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Sample records for selective lysosomal targeting

  1. Targeting the lysosome in cancer

    PubMed Central

    Piao, Shengfu; Amaravadi, Ravi K.

    2016-01-01

    Lysosomes are membrane-bound intracellular organelles that receive macromolecules delivered by endocytosis, phagocytosis, and autophagy for degradation and recycling. Over the last decade, advances in lysosome research have established a broad role for the lysosome in the pathophysiology of disease. In this review, we highlight the recent discoveries in lysosome biology, with an emphasis on their implications for cancer therapy. We focus on targeting the lysosome in cancer by exploring lysosomal biogenesis and its role in the crosstalk between apoptosis and autophagy. We also discuss how lysosomal inhibition could emerge as a new therapeutic strategy to overcome drug resistance in cancer. PMID:26599426

  2. Select microtubule inhibitors increase lysosome acidity and promote lysosomal disruption in acute myeloid leukemia (AML) cells.

    PubMed

    Bernard, Dannie; Gebbia, Marinella; Prabha, Swayam; Gronda, Marcela; MacLean, Neil; Wang, Xiaoming; Hurren, Rose; Sukhai, Mahadeo A; Cho, Eunice E; Manolson, Morris F; Datti, Alessandro; Wrana, Jeffrey; Minden, Mark D; Al-Awar, Rima; Aman, Ahmed; Nislow, Corey; Giaever, Guri; Schimmer, Aaron D

    2015-07-01

    To identify new biological vulnerabilities in acute myeloid leukemia, we screened a library of natural products for compounds cytotoxic to TEX leukemia cells. This screen identified the novel small molecule Deoxysappanone B 7,4' dimethyl ether (Deox B 7,4), which possessed nanomolar anti-leukemic activity. To determine the anti-leukemic mechanism of action of Deox B 7,4, we conducted a genome-wide screen in Saccharomyces cerevisiae and identified enrichment of genes related to mitotic cell cycle as well as vacuolar acidification, therefore pointing to microtubules and vacuolar (V)-ATPase as potential drug targets. Further investigations into the mechanisms of action of Deox B 7,4 and a related analogue revealed that these compounds were reversible microtubule inhibitors that bound near the colchicine site. In addition, Deox B 7,4 and its analogue increased lysosomal V-ATPase activity and lysosome acidity. The effects on microtubules and lysosomes were functionally important for the anti-leukemic effects of these drugs. The lysosomal effects were characteristic of select microtubule inhibitors as only the Deox compounds and nocodazole, but not colchicine, vinca alkaloids or paclitaxel, altered lysosome acidity and induced lysosomal disruption. Thus, our data highlight a new mechanism of action of select microtubule inhibitors on lysosomal function. PMID:25832785

  3. Hexamethylene amiloride engages a novel reactive oxygen species- and lysosome-dependent programmed necrotic mechanism to selectively target breast cancer cells.

    PubMed

    Rowson-Hodel, Ashley R; Berg, Anastasia L; Wald, Jessica H; Hatakeyama, Jason; VanderVorst, Kacey; Curiel, Daniel A; Leon, Leonardo J; Sweeney, Colleen; Carraway, Kermit L

    2016-05-28

    Anticancer chemotherapeutics often rely on induction of apoptosis in rapidly dividing cells. While these treatment strategies are generally effective in debulking the primary tumor, post-therapeutic recurrence and metastasis are pervasive concerns with potentially devastating consequences. We demonstrate that the amiloride derivative 5-(N,N-hexamethylene) amiloride (HMA) harbors cytotoxic properties particularly attractive for a novel class of therapeutic agent. HMA is potently and specifically cytotoxic toward breast cancer cells, with remarkable selectivity for transformed cells relative to non-transformed or primary cells. Nonetheless, HMA is similarly cytotoxic to breast cancer cells irrespective of their molecular profile, proliferative status, or species of origin, suggesting that it engages a cell death mechanism common to all breast tumor subtypes. We observed that HMA induces a novel form of caspase- and autophagy-independent programmed necrosis relying on the orchestration of mitochondrial and lysosomal pro-death mechanisms, where its cytotoxicity was attenuated with ROS-scavengers or lysosomal cathepsin inhibition. Overall, our findings suggest HMA may efficiently target the heterogeneous populations of cancer cells known to reside within a single breast tumor by induction of a ROS- and lysosome-mediated form of programmed necrosis. PMID:26944316

  4. Screening and Optimization of Ligand Conjugates for Lysosomal Targeting

    PubMed Central

    Meerovich, Igor; Koshkaryev, Alexander; Thekkedath, Ritesh; Torchilin, Vladimir P.

    2011-01-01

    The use of lysosome-targeted liposomes may significantly improve the delivery of therapeutic enzymes and chaperones into lysosomes for the treatment of lysosomal storage disorders. The aim of this research was to synthesize new potentially lysosomotropic ligands on a base of Neutral Red and rhodamine B and to study their ability to enhance specific lysosomal delivery of surface-modified liposomes loaded with a model compound, fluorescein isothiocyanate-dextran (FD). The delivery of these liposomes and their content to lysosomes in HeLa cells was investigated by confocal immunofluorescent microscopy, subcellular fractionation and flow cytometry. Confocal microscopy demonstrated that liposomes modified with derivatives of rhodamine B provide good rate of co-localization well the specific lysosomal markers. The comparison of fluorescence of FD in lysosomes isolated by subcellular fractionation also showed that the efficiency of lysosomal delivery of liposomal load by liposomes modified with some of synthesized ligands was significantly higher compared with plain liposomes. These results were additionally confirmed by the flow cytometry of the intact cells treated with liposomes loaded with with 5-dodecanoylaminofluorescein di-β-D-galactopyranoside, a specific substrate for the intralysosomal β-galactosidase, using a number of cell lines, including macrophages with induced phenotype of lysosomal enzyme deficiency; two of the synthesized ligands – rhodamine B DSPE-PEG2k-amide and 6-(3-(DSPE-PEG2k)-thioureido) rhodamine B – demonstrated enhanced lysosomal delivery, in some cases, higher than that for commercially available rhodamine B octadecyl ester, with the best results (the enhancement of the lysosomal delivery up to 75% greater in comparison to plain liposomes) shown for the cells with induced lysosomal enzyme deficiency phenotype. Use of liposomes modified with rhodamine B derivatives may be advantageous for the development of drug delivery systems for the

  5. Induced oligomerization targets Golgi proteins for degradation in lysosomes

    PubMed Central

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D.

    2015-01-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130’s cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. PMID:26446839

  6. Induced oligomerization targets Golgi proteins for degradation in lysosomes.

    PubMed

    Tewari, Ritika; Bachert, Collin; Linstedt, Adam D

    2015-12-01

    Manganese protects cells against forms of Shiga toxin by down-regulating the cycling Golgi protein GPP130. Down-regulation occurs when Mn binding causes GPP130 to oligomerize and traffic to lysosomes. To determine how GPP130 is redirected to lysosomes, we tested the role of GGA1 and clathrin, which mediate sorting in the canonical Golgi-to-lysosome pathway. GPP130 oligomerization was induced using either Mn or a self-interacting version of the FKBP domain. Inhibition of GGA1 or clathrin specifically blocked GPP130 redistribution, suggesting recognition of the aggregated GPP130 by the GGA1/clathrin-sorting complex. Unexpectedly, however, GPP130's cytoplasmic domain was not required, and redistribution also occurred after removal of GPP130 sequences needed for its normal cycling. Therefore, to test whether aggregate recognition might be a general phenomenon rather than one involving a specific GPP130 determinant, we induced homo-oligomerization of two unrelated Golgi-targeted constructs using the FKBP strategy. These were targeted to the cis- and trans-Golgi, respectively, using domains from mannosidase-1 and galactosyltransferase. Significantly, upon oligomerization, each redistributed to peripheral punctae and was degraded. This occurred in the absence of detectable UPR activation. These findings suggest the unexpected presence of quality control in the Golgi that recognizes aggregated Golgi proteins and targets them for degradation in lysosomes. PMID:26446839

  7. Neuronal-Targeted TFEB Accelerates Lysosomal Degradation of APP, Reducing Aβ Generation and Amyloid Plaque Pathogenesis

    PubMed Central

    Xiao, Qingli; Yan, Ping; Ma, Xiucui; Liu, Haiyan; Perez, Ronaldo; Zhu, Alec; Gonzales, Ernesto; Tripoli, Danielle L.; Czerniewski, Leah; Ballabio, Andrea; Cirrito, John R.

    2015-01-01

    In AD, an imbalance between Aβ production and removal drives elevated brain Aβ levels and eventual amyloid plaque deposition. APP undergoes nonamyloidogenic processing via α-cleavage at the plasma membrane, amyloidogenic β- and γ-cleavage within endosomes to generate Aβ, or lysosomal degradation in neurons. Considering multiple reports implicating impaired lysosome function as a driver of increased amyloidogenic processing of APP, we explored the efficacy of targeting transcription factor EB (TFEB), a master regulator of lysosomal pathways, to reduce Aβ levels. CMV promoter-driven TFEB, transduced via stereotactic hippocampal injections of adeno-associated virus particles in APP/PS1 mice, localized primarily to neuronal nuclei and upregulated lysosome biogenesis. This resulted in reduction of APP protein, the α and β C-terminal APP fragments (CTFs), and in the steady-state Aβ levels in the brain interstitial fluid. In aged mice, total Aβ levels and amyloid plaque load were selectively reduced in the TFEB-transduced hippocampi. TFEB transfection in N2a cells stably expressing APP695, stimulated lysosome biogenesis, reduced steady-state levels of APP and α- and β-CTFs, and attenuated Aβ generation by accelerating flux through the endosome-lysosome pathway. Cycloheximide chase assays revealed a shortening of APP half-life with exogenous TFEB expression, which was prevented by concomitant inhibition of lysosomal acidification. These data indicate that TFEB enhances flux through lysosomal degradative pathways to induce APP degradation and reduce Aβ generation. Activation of TFEB in neurons is an effective strategy to attenuate Aβ generation and attenuate amyloid plaque deposition in AD. SIGNIFICANCE STATEMENT A key driver for AD pathogenesis is the net balance between production and clearance of Aβ, the major component of amyloid plaques. Here we demonstrate that lysosomal degradation of holo-APP influences Aβ production by limiting the availability of

  8. Protein Networks Supporting AP-3 Function in Targeting Lysosomal Membrane Proteins

    PubMed Central

    Baust, Thorsten; Anitei, Mihaela; Czupalla, Cornelia; Parshyna, Iryna; Bourel, Line; Thiele, Christoph; Krause, Eberhard

    2008-01-01

    The AP-3 adaptor complex targets selected transmembrane proteins to lysosomes and lysosome-related organelles. We reconstituted its preferred interaction with liposomes containing the ADP ribosylation factor (ARF)-1 guanosine triphosphatase (GTPase), specific cargo tails, and phosphatidylinositol-3 phosphate, and then we performed a proteomic screen to identify new proteins supporting its sorting function. We identified ≈30 proteins belonging to three networks regulating either AP-3 coat assembly or septin polymerization or Rab7-dependent lysosomal transport. RNA interference shows that, among these proteins, the ARF-1 exchange factor brefeldin A-inhibited exchange factor 1, the ARF-1 GTPase-activating protein 1, the Cdc42-interacting Cdc42 effector protein 4, an effector of septin-polymerizing GTPases, and the phosphatidylinositol-3 kinase IIIC3 are key components regulating the targeting of lysosomal membrane proteins to lysosomes in vivo. This analysis reveals that these proteins, together with AP-3, play an essential role in protein sorting at early endosomes, thereby regulating the integrity of these organelles. PMID:18287518

  9. Targeting the Autophagy/Lysosomal Degradation Pathway in Parkinson's Disease.

    PubMed

    Rivero-Ríos, Pilar; Madero-Pérez, Jesús; Fernández, Belén; Hilfiker, Sabine

    2016-01-01

    Autophagy is a cellular quality control mechanism crucial for neuronal homeostasis. Defects in autophagy are critically associated with mechanisms underlying Parkinson's disease (PD), a common and debilitating neurodegenerative disorder. Autophagic dysfunction in PD can occur at several stages of the autophagy/lysosomal degradative machinery, contributing to the formation of intracellular protein aggregates and eventual neuronal cell death. Therefore, autophagy inducers may comprise a promising new therapeutic approach to combat neurodegeneration in PD. Several currently available FDA-approved drugs have been shown to enhance autophagy, which may allow for their repurposing for use in novel clinical conditions including PD. This review summarizes our current knowledge of deficits in the autophagy/lysosomal degradation pathways associated with PD, and highlight current approaches which target this pathway as possible means towards novel therapeutic strategies. PMID:26517050

  10. Lysine fatty acylation promotes lysosomal targeting of TNF-α

    PubMed Central

    Jiang, Hong; Zhang, Xiaoyu; Lin, Hening

    2016-01-01

    Tumor necrosis factor-α (TNF-α) is a proinflammation cytokine secreted by various cells. Understanding its secretive pathway is important to understand the biological functions of TNF-α and diseases associated with TNF-α. TNF-α is one of the first proteins known be modified by lysine fatty acylation (e.g. myristoylation). We previously demonstrated that SIRT6, a member of the mammalian sirtuin family of enzymes, can remove the fatty acyl modification on TNF-α and promote its secretion. However, the mechanistic details about how lysine fatty acylation regulates TNF-α secretion have been unknown. Here we present experimental data supporting that lysine fatty acylation promotes lysosomal targeting of TNF-α. The result is an important first step toward understanding the biological functions of lysine fatty acylation. PMID:27079798

  11. Iron-binding drugs targeted to lysosomes: a potential strategy to treat inflammatory lung disorders.

    PubMed

    Persson, H Lennart; Richardson, Des R

    2005-08-01

    In many inflammatory lung disorders, an abnormal assimilation of redox-active iron will exacerbate oxidative tissue damage. It may be that the most important cellular pool of redox-active iron exists within lysosomes, making these organelles vulnerable to oxidative stress. In experiments employing respiratory epithelial cells and macrophages, the chelation of intra-lysosomal iron efficiently prevented lysosomal rupture and the ensuing cell death induced by hydrogen peroxide, ionising radiation or silica particles. Furthermore, cell-permeable iron-binding agents (weak bases) that accumulate within lysosomes due to proton trapping were much more efficient for cytoprotection than the chelator, desferrioxamine. On a molar basis, the weak base alpha-lipoic acid plus was 5000 times more effective than desferrioxamine at preventing lysosomal rupture and apoptotic cell death in cell cultures exposed to hydrogen peroxide. Thus, iron-chelating therapy that targets the lysosome might be a future treatment strategy for inflammatory pulmonary diseases. PMID:16050792

  12. A Lysosome-Targeting AIEgen for Autophagy Visualization.

    PubMed

    Leung, Chris Wai Tung; Wang, Zhiming; Zhao, Engui; Hong, Yuning; Chen, Sijie; Kwok, Ryan Tsz Kin; Leung, Anakin Chun Sing; Wen, Rongsen; Li, Bingshi; Lam, Jacky Wing Yip; Tang, Ben Zhong

    2016-02-18

    In this work, a morpholine-functionalized aggregation-induced emission luminogen (AIEgen), AIE-LysoY, is reported for lysosomal imaging and autophagy visualization. To attain outstanding imaging contrast, AIE-LysoY is equipped with excited state intramolecular proton transfer (ESIPT) characteristic. AIE-LysoY provides a new platform for lysosome visualization with good biocompatibility, large Stokes shift, superior signal-to-noise ratio, and high photostability. PMID:26688031

  13. Design of a simultaneous target and location-activatable fluorescent probe for visualizing hydrogen sulfide in lysosomes.

    PubMed

    Yang, Sheng; Qi, Yue; Liu, Changhui; Wang, Yijun; Zhao, Yirong; Wang, Lili; Li, Jishan; Tan, Weihong; Yang, Ronghua

    2014-08-01

    Molecular tools capable of providing information on a target analyte in an organelle of interest are especially appreciated. Traditionally, organelle-targetable probes are designed by incorporating an organelle-specific guiding unit to target the probe molecules into the organelle. The imperfect targeting function of the guiding unit and nonspecific distribution of the analyte in cytosol and each organelle would lead to low spatiotemporal resolution and limited sensitivity. To solve this problem, we report herein a new approach for detection of a target analyte in a specific organelle by engineering a target and location dual-controlled molecular switch. For this proof-of-concept study, fluorescent detection of H2S in lysosomes was performed with a simultaneous H2S and proton-activatable probe based on the acidic environment of lysosomes. The new synthesized fluorescent sensor, "SulpHensor", which contains a spirolactam moiety to bind hydrogen protons and an azide group to react with H2S, displays highly sensitive and selective fluorescence response to H2S under lysosomal pH environment but is out of operation in neutral cytosol and other organelles. Fluorescence imaging shows that SulpHensor is membrane-permeable and suitable for visualization of both the exogenous and endogenous H2S in lysosomes of living cells. The good performance of our proposed approach for H2S sensing demonstrates that this strategy might open up new opportunities for the development of efficient subcellular molecular tools for bioanalytical and biomedical applications. PMID:24975419

  14. Two motifs target Batten disease protein CLN3 to lysosomes in transfected nonneuronal and neuronal cells.

    PubMed

    Kyttälä, Aija; Ihrke, Gudrun; Vesa, Jouni; Schell, Michael J; Luzio, J Paul

    2004-03-01

    Batten disease is a neurodegenerative disorder resulting from mutations in CLN3, a polytopic membrane protein, whose predominant intracellular destination in nonneuronal cells is the lysosome. The topology of CLN3 protein, its lysosomal targeting mechanism, and the development of Batten disease are poorly understood. We provide experimental evidence that both the N and C termini and one large loop domain of CLN3 face the cytoplasm. We have identified two lysosomal targeting motifs that mediate the sorting of CLN3 in transfected nonneuronal and neuronal cells: an unconventional motif in the long C-terminal cytosolic tail consisting of a methionine and a glycine separated by nine amino acids [M(X)9G], and a more conventional dileucine motif, located in the large cytosolic loop domain and preceded by an acidic patch. Each motif on its own was sufficient to mediate lysosomal targeting, but optimal efficiency required both. Interestingly, in primary neurons, CLN3 was prominently seen both in lysosomes in the cell body and in endosomes, containing early endosomal antigen-1 along neuronal processes. Because there are few lysosomes in axons and peripheral parts of dendrites, the presence of CLN3 in endosomes of neurons may be functionally important. Endosomal association of the protein was independent of the two lysosomal targeting motifs. PMID:14699076

  15. Receptor Crosslinking: A General Method to Trigger Internalization and Lysosomal Targeting of Therapeutic Receptor:Ligand Complexes

    PubMed Central

    Moody, Paul R; Sayers, Edward J; Magnusson, Johannes P; Alexander, Cameron; Borri, Paola; Watson, Peter; Jones, Arwyn T

