue, a biodistribution study is required in order to determine whether the behaviour of the nanoparticles after intravenous injection is safe (no accumulation in healthy tissue, no uptake by phagocytic cell-rich organs (liver, spleen) and renal clearance). The biodistribution of Au@DTDTPA nanoparticles which are composed of a gold core and a DTDTPA (dithiolated polyaminocarboxylate) shell can be established by X-ray imaging (owing to the X-ray absorption of the gold core) and by magnetic resonance imaging (MRI) since the DTDTPA shell was designed for the immobilization of paramagnetic gadolinium ions. However scintigraphy appears better suited for a biodistribution study owing to a great sensitivity. The successful immobilization of radioelements (99mTc, 111In) in the DTDTPA shell, instead of gadolinium ions, renders possible the follow up of Au@DTDTPA by scintigraphy which showed that Au@DTDTPA nanoparticles exhibit a safe behaviour after intravenous injection to healthy rats. Electronic supplementary information (ESI) available: Planar scintigraphy image. See DOI: 10.1039/c3nr00012e

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

PubMed

Gupta, Amita; Shrivastava, Nimisha; Grover, Payal; Singh, Ajay; Mathur, Kapil; Verma, Vaishali; Kaur, Charanpreet; Chaudhary, Vijay K

2013-01-01

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

An Improved Ivermectin-activated Chloride Channel Receptor for Inhibiting Electrical Activity in Defined Neuronal Populations*

PubMed Central

Lynagh, Timothy; Lynch, Joseph W.

2010-01-01

The ability to silence the electrical activity of defined neuronal populations in vivo is dramatically advancing our understanding of brain function. This technology may eventually be useful clinically for treating a variety of neuropathological disorders caused by excessive neuronal activity. Several neuronal silencing methods have been developed, with the bacterial light-activated halorhodopsin and the invertebrate allatostatin-activated G protein-coupled receptor proving the most successful to date. However, both techniques may be difficult to implement clinically due to their requirement for surgically implanted stimulus delivery methods and their use of nonhuman receptors. A third silencing method, an invertebrate glutamate-gated chloride channel receptor (GluClR) activated by ivermectin, solves the stimulus delivery problem as ivermectin is a safe, well tolerated drug that reaches the brain following systemic administration. However, the limitations of this method include poor functional expression, possibly due to the requirement to coexpress two different subunits in individual neurons, and the nonhuman origin of GluClR. Here, we describe the development of a modified human α1 glycine receptor as an improved ivermectin-gated silencing receptor. The crucial development was the identification of a mutation, A288G, which increased ivermectin sensitivity almost 100-fold, rendering it similar to that of GluClR. Glycine sensitivity was eliminated via the F207A mutation. Its large unitary conductance, homomeric expression, and human origin may render the F207A/A288G α1 glycine receptor an improved silencing receptor for neuroscientific and clinical purposes. As all known highly ivermectin-sensitive GluClRs contain an endogenous glycine residue at the corresponding location, this residue appears essential for exquisite ivermectin sensitivity. PMID:20308070

Maternal Sensitivity Predicts Fewer Sleep Problems at Early Adolescence for Toddlers with Negative Emotionality: A Case of Differential Susceptibility.

PubMed

Conway, Anne; Modrek, Anahid; Gorroochurn, Prakash

2018-02-01

Theory underscores the importance of parenting in sleep development, but few studies have examined whether links vary by temperament. To address this gap, we tested whether potential links between early maternal sensitivity and early adolescent sleep problems varied by child negative emotionality and delay of gratification. Using data from the National Institute of Child Health and Human Development Study of Early Child Care and Youth Development (N = 820), we found that high maternal sensitivity predicted fewer bedtime problems and longer sleep duration at 6th grade for toddlers with high negative emotionality, whereas low maternal sensitivity predicted the reverse. No differences were observed for low negative emotionality. Moreover, delay of gratification predicted fewer bedtime problems at 6th grade, but did not moderate associations between maternal sensitivity, negative emotionality, and sleep. Findings demonstrate that high, but not low, negative emotionality renders toddlers differentially susceptible and receptive to maternal sensitivity in relation to sleep.

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.

Intercellular Calcium Waves in HeLa Cells Expressing GFP-labeled Connexin 43, 32, or 26

PubMed Central

Paemeleire, Koen; Martin, Patricia E. M.; Coleman, Sharon L.; Fogarty, Kevin E.; Carrington, Walter A.; Leybaert, Luc; Tuft, Richard A.; Evans, W. Howard; Sanderson, Michael J.

2000-01-01

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca2+ waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca2+]i associated with Ca2+ waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca2+-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca2+ waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca2+]i changes were characterized by initiating Ca2+ puffs associated with the perinuclear ER. By contrast, in Cx–GFP-transfected cells and in the presence of apyrase, [Ca2+]i changes were propagated without initiating perinuclear Ca2+ puffs and were communicated between cells at the sites of the Cx–GFP gap junctions. The efficiency of Cx expression determined the extent of Ca2+ wave propagation. These results demonstrate that intercellular Ca2+ waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other. PMID:10793154

AMP-activated protein kinase couples 3-bromopyruvate-induced energy depletion to apoptosis via activation of FoxO3a and upregulation of proapoptotic Bcl-2 proteins.

PubMed

Bodur, Cagri; Karakas, Bahriye; Timucin, Ahmet Can; Tezil, Tugsan; Basaga, Huveyda

2016-11-01

Most tumors primarily rely on glycolysis rather than mitochondrial respiration for ATP production. This phenomenon, also known as Warburg effect, renders tumors more sensitive to glycolytic disturbances compared to normal cells. 3-bromopyruvate is a potent inhibitor of glycolysis that shows promise as an anticancer drug candidate. Although investigations revealed that 3-BP triggers apoptosis through ATP depletion and subsequent AMPK activation, the underlying molecular mechanisms coupling AMPK to apoptosis are poorly understood. We showed that 3-BP leads to a rapid ATP depletion which was followed by growth inhibition and Bax-dependent apoptosis in HCT116 cells. Apoptosis was accompanied with activation of caspase-9 and -3 while pretreatment with a general caspase inhibitor attenuated cell death. AMPK, p38, JNK, and Akt were phosphorylated immediately upon treatment. Pharmacological inhibition and silencing of AMPK largely inhibited 3-BP-induced apoptosis and reversed phosphorylation of JNK. Transcriptional activity of FoxO3a was dramatically increased subsequent to AMPK-mediated phosphorylation of FoxO3a at Ser413. Cell death analysis of cells transiently transfected with wt or AMPK-phosphorylation-deficient FoxO3 expression plasmids verified the contributory role of AMPK-FoxO3a axis in 3-BP-induced apoptosis. In addition, expression of proapoptotic Bcl-2 proteins Bim and Bax were upregulated in an AMPK-dependent manner. Bim was transcriptionally activated in association with FoxO3a activity, while Bax upregulation was abolished in p53-null cells. Together, these data suggest that AMPK couples 3-BP-induced metabolic disruption to intrinsic apoptosis via modulation of FoxO3a-Bim axis and Bax expression. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Role of transcription factor KLF11 and its diabetes-associated gene variants in pancreatic beta cell function

PubMed Central

Neve, Bernadette; Fernandez-Zapico, Martin E.; Ashkenazi-Katalan, Vered; Dina, Christian; Hamid, Yasmin H.; Joly, Erik; Vaillant, Emmanuel; Benmezroua, Yamina; Durand, Emmanuelle; Bakaher, Nicolas; Delannoy, Valerie; Vaxillaire, Martine; Cook, Tiffany; Dallinga-Thie, Geesje M.; Jansen, Hans; Charles, Marie-Aline; Clément, Karine; Galan, Pilar; Hercberg, Serge; Helbecque, Nicole; Charpentier, Guillaume; Prentki, Marc; Hansen, Torben; Pedersen, Oluf; Urrutia, Raul; Melloul, Danielle; Froguel, Philippe

2005-01-01

KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first time, to our knowledge, the characterization of KLF11 as a glucose-inducible regulator of the insulin gene. A combination of random oligonucleotide binding, EMSA, luciferase reporter, and chromatin immunoprecipitation assays shows that KLF11 binds to the insulin promoter and regulates its activity in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1,776 normoglycemic subjects show a frequent polymorphic Gln62Arg variant that significantly associates with type 2 diabetes mellitus in North European populations (OR = 1.29, P = 0.00033). Moreover, this variant alters the corepressor mSin3A-binding activity of KLF11, impairs the activation of the insulin promoter and shows lower levels of insulin expression in pancreatic beta cells. In addition, subjects carrying the Gln62Arg allele show decreased plasma insulin after an oral glucose challenge. Interestingly, all three nonsynonymous KLF11 variants show increased repression of the catalase 1 promoter, suggesting a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes. PMID:15774581

Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies.

PubMed

Reynolds, K A; Roll, K; Fujioka, R S; Gerba, C P; Pepper, I L

1998-06-01

The consequence of point and nonpoint pollution sources, discharged into marine waters, on public recreational beaches in Mamala Bay, Hawaii was evaluated using virus cell culture and direct reverse transcriptase-polymerase chain reaction (RT-PCR). Twelve sites, nine marine, two freshwater (one stream and one canal), and one sewage, were assessed either quarterly or monthly for 1 year to detect the presence of human enteric viruses. Water samples were concentrated from initial volumes of 400 L to final volumes of 30 mL using Filterite electronegative cartridge filters and a modified beef extract elution procedure. Cell culture was applied using the Buffalo Green Monkey kidney cell line to analyze samples for enteroviruses. Positive samples were also evaluated by RT-PCR, using enterovirus-specific primers. Levels of RT-PCR inhibition varied with each concentrated sample. Resin column purification increased PCR detection sensitivity by at least one order of magnitude in a variety of sewage outfall and recreational marine water samples but not in the freshwater canal samples. Using cell culture, viable enteroviruses were found in 50 and 17% of all outfall and canal samples, respectively. Samples were positive at beaches 8% of the time. These data illustrate the potential public health hazard associated with recreational waters. Using direct PCR, viruses were detected at the outfall but were not found in any beach or canal samples, in part, owing to substances that inhibit PCR. Therefore, conventional cell culture is the most effective means of detecting low levels of infectious enteroviruses in environmental waters, whereas direct RT-PCR is rendered less effective by inhibitory compounds and low equivalent reaction volumes.

Human Papillomavirus Type 18 E6 Protein Binds the Cellular PDZ Protein TIP-2/GIPC, Which Is Involved in Transforming Growth Factor β Signaling and Triggers Its Degradation by the Proteasome

PubMed Central

Favre-Bonvin, Arnaud; Reynaud, Caroline; Kretz-Remy, Carole; Jalinot, Pierre

2005-01-01

Several viral proteins expressed by DNA or RNA transforming viruses have the particular property of binding via their C-terminal end to various cellular proteins with PDZ domains. This study is focused on the PDZ protein TIP-2/GIPC, which was originally identified in two-hybrid screens performed with two different baits: the human T-cell leukemia virus type 1 Tax oncoprotein and the regulator of G signaling RGS-GAIP. Further studies have shown that TIP-2/GIPC is also able to associate with the cytoplasmic domains of various transmembrane proteins. In this report we show that TIP-2/GIPC interacts with the E6 protein of human papillomavirus type 18 (HPV-18). This event triggers polyubiquitination and proteasome-mediated degradation of the cellular protein. In agreement with this observation, silencing of E6 by RNA interference in HeLa cells causes an increase in the intracellular TIP-2/GIPC level. This PDZ protein has been previously found to be involved in transforming growth factor β (TGF-β) signaling by favoring expression of the TGF-β type III receptor at the cell membrane. In line with this activity of TIP-2/GIPC, we observed that depletion of this protein in HeLa cells hampers induction of the Id3 gene by TGF-β treatment and also diminishes the antiproliferative effect of this cytokine. Conversely, silencing of E6 increases the expression of Id3 and blocks proliferation of HeLa cells. These results support the notion that HPV-18 E6 renders cells less sensitive to the cytostatic effect of TGF-β by lowering the intracellular amount of TIP-2/GIPC. PMID:15767424

A high throughput spectral image microscopy system

NASA Astrophysics Data System (ADS)

Gesley, M.; Puri, R.

2018-01-01

A high throughput spectral image microscopy system is configured for rapid detection of rare cells in large populations. To overcome flow cytometry rates and use of fluorophore tags, a system architecture integrates sample mechanical handling, signal processors, and optics in a non-confocal version of light absorption and scattering spectroscopic microscopy. Spectral images with native contrast do not require the use of exogeneous stain to render cells with submicron resolution. Structure may be characterized without restriction to cell clusters of differentiation.

What Is the Tree of Life?

PubMed

Doolittle, W Ford; Brunet, Tyler D P

2016-04-01

A universal Tree of Life (TOL) has long been a goal of molecular phylogeneticists, but reticulation at the level of genes and possibly at the levels of cells and species renders any simple interpretation of such a TOL, especially as applied to prokaryotes, problematic.

Biallelic Germline Transcription at the κ Immunoglobulin Locus

PubMed Central

Singh, Nandita; Bergman, Yehudit; Cedar, Howard; Chess, Andrew

2003-01-01

Rearrangement of antigen receptor genes generates a vast array of antigen receptors on lymphocytes. The establishment of allelic exclusion in immunoglobulin genes requires differential treatment of the two sequence identical alleles. In the case of the κ immunoglobulin locus, changes in chromatin structure, methylation, and replication timing of the two alleles are all potentially involved in regulating rearrangement. Additionally, germline transcription of the κ locus which precedes rearrangement has been proposed to reflect an opening of the chromatin structure rendering it available for rearrangement. As the initial restriction of rearrangement to one allele is critical to the establishment of allelic exclusion, a key question is whether or not germline transcription at the κ locus is monoallelic or biallelic. We have used a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay and an RNA–fluorescence in situ hybridization (FISH) to show that germline transcription of the κ locus is biallelic in wild-type immature B cells and in recombination activating gene (RAG)−/−, μ+ B cells. Therefore, germline transcription is unlikely to dictate which allele will be rearranged first and rather reflects a general opening on both alleles that must be accompanied by a mechanism allowing one of the two alleles to be rearranged first. PMID:12629064

Polarization: A Key Difference between Man-made and Natural Electromagnetic Fields, in regard to Biological Activity

PubMed Central

Panagopoulos, Dimitris J.; Johansson, Olle; Carlo, George L.

2015-01-01

In the present study we analyze the role of polarization in the biological activity of Electromagnetic Fields (EMFs)/Electromagnetic Radiation (EMR). All types of man-made EMFs/EMR - in contrast to natural EMFs/EMR - are polarized. Polarized EMFs/EMR can have increased biological activity, due to: 1) Ability to produce constructive interference effects and amplify their intensities at many locations. 2) Ability to force all charged/polar molecules and especially free ions within and around all living cells to oscillate on parallel planes and in phase with the applied polarized field. Such ionic forced-oscillations exert additive electrostatic forces on the sensors of cell membrane electro-sensitive ion channels, resulting in their irregular gating and consequent disruption of the cell’s electrochemical balance. These features render man-made EMFs/EMR more bioactive than natural non-ionizing EMFs/EMR. This explains the increasing number of biological effects discovered during the past few decades to be induced by man-made EMFs, in contrast to natural EMFs in the terrestrial environment which have always been present throughout evolution, although human exposure to the latter ones is normally of significantly higher intensities/energy and longer durations. Thus, polarization seems to be a trigger that significantly increases the probability for the initiation of biological/health effects. PMID:26456585

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.

Polarization: A Key Difference between Man-made and Natural Electromagnetic Fields, in regard to Biological Activity

NASA Astrophysics Data System (ADS)

Panagopoulos, Dimitris J.; Johansson, Olle; Carlo, George L.

2015-10-01

In the present study we analyze the role of polarization in the biological activity of Electromagnetic Fields (EMFs)/Electromagnetic Radiation (EMR). All types of man-made EMFs/EMR - in contrast to natural EMFs/EMR - are polarized. Polarized EMFs/EMR can have increased biological activity, due to: 1) Ability to produce constructive interference effects and amplify their intensities at many locations. 2) Ability to force all charged/polar molecules and especially free ions within and around all living cells to oscillate on parallel planes and in phase with the applied polarized field. Such ionic forced-oscillations exert additive electrostatic forces on the sensors of cell membrane electro-sensitive ion channels, resulting in their irregular gating and consequent disruption of the cell’s electrochemical balance. These features render man-made EMFs/EMR more bioactive than natural non-ionizing EMFs/EMR. This explains the increasing number of biological effects discovered during the past few decades to be induced by man-made EMFs, in contrast to natural EMFs in the terrestrial environment which have always been present throughout evolution, although human exposure to the latter ones is normally of significantly higher intensities/energy and longer durations. Thus, polarization seems to be a trigger that significantly increases the probability for the initiation of biological/health effects.

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.

Rugged, Portable, Real-Time Optical Gaseous Analyzer for Hydrogen Fluoride

NASA Technical Reports Server (NTRS)

Pilgrim, Jeffrey; Gonzales, Paula

2012-01-01

Hydrogen fluoride (HF) is a primary evolved combustion product of fluorinated and perfluorinated hydrocarbons. HF is produced during combustion by the presence of impurities and hydrogen- containing polymers including polyimides. This effect is especially dangerous in closed occupied volumes like spacecraft and submarines. In these systems, combinations of perfluorinated hydrocarbons and polyimides are used for insulating wiring. HF is both highly toxic and short-lived in closed environments due to its reactivity. The high reactivity also makes HF sampling problematic. An infrared optical sensor can detect promptly evolving HF with minimal sampling requirements, while providing both high sensitivity and high specificity. A rugged optical path length enhancement architecture enables both high HF sensitivity and rapid environmental sampling with minimal gaseous contact with the low-reactivity sensor surfaces. The inert optical sample cell, combined with infrared semiconductor lasers, is joined with an analog and digital electronic control architecture that allows for ruggedness and compactness. The combination provides both portability and battery operation on a simple camcorder battery for up to eight hours. Optical detection of gaseous HF is confounded by the need for rapid sampling with minimal contact between the sensor and the environmental sample. A sensor is required that must simultaneously provide the required sub-parts-permillion detection limits, but with the high specificity and selectivity expected of optical absorption techniques. It should also be rugged and compact for compatibility with operation onboard spacecraft and submarines. A new optical cell has been developed for which environmental sampling is accomplished by simply traversing the few mm-thick cell walls into an open volume where the measurement is made. A small, low-power fan or vacuum pump may be used to push or pull the gaseous sample into the sample volume for a response time of a few seconds. The optical cell simultaneously provides for an enhanced optical interaction path length between the environmental sample and the infrared laser. Further, the optical cell itself is comprised of inert materials that render it immune to attack by HF. In some cases, the sensor may be configured so that the optoelectronic devices themselves are protected and isolated from HF by the optical cell. The optical sample cell is combined with custom-developed analog and digital control electronics that provide rugged, compact operation on a platform that can run on a camcorder battery. The sensor is inert with respect to acidic gases like HF, while providing the required sensitivity, selectivity, and response time. Certain types of combustion events evolve copious amounts of HF, very little of other gases typically associated with combustion (e.g., carbon monoxide), and very low levels of aerosols and particulates (which confound traditional smoke detectors). The new sensor platform could warn occupants early enough to take the necessary countermeasures.

N719 dye-sensitized organophotocatalysis: enantioselective tandem Michael addition/oxyamination of aldehydes.

PubMed

Yoon, Hyo-Sang; Ho, Xuan-Huong; Jang, Jiyeon; Lee, Hwa-Jung; Kim, Seung-Joo; Jang, Hye-Young

2012-07-06

A remarkably efficient photosensitizer, N719 dye, was used in asymmetric tandem Michael addition/oxyamination of aldehydes, rendering α,β-substituted aldehydes in good yields with excellent levels of enantioselectivity and diastereoselectivity. This is the first report of a multiorganocatalytic reaction involving iminium catalysis and photoinduced singly occupied molecular orbital (SOMO) catalysis. This reaction is expected to expand the scope of tandem organocatalytic reactions.

What Is the Tree of Life?

PubMed Central

Doolittle, W. Ford; Brunet, Tyler D. P.

2016-01-01

A universal Tree of Life (TOL) has long been a goal of molecular phylogeneticists, but reticulation at the level of genes and possibly at the levels of cells and species renders any simple interpretation of such a TOL, especially as applied to prokaryotes, problematic. PMID:27078870

High-Efficiency and High-Color-Rendering-Index Semitransparent Polymer Solar Cells Induced by Photonic Crystals and Surface Plasmon Resonance.