    2015-01-01

    A major unmet clinical need is a universal method for subcellular targeting of bioactive molecules to lysosomes. Delivery to this organelle enables either degradation of oncogenic receptors that are overexpressed in cancers, or release of prodrugs from antibody–drug conjugates. Here, we describe a general method that uses receptor crosslinking to trigger endocytosis and subsequently redirect trafficking of receptor:cargo complexes from their expected route, to lysosomes. By incubation of plasma membrane receptors with biotinylated cargo and subsequent addition of streptavidin to crosslink receptor:cargo–biotin complexes, we achieved rapid and selective lysosomal targeting of transferrin, an anti-MHC class I antibody, and the clinically approved anti-Her2 antibody trastuzumab. These three protein ligands each target a receptor with a distinct cellular function and intracellular trafficking profile. Importantly, we confirmed that crosslinking of trastuzumab increased lysosomal degradation of its cognate oncogenic receptor Her2 in breast cancer cell lines SKBR3 and BT474. These data suggest that crosslinking could be exploited for a wide range of target receptors, for navigating therapeutics through the endolysosomal pathway, for significant therapeutic benefit. PMID:26412588

  16. An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease

    PubMed Central

    Kizuka, Yasuhiko; Kitazume, Shinobu; Fujinawa, Reiko; Saito, Takashi; Iwata, Nobuhisa; Saido, Takaomi C; Nakano, Miyako; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro; Staufenbiel, Matthias; Hatsuta, Hiroyuki; Murayama, Shigeo; Manya, Hiroshi; Endo, Tamao; Taniguchi, Naoyuki

    2015-01-01

    The β-site amyloid precursor protein cleaving enzyme-1 (BACE1), an essential protease for the generation of amyloid-β (Aβ) peptide, is a major drug target for Alzheimer's disease (AD). However, there is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N-acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT-III (Mgat3), revealed that cleavage of Aβ-precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in Aβ plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin-2, are normally cleaved in GnT-III-deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT-III-deficient mice remain healthy, GnT-III may be a novel and promising drug target for AD therapeutics. PMID:25592972

  17. Iowa Mutant Apolipoprotein A-I (ApoA-IIowa) Fibrils Target Lysosomes.

    PubMed

    Kameyama, Hirokazu; Nakajima, Hiroyuki; Nishitsuji, Kazuchika; Mikawa, Shiho; Uchimura, Kenji; Kobayashi, Norihiro; Okuhira, Keiichiro; Saito, Hiroyuki; Sakashita, Naomi

    2016-01-01

    The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIowa) is the first mutation that was associated with familial AApoA1 amyloidosis. The N-terminal fragments (amino acid residues 1-83) of apoA-I containing this mutation deposit as amyloid fibrils in patients' tissues and organs, but the mechanisms of cellular degradation and cytotoxicity have not yet been clarified. In this study, we demonstrated degradation of apoA-IIowa fibrils via the autophagy-lysosomal pathway in human embryonic kidney 293 cells. ApoA-IIowa fibrils induced an increase in lysosomal pH and the cytosolic release of the toxic lysosomal protease cathepsin B. The mitochondrial dysfunction caused by apoA-IIowa fibrils depended on cathepsin B and was ameliorated by increasing the degradation of apoA-IIowa fibrils. Thus, although apoA-IIowa fibril transport to lysosomes and fibril degradation in lysosomes may have occurred, the presence of an excess number of apoA-IIowa fibrils, more than the lysosomes could degrade, may be detrimental to cells. Our results thus provide evidence that the target of apoA-IIowa fibrils is lysosomes, and we thereby gained a novel insight into the mechanism of AApoA1 amyloidosis. PMID:27464946

  18. Iowa Mutant Apolipoprotein A-I (ApoA-IIowa) Fibrils Target Lysosomes

    PubMed Central

    Kameyama, Hirokazu; Nakajima, Hiroyuki; Nishitsuji, Kazuchika; Mikawa, Shiho; Uchimura, Kenji; Kobayashi, Norihiro; Okuhira, Keiichiro; Saito, Hiroyuki; Sakashita, Naomi

    2016-01-01

    The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIowa) is the first mutation that was associated with familial AApoA1 amyloidosis. The N-terminal fragments (amino acid residues 1–83) of apoA-I containing this mutation deposit as amyloid fibrils in patients’ tissues and organs, but the mechanisms of cellular degradation and cytotoxicity have not yet been clarified. In this study, we demonstrated degradation of apoA-IIowa fibrils via the autophagy-lysosomal pathway in human embryonic kidney 293 cells. ApoA-IIowa fibrils induced an increase in lysosomal pH and the cytosolic release of the toxic lysosomal protease cathepsin B. The mitochondrial dysfunction caused by apoA-IIowa fibrils depended on cathepsin B and was ameliorated by increasing the degradation of apoA-IIowa fibrils. Thus, although apoA-IIowa fibril transport to lysosomes and fibril degradation in lysosomes may have occurred, the presence of an excess number of apoA-IIowa fibrils, more than the lysosomes could degrade, may be detrimental to cells. Our results thus provide evidence that the target of apoA-IIowa fibrils is lysosomes, and we thereby gained a novel insight into the mechanism of AApoA1 amyloidosis. PMID:27464946

  19. Activation of Membrane NADPH Oxidase Associated with Lysosome-Targeted Acid Sphingomyelinase in Coronary Endothelial Cells

    PubMed Central

    Bao, Jun-Xiang; Jin, Si; Zhang, Fan; Wang, Zheng-Chao; Li, Ningjun

    2010-01-01

    Abstract This study explored the mechanism mediating the aggregation of membrane NADPH oxidase (NOX) subunits and subsequent activation of this enzyme in bovine coronary arterial endothelial cells (CAECs). With confocal microscopy, we found that FasL stimulated lipid rafts (LRs) clustering with NOX subunit aggregation and acid sphingomyelinase (ASM) gathering, which was blocked by the siRNA of sortilin, an intracellular protein responsible for the binding and targeting of ASM to lysosomes. Correspondingly, FasL-induced O2·− production through NOX in LRs fractions was abolished by sortilin siRNA. Further, with flow-cytometry and fluorescence resonance energy transfer (FRET) analysis, we surprisingly demonstrated that after FasL stimulation, sortilin was exposed to cell membranes from lysosomes together with Lamp-1 and ASM, and these lysosomal components were aggregated and form a signaling complex in cell membranes. With co-immunoprecipitation, lysosomal sortilin and ASM were found to interact more strongly when CAECs were stimulated by FasL. Functionally, inhibition of either sortilin expression, lysosome function, LRs clustering, or NOX activity significantly attenuated FasL-induced decrease in nitric oxide (NO) levels. It is concluded that lysosome-targeted ASM, through sortilin, is able to traffic to and expose to cell-membrane surface, which may lead to LRs clustering and NOX activation in CAECs. Antioxid. Redox Signal. 12, 703–712. PMID:19761405

  20. Disruption of lysosomal targeting is associated with insecticidal potency of juvenile hormone esterase

    PubMed Central

    Bonning, Bryony C.; Ward, Vernon K.; van Meer, Marnix M. M.; Booth, Tim F.; Hammock, Bruce D.

    1997-01-01

    Juvenile hormone esterase (JHE; EC 3.1.1.1), which is intrinsically involved in regulation of development of some insect larvae, is rapidly removed from the hemolymph by the pericardial cells. Lys-29 and Lys-524, which are implicated in the degradation of JHE, were mutated to Arg. Neither the half-life of the modified JHE in the hemolymph nor the catalytic parameters were changed significantly, but when combined, these mutations resulted in apparent failure of lysosomal targeting in the pericardial cell complex. A hypothesis for the mechanism of reduced efficiency of lysosomal targeting is presented. Infection of larvae with a recombinant baculovirus expressing the modified JHE resulted in a 50% reduction in feeding damage compared with larvae infected with the wild-type virus, thus demonstrating improved properties as a biological insecticide. These data demonstrate that alteration of specific residues of JHE that disrupted lysosomal targeting, dramatically increased the insecticidal activity of this protein. PMID:9177159

  1. Distinguishing normal cells from cancer cells via lysosome-targetable pH biomarkers with benzo[a]phenoxazine skeleton.

    PubMed

    Zhan, Yan-Hua; Li, Xiao-Jun; Sun, Ru; Xu, Yu-Jie; Ge, Jian-Feng

    2016-08-24

    In this paper, the design of a lysosome-targetable pH probe that has a fluorescent OFF (pH = 4) to ON (pH = 5-6) response is described to identify lysosomes in normal cells. The mechanism of photoinduced electron transfer with a fluorophore-based reaction (FBR-PET) was proposed. Benzo[a]phenoxazines with electro-donating aryl groups were selected, its (2,5-dimethoxyphenyl)imino-, (2-hydroxyphenyl)imino- and (2-hydroxy-5-methoxyphenyl)- imino-derivatives (probes 1a-c) were prepared and their optical responses towards pH were evaluated; their fluorescence pH titration experiments gave regularly changes with the increasing electro-donating abilities at the linked aryl groups, the (2-hydroxy-5-methoxyphenyl)iminobenzo[a]phenoxazine (probe 1c) exhibited a nearly OFF-ON response at 580-800 nm. All probes were reversible, and they showed excellent selectivity toward the proton over other competitive species. Fluorescence confocal images were performed with HeLa, KB cancer cells and V79 normal cells, probes 1a-c are all lysosome-targetable pH probes, and benzo[a]phenoxazine with (2-hydroxy-5-methoxyphenyl)imino-group (probe 1c) has potential applications in selective differentiation of normal cells from cancer cells. PMID:27497010

  2. Toll-like receptor 4 is not targeted to the lysosome in cystic fibrosis airway epithelial cells

    PubMed Central

    Kelly, Catriona; Canning, Paul; Buchanan, Paul J.; Williams, Mark T.; Brown, Vanessa; Gruenert, Dieter C.; Elborn, J. Stuart; Ennis, Madeleine

    2013-01-01

    The innate immune response to bacterial infection is mediated through Toll-like receptors (TLRs), which trigger tightly regulated signaling cascades through transcription factors including NF-κB. LPS activation of TLR4 triggers internalization of the receptor-ligand complex which is directed toward lysosomal degradation or endocytic recycling. Cystic fibrosis (CF) patients display a robust and uncontrolled inflammatory response to bacterial infection, suggesting a defect in regulation. This study examined the intracellular trafficking of TLR4 in CF and non-CF airway epithelial cells following stimulation with LPS. We employed cells lines [16hBE14o-, CFBE41o- (CF), and CFTR-complemented CFBE41o-] and confirmed selected experiments in primary nasal epithelial cells from non-CF controls and CF patients (F508del homozygous). In control cells, TLR4 expression (surface and cytoplasmic) was reduced after LPS stimulation but remained unchanged in CF cells and was accompanied by a heightened inflammatory response 24 h after stimulation. All cells expressed markers of the early (EEA1) and late (Rab7b) endosomes at basal levels. However, only CF cells displayed persistent expression of Rab7b following LPS stimulation. Rab7 variants may directly internalize bacteria to the Golgi for recycling or to the lysosome for degradation. TLR4 colocalized with the lysosomal marker LAMP1 in 16 hBE14o- cells, suggesting that TLR4 is targeted for lysosomal degradation in these cells. However, this colocalization was not observed in CFBE41o- cells, where persistent expression of Rab7 and release of proinflammatory cytokines was detected. Consistent with the apparent inability of CF cells to target TLR4 toward the lysosome for degradation, we observed persistent surface and cytoplasmic expression of this pathogen recognition receptor. This defect may account for the prolonged cycle of chronic inflammation associated with CF. PMID:23316065

  3. Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

    SciTech Connect

    Haylett, T.; Thilo, L.

    1986-10-01

    Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D/sub 1/, was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from <0.1% to a steady-state level of approx.2.5% of the total label. As analyzed by NaDodSO/sub 4/ PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only approx.1% of internalized membrane is recycled via a membrane pool of secondary lysosomes.

  4. Engineering Lysosome-Targeting BODIPY Nanoparticles for Photoacoustic Imaging and Photodynamic Therapy under Near-Infrared Light.

    PubMed

    Hu, Wenbo; Ma, Hengheng; Hou, Bing; Zhao, Hui; Ji, Yu; Jiang, Rongcui; Hu, Xiaoming; Lu, Xiaomei; Zhang, Lei; Tang, Yufu; Fan, Quli; Huang, Wei

    2016-05-18

    Developing lysosome-targeting organic nanoparticles combined with photoacoustic imaging (PAI) and photodynamic therapy (PDT) functions toward personalized medicine are highly desired yet challenging. Here, for the first time, lysosome-targeting BODIPY nanoparticles were engineered by encapsulating near-infrared (NIR) absorbed BODIPY dye within amphiphilic DSPE-mPEG5000 for high-performing lysosomal PAI and acid-activatable PDT against cancer cells under NIR light. PMID:27123534

  5. TNFα Post-Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes.

    PubMed

    Hennigar, Stephen R; Kelleher, Shannon L

    2015-10-01

    Mammary epithelial cells undergo widespread lysosomal-mediated cell death (LCD) during early mammary gland involution. Recently, we demonstrated that tumor necrosis factor-α (TNFα), a cytokine released during early involution, redistributes the zinc (Zn) transporter ZnT2 to accumulate Zn in lysosomes and activate LCD and involution. The objective of this study is to determine how TNFα retargets ZnT2 to lysosomes. We tested the hypothesis that TNFα signaling dephosphorylates ZnT2 to uncover a highly conserved dileucine motif (L294L) in the C-terminus of ZnT2, allowing adaptor protein complex-3 (AP-3) to bind and traffic ZnT2 to lysosomes. Confocal micrographs showed that TNFα redistributed wild-type (WT) ZnT2 from late endosomes (Pearson's coefficient = 0.202 ± 0.05 and 0.097 ± 0.03; P<0.05) to lysosomes (0.292 ± 0.03 and 0.649 ± 0.03; P<0.0001), which increased lysosomal Zn (P<0.0001) and activated LCD (P<0.0001) compared to untreated cells. Mutation of the dileucine motif (L294V) eliminated the ability of TNFα to redistribute ZnT2 from late endosomes to lysosomes, increase lysosomal Zn, or activate LCD. Moreover, TNFα increased (P<0.05) AP-3 binding to wt ZnT2 but not to L294V immunoprecipitates. Finally, using phospho- and dephospho-mimetics of predicted phosphorylation sites (T281, T288, and S296), we found that dephosphorylated S296 was required to target ZnT2 to accumulate Zn in lysosomes and activate LCD. Our findings suggest that women with variation in the C-terminus of ZnT2 may be at risk for inadequate involution and breast disease due the inability to traffic ZnT2 to lysosomes. PMID:25808614

  6. Identification of a Novel Lysosomal Trafficking Peptide using Phage Display Biopanning Coupled with Endocytic Selection Pressure

    PubMed Central

    2015-01-01

    Methods to select ligands that accumulate specifically in cancer cells and traffic through a defined endocytic pathway may facilitate rapid pairing of ligands with linkers suitable for drug conjugate therapies. We performed phage display biopanning on cancer cells that are treated with selective inhibitors of a given mechanism of endocytosis. Using chlorpromazine to inhibit clathrin-mediated endocytosis in H1299 nonsmall cell lung cancer cells, we identified two clones, ATEPRKQYATPRVFWTDAPG (15.1) and a novel peptide LQWRRDDNVHNFGVWARYRL (H1299.3). The peptides segregate by mechanism of endocytosis and subsequent location of subcellular accumulation. The H1299.3 peptide primarily utilizes clathrin-mediated endocytosis and colocalizes with Lamp1, a lysosomal marker. Conversely, the 15.1 peptide is clathrin-independent and localizes to a perinuclear region. Thus, this novel phage display scheme allows for selection of peptides that selectively internalize into cells via a known mechanism of endocytosis. These types of selections may allow for better matching of linker with targeting ligand by selecting ligands that internalize and traffic to known subcellular locations. PMID:25188559

  7. Marked enhancement of lysosomal targeting and efficacy of ErbB2-targeted drug delivery by HSP90 inhibition

    PubMed Central

    Mohapatra, Bhopal; Luan, Haitao; Soni, Kruti; Zhang, Jinjin; Storck, Matthew A.; Feng, Dan; Bielecki, Timothy A.; Band, Vimla; Cohen, Samuel M.; Bronich, Tatiana K.; Band, Hamid

    2016-01-01

    Targeted delivery of anticancer drugs to tumor cells using monoclonal antibodies against oncogenic cell surface receptors is an emerging therapeutic strategy. These strategies include drugs directly conjugated to monoclonal antibodies through chemical linkers (Antibody-Drug Conjugates, ADCs) or those encapsulated within nanoparticles that in turn are conjugated to targeting antibodies (Antibody-Nanoparticle Conjugates, ANPs). The recent FDA approval of the ADC Trastuzumab-TDM1 (Kadcyla®; Genentech; San Francisco) for the treatment of ErbB2-overexpressing metastatic breast cancer patients has validated the strong potential of these strategies. Even though the activity of ANPs and ADCs is dependent on lysosomal traffic, the roles of the endocytic route traversed by the targeted receptor and of cancer cell-specific alterations in receptor dynamics on the efficiency of drug delivery have not been considered in these new targeted therapies. For example, constitutive association with the molecular chaperone HSP90 is thought to either retard ErbB2 endocytosis or to promote its recycling, traits undesirable for targeted therapy with ANPs and ADCs. HSP90 inhibitors are known to promote ErbB2 ubiquitination, targeting to lysosome and degradation. We therefore hypothesized that ErbB2-targeted drug delivery using Trastuzumab-conjugated nanoparticles could be significantly improved by HSP90 inhibitor-promoted lysosomal traffic of ErbB2. Studies reported here validate this hypothesis and demonstrate, both in vitro and in vivo, that HSP90 inhibition facilitates the intracellular delivery of Trastuzumab-conjugated ANPs carrying a model chemotherapeutic agent, Doxorubicin, specifically into ErbB2-overexpressing breast cancer cells, resulting in improved antitumor activity. These novel findings highlight the need to consider oncogene-specific alterations in receptor traffic in the design of targeted drug delivery strategies. We suggest that combination of agents that enhance

  8. Marked enhancement of lysosomal targeting and efficacy of ErbB2-targeted drug delivery by HSP90 inhibition.

    PubMed

    Raja, Srikumar M; Desale, Swapnil S; Mohapatra, Bhopal; Luan, Haitao; Soni, Kruti; Zhang, Jinjin; Storck, Matthew A; Feng, Dan; Bielecki, Timothy A; Band, Vimla; Cohen, Samuel M; Bronich, Tatiana K; Band, Hamid

    2016-03-01

    Targeted delivery of anticancer drugs to tumor cells using monoclonal antibodies against oncogenic cell surface receptors is an emerging therapeutic strategy. These strategies include drugs directly conjugated to monoclonal antibodies through chemical linkers (Antibody-Drug Conjugates, ADCs) or those encapsulated within nanoparticles that in turn are conjugated to targeting antibodies (Antibody-Nanoparticle Conjugates, ANPs). The recent FDA approval of the ADC Trastuzumab-TDM1 (Kadcyla; Genentech; San Francisco) for the treatment of ErbB2-overexpressing metastatic breast cancer patients has validated the strong potential of these strategies. Even though the activity of ANPs and ADCs is dependent on lysosomal traffic, the roles of the endocytic route traversed by the targeted receptor and of cancer cell-specific alterations in receptor dynamics on the efficiency of drug delivery have not been considered in these new targeted therapies. For example, constitutive association with the molecular chaperone HSP90 is thought to either retard ErbB2 endocytosis or to promote its recycling, traits undesirable for targeted therapy with ANPs and ADCs. HSP90 inhibitors are known to promote ErbB2 ubiquitination, targeting to lysosome and degradation. We therefore hypothesized that ErbB2-targeted drug delivery using Trastuzumab-conjugated nanoparticles could be significantly improved by HSP90 inhibitor-promoted lysosomal traffic of ErbB2. Studies reported here validate this hypothesis and demonstrate, both in vitro and in vivo, that HSP90 inhibition facilitates the intracellular delivery of Trastuzumab-conjugated ANPs carrying a model chemotherapeutic agent, Doxorubicin, specifically into ErbB2-overexpressing breast cancer cells, resulting in improved antitumor activity. These novel findings highlight the need to consider oncogene-specific alterations in receptor traffic in the design of targeted drug delivery strategies. We suggest that combination of agents that enhance receptor

  9. Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis

    PubMed Central

    Napolitano, Gennaro; Johnson, Jennifer L; He, Jing; Rocca, Celine J; Monfregola, Jlenia; Pestonjamasp, Kersi; Cherqui, Stephanie; Catz, Sergio D

    2015-01-01

    Metabolite accumulation in lysosomal storage disorders (LSDs) results in impaired cell function and multi-systemic disease. Although substrate reduction and lysosomal overload-decreasing therapies can ameliorate disease progression, the significance of lysosomal overload-independent mechanisms in the development of cellular dysfunction is unknown for most LSDs. Here, we identify a mechanism of impaired chaperone-mediated autophagy (CMA) in cystinosis, a LSD caused by defects in the cystine transporter cystinosin (CTNS) and characterized by cystine lysosomal accumulation. We show that, different from other LSDs, autophagosome number is increased, but macroautophagic flux is not impaired in cystinosis while mTOR activity is not affected. Conversely, the expression and localization of the CMA receptor LAMP2A are abnormal in CTNS-deficient cells and degradation of the CMA substrate GAPDH is defective in Ctns−/− mice. Importantly, cysteamine treatment, despite decreasing lysosomal overload, did not correct defective CMA in Ctns−/− mice or LAMP2A mislocalization in cystinotic cells, which was rescued by CTNS expression instead, suggesting that cystinosin is important for CMA activity. In conclusion, CMA impairment contributes to cell malfunction in cystinosis, highlighting the need for treatments complementary to current therapies that are based on decreasing lysosomal overload. PMID:25586965

  10. Targeted disruption of the mouse cation-dependent mannose 6-phosphate receptor results in partial missorting of multiple lysosomal enzymes.

    PubMed Central

    Ludwig, T; Ovitt, C E; Bauer, U; Hollinshead, M; Remmler, J; Lobel, P; Rüther, U; Hoflack, B

    1993-01-01

    In mammalian cells two mannose 6-phosphate receptors (MPRs) are involved in lysosomal enzyme transport. To understand the precise function of the cation-dependent mannose 6-phosphate receptor (CD-MPR), one allele of the corresponding gene has been disrupted in mouse embryonic stem cells and homozygous mice lacking this receptor have been generated. The homozygous mice appear normal, suggesting that other targeting mechanisms can partially compensate for the loss of the CD-MPR in vivo. However, homozygous receptor-deficient cells and animals clearly exhibit defects in targeting of multiple lysosomal enzymes when compared with wild-types. Increased levels of phosphorylated lysosomal enzymes were present in body fluids of homozygous animals. In thymocytes from homozygous mice or in primary cultures of fibroblasts from homozygous embryos, there is a marked increase in the amount of phosphorylated lysosomal enzymes that are secreted into the extracellular medium. The cultured fibroblasts have decreased intracellular levels of multiple lysosomal enzymes and accumulate macromolecules within their endosomal/lysosomal system. Taken together, these results clearly indicate that the CD-MPR is required for efficient intracellular targeting of multiple lysosomal enzymes. Images PMID:8262065

  11. (-)-Oleocanthal rapidly and selectively induces cancer cell death via lysosomal membrane permeabilization

    PubMed Central

    LeGendre, Onica; Breslin, Paul AS; Foster, David A

    2015-01-01

    (-)-Oleocanthal (OC), a phenolic compound present in extra-virgin olive oil (EVOO), has been implicated in the health benefits associated with diets rich in EVOO. We investigated the effect of OC on human cancer cell lines in culture and found that OC induced cell death in all cancer cells examined as rapidly as 30 minutes after treatment in the absence of serum. OC treatment of non-transformed cells suppressed their proliferation but did not cause cell death. OC induced both primary necrotic and apoptotic cell death via induction of lysosomal membrane permeabilization (LMP). We provide evidence that OC promotes LMP by inhibiting acid sphingomyelinase (ASM) activity, which destabilizes the interaction between proteins required for lysosomal membrane stability. The data presented here indicate that cancer cells, which tend to have fragile lysosomal membranes compared to non-cancerous cells, are susceptible to cell death induced by lysosomotropic agents. Therefore, targeting lysosomal membrane stability represents a novel approach for the induction of cancer-specific cell death. PMID:26380379

  12. Targeting, Endocytosis, and Lysosomal Delivery of Active Enzymes to Model Human Neurons by ICAM-1-Targeted Nanocarriers

    PubMed Central

    Hsu, Janet; Hoenicka, Janet; Muro, Silvia

    2016-01-01

    Purpose Delivery of therapeutics to neurons is paramount to treat neurological conditions, including many lysosomal storage disorders. However, key aspects of drug-carrier behavior in neurons are relatively unknown: the occurrence of non-canonical endocytic pathways (present in other cells); whether carriers that traverse the blood-brain barrier are, contrarily, retained within neurons; if neuron-surface receptors are accessible to bulky carriers compared to small ligands; or if there are differences regarding neuronal compartments (neuron body vs. neurites) pertaining said parameters. We have explored these questions using model polymer nanocarriers targeting intercellular adhesion molecule-1 (ICAM-1). Methods Differentiated human neuroblastoma cells were incubated with anti-ICAM-coated polystyrene nanocarriers and analyzed by fluorescence microscopy. Results ICAM-1 expression and nanocarrier binding was enhanced in altered (TNFα) vs. control conditions. While small ICAM-1 ligands (anti-ICAM) preferentially accessed the cell body, anti-ICAM nanocarriers bound with faster kinetics to neurites, yet reached similar saturation over time. Anti-ICAM nanocarriers were also endocytosed with faster kinetics and lower saturation levels in neurites. Non-classical cell adhesion molecule (CAM) endocytosis ruled uptake, and neurite-to-cell body transport was inferred. Nanocarriers trafficked to lysosomes, delivering active enzymes (dextranase) with substrate reduction in a lysosomal-storage disease model. Conclusion ICAM-1-targeting holds potential for intracellular delivery of therapeutics to neurons. PMID:25319100

  13. Two Novel Human Cytomegalovirus NK Cell Evasion Functions Target MICA for Lysosomal Degradation

    PubMed Central

    Fielding, Ceri A.; Aicheler, Rebecca; Stanton, Richard J.; Wang, Eddie C. Y.; Han, Song; Seirafian, Sepehr; Davies, James; McSharry, Brian P.; Weekes, Michael P.; Antrobus, P. Robin; Prod'homme, Virginie; Blanchet, Fabien P.; Sugrue, Daniel; Cuff, Simone; Roberts, Dawn; Davison, Andrew J.; Lehner, Paul J.; Wilkinson, Gavin W. G.; Tomasec, Peter

    2014-01-01

    NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αβ and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1–6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12–US21; a genetic arrangement, which is suggestive of an ‘accordion’ expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on

  14. Visualization of Endogenous and Exogenous Hydrogen Peroxide Using A Lysosome-Targetable Fluorescent Probe

    NASA Astrophysics Data System (ADS)

    Kim, Dabin; Kim, Gyoungmi; Nam, Sang-Jip; Yin, Jun; Yoon, Juyoung

    2015-02-01

    Reactive oxygen species (ROS) play crucial roles in diverse physiological processes; therefore, the efficient detection of ROS is very crucial. In this study, we report a boronate-based hydrogen peroxide (H2O2) probe having naphthalimide fluorophore. This probe also contained a morpholine moiety as a directing group for lysosome. The recognition property indicated that the probe exhibited high selectivity towards H2O2 not only in the solution but also in the living cells. Furthermore, it was used to monitor the level of endogenous and exogenous H2O2. These results support that the probe can function as an efficient indicator to detect H2O2.

  15. Site-1 protease-activated formation of lysosomal targeting motifs is independent of the lipogenic transcription control[S

    PubMed Central

    Klünder, Sarah; Heeren, Jörg; Markmann, Sandra; Santer, René; Braulke, Thomas; Pohl, Sandra

    2015-01-01

    Site-1 protease (S1P) cleaves membrane-bound lipogenic sterol regulatory element-binding proteins (SREBPs) and the α/β-subunit precursor protein of the N-acetylglucosamine-1-phosphotransferase forming mannose 6-phosphate (M6P) targeting markers on lysosomal enzymes. The translocation of SREBPs from the endoplasmic reticulum (ER) to the Golgi-resident S1P depends on the intracellular sterol content, but it is unknown whether the ER exit of the α/β-subunit precursor is regulated. Here, we investigated the effect of cholesterol depletion (atorvastatin treatment) and elevation (LDL overload) on ER-Golgi transport, S1P-mediated cleavage of the α/β-subunit precursor, and the subsequent targeting of lysosomal enzymes along the biosynthetic and endocytic pathway to lysosomes. The data showed that the proteolytic cleavage of the α/β-subunit precursor into mature and enzymatically active subunits does not depend on the cholesterol content. In either treatment, lysosomal enzymes are normally decorated with M6P residues, allowing the proper sorting to lysosomes. In addition, we found that, in fibroblasts of mucolipidosis type II mice and Niemann-Pick type C patients characterized by aberrant cholesterol accumulation, the proteolytic cleavage of the α/β-subunit precursor was not impaired. We conclude that S1P substrate-dependent regulatory mechanisms for lipid synthesis and biogenesis of lysosomes are different. PMID:26108224

  16. SETI target selection.

    NASA Astrophysics Data System (ADS)

    Latham, D. W.; Soderblom, D. R.

    1995-06-01

    The NASA High Resolution Microwave Survey consists of two complementary elements: a Sky Survey of the entire sky to a moderate level of sensitivity; and a Targeted Search of nearby stars, one at a time, to a much deeper level of sensitivity. The authors propose strategies for target selection with two goals: to improve the chances of successful detection of signals from technical civilizations that inhabit planets around solar-type stars, and to minimize the chances of missing signals from unexpected sites.

  17. A comparative study of lysosome-targetable pH probes based on phenoxazinium attached with aliphatic and aromatic amines.

    PubMed

    Wang, Xiu-Li; Li, Xiao-Jun; Sun, Ru; Xu, Yu-Jie; Ge, Jian-Feng

    2016-05-10

    In this paper, phenoxazinium was used as a fluorophore for the design of pH probes by the photoinduced electron transfer (PET) mechanism. Phenoxazinium with an aliphatic morpholinyl group (probe ) gave increased emission at 665 nm with pH ranging from 7.4 to 4.4; meanwhile, the other one with an aromatic diethylaminophenyl group (probe ) gave nearly OFF-ON emission at 679 nm with pH ranging from 7.4 to 4.2. They both were reversible pH probes with good selectivity. Their optical properties, especially the PET mechanism, were illustrated by (TD)DFT theory. Fluorescence confocal imaging of probes and a typical phenoxazinium dye (Oxazine 1) was also performed, and the results indicated that probes are lysosome-targetable biomarkers. PMID:27048759

  18. Identification of the amino acid sequence that targets peroxiredoxin 6 to lysosome-like structures of lung epithelial cells.

    PubMed

    Sorokina, Elena M; Feinstein, Sheldon I; Milovanova, Tatyana N; Fisher, Aron B

    2009-11-01

    Peroxiredoxin 6 (Prdx6), an enzyme with glutathione peroxidase and PLA2 (aiPLA2) activities, is highly expressed in respiratory epithelium, where it participates in phospholipid turnover and antioxidant defense. Prdx6 has been localized by immunocytochemistry and subcellular fractionation to acidic organelles (lung lamellar bodies and lysosomes) and cytosol. On the basis of their pH optima, we have postulated that protein subcellular localization determines the balance between the two activities of Prdx6. Using green fluorescent protein-labeled protein expression in alveolar epithelial cell lines, we showed Prdx6 localization to organellar structures resembling lamellar bodies in mouse lung epithelial (MLE-12) cells and lysosomes in A549 cells. Localization within lamellar bodies/lysosomes was in the luminal compartment. Targeting to lysosome-like organelles was abolished by the deletion of amino acids 31-40 from the Prdx6 NH2-terminal region; deletion of the COOH-terminal region had no effect. A green fluorescent protein-labeled peptide containing only amino acids 31-40 showed lysosomal targeting that was abolished by mutation of S32 or G34 within the peptide. Studies with mutated protein indicated that lipid binding was not necessary for Prdx6 targeting. This peptide sequence has no homology to known organellar targeting motifs. These studies indicate that the localization of Prdx6 in acidic organelles and consequent PLA2 activity depend on a novel 10-aa peptide located at positions 31-40 of the protein. PMID:19700648

  19. Burglar Target Selection

    PubMed Central

    Townsley, Michael; Bernasco, Wim; Ruiter, Stijn; Johnson, Shane D.; White, Gentry; Baum, Scott

    2015-01-01

    Objectives: This study builds on research undertaken by Bernasco and Nieuwbeerta and explores the generalizability of a theoretically derived offender target selection model in three cross-national study regions. Methods: Taking a discrete spatial choice approach, we estimate the impact of both environment- and offender-level factors on residential burglary placement in the Netherlands, the United Kingdom, and Australia. Combining cleared burglary data from all study regions in a single statistical model, we make statistical comparisons between environments. Results: In all three study regions, the likelihood an offender selects an area for burglary is positively influenced by proximity to their home, the proportion of easily accessible targets, and the total number of targets available. Furthermore, in two of the three study regions, juvenile offenders under the legal driving age are significantly more influenced by target proximity than adult offenders. Post hoc tests indicate the magnitudes of these impacts vary significantly between study regions. Conclusions: While burglary target selection strategies are consistent with opportunity-based explanations of offending, the impact of environmental context is significant. As such, the approach undertaken in combining observations from multiple study regions may aid criminology scholars in assessing the generalizability of observed findings across multiple environments. PMID:25866418

  20. Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer’s-like axonal dystrophy

    PubMed Central

    Lee, Sooyeon; Sato, Yutaka; Nixon, Ralph A.

    2012-01-01

    In the hallmark neuritic dystrophy of Alzheimer’s disease (AD), autophagic vacuoles containing incompletely digested proteins selectively accumulate in focal axonal swellings, reflecting defects in both axonal transport and autophagy. Here, we investigated the possibility that impaired lysosomal proteolysis could be a basis for both defects leading to neuritic dystrophy. In living primary mouse cortical neurons expressing fluorescence-tagged markers, LC3-positive autophagosomes forming in axons rapidly acquired the endo-lysosomal markers, Rab7 and LAMP1, and underwent exclusive retrograde movement. Proteolytic clearance of these transported autophagic vacuoles was initiated upon fusion with bi-directionally moving lysosomes that increase in number at more proximal axon levels and in the perikaryon. Disrupting lysosomal proteolysis by either inhibiting cathepsins directly or by suppressing lysosomal acidification slowed the axonal transport of autolysosomes, late endosomes and lysosomes and caused their selective accumulation within dystrophic axonal swellings. Mitochondria and other organelles lacking cathepsins moved normally under these conditions, indicating that the general functioning of the axonal transport system was preserved. Dystrophic swellings induced by lysosomal proteolysis inhibition resembled in composition those in several mouse models of AD and also acquired other AD-like features, including immunopositivity for ubiquitin, APP, and neurofilament protein hyperphosphorylation. Restoration of lysosomal proteolysis reversed the affected movements of proteolytic Rab7 vesicles, which in turn, largely cleared autophagic substrates and reversed the axonal dystrophy. These studies identify the AD-associated defects in neuronal lysosomal proteolysis as a possible basis for the selective transport abnormalities and highly characteristic pattern of neuritic dystrophy associated with AD. PMID:21613495

  1. Targeting HER2+ breast cancer cells: lysosomal accumulation of anti-HER2 antibodies is influenced by antibody binding site and conjugation to polymeric nanoparticles.

    PubMed

    Owen, Shawn C; Patel, Nish; Logie, Jennifer; Pan, Guohua; Persson, Helena; Moffat, Jason; Sidhu, Sachdev S; Shoichet, Molly S

    2013-12-10

    Humanized monoclonal antibodies (mAb) against HER2 are being engineered to treat cancer. We utilized phage-display technology to generate a novel anti-HER2 mAb (named 73JIgG) that binds an epitope of HER2 distinct from that of trastuzumab. Although these mAbs bind to the same cell surface receptor, they have different cell distribution profiles. After 3h of incubation, almost 10% of the total 73JIgG reaches the lysosome compared to less than 3% of trastuzumab. Interestingly, 73JIgG disassociates from HER2 whereas trastuzumab remains bound to the receptor. Importantly, HER2 distribution is not affected by the antibody binding epitope, thus negating this mechanism as the reason for the difference in intracellular trafficking of 73JIgG versus trastuzumab. Each of trastuzumab and 73JIgG was chemically-modified with either a small molecule or polymeric nanoparticle to better understand the influence of conjugation on cellular localization. Relative to antibody alone, antibody-nanoparticle conjugates resulted in a higher concentration of antibodies in the lysosome whereas antibody-small molecule conjugates did not affect cell trafficking to the lysosome. Given the importance of lysosomal targeting, these results demonstrate the importance of understanding the influence of the antibody-conjugate on cell trafficking for ultimate optimization of treatment selection. PMID:23880472

  2. A Requirement for Bid for Induction of Apoptosis by Photodynamic Therapy with a Lysosome- but not a Mitochondrion-Targeted Photosensitizer

    PubMed Central

    Chiu, Song-mao; Xue, Liang-yan; Lam, Minh; Rodriguez, Myriam E.; Zhang, Ping; Kenney, Malcolm E.; Nieminen, Anna-Liisa; Oleinick, Nancy L.