PubMed

Shen, Ping; Wang, Guoxin; Kang, Bonan; Guo, Wenbin; Shen, Liang

2018-02-21

Semitransparent polymer solar cells (ST-PSCs) show attractive potential in power-generating windows or building-integrated photovoltaics. However, the development of ST-PSCs is lagging behind opaque PSCs because of the contradiction between device efficiency and transmission. Herein, Ag/Au alloy nanoparticles and photonic crystals (PCs) were simultaneously introduced into ST-PSCs, acting compatibly as localized surface plasmon resonances and distributed Bragg reflectors to enhance light absorption and transmission. As a result, ST-PSCs based on a hybrid PTB7-Th:PC 71 BM active layer contribute an efficiency as high as 7.13 ± 0.15% and an average visible transmission beyond 20%, which are superior to most of the reported results. Furthermore, PCs can partly compensate valley range of transmission by balancing reflection and transmission regions, yielding a high color rendering index of 95. We believe that the idea of two light management methods compatibly enhancing the performance of ST-PSCs can offer a promising path to develop photovoltaic applications.

Systemic IL-2/anti-IL-2Ab complex combined with sublingual immunotherapy suppresses experimental food allergy in mice through induction of mucosal regulatory T cells.

PubMed

Smaldini, P L; Trejo, F; Cohen, J L; Piaggio, E; Docena, G H

2018-04-01

Therapeutic tolerance restoration has been proven to modify food allergy in patients and animal models and although sublingual immunotherapy (SLIT) has showed promise, combined therapy may be necessary to achieve a strong and long-term tolerance. In this work, we combined SLIT with systemic administration of IL-2 associated with an anti-IL-2 monoclonal antibody (IL-2/anti-IL-2Ab complex or IL-2C) to reverse the IgE-mediated experimental allergy. Balb/c mice were sensitized with cholera toxin and milk proteins and orally challenged with allergen to elicit hypersensitivity reactions. Then, allergic mice were treated with a sublingual administration of very low amounts of milk proteins combined with intraperitoneal injection of low doses of IL-2C. The animals were next re-exposed to allergens and mucosal as well as systemic immunological parameters were assessed in vivo and in vitro. The treatment reduced serum specific IgE, IL-5 secretion by spleen cells and increased IL-10 and TGF-β in the lamina propria of buccal and duodenal mucosa. We found an augmented frequency of IL-10-secreting CD4 + CD25 + Foxp3 + regulatory T cells (Treg) in the submaxilar lymph nodes and buccal lamina propria. Tregs were sorted, characterized and adoptively transferred to naïve mice, which were subsequently sensitized. No allergy was experienced in these mice and we encouragingly discovered a faster and more efficient tolerance induction with the combined therapy compared with SLIT. The combination of two therapeutic strategies rendered Treg-mediated tolerance more efficient compared to individual treatments and reversed the established IgE-mediated food allergy. This approach highlights the ability of IL-2C to expand Tregs, and it may represent a promising disease-modifying therapy for managing food allergy. © 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: a mechanistic study.

PubMed

Proks, Peter; de Wet, Heidi; Ashcroft, Frances M

2014-11-01

Sulfonylureas, which stimulate insulin secretion from pancreatic β-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K(+) (KATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on β-cell and cardiac types of KATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the KATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on β-cell KATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas. © 2014 Proks et al.

Nuclear Shield: A Multi-Enzyme Task-Force for Nucleus Protection

PubMed Central

Pallottini, Valentina; Canuti, Lorena; De Canio, Michele; Urbani, Andrea; Marzano, Valeria; Cornetta, Tommaso; Stano, Pasquale; Giovanetti, Anna; Stella, Lorenzo; Canini, Antonella; Federici, Giorgio; Ricci, Giorgio

2010-01-01

Background In eukaryotic cells the nuclear envelope isolates and protects DNA from molecules that could damage its structure or interfere with its processing. Moreover, selected protection enzymes and vitamins act as efficient guardians against toxic compounds both in the nucleoplasm and in the cytosol. The observation that a cytosolic detoxifying and antioxidant enzyme i.e. glutathione transferase is accumulated in the perinuclear region of the rat hepatocytes suggests that other unrecognized modalities of nuclear protection may exist. Here we show evidence for the existence of a safeguard enzyme machinery formed by an hyper-crowding of cationic enzymes and proteins encompassing the nuclear membrane and promoted by electrostatic interactions. Methodology/Principal Findings Electron spectroscopic imaging, zeta potential measurements, isoelectrofocusing, comet assay and mass spectrometry have been used to characterize this surprising structure that is present in the cells of all rat tissues examined (liver, kidney, heart, lung and brain), and that behaves as a “nuclear shield”. In hepatocytes, this hyper-crowding structure is about 300 nm thick, it is mainly formed by cationic enzymes and the local concentration of key protection enzymes, such as glutathione transferase, catalase and glutathione peroxidase is up to seven times higher than in the cytosol. The catalytic activity of these enzymes, when packed in the shield, is not modified and their relative concentrations vary remarkably in different tissues. Removal of this protective shield renders chromosomes more sensitive to damage by oxidative stress. Specific nuclear proteins anchored to the outer nuclear envelope are likely involved in the shield formation and stabilization. Conclusions/Significance The characterization of this previously unrecognized nuclear shield in different tissues opens a new interesting scenario for physiological and protection processes in eukaryotic cells. Selection and accumulation of protection enzymes near sensitive targets represents a new safeguard modality which deeply differs from the adaptive response which is based on expression of specific enzymes. PMID:21170318

A model for the relative biological effectiveness of protons: the tissue specific parameter α/β of photons is a predictor for the sensitivity to LET changes.

PubMed

Wedenberg, Minna; Lind, Bengt K; Hårdemark, Björn

2013-04-01

The biological effects of particles are often expressed in relation to that of photons through the concept of relative biological effectiveness, RBE. In proton radiotherapy, a constant RBE of 1.1 is usually assumed. However, there is experimental evidence that RBE depends on various factors. The aim of this study is to develop a model to predict the RBE based on linear energy transfer (LET), dose, and the tissue specific parameter α/β of the linear-quadratic model for the reference radiation. Moreover, the model should capture the basic features of the RBE using a minimum of assumptions, each supported by experimental data. The α and β parameters for protons were studied with respect to their dependence on LET. An RBE model was proposed where the dependence of LET is affected by the (α/β)phot ratio of photons. Published cell survival data with a range of well-defined LETs and cell types were selected for model evaluation rendering a total of 10 cell lines and 24 RBE values. A statistically significant relation was found between α for protons and LET. Moreover, the strength of that relation varied significantly with (α/β)phot. In contrast, no significant relation between β and LET was found. On the whole, the resulting RBE model provided a significantly improved fit (p-value < 0.01) to the experimental data compared to the standard constant RBE. By accounting for the α/β ratio of photons, clearer trends between RBE and LET of protons were found, and our results suggest that late responding tissues are more sensitive to LET changes than early responding tissues and most tumors. An advantage with the proposed RBE model in optimization and evaluation of treatment plans is that it only requires dose, LET, and (α/β)phot as input parameters. Hence, no proton specific biological parameters are needed.

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.

ptf1a+, ela3l− cells are developmentally maintained progenitors for exocrine regeneration following extreme loss of acinar cells in zebrafish larvae

PubMed Central

Schmitner, Nicole; Kohno, Kenji

2017-01-01

ABSTRACT The exocrine pancreas displays a significant capacity for regeneration and renewal. In humans and mammalian model systems, the partial loss of exocrine tissue, such as after acute pancreatitis or partial pancreatectomy induces rapid recovery via expansion of surviving acinar cells. In mouse it was further found that an almost complete removal of acinar cells initiates regeneration from a currently not well-defined progenitor pool. Here, we used the zebrafish as an alternative model to study cellular mechanisms of exocrine regeneration following an almost complete removal of acinar cells. We introduced and validated two novel transgenic approaches for genetically encoded conditional cell ablation in the zebrafish, either by caspase-8-induced apoptosis or by rendering cells sensitive to diphtheria toxin. By using the ela3l promoter for exocrine-specific expression, we show that both approaches allowed cell-type-specific removal of >95% of acinar tissue in larval and adult zebrafish without causing any signs of unspecific side effects. We find that zebrafish larvae are able to recover from a virtually complete acinar tissue ablation within 2 weeks. Using short-term lineage-tracing experiments and EdU incorporation assays, we exclude duct-associated Notch-responsive cells as the source of regeneration. Rather, a rare population of slowly dividing ela3l-negative cells expressing ptf1a and CPA was identified as the origin of the newly forming exocrine cells. Cells are actively maintained, as revealed by a constant number of these cells at different larval stages and after repeated cell ablation. These cells establish ela3l expression about 4-6 days after ablation without signs of increased proliferation in between. With onset of ela3l expression, cells initiate rapid proliferation, leading to fast expansion of the ela3l-positive population. Finally, we show that this proliferation is blocked by overexpression of the Wnt-signaling antagonist dkk1b. In conclusion, we show a conserved requirement for Wnt signaling in exocrine tissue expansion and reveal a potential novel progenitor or stem cell population as a source for exocrine neogenesis after complete loss of acinar cells. PMID:28138096

Signal transduction in primary human T lymphocytes in altered gravity during parabolic flight and clinostat experiments.

PubMed

Tauber, Svantje; Hauschild, Swantje; Paulsen, Katrin; Gutewort, Annett; Raig, Christiane; Hürlimann, Eva; Biskup, Josefine; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Pantaleo, Antonella; Cogoli, Augusto; Pippia, Proto; Layer, Liliana E; Thiel, Cora S; Ullrich, Oliver

2015-01-01

Several limiting factors for human health and performance in microgravity have been clearly identified arising from the immune system, and substantial research activities are required in order to provide the basic information for appropriate integrated risk management. The gravity-sensitive nature of cells of the immune system renders them an ideal biological model in search for general gravity-sensitive mechanisms and to understand how the architecture and function of human cells is related to the gravitational force and therefore adapted to life on Earth. We investigated the influence of altered gravity in parabolic flight and 2D clinostat experiments on key proteins of activation and signaling in primary T lymphocytes. We quantified components of the signaling cascade 1.) in non-activated T lymphocytes to assess the "basal status" of the cascade and 2.) in the process of activation to assess the signal transduction. We found a rapid decrease of CD3 and IL-2R surface expression and reduced p-LAT after 20 seconds of altered gravity in non-activated primary T lymphocytes during parabolic flight. Furthermore, we observed decreased CD3 surface expression, reduced ZAP-70 abundance and increased histone H3-acetylation in activated T lymphocytes after 5 minutes of clinorotation and a transient downregulation of CD3 and stable downregulation of IL-2R during 60 minutes of clinorotation. CD3 and IL-2R are downregulated in primary T lymphocytes in altered gravity. We assume that a gravity condition around 1g is required for the expression of key surface receptors and appropriate regulation of signal molecules in T lymphocytes. © 2015 S. Karger AG, Basel.

Photoswitching Near-Infrared Fluorescence from Polymer Nanoparticles Catapults Signals over the Region of Noises and Interferences for Enhanced Sensitivity.

PubMed

Wang, Jie; Lv, Yanlin; Wan, Wei; Wang, Xuefei; Li, Alexander D Q; Tian, Zhiyuan

2016-02-01

As a very sensitive technique, photoswitchable fluorescence not only gains ultrasensitivity but also imparts many novel and unexpected applications. Applications of near-infrared (NIR) fluorescence have demonstrated low background noises, high tissue-penetrating ability, and an ability to reduce photodamage to live cells. Because of these desired features, NIR-fluorescent dyes have been the premium among fluorescent dyes, and probes with photoswitchable NIR fluorescence are even more desirable for enhanced signal quality in the emerging optical imaging modalities but rarely used because they are extremely challenging to design and construct. Using a spiropyran derivative functioning as both a photoswitch and a fluorophore to launch its periodically modulated red fluorescence excitation energy into a NIR acceptor, we fabricated core-shell polymer nanoparticles exhibiting a photoswitchable fluorescence signal within the biological window (∼700-1000 nm) with a peak maximum of 776 nm. Live cells constantly synthesize new molecules, including fluorescent molecules, and also endocytose exogenous particles, including fluorescent particles. Upon excitation at different wavelengths, these fluorescent species bring about background noises and interferences covering nearly the whole visible region and therefore render many intracellular targets unaddressable. The oscillating NIR fluorescence signal with an on/off ratio of up to 67 that the polymer nanoparticles display is beyond the typical background noises and interferences, thus producing superior sharpness, reliability, and signal-to-noise ratios in cellular imaging. Taking these salient features, we anticipate that these types of nanoparticles will be useful for in vivo imaging of biological tissue and other complex specimens, where two-photon activation and excitation are used in combination with NIR-fluorescence photoswitching.

Discovery of black dye crystal structure polymorphs: Implications for dye conformational variation in dye-sensitized solar cells

DOE PAGES

Cole, Jacqueline M.; Low, Kian Sing; Gong, Yun

2015-11-24

Here, we present the discovery of a new crystal structure polymorph (1) and pseudopolymorph (2) of the Black Dye, one of the world’s leading dyes for dye-sensitized solar cells, DSSCs (10.4% device performance efficiency). This reveals that Black Dye molecules can adopt multiple low-energy conformers. This is significant since it challenges existing models of the Black Dye···TiO 2 adsorption process that renders a DSSC working electrode; these have assumed a single molecular conformation that refers to the previously reported Black Dye crystal structure (3). The marked structural differences observed between 1, 2, and 3 make the need for modeling multiplemore » conformations more acute. Additionally, the ordered form of the Black Dye (1) provides a more appropriate depiction of its anionic structure, especially regarding its anchoring group and NCS bonding descriptions. The tendency toward NCS ligand isomerism, evidenced via the disordered form 2, has consequences for electron injection and electron recombination in Black Dye embedded DSSC devices. Dyes 2 and 3 differ primarily by the absence or presence of a solvent of crystallization, respectively; solvent environment effects on the dye are thereby elucidated. This discovery of multiple Black Dye conformers from diffraction, with atomic-level definition, complements recently reported nanoscopic evidence for multiple dye conformations existing at a dye···TiO 2 interface, for a chemically similar DSSC dye; those results emanated from imaging and spectroscopy, but were unresolved at the submolecular level. Taken together, these findings lead to the general notion that multiple dye conformations should be explicitly considered when modeling dye···TiO 2 interfaces in DSSCs, at least for ruthenium-based dye complexes.« less

ATM is required for SOD2 expression and homeostasis within the mammary gland.

PubMed

Dyer, Lisa M; Kepple, Jessica D; Ai, Lingbao; Kim, Wan-Ju; Stanton, Virginia L; Reinhard, Mary K; Backman, Lindsey R F; Streitfeld, W Scott; Babu, Nivetha Ramesh; Treiber, Nicolai; Scharffetter-Kochanek, Karin; McKinnon, Peter J; Brown, Kevin D

2017-12-01

ATM activates the NF-κB transcriptional complex in response to genotoxic and oxidative stress. The purpose of this study was to examine if the NF-κB target gene and critical antioxidant SOD2 (MnSOD) in cultured mammary epithelium is also ATM-dependent, and what phenotypes arise from deletion of ATM and SOD2 within the mammary gland. SOD2 expression was studied in human mammary epithelial cells and MCF10A using RNAi to knockdown ATM or the NF-κB subunit RelA. To study ATM and SOD2 function in mammary glands, mouse lines containing Atm or Sod2 genes containing LoxP sites were mated with mice harboring Cre recombinase under the control of the whey acidic protein promoter. Quantitative PCR was used to measure gene expression, and mammary gland structure was studied using histology. SOD2 expression is ATM- and RelA-dependent, ATM knockdown renders cells sensitive to pro-oxidant exposure, and SOD mimetics partially rescue this sensitivity. Mice with germline deletion of Atm fail to develop mature mammary glands, but using a conditional knockout approach, we determined that Atm deletion significantly diminished the expression of Sod2. We also observed that these mice (termed Atm Δ/Δ ) displayed a progressive lactation defect as judged by reduced pup growth rate, aberrant lobulo-alveolar structure, diminished milk protein gene expression, and increased apoptosis within lactating glands. This phenotype appears to be linked to dysregulated Sod2 expression as mammary gland-specific deletion of Sod2 phenocopies defects observed in Atm Δ/Δ dams. We conclude that ATM is required to promote expression of SOD2 within the mammary epithelium, and that both ATM and SOD2 play a crucial role in mammary gland homeostasis.

Purification and characterization of a Shiga toxin A subunit-CD4 fusion protein cytotoxic to human immunodeficiency virus-infected cells.

PubMed

al-Jaufy, A Y; King, S R; Jackson, M P

1995-08-01

In a previous paper, we reported that a chimeric toxin composed of the enzymatic domain of the Shiga toxin A polypeptide (StxA1) genetically fused to the human CD4 (hCD4) molecule selectively kills cells infected with human immunodeficiency virus type 1 (HIV-1). Although other hCD4-containing chimeras cytotoxic to HIV-infected cells have been developed, there is limited information regarding their receptor binding and internalization. Therefore, the goals of this study were to purify the StxA1-hCD4 fusion protein, identify the receptor(s), and investigate the cytosolic trafficking route used by the chimeric toxin. Sufficient quantities of the StxA1-hCD4 hybrid were isolated for this investigation by using the pET expression and purification system. Cos-1 cells were rendered sensitive to the StxA1-hCD4 chimera by transfection with the env gene, which encodes HIV-1 envelope glycoproteins. The entry and translocation pathway used by the StxA1-hCD4 hybrid toxin was investigated by assessing the protective capacities of chemical reagents which interfere with microfilament movement, acidification of endosomes, and the integrity of the Golgi apparatus. Our findings indicated that the chimera uses HIV-1 glycoprotein gp120, and perhaps gp41, as a receptor which directs its entry through receptor cycling. Uptake is pH independent, and the StxA1-hCD4 hybrid is apparently translocated to the Golgi complex as with other bipartite toxins.

Novel MntR-Independent Mechanism of Manganese Homeostasis in Escherichia coli by the Ribosome-Associated Protein HflX

PubMed Central

Kaur, Gursharan; Sengupta, Sandeepan; Kumar, Vineet; Kumari, Aruna; Ghosh, Aditi; Parrack, Pradeep

2014-01-01

Manganese is a micronutrient required for activities of several important enzymes under conditions of oxidative stress and iron starvation. In Escherichia coli, the manganese homeostasis network primarily constitutes a manganese importer (MntH) and an exporter (MntP), which are regulated by the MntR dual regulator. In this study, we find that deletion of E. coli hflX, which encodes a ribosome-associated GTPase with unknown function, renders extreme manganese sensitivity characterized by arrested cell growth, filamentation, lower rate of replication, and DNA damage. We demonstrate that perturbation by manganese induces unprecedented influx of manganese in ΔhflX cells compared to that in the wild-type E. coli strain. Interestingly, our study indicates that the imbalance in manganese homeostasis in the ΔhflX strain is independent of the MntR regulon. Moreover, the influx of manganese leads to a simultaneous influx of zinc and inhibition of iron import in ΔhflX cells. In order to review a possible link of HflX with the λ phage life cycle, we performed a lysis-lysogeny assay to show that the Mn-perturbed ΔhflX strain reduces the frequency of lysogenization of the phage. This observation raises the possibility that the induced zinc influx in the manganese-perturbed ΔhflX strain stimulates the activity of the zinc-metalloprotease HflB, the key determinant of the lysis-lysogeny switch. Finally, we propose that manganese-mediated autophosphorylation of HflX plays a central role in manganese, zinc, and iron homeostasis in E. coli cells. PMID:24794564

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.

Hierarchical structures consisting of SiO2 nanorods and p-GaN microdomes for efficiently harvesting solar energy for InGaN quantum well photovoltaic cells.

PubMed

Ho, Cheng-Han; Lien, Der-Hsien; Chang, Hung-Chih; Lin, Chin-An; Kang, Chen-Fang; Hsing, Meng-Kai; Lai, Kun-Yu; He, Jr-Hau

2012-12-07

We experimentally and theoretically demonstrated the hierarchical structure of SiO(2) nanorod arrays/p-GaN microdomes as a light harvesting scheme for InGaN-based multiple quantum well solar cells. The combination of nano- and micro-structures leads to increased internal multiple reflection and provides an intermediate refractive index between air and GaN. Cells with the hierarchical structure exhibit improved short-circuit current densities and fill factors, rendering a 1.47 fold efficiency enhancement as compared to planar cells.

Microcapsules Containing pH-Responsive, Fluorescent Polymer-Integrated MoS2: An Effective Platform for in Situ pH Sensing and Photothermal Heating.

PubMed

Park, Chan Ho; Lee, Sangmin; Pornnoppadol, Ghasidit; Nam, Yoon Sung; Kim, Shin-Hyun; Kim, Bumjoon J

2018-03-14

We report the design of a novel microcapsule platform for in situ pH sensing and photothermal heating, which involves the encapsulation of pH-responsive polymer-coated molybdenum disulfide (MoS 2 ) nanosheets (NSs) in microcapsules with an aqueous core and a semipermeable polymeric shell. The MoS 2 NSs were functionalized with pH-responsive polymers having fluorescent groups at the distal end to provide pH-sensitive Förster resonance energy transfer (FRET) effect. The pH-responsive polymers were carefully designed to produce a dramatic change in the polymer conformation, which translated to a change in the FRET efficiency near pH 7.0 in response to subtle pH changes, enabling the detection of cancer cells. The pH-sensitive MoS 2 NSs were microfluidically encapsulated within semipermeable membranes to yield microcapsules with a uniform size and composition. The microcapsules retained the MoS 2 NSs without leakage while allowing the diffusion of small ions and water through the membrane. At the same time, the membranes excluded adhesive proteins and lipids in the surrounding media, protecting the encapsulated MoS 2 NSs from deactivation and enabling in situ pH monitoring. Moreover, the encapsulated MoS 2 NSs showed high-performance photothermal heating, rendering the dual-functional microcapsules highly suitable for cancer diagnosis and treatment.

Epigallocatechin gallate incorporation into lignin enhances the alkaline delignification and enzymatic saccharification of cell walls

USDA-ARS?s Scientific Manuscript database

Epigallocatechin gallate (EGCG) was evaluated as a potential lignin bioengineering target for rendering biomass more amenable to processing for biofuel production. In vitro peroxidase-catalyzed polymerization experiments revealed that both gallate and pyrogalloyl (B-ring) moieties in EGCG underwent ...

Protective effects of the thioredoxin and glutaredoxin systems in dopamine-induced cell death.

PubMed

Arodin, Lisa; Miranda-Vizuete, Antonio; Swoboda, Peter; Fernandes, Aristi P

2014-08-01

Although the etiology of sporadic Parkinson disease (PD) is unknown, it is well established that oxidative stress plays an important role in the pathogenic mechanism. The thioredoxin (Trx) and glutaredoxin (Grx) systems are two central systems upholding the sulfhydryl homeostasis by reducing disulfides and mixed disulfides within the cell and thereby protecting against oxidative stress. By examining the expression of redox proteins in human postmortem PD brains, we found the levels of Trx1 and thioredoxin reductase 1 (TrxR1) to be significantly decreased. The human neuroblastoma cell line SH-SY5Y and the nematode Caenorhabditis elegans were used as model systems to explore the potential protective effects of the redox proteins against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. 6-OHDA is highly prone to oxidation, resulting in the formation of the quinone of 6-OHDA, a highly reactive species and powerful neurotoxin. Treatment of human cells with 6-OHDA resulted in an increased expression of Trx1, TrxR1, Grx1, and Grx2, and small interfering RNA for these genes significantly increased the cytotoxic effects exerted by the 6-OHDA neurotoxin. Evaluation of the dopaminergic neurons in C. elegans revealed that nematodes lacking trxr-1 were significantly more sensitive to 6-OHDA, with significantly increased neuronal degradation. Importantly, both the Trx and the Grx systems were also found to directly mediate reduction of the 6-OHDA-quinone in vitro and thus render its cytotoxic effects. In conclusion, our results suggest that the two redox systems are important for neuronal survival in dopamine-induced cell death. Copyright © 2014 Elsevier Inc. All rights reserved.

A new concept of skin-associated lymphoid tissue (SALT): UVB light impaired cutaneous immunity reveals a prominent role for cutaneous nerves.

PubMed

Streilein, J W; Alard, P; Niizeki, H

1999-03-01

More than 20 years have passed since the concept that the skin has its own associated immune system was first proposed by Streilein. This proposal was advanced in part on evidence that cutaneous contact hypersensitivity (CH) reactions are closely correlated with Langerhans cells (LC). Recent reports have demonstrated that LC have neural connectivity with cutaneous nerve termini containing calcitonin gene-related peptide (CGRP), suggesting that a link exists between innervation and immune responses in the skin. Here we discuss the neural components which have recently been found to be participants in skin-associated lymphoid tissue (SALT). In part, discovery of a functional link between the nervous system and SALT is based on studies in which cutaneous immunity was impaired by ultraviolet-B radiation (UVR). The deleterious effects of UVR on cutaneous immunity include failed CH induction and promotion of hapten-specific tolerance, effects that are mediated by tumor necrosis factor-alpha and interleukin-10, respectively. The source of these cytokines after UVR appears to be dermal mast cells. Evidence indicates that mast cells are triggered to release these cytokines in response to CGRP, which is released from UVR-damaged cutaneous nerve endings. Moreover, a substance P agonist was able to reverse the deleterious effects of UVR on CH induction, rendering the mice able to develop intense CH. These observations indicate that two cell types not originally included in the SALT concept are critical to the functional integrity of cutaneous immunity: mast cells and cutaneous nerves. We propose that cutaneous nerves dictate whether antigen applied to or arising within skin will lead to sensitivity or tolerance.

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

Decreased ATP synthesis is phenotypically expressed during increased energy demand in fibroblasts containing mitochondrial tRNA mutations.

PubMed

James, A M; Sheard, P W; Wei, Y H; Murphy, M P

1999-01-01

Mutations in the tRNA genes of mitochondrial DNA (mtDNA) cause the debilitating MELAS (mitochondrial, myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fibres) syndromes. These mtDNA mutations affect respiratory chain function, apparently without decreasing cellular ATP concentration [Moudy et al. (1995) PNAS, 92, 729-733]. To address this issue, we investigated the role of mitochondrial ATP synthesis in fibroblasts from MELAS and MERRF patients. The maximum rate of mitochondrial ATP synthesis was decreased by 60-88%, as a consequence of the decrease in the proton electrochemical potential gradient of MELAS and MERRF mitochondria. However, in quiescent fibroblasts neither ATP concentration or the ATP/ADP ratio was affected by the lowered rate of ATP synthesis. We hypothesized that the low ATP demand of quiescent fibroblasts masked the mitochondrial ATP synthesis defect and that this defect might become apparent during higher ATP use. To test this we simulated high energy demand by titrating cells with gramicidin, an ionophore that stimulates ATP hydrolysis by the plasma membrane Na+/K+-ATPase. We found a threshold gramicidin concentration in control cells at which both the ATP/ADP ratio and the plasma membrane potential decreased dramatically, due to ATP demand by the Na+/K+-ATPase outstripping mitochondrial ATP synthesis. In MELAS and MERRF fibroblasts the corresponding threshold concentrations of gramicidin were 2-20-fold lower than those for control cells. This is the first demonstration that cells containing mtDNA mutations are particularly sensitive to increased ATP demand and this has several implications for how mitochondrial dysfunction contributes to disease pathophysiology. In particular, the increased susceptibility to plasma membrane depolarization will render neurons with dysfunctional mitochondria susceptible to excitotoxic cell death.

Which AML subsets benefit from leukemic cell priming during chemotherapy? Long-term analysis of the ALFA-9802 GM-CSF study.

PubMed

Thomas, Xavier; Raffoux, Emmanuel; Renneville, Aline; Pautas, Cecile; de Botton, Stephane; Terre, Christine; Gardin, Claude; Hayette, Sandrine; Preudhomme, Claude; Dombret, Herve

2010-04-01

: Priming with granulocytic hematopoietic growth factors may modulate cell cycle kinetics of leukemic cells and render them more susceptible to phase-specific chemotherapeutic agents. In a first report, we have shown that priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) may enhance complete remission (CR) rate and event-free survival (EFS) in younger adults with acute myeloid leukemia (AML). : In this randomized trial, 259 patients with AML were randomized at baseline to receive or not receive GM-CSF concurrently with all cycles of chemotherapy. The effects of GM-CSF on survival were reported herein with a long-term follow-up and studied according to distinct biological subgroups defined on cytogenetics and molecular markers. : The EFS rate was better in the GM-CSF group (43% vs 34%; P = .04). GM-CSF did not improve the outcome in patients from good risk subgroups, while patients from poor risk subgroups benefited from GM-CSF therapy. In this population, the difference in terms of EFS probability was mainly observed in patients with high initial white blood cell count and in those with FLT3-ITD or MLL rearrangement. When combining these 2 molecular abnormalities for comparison of the effect of GM-CSF priming, the difference in terms of EFS was highly significant (5-year EFS, 39% with GM-CSF vs 8% without GM-CSF; P = .007). : Sensitization of leukemic cells and their progenitors by GM-CSF appears as a plausible strategy for improving the outcome of patients with newly diagnosed AML. Patients with poor-prognosis FLT3-ITD or MLL rearrangement might be a good target population to further investigate priming strategies. Cancer 2010. (c) 2010 American Cancer Society.

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.

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

Nonfeed application of rendered animal proteins for microbial production of eicosapentaenoic acid by the fungus Pythium irregulare.

PubMed

Liang, Yi; Garcia, Rafael A; Piazza, George J; Wen, Zhiyou

2011-11-23

Rendered animal proteins are well suited for animal nutrition applications, but the market is maturing, and there is a need to develop new uses for these products. The objective of this study is to explore the possibility of using animal proteins as a nutrient source for microbial production of omega-3 polyunsaturated fatty acids by the microalga Schizochytrium limacinum and the fungus Pythium irregulare. To be absorbed by the microorganisms, the proteins needed to be hydrolyzed into small peptides and free amino acids. The utility of the protein hydrolysates for microorganisms depended on the hydrolysis method used and the type of microorganism. The enzymatic hydrolysates supported better cell growth performance than the alkali hydrolysates did. P. irregulare displayed better overall growth performance on the experimental hydrolysates compared to S. limacinum. When P. irregulare was grown in medium containing 10 g/L enzymatic hydrolysate derived from meat and bone meal or feather meal, the performance of cell growth, lipid synthesis, and omega-3 fatty acid production was comparable to the that of culture using commercial yeast extract. The fungal biomass derived from the animal proteins had 26-29% lipid, 32-34% protein, 34-39% carbohydrate, and <2% ash content. The results show that it is possible to develop a nonfeed application for rendered animal protein by hydrolysis of the protein and feeding to industrial microorganisms which can produce omega-3 fatty acids for making omega-3-fortified foods or feeds.

Estrogen and testosterone in concert with EFNB3 regulate vascular smooth muscle cell contractility and blood pressure.

PubMed

Wang, Yujia; Wu, Zenghui; Thorin, Eric; Tremblay, Johanne; Lavoie, Julie L; Luo, Hongyu; Peng, Junzheng; Qi, Shijie; Wu, Tao; Chen, Fei; Shen, Jianzhong; Hu, Shenjiang; Wu, Jiangping

2016-04-01

EPH kinases and their ligands, ephrins (EFNs), have vital and diverse biological functions, although their function in blood pressure (BP) control has not been studied in detail. In the present study, we report that Efnb3 gene knockout (KO) led to increased BP in female but not male mice. Vascular smooth muscle cells (VSMCs) were target cells for EFNB3 function in BP regulation. The deletion of EFNB3 augmented contractility of VSMCs from female but not male KO mice, compared with their wild-type (WT) counterparts. Estrogen augmented VSMC contractility while testosterone reduced it in the absence of EFNB3, although these sex hormones had no effect on the contractility of VSMCs from WT mice. The effect of estrogen on KO VSMC contractility was via a nongenomic pathway involving GPER, while that of testosterone was likely via a genomic pathway, according to VSMC contractility assays and GPER knockdown assays. The sex hormone-dependent contraction phenotypes in KO VSMCs were reflected in BP in vivo. Ovariectomy rendered female KO mice normotensive. At the molecular level, EFNB3 KO in VSMCs resulted in reduced myosin light chain kinase phosphorylation, an event enhancing sensitivity to Ca(2+)flux in VSMCs. Our investigation has revealed previously unknown EFNB3 functions in BP regulation and show that EFNB3 might be a hypertension risk gene in certain individuals. Copyright © 2016 the American Physiological Society.

A noninvasive optical approach for assessing chloride extrusion activity of the K-Cl cotransporter KCC2 in neuronal cells.

PubMed

Ludwig, Anastasia; Rivera, Claudio; Uvarov, Pavel

2017-01-31

Cation-chloride cotransporters (CCCs) are indispensable for maintaining chloride homeostasis in multiple cell types, but K-Cl cotransporter KCC2 is the only CCC member with an exclusively neuronal expression in mammals. KCC2 is critical for rendering fast hyperpolarizing responses of ionotropic γ-aminobutyric acid and glycine receptors in adult neurons, for neuronal migration in the developing central nervous system, and for the formation and maintenance of small dendritic protrusions-dendritic spines. Deficit in KCC2 expression and/or activity is associated with epilepsy and neuropathic pain, and effective strategies are required to search for novel drugs augmenting KCC2 function. We revised current methods to develop a noninvasive optical approach for assessing KCC2 transport activity using a previously characterized genetically encoded chloride sensor. Our protocol directly assesses dynamics of KCC2-mediated chloride efflux and allows measuring genuine KCC2 activity with good spatial and temporal resolution. As a proof of concept, we used this approach to compare transport activities of the two known KCC2 splice isoforms, KCC2a and KCC2b, in mouse neuronal Neuro-2a cells. Our noninvasive optical protocol proved to be efficient for assessment of furosemide-sensitive chloride fluxes. Transport activities of the N-terminal splice isoforms KCC2a and KCC2b obtained by the novel approach matched to those reported previously using standard methods for measuring chloride fluxes.

Classification of biosensor time series using dynamic time warping: applications in screening cancer cells with characteristic biomarkers.

PubMed

Rai, Shesh N; Trainor, Patrick J; Khosravi, Farhad; Kloecker, Goetz; Panchapakesan, Balaji

2016-01-01

The development of biosensors that produce time series data will facilitate improvements in biomedical diagnostics and in personalized medicine. The time series produced by these devices often contains characteristic features arising from biochemical interactions between the sample and the sensor. To use such characteristic features for determining sample class, similarity-based classifiers can be utilized. However, the construction of such classifiers is complicated by the variability in the time domains of such series that renders the traditional distance metrics such as Euclidean distance ineffective in distinguishing between biological variance and time domain variance. The dynamic time warping (DTW) algorithm is a sequence alignment algorithm that can be used to align two or more series to facilitate quantifying similarity. In this article, we evaluated the performance of DTW distance-based similarity classifiers for classifying time series that mimics electrical signals produced by nanotube biosensors. Simulation studies demonstrated the positive performance of such classifiers in discriminating between time series containing characteristic features that are obscured by noise in the intensity and time domains. We then applied a DTW distance-based k -nearest neighbors classifier to distinguish the presence/absence of mesenchymal biomarker in cancer cells in buffy coats in a blinded test. Using a train-test approach, we find that the classifier had high sensitivity (90.9%) and specificity (81.8%) in differentiating between EpCAM-positive MCF7 cells spiked in buffy coats and those in plain buffy coats.

A Novel Approach to Visualizing Dark Matter Simulations.

PubMed

Kaehler, R; Hahn, O; Abel, T

2012-12-01

In the last decades cosmological N-body dark matter simulations have enabled ab initio studies of the formation of structure in the Universe. Gravity amplified small density fluctuations generated shortly after the Big Bang, leading to the formation of galaxies in the cosmic web. These calculations have led to a growing demand for methods to analyze time-dependent particle based simulations. Rendering methods for such N-body simulation data usually employ some kind of splatting approach via point based rendering primitives and approximate the spatial distributions of physical quantities using kernel interpolation techniques, common in SPH (Smoothed Particle Hydrodynamics)-codes. This paper proposes three GPU-assisted rendering approaches, based on a new, more accurate method to compute the physical densities of dark matter simulation data. It uses full phase-space information to generate a tetrahedral tessellation of the computational domain, with mesh vertices defined by the simulation's dark matter particle positions. Over time the mesh is deformed by gravitational forces, causing the tetrahedral cells to warp and overlap. The new methods are well suited to visualize the cosmic web. In particular they preserve caustics, regions of high density that emerge, when several streams of dark matter particles share the same location in space, indicating the formation of structures like sheets, filaments and halos. We demonstrate the superior image quality of the new approaches in a comparison with three standard rendering techniques for N-body simulation data.

CD8+ memory T-cell inflation renders compromised CD4+ T-cell-dependent CD8+ T-cell immunity via naïve T-cell anergy.

PubMed

Xu, Aizhang; Freywald, Andrew; Xie, Yufeng; Li, Zejun; Xiang, Jim

2017-01-01

Whether inflation of CD8 + memory T (mT) cells, which is often derived from repeated prime-boost vaccinations or chronic viral infections in the elderly, would affect late CD8 + T-cell immunity is a long-standing paradox. We have previously established an animal model with mT-cell inflation by transferring ConA-stimulated monoclonal CD8 + T cells derived from Ova-specific T-cell-receptor transgenic OTI mice into irradiation-induced lymphopenic B6 mice. In this study, we also established another two animal models with mT-cell inflation by transferring, 1) ConA-stimulated monoclonal CD8 + T cells derived from lymphocytic choriomeningitis virus glycoprotein-specific T-cell-receptor transgenic P14 mice, and 2) ConA-stimulated polyclonal CD8 + T cells derived from B6.1 mice into B6 mice with irradiation-induced lymphopenia. We vaccinated these mice with recombinant Ova-expressing Listeria monocytogenes and Ova-pulsed dendritic cells, which stimulated CD4 + T cell-independent and CD4 + T-cell-dependent CD8 + T-cell responses, respectively, and assessed Ova-specific CD8 + T-cell responses by flow cytometry. We found that Ova-specific CD8 + T-cell responses derived from the latter but not the former vaccination were significantly reduced in mice with CD8 + mT-cell inflation compared to wild-type B6 mice. We determined that naïve CD8 + T cells purified from splenocytes of mice with mT-cell inflation had defects in cell proliferation upon stimulation in vitro and in vivo and upregulated T-cell anergy-associated Itch and GRAIL molecules. Taken together, our data reveal that CD8 + mT-cell inflation renders compromised CD4 + T-cell-dependent CD8 + T-cell immunity via naïve T-cell anergy, and thus show promise for the design of efficient vaccines for elderly patients with CD8 + mT-cell inflation.

[Correcting influence of vitamin E short chain derivatives on lipid peroxidation, liver cell membrane, and chromatin structure when rats are exposed to embichin].

PubMed

Kovalenko, V M; Byshovets', T F; Hubs'kyĭ, Iu I; Levyts'kyĭ, Ie L; Shaiakhmetova, H M; Marchenko, O M; Voloshyna, O S; Saĭfetdinova, H A; Okhrimenko, V O; Donchenko, H V

2000-01-01

Embikhin causes activation of LPO processes in endoplasmic reticulum and in nuclear chromatine fractions of rat liver cells. The latter is accompanied by the impairment of repressive and active nuclear chromatine fractions structure. Derivate of vitamin E in these conditions renders correcting action on parameters of lipid peroxidation in the investigated subcellular structures, testifying its positive influence on the cell heredity apparatus state. The normalizing action of tocopherol derivative on cytochromes P450 and b5 levels is shown.

Inorganic Halogen Oxidizers.

DTIC Science & Technology

1984-02-22

XeFfi 1s capable of deliver- ing the highest known Derformanrp fnr any NF F? gas generator composi- tion. The difficulty of synthesizing the compound ...the high cost of Xe and the shock sensitivity of the Xe(L hydrolysis product, however, render this compound Impractical at the present time. A...discovered for F0C10„ which allowed us to carry out a thorough characterization of this Interesting compound (see Appendix L). We have also

Application of bacteriophages to reduce biofilms formed by hydrogen sulfide producing bacteria on surfaces in a rendering plant.

PubMed

Gong, Chao; Jiang, Xiuping

2015-08-01

Hydrogen sulfide producing bacteria (SPB) in raw animal by-products are likely to grow and form biofilms in the rendering processing environments, resulting in the release of harmful hydrogen sulfide (H2S) gas. The objective of this study was to reduce SPB biofilms formed on different surfaces typically found in rendering plants by applying a bacteriophage cocktail. Using a 96-well microplate method, we determined that 3 SPB strains of Citrobacter freundii and Hafnia alvei are strong biofilm formers. Application of 9 bacteriophages (10(7) PFU/mL) from families of Siphoviridae and Myoviridae resulted in a 33%-70% reduction of biofilm formation by each SPB strain. On stainless steel and plastic templates, phage treatment (10(8) PFU/mL) reduced the attached cells of a mixed SPB culture (no biofilm) by 2.3 and 2.7 log CFU/cm(2) within 6 h at 30 °C, respectively, as compared with 2 and 1.5 log CFU/cm(2) reductions of SPB biofilms within 6 h at 30 °C. Phage treatment was also applied to indigenous SPB biofilms formed on the environmental surface, stainless steel, high-density polyethylene plastic, and rubber templates in a rendering plant. With phage treatment (10(9) PFU/mL), SPB biofilms were reduced by 0.7-1.4, 0.3-0.6, and 0.2-0.6 log CFU/cm(2) in spring, summer, and fall trials, respectively. Our study demonstrated that bacteriophages could effectively reduce the selected SPB strains either attached to or in formed biofilms on various surfaces and could to some extent reduce the indigenous SPB biofilms on the surfaces in the rendering environment.