    2010-01-01

    Photodynamic therapy (PDT) with lysosome-targeted photosensitizers induces the intrinsic pathway of apoptosis via the cleavage and activation of the BH3-only protein Bid by proteolytic enzymes released from photo-disrupted lysosomes. To investigate the role of Bid in apoptosis induction and the role of damaged lysosomes on cell killing by lysosome-targeted PDT, we compared the responses of wild type and Bid-knock-out murine embryonic fibroblasts toward a mitochondrion/endoplasmic reticulum-binding photosensitizer, Pc 4, and a lysosome-targeted sensitizer, Pc 181. Whereas apoptosis and overall cell killing were induced equally well by Pc 4-PDT in both cell lines, Bid−/− cells were relatively resistant to induction of apoptosis and to overall killing following PDT with Pc 181, particularly at low PDT doses. Thus, Bid is critical for the induction of apoptosis caused by PDT with the lysosome-specific sensitizers, but dispensable for PDT targeted to other membranes. PMID:20553412

  3. Visualization of Endogenous and Exogenous Hydrogen Peroxide Using A Lysosome-Targetable Fluorescent Probe

    PubMed Central

    Kim, Dabin; Kim, Gyoungmi; Nam, Sang-Jip; Yin, Jun; Yoon, Juyoung

    2015-01-01

    Reactive oxygen species (ROS) play crucial roles in diverse physiological processes; therefore, the efficient detection of ROS is very crucial. In this study, we report a boronate-based hydrogen peroxide (H2O2) probe having naphthalimide fluorophore. This probe also contained a morpholine moiety as a directing group for lysosome. The recognition property indicated that the probe exhibited high selectivity towards H2O2 not only in the solution but also in the living cells. Furthermore, it was used to monitor the level of endogenous and exogenous H2O2. These results support that the probe can function as an efficient indicator to detect H2O2. PMID:25684681

  4. SETI target selection.

    PubMed

    Latham, D W; Soderblom, D R

    1995-01-01

    The NASA High Resolution Microwave Survey consists of two complementary elements: a Sky Survey of the entire sky to a moderate level of sensitivity; and a Targeted Search of nearby stars, one at a time, to a much deeper level of sensitivity. In this paper we propose strategies for target selection. We have two goals: to improve the chances of successful detection of signals from technical civilizations that inhabit planets around solar-type stars, and to minimize the chances of missing signals from unexpected sites. For the main Targeted Search survey of approximately 1000 nearby solar-type stars, we argue that the selection criteria should be heavily biased by what we know about the origin and evolution of life here on Earth. We propose that observations of stars with stellar companions orbiting near the habitable zone should be de-emphasized, because such companions would prevent the formation of habitable planets. We also propose that observations of stars younger than about three billion years should be de-emphasized in favor of older stars, because our own technical civilization took longer than three billion years to evolve here on Earth. To provide the information needed for the preparation of specific target lists, we have undertaken an inventory of a large sample of solar-type stars out to a distance of 60 pc, with the goal of characterizing the relevant astrophysical properties of these stars, especially their ages and companionship. To complement the main survey, we propose that a modest sample of the nearest stars should be observed without any selection biases whatsoever. Finally, we argue that efforts to identify stars with planetary systems should be expanded. If found, such systems should receive intensive scrutiny. PMID:11540737

  5. CDTI target selection criteria

    NASA Technical Reports Server (NTRS)

    Britt, C. L.; Davis, C. M.; Jackson, C. B.; Mcclellan, V. A.

    1984-01-01

    A Cockpit Display of Traffic Information (CDTI) is a cockpit instrument which provides information to the aircrew on the relative location of aircraft traffic in the vicinity of their aircraft (township). In addition, the CDTI may provide information to assist in navigation and in aircraft control. It is usually anticipated that the CDTI will be integrated with a horizontal situation indicator used for navigational purposes and/or with a weather radar display. In this study, several sets of aircraft traffic data are analyzed to determine statistics on the number of targets that will be displayed on a CDTI using various target selection criteria. Traffic data were obtained from an Atlanta Terminal Area Simulation and from radar tapes recorded at the Atlanta and Miami terminal areas. Results are given in the form of plots showing the average percentage of time (or probability) that an aircraft equipped with a CDTI would observe from 0 to 10 other aircraft on the display for range settings on the CDTI up to 30 n. mi. and using various target discrimination techniques.

  6. Lipid-Induced Endoplasmic Reticulum Stress Impairs Selective Autophagy at the Step of Autophagosome-Lysosome Fusion in Hepatocytes.

    PubMed

    Miyagawa, Koichiro; Oe, Shinji; Honma, Yuichi; Izumi, Hiroto; Baba, Ryoko; Harada, Masaru

    2016-07-01

    Blockage of hepatic autophagic degradation system occurs in obesity and is associated with the development of nonalcoholic fatty liver disease. However, the mechanism of this blockage remains unclear. We found a high-fat diet induced accumulation of autophagosomes in the mice livers. However, autophagy substrates such as p62 and ubiquitinated proteins also accumulated in the livers in this model. These findings indicate the possibility that a high-fat diet impairs autophagic flux in the liver. Then, to assess the autophagic flux in more detail, we performed analyses of autophagic flux in cultured hepatocytes exposed to monounsaturated fatty acids (FAs) or saturated FAs (SFAs). SFAs but not monounsaturated FAs suppressed degradation of contents in the autophagosomes. We analyzed each stage of the autophagy pathway (ie, autophagosome formation, autophagosome-lysosome fusion, lysosomal degradation) in cultured hepatocytes treated with monounsaturated FAs or SFAs and found that SFAs impaired autophagosome-lysosome fusion. This impairment occurred in an endoplasmic reticulum stress-dependent manner. Moreover, ubiquitin and p62-positive inclusions observed in high-fat diet-fed mice livers and SFA-treated cells were sequestered within autophagosomes. We also found that SFA-induced accumulation of Ser351-phosphorylated p62, which is indispensable for selective autophagy, further increased on administration of a lysosomal proteinase inhibitor. Although lipid-induced endoplasmic reticulum stress interferes with the autophagosome-lysosome fusion, selective autophagic sequestration of aggregated proteins is not inhibited. PMID:27157992

  7. Targeting (cellular) lysosomal acid ceramidase by B13: Design, synthesis and evaluation of novel DMG-B13 ester prodrugs

    PubMed Central

    Bai, Aiping; Szulc, Zdzislaw, M.; Bielawski, Jacek; Pierce, Jason S.; Rembisa, Barbara; Terzieva, Silva; Mao, Cungui; Xu, Ruijuan; Wu, Bill; Clarke, Christopher J.; Newcomb, Benjamin; Liu, Xiang; Norris, James; Hannun, Yusuf A.; Bielawska, Alicja

    2015-01-01

    Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its N, N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13, LCL522 (3-O-DMG-B13•HCl) and LCL596 (1-O-DMG-B13•HCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-B13•2HCl) conjugates, were designed and synthesized through N, N-dimethyl glycine (DMG) esterification of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80-97% Control/1h) by DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase. PMID:25456083

  8. Detection of Misdistribution of Tyrosinase from Melanosomes to Lysosomes and Its Upregulation under Psoralen/Ultraviolet A with a Melanosome-Targeting Tyrosinase Fluorescent Probe.

    PubMed

    Zhou, Jin; Shi, Wen; Li, Lihong; Gong, Qiuyu; Wu, Xiaofeng; Li, Xiaohua; Ma, Huimin

    2016-04-19

    Tyrosinase is regarded as an important biomarker of melanoma cancer, and its metabolism is closely related to some severe skin diseases such as vitiligo. Since tyrosinase is mainly located in the melanosomes of melanocytes, a probe that can specifically detect and image tysosinase in melanosomes would be in urgent demand to study the behavior of the enzyme in cells, but unfortunately, no melanosome-targeting tyrosinase fluorescent probe has been reported so far to the best of our knowledge. In this work, we have developed such a new probe, Mela-TYR, which bears morpholine as a melanosome-targeting group and 4-aminophenol as a tyrosinase reaction group. The probe exhibits not only a highly sensitive and selective off-on response to tyrosinase via oxidization cleavage, but also an accurate targeting ability toward the acidic organelles of melanosomes and lyososomes, which is validated by colocalization experiments with mCherry-tagged melanosomes as well as DND-99 (a commercial dye). The probe has been used to image the relative contents of tyrosinase in different cells. Notably, because of the tyrosinase deficiency in normal lysosomes, the probe only fluoresces in melanosomes in principle although it can accumulate in other acidic organelles like lysosomes. By virtue of this property, the misdistribution of tyrosinase from melanosomes to lysosomes in murine melanoma B16 cells under the stimulation of inulavosin is imaged in real time for the first time. Moreover, the upregulation of melanosomal tyrosinase in live B16 cells under the stimulation of psoralen/ultraviolet A is detected with our probe, and this upregulation is further verified by standard colorimetric assay. The probe provides a simple, visual method to study the metabolism of tyrosinase in cells and shows great potential in clinical diagnosis and treatments of tyrosinase-associated diseases. PMID:27021123

  9. Chaperone-Mediated Autophagy Targets IFNAR1 for Lysosomal Degradation in Free Fatty Acid Treated HCV Cell Culture

    PubMed Central

    Kurt, Ramazan; Chandra, Partha K.; Aboulnasr, Fatma; Panigrahi, Rajesh; Ferraris, Pauline; Aydin, Yucel; Reiss, Krzysztof; Wu, Tong; Balart, Luis A.; Dash, Srikanta

    2015-01-01

    -treated cell culture. Pharmacological inhibitors of lysosomal degradation, such as ammonium chloride and bafilomycin, prevented IFNAR1 degradation in FFA-treated HCV cell culture. Activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased IFNAR1 levels in Huh-7.5 cells. Co-immunoprecipitation, colocalization and siRNA knockdown experiments revealed that IFNAR1 but not IFNLR1 interacts with HSC70 and LAMP2A, which are core components of chaperone-mediated autophagy (CMA). Conclusion Our study presents evidence indicating that chaperone-mediated autophagy targets IFNAR1 degradation in the lysosome in FFA-treated HCV cell culture. These results provide a mechanism for why HCV induced autophagy response selectively degrades type I but not the type III IFNAR1. PMID:25961570

  10. Mannose 6-phosphate-dependent targeting of lysosomal enzymes is required for normal craniofacial and dental development.

    PubMed

    Koehne, Till; Markmann, Sandra; Schweizer, Michaela; Muschol, Nicole; Friedrich, Reinhard E; Hagel, Christian; Glatzel, Markus; Kahl-Nieke, Bärbel; Amling, Michael; Schinke, Thorsten; Braulke, Thomas

    2016-09-01

    Mucolipidosis II (MLII) is a severe systemic genetic disorder caused by defects in mannose 6-phosphate-dependent targeting of multiple lysosomal hydrolases and subsequent lysosomal accumulation of non-degraded material. MLII patients exhibit marked facial coarseness and gingival overgrowth soon after birth, accompanied with delayed tooth eruption and dental infections. To examine the pathomechanisms of early craniofacial and dental abnormalities, we analyzed mice with an MLII patient mutation that mimic the clinical and biochemical symptoms of MLII patients. The mouse data were compared with clinical and histological data of gingiva and teeth from MLII patients. Here, we report that progressive thickening and porosity of calvarial and mandibular bones, accompanied by elevated bone loss due to 2-fold higher number of osteoclasts cause the characteristic craniofacial phenotype in MLII. The analysis of postnatal tooth development by microcomputed tomography imaging and histology revealed normal dentin and enamel formation, and increased cementum thickness accompanied with accumulation of storage material in cementoblasts of MLII mice. Massive accumulation of storage material in subepithelial cells as well as disorganization of collagen fibrils led to gingival hypertrophy. Electron and immunofluorescence microscopy, together with (35)S-sulfate incorporation experiments revealed the accumulation of non-degraded material, non-esterified cholesterol and glycosaminoglycans in gingival fibroblasts, which was accompanied by missorting of various lysosomal proteins (α-fucosidase 1, cathepsin L and Z, Npc2, α-l-iduronidase). Our study shows that MLII mice closely mimic the craniofacial and dental phenotype of MLII patients and reveals the critical role of mannose 6-phosphate-dependent targeting of lysosomal proteins for alveolar bone, cementum and gingiva homeostasis. PMID:27239697

  11. Nedd4 Mediates Agonist-dependent Ubiquitination, Lysosomal Targeting, and Degradation of the β2-Adrenergic Receptor*S⃞

    PubMed Central

    Shenoy, Sudha K.; Xiao, Kunhong; Venkataramanan, Vidya; Snyder, Peter M.; Freedman, Neil J.; Weissman, Allan M.

    2008-01-01

    Agonist-stimulated β2-adrenergic receptor (β2AR) ubiquitination is a major factor that governs both lysosomal trafficking and degradation of internalized receptors, but the identity of the E3 ubiquitin ligase regulating this process was unknown. Among the various catalytically inactive E3 ubiquitin ligase mutants that we tested, a dominant negative Nedd4 specifically inhibited isoproterenol-induced ubiquitination and degradation of the β2AR in HEK-293 cells. Moreover, siRNA that down-regulates Nedd4 expression inhibited β2AR ubiquitination and lysosomal degradation, whereas siRNA targeting the closely related E3 ligases Nedd4-2 or AIP4 did not. Interestingly, β2AR as well as β-arrestin2, the endocytic and signaling adaptor for the β2AR, interact robustly with Nedd4 upon agonist stimulation. However, β2AR-Nedd4 interaction is ablated when β-arrestin2 expression is knocked down by siRNA transfection, implicating an essential E3 ubiquitin ligase adaptor role for β-arrestin2 in mediating β2AR ubiquitination. Notably, β-arrestin2 interacts with two different E3 ubiquitin ligases, namely, Mdm2 and Nedd4 to regulate distinct steps in β2AR trafficking. Collectively, our findings indicate that the degradative fate of the β2AR in the lysosomal compartments is dependent upon β-arrestin2-mediated recruitment of Nedd4 to the activated receptor and Nedd4-catalyzed ubiquitination. PMID:18544533

  12. Lysosomal Physiology

    PubMed Central

    Xu, Haoxing; Ren, Dejian

    2015-01-01

    Lysosomes are acidic compartments filled with more than 60 different types of hydrolases. They mediate the degradation of extracellular particles from endocytosis and of intracellular components from autophagy. The digested products are transported out of the lysosome via specific catabolite exporters or via vesicular membrane trafficking. Lysosomes also contain more than 50 membrane proteins and are equipped with the machinery to sense nutrient availability, which determines the distribution, number, size, and activity of lysosomes to control the specificity of cargo flux and timing (the initiation and termination) of degradation. Defects in degradation, export, or trafficking result in lysosomal dysfunction and lysosomal storage diseases (LSDs). Lysosomal channels and transporters mediate ion flux across perimeter membranes to regulate lysosomal ion homeostasis, membrane potential, catabolite export, membrane trafficking, and nutrient sensing. Dysregulation of lysosomal channels underlies the pathogenesis of many LSDs and possibly that of metabolic and common neurodegenerative diseases. PMID:25668017

  13. Lysosomal physiology.

    PubMed

    Xu, Haoxing; Ren, Dejian

    2015-01-01

    Lysosomes are acidic compartments filled with more than 60 different types of hydrolases. They mediate the degradation of extracellular particles from endocytosis and of intracellular components from autophagy. The digested products are transported out of the lysosome via specific catabolite exporters or via vesicular membrane trafficking. Lysosomes also contain more than 50 membrane proteins and are equipped with the machinery to sense nutrient availability, which determines the distribution, number, size, and activity of lysosomes to control the specificity of cargo flux and timing (the initiation and termination) of degradation. Defects in degradation, export, or trafficking result in lysosomal dysfunction and lysosomal storage diseases (LSDs). Lysosomal channels and transporters mediate ion flux across perimeter membranes to regulate lysosomal ion homeostasis, membrane potential, catabolite export, membrane trafficking, and nutrient sensing. Dysregulation of lysosomal channels underlies the pathogenesis of many LSDs and possibly that of metabolic and common neurodegenerative diseases. PMID:25668017

  14. Lysosomal storage disorders: The cellular impact of lysosomal dysfunction

    PubMed Central

    2012-01-01

    Lysosomal storage diseases (LSDs) are a family of disorders that result from inherited gene mutations that perturb lysosomal homeostasis. LSDs mainly stem from deficiencies in lysosomal enzymes, but also in some non-enzymatic lysosomal proteins, which lead to abnormal storage of macromolecular substrates. Valuable insights into lysosome functions have emerged from research into these diseases. In addition to primary lysosomal dysfunction, cellular pathways associated with other membrane-bound organelles are perturbed in these disorders. Through selective examples, we illustrate why the term “cellular storage disorders” may be a more appropriate description of these diseases and discuss therapies that can alleviate storage and restore normal cellular function. PMID:23185029

  15. Biosynthesis, targeting, and processing of lysosomal proteins: pulse-chase labeling and immune precipitation.

    PubMed

    Pohl, Sandra; Hasilik, Andrej

    2015-01-01

    Incorporation of radioactive precursors of amino acids and/or modifier groups into proteins, isolation and sizing of polypeptide species of interest, and finally their detection and characterization provide a robust handle to examine the life cycle and varied modifications of any protein. A prerequisite in application of these techniques to lysosomal enzymes is the availability of an avid and specific antibody, because lysosomal proteins represent a very minor fraction of the cellular protein and must be purified without a significant loss many 1000-fold as conveniently as possible. Pulse-chase labeling and good knowledge on organelle-specific modifications of lysosomal proteins may enhance the information that can be obtained from such experiments. We describe procedures for pulse-chase labeling experiments that have proven to work with a commercially available antibody against a mouse and a human lysosomal protease and can be used as a reference in establishing the technique in any laboratory that has an access to a certified isotope facility and the knowledge to handle radioactivity safely. We discuss the crucial steps and refer to alternatives described in the literature. The present model protein cathepsin Z is synthesized as a larger proenzyme that contains two N-linked oligosaccharides and matures to a shorter single chain enzyme retaining the processed oligosaccharides. A pulse-chase experiment demonstrates the conversion of the precursor into the mature form. In addition, results on deglycosylation of metabolically labeled cathepsin Z are shown and the alterations in the apparent size of the glycopeptides are explained. PMID:25665441

  16. Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins.

    PubMed Central

    Köster, A; Saftig, P; Matzner, U; von Figura, K; Peters, C; Pohlmann, R

    1993-01-01

    Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo. Images PMID:8262064

  17. Cathepsin-Mediated Alterations in TGFß-Related Signaling Underlie Disrupted Cartilage and Bone Maturation Associated With Impaired Lysosomal Targeting.

    PubMed

    Flanagan-Steet, Heather; Aarnio, Megan; Kwan, Brian; Guihard, Pierre; Petrey, Aaron; Haskins, Mark; Blanchard, Frederic; Steet, Richard

    2016-03-01

    Hypersecretion of acid hydrolases is a hallmark feature of mucolipidosis II (MLII), a lysosomal storage disease caused by loss of carbohydrate-dependent lysosomal targeting. Inappropriate extracellular action of these hydrolases is proposed to contribute to skeletal pathogenesis, but the mechanisms that connect hydrolase activity to the onset of disease phenotypes remain poorly understood. Here we link extracellular cathepsin K activity to abnormal bone and cartilage development in MLII animals by demonstrating that it disrupts the balance of TGFß-related signaling during chondrogenesis. TGFß-like Smad2,3 signals are elevated and BMP-like Smad1,5,8 signals reduced in both feline and zebrafish MLII chondrocytes and osteoblasts, maintaining these cells in an immature state. Reducing either cathepsin K activity or expression of the transcriptional regulator Sox9a in MLII zebrafish significantly improved phenotypes. We further identify components of the large latent TGFß complex as novel targets of cathepsin K at neutral pH, providing a possible mechanism for enhanced Smad2,3 activation in vivo. These findings highlight the complexity of the skeletal disease associated with MLII and bring new insight to the role of secreted cathepsin proteases in cartilage development and growth factor regulation. © 2015 American Society for Bone and Mineral Research. PMID:26404503

  18. Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targeting of rat lgp120 (lamp-I) in MDCK cells

    PubMed Central

    1995-01-01

    Rat lysosomal glycoprotein 120 (lgp120; lamp-I) is a transmembrane protein that is directly delivered from the trans-Golgi network (TGN) to the endosomal/lysosomal system without prior appearance on the cell surface. Its short cytosolic domain of 11 residues encodes determinants for direct lysosomal sorting, endocytosis and, in polarized cells, basolateral targeting. We now characterize the structural requirements in the cytosolic domain required for sorting of lgp120 into the different pathways. Our results show that the cytoplasmic tail is sufficient to mediate direct transport from the trans-Golgi network (TGN) to lysosomes and that a G7-Y8-X-X-I11 motif is crucial for this sorting event. While G7 is only critical for direct lysosomal sorting in the TGN, Y8 and I11 are equally important for lysosomal sorting, endocytosis, and basolateral targeting. Thus, a small motif of five amino acids in the cytoplasmic tail of lgp120 can be recognized by the sorting machinery at several cellular locations and direct the protein into a variety of intracellular pathways. PMID:7844146

  19. Novel Mechanism of Cytotoxicity for the Selective Selenosemicarbazone, 2-Acetylpyridine 4,4-Dimethyl-3-selenosemicarbazone (Ap44mSe): Lysosomal Membrane Permeabilization.