The influence of antigen targeting to sub-cellular compartments on the anti-allergic potential of a DNA vaccine.

PubMed

Weinberger, Esther E; Isakovic, Almedina; Scheiblhofer, Sandra; Ramsauer, Christina; Reiter, Katrin; Hauser-Kronberger, Cornelia; Thalhamer, Josef; Weiss, Richard

2013-12-09

Gene vaccines offer attractive rationales for prophylactic as well as therapeutic treatments of type I allergies. DNA and mRNA vaccines have been shown to prevent from allergic sensitization and to counterbalance established allergic immune reactions. Recent advances in gene vaccine manipulation offer additional opportunities for modulation of T helper cell profiles by specific targeting of cellular compartments. DNA vaccines encoding the major birch pollen allergen Bet v 1.0101 were equipped with different leader sequences to shuttle the antigen to lysosomes (LIMP-II), to trigger cellular secretion (hTPA), or to induce proteasomal degradation via forced ubiquitination (ubi). Mice were pre-vaccinated with these constructs and the protective efficacy was tested by subcutaneous Th2-promoting challenges, followed by allergen inhalation. IgG antibody subclass distribution and allergen-specific IgE as well as cytokine profiles from re-stimulated splenocytes and from BALFs were assessed. The cellular composition of BALFs, and lung resistance and compliance were determined. Immunization with all targeting variants protected from allergic sensitization, i.e. IgE induction, airway hyperresponsiveness, lung inflammation, and systemic and local Th2 cytokine expression. Surprisingly, protection did not clearly correlate with the induction of a systemic Th1 cytokine profile, but rather with proliferating CD4+ CD25+ FoxP3+ T regulatory cells in splenocyte cultures. Targeting the allergen to proteasomal or lysosomal degradation severely down-regulated antibody induction after vaccination, while T cell responses remained unaffected. Although secretion of antigen promoted the highest numbers of Th1 cells, this vaccine type was the least efficient in suppressing the establishment of an allergic immune response. This comparative analysis highlights the modulatory effect of antigen targeting on the resulting immune response, with a special emphasis on prophylactic anti-allergy DNA vaccination. Targeting the antigen to proteasomal or lysosomal degradation reduces the availability of native allergen, thereby rendering the vaccine hypoallergenic without compromising efficacy, an important feature for a therapeutic setting. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

An endogenous 55 kDa TNF receptor mediates cell death in a neural cell line.

PubMed

Sipe, K J; Srisawasdi, D; Dantzer, R; Kelley, K W; Weyhenmeyer, J A

1996-06-01

Tumor necrosis factor-alpha (TNF) is associated with developmental and injury-related events in the central nervous system (CNS). In the present study, we have examined the role of TNF on neurons using the clonal murine neuroblastoma line, N1E-115 (N1E). N1E cells represent a well-defined model for studying neuronal development since they can be maintained as either undifferentiated, mitotically active neuroblasts or as differentiated, mature neurons. Northern and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that both undifferentiated and differentiated N1Es express transcripts for the 55 kDa TNF receptor (TNFR), but not the 75 kDa TNFR. The biological activity of the expressed TNF receptor was demonstrated by a dose dependent cytotoxicity to either recombinant murine or human TNF when the cells were incubated with the transcriptional inhibitor actinomycin D. The lack of the 75 kDa receptor mRNA expression and the dose dependent response to rHuTNF, an agonist specific for the murine 55 kDa receptor, suggest that the TNF induced cytotoxicity is mediated through the 55 kDa receptor in both the undifferentiated and differentiated N1Es. Light microscopic observations, flow cytometric analysis of hypodiploid DNA, and electrophoretic analysis of nucleosomal DNA fragmentation of N1Es treated with actinomycin D and TNF revealed features characteristic of both necrotic and apoptotic cell death. These findings demonstrate that blast and mature N1E cells express the 55 kDa TNF receptor which is responsible for inducing both necrotic and apoptotic death in these cells. The observation that actinomycin D renders N1E cells susceptible to the cytotoxic effects of TNF indicates that a sensitization step, such as removal of an endogenous protective factor or viral-mediated inhibition of transcription, may be necessary for TNF cytotoxicity in neurons.

Thermotolerance induced at a mild temperature of 40°C alleviates heat shock-induced ER stress and apoptosis in HeLa cells.

PubMed

Bettaieb, Ahmed; Averill-Bates, Diana A

2015-01-01

Hyperthermia (39-45°C) has emerged as an alternate prospect for cancer therapy in combination with radiation and chemotherapy. Despite promising progress in the clinic, molecular mechanisms involved in hyperthermia-induced cell death are not clear. Hyperthermia causes protein denaturation/aggregation, which results in cell death by apoptosis and/or necrosis. Hyperthermia also induces thermotolerance, which renders cells resistant to subsequent exposure to lethal heat shock. This study investigates the role of both lethal (42-43°C) and mild (40°C) hyperthermia in regulating ER stress and ER stress-induced apoptosis in HeLa cells. The ability of mild thermotolerance induced at 40°C to alleviate either or both of these processes is also determined. Hyperthermia (42-43°C) induced ER stress, revealed by phosphorylation of PERK, eIF2α and IRE1α, cleavage of ATF6 and increased expression of BiP and sXBP1. Real-time PCR revealed that mRNA levels of ATF6, ATF4, BiP, sXBP1 and CHOP increased in cells exposed to hyperthermia. Moreover, hyperthermia caused disruption of calcium homeostasis and activated the calpain-calpastatin proteolytic system and ER resident caspase 4. Pre-exposure to mild hyperthermia (40°C) alleviated the induction of cytotoxicity and ER stress by hyperthermia (42-43°C) and protected cells against ER stress-induced apoptosis. ShRNA-mediated depletion of Hsp72 abrogated protective effects of mild thermotolerance (40°C) against heat-shock induced ER stress and sensitized cells to ER stress-mediated apoptosis. Our findings show that Hsp72 contributes to the protective effects of mild hyperthermia (40°C) against hyperthermia-induced ER stress and apoptosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Acceleration and sensitivity analysis of lattice kinetic Monte Carlo simulations using parallel processing and rate constant rescaling

NASA Astrophysics Data System (ADS)

Núñez, M.; Robie, T.; Vlachos, D. G.

2017-10-01

Kinetic Monte Carlo (KMC) simulation provides insights into catalytic reactions unobtainable with either experiments or mean-field microkinetic models. Sensitivity analysis of KMC models assesses the robustness of the predictions to parametric perturbations and identifies rate determining steps in a chemical reaction network. Stiffness in the chemical reaction network, a ubiquitous feature, demands lengthy run times for KMC models and renders efficient sensitivity analysis based on the likelihood ratio method unusable. We address the challenge of efficiently conducting KMC simulations and performing accurate sensitivity analysis in systems with unknown time scales by employing two acceleration techniques: rate constant rescaling and parallel processing. We develop statistical criteria that ensure sufficient sampling of non-equilibrium steady state conditions. Our approach provides the twofold benefit of accelerating the simulation itself and enabling likelihood ratio sensitivity analysis, which provides further speedup relative to finite difference sensitivity analysis. As a result, the likelihood ratio method can be applied to real chemistry. We apply our methodology to the water-gas shift reaction on Pt(111).

STUDIES ON DECREASING THE REACTION OF NORMAL SKIN TO DESTRUCTIVE DOSES OF X-RAYS BY PHARMACOLOGICAL MEANS AND ON THE MECHANISM INVOLVED

PubMed Central

Auer, John; Witherbee, William D.

1921-01-01

When a fixed area of the ears of rabbits is subjected to the action of a standard destructive dose of x-rays (30 skin units) the type of reaction resulting depends upon the previous treatment of the rabbit. (1) In normal rabbits a mild acute inflammation develops in the x-rayed area which leads at once to a perforating gangrene within an average of 15 days. (2) If rabbits are x-rayed and about 2 weeks later injected with horse serum for the first time, a mild acute inflammation appears which heals for a time; then a second, subacute inflammation sets in which leads to a perforating gangrene. The average time of the process from the first inflammation to gangrene is 32 days. (3) If rabbits are sensitized with horse serum and 10 days later are exposed locally to the standard dose of x-rays, the ensuing ear reaction is either similar to the second reaction described above, except that it may last up to 110 days, or the first inflammation leads to a healing which may be apparently permanent (340 + days). (4) If rabbits are first sensitized with horse serum, exposed locally to the standard dose of x-rays 10 days later, and 13 days after the x-ray treatment reinjected with horse serum, the reaction of the x-rayed area of the ears is in general similar to the second reaction described above (inflammation—healing—inflammation—gangrene). The average time of the whole process is about 42 days. On the basis of the general hypothesis that an anaphylactic reaction is initiated in the body when the specific antibody meets its antigen, and that both antibody and antigen are rendered more or less functionally inert by their interaction, the following inferences may be drawn from our experimental results. (1) The protection from the effects of a standard destructive dose of x-rays which a previous sensitization confers, is referable to the presence of anaphylactic antibodies in the x-rayed area. (2) This protection is largely due to the anaphylactic antibodies which are anchored in the x-rayed area, and not to those which are free in the circulation. (3) An anaphylactic reaction renders the anchored anaphylactic antibodies largely impotent as protective factors against the standard destructive x-ray dose, even though sensitization preceded exposure to the x-rays. (4) An area treated with the standard destructive dose of x-rays is unable to produce or to anchor a sufficient amount of anaphylactic antibodies for protection from necrosis, when the x-ray treatment precedes the sensitization, or when the locally anchored anaphylactic antibodies are rendered functionally inactive by a general anaphylactic reaction. It is possible that the procedure of increasing the resistance of the skin to a destructive dose of x-rays by means of a previous sensitization with protein may be applicable in the treatment of certain types of inoperable disease, when it is important to use massive doses of x-rays. Animals which have been sensitized, or sensitized and reinjected with any undenatured alien protein, should not be reemployed as normal controls in any investigation unless trial has shown that these proteinized animals react quantitatively and qualitatively like normal animals. The presence of an abnormal reactor in a group of supposedly normal animals may be an indication of a previous proteinization. PMID:19868536

Phase-contrast tomography of neuronal tissues: from laboratory- to high resolution synchrotron CT

NASA Astrophysics Data System (ADS)

Töpperwien, Mareike; Krenkel, Martin; Müller, Kristin; Salditt, Tim

2016-10-01

Assessing the three-dimensional architecture of neuronal tissues with sub-cellular resolution presents a significant analytical challenge. Overcoming the limitations associated with serial slicing, phase-contrast x-ray tomography has the potential to contribute to this goal. Even compact laboratory CT at an optimized liquid-metal jet micro- focus source combined with suitable phase-retrieval algorithms and preparation protocols can yield renderings with single cell sensitivity in millimeter sized brain areas of mouse. Here, we show the capabilities of the setup by imaging a Golgi-Cox impregnated mouse brain. Towards higher resolution we extend these studies at our recently upgraded waveguide-based cone-beam holo-tomography instrument GINIX at DESY. This setup allows high resolution recordings with adjustable field of view and resolution, down to the voxel sizes in the range of a few ten nanometers. The recent results make us confident that important issues of neuronal connectivity can be addressed by these methods, and that 3D (virtual) histology with nanoscale resolution will become an attractive modality for neuroscience research.

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

An enhanced mangiferaindica for dye sensitized solar cell application

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

Uno, U. E., E-mail: moses.emetere@covenantuniversity.edu.ng; Emetere, M. E., E-mail: uno-essang@yahoo.co.uk; Fadipe, L. A.

Titanium dioxide (T1O2) is preferred to Zinc oxide as mesoporous oxide layer because it raised the efficiency of DSSCs from 1% to 7%. The chemistry of the process however seem rigorous to allow the light induced electron injection from the adsorbed dye into the nanocrystallites i.e. which renders the TiO{sub 2} conductive. The DSSC fabricated consist of 2.25 cm{sup 2} active area of titanium dioxide coated on FTO glass (fluorine tin oxide) immersed in ethanol solution of natural dye extracted as an anode (electrode) and counter electrode. These two electrodes were coupled together and the space between them was filledmore » with the Iodolyte AN-50 as solid electrolyte or redox mediator. The photo electrochemical parameters of the dye extracted (Mango fruit Peel) from the results obtained are short circuit current (Isc)= 1.22×10{sup −2}, current density (Jsc)=4.07×10{sup −2}, open circuit voltage (voc) =0.53V, fill factor (FF) of 0.16 and the overall conversion efficiency (Eff) =0.345%.« less

β-Catenin activity in the dermal papilla of the hair follicle regulates pigment-type switching

PubMed Central

Enshell-Seijffers, David; Lindon, Catherine; Wu, Eleanor; Taketo, Makoto M.; Morgan, Bruce A.

2010-01-01

The switch between black and yellow pigment is mediated by the interaction between Melanocortin receptor 1 (Mc1r) and its antagonist Agouti, but the genetic and developmental mechanisms that modify this interaction to obtain different coat color in distinct environments are poorly understood. Here, the role of Wnt/β-catenin signaling in the regulation of pigment-type switching was studied. Loss and gain of function of β-catenin in the dermal papilla (DP) of the hair follicle results in yellow and black animals, respectively. β-Catenin activity in the DP suppresses Agouti expression and activates Corin, a negative regulator of Agouti activity. In addition, β-catenin activity in the DP regulates melanocyte activity by a mechanism that is independent of both Agouti and Corin. The coordinate and inverse regulation of Agouti and Corin renders pelage pigmentation sensitive to changes in β-catenin activity in the DP that do not alter pelage structure. As a result, the signals that specify two biologically distinct quantitative traits are partially uncoupled despite their common regulation by the β-catenin pathway in the same cells. PMID:21098273

Characterization of a candidate tetravalent vaccine based on 2'-O-methyltransferase mutants

PubMed Central

Züst, Roland; Li, Shi-Hua; Xie, Xuping; Velumani, Sumathy; Chng, Melissa; Toh, Ying-Xiu; Zou, Jing; Dong, Hongping; Shan, Chao; Pang, Jassia; Qin, Cheng-Feng; Newell, Evan W.; Shi, Pei-Yong

2018-01-01

Dengue virus (DENV) is one of the most widespread arboviruses. The four DENV serotypes infect about 400 million people every year, causing 96 million clinical dengue cases, of which approximately 500’000 are severe and potentially life-threatening. The only licensed vaccine has a limited efficacy and is only recommended in regions with high endemicity. We previously reported that 2’-O-methyltransferase mutations in DENV-1 and DENV-2 block their capacity to inhibit type I IFNs and render the viruses attenuated in vivo, making them amenable as vaccine strains; here we apply this strategy to all four DENV serotypes to generate a tetravalent, non-chimeric live-attenuated dengue vaccine. 2’-O-methyltransferase mutants of all four serotypes are highly sensitive to type I IFN inhibition in human cells. The tetravalent formulation is attenuated and immunogenic in mice and cynomolgus macaques and elicits a response that protects from virus challenge. These results show the potential of 2’-O-methyltransferase mutant viruses as a safe, tetravalent, non-chimeric dengue vaccine. PMID:29298302

Development of satellite borne nickel hydrogen battery experiment equipment for ETS-6

NASA Astrophysics Data System (ADS)

Kuwashima, Saburou; Kamimori, Norimitsu; Kusawake, Hiroaki; Takahashi, Kazumichi

1992-08-01

An overview of the support rendered for the Engineering Test Satellite-6 (ETS-6) system integration test and protoflight test by the ETS-6 borne experimental nickel hydrogen battery development part is presented. Articles in the ETS-6 specifications and procedures related to the experimental battery were prepared or supported in preparation because of the battery's special characteristics such as its automatic control dependency on the bus voltage, thermal sensitivity equivalent to that of other batteries and so forth. System tests were witnessed and the acquired data were evaluated. Charging characteristics from 0 V were verified at trickle charging rate, using a flight scale model of Nickel Hydrogen (Ni-H2) Battery (NHB) after long term storage and an engineering model of the Ni-H2 Battery Controller (NHC). Requests for approval were submitted to the related self governing bodies in accordance with the Explosives Control Law when NHB's were charged and discharged. Installation and calibration data acquisition of the inner pressure sensors for the Ni-H2 battery cells for the flight model NHB were conducted and the battery assembly was started.

Low-temperature electrodeposition approach leading to robust mesoscopic anatase TiO2 films

NASA Astrophysics Data System (ADS)

Patra, Snehangshu; Andriamiadamanana, Christian; Tulodziecki, Michal; Davoisne, Carine; Taberna, Pierre-Louis; Sauvage, Frédéric

2016-02-01

Anatase TiO2, a wide bandgap semiconductor, likely the most worldwide studied inorganic material for many practical applications, offers unequal characteristics for applications in photocatalysis and sun energy conversion. However, the lack of controllable, cost-effective methods for scalable fabrication of homogeneous thin films of anatase TiO2 at low temperatures (ie. < 100 °C) renders up-to-date deposition processes unsuited to flexible plastic supports or to smart textile fibres, thus limiting these wearable and easy-to-integrate emerging technologies. Here, we present a very versatile template-free method for producing robust mesoporous films of nanocrystalline anatase TiO2 at temperatures of/or below 80 °C. The individual assembly of the mesoscopic particles forming ever-demonstrated high optical quality beads of TiO2 affords, with this simple methodology, efficient light capture and confinement into the photo-anode, which in flexible dye-sensitized solar cell technology translates into a remarkable power conversion efficiency of 7.2% under A.M.1.5G conditions.

Low-temperature electrodeposition approach leading to robust mesoscopic anatase TiO2 films

PubMed Central

Patra, Snehangshu; Andriamiadamanana, Christian; Tulodziecki, Michal; Davoisne, Carine; Taberna, Pierre-Louis; Sauvage, Frédéric

2016-01-01

Anatase TiO2, a wide bandgap semiconductor, likely the most worldwide studied inorganic material for many practical applications, offers unequal characteristics for applications in photocatalysis and sun energy conversion. However, the lack of controllable, cost-effective methods for scalable fabrication of homogeneous thin films of anatase TiO2 at low temperatures (ie. < 100 °C) renders up-to-date deposition processes unsuited to flexible plastic supports or to smart textile fibres, thus limiting these wearable and easy-to-integrate emerging technologies. Here, we present a very versatile template-free method for producing robust mesoporous films of nanocrystalline anatase TiO2 at temperatures of/or below 80 °C. The individual assembly of the mesoscopic particles forming ever-demonstrated high optical quality beads of TiO2 affords, with this simple methodology, efficient light capture and confinement into the photo-anode, which in flexible dye-sensitized solar cell technology translates into a remarkable power conversion efficiency of 7.2% under A.M.1.5G conditions. PMID:26911529

A gene cassette for adapting Escherichia coli strains as hosts for att-Int-mediated rearrangement and pL expression vectors.

PubMed

Balakrishnan, R; Bolten, B; Backman, K C

1994-01-28

A cassette of genes from bacteriophage lambda, when carried on a derivative of bacteriophage Mu, renders strains of Escherichia coli (and in principle other Mu-sensitive bacteria) capable of supporting lambda-based expression vectors, such as rearrangement vectors and pL vectors. The gene cassette contains a temperature-sensitive allele of the repressor gene, cIts857, and a shortened leftward operon comprising, oLpL, N, xis and int. Transfection and lysogenization of this cassette into various host bacteria is mediated by phage Mu functions. Examples of regulated expression of the gene encoding T4 DNA ligase are presented.

On-fiber plasmonic interferometer for multi-parameter sensing

DOE PAGES

Zhang, Zhijian; Chen, Yongyao; Liu, Haijun; ...

2015-01-01

We demonstrate a novel miniature multi-parameter sensing device based on a plasmonic interferometer fabricated on a fiber facet in the optical communication wavelength range. This device enables the coupling between surface plasmon resonance and plasmonic interference in the structure, which are the two essential mechanisms for multi-parameter sensing. We experimentally show that these two mechanisms have distinctive responses to temperature and refractive index, rendering the device the capability of simultaneous temperature and refractive index measurement on an ultra-miniature form factor. A high refractive index sensitivity of 220 nm per refractive index unit (RIU) and a high temperature sensitivity of –60more » pm/ °C is achieved with our device.« less

Mechanisms of Resistance to Chemotherapies Targeting BRCA-Mutant Breast Cancer

DTIC Science & Technology

2016-12-01

such as 53BP1, restored normal HR activity in BRCA1 deficient cells and rendered these cells resistant to chemotherapeutic agents. Under the aegis...NHEJ (defined as NHEJ conducted during S phase which typically results in genome instability). We found that the anti-resection activities of 53BP1...for the recruitment of the PALB2/BRCA2 complex that may be dependent on the ubiquitin ligase activity of RNF168. Specifically, we are interested in

Novel Electromagnetic - Ultrasound Synergistic Technique for Treatment of Cancer

DTIC Science & Technology

2013-11-01

vaporized droplets [1, 4]. Emulsions of perfluorocarbon droplets (lipid coated , C5F12, Ø 2.0±0.1 µm, ~99% < 8 µm Ø) were used to create thermal agents...Hyperthermia and thermal ablation are thermal therapies in which the cytotoxic effects of elevated temperatures in tissue are induced to achieve cell death...or render the cells more vulnerable to ionizing radiation and chemical toxins. In application of a thermal ablation approach, high intensity focused

Development of tumor-targeted near infrared probes for fluorescence guided surgery.

PubMed

Kelderhouse, Lindsay E; Chelvam, Venkatesh; Wayua, Charity; Mahalingam, Sakkarapalayam; Poh, Scott; Kularatne, Sumith A; Low, Philip S

2013-06-19

Complete surgical resection of malignant disease is the only reliable method to cure cancer. Unfortunately, quantitative tumor resection is often limited by a surgeon's ability to locate all malignant disease and distinguish it from healthy tissue. Fluorescence-guided surgery has emerged as a tool to aid surgeons in the identification and removal of malignant lesions. While nontargeted fluorescent dyes have been shown to passively accumulate in some tumors, the resulting tumor-to-background ratios are often poor, and the boundaries between malignant and healthy tissues can be difficult to define. To circumvent these problems, our laboratory has developed high affinity tumor targeting ligands that bind to receptors that are overexpressed on cancer cells and deliver attached molecules selectively into these cells. In this study, we explore the use of two tumor-specific targeting ligands (i.e., folic acid that targets the folate receptor (FR) and DUPA that targets prostate specific membrane antigen (PSMA)) to deliver near-infrared (NIR) fluorescent dyes specifically to FR and PSMA expressing cancers, thereby rendering only the malignant cells highly fluorescent. We report here that all FR- and PSMA-targeted NIR probes examined bind cultured cancer cells in the low nanomolar range. Moreover, upon intravenous injection into tumor-bearing mice with metastatic disease, these same ligand-NIR dye conjugates render receptor-expressing tumor tissues fluorescent, enabling their facile resection with minimal contamination from healthy tissues.

Recognition of the Species of Origin of Cells in Culture by Mixed Agglutination

PubMed Central

Coombs, R. R. A.; Daniel, Mary R.; Gurner, B. W.; Kelus, A.

1961-01-01

Preliminary experiment on the mixed agglutination reaction suggests that this reaction will afford a useful method for identifying the species of origin of cells maintained in culture. The reaction depends on the presence of antigens characteristic of the species, common to both tissue cells and red cells. Culture cells derived from man, ox, pig and rat could be distinguished one from the other. Fibroblasts of the mouse may be differentiated from those of the rat by means of a rat anti-mouse red-cell serum or a mouse anti-rat red-cell serum. Experiments are reported on trial absorption procedures to render the sera completely species-specific in their reactions. ImagesFIG. 1 PMID:13695283

Anticarcinogenesis by dietary phytochemicals: cytoprotection by Nrf2 in normal cells and cytotoxicity by modulation of transcription factors NF-kappa B and AP-1 in abnormal cancer cells.

PubMed

Gopalakrishnan, Avanthika; Tony Kong, Ah-Ng

2008-04-01

Cancer statistics from the American Cancer Society and other sources are a stark reminder of our failure to combat this deadly disease. Chemoprevention entails the use of specific naturally occurring dietary or synthetic agents to thwart cancer development and progression. Some of these agents are believed to do so by protecting the cells or tissues from the malicious attack of exogenous carcinogens and/or endogenous reactive oxygen/nitrogen species (RONS) by inducing several detoxifying/antioxidant enzymes that appear to form stable conjugates such as glutathione, glucuronides or sulfates thus rendering the carcinogenic species harmless. This process of inducing the cellular defense enzymes is believed to be mediated by the antioxidant response elements (ARE) within the promoter regions of these genes. Nrf2, a redox sensitive transcription factor has been documented to play a central role in ARE-driven gene expression. Nrf2, under normal unstimulated conditions, remains sequestered in the cytosol by Keap1. The putative chemopreventive agents disrupt the Nrf2-Keap1 association, thereby releasing Nrf2 which then translocates to the nucleus and drives the gene expression of detoxifying enzymes. The role of other transcription factors such as NF-kappaB and AP-1 in carcinogenesis is well established. By modulating the activity of these transcription factors and their upstream signaling molecules, naturally occurring dietary phytochemicals appear to cause apoptosis in abnormal cells that over-express these factors, thereby inhibiting the promotion and progression. This review discusses the most current and up to date understanding of the possible signaling mechanisms by which these naturally dietary phytochemicals can differentially modulate signal transduction cascades such that they can bring about apoptosis/cell death in abnormal cancer cells but at the same time induce defensive enzymes to protect against carcinogenesis in normal cells.

Radiosensitivity of Patient-Derived Glioma Stem Cell 3-Dimensional Cultures to Photon, Proton, and Carbon Irradiation

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

Chiblak, Sara; Tang, Zili; Molecular and Translational Radiation Oncology, Heidelberg Ion Therapy Center, Heidelberg Institute of Radiation Oncology, University of Heidelberg Medical School and National Center for Tumor Diseases, German Cancer Research Center, Heidelberg

Purpose: To investigate the radiosensitivity of primary glioma stem cell (GSC) cultures with different CD133 status in a 3-dimensional (3D) model after photon versus proton versus carbon irradiation. Methods and Materials: Human primary GSC spheroid cultures were established from tumor specimens of six consented glioblastoma patients. Human U87MG was used as a classical glioblastoma radioresistant cell line. Cell suspensions were generated by mechanical dissociation of GSC spheroids and embedded in a semi-solid 3D matrix before irradiation. Spheroid-like colonies were manually counted by microscopy. Cells were also recovered and quantified by fluorescence. CD133 expression and DNA damage were evaluated by flow cytometry.more » Results: The fraction of CD133{sup +} cells varied between 0.014% and 96% in the six GSC cultures and showed a nonsignificant correlation with plating efficiency and survival fractions. The 4 most photon-radioresistant GSC cultures were NCH644, NCH421k, NCH441, and NCH636. Clonogenic survival for proton irradiation revealed relative biologic effectiveness (RBE) in the range of 0.7-1.20. However, carbon irradiation rendered the photon-resistant GSC cultures sensitive, with average RBE of 1.87-3.44. This effect was partly attributed to impaired capability of GSC to repair carbon ion–induced DNA double-strand breaks as determined by residual DNA repair foci. Interestingly, radiosensitivity of U87 cells was comparable to GSC cultures using clonogenic survival as the standard readout. Conclusions: Carbon irradiation is effective in GSC eradication with similar RBE ranges approximately 2-3 as compared with non-stem GSC cultures (U87). Our data strongly suggest further exploration of GSC using classic radiobiology endpoints such as the here-used 3D clonogenic survival assay and integration of additional GSC-specific markers.« less

The Role of FcRn in Antigen Presentation

PubMed Central

Baker, Kristi; Rath, Timo; Pyzik, Michal; Blumberg, Richard S.

2014-01-01

Immunoglobulins are unique molecules capable of simultaneously recognizing a diverse array of antigens and themselves being recognized by a broad array of receptors. The abundance specifically of the IgG subclass and the variety of signaling receptors to which it binds render this an important immunomodulatory molecule. In addition to the classical Fcγ receptors that bind IgG at the cell surface, the neonatal Fc receptor (FcRn) is a lifelong resident of the endolysosomal system of most hematopoietic cells where it determines the intracellular fate of both IgG and IgG-containing immune complexes (IgG IC). Cross-linking of FcRn by multivalent IgG IC within antigen presenting cells such as dendritic cells initiates specific mechanisms that result in trafficking of the antigen-bearing IgG IC into compartments from which the antigen can successfully be processed into peptide epitopes compatible with loading onto both major histocompatibility complex class I and II molecules. In turn, this enables the synchronous activation of both CD4+ and CD8+ T cell responses against the cognate antigen, thereby bridging the gap between the humoral and cellular branches of the adaptive immune response. Critically, FcRn-driven T cell priming is efficient at very low doses of antigen due to the exquisite sensitivity of the IgG-mediated antigen delivery system through which it operates. FcRn-mediated antigen presentation has important consequences in tissue compartments replete with IgG and serves not only to determine homeostatic immune activation at a variety of sites but also to induce inflammatory responses upon exposure to antigens perceived as foreign. Therapeutically targeting the pathway by which FcRn enables T cell activation in response to IgG IC is thus a highly attractive prospect not only for the treatment of diseases that are driven by immune complexes but also for manipulating local immune responses against defined antigens such as those present during infections and cancer. PMID:25221553

Connections between cadherin-catenin proteins, spindle misorientation, and cancer

PubMed Central

Shahbazi, Marta N; Perez-Moreno, Mirna

2015-01-01

Cadherin-catenin mediated adhesion is an important determinant of tissue architecture in multicellular organisms. Cancer progression and maintenance is frequently associated with loss of their expression or functional activity, which not only leads to decreased cell-cell adhesion, but also to enhanced tumor cell proliferation and loss of differentiated characteristics. This review is focused on the emerging implications of cadherin-catenin proteins in the regulation of polarized divisions through their connections with the centrosomes, cytoskeleton, tissue tension and signaling pathways; and illustrates how alterations in cadherin-catenin levels or functional activity may render cells susceptible to transformation through the loss of their proliferation-differentiation balance. PMID:26451345

NKG2D and its ligands in cancer.

PubMed

Dhar, Payal; Wu, Jennifer D

2018-04-01

NKG2D is an activating immune receptor expressed by NK and effector T cells. Induced expression of NKG2D ligand on tumor cell surface during oncogenic insults renders cancer cells susceptible to immune destruction. In advanced human cancers, tumor cells shed NKG2D ligand to produce an immune soluble form as a means of immune evasion. Soluble NKG2D ligands have been associated with poor clinical prognosis in cancer patients. Harnessing NKG2D pathway is considered a viable avenue in cancer immunotherapy over recent years. In this review, we will discuss the progress and perspectives. Copyright © 2018. Published by Elsevier Ltd.

Preliminary Study on Intrasplenic Implantation of Artificial Cell Bioencapsulated Stem Cells to Increase the Survival of 90% Hepatectomized Rats

PubMed Central

Liu, Zun Chang; Chang, Thomas M.S.

2012-01-01

We implanted artificial cell bioencapsulated bone marrow mesenchymal stem cells into the spleens of 90% hepatectomized (PH) rats. The resulting 14 days survival rate was 91%. This is compared to a survival rate of 21% in 90% hepatectomized rats and 25% for those receiving free MSCs transplanted the same way. Unlike free MSCs, the bioencapsulated MSCs are retained in the spleens and their hepatotrophic factors can continue to drain directly into the liver without dilution resulting in improved hepatic regeneration. In addition, with time the transdifferentiation of MSCs into hepatocyte-like cells in the spleen renders the spleen as a ectopic liver support. PMID:19132579

Splenic red pulp macrophages are intrinsically superparamagnetic and contaminate magnetic cell isolates.

PubMed

Franken, Lars; Klein, Marika; Spasova, Marina; Elsukova, Anna; Wiedwald, Ulf; Welz, Meike; Knolle, Percy; Farle, Michael; Limmer, Andreas; Kurts, Christian

2015-08-11

A main function of splenic red pulp macrophages is the degradation of damaged or aged erythrocytes. Here we show that these macrophages accumulate ferrimagnetic iron oxides that render them intrinsically superparamagnetic. Consequently, these cells routinely contaminate splenic cell isolates obtained with the use of MCS, a technique that has been widely used in immunological research for decades. These contaminations can profoundly alter experimental results. In mice deficient for the transcription factor SpiC, which lack red pulp macrophages, liver Kupffer cells take over the task of erythrocyte degradation and become superparamagnetic. We describe a simple additional magnetic separation step that avoids this problem and substantially improves purity of magnetic cell isolates from the spleen.

Mammalian sex chromosomes. III. Activity of pseudoautosomal steroid sulfatase enzyme during spermatogenesis in Mus musculus.

PubMed

Raman, R; Das, P

1991-09-01

Parallel to the inactivation of the X chromosome in somatic cells of female, the male X in mammals is rendered inactive during spermatogenesis. Pseudoautosomal genes, those present on the X-Y meiotically pairable region of male, escape inactivation in female soma. It is suggested, but not demonstrated, that they may also be refractory to the inactivation signal in male germ cells. We have assayed activity of the enzyme steroid sulfatase, product of a pseudoautosomal gene, in testicular cells of the mouse and shown its presence in premeiotic, meiotic (pachytene), and postmeiotic (spermatid) cell types. It appears that, as in females, pseudoautosomal genes may escape inactivation in male germ cells also.

Public Health Implications of the 1540 nm Laser on the Cornea

DTIC Science & Technology

2007-05-30

film layer. The epithelium has numerous nerve endings that make the cornea extremely sensitive to pain when an injury occurs. This pain reflex helps...used in research by using techniques and procedures that reduce distress and pain The Three R’s have lead researchers to look for alternative models...cause severe pain and render a person incapable of working. Injury to the cornea may cause temporary to permanent vision impairment depending on how

Collection of ions

DOEpatents

Orr, Christopher Henry; Luff, Craig Janson; Dockray, Thomas; Macarthur, Duncan Whittemore; Bounds, John Alan; Koster, James E.

2001-01-01

The apparatus and method provide an improved technique for detecting ions as the area from which ions are attracted to a detector is increased, consequently increasing the number of ions detected. This is achieved by providing the outer electrodes of the detector connected to the electrical potential, together with alternate intermediate electrodes. The other intermediate electrodes and preferably the housing are grounded. The technique renders such detection techniques more sensitive and gives them a lower threshold at which they can function.

Wheat-based foods and non celiac gluten/wheat sensitivity: Is drastic processing the main key issue?

PubMed

Fardet, Anthony

2015-12-01

While gluten and wheat must be absolutely avoided in coeliac disease and allergy, respectively, nutritional recommendations are largely more confused about non-coeliac wheat/gluten sensitivity (NCWGS). Today, some even recommend avoiding all cereal-based foods. In this paper, the increased NCWGS prevalence is hypothesized to parallel the use of more and more drastic processes applied to the original wheat grain. First, a parallel between gluten-related disorders and wheat processing and consumption evolution is briefly proposed. Notably, increased use of exogenous vital gluten is considered. Drastic processing in wheat technology are mainly grain fractionation and refining followed by recombination and salt, sugars and fats addition, being able to render ultra-processed cereal-based foods more prone to trigger chronic low-grade inflammation. Concerning bread, intensive kneading and the choice of wheat varieties with high baking quality may have rendered gluten less digestible, moving digestion from pancreatic to intestinal proteases. The hypothesis of a gluten resistant fraction reaching colon and interacting with microflora is also considered in relation with increased inflammation. Besides, wheat flour refining removes fiber co-passenger which have potential anti-inflammatory property able to protect digestive epithelium. Finally, some research tracks are proposed, notably the comparison of NCWGS prevalence in populations consuming ultra-versus minimally-processed cereal-based foods. Copyright © 2015 Elsevier Ltd. All rights reserved.

RenderToolbox3: MATLAB tools that facilitate physically based stimulus rendering for vision research.

PubMed

Heasly, Benjamin S; Cottaris, Nicolas P; Lichtman, Daniel P; Xiao, Bei; Brainard, David H

2014-02-07

RenderToolbox3 provides MATLAB utilities and prescribes a workflow that should be useful to researchers who want to employ graphics in the study of vision and perhaps in other endeavors as well. In particular, RenderToolbox3 facilitates rendering scene families in which various scene attributes and renderer behaviors are manipulated parametrically, enables spectral specification of object reflectance and illuminant spectra, enables the use of physically based material specifications, helps validate renderer output, and converts renderer output to physical units of radiance. This paper describes the design and functionality of the toolbox and discusses several examples that demonstrate its use. We have designed RenderToolbox3 to be portable across computer hardware and operating systems and to be free and open source (except for MATLAB itself). RenderToolbox3 is available at https://github.com/DavidBrainard/RenderToolbox3.

The Polar and Electrical Nature of Dye Binding Sites on Human Red Blood Cell Membranes.

DTIC Science & Technology

positive charges at the binding sites. By increasing the concentration of the anionic BPB (or by the addition of the anionic detergent sodium lauryl ... sulfate ) these positive charges appear to be successively titrated, rendering the membrane binding sites electrically neutral at this pH. The average

I: Biomarker quantification in fish exposed to crude oil as input to species sensitivity distributions and threshold values for environmental monitoring.

PubMed

Sanni, Steinar; Björkblom, Carina; Jonsson, Henrik; Godal, Brit F; Liewenborg, Birgitta; Lyng, Emily; Pampanin, Daniela M

2017-04-01

The aim of this study was to determine a suitable set of biomarker based methods for environmental monitoring in sub-arctic and temperate offshore areas using scientific knowledge on the sensitivity of fish species to dispersed crude oil. Threshold values for environmental monitoring and risk assessment were obtained based on a quantitative comparison of biomarker responses. Turbot, halibut, salmon and sprat were exposed for up to 8 weeks to five different sub-lethal concentrations of dispersed crude oil. Biomarkers assessing PAH metabolites, oxidative stress, detoxification system I activity, genotoxicity, immunotoxicity, endocrine disruption, general cellular stress and histological changes were measured. Results showed that PAH metabolites, CYP1A/EROD, DNA adducts and histopathology rendered the most robust results across the different fish species, both in terms of sensitivity and dose-responsiveness. The reported results contributed to forming links between biomonitoring and risk assessment procedures by using biomarker species sensitivity distributions. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Crystal Structure of Peroxiredoxin Asp f3 Provides Mechanistic Insight into Oxidative Stress Resistance and Virulence of Aspergillus fumigatus.

PubMed

Hillmann, Falk; Bagramyan, Karine; Straßburger, Maria; Heinekamp, Thorsten; Hong, Teresa B; Bzymek, Krzysztof P; Williams, John C; Brakhage, Axel A; Kalkum, Markus

2016-09-14

Invasive aspergillosis and other fungal infections occur in immunocompromised individuals, including patients who received blood-building stem cell transplants, patients with chronic granulomatous disease (CGD), and others. Production of reactive oxygen species (ROS) by immune cells, which incidentally is defective in CGD patients, is considered to be a fundamental process in inflammation and antifungal immune response. Here we show that the peroxiredoxin Asp f3 of Aspergillus fumigatus inactivates ROS. We report the crystal structure and the catalytic mechanism of Asp f3, a two-cysteine type peroxiredoxin. The latter exhibits a thioredoxin fold and a homodimeric structure with two intermolecular disulfide bonds in its oxidized state. Replacement of the Asp f3 cysteines with serine residues retained its dimeric structure, but diminished Asp f3's peroxidase activity, and extended the alpha-helix with the former peroxidatic cysteine residue C61 by six residues. The asp f3 deletion mutant was sensitive to ROS, and this phenotype was rescued by ectopic expression of Asp f3. Furthermore, we showed that deletion of asp f3 rendered A. fumigatus avirulent in a mouse model of pulmonary aspergillosis. The conserved expression of Asp f3 homologs in medically relevant molds and yeasts prompts future evaluation of Asp f3 as a potential therapeutic target.

Glowing locked nucleic acids: brightly fluorescent probes for detection of nucleic acids in cells.