    PubMed

    Al-Eisawi, Zaynab; Stefani, Christian; Jansson, Patric J; Arvind, Akanksha; Sharpe, Philip C; Basha, Maram T; Iskander, George M; Kumar, Naresh; Kovacevic, Zaklina; Lane, Darius J R; Sahni, Sumit; Bernhardt, Paul V; Richardson, Des R; Kalinowski, Danuta S

    2016-01-14

    Selenosemicarbazones show marked antitumor activity. However, their mechanism of action remains unknown. We examined the medicinal chemistry of the selenosemicarbazone, 2-acetylpyridine 4,4-dimethyl-3-selenosemicarbazone (Ap44mSe), and its iron and copper complexes to elucidate its mechanisms of action. Ap44mSe demonstrated a pronounced improvement in selectivity toward neoplastic relative to normal cells compared to its parent thiosemicarbazone. It also effectively depleted cellular Fe, resulting in transferrin receptor-1 up-regulation, ferritin down-regulation, and increased expression of the potent metastasis suppressor, N-myc downstream regulated gene-1. Significantly, Ap44mSe limited deleterious methemoglobin formation, highlighting its usefulness in overcoming toxicities of clinically relevant thiosemicarbazones. Furthermore, Cu-Ap44mSe mediated intracellular reactive oxygen species generation, which was attenuated by the antioxidant, N-acetyl-L-cysteine, or Cu sequestration. Notably, Ap44mSe forms redox active Cu complexes that target the lysosome to induce lysosomal membrane permeabilization. This investigation highlights novel structure-activity relationships for future chemotherapeutic design and underlines the potential of Ap44mSe as a selective anticancer/antimetastatic agent. PMID:26645570

  20. Assessment of a targeted resequencing assay as a support tool in the diagnosis of lysosomal storage disorders

    PubMed Central

    2014-01-01

    Background With over 50 different disorders and a combined incidence of up to 1/3000 births, lysosomal storage diseases (LSDs) constitute a major public health problem and place an enormous burden on affected individuals and their families. Many factors make LSD diagnosis difficult, including phenotype and penetrance variability, shared signs and symptoms, and problems inherent to biochemical diagnosis. Developing a powerful diagnostic tool could mitigate the protracted diagnostic process for these families, lead to better outcomes for current and proposed therapies, and provide the basis for more appropriate genetic counseling. Methods We have designed a targeted resequencing assay for the simultaneous testing of 57 lysosomal genes, using in-solution capture as the enrichment method and two different sequencing platforms. A total of 84 patients with high to moderate-or low suspicion index for LSD were enrolled in different centers in Spain and Portugal, including 18 positive controls. Results We correctly diagnosed 18 positive blinded controls, provided genetic diagnosis to 25 potential LSD patients, and ended with 18 diagnostic odysseys. Conclusion We report the assessment of a next–generation-sequencing-based approach as an accessory tool in the diagnosis of LSDs, a group of disorders which have overlapping clinical profiles and genetic heterogeneity. We have also identified and quantified the strengths and limitations of next generation sequencing (NGS) technology applied to diagnosis. PMID:24767253

  1. Targeted Polymeric Nanoparticles for Brain Delivery of High Molecular Weight Molecules in Lysosomal Storage Disorders

    PubMed Central

    Belletti, Daniela; D’Avanzo, Francesca; Pederzoli, Francesca; Ruozi, Barbara; Marin, Oriano; Vandelli, Maria Angela; Forni, Flavio; Scarpa, Maurizio; Tomanin, Rosella; Tosi, Giovanni

    2016-01-01

    Lysosomal Storage Disorders (LSDs) are a group of metabolic syndromes, each one due to the deficit of one lysosomal enzyme. Many LSDs affect most of the organ systems and overall about 75% of the patients present neurological impairment. Enzyme Replacement Therapy, although determining some systemic clinical improvements, is ineffective on the CNS disease, due to enzymes' inability to cross the blood-brain barrier (BBB). With the aim to deliver the therapeutic enzymes across the BBB, we here assayed biodegradable and biocompatible PLGA-nanoparticles (NPs) in two murine models for LSDs, Mucopolysaccharidosis type I and II (MPS I and MPS II). PLGA-NPs were modified with a 7-aminoacid glycopeptide (g7), yet demonstrated to be able to deliver low molecular weight (MW) molecules across the BBB in rodents. We specifically investigated, for the first time, the g7-NPs ability to transfer a model drug (FITC-albumin) with a high MW, comparable to the enzymes to be delivered for LSDs brain therapy. In vivo experiments, conducted on wild-type mice and knockout mouse models for MPS I and II, also included a whole series of control injections to obtain a broad preliminary view of the procedure efficiency. Results clearly showed efficient BBB crossing of albumin in all injected mice, underlying the ability of NPs to deliver high MW molecules to the brain. These results encourage successful experiments with enzyme-loaded g7-NPs to deliver sufficient amounts of the drug to the brain district on LSDs, where exerting a corrective effect on the pathological phenotype. PMID:27228099

  2. Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein

    PubMed Central

    Kiss, Katalin; Kucsma, Nora; Brozik, Anna; Tusnady, Gabor E.; Bergam, Ptissam; vanNiel, Guillaume; Szakacs, Gergely

    2015-01-01

    ATP-binding cassette, subfamily B (ABCB) 6 is a homodimeric ATP-binding cassette (ABC) transporter present in the plasma membrane and in the intracellular organelles. The intracellular localization of ABCB6 has been a matter of debate, as it has been suggested to reside in the mitochondria and the endo-lysosomal system. Using a variety of imaging modalities, including confocal microscopy and EM, we confirm the endo-lysosomal localization of ABCB6 and show that the protein is internalized from the plasma membrane through endocytosis, to be distributed to multivesicular bodies and lysosomes. In addition to the canonical nucleotide-binding domain (NBD) and transmembrane domain (TMD), ABCB6 contains a unique N-terminal TMD (TMD0), which does not show sequence homology to known proteins. We investigated the functional role of these domains through the molecular dissection of ABCB6. We find that the folding, dimerization, membrane insertion and ATP binding/hydrolysis of the core–ABCB6 complex devoid of TMD0 are preserved. However, in contrast with the full-length transporter, the core–ABCB6 construct is retained at the plasma membrane and does not appear in Rab5-positive endosomes. TMD0 is directly targeted to the lysosomes, without passage to the plasma membrane. Collectively, our results reveal that TMD0 represents an independently folding unit, which is dispensable for catalysis, but has a crucial role in the lysosomal targeting of ABCB6. PMID:25627919

  3. A ruthenium(II) complex-based lysosome-targetable multisignal chemosensor for in vivo detection of hypochlorous acid.

    PubMed

    Cao, Liyan; Zhang, Run; Zhang, Wenzhu; Du, Zhongbo; Liu, Chunjun; Ye, Zhiqiang; Song, Bo; Yuan, Jingli

    2015-11-01

    Although considerable efforts have been made for the development of ruthenium(II) complex-based chemosensors and bioimaging reagents, the multisignal chemosensor using ruthenium(II) complexes as the reporter is scarce. In addition, the mechanisms of cellular uptake of ruthenium(II)-based chemosensors and their intracellular distribution are ill-defined. Herein, a new ruthenium(II) complex-based multisignal chemosensor, Ru-Fc, is reported for the highly sensitive and selective detection of lysosomal hypochlorous acid (HOCl). Ru-Fc is weakly luminescent because the MLCT (metal-to-ligand charge transfer) state is corrupted by the efficient PET (photoinduced electron transfer) process from Fc (ferrocene) moiety to Ru(II) center. The cleavage of Fc moiety by a HOCl-induced specific reaction leads to elimination of PET, which re-establishes the MLCT state of the Ru(II) complex, accompanied by remarkable photoluminescence (PL) and electrochemiluminescence (ECL) enhancements. The result of MTT assay showed that the proposed chemosensor, Ru-Fc, was low cytotoxicity. The applicability of Ru-Fc for the quantitative detection of HOCl in live cells was demonstrated by the confocal microscopy imaging and flow cytometry analysis. Dye colocalization studies confirmed very precise distribution of the Ru(II) complex in lysosomes, and inhibition studies revealed that the caveolae-mediated endocytosis played an important role during the cellular internalization of Ru-Fc. By using Ru-Fc as a chemosensor, the imaging of the endogenous HOCl generated in live macrophage cells during the stimulation was achieved. Furthermore, the practical applicability of Ru-Fc was demonstrated by the visualizing of HOCl in laboratory model animals, Daphnia magna and zebrafish. PMID:26256295

  4. Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.

    PubMed

    Yoon, Jihee; Sekhon, Simranjeet Singh; Kim, Yang-Hoon; Min, Jiho

    2016-06-01

    Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions. PMID:27221740

  5. Strategies for SETI target selection

    NASA Astrophysics Data System (ADS)

    Latham, David W.; Soderblom, David R.

    1993-08-01

    The NASA High Resolution Microwave Survey consists of two complementary elements: a Sky Survey of the entire sky to a moderate level of sensitivity; and a Targeted Search of nearby stars, one at a time, to a much deeper level of sensitivity. In this paper we present a strategy for target selection and observing. The strategy has two goals: to improve the chances of successful detection of signals from technical civilizations that inhabit planets around solar- type stars, and to minimize the chances of missing signals from unexpected sites. For the main Targeted Search survey of approximately 1000 nearby solar-type stars, we argue that the selection criteria should be heavily biased by what we know about the origin and evolution of life here on earth. We propose that observations of stars with stellar companions orbiting near the habitable zone should be de-emphasized, because such companions would prevent the formation of habitable planets. We also propose that observations of stars younger than about three billion years should be de-emphasized in favor of older stars, because our own technical civilization took longer than three billion years to evolve here on earth.

  6. Ubiquitination of lysine-331 by Kaposi's sarcoma-associated herpesvirus protein K5 targets HFE for lysosomal degradation

    PubMed Central

    Rhodes, David A.; Boyle, Louise H.; Boname, Jessica M.; Lehner, Paul J.; Trowsdale, John

    2010-01-01

    The nonclassical MHC class I-related (MHC-I) molecule HFE controls cellular iron homeostasis by a mechanism that has not been fully elucidated. We examined the regulation of HFE by K5, the E3 ubiquitin ligase encoded by Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8), that is known to down-regulate classical MHC-I. K5 down-regulated HFE efficiently, using polyubiquitination of the membrane proximal lysine in the HFE cytoplasmic tail (K331), to target the molecule for degradation via ESCRT1/TSG101-dependent sorting from endosomes to multivesicular bodies (MVBs)/lysosomes. In the primary effusion lymphoma cell line BC-3, which carries latent KSHV, HFE was degraded rapidly upon virus reactivation. HFE was ubiquitinated on lysine-331 in unactivated BC-3 cells, conditions where K5 was not detectable, consistent with an endogenous E3 ubiquitin ligase controlling HFE expression. The results show regulated expression of HFE by ubiquitination, consistent with a role in cellular iron homeostasis, a molecular mechanism targeted by KSHV to achieve a positive iron balance. PMID:20805500

  7. The Biogenesis of Lysosomes and Lysosome-Related Organelles

    PubMed Central

    Luzio, J. Paul; Hackmann, Yvonne; Dieckmann, Nele M.G.; Griffiths, Gillian M.

    2014-01-01

    Lysosomes were once considered the end point of endocytosis, simply used for macromolecule degradation. They are now recognized to be dynamic organelles, able to fuse with a variety of targets and to be re-formed after fusion events. They are also now known to be the site of nutrient sensing and signaling to the cell nucleus. In addition, lysosomes are secretory organelles, with specialized machinery for regulated secretion of proteins in some cell types. The biogenesis of lysosomes and lysosome-related organelles is discussed, taking into account their dynamic nature and multiple roles. PMID:25183830

  8. Dual Site-Controlled and Lysosome-Targeted Intramolecular Charge Transfer-Photoinduced Electron Transfer-Fluorescence Resonance Energy Transfer Fluorescent Probe for Monitoring pH Changes in Living Cells.

    PubMed

    Dong, Baoli; Song, Xuezhen; Wang, Chao; Kong, Xiuqi; Tang, Yonghe; Lin, Weiying

    2016-04-01

    Acidic pH is a critical physiological factor for controlling the activities and functions of lysosome. Herein, we report a novel dual site-controlled and lysosome-targeted intramolecular charge transfer-photoinduced electron transfer-Fluorescence resonance energy transfer (ICT-PET-FRET) fluorescent probe (CN-pH), which was essentially the combination of a turn-on pH probe (CN-1) and a turn-off pH probe (CN-2) by a nonconjugated linker. Coumarin and naphthalimide fluorophores were selected as donor and acceptor to construct the FRET platform. Hydroxyl group and morpholine were simultaneously employed as the two pH sensing sites and controlled the fluorescence of coumarin and naphthalimide units by ICT and PET, respectively. The sensing mechanism of CN-pH to pH was essentially an integration of ICT, PET, and FRET processes. Meanwhile, the morpholine also can serve as a lysosome-targeted group. By combining the two data analysis approaches of the ratios of the two emission intensities (R) and the reverse ratio R' (R' = 1/R), the fluorescent ratio of CN-pH can show proportional relationship to pH values in a very broad range from pH 4.0 to 8.0 with high sensitivity. The probe has been successfully applied for the fluorescence imaging of the lysosomal pH values, as well as ratiometrically visualizing chloroquine-stimulated changes of intracellular pH in living cells. These features demonstrate that the probe can afford practical application in biological systems. PMID:26987045

  9. Fusion to the Lysosome Targeting Signal of the Invariant Chain Alters the Processing and Enhances the Immunogenicity of HIV-1 Reverse Transcriptase

    PubMed Central

    Starodubova, E. S.; Isaguliants, M. G.; Kuzmenko, Y. V.; Latanova, A. A.; Krotova, O. A.; Karpov, V. L.

    2014-01-01

    Intracellular processing of the antigen encoded by a DNA vaccine is one of the key steps in generating an immune response. Immunization with DNA constructs targeted to the endosomal-lysosomal compartments and to the MHC class II pathway can elicit a strong immune response. Herein, the weakly immunogenic reverse transcriptase of HIV-1 was fused to the minimal lysosomal targeting motif of the human MHC class II invariant chain. The motif fused to the N-terminus shifted the enzyme intracellular localization and accelerated its degradation. Degradation of the chimeric protein occurred predominantly in the lysosomal compartment. BALB/c mice immunized with the plasmid encoding the chimeric protein demonstrated an enhanced immune response, in the form of an increased antigen-specific production of Th1 cytokines, INF-γ and IL-2, by mouse splenocytes. Moreover, the majority of the splenocytes secreted both cytokines; i.e., were polyfunctional. These findings suggest that retargeting of the antigen to the lysosomes enhances the immune response to DNA vaccine candidates with low intrinsic immunogenicity. PMID:24772328

  10. Characterization of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Trafficking Reveals a Novel Lysosomal Targeting Mechanism via Amyloid Precursor-like Protein 2 (APLP2)

    PubMed Central

    DeVay, Rachel M.; Shelton, David L.; Liang, Hong

    2013-01-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor protein levels by diverting it to lysosomes. Monoclonal antibody therapeutics aimed to neutralize PCSK9 have been shown to successfully lower serum LDL levels; however, we previously found that such therapeutic antibodies are subject to PCSK9-mediated clearance. In this study, we discovered that PCSK9 interacts via its C-terminal domain directly and in a pH-dependent manner with amyloid precursor protein as well as its closely related family member, amyloid precursor protein-like protein 2. Furthermore, we determined that amyloid precursor protein-like protein-2, but not amyloid precursor protein, is involved in mediating postendocytic delivery of PCSK9 to lysosomes and is therefore important for PCSK9 function. Based on our data, we propose a model for a lysosomal transport complex by which a soluble protein can target another protein for degradation from the luminal side of the membrane by bridging it to a lysosomally targeted transmembrane protein. PMID:23430252

  11. The mechanical activation of mTOR signaling: an emerging role for late endosome/lysosomal targeting.

    PubMed

    Jacobs, Brittany L; Goodman, Craig A; Hornberger, Troy A

    2014-02-01

    It is well recognized that mechanical signals play a critical role in the regulation of skeletal muscle mass, and the maintenance of muscle mass is essential for mobility, disease prevention and quality of life. Furthermore, over the last 15 years it has become established that signaling through a protein kinase called the mammalian (or mechanistic) target of rapamycin (mTOR) is essential for mechanically-induced changes in protein synthesis and muscle mass, however, the mechanism(s) via which mechanical stimuli regulate mTOR signaling have not been defined. Nonetheless, advancements are being made, and an emerging body of evidence suggests that the late endosome/lysosomal (LEL) system might play a key role in this process. Therefore, the purpose of this review is to summarize this body of evidence. Specifically, we will first explain why the Ras homologue enriched in brain (Rheb) and phosphatidic acid (PA) are considered to be direct activators of mTOR signaling. We will then describe the process of endocytosis and its involvement in the formation of LEL structures, as well as the evidence which indicates that mTOR and its direct activators (Rheb and PA) are all enriched at the LEL. Finally, we will summarize the evidence that has implicated the LEL in the regulation of mTOR by various growth regulatory inputs such as amino acids, growth factors and mechanical stimuli. PMID:24162376

  12. Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection

    PubMed Central

    Lu, Feiran; Liang, Qiren; Abi-Mosleh, Lina; Das, Akash; De Brabander, Jef K; Goldstein, Joseph L; Brown, Michael S

    2015-01-01

    Niemann-Pick C1 (NPC1) is a lysosomal membrane protein that exports cholesterol derived from receptor-mediated uptake of LDL, and it also mediates cellular entry of Ebola virus. Cholesterol export is inhibited by nanomolar concentrations of U18666A, a cationic sterol. To identify the target of U18666A, we synthesized U-X, a U18666A derivative with a benzophenone that permits ultraviolet-induced crosslinking. When added to CHO cells, U-X crosslinked to NPC1. Crosslinking was blocked by U18666A derivatives that block cholesterol export, but not derivatives lacking blocking activity. Crosslinking was prevented by point mutation in the sterol-sensing domain (SSD) of NPC1, but not by point mutation in the N-terminal domain (NTD). These data suggest that the SSD contains a U18666A-inhibitable site required for cholesterol export distinct from the cholesterol-binding site in the NTD. Inasmuch as inhibition of Ebola requires 100-fold higher concentrations of U18666A, the high affinity U16888A-binding site is likely not required for virus entry. DOI: http://dx.doi.org/10.7554/eLife.12177.001 PMID:26646182

  13. Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection.