PubMed

Østergaard, Michael E; Cheguru, Pallavi; Papasani, Madhusudhan R; Hill, Rodney A; Hrdlicka, Patrick J

2010-10-13

Fluorophore-modified oligonucleotides have found widespread use in genomics and enable detection of single-nucleotide polymorphisms, real-time monitoring of PCR, and imaging of mRNA in living cells. Hybridization probes modified with polarity-sensitive fluorophores and molecular beacons (MBs) are among the most popular approaches to produce hybridization-induced increases in fluorescence intensity for nucleic acid detection. In the present study, we demonstrate that the 2'-N-(pyren-1-yl)carbonyl-2'-amino locked nucleic acid (LNA) monomer X is a highly versatile building block for generation of efficient hybridization probes and quencher-free MBs. The hybridization and fluorescence properties of these Glowing LNA probes are efficiently modulated and optimized by changes in probe backbone chemistry and architecture. Correctly designed probes are shown to exhibit (a) high affinity toward RNA targets, (b) excellent mismatch discrimination, (c) high biostability, and (d) pronounced hybridization-induced increases in fluorescence intensity leading to formation of brightly fluorescent duplexes with unprecedented emission quantum yields (Φ(F) = 0.45-0.89) among pyrene-labeled oligonucleotides. Finally, specific binding between messenger RNA and multilabeled quencher-free MBs based on Glowing LNA monomers is demonstrated (a) using in vitro transcription assays and (b) by quantitative fluorometric assays and direct microscopic observation of probes bound to mRNA in its native form. These features render Glowing LNA as promising diagnostic probes for biomedical applications.

Toxoplasma gondii Relies on Both Host and Parasite Isoprenoids and Can Be Rendered Sensitive to Atorvastatin

PubMed Central

Li, Zhu-Hong; Ramakrishnan, Srinivasan; Striepen, Boris; Moreno, Silvia N. J.

2013-01-01

Intracellular pathogens have complex metabolic interactions with their host cells to ensure a steady supply of energy and anabolic building blocks for rapid growth. Here we use the obligate intracellular parasite Toxoplasma gondii to probe this interaction for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Synthesis of precursors for isoprenoids in Apicomplexa occurs in the apicoplast and is essential. To synthesize longer isoprenoids from these precursors, T. gondii expresses a bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase (TgFPPS). In this work we construct and characterize T. gondii null mutants for this enzyme. Surprisingly, these mutants have only a mild growth phenotype and an isoprenoid composition similar to wild type parasites. However, when extracellular, the loss of the enzyme becomes phenotypically apparent. This strongly suggests that intracellular parasite salvage FPP and/or geranylgeranyl diphosphate (GGPP) from the host. We test this hypothesis using inhibitors of host cell isoprenoid synthesis. Mammals use the mevalonate pathway, which is susceptible to statins. We document strong synergy between statin treatment and pharmacological or genetic interference with the parasite isoprenoid pathway. Mice can be cured with atorvastatin (Lipitor) from a lethal infection with the TgFPPs mutant. We propose a double-hit strategy combining inhibitors of host and parasite pathways as a novel therapeutic approach against Apicomplexan parasites. PMID:24146616

Autophagy-deficient breast cancer shows early tumor recurrence and escape from dormancy

PubMed Central

Aqbi, Hussein F.; Tyutyunyk-Massey, Liliya; Keim, Rebecca C.; Butler, Savannah E.; Thekkudan, Theresa; Joshi, Supriya; Smith, Timothy M.; Bandyopadhyay, Dipankar; Idowu, Michael O.; Bear, Harry D.; Payne, Kyle K.; Gewirtz, David A.; Manjili, Masoud H.

2018-01-01

Breast cancer patients who initially respond to cancer therapies often succumb to distant recurrence of the disease. It is not clear why people with the same type of breast cancer respond to treatments differently; some escape from dormancy and relapse earlier than others. In addition, some tumor clones respond to immunotherapy while others do not. We investigated how autophagy plays a role in accelerating or delaying recurrence of neu-overexpressing mouse mammary carcinoma (MMC) following adriamycin (ADR) treatment, and in affecting response to immunotherapy. We explored two strategies: 1) transient blockade of autophagy with chloroquine (CQ), which blocks fusion of autophagosomes and lysosomes during ADR treatment, and 2) permanent inhibition of autophagy by a stable knockdown of ATG5 (ATG5KD), which inhibits the formation of autophagosomes in MMC during and after ADR treatment. We found that while CQ prolonged tumor dormancy, but that stable knockdown of autophagy resulted in early escape from dormancy and recurrence. Interestingly, ATG5KD MMC contained an increased frequency of ADR-induced polyploid-like cells and rendered MMC resistant to immunotherapy. On the other hand, a transient blockade of autophagy did not affect the sensitivity of MMC to immunotherapy. Our observations suggest that while chemotherapy-induced autophagy may facilitate tumor relapse, cell-intrinsic autophagy delays tumor relapse, in part, by inhibiting the formation of polyploid-like tumor dormancy. PMID:29774126

Toxoplasma gondii relies on both host and parasite isoprenoids and can be rendered sensitive to atorvastatin.

PubMed

Li, Zhu-Hong; Ramakrishnan, Srinivasan; Striepen, Boris; Moreno, Silvia N J

2013-01-01

Intracellular pathogens have complex metabolic interactions with their host cells to ensure a steady supply of energy and anabolic building blocks for rapid growth. Here we use the obligate intracellular parasite Toxoplasma gondii to probe this interaction for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Synthesis of precursors for isoprenoids in Apicomplexa occurs in the apicoplast and is essential. To synthesize longer isoprenoids from these precursors, T. gondii expresses a bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase (TgFPPS). In this work we construct and characterize T. gondii null mutants for this enzyme. Surprisingly, these mutants have only a mild growth phenotype and an isoprenoid composition similar to wild type parasites. However, when extracellular, the loss of the enzyme becomes phenotypically apparent. This strongly suggests that intracellular parasite salvage FPP and/or geranylgeranyl diphosphate (GGPP) from the host. We test this hypothesis using inhibitors of host cell isoprenoid synthesis. Mammals use the mevalonate pathway, which is susceptible to statins. We document strong synergy between statin treatment and pharmacological or genetic interference with the parasite isoprenoid pathway. Mice can be cured with atorvastatin (Lipitor) from a lethal infection with the TgFPPs mutant. We propose a double-hit strategy combining inhibitors of host and parasite pathways as a novel therapeutic approach against Apicomplexan parasites.

Diabetes Insipidus in Mice with a Mutation in Aquaporin-2

PubMed Central

Lloyd, David J; Hall, Frank Wesley; Tarantino, Lisa M; Gekakis, Nicholas

2005-01-01

Congenital nephrogenic diabetes insipidus (NDI) is a disease characterized by failure of the kidney to concentrate urine in response to vasopressin. Human kindreds with nephrogenic diabetes insipidus have been found to harbor mutations in the vasopressin receptor 2 (Avpr2) gene or the vasopressin-sensitive water channel aquaporin-2 (Aqp2) gene. Development of a treatment is rendered difficult due to the lack of a viable animal model. Through forward genetic screening of ethylnitrosourea-mutagenized mice, we report the identification and characterization of a mouse model of NDI, with an F204V mutation in the Aqp2 gene. Unlike previously attempted murine models of NDI, our mice survive to adulthood and more exactly recapitulate the human disorder. Previous in vitro experiments using renal cell lines suggest recessive Aqp2 mutations result in improper trafficking of the mutant water pore. Using these animals, we have directly proven this hypothesis of improper AQP2 translocation as the molecular defect in nephrogenic diabetes insipidus in the intact organism. Additionally, using a renal cell line we show that the mutated protein, AQP2-F204V, is retained in the endoplasmic reticulum and that this abnormal localization can be rescued by wild-type protein. This novel mouse model allows for further mechanistic studies as well as testing of pharmacological and gene therapies for NDI. PMID:16121255

Malignant granular cell tumors: the role of electron microscopy in the definitive diagnosis of an extremely aggressive soft tissue neoplasm.

PubMed

Knowles, Kurt J; Al-Delfi, Firas; Abdulsattar, Jehan; Lacour, Robin; Black, Destin; Chaudhery, Shabnum; Turbat-Herrera, Elba A

2018-01-01

Granular cell tumors (GCTs) are rare soft tissue neoplasms which may be multicentric. The vast majority are benign, however approximately 100 malignant GCTs have been reported, with only 8 originating in the vulva. Malignant GCTs are very aggressive with very poor survival rates. As the diagnosis of malignant GCT carries an extremely poor prognosis, the utilization of EM ensures that the most accurate diagnosis possible can be rendered.

Sine Oculis Homeobox Homolog 1 Regulates Mitochondrial Apoptosis Pathway Via Caspase-7 In Gastric Cancer Cells.

PubMed

Du, Peizhun; Zhao, Jing; Wang, Jing; Liu, Yongchao; Ren, Hong; Patel, Rajan; Hu, Cheng'en; Zhang, Wenhong; Huang, Guangjian

2017-01-01

Sine oculis homeobox homolog 1 (Six1) is crucial in normal organ development. Recently, Six1 is reported to display aberrant expression in various cancers and plays important roles in cancer development. However, the regulatory mechanism of Six1 in gastric cancer is largely unknown. In the current study, we found that Six1 was increased in gastric cancer tissues, and its upregulation significantly associated with lymph node metastasis (p=0.042) and poor differentiation (p=0.039). Next, we took advantage of public available microarray data to assess Six1 prognostic value with online K-M Plotter software in gastric cancer, which demonstrated that patients with higher Six1 expression had shorter survival time (p=0.02). To explore the underlying mechanism of Six1, we silenced its upregulation in gastric cells to detect cellular functions. Our results indicated that knock-down Six1 could decrease colony formation number and rendered cells sensitive to 5- Fluorouracil drug treatment. The flow cytometry analyses showed that Six1 silence could promote apoptosis but had little effect on cell cycle transition. Along this clue, we tested mitochondrial membrane potential with JC-1 assay, which suggested that Six1 inhibition could trigger mitochondrial apoptosis. Our subsequent results revealed that Six1 knock-down could reduce the level of anti-apoptotic protein Bcl-2, and caspase-7 but not caspase-3 was involved to execute the mitochondrial apoptosis pathway. Taken together, we find Six1 has oncogenic role in gastric cancer development, and silenced Six1 expression can promote mitochondrial apoptosis by repressing Bcl-2 and activating executor caspase-7. These findings suggest that Six1 may become a valuable prognostic and therapeutic target in gastric cancer.

Sine Oculis Homeobox Homolog 1 Regulates Mitochondrial Apoptosis Pathway Via Caspase-7 In Gastric Cancer Cells

PubMed Central

Du, Peizhun; Zhao, Jing; Wang, Jing; Liu, Yongchao; Ren, Hong; Patel, Rajan; Hu, Cheng'en; Zhang, Wenhong; Huang, Guangjian

2017-01-01

Sine oculis homeobox homolog 1 (Six1) is crucial in normal organ development. Recently, Six1 is reported to display aberrant expression in various cancers and plays important roles in cancer development. However, the regulatory mechanism of Six1 in gastric cancer is largely unknown. In the current study, we found that Six1 was increased in gastric cancer tissues, and its upregulation significantly associated with lymph node metastasis (p=0.042) and poor differentiation (p=0.039). Next, we took advantage of public available microarray data to assess Six1 prognostic value with online K-M Plotter software in gastric cancer, which demonstrated that patients with higher Six1 expression had shorter survival time (p=0.02). To explore the underlying mechanism of Six1, we silenced its upregulation in gastric cells to detect cellular functions. Our results indicated that knock-down Six1 could decrease colony formation number and rendered cells sensitive to 5- Fluorouracil drug treatment. The flow cytometry analyses showed that Six1 silence could promote apoptosis but had little effect on cell cycle transition. Along this clue, we tested mitochondrial membrane potential with JC-1 assay, which suggested that Six1 inhibition could trigger mitochondrial apoptosis. Our subsequent results revealed that Six1 knock-down could reduce the level of anti-apoptotic protein Bcl-2, and caspase-7 but not caspase-3 was involved to execute the mitochondrial apoptosis pathway. Taken together, we find Six1 has oncogenic role in gastric cancer development, and silenced Six1 expression can promote mitochondrial apoptosis by repressing Bcl-2 and activating executor caspase-7. These findings suggest that Six1 may become a valuable prognostic and therapeutic target in gastric cancer. PMID:28367243

A Targeted Quantitative Proteomics Strategy for Global Kinome Profiling of Cancer Cells and Tissues*

PubMed Central

Xiao, Yongsheng; Guo, Lei; Wang, Yinsheng

2014-01-01

Kinases are among the most intensively pursued enzyme superfamilies as targets for anti-cancer drugs. Large data sets on inhibitor potency and selectivity for more than 400 human kinases became available recently, offering the opportunity to design rationally novel kinase-based anti-cancer therapies. However, the expression levels and activities of kinases are highly heterogeneous among different types of cancer and even among different stages of the same cancer. The lack of effective strategy for profiling the global kinome hampers the development of kinase-targeted cancer chemotherapy. Here, we introduced a novel global kinome profiling method, based on our recently developed isotope-coded ATP-affinity probe and a targeted proteomic method using multiple-reaction monitoring (MRM), for assessing simultaneously the expression of more than 300 kinases in human cells and tissues. This MRM-based assay displayed much better sensitivity, reproducibility, and accuracy than the discovery-based shotgun proteomic method. Approximately 250 kinases could be routinely detected in the lysate of a single cell line. Additionally, the incorporation of iRT into MRM kinome library rendered our MRM kinome assay easily transferrable across different instrument platforms and laboratories. We further employed this approach for profiling kinase expression in two melanoma cell lines, which revealed substantial kinome reprogramming during cancer progression and demonstrated an excellent correlation between the anti-proliferative effects of kinase inhibitors and the expression levels of their target kinases. Therefore, this facile and accurate kinome profiling assay, together with the kinome-inhibitor interaction map, could provide invaluable knowledge to predict the effectiveness of kinase inhibitor drugs and offer the opportunity for individualized cancer chemotherapy. PMID:24520089

Mechanism-based facilitated maturation of human pluripotent stem cell-derived cardiomyocytes

PubMed Central

Lieu, Deborah K.; Fu, Ji-Dong; Chiamvimonvat, Nipavan; Chan Tung, Kelvin W.; McNerney, Gregory P.; Huser, Thomas; Keller, Gordon; Kong, Chi-Wing

2013-01-01

Background Human embryonic stem cells (hESCs) can be efficiently and reproducibly directed into cardiomyocytes (CMs) using stage-specific induction protocols. However, their functional properties and suitability for clinical and other applications have not been evaluated. Methods and Results Here we showed that CMs derived from multiple pluripotent human stem cell lines (hESC: H1, HES2) and types (induced pluripotent stem cell or iPSC) using different in vitro differentiation protocols (embryoid body formation, endodermal induction, directed differentiation) commonly displayed immature, pro-arrhythmic action potential (AP) properties such as high-degree of automaticity, depolarized resting membrane potential (RMP), Phase 4- depolarization and delayed after-depolarization (DAD). Among the panoply of sarcolemmal ionic currents investigated (INa+/ICaL2+/IKr+/INCX+/If+/Ito+/IK1-/IKs-), we pinpointed the lack of the Kir2.1-encoded inwardly rectifying K+ current (IK1) as the single mechanistic contributor to the observed immature electrophysiological properties in hESC-CMs. Forced expression of Kir2.1 in hESC-CMs led to robust expression of Ba2+-sensitive IK1 and more importantly, completely ablated all the pro-arrhythmic AP traits, rendering the electrophysiological phenotype indistinguishable from the adult counterparts. These results provided the first link of a complex developmentally arrested phenotype to a major effector gene, and importantly, further led us to develop a biomimetic culturing strategy for enhancing maturation. Conclusions By providing the environmental cues that are missing in conventional culturing method, this approach did not require any genetic or pharmacological interventions. Our findings can facilitate clinical applications, drug discovery and cardiotoxicity screening by improving the yield, safety and efficacy of derived CMs. PMID:23392582

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

Potential mechanisms of development-dependent adverse effects of the herbicide paraquat in 3D rat brain cell cultures.

PubMed

Sandström, J; Broyer, A; Zoia, D; Schilt, C; Greggio, C; Fournier, M; Do, K Q; Monnet-Tschudi, F

2017-05-01

Exposure to environmental toxicants during vulnerable windows of brain development is suspected to raise the prevalence for neurological dysfunctions at later stages in life. Differentiation processes and changes in morphology, as well as a lack of physiological barriers, might be reasons that render a developing brain more susceptible to neurotoxicants than an adult. However, also the intrinsic capacity of cells to combat toxicant induced cellular stress might differ between the immature- and mature brain. In order to study whether this intrinsic protection capacity differs between immature and maturated brain cells we chose to study the maturation-dependent adverse effects of the known neurotoxicant Paraquat Dichloride (PQ) in 3D rat brain cell cultures. This in vitro system consists of the major brain cell types - neurons, astrocytes, oligodendrocytes and microglia - and over the time in vitro cultured cells undergo differentiation and maturation into a tissue-like organization. PQ was applied repeatedly over ten days in the sub-micromolar range, and effects were evaluated on neurons and glial cells. We observed that despite a higher PQ-uptake in mature cultures, PQ-induced adverse effects on glutamatergic-, GABAergic- and dopaminergic neurons, as assessed by gene expression and enzymatic activity, were more pronounced in immature cultures. This was associated with a stronger astrogliosis in immature- as compared to mature cultures, as well as perturbations of the glutathione-mediated defense against oxidative stress. Furthermore we observed evidence of microglial activation only in mature cultures, whereas immature cultures appeared to down-regulate markers for neuroprotective M2-microglial phenotype upon PQ-exposure. Taken together our results indicate that immature brain cell cultures have less intrinsic capacity to cope with cellular stress elicited by PQ as compared to mature cells. This may render immature brain cells more susceptible to the adverse effects of PQ. Copyright © 2017 Elsevier B.V. All rights reserved.

Silicon photonic dual-gas sensor for H2 and CO2 detection.

PubMed

Mi, Guangcan; Horvath, Cameron; Van, Vien

2017-07-10

We report a silicon photonic dual-gas sensor based on a wavelength-multiplexed microring resonator array for simultaneous detection of H 2 and CO 2 gases. The sensor uses Pd as the sensing layer for H 2 gas and a novel functional material based on the Polyhexamethylene Biguanide (PHMB) polymer for CO 2 gas sensing. Gas sensing experiments showed that the PHMB-functionalized microring exhibited high sensitivity to CO 2 gas and excellent selectivity against H 2 . However, the Pd-functionalized microring was found to exhibit sensitivity to both H 2 and CO 2 gases, rendering it ineffective for detecting H 2 in a gas mixture containing CO 2 . We show that the dual-gas sensing scheme can allow for accurate measurement of H 2 concentration in the presence of CO 2 by accounting for the cross-sensitivity of Pd to the latter.

Spectral optimization simulation of white light based on the photopic eye-sensitivity curve

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

Dai, Qi, E-mail: qidai@tongji.edu.cn; Institute for Advanced Study, Tongji University, 1239 Siping Road, Shanghai 200092; Key Laboratory of Ecology and Energy-saving Study of Dense Habitat

Spectral optimization simulation of white light is studied to boost maximum attainable luminous efficacy of radiation at high color-rendering index (CRI) and various color temperatures. The photopic eye-sensitivity curve V(λ) is utilized as the dominant portion of white light spectra. Emission spectra of a blue InGaN light-emitting diode (LED) and a red AlInGaP LED are added to the spectrum of V(λ) to match white color coordinates. It is demonstrated that at the condition of color temperature from 2500 K to 6500 K and CRI above 90, such white sources can achieve spectral efficacy of 330–390 lm/W, which is higher than the previously reportedmore » theoretical maximum values. We show that this eye-sensitivity-based approach also has advantages on component energy conversion efficiency compared with previously reported optimization solutions.« less

Molecular engineering of fluorescein dyes as complementary absorbers in dye co-sensitized solar cells

DOE PAGES

Pepe, Giulio; Cole, Jacqueline M.; Waddell, Paul G.; ...