    PubMed

    Lu, Feiran; Liang, Qiren; Abi-Mosleh, Lina; Das, Akash; De Brabander, Jef K; Goldstein, Joseph L; Brown, Michael S

    2015-01-01

    Niemann-Pick C1 (NPC1) is a lysosomal membrane protein that exports cholesterol derived from receptor-mediated uptake of LDL, and it also mediates cellular entry of Ebola virus. Cholesterol export is inhibited by nanomolar concentrations of U18666A, a cationic sterol. To identify the target of U18666A, we synthesized U-X, a U18666A derivative with a benzophenone that permits ultraviolet-induced crosslinking. When added to CHO cells, U-X crosslinked to NPC1. Crosslinking was blocked by U18666A derivatives that block cholesterol export, but not derivatives lacking blocking activity. Crosslinking was prevented by point mutation in the sterol-sensing domain (SSD) of NPC1, but not by point mutation in the N-terminal domain (NTD). These data suggest that the SSD contains a U18666A-inhibitable site required for cholesterol export distinct from the cholesterol-binding site in the NTD. Inasmuch as inhibition of Ebola requires 100-fold higher concentrations of U18666A, the high affinity U16888A-binding site is likely not required for virus entry. PMID:26646182

  14. ChemCam Automated Target Selection Status

    NASA Technical Reports Server (NTRS)

    Castano, Becky; Bornstein, Ben

    2006-01-01

    Usage Scenarios: I) Identify candidate targets: a) Locate rocks; b) Select points on rocks. II) Identify and prioritize candidate targets: a) Locate rocks; b) Select points on rocks; c) Extract rock properties; and d) Prioritize points based on rock properties.

  15. Quasar target selection fiber efficiency

    SciTech Connect

    Newberg, H.; Yanny, B.

    1996-05-01

    We present estimates of the efficiency for finding QSOs as a function of limiting magnitude and galactic latitude. From these estimates, we have formulated a target selection strategy that should net 80,000 QSOs in the north galactic cap with an average of 70 fibers per plate, not including fibers reserved for high-redshift quasars. With this plan, we expect 54% of the targets to be QSOs. The North Galactic Cap is divided into two zones of high and low stellar density. We use about five times as many fibers for QSO candidates in the half of the survey with the lower stellar density as we use in the half with higher stellar density. The current plan assigns 15% of the fibers to FIRST radio sources; if these are not available, those fibers would be allocated to lower probability QSO sources, dropping the total number of QSOs by a small factor (5%). We will find about 17,000 additional quasars in the southern strips, and maybe a few more at very high redshift. Use was made of two data sets: the star and quasar simulated test data generated by Don Schneider, and the data from UJFN plate surveys by Koo (1986) and Kron (1980). This data was compared to results from the Palomar-Green Survey and a recent survey by Pat Osmer and collaborators.

  16. TFEB regulates lysosomal proteostasis.

    PubMed

    Song, Wensi; Wang, Fan; Savini, Marzia; Ake, Ashley; di Ronza, Alberto; Sardiello, Marco; Segatori, Laura

    2013-05-15

    Loss-of-function diseases are often caused by destabilizing mutations that lead to protein misfolding and degradation. Modulating the innate protein homeostasis (proteostasis) capacity may lead to rescue of native folding of the mutated variants, thereby ameliorating the disease phenotype. In lysosomal storage disorders (LSDs), a number of highly prevalent alleles have missense mutations that do not impair the enzyme's catalytic activity but destabilize its native structure, resulting in the degradation of the misfolded protein. Enhancing the cellular folding capacity enables rescuing the native, biologically functional structure of these unstable mutated enzymes. However, proteostasis modulators specific for the lysosomal system are currently unknown. Here, we investigate the role of the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and function, in modulating lysosomal proteostasis in LSDs. We show that TFEB activation results in enhanced folding, trafficking and lysosomal activity of a severely destabilized glucocerebrosidase (GC) variant associated with the development of Gaucher disease (GD), the most common LSD. TFEB specifically induces the expression of GC and of key genes involved in folding and lysosomal trafficking, thereby enhancing both the pool of mutated enzyme and its processing through the secretory pathway. TFEB activation also rescues the activity of a β-hexosaminidase mutant associated with the development of another LSD, Tay-Sachs disease, thus suggesting general applicability of TFEB-mediated proteostasis modulation to rescue destabilizing mutations in LSDs. In summary, our findings identify TFEB as a specific regulator of lysosomal proteostasis and suggest that TFEB may be used as a therapeutic target to rescue enzyme homeostasis in LSDs. PMID:23393155

  17. Sphingomyelins suppress the targeted disruption of lysosomes/endosomes by the photosensitizer NPe6 during photodynamic therapy.

    PubMed

    Caruso, Joseph A; Mathieu, Patricia A; Reiners, John J

    2005-12-01

    Recent studies have described a biochemical pathway whereby lysosome disruption and the released proteases initiate the intrinsic apoptotic pathway. Irradiation of murine hepatoma 1c1c7 cells preloaded with the lysosomal photosensitizer NPe6 (N-aspartyl chlorin e6) caused a rapid loss of Acridine Orange staining of acidic organelles, release of cathepsin D from late endosomes/lysosomes and the activation of procaspase-3. Pretreatment of NPe6-loaded cultures with 10-50 microM 3-O-MeSM (3-O-methylsphingomyelin) caused a concentration-dependent suppression of apoptosis following irradiation. This suppression reflected a stabilization of lysosomes/endosomes, as opposed to an inhibition of the accumulation of photosensitizer in these organelles. Exogenously added sphingomyelin, at comparable concentrations, offered some protection, but less than 3-O-MeSM. Fluorescence microscopy showed that 3-O-MeSM competed with NBD-C6-sphingomyelin (6-{[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl} sphingosyl phosphocholine) for co-localization with LysoTracker Red in acidic organelles. Pre-treatment of 1c1c7 cultures with 3-O-MeSM also suppressed the induction of apoptosis by TNFalpha (tumour necrosis factor alpha), but offered no protection against HA14-1 [ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate], staurosporine, tunicamycin or thapsigargin. These results suggest that exogenously added 3-O-MeSM is trafficked to and stabilizes late endosomes/lysosomes against oxidant-induced damage, and further implicate a role for lysosomal proteases in the apoptotic processes initiated by TNFalpha and lysosomal photosensitizers. PMID:15943580

  18. Sphingomyelins suppress the targeted disruption of lysosomes/endosomes by the photosensitizer NPe6 during photodynamic therapy

    PubMed Central

    2005-01-01

    Recent studies have described a biochemical pathway whereby lysosome disruption and the released proteases initiate the intrinsic apoptotic pathway. Irradiation of murine hepatoma 1c1c7 cells preloaded with the lysosomal photosensitizer NPe6 (N-aspartyl chlorin e6) caused a rapid loss of Acridine Orange staining of acidic organelles, release of cathepsin D from late endosomes/lysosomes and the activation of procaspase-3. Pretreatment of NPe6-loaded cultures with 10–50 μM 3-O-MeSM (3-O-methylsphingomyelin) caused a concentration-dependent suppression of apoptosis following irradiation. This suppression reflected a stabilization of lysosomes/endosomes, as opposed to an inhibition of the accumulation of photosensitizer in these organelles. Exogenously added sphingomyelin, at comparable concentrations, offered some protection, but less than 3-O-MeSM. Fluorescence microscopy showed that 3-O-MeSM competed with NBD-C6-sphingomyelin (6-{[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl} sphingosyl phosphocholine) for co-localization with LysoTracker Red in acidic organelles. Pre-treatment of 1c1c7 cultures with 3-O-MeSM also suppressed the induction of apoptosis by TNFα (tumour necrosis factor α), but offered no protection against HA14-1 [ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate], staurosporine, tunicamycin or thapsigargin. These results suggest that exogenously added 3-O-MeSM is trafficked to and stabilizes late endosomes/lysosomes against oxidant-induced damage, and further implicate a role for lysosomal proteases in the apoptotic processes initiated by TNFα and lysosomal photosensitizers. PMID:15943580

  19. Lysosomal Adaptation: How the Lysosome Responds to External Cues

    PubMed Central

    Settembre, Carmine; Ballabio, Andrea

    2014-01-01

    Recent evidence indicates that the importance of the lysosome in cell metabolism and organism physiology goes far beyond the simple disposal of cellular garbage. This dynamic organelle is situated at the crossroad of the most important cellular pathways and is involved in sensing, signaling, and transcriptional mechanisms that respond to environmental cues, such as nutrients. Two main mediators of these lysosomal adaptation mechanisms are the mTORC1 kinase complex and the transcription factor EB (TFEB). These two factors are linked in a lysosome-to-nucleus signaling pathway that provides the lysosome with the ability to adapt to extracellular cues and control its own biogenesis. Modulation of lysosomal function by acting on TFEB has a profound impact on cellular clearance and energy metabolism and is a promising therapeutic target for a large variety of disease conditions. PMID:24799353

  20. A C-terminally truncated mouse Best3 splice variant targets and alters the ion balance in lysosome-endosome hybrids and the endoplasmic reticulum

    PubMed Central

    Wu, Lichang; Sun, Yu; Ma, Liqiao; Zhu, Jun; Zhang, Baoxia; Pan, Qingjie; Li, Yuyin; Liu, Huanqi; Diao, Aipo; Li, Yinchuan

    2016-01-01

    The Bestrophin family has been characterized as Cl− channels in mammals and Na+ channels in bacteria, but their exact physiological roles remian unknown. In this study, a natural C-terminally truncated variant of mouse Bestrophin 3 (Best3V2) expression in myoblasts and muscles is demonstrated. Unlike full-length Best3, Best3V2 targets the two important intracellular Ca stores: the lysosome and the ER. Heterologous overexpression leads to lysosome swelling and renders it less acidic. Best3V2 overexpression also results in compromised Ca2+ release from the ER. Knocking down endogenous Best3 expression in myoblasts makes these cells more excitable in response to Ca2+ mobilizing reagents, such as caffeine. We propose that Best3V2 in myoblasts may work as a tuner to control Ca2+ release from intracellular Ca2+ stores. PMID:27265833

  1. A C-terminally truncated mouse Best3 splice variant targets and alters the ion balance in lysosome-endosome hybrids and the endoplasmic reticulum.

    PubMed

    Wu, Lichang; Sun, Yu; Ma, Liqiao; Zhu, Jun; Zhang, Baoxia; Pan, Qingjie; Li, Yuyin; Liu, Huanqi; Diao, Aipo; Li, Yinchuan

    2016-01-01

    The Bestrophin family has been characterized as Cl(-) channels in mammals and Na(+) channels in bacteria, but their exact physiological roles remian unknown. In this study, a natural C-terminally truncated variant of mouse Bestrophin 3 (Best3V2) expression in myoblasts and muscles is demonstrated. Unlike full-length Best3, Best3V2 targets the two important intracellular Ca stores: the lysosome and the ER. Heterologous overexpression leads to lysosome swelling and renders it less acidic. Best3V2 overexpression also results in compromised Ca(2+) release from the ER. Knocking down endogenous Best3 expression in myoblasts makes these cells more excitable in response to Ca(2+) mobilizing reagents, such as caffeine. We propose that Best3V2 in myoblasts may work as a tuner to control Ca(2+) release from intracellular Ca(2+) stores. PMID:27265833

  2. Lysosomal Trafficking Regulator (LYST).

    PubMed

    Ji, Xiaojie; Chang, Bo; Naggert, Jürgen K; Nishina, Patsy M

    2016-01-01

    Regulation of vesicle trafficking to lysosomes and lysosome-related organelles (LROs) as well as regulation of the size of these organelles are critical to maintain their functions. Disruption of the lysosomal trafficking regulator (LYST) results in Chediak-Higashi syndrome (CHS), a rare autosomal recessive disorder characterized by oculocutaneous albinism, prolonged bleeding, severe immunodeficiency, recurrent bacterial infection, neurologic dysfunction and hemophagocytic lympohistiocytosis (HLH). The classic diagnostic feature of the syndrome is enlarged LROs in all cell types, including lysosomes, melanosomes, cytolytic granules and platelet dense bodies. The most striking CHS ocular pathology observed is an enlargement of melanosomes in the retinal pigment epithelium (RPE), which leads to aberrant distribution of eye pigmentation, and results in photophobia and decreased visual acuity. Understanding the molecular function of LYST and identification of its interacting partners may provide therapeutic targets for CHS and other diseases associated with the regulation of LRO size and/or vesicle trafficking, such as asthma, urticaria and Leishmania amazonensis infections. PMID:26427484

  3. Effect of Acute Emotional Stress on Proteomic Profile of Selected Brain Areas and Lysosomal Proteolysis in Rats with Different Behavioral Activity.

    PubMed

    Sharanova, N E; Kirbaeva, N V; Toropygin, I Yu; Khryapova, E V; Koplik, E V; Soto, C Kh; Pertsov, S S; Vasiliev, A V

    2016-07-01

    We compared proteome profiles of selected brain areas (cortex, amygdala, hippocampus, and reticular formation) and measured cathepsins B and D activity in liver lysosomal fraction in rats with different behavioral activity under conditions of emotional stress. In passive rats, the expression of some proteins in various brain regions was changed and baseline cathepsin B activity was higher than in active animals. Taken together, the results attest to differences in the adaptive response formation in rats, depending on behavioral features. PMID:27502534

  4. Lysosome-associated membrane glycoprotein (LAMP) – preliminary study on a hidden antigen target for vaccination against schistosomiasis

    PubMed Central

    Nawaratna, Sujeevi S. K.; Gobert, Geoffrey N.; Willis, Charlene; Mulvenna, Jason; Hofmann, Andreas; McManus, Donald P.; Jones, Malcolm K.

    2015-01-01

    Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16–25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection. PMID:26472258

  5. Lysosome-associated membrane glycoprotein (LAMP)--preliminary study on a hidden antigen target for vaccination against schistosomiasis.

    PubMed

    Nawaratna, Sujeevi S K; Gobert, Geoffrey N; Willis, Charlene; Mulvenna, Jason; Hofmann, Andreas; McManus, Donald P; Jones, Malcolm K

    2015-01-01

    Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16-25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection. PMID:26472258

  6. Selective Targeting to Glioma with Nucleic Acid Aptamers

    PubMed Central

    Aptekar, Shraddha; Arora, Mohit; Lawrence, Clare Louise; Lea, Robert William; Ashton, Katherine; Dawson, Tim; Alder, Jane Elizabeth; Shaw, Lisa

    2015-01-01

    Malignant glioma is characterised by a rapid growth rate and high capacity for invasive infiltration to surrounding brain tissue; hence, diagnosis and treatment is difficult and patient survival is poor. Aptamers contribute a promising and unique technology for the in vitro imaging of live cells and tissues, with a potentially bright future in clinical diagnostics and therapeutics for malignant glioma. The binding selectivity, uptake capacity and binding target of two DNA aptamers, SA43 and SA44, were investigated in glioma cells and patient tissues. The binding assay showed that SA43 and SA44 bound with strong affinity (Kd, 21.56 ± 4.60 nM and Kd, 21.11 ± 3.30 nM respectively) to the target U87MG cells. Quantitative analysis by flow cytometry showed that the aptamers were able to actively internalise in U87MG and 1321N1 glioma cells compared to the non-cancerous and non-glioma cell types. Confocal microscopy confirmed staining in the cytoplasm, and co-localisation studies with endoplasmic reticulum, Golgi apparatus and lysosomal markers suggested internalisation and compartmentalisation within the endomembrane system. Both aptamers selectively bound to Ku 70 and Ku 80 DNA repair proteins as determined by aptoprecipitation (AP) followed by mass spectrometry analysis and confirmation by Western blot. In addition, aptohistochemical (AHC) staining on paraffin embedded, formalin fixed patient tissues revealed that the binding selectivity was significantly higher for SA43 aptamer in glioma tissues (grade I, II, III and IV) compared to the non-cancerous tissues, whereas SA44 did not show selectivity towards glioma tissues. The results indicate that SA43 aptamer can differentiate between glioma and non-cancerous cells and tissues and therefore, shows promise for histological diagnosis of glioma. PMID:26252900

  7. Similarities and differences in the biogenesis, processing and lysosomal targeting between zebrafish and human pro-Cathepsin D: functional implications.

    PubMed

    Follo, Carlo; Ozzano, Matteo; Montalenti, Claudia; Ekkapongpisit, Maneerat; Isidoro, Ciro

    2013-02-01

    The lysosomal protease Cathepsin D (CD) plays a role in neurodegenerative diseases, cancer, and embryo-fetus abnormalities. It is therefore of interest to know how this protein is synthesized in animal species used for modeling human diseases. Zebrafish (Danio rerio) is emerging as a valuable 'in vivo' vertebrate model for several human diseases. We have characterized the biogenetic pathways of zebrafish and human CD transgenically expressed in both human SH-SY5Y cells and zebrafish PAC2 cells. Differently from human CD, zebrafish CD was synthesized as a mono-glycosylated precursor (pro-CD) that was eventually processed into a single-chain mature polypeptide. In PAC2 cells, ammonium chloride and chloroquine impaired the N-glycosylation, and greatly stimulated the secretion, of pro-CD; still, a portion of un-glycosylated pro-CD reached the lysosomes and was processed to mature CD. The treatment with tunicamycin, which abrogates N-glycosylation, resulted in a similar effect. Zebrafish pro-CD was correctly processed when expressed in human cells, and its glycosylation, transport and maturation were not impaired by ammonium chloride. On the contrary, the transport and processing of human pro-CD expressed in zebrafish cells were profoundly altered: while the intermediate single-chain was not detectable, a small amount of double-chain mature CD still formed. This fact indicates that the enzyme machinery for single- to double-chain processing of mammal CD is present in zebrafish. Our data highlight the respective impact of the information imparted by the primary sequence and of the cellular transport and processing machineries in the biogenesis of lysosomal CD. PMID:23107604

  8. Target Selection for the TESS Mission

    NASA Astrophysics Data System (ADS)

    Pepper, Joshua; Stassun, Keivan

    2015-12-01

    The goal of the TESS mission is to discover small, rocky planets transiting bright stars. To reach that goal, we have constructed a compiled catalog of stars from which to select TESS targets. The catalog contains all dwarf stars in the sky with spectral types F5 and later, and I < 12, along with selected sets of fainter M stars. Provisions are being made to augment the target list with stars that fall outside the nominal spectral type and magnitude limits, and to permit dynamic updating of the catalog to accommodate new survey data being released (e.g. Gaia). I will describe the overall target selection strategy, the current target catalog, and how we intend to further expand and refine the TESS target lists.

  9. Lysosomal dysfunction causes neurodegeneration in mucolipidosis II ‘knock-in’ mice

    PubMed Central

    Kollmann, K.; Damme, M.; Markmann, S.; Morelle, W.; Schweizer, M.; Hermans-Borgmeyer, I.; Röchert, A. K.; Pohl, S.; Lübke, T.; Michalski, J.-C.; Käkelä, R.; Walkley, S. U.

    2012-01-01

    Mucolipidosis II is a neurometabolic lysosomal trafficking disorder of infancy caused by loss of mannose 6-phosphate targeting signals on lysosomal proteins, leading to lysosomal dysfunction and accumulation of non-degraded material. However, the identity of storage material and mechanisms of neurodegeneration in mucolipidosis II are unknown. We have generated ‘knock-in’ mice with a common mucolipidosis II patient mutation that show growth retardation, progressive brain atrophy, skeletal abnormalities, elevated lysosomal enzyme activities in serum, lysosomal storage in fibroblasts and brain and premature death, closely mimicking the mucolipidosis II disease in humans. The examination of affected mouse brains at different ages by immunohistochemistry, ultrastructural analysis, immunoblotting and mass spectrometric analyses of glycans and anionic lipids revealed that the expression and proteolytic processing of distinct lysosomal proteins such as α-l-fucosidase, β-hexosaminidase, α-mannosidase or Niemann–Pick C2 protein are more significantly impacted by the loss of mannose 6-phosphate residues than enzymes reaching lysosomes independently of this targeting mechanism. As a consequence, fucosylated N-glycans, GM2 and GM3 gangliosides, cholesterol and bis(monoacylglycero)phosphate accumulate progressively in the brain of mucolipidosis II mice. Prominent astrogliosis and the accumulation of organelles and storage material in focally swollen axons were observed in the cerebellum and were accompanied by a loss of Purkinje cells. Moreover, an increased neuronal level of the microtubule-associated protein 1 light chain 3 and the formation of p62-positive neuronal aggregates indicate an impairment of constitutive autophagy in the mucolipidosis II brain. Our findings demonstrate the essential role of mannose 6-phosphate for selected lysosomal proteins to maintain the capability for degradation of sequestered components in lysosomes and autophagolysosomes and prevent

  10. Lysosomotropism of basic cathepsin K inhibitors contributes to increased cellular potencies against off-target cathepsins and reduced functional selectivity.

    PubMed

    Falgueyret, Jean-Pierre; Desmarais, Sylvie; Oballa, Renata; Black, W Cameron; Cromlish, Wanda; Khougaz, Karine; Lamontagne, Sonia; Massé, Frederic; Riendeau, Denis; Toulmond, Sylvie; Percival, M David

    2005-12-01

    The lysosomal cysteine protease cathepsin K is a target for osteoporosis therapy. The aryl-piperazine-containing cathepsin K inhibitor CRA-013783/L-006235 (1) displays greater than 4000-fold selectivity against the lysosomal/endosomal antitargets cathepsin B, L, and S. However, 1 and other aryl-piperazine-containing analogues, including balicatib (10), are approximately 10-100-fold more potent in cell-based enzyme occupancy assays than against each purified enzyme. This phenomenon arises from their basic, lipophilic nature, which results in lysosomal trapping. Consistent with its lysosomotropic nature, 1 accumulates in cells and in rat tissues of high lysosome content. In contrast, nonbasic aryl-morpholino-containing analogues do not exhibit lysosomotropic properties. Increased off-target activities of basic cathepsin K inhibitors were observed in a cell-based cathepsin S antigen presentation assay. No potency increases of basic inhibitors in a functional cathepsin K bone resorption whole cell assay were detected. Therefore, basic cathepsin K inhibitors, such as 1, suffer from reduced functional selectivities compared to those predicted using purified enzyme assays. PMID:16302795

  11. Imidazoacridinone-dependent lysosomal photodestruction: a pharmacological Trojan horse approach to eradicate multidrug-resistant cancers.

    PubMed

    Adar, Y; Stark, M; Bram, E E; Nowak-Sliwinska, P; van den Bergh, H; Szewczyk, G; Sarna, T; Skladanowski, A; Griffioen, A W; Assaraf, Y G

    2012-01-01

    Multidrug resistance (MDR) remains a primary hindrance to curative cancer therapy. Thus, introduction of novel strategies to overcome MDR is of paramount therapeutic significance. Sequestration of chemotherapeutics in lysosomes is an established mechanism of drug resistance. Here, we show that MDR cells display a marked increase in lysosome number. We further demonstrate that imidazoacridinones (IAs), which are cytotoxic fluorochromes, undergo a dramatic compartmentalization in lysosomes because of their hydrophobic weak base nature. We hence developed a novel photoactivation-based pharmacological Trojan horse approach to target and eradicate MDR cancer cells based on photo-rupture of IA-loaded lysosomes and tumor cell lysis via formation of reactive oxygen species. Illumination of IA-loaded cells resulted in lysosomal photodestruction and restoration of parental cell drug sensitivity. Lysosomal photodestruction of MDR cells overexpressing the key MDR efflux transporters ABCG2, ABCB1 or ABCC1 resulted in 10- to 52-fold lower IC(50) values of various IAs, thereby restoring parental cell sensitivity. Finally, in vivo application of this photodynamic therapy strategy after i.v. injection of IAs in human ovarian tumor xenografts in the chorioallantoic membrane model revealed selective destruction of tumors and their associated vasculature. These findings identify lysosomal sequestration of IAs as an Achilles heel of MDR cells that can be harnessed to eradicate MDR tumor cells via lysosomal photodestruction. PMID:22476101

  12. Gamma-interferon causes a selective induction of the lysosomal proteases, cathepsins B and L, in macrophages

    NASA Technical Reports Server (NTRS)

    Lah, T. T.; Hawley, M.; Rock, K. L.; Goldberg, A. L.

    1995-01-01

    Previous studies have indicated that acid-optimal cysteine proteinase(s) in the endosomal-lysosomal compartments, cathepsins, play a critical role in the proteolytic processing of endocytosed proteins to generate the antigenic peptides presented to the immune system on major histocompatibility complex (MHC) class II molecules. The presentation of these peptides and the expression of MHC class II molecules by macrophages and lymphocytes are stimulated by gamma-interferon (gamma-IFN). We found that treatment of human U-937 monocytes with gamma-IFN increased the activities and the content of the two major lysosomal cysteine proteinases, cathepsins B and L. Assays of protease activity, enzyme-linked immunosorbant assays (ELISA) and immunoblotting showed that this cytokine increased the amount of cathepsin B 5-fold and cathepsin L 3-fold in the lysosomal fraction. By contrast, the aspartic proteinase, cathepsin D, in this fraction was not significantly altered by gamma-IFN treatment. An induction of cathepsins B and L was also observed in mouse macrophages, but not in HeLa cells. These results suggest coordinate regulation in monocytes of the expression of cathepsins B and L and MHC class II molecules. Presumably, this induction of cysteine proteases contributes to the enhancement of antigen presentation by gamma-IFN.

  13. Target selection for FDA-approved medicines.

    PubMed

    Kinch, Michael S; Hoyer, Denton; Patridge, Eric; Plummer, Mark

    2015-07-01

    The biopharmaceutical industry translates fundamental understanding of disease into new medicines. As part of a comprehensive analysis of FDA-approved new molecular entities (NMEs), we assessed the mechanistic basis of drug efficacy, with emphasis on target selection. Three target families capture almost half of all NMEs and the leading ten families capture more than three-quarters of NME approvals. Target families were related to their clinical application and identify dynamic trends in targeting over time. These data suggest increasing attention toward novel target families, which presumably reflects increased understanding of disease etiology. We also suggest the need to balance the ongoing emphasis on target-based drug discovery with phenotypic approaches to drug discovery. PMID:25462532

  14. [Application of lysosomal detection in marine pollution monitoring: research progress].

    PubMed

    Weng, You-Zhu; Fang, Yong-Qiang; Zhang, Yu-Sheng

    2013-11-01

    Lysosome is an important organelle existing in eukaryotic cells. With the development of the study on the structure and function of lysosome in recent years, lysosome is considered as a target of toxic substances on subcellular level, and has been widely applied abroad in marine pollution monitoring. This paper summarized the biological characteristics of lysosomal marker enzyme, lysosome-autophagy system, and lysosomal membrane, and introduced the principles and methods of applying lysosomal detection in marine pollution monitoring. Bivalve shellfish digestive gland and fish liver are the most sensitive organs for lysosomal detection. By adopting the lysosomal detection techniques such as lysosomal membrane stability (LMS) test, neutral red retention time (NRRT) assay, morphological measurement (MM) of lysosome, immunohistochemical (Ih) assay of lysosomal marker enzyme, and electron microscopy (EM), the status of marine pollution can be evaluated. It was suggested that the lysosome could be used as a biomarker for monitoring marine environmental pollution. The advantages and disadvantages of lysosomal detection and some problems worthy of attention were analyzed, and the application prospects of lysosomal detection were discussed. PMID:24564165

  15. Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation.

    PubMed

    Dominguez-Brauer, Carmen; Hao, Zhenyue; Elia, Andrew J; Fortin, Jérôme M; Nechanitzky, Robert; Brauer, Patrick M; Sheng, Yi; Mana, Miyeko D; Chio, Iok In Christine; Haight, Jillian; Pollett, Aaron; Cairns, Robert; Tworzyanski, Leanne; Inoue, Satoshi; Reardon, Colin; Marques, Ana; Silvester, Jennifer; Cox, Maureen A; Wakeham, Andrew; Yilmaz, Omer H; Sabatini, David M; van Es, Johan H; Clevers, Hans; Sato, Toshiro; Mak, Tak W

    2016-08-01

    The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche. PMID:27184401

  16. Subverting lysosomal function in Trypanosoma brucei

    PubMed Central

    Alsford, Sam

    2016-01-01

    In this issue of Microbial Cell, Koh and colleagues present data highlighting the utility of the lysosomotropic compound L-leucyl-L-leucyl methyl ester (LeuLeu-OMe) as an anti-Trypanosoma brucei agent, adding to the range of compounds that either directly target lysosomal enzymes or that can be used to subvert the function of the lysosome for parasite destruction.

  17. Regulated lysosomal exocytosis mediates cancer progression

    PubMed Central

    Machado, Eda; White-Gilbertson, Shai; van de Vlekkert, Diantha; Janke, Laura; Moshiach, Simon; Campos, Yvan; Finkelstein, David; Gomero, Elida; Mosca, Rosario; Qiu, Xiaohui; Morton, Christopher L.; Annunziata, Ida; d’Azzo, Alessandra

    2015-01-01

    Understanding how tumor cells transition to an invasive and drug-resistant phenotype is central to cancer biology, but the mechanisms underlying this transition remain unclear. We show that sarcomas gain these malignant traits by inducing lysosomal exocytosis, a ubiquitous physiological process. During lysosomal exocytosis, the movement of exocytic lysosomes along the cytoskeleton and their docking at the plasma membrane involve LAMP1, a sialylated membrane glycoprotein and target of the sialidase NEU1. Cleavage of LAMP1 sialic acids by NEU1 limits the extent of lysosomal exocytosis. We found that by down-regulation of NEU1 and accumulation of oversialylated LAMP1, tumor cells exacerbate lysosomal exocytosis of soluble hydrolases and exosomes. This facilitates matrix invasion and propagation of invasive signals, and purging of lysosomotropic chemotherapeutics. In Arf−⁄− mice, Neu1 haploinsufficiency fostered the development of invasive, pleomorphic sarcomas, expressing epithelial and mesenchymal markers, and lysosomal exocytosis effectors, LAMP1 and Myosin-11. These features are analogous to those of metastatic, pleomorphic human sarcomas, where low NEU1 levels correlate with high expression of lysosomal exocytosis markers. In a therapeutic proof of principle, we demonstrate that inhibiting lysosomal exocytosis reversed invasiveness and chemoresistance in aggressive sarcoma cells. Thus, we reveal that this unconventional, lysosome-regulated pathway plays a primary role in tumor progression and chemoresistance. PMID:26824057

  18. Lysosomes as mediators of drug resistance in cancer.

    PubMed

    Zhitomirsky, Benny; Assaraf, Yehuda G

    2016-01-01

    Drug resistance remains a leading cause of chemotherapeutic treatment failure and cancer-related mortality. While some mechanisms of anticancer drug resistance have been well characterized, multiple mechanisms remain elusive. In this respect, passive ion trapping-based lysosomal sequestration of multiple hydrophobic weak-base chemotherapeutic agents was found to reduce the accessibility of these drugs to their target sites, resulting in a markedly reduced cytotoxic effect and drug resistance. Recently we have demonstrated that lysosomal sequestration of hydrophobic weak base drugs triggers TFEB-mediated lysosomal biogenesis resulting in an enlarged lysosomal compartment, capable of enhanced drug sequestration. This study further showed that cancer cells with an increased number of drug-accumulating lysosomes are more resistant to lysosome-sequestered drugs, suggesting a model of drug-induced lysosome-mediated chemoresistance. In addition to passive drug sequestration of hydrophobic weak base chemotherapeutics, other mechanisms of lysosome-mediated drug resistance have also been reported; these include active lysosomal drug sequestration mediated by ATP-driven transporters from the ABC superfamily, and a role for lysosomal copper transporters in cancer resistance to platinum-based chemotherapeutics. Furthermore, lysosomal exocytosis was suggested as a mechanism to facilitate the clearance of chemotherapeutics which highly accumulated in lysosomes, thus providing an additional line of resistance, supplementing the organelle entrapment of chemotherapeutics away from their target sites. Along with these mechanisms of lysosome-mediated drug resistance, several approaches were recently developed for the overcoming of drug resistance or exploiting lysosomal drug sequestration, including lysosomal photodestruction and drug-induced lysosomal membrane permeabilization. In this review we explore the current literature addressing the role of lysosomes in mediating cancer drug

  19. Structural Implications for Selective Targeting of PARPs

    PubMed Central

    Steffen, Jamin D.; Brody, Jonathan R.; Armen, Roger S.; Pascal, John M.

    2013-01-01

    Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that use NAD+ as a substrate to synthesize polymers of ADP-ribose (PAR) as post-translational modifications of proteins. PARPs have important cellular roles that include preserving genomic integrity, telomere maintenance, transcriptional regulation, and cell fate determination. The diverse biological roles of PARPs have made them attractive therapeutic targets, which have fueled the pursuit of small molecule PARP inhibitors. The design of PARP inhibitors has matured over the past several years resulting in several lead candidates in clinical trials. PARP inhibitors are mainly used in clinical trials to treat cancer, particularly as sensitizing agents in combination with traditional chemotherapy to reduce side effects. An exciting aspect of PARP inhibitors is that they are also used to selectivity kill tumors with deficiencies in DNA repair proteins (e.g., BRCA1/2) through an approach termed “synthetic lethality.” In the midst of the tremendous efforts that have brought PARP inhibitors to the forefront of modern chemotherapy, most clinically used PARP inhibitors bind to conserved regions that permits cross-selectivity with other PARPs containing homologous catalytic domains. Thus, the differences between therapeutic effects and adverse effects stemming from pan-PARP inhibition compared to selective inhibition are not well understood. In this review, we discuss current literature that has found ways to gain selectivity for one PARP over another. We furthermore provide insights into targeting other domains that make up PARPs, and how new classes of drugs that target these domains could provide a high degree of selectivity by affecting specific cellular functions. A clear understanding of the inhibition profiles of PARP inhibitors will not only enhance our understanding of the biology of individual PARPs, but may provide improved therapeutic options for patients. PMID:24392349

  20. Target selection for the HRIBF Project

    SciTech Connect

    Dellwo, J.; Alton, G.D.; Batchelder, J.C.

    1994-12-31

    Experiments are in progress at the Oak Ridge National Laboratory (ORNL) which are designed to select the most appropriate target materials for generating particular radioactive ion beams for the Holifield Radioactive Ion Beam Facility (HRIBF). The 25-MV tandem accelerator is used to implant stable complements of interesting radioactive elements into refractory targets mounted in a high-temperature FEBIAD ion source which is on-line at the UNISOR facility. These experiments permit selection of the target material most appropriate for the rapid release of the element of interest, as well as realistic estimates of the efficiency of the FEBIAD source. From diffusion release data information on the release times and diffusion coefficients can be derived. Diffusion coefficients for CI implanted into and diffused from CeS and Zr{sub 5}Si{sub 3} and As, Br, and Se implanted into and diffused from Zr{sub 5}Ge{sub 3} have been derived from the resulting intensity versus time profiles.

  1. Spectral Signatures of Saccade Target Selection.

    PubMed

    Carl, Christine; Hipp, Joerg F; König, Peter; Engel, Andreas K

    2016-01-01

    Action generation relies on a widely distributed network of brain areas. However, little is known about the spatiotemporal dynamics of neuronal activity in the network that gives rise to voluntary action in humans. Here, we used magnetoencephalography (MEG) and source analysis (n = 15, 7 female subjects) to investigate the spectral signatures of human cortical networks engaged in active and intrinsically motivated viewing behavior. We compared neuronal activity of externally cued saccades with saccades to freely chosen targets. For planning and execution of both saccade types, we found an increase in gamma band (~64-128 Hz) activity and a concurrent decrease in beta band (~12-32 Hz) activity in saccadic control areas, including the intraparietal sulcus and the frontal eye fields. Guided compared to voluntary actions were accompanied by stronger transient increases in the gamma and low frequency (<16 Hz) range immediately following the instructional cue. In contrast, action selection between competing alternatives was reflected by stronger sustained fronto-parietal gamma increases that occurred later in time and persisted until movement execution. This sustained enhancement for free target selection was accompanied by a spatially widespread reduction of lower frequency power (~8-45 Hz) in parietal and extrastriate areas. Our results suggest that neuronal population activity in the gamma frequency band in a distributed network of fronto-parietal areas reflects the intrinsically driven process of selection among competing behavioral alternatives. PMID:25690830

  2. MaNGA: Target selection and Optimization

    NASA Astrophysics Data System (ADS)

    Wake, David

    2016-01-01

    The 6-year SDSS-IV MaNGA survey will measure spatially resolved spectroscopy for 10,000 nearby galaxies using the Sloan 2.5m telescope and the BOSS spectrographs with a new fiber arrangement consisting of 17 individually deployable IFUs. We present the simultaneous design of the target selection and IFU size distribution to optimally meet our targeting requirements. The requirements for the main samples were to use simple cuts in redshift and magnitude to produce an approximately flat number density of targets as a function of stellar mass, ranging from 1x109 to 1x1011 M⊙, and radial coverage to either 1.5 (Primary sample) or 2.5 (Secondary sample) effective radii, while maximizing S/N and spatial resolution. In addition we constructed a "Color-Enhanced" sample where we required 25% of the targets to have an approximately flat number density in the color and mass plane. We show how these requirements are met using simple absolute magnitude (and color) dependent redshift cuts applied to an extended version of the NASA Sloan Atlas (NSA), how this determines the distribution of IFU sizes and the resulting properties of the MaNGA sample.

  3. MaNGA: Target selection and Optimization

    NASA Astrophysics Data System (ADS)

    Wake, David

    2015-01-01

    The 6-year SDSS-IV MaNGA survey will measure spatially resolved spectroscopy for 10,000 nearby galaxies using the Sloan 2.5m telescope and the BOSS spectrographs with a new fiber arrangement consisting of 17 individually deployable IFUs. We present the simultaneous design of the target selection and IFU size distribution to optimally meet our targeting requirements. The requirements for the main samples were to use simple cuts in redshift and magnitude to produce an approximately flat number density of targets as a function of stellar mass, ranging from 1x109 to 1x1011 M⊙, and radial coverage to either 1.5 (Primary sample) or 2.5 (Secondary sample) effective radii, while maximizing S/N and spatial resolution. In addition we constructed a 'Color-Enhanced' sample where we required 25% of the targets to have an approximately flat number density in the color and mass plane. We show how these requirements are met using simple absolute magnitude (and color) dependent redshift cuts applied to an extended version of the NASA Sloan Atlas (NSA), how this determines the distribution of IFU sizes and the resulting properties of the MaNGA sample.