2016-09-22

Fluorescein dye derivatives exhibit extended optical absorption up to 500 nm, rendering these compounds suitable as co-absorbers in dye-sensitized solar cells (DSCs). A molecular engineering approach is presented, which embraces this intrinsic optical attribute of fluoresceins, while modifying the dye chemistry to enhance their light harvesting efficiency, in order to effectively tailor them for DSC applications. This approach first realizes relationships between the molecular structure and the optoelectronic properties for a series of five a priori known (parent) fluorescein dyes: 5-carboxyfluorescein (1), a mixture of m-carboxyfluorescein where m = 5 or 6 (2), 5-carboxyfluorescein diacetate (3), 6-carboxyfluorescein diacetate (4), amore » mixture of n-carboxy-2',7'-dichlorofluorescein diacetate where n = 5 or 6 (5). The first step in this approach combines, where available, experimental and computational methods so that electronic structure calculations can also be validated for representative fluorescein dyes. Such calculations can then be used reliably to predict the structure and properties of fluorescein dyes for cases where experimental data are lacking. Structure-function relationships established from this initial step inform the selection of parent dye 1 that is taken forward to the second step in molecular engineering: in silico chemical derivation to re-functionalize 1 for DSC applications. For this purpose, computational calculations are used to extend the charge conjugation in 1 between its donor and acceptor moieties. These structural modifications result in a bathochromic shift of the lowest excitation by ~1.3-1.9 eV (100-170 nm), making the dye optically absorb in the visible region. Further calculations on dye molecules adsorbed onto the surface of a TiO 2 cluster are used to investigate the dye sensitization behavior via dye adsorption energies and anchoring modes. The results of this theoretical investigation lead to two molecularly engineered fluoresceins being proposed to act as co-sensitizers together with a rhodamine dye. This combination of three dyes ensures chemical compatibility, panchromatic absorption, and restores optical absorption dipping otherwise observed in a DSC device at ~350-400 nm owing to the I-/I- 3 electrolyte. Altogether, the results of this study demonstrate that molecular engineering can be used to identify suitable chemical modifications for organic dyes with improved light harvesting properties for photovoltaic applications.« less

Molecular engineering of fluorescein dyes as complementary absorbers in dye co-sensitized solar cells

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

Pepe, Giulio; Cole, Jacqueline M.; Waddell, Paul G.

Fluorescein dye derivatives exhibit extended optical absorption up to 500 nm, rendering these compounds suitable as co-absorbers in dye-sensitized solar cells (DSCs). A molecular engineering approach is presented, which embraces this intrinsic optical attribute of fluoresceins, while modifying the dye chemistry to enhance their light harvesting efficiency, in order to effectively tailor them for DSC applications. This approach first realizes relationships between the molecular structure and the optoelectronic properties for a series of five a priori known (parent) fluorescein dyes: 5-carboxyfluorescein (1), a mixture of m-carboxyfluorescein where m = 5 or 6 (2), 5-carboxyfluorescein diacetate (3), 6-carboxyfluorescein diacetate (4), amore » mixture of n-carboxy-2',7'-dichlorofluorescein diacetate where n = 5 or 6 (5). The first step in this approach combines, where available, experimental and computational methods so that electronic structure calculations can also be validated for representative fluorescein dyes. Such calculations can then be used reliably to predict the structure and properties of fluorescein dyes for cases where experimental data are lacking. Structure-function relationships established from this initial step inform the selection of parent dye 1 that is taken forward to the second step in molecular engineering: in silico chemical derivation to re-functionalize 1 for DSC applications. For this purpose, computational calculations are used to extend the charge conjugation in 1 between its donor and acceptor moieties. These structural modifications result in a bathochromic shift of the lowest excitation by ~1.3-1.9 eV (100-170 nm), making the dye optically absorb in the visible region. Further calculations on dye molecules adsorbed onto the surface of a TiO 2 cluster are used to investigate the dye sensitization behavior via dye adsorption energies and anchoring modes. The results of this theoretical investigation lead to two molecularly engineered fluoresceins being proposed to act as co-sensitizers together with a rhodamine dye. This combination of three dyes ensures chemical compatibility, panchromatic absorption, and restores optical absorption dipping otherwise observed in a DSC device at ~350-400 nm owing to the I-/I- 3 electrolyte. Altogether, the results of this study demonstrate that molecular engineering can be used to identify suitable chemical modifications for organic dyes with improved light harvesting properties for photovoltaic applications.« less

Method of making biocompatible electrodes

DOEpatents

Wollam, John S.

1992-01-01

A process of improving the sensing function of biocompatible electrodes and the product so made are disclosed. The process is designed to alter the surfaces of the electrodes at their tips to provide increased surface area and therefore decreased contact resistance at the electrode-tissue interface for increased sensitivity and essentially includes rendering the tips atomically clean by exposing them to bombardment by ions of an inert gas, depositing an adhesion layer on the cleaned tips, forming a hillocked layer on the adhesion layer by increasing the temperature of the tips, and applying a biocompatible coating on the hillocked layer. The resultant biocompatible electrode is characterized by improved sensitivity, minimum voltage requirement for organ stimulation and a longer battery life for the device in which it is employed.

Virus inactivation by nucleic acid extraction reagents.

PubMed

Blow, Jamie A; Dohm, David J; Negley, Diane L; Mores, Christopher N

2004-08-01

Many assume that common methods to extract viral nucleic acids are able to render a sample non-infectious. It may be that inactivation of infectious virus is incomplete during viral nucleic acid extraction methods. Accordingly, two common viral nucleic acid extraction techniques were evaluated for the ability to inactivate high viral titer specimens. In particular, the potential for TRIzol LS Reagent (Invitrogen Corp., Carlsbad, CA) and AVL Buffer (Qiagen, Valencia, CA) were examined to render suspensions of alphaviruses, flaviviruses, filoviruses and a bunyavirus non-infectious to tissue culture assay. The dilution series for both extraction reagents consistently caused cell death through a 100-fold dilution. Except for the DEN subtype 4 positive control, all viruses had titers of at least 10(6)pfu/ml. No plaques were detected in any extraction reagent plus virus combination in this study, therefore, the extraction reagents appeared to inactivate completely each of the high-titer viruses used in this study. These results support the reliance upon either TRIzol LS Reagent or AVL Buffer to render clinical or environmental samples non-infectious, which has implications for the handling and processing of samples under austere field conditions and low level containment.

Hydrothermal synthesis of NiWO4 crystals for high performance non-enzymatic glucose biosensors

NASA Astrophysics Data System (ADS)

Mani, Sivakumar; Vediyappan, Veeramani; Chen, Shen-Ming; Madhu, Rajesh; Pitchaimani, Veerakumar; Chang, Jia-Yaw; Liu, Shang-Bin

2016-04-01

A facile hydrothermal route for the synthesis of ordered NiWO4 nanocrystals, which show promising applications as high performance non-enzymatic glucose sensor is reported. The NiWO4-modified electrodes showed excellent sensitivity (269.6 μA mM-1 cm-2) and low detection limit (0.18 μM) for detection of glucose with desirable selectivity, stability, and tolerance to interference, rendering their prospective applications as cost-effective, enzyme-free glucose sensors.

Hydrothermal synthesis of NiWO4 crystals for high performance non-enzymatic glucose biosensors.

PubMed

Mani, Sivakumar; Vediyappan, Veeramani; Chen, Shen-Ming; Madhu, Rajesh; Pitchaimani, Veerakumar; Chang, Jia-Yaw; Liu, Shang-Bin

2016-04-18

A facile hydrothermal route for the synthesis of ordered NiWO4 nanocrystals, which show promising applications as high performance non-enzymatic glucose sensor is reported. The NiWO4-modified electrodes showed excellent sensitivity (269.6 μA mM(-1 )cm(-2)) and low detection limit (0.18 μM) for detection of glucose with desirable selectivity, stability, and tolerance to interference, rendering their prospective applications as cost-effective, enzyme-free glucose sensors.

Properties of new Escherichia coli Hfr strains constructed by integration of pSC101-derived conjugative plasmids.

PubMed Central

François, V; Conter, A; Louarn, J M

1990-01-01

Conjugative temperature-sensitive plasmids were derived from pSC101. These plasmids are useful in genetic analysis for two reasons: (i) they render possible the construction of new Hfr lines by plasmid integration at predetermined chromosomal loci via Tn10 inverse transposition, and (ii) the Hfr characters are transducible via bacteriophage P1. We also showed that replication from pSC101 origin is deleterious for the plasmid-chromosome fusion. PMID:2155201

SMAD4 loss enables EGF, TGFβ1 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells.

PubMed

Moz, Stefania; Basso, Daniela; Bozzato, Dania; Galozzi, Paola; Navaglia, Filippo; Negm, Ola H; Arrigoni, Giorgio; Zambon, Carlo-Federico; Padoan, Andrea; Tighe, Paddy; Todd, Ian; Franchin, Cinzia; Pedrazzoli, Sergio; Punzi, Leonardo; Plebani, Mario

2016-10-25

Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor β1 (TGFβ1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGFβ1 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGFβ1 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/β-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGFβ1 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-κB and PI3K/AKT in response to TGFβ1 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/β-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGFβ1 and S100A8/A9 mainly inhibit NF-κB and PI3K/AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner.

SMAD4 loss enables EGF, TGFβ1 and S100A8/A9 induced activation of critical pathways to invasion in human pancreatic adenocarcinoma cells

PubMed Central

Moz, Stefania; Basso, Daniela; Bozzato, Dania; Galozzi, Paola; Navaglia, Filippo; Negm, Ola H.; Arrigoni, Giorgio; Zambon, Carlo-Federico; Padoan, Andrea; Tighe, Paddy; Todd, Ian; Franchin, Cinzia; Pedrazzoli, Sergio; Punzi, Leonardo; Plebani, Mario

2016-01-01

Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor β1 (TGFβ1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGFβ1 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGFβ1 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/β-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGFβ1 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-κB and PI3K/AKT in response to TGFβ1 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/β-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGFβ1 and S100A8/A9 mainly inhibit NF-κB and PI3K/AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner. PMID:27655713

ptf1a+ , ela3l- cells are developmentally maintained progenitors for exocrine regeneration following extreme loss of acinar cells in zebrafish larvae.

PubMed

Schmitner, Nicole; Kohno, Kenji; Meyer, Dirk

2017-03-01

The exocrine pancreas displays a significant capacity for regeneration and renewal. In humans and mammalian model systems, the partial loss of exocrine tissue, such as after acute pancreatitis or partial pancreatectomy induces rapid recovery via expansion of surviving acinar cells. In mouse it was further found that an almost complete removal of acinar cells initiates regeneration from a currently not well-defined progenitor pool. Here, we used the zebrafish as an alternative model to study cellular mechanisms of exocrine regeneration following an almost complete removal of acinar cells. We introduced and validated two novel transgenic approaches for genetically encoded conditional cell ablation in the zebrafish, either by caspase-8-induced apoptosis or by rendering cells sensitive to diphtheria toxin. By using the ela3l promoter for exocrine-specific expression, we show that both approaches allowed cell-type-specific removal of >95% of acinar tissue in larval and adult zebrafish without causing any signs of unspecific side effects. We find that zebrafish larvae are able to recover from a virtually complete acinar tissue ablation within 2 weeks. Using short-term lineage-tracing experiments and EdU incorporation assays, we exclude duct-associated Notch-responsive cells as the source of regeneration. Rather, a rare population of slowly dividing ela3l- negative cells expressing ptf1a and CPA was identified as the origin of the newly forming exocrine cells. Cells are actively maintained, as revealed by a constant number of these cells at different larval stages and after repeated cell ablation. These cells establish ela3l expression about 4-6 days after ablation without signs of increased proliferation in between. With onset of ela3l expression, cells initiate rapid proliferation, leading to fast expansion of the ela3l -positive population. Finally, we show that this proliferation is blocked by overexpression of the Wnt-signaling antagonist dkk1b In conclusion, we show a conserved requirement for Wnt signaling in exocrine tissue expansion and reveal a potential novel progenitor or stem cell population as a source for exocrine neogenesis after complete loss of acinar cells. © 2017. Published by The Company of Biologists Ltd.

Lapatinib–induced NF-kappaB activation sensitizes triple-negative breast cancer cells to proteasome inhibitors

PubMed Central

2013-01-01

Introduction Triple-negative breast cancer (TNBC), a subtype of breast cancer with negative expressions of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), is frequently diagnosed in younger women and has poor prognosis for disease-free and overall survival. Due to the lack of known oncogenic drivers for TNBC proliferation, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are urgently needed. Methods Triple-negative breast cancer cell lines were treated with proteasome inhibitors in combination with lapatinib (a dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor). Their in vitro and in vivo viability was examined by MTT assay, clonogenic analysis, and orthotopic xenograft mice model. Luciferase reporter gene, immunoblot, and RT-qPCR, immunoprecipitation assays were used to investigate the molecular mechanisms of action. Results Our data showed that nuclear factor (NF)-κB activation was elicited by lapatinib, independent of EGFR/HER2 inhibition, in TNBCs. Lapatinib-induced constitutive activation of NF-κB involved Src family kinase (SFK)-dependent p65 and IκBα phosphorylations, and rendered these cells more vulnerable to NF-κB inhibition by p65 small hairpin RNA. Lapatinib but not other EGFR inhibitors synergized the anti-tumor activity of proteasome inhibitors both in vitro and in vivo. Our results suggest that treatment of TNBCs with lapatinib may enhance their oncogene addiction to NF-κB, and thus augment the anti-tumor activity of proteasome inhibitors. Conclusions These findings suggest that combination therapy of a proteasome inhibitor with lapatinib may benefit TNBC patients. PMID:24216290

Lapatinib-induced NF-kappaB activation sensitizes triple-negative breast cancer cells to proteasome inhibitors.

PubMed

Chen, Yun-Ju; Yeh, Ming-Hsin; Yu, Meng-Chieh; Wei, Ya-Ling; Chen, Wen-Shu; Chen, Jhen-Yu; Shih, Chih-Yu; Tu, Chih-Yen; Chen, Chia-Hung; Hsia, Te-Chun; Chien, Pei-Hsuan; Liu, Shu-Hui; Yu, Yung-Luen; Huang, Wei-Chien

2013-11-12

Triple-negative breast cancer (TNBC), a subtype of breast cancer with negative expressions of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), is frequently diagnosed in younger women and has poor prognosis for disease-free and overall survival. Due to the lack of known oncogenic drivers for TNBC proliferation, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are urgently needed. Triple-negative breast cancer cell lines were treated with proteasome inhibitors in combination with lapatinib (a dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor). Their in vitro and in vivo viability was examined by MTT assay, clonogenic analysis, and orthotopic xenograft mice model. Luciferase reporter gene, immunoblot, and RT-qPCR, immunoprecipitation assays were used to investigate the molecular mechanisms of action. Our data showed that nuclear factor (NF)-κB activation was elicited by lapatinib, independent of EGFR/HER2 inhibition, in TNBCs. Lapatinib-induced constitutive activation of NF-κB involved Src family kinase (SFK)-dependent p65 and IκBα phosphorylations, and rendered these cells more vulnerable to NF-κB inhibition by p65 small hairpin RNA. Lapatinib but not other EGFR inhibitors synergized the anti-tumor activity of proteasome inhibitors both in vitro and in vivo. Our results suggest that treatment of TNBCs with lapatinib may enhance their oncogene addiction to NF-κB, and thus augment the anti-tumor activity of proteasome inhibitors. These findings suggest that combination therapy of a proteasome inhibitor with lapatinib may benefit TNBC patients.

Xenobiotic-metabolizing enzymes in the skin of rat, mouse, pig, guinea pig, man, and in human skin models.

PubMed

Oesch, F; Fabian, E; Guth, K; Landsiedel, R

2014-12-01

The exposure of the skin to medical drugs, skin care products, cosmetics, and other chemicals renders information on xenobiotic-metabolizing enzymes (XME) in the skin highly interesting. Since the use of freshly excised human skin for experimental investigations meets with ethical and practical limitations, information on XME in models comes in the focus including non-human mammalian species and in vitro skin models. This review attempts to summarize the information available in the open scientific literature on XME in the skin of human, rat, mouse, guinea pig, and pig as well as human primary skin cells, human cell lines, and reconstructed human skin models. The most salient outcome is that much more research on cutaneous XME is needed for solid metabolism-dependent efficacy and safety predictions, and the cutaneous metabolism comparisons have to be viewed with caution. Keeping this fully in mind at least with respect to some cutaneous XME, some models may tentatively be considered to approximate reasonable closeness to human skin. For dermal absorption and for skin irritation among many contributing XME, esterase activity is of special importance, which in pig skin, some human cell lines, and reconstructed skin models appears reasonably close to human skin. With respect to genotoxicity and sensitization, activating XME are not yet judgeable, but reactive metabolite-reducing XME in primary human keratinocytes and several reconstructed human skin models appear reasonably close to human skin. For a more detailed delineation and discussion of the severe limitations see the "Overview and Conclusions" section in the end of this review.

The insulin-like growth factor pathway is altered in Spinocerebellar ataxia type 1 and type 7

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

Gatchel, Jennifer R.; Watase, Kei; Thaller, Christina

2008-01-29

Polyglutamine diseases are inherited neurodegenerative disorders caused by expansion of CAG trinucleotide repeats encoding a polyglutamine tract in the disease-causing proteins. There are nine of these disorders each having distinct features but also clinical and pathological similarities. In particular, spinocerebellar ataxia type 1 and 7 (SCA1 and SCA7) patients manifest cerebellar ataxia with corresponding degeneration of Purkinje cells. Given this common phenotype, we asked whether the two disorders share common molecular pathogenic events. To address this question we studied two genetically accurate mouse models of SCA1 and SCA7—Sca1154Q/2Q and Sca7266Q/5Q knock-in mice—that express the glutamine-expanded proteins from the respective endogenousmore » loci. We found common transcriptional changes in early symptomatic mice, with downregulation of Insulin-like growth factor binding protein 5 (Igfbp5) representing one of the most robust transcriptional changes that closely correlates with disease state. Interestingly, down-regulation of Igfbp5 occurred in granule neurons through a non-cell autonomous mechanism and was concomitant with activation of the Insulin-like growth factor I (Igf-I) pathway, and, in particular, the Igf-I receptor, expressed in part on Purkinje cells (PC). These data define a possible common pathogenic response in SCA1 and SCA7 and reveal the importance of neuron-neuron interactions in SCA1 and SCA7 pathogenesis. The sensitivity of Igfbp5 levels to disease state could render it and other components of its effector pathway useful as biomarkers in this class of diseases.« less

Suppression of HER2/HER3-mediated growth of breast cancer cells with combinations of GDC-0941 PI3K inhibitor, trastuzumab, and pertuzumab.

PubMed

Yao, Evelyn; Zhou, Wei; Lee-Hoeflich, Si Tuen; Truong, Tom; Haverty, Peter M; Eastham-Anderson, Jeffrey; Lewin-Koh, Nicholas; Gunter, Bert; Belvin, Marcia; Murray, Lesley J; Friedman, Lori S; Sliwkowski, Mark X; Hoeflich, Klaus P

2009-06-15

Oncogenic activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is prevalent in breast cancer and has been associated with resistance to HER2 inhibitors in the clinic. We therefore investigated the combinatorial activity of GDC-0941, a novel class I PI3K inhibitor, with standard-of-care therapies for HER2-amplified breast cancer. Three-dimensional laminin-rich extracellular matrix cultures of human breast cancer cells were utilized to provide a physiologically relevant approach to analyze the efficacy and molecular mechanism of combination therapies ex vivo. Combination studies were done using GDC-0941 with trastuzumab (Herceptin), pertuzumab, lapatinib (Tykerb), and docetaxel, the principal therapeutic agents that are either approved or being evaluated for treatment of early HER2-positive breast cancer. Significant GDC-0941 activity (EC(50) <1 micromol/L) was observed for >70% of breast cancer cell lines that were examined in three-dimensional laminin-rich extracellular matrix culture. Differential responsiveness to GDC-0941 as a single agent was observed for luminal breast cancer cells upon stimulation with the HER3 ligand, heregulin. Combined treatment of GDC-0941, trastuzumab, and pertuzumab resulted in growth inhibition, altered acinar morphology, and suppression of AKT mitogen-activated protein kinase (MAPK) / extracellular signed-regulated kinase (ERK) kinase and MEK effector signaling pathways for HER2-amplified cells in both normal and heregulin-supplemented media. The GDC-0941 and lapatinib combination further showed that inhibition of HER2 activity was essential for maximum combinatorial efficacy. PI3K inhibition also rendered HER2-amplified BT-474M1 cells and tumor xenografts more sensitive to docetaxel. GDC-0941 is efficacious in preclinical models of breast cancer. The addition of GDC-0941 to HER2-directed treatment could augment clinical benefit in breast cancer patients.

STAT3/IRF1 Pathway Activation Sensitizes Cervical Cancer Cells to Chemotherapeutic Drugs.

PubMed

Walch-Rückheim, Barbara; Pahne-Zeppenfeld, Jennifer; Fischbach, Jil; Wickenhauser, Claudia; Horn, Lars Christian; Tharun, Lars; Büttner, Reinhard; Mallmann, Peter; Stern, Peter; Kim, Yoo-Jin; Bohle, Rainer Maria; Rübe, Christian; Ströder, Russalina; Juhasz-Böss, Ingolf; Solomayer, Erich-Franz; Smola, Sigrun

2016-07-01

Neoadjuvant radio/chemotherapy regimens can markedly improve cervical cancer outcome in a subset of patients, while other patients show poor responses, but may encounter severe adverse effects. Thus, there is a strong need for predictive biomarkers to improve clinical management of cervical cancer patients. STAT3 is considered as a critical antiapoptotic factor in various malignancies. We therefore investigated STAT3 activation during cervical carcinogenesis and its impact on the response of cervical cancer cells to chemotherapeutic drugs. Tyr705-phosphorylated STAT3 increased from low-grade cervical intraepithelial neoplasia (CIN1) to precancerous CIN3 lesions. Notably, pTyr705-STAT3 activation significantly declined from CIN3 to invasive cancer, also when compared in the same clinical biopsy. pTyr705-STAT3 was also low or absent in cultured human cervical cancer cell lines, consistent with the in vivo expression data. Unexpectedly, IL6-type cytokine signaling inducing STAT3 activation rendered cervical cancer cells significantly more susceptible to chemotherapeutic drugs, that is, cisplatin or etoposide. This chemosensitization was STAT3-dependent and we identified IFN regulatory factor-1 (IRF1) as the STAT3-inducible mediator required for cell death enhancement. In line with these data, pTyr705-STAT3 significantly correlated with nuclear IRF1 expression in cervical cancer in vivo Importantly, high IRF1 expression in pretreatment cervical cancer biopsy cells was associated with a significantly better response to neoadjuvant radio/chemotherapy of the patients. In summary, our study has identified a key role of the STAT3/IRF1 pathway for chemosensitization in cervical cancer. Our results suggest that pretherapeutic IRF1 expression should be evaluated as a novel predictive biomarker for neoadjuvant radio/chemotherapy responses. Cancer Res; 76(13); 3872-83. ©2016 AACR. ©2016 American Association for Cancer Research.

Estrogen and insulin transport through the blood-brain barrier

PubMed Central

May, Aaron A.; Bedel, Nicholas D.; Shen, Ling; Woods, Stephen C.; Liu, Min

2016-01-01

The metabolic syndrome is associated with insulin resistance and reduced transport of insulin through the blood-brain barrier (BBB). Reversal of high-fat diet-induced obesity (HFD-DIO) by dietary intervention improves the transport of insulin through the BBB and the sensitivity of insulin in the brain. Although both insulin and estrogen (E2), when given alone, reduce food intake and body weight via the brain, E2 actually renders the brain relatively insensitive to insulin’s catabolic action. The objective of these studies was to determine if E2 influences the ability of insulin to be transported into the brain, since both E2 and insulin receptors are found in BBB endothelial cells. E2 (acute or chronic) was systemically administered to ovariectomized (OVX) female rats and male rats fed a chow or a high-fat diet. Food intake, body weight and other metabolic parameters were assessed along with insulin entry into the cerebrospinal fluid (CSF). Acute E2 treatment in OVX female and male rats reduced body weight and food intake, and chronic E2 treatment prevented or partially reversed high-fat diet-induced obesity. However, none of these conditions increased insulin transport into the CNS; rather, chronic E2 treatment was associated less-effective insulin transport into the CNS relative to weight-matched controls. Thus, the reduction of brain insulin sensitivity by E2 is unlikely to be mediated by increasing the amount of insulin entering the CNS. PMID:27182046

Human Leukocyte Antigen Class II Transgenic Mouse Model Unmasks the Significant Extrahepatic Pathology in Toxic Shock Syndrome

PubMed Central

Tilahun, Ashenafi Y.; Marietta, Eric V.; Wu, Tsung-Teh; Patel, Robin; David, Chella S.; Rajagopalan, Govindarajan

2011-01-01

Among the exotoxins produced by Staphylococcus aureus and Streptococcus pyogenes, the superantigens (SAgs) are the most potent T-cell activators known to date. SAgs are implicated in several serious diseases including toxic shock syndrome (TSS), Kawasaki disease, and sepsis. However, the immunopathogenesis of TSS and other diseases involving SAgs are still not completely understood. The commonly used conventional laboratory mouse strains do not respond robustly to SAgs in vivo. Therefore, they must be artificially rendered susceptible to TSS by using sensitizing agents such as d-galactosamine (d-galN), which skews the disease exclusively to the liver and, hence, is not representative of the disease in humans. SAg-induced TSS was characterized using transgenic mice expressing HLA class II molecules that are extremely susceptible to TSS without d-galN. HLA-DR3 transgenic mice recapitulated TSS in humans with extensive multiple-organ inflammation affecting the lung, liver, kidneys, heart, and small intestines. Heavy infiltration with T lymphocytes (both CD4+ and CD8+), neutrophils, and macrophages was noted. In particular, the pathologic changes in the small intestines were extensive and accompanied by significantly altered absorptive functions of the enterocytes. In contrast to massive liver failure alone in the d-galN sensitization model of TSS, findings of the present study suggest that gut dysfunction might be a key pathogenic event that leads to high morbidity and mortality in humans with TSS. PMID:21641398

Temperature-Sensitive Salmonella enterica Serovar Enteritidis PT13a Expressing Essential Proteins of Psychrophilic Bacteria.

PubMed

Duplantis, Barry N; Puckett, Stephanie M; Rosey, Everett L; Ameiss, Keith A; Hartman, Angela D; Pearce, Stephanie C; Nano, Francis E

2015-10-01

Synthetic genes based on deduced amino acid sequences of the NAD-dependent DNA ligase (ligA) and CTP synthetase (pyrG) of psychrophilic bacteria were substituted for their native homologues in the genome of Salmonella enterica serovar Enteritidis phage type 13a (PT13a). The resulting strains were rendered temperature sensitive (TS) and did not revert to temperature resistance at a detectable level. At permissive temperatures, TS strains grew like the parental strain in broth medium and in macrophage-like cells, but their growth was slowed or stopped when they were shifted to a restrictive temperature. When injected into BALB/c mice at the base of the tail, representing a cool site of the body, the strains with restrictive temperatures of 37, 38.5, and 39°C persisted for less than 1 day, 4 to 7 days, and 20 to 28 days, respectively. The wild-type strain persisted at the site of inoculation for at least 28 days. The wild-type strain, but not the TS strains, was also found in spleen-plus-liver homogenates within 1 day of inoculation of the tail and was detectable in these organs for at least 28 days. Intramuscular vaccination of White Leghorn chickens with the PT13a strain carrying the psychrophilic pyrG gene provided some protection against colonization of the reproductive tract and induced an anti-S. enterica antibody response. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Colloid Surface Chemistry Critically Affects Multiple Particle Tracking Measurements of Biomaterials

PubMed Central

Valentine, M. T.; Perlman, Z. E.; Gardel, M. L.; Shin, J. H.; Matsudaira, P.; Mitchison, T. J.; Weitz, D. A.

2004-01-01

Characterization of the properties of complex biomaterials using microrheological techniques has the promise of providing fundamental insights into their biomechanical functions; however, precise interpretations of such measurements are hindered by inadequate characterization of the interactions between tracers and the networks they probe. We here show that colloid surface chemistry can profoundly affect multiple particle tracking measurements of networks of fibrin, entangled F-actin solutions, and networks of cross-linked F-actin. We present a simple protocol to render the surface of colloidal probe particles protein-resistant by grafting short amine-terminated methoxy-poly(ethylene glycol) to the surface of carboxylated microspheres. We demonstrate that these poly(ethylene glycol)-coated tracers adsorb significantly less protein than particles coated with bovine serum albumin or unmodified probe particles. We establish that varying particle surface chemistry selectively tunes the sensitivity of the particles to different physical properties of their microenvironments. Specifically, particles that are weakly bound to a heterogeneous network are sensitive to changes in network stiffness, whereas protein-resistant tracers measure changes in the viscosity of the fluid and in the network microstructure. We demonstrate experimentally that two-particle microrheology analysis significantly reduces differences arising from tracer surface chemistry, indicating that modifications of network properties near the particle do not introduce large-scale heterogeneities. Our results establish that controlling colloid-protein interactions is crucial to the successful application of multiple particle tracking techniques to reconstituted protein networks, cytoplasm, and cells. PMID:15189896

Temperature-Sensitive Salmonella enterica Serovar Enteritidis PT13a Expressing Essential Proteins of Psychrophilic Bacteria

PubMed Central

Duplantis, Barry N.; Puckett, Stephanie M.; Rosey, Everett L.; Ameiss, Keith A.; Hartman, Angela D.; Pearce, Stephanie C.

2015-01-01

Synthetic genes based on deduced amino acid sequences of the NAD-dependent DNA ligase (ligA) and CTP synthetase (pyrG) of psychrophilic bacteria were substituted for their native homologues in the genome of Salmonella enterica serovar Enteritidis phage type 13a (PT13a). The resulting strains were rendered temperature sensitive (TS) and did not revert to temperature resistance at a detectable level. At permissive temperatures, TS strains grew like the parental strain in broth medium and in macrophage-like cells, but their growth was slowed or stopped when they were shifted to a restrictive temperature. When injected into BALB/c mice at the base of the tail, representing a cool site of the body, the strains with restrictive temperatures of 37, 38.5, and 39°C persisted for less than 1 day, 4 to 7 days, and 20 to 28 days, respectively. The wild-type strain persisted at the site of inoculation for at least 28 days. The wild-type strain, but not the TS strains, was also found in spleen-plus-liver homogenates within 1 day of inoculation of the tail and was detectable in these organs for at least 28 days. Intramuscular vaccination of White Leghorn chickens with the PT13a strain carrying the psychrophilic pyrG gene provided some protection against colonization of the reproductive tract and induced an anti-S. enterica antibody response. PMID:26187965

Stem Cell Microencapsulation for Phenotypic Control, Bioprocessing, and Transplantation

PubMed Central

Wilson, Jenna L.

2014-01-01

Cell microencapsulation has been utilized for decades as a means to shield cells from the external environment while simultaneously permitting transport of oxygen, nutrients, and secretory molecules. In designing cell therapies, donor primary cells are often difficult to obtain and expand to appropriate numbers, rendering stem cells an attractive alternative due to their capacities for self-renewal, differentiation, and trophic factor secretion. Microencapsulation of stem cells offers several benefits, namely the creation of a defined microenvironment which can be designed to modulate stem cell phenotype, protection from hydrodynamic forces and prevention of agglomeration during expansion in suspension bioreactors, and a means to transplant cells behind a semi-permeable barrier, allowing for molecular secretion while avoiding immune reaction. This review will provide an overview of relevant microencapsulation processes and characterization in the context of maintaining stem cell potency, directing differentiation, investigating scalable production methods, and transplanting stem cells for clinically relevant disorders. PMID:23239279

Cornea and ocular lens visualized with three-dimensional confocal microscopy

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1992-08-01

This paper demonstrates the advantages of three-dimensional reconstruction of the cornea and the ocular crystalline lens by confocal microscopy and volume rendering computer techniques. The advantages of noninvasive observation of ocular structures in living, unstained, unfixed tissue include the following: the tissue is in a natural living state without the artifacts of fixation, mechanical sectioning, and staining; the three-dimensional structure can be observed from any view point and quantitatively analyzed; the dynamics of morphological changes can be studied; and the use of confocal microscopic observation results in a reduction of the number of animals required for ocular morphometric studies. The main advantage is that the dynamic morphology of ocular structures can be investigated in living ocular tissue. 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 488 nm wavelength. The microscope objective was a Leitz 25X, 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 under sampling resulted in a three-dimensional visualization rendering in which the corneal thickness (z-axis) is compressed. 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, free nerve endings in the basal epithelial cells, 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.

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

Sphere-enhanced microwave ablation (sMWA) versus bland microwave ablation (bMWA): technical parameters, specific CT 3D rendering and histopathology.

PubMed

Gockner, T L; Zelzer, S; Mokry, T; Gnutzmann, D; Bellemann, N; Mogler, C; Beierfuß, A; Köllensperger, E; Germann, G; Radeleff, B A; Stampfl, U; Kauczor, H U; Pereira, P L; Sommer, C M

2015-04-01

This study was designed to compare technical parameters during ablation as well as CT 3D rendering and histopathology of the ablation zone between sphere-enhanced microwave ablation (sMWA) and bland microwave ablation (bMWA). In six sheep-livers, 18 microwave ablations were performed with identical system presets (power output: 80 W, ablation time: 120 s). In three sheep, transarterial embolisation (TAE) was performed immediately before microwave ablation using spheres (diameter: 40 ± 10 μm) (sMWA). In the other three sheep, microwave ablation was performed without spheres embolisation (bMWA). Contrast-enhanced CT, sacrifice, and liver harvest followed immediately after microwave ablation. Study goals included technical parameters during ablation (resulting power output, ablation time), geometry of the ablation zone applying specific CT 3D rendering with a software prototype (short axis of the ablation zone, volume of the largest aligned ablation sphere within the ablation zone), and histopathology (hematoxylin-eosin, Masson Goldner and TUNEL). Resulting power output/ablation times were 78.7 ± 1.0 W/120 ± 0.0 s for bMWA and 78.4 ± 1.0 W/120 ± 0.0 s for sMWA (n.s., respectively). Short axis/volume were 23.7 ± 3.7 mm/7.0 ± 2.4 cm(3) for bMWA and 29.1 ± 3.4 mm/11.5 ± 3.9 cm(3) for sMWA (P < 0.01, respectively). Histopathology confirmed the signs of coagulation necrosis as well as early and irreversible cell death for bMWA and sMWA. For sMWA, spheres were detected within, at the rim, and outside of the ablation zone without conspicuous features. Specific CT 3D rendering identifies a larger ablation zone for sMWA compared with bMWA. The histopathological signs and the detectable amount of cell death are comparable for both groups. When comparing sMWA with bMWA, TAE has no effect on the technical parameters during ablation.

Gravity and the cell: Intracellular structures and Stokes sedimentation

NASA Technical Reports Server (NTRS)

Todd, P.

1977-01-01

Plant and certain animal embryos appear to be responsive to the gravity vector during early stages of development. The convection of particle sedimentation as the basis for the sensing of gravity is investigated using the cells of wheat seedlings, amphibian embryos, and mammals. Exploration of the mammalian cell for sedimenting particles reveals that their existence is unlikely, especially in the presence of a network of microtubules and microfilaments considered to be responsible for intracellular organization. Destruction of these structures renders the cell susceptible to accelerations several times g. Large dense particles, such as chromosomes, nucleoli, and cytoplasmic organelles are acted upon by forces much larger than that due to gravity, and their positions in the cell appear to be insensitive to gravity.

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.

Molecular Characterization of Macrophage-Biomaterial Interactions

PubMed Central

Moore, Laura Beth; Kyriakides, Themis R.

2015-01-01

Implantation of biomaterials in vascularized tissues elicits the sequential engagement of molecular and cellular elements that constitute the foreign body response. Initial events include the non-specific adsorption of proteins to the biomaterial surface that render it adhesive for cells such as neutrophils and macrophages. The latter undergo unique activation and in some cases undergo cell-cell fusion to form foreign body giant cells that contribute to implant damage and fibrotic encapsulation. In this review, we discuss the molecular events that contribute to macrophage activation and fusion with a focus on the role of the inflammasome, signaling pathways such as JAK/STAT and NF-κB, and the putative involvement of micro RNAs in the regulation of these processes. PMID:26306446

Molecular Characterization of Macrophage-Biomaterial Interactions.

PubMed

Moore, Laura Beth; Kyriakides, Themis R

2015-01-01

Implantation of biomaterials in vascularized tissues elicits the sequential engagement of molecular and cellular elements that constitute the foreign body response. Initial events include the non-specific adsorption of proteins to the biomaterial surface that render it adhesive for cells such as neutrophils and macrophages. The latter undergo unique activation and in some cases undergo cell-cell fusion to form foreign body giant cells that contribute to implant damage and fibrotic encapsulation. In this review, we discuss the molecular events that contribute to macrophage activation and fusion with a focus on the role of the inflammasome, signaling pathways such as JAK/STAT and NF-κB, and the putative involvement of micro RNAs in the regulation of these processes.

A Parallel Pipelined Renderer for the Time-Varying Volume Data

NASA Technical Reports Server (NTRS)

Chiueh, Tzi-Cker; Ma, Kwan-Liu

1997-01-01

This paper presents a strategy for efficiently rendering time-varying volume data sets on a distributed-memory parallel computer. Time-varying volume data take large storage space and visualizing them requires reading large files continuously or periodically throughout the course of the visualization process. Instead of using all the processors to collectively render one volume at a time, a pipelined rendering process is formed by partitioning processors into groups to render multiple volumes concurrently. In this way, the overall rendering time may be greatly reduced because the pipelined rendering tasks are overlapped with the I/O required to load each volume into a group of processors; moreover, parallelization overhead may be reduced as a result of partitioning the processors. We modify an existing parallel volume renderer to exploit various levels of rendering parallelism and to study how the partitioning of processors may lead to optimal rendering performance. Two factors which are important to the overall execution time are re-source utilization efficiency and pipeline startup latency. The optimal partitioning configuration is the one that balances these two factors. Tests on Intel Paragon computers show that in general optimal partitionings do exist for a given rendering task and result in 40-50% saving in overall rendering time.

Role of NK cells in immunotherapy and virotherapy of solid tumors.

PubMed

Cantoni, Claudia; Grauwet, Korneel; Pietra, Gabriella; Parodi, Monica; Mingari, Maria Cristina; Maria, Andrea De; Favoreel, Herman; Vitale, Massimo

2015-01-01

Although natural killer (NK) cells are endowed with powerful cytolytic activity against cancer cells, their role in different therapies against solid tumors has not yet been fully elucidated. Their interactions with various elements of the tumor microenvironment as well as their possible effects in contributing to and/or limiting oncolytic virotherapy render this potential immunotherapeutic tool still difficult to exploit at the bedside. Here, we will review the current literature with the aim of providing new hints to manage this powerful cell type in future innovative therapies, such as the use of NK cells in combination with new cytokines, specific mAbs (inducing ADCC), Tyr-Kinase inhibitors, immunomodulatory drugs and/or the design of oncolytic viruses aimed at optimizing the effect of NK cells in virotherapy.

Deficiency in the nuclear factor E2-related factor 2 renders pancreatic β-cells vulnerable to arsenic-induced cell damage

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

Yang, Bei; Department of Histology and Embryology, College of Basic Medical Sciences, China Medical University, Shenyang 110001; Fu, Jingqi

2012-11-01

Chronic human exposure to inorganic arsenic (iAs), a potent environmental oxidative stressor, is associated with increased prevalence of type 2 diabetes, where impairment of pancreatic β-cell function is a key pathogenic factor. Nuclear factor E2-related factor 2 (Nrf2) is a central transcription factor regulating cellular adaptive response to oxidative stress. However, persistent activation of Nrf2 in response to chronic oxidative stress, including inorganic arsenite (iAs{sup 3+}) exposure, blunts glucose-triggered reactive oxygen species (ROS) signaling and impairs glucose-stimulated insulin secretion (GSIS). In the current study, we found that MIN6 pancreatic β-cells with stable knockdown of Nrf2 (Nrf2-KD) by lentiviral shRNA andmore » pancreatic islets isolated from Nrf2-knockout (Nrf2−/−) mice exhibited reduced expression of several antioxidant and detoxification enzymes in response to acute iAs{sup 3+} exposure. As a result, Nrf2-KD MIN6 cells and Nrf2−/− islets were more susceptible to iAs{sup 3+} and monomethylarsonous acid (MMA{sup 3+})-induced cell damage, as measured by decreased cell viability, augmented apoptosis and morphological change. Pretreatment of MIN6 cells with Nrf2 activator tert-butylhydroquinone protected the cells from iAs{sup 3+}-induced cell damage in an Nrf2-dependent fashion. In contrast, antioxidant N‐acetyl cysteine protected Nrf2-KD MIN6 cells against acute cytotoxicity of iAs{sup 3+}. The present study demonstrates that Nrf2-mediated antioxidant response is critical in the pancreatic β-cell defense mechanism against acute cytotoxicity by arsenic. The findings here, combined with our previous results on the inhibitory effect of antioxidants on ROS signaling and GSIS, suggest that Nrf2 plays paradoxical roles in pancreatic β-cell dysfunction induced by environmental arsenic exposure. -- Highlights: ► Lack of Nrf2 reduced expression of antioxidant genes induced by iAs{sup 3+} in β-cells. ► Deficiency of Nrf2 in β-cells sensitized to iAs{sup 3+} and MMA{sup 3+}-induced cytotoxicity. ► Nrf2 activation protected β-cells from acute iAs{sup 3+} cytotoxicity.« less

SCL, LMO1 and Notch1 Reprogram Thymocytes into Self-Renewing Cells

PubMed Central

Rojas-Sutterlin, Shanti; Herblot, Sabine; Hébert, Josée; Sauvageau, Guy; Lemieux, Sébastien; Lécuyer, Eric; Veiga, Diogo F. T.; Hoang, Trang

2014-01-01

The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network described here may be relevant to a majority of human T-ALL. PMID:25522233