  4. An efficient ratiometric fluorescent probe for tracking dynamic changes in lysosomal pH.

    PubMed

    Wang, Qianqian; Zhou, Liyi; Qiu, Liping; Lu, Danqing; Wu, Yongxiang; Zhang, Xiao-Bing

    2015-08-21

    Lysosomes are acidic organelles (approximately pH 4.5-5.5) and tracking the changes in lysosomal pH is of great biological importance. To address this issue, quite a few of fluorescent probes have been developed. However, few of these probes can realize the tracking of dynamic changes in lysosomal pH. Herein, we report a new lysosome-targeted ratiometric fluorescent probe (FR-Lys) by hybridizing morpholine with a xanthane derivative and an o-hydroxy benzoxazole group. In this probe, the morpholine group serves as a targeting unit for lysosome, the xanthane derivative exhibits a pH-modulated open/close reaction of the spirocycle, while the o-hydroxy benzoxazole moiety shows a pH modulated excited-state intramolecular proton transfer (ESIPT) process. Such a design affords the probe a ratiometric fluorescence response towards pH with pH values ranging from 4.0 to 6.3. The response of the probe to pH was fast and reversible with high selectivity. Moreover, this probe possesses further advantages such as easy synthesis, high photostability and low cytotoxicity. These features are favorable for tracking dynamic pH changes in biosystems. It was then applied for dynamic imaging pH changes in lysosomes with satisfactory results. PMID:26107774

  5. 78 kDa receptor for Man6P-independent lysosomal enzyme targeting: Biosynthetic transport from endoplasmic reticulum to 'high-density vesicles'

    SciTech Connect

    Gonzalez-Noriega, Alfonso . E-mail: gonor@biomedicas.unam.mx; Ortega Cuellar, Daniel D.; Michalak, Colette

    2006-04-15

    Recent work has shown that the cation-independent mannose 6-phosphate and the 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments. While the mannose 6-phosphate receptor is enriched in the Percoll fractions that contain Golgi apparatus, most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient. This report presents the biosynthetic transport of the 78 kDa receptor. Newly synthesized 78 kDa receptor was transported to Golgi from endoplasmic reticulum with a half life of 5 min. From the Golgi apparatus, the receptor takes two routes; about 15-25% is transported to the plasma membrane, and the rest migrates to late endosomes, subsequently to prelysosomes and finally to the dense vesicles. The 78 kDa receptor starts appearing at the dense vesicles 120 min after biosynthesis and reaches a maximum of 40-50% of the total receptor. Treatment of cells with NH{sub 4}Cl causes depletion of the receptor from the dense vesicles and prelysosomes and corresponding augmentation in endosomes and plasma membrane. These results suggest that the 78 kDa receptor cycles between compartments and that the dense vesicles seem to represent the most distal compartment in the biosynthetic pathway of this receptor.

  6. Optogenetic Acidification of Synaptic Vesicles and Lysosomes

    PubMed Central

    Grauel, M. Katharina; Wozny, Christian; Bentz, Claudia; Blessing, Anja; Rosenmund, Tanja; Jentsch, Thomas J.; Schmitz, Dietmar; Hegemann, Peter; Rosenmund, Christian

    2016-01-01

    Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes. PMID:26551543

  7. Calpain 1 induce lysosomal permeabilization by cleavage of lysosomal associated membrane protein 2.

    PubMed

    Villalpando Rodriguez, Gloria E; Torriglia, Alicia

    2013-10-01

    In light induced retinal degeneration (LIRD) photoreceptor cell death is mediated by caspase independent mechanisms. The activation of LEI/L-DNase II pathway in this model, is due to cathepsin D release from lysosomes, although the underlying mechanism remains poorly understood. In this paper we studied the involvement of calpains in lysosomal permeabilization. We investigated, for the first time, the calpain targets at lysosomal membrane level. We found that calpain 1 is responsible for lysosomal permeabilization by cleavage of the lysosomal associated membrane protein 2 (LAMP 2). Moreover, LAMP 2 degradation and lysosomal permeabilization were rescued by calpain inhibition and the use of MEF(-/-)lamp 2 cells indicates that the cleavage of LAMP 2A is essential for this permeabilization. Finally, we found that LAMP 2 is cleaved in LIRD, suggesting that the mechanism of calpain induced lysosomal permeabilization is not exclusive of a single cell death model. Overall, these data shed new light on understanding the mechanisms of lysosomal and caspase-independent cell death and point to the original targets for development of the new therapeutic protocols. PMID:23747342

  8. Bright and photostable fluorescent probe with aggregation-induced emission characteristics for specific lysosome imaging and tracking.

    PubMed

    Ouyang, Jiang; Zang, Qiguang; Chen, Wansong; Wang, Liqiang; Li, Shuo; Liu, Ren-Yu; Deng, Yuanyuan; Liu, Zhao-Qian; Li, Juan; Deng, Liu; Liu, You-Nian

    2016-10-01

    We develop a new lysosome-targeting AIE fluorescent probe tetraphenylethene-morpholine (TPE-MPL), by incorporating a typical lysosome-targeting moiety of morpholine into a stable tetraphenylethene skeleton. Due to both the AIE and antenna effects, TPE-MPL possesses superior photostability, appreciable tolerance to microenvironment change and high lysosome targeting ability. Our findings confirm that TPE-MPL is a well-suited imaging agent for targeting lysosome and tracking dynamic movement of lysosome. Moreover, due to its synthetic accessibility, TPE-MPL could be further modified as a dual-functional probe for lysosome, thereby gain further insight into the role of lysosome in biomedical applications. PMID:27474306

  9. Rabies DNA vaccine encoding lysosome-targeted glycoprotein supplemented with Emulsigen-D confers complete protection in preexposure and postexposure studies in BALB/c mice.

    PubMed

    Kaur, Manpreet; Saxena, Ankur; Rai, Anant; Bhatnagar, Rakesh

    2010-01-01

    The worldwide incidence of rabies and the inability of currently used vaccination strategies to provide highly potent and cost-effective therapy indicate the need for an improved rabies vaccine. Thus, DNA vaccine based on lysosome-targeted glycoprotein of the rabies virus was evaluated in BALB/c mice. It imparted partial protection (60%) against challenge with 20 LD(50) of the challenge virus standard (CVS) strain of rabies virus. To improve the outcome of vaccination, to ultimately enhance the immune response, we investigated different routes for DNA vaccine delivery, varied doses of DNA, and the influence of adjuvant supplementation. The highest immune response pertaining to IgG antibody titer, with a predominantly IgG1/IgG2a subclass distribution, effective cellular immunity, and a high level of rabies virus neutralizing antibodies (RVNAs) was attained by the optimized DNA vaccine formulation comprising intramuscular administration of 100 microg of DNA vaccine supplemented with Emulsigen-D. In preexposure prophylaxis, a 3-dose regimen of this formulation generated a high RVNA titer (32 IU/ml) and conferred complete protection against challenge with 20 LD(50) of CVS. For postexposure efficacy analysis, rabies was experimentally induced with 50 LD(50) of CVS. Subsequent therapy with 5 doses of the formulation completely prevented rabies in BALB/c mice, which maintained protective RVNA titers of 4 IU/ml. The World Health Organization recommended rabies protective titer threshold is 0.5 IU/ml. Thus, this optimized DNA vaccine formulation provides an avenue for preventing and controlling rabies. PMID:19741168

  10. Lysosomes and autophagy in aquatic animals.

    PubMed

    Moore, Michael N; Kohler, Angela; Lowe, David; Viarengo, Aldo

    2008-01-01

    The lysosomal-autophagic system appears to be a common target for many environmental pollutants, as lysosomes accumulate many toxic metals and organic xenobiotics, which perturb normal function and damage the lysosomal membrane. In fact, autophagic reactions frequently involving reduced lysosomal membrane integrity or stability appear to be effective generic indicators of cellular well-being in eukaryotes: in social amoebae (slime mold), mollusks and fish, autophagy/membrane destabilization is correlated with many stress and toxicological responses and pathological reactions. Prognostic use of adverse lysosomal and autophagic reactions to environmental pollutants can be used for predicting cellular dysfunction and health in aquatic animals, such as shellfish and fish, which are extensively used as sensitive bioindicators in monitoring ecosystem health; and also represent a significant food resource for at least 20% of the global human population. Explanatory frameworks for prediction of pollutant impact on health have been derived encompassing a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells and tissues. Methods are described for tracking in vivo autophagy of fluorescently labeled cytoplasmic proteins, measuring degradation of radiolabeled intracellular proteins and morphometric measurement of lysosomal/cytoplasmic volume ratio. Additional methods for the determination of lysosomal membrane stability in lower animals are also described, which can be applied to frozen tissue sections, protozoans and isolated cells in vivo. Experimental and simulated results have also indicated that nutritional deprivation (analogous in marine mussels to caloric restriction)-induced autophagy has a protective function against toxic effects mediated by reactive oxygen species (ROS). Finally, coupled measurement of lysosomal-autophagic reactions and simulation modelling is proposed as a practical toolbox for predicting toxic

  11. Lysosomotropic agents selectively target chronic lymphocytic leukemia cells due to altered sphingolipid metabolism.

    PubMed

    Dielschneider, R F; Eisenstat, H; Mi, S; Curtis, J M; Xiao, W; Johnston, J B; Gibson, S B

    2016-06-01

    Lysosome membrane permeabilization (LMP) mediates cell death in a variety of cancer cells. However, little is known about lysosomes and LMP in chronic lymphocytic leukemia (CLL). Owing to drug resistance and toxicity in CLL patients, better treatment strategies are required. Our results show that CLL cells were sensitive to the lysosomotropic agent siramesine. Furthermore, this drug was more effective in CLL cells, regardless of prognostic factors, compared with normal B cells. Siramesine caused LMP, lipid peroxidation and transcription factor EB nuclear translocation followed by mitochondrial membrane potential loss and reactive oxygen species release. Siramesine-induced cell death was blocked by lipid antioxidants, but not by soluble antioxidants or protease inhibitors. To determine whether CLL cells had altered lysosomes, we investigated sphingolipid metabolism as the lysosome is a hub for lipid metabolism. We found that CLL cells had more lysosomes, increased sphingosine-1-phosphate phosphatase 1 (SPP1) expression, and increased levels of sphingosine compared with normal B cells. Raising sphingosine levels increased LMP and cell death in CLL cells, but not in normal B cells. Together, these results show that excess sphingosine in CLL cells could contribute to their sensitivity toward LMP. Thus, targeting the lysosome could be a novel therapeutic strategy in CLL. PMID:26859075

  12. Altered lysosomal positioning affects lysosomal functions in a cellular model of Huntington's disease.

    PubMed

    Erie, Christine; Sacino, Matthew; Houle, Lauren; Lu, Michael L; Wei, Jianning

    2015-08-01

    Huntington's disease (HD) is a hereditary and devastating neurodegenerative disorder caused by a mutation in the huntingtin protein. Understanding the functions of normal and mutant huntingtin protein is the key to revealing the pathogenesis of HD and developing therapeutic targets. Huntingtin plays an important role in vesicular and organelle trafficking. Lysosomes are dynamic organelles that integrate several degradative pathways and regulate the activity of mammalian target of rapamycin complex 1 (mTORC1). In the present study, we found that the perinuclear accumulation of lysosomes was increased in a cellular model of HD derived from HD knock-in mice and primary fibroblasts from an HD patient. This perinuclear lysosomal accumulation could be reversed when normal huntingtin was overexpressed in HD cells. When we further investigated the functional significance of the increased perinuclear lysosomal accumulation in HD cells, we demonstrated that basal mTORC1 activity was increased in HD cells. In addition, autophagic influx was also increased in HD cells in response to serum deprivation, which leads to premature fusion of lysosomes with autophagosomes. Taken together, our data suggest that the increased perinuclear accumulation of lysosomes may play an important role in HD pathogenesis by altering lysosomal-dependent functions. PMID:25997742

  13. Target selection: invasion, mapping and cell choice.

    PubMed

    Holt, C E; Harris, W A

    1998-02-01

    Recent research has shown that changes in the concentration of particular molecules lead axons to invade their target, and that concentration changes in other molecules at the borders of the target prevent axons from leaving the target area. After invasion, topographic and lamina-specific cues guide axons to the correct location within the target field. At the level of a single cell or part of a cell, the evidence raises the possibility that axon targeting might be a combinatorial affair whereby specific axons compare the relative concentrations of several molecules on the surface of postsynaptic cells in order to choose a particular target. Both proteins and carbohydrates of various classes play major roles in these processes. PMID:9568397

  14. Enhancing Academic Performance: Issues in Target Selection.

    ERIC Educational Resources Information Center

    Hoge, Robert D.; Andrews, D. A.

    1987-01-01

    Learning of subject matter and acquisition of academically relevant skills are important goals in enhancing academic achievement in the classroom. The results of 22 experiments reviewed in this article support the validity of the academic performance targets but not classroom behavior targets. Some limitations on these conclusions are discussed.…

  15. Decisions in motion: vestibular contributions to saccadic target selection.

    PubMed

    Rincon-Gonzalez, L; Selen, L P J; Halfwerk, K; Koppen, M; Corneil, B D; Medendorp, W P

    2016-09-01

    The natural world continuously presents us with many opportunities for action, and thus a process of target selection must precede action execution. While there has been considerable progress in understanding target selection in stationary environments, little is known about target selection when we are in motion. Here we investigated the effect of self-motion signals on saccadic target selection in a dynamic environment. Human subjects were sinusoidally translated (f = 0.6 Hz, 30-cm peak-to-peak displacement) along an interaural axis with a vestibular sled. During the motion two visual targets were presented asynchronously but equidistantly on either side of fixation. Subjects had to look at one of these targets as quickly as possible. With an adaptive approach, the time delay between these targets was adjusted until the subject selected both targets equally often. We determined this balanced time delay for different phases of the motion in order to distinguish the effects of body acceleration and velocity on saccadic target selection. Results show that acceleration (or position, as these are indistinguishable during sinusoidal motion), but not velocity, affects target selection for saccades. Subjects preferred to look at targets in the direction of the acceleration-the leftward target was preferred when the sled accelerated to the left, and vice versa. Saccadic reaction times mimicked this selection bias by being reliably shorter to targets in the direction of acceleration. Our results provide evidence that saccade target selection mechanisms are modulated by self-motion signals, which could be derived directly from the otolith system. PMID:27281751

  16. Regulation of lysosomal ion homeostasis by channels and transporters.

    PubMed

    Xiong, Jian; Zhu, Michael X

    2016-08-01

    Lysosomes are the major organelles that carry out degradation functions. They integrate and digest materials compartmentalized by endocytosis, phagocytosis or autophagy. In addition to more than 60 hydrolases residing in the lysosomes, there are also ion channels and transporters that mediate the flux or transport of H(+), Ca(2+), Na(+), K(+), and Cl(-) across the lysosomal membranes. Defects in ionic exchange can lead to abnormal lysosome morphology, defective vesicle trafficking, impaired autophagy, and diseases such as neurodegeneration and lysosomal storage disorders. The latter are characterized by incomplete lysosomal digestion and accumulation of toxic materials inside enlarged intracellular vacuoles. In addition to degradation, recent studies have revealed the roles of lysosomes in metabolic pathways through kinases such as mechanistic target of rapamycin (mTOR) and transcriptional regulation through calcium signaling molecules such as transcription factor EB (TFEB) and calcineurin. Owing to the development of new approaches including genetically encoded fluorescence probes and whole endolysosomal patch clamp recording techniques, studies on lysosomal ion channels have made remarkable progress in recent years. In this review, we will focus on the current knowledge of lysosome-resident ion channels and transporters, discuss their roles in maintaining lysosomal function, and evaluate how their dysfunction can result in disease. PMID:27430889

  17. Intracellular Protein Degradation: From a Vague Idea through the Lysosome and the Ubiquitin-Proteasome System and onto Human Diseases and Drug Targeting

    PubMed Central

    Ciechanover, Aaron

    2012-01-01

    Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code was transcribed to RNA and translated to proteins, but how proteins were degraded had remained a neglected research area. With the discovery of the lysosome by Christian de Duve it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis was largely non-lysosomal, but the mechanisms involved have remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs. PMID:23908826

  18. Aging. Lysosomal signaling molecules regulate longevity in Caenorhabditis elegans.

    PubMed

    Folick, Andrew; Oakley, Holly D; Yu, Yong; Armstrong, Eric H; Kumari, Manju; Sanor, Lucas; Moore, David D; Ortlund, Eric A; Zechner, Rudolf; Wang, Meng C

    2015-01-01

    Lysosomes are crucial cellular organelles for human health that function in digestion and recycling of extracellular and intracellular macromolecules. We describe a signaling role for lysosomes that affects aging. In the worm Caenorhabditis elegans, the lysosomal acid lipase LIPL-4 triggered nuclear translocalization of a lysosomal lipid chaperone LBP-8, which promoted longevity by activating the nuclear hormone receptors NHR-49 and NHR-80. We used high-throughput metabolomic analysis to identify several lipids in which abundance was increased in worms constitutively overexpressing LIPL-4. Among them, oleoylethanolamide directly bound to LBP-8 and NHR-80 proteins, activated transcription of target genes of NHR-49 and NHR-80, and promoted longevity in C. elegans. These findings reveal a lysosome-to-nucleus signaling pathway that promotes longevity and suggest a function of lysosomes as signaling organelles in metazoans. PMID:25554789

  19. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    SciTech Connect

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose; Okada, Masato

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. Black-Right-Pointing-Pointer We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. Black-Right-Pointing-Pointer The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. Black-Right-Pointing-Pointer Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. Black-Right-Pointing-Pointer The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.

  20. A six-membered-ring incorporated Si-rhodamine for imaging of copper(ii) in lysosomes.

    PubMed

    Wang, Baogang; Cui, Xiaoyan; Zhang, Zhiqiang; Chai, Xiaoyun; Ding, Hao; Wu, Qiuye; Guo, Zhongwu; Wang, Ting

    2016-07-12

    The regulation of copper homeostasis in lysosomes of living cells is closely related to various physiological and pathological processes. Thus, it is of urgent need to develop a fluorescent probe for selectively and sensitively monitoring the location and concentration of lysosomal Cu(2+). Herein, a six-membered ring, thiosemicarbazide, was incorporated into a Si-rhodamine (SiR) scaffold for the first time, affording a SiR-based fluorescent probe SiRB-Cu. Through the effective Cu(2+)-triggered ring-opening process, the probe exhibits fast NIR chromogenic and fluorogenic responses to Cu(2+) within 2 min as the result of formation of a highly fluorescent product SiR-NCS. Compared with a five-membered ring, the expanded ring retains great tolerance to H(+), ensuring the superior sensitivity with a detection limit as low as 7.7 nM and 200-fold enhancement of relative fluorescence in the presence of 1.0 equiv. of Cu(2+) in pH = 5.0 solution, the physiological pH of lysosome. Moreover, the thiosemicarbazide moiety acts not only as the chelating and reactive site, but also as an efficient lysosome-targeting group, leading to the proactive accumulation of the probe into lysosomes. Taking advantage of these distinct properties, SiRB-Cu provides a functional probe suitable for imaging exogenous and endogenous lysosomal Cu(2+) with high imaging contrast and fidelity. PMID:27314426

  1. Tuning target selection algorithms to improve galaxy redshift estimates

    NASA Astrophysics Data System (ADS)

    Hoyle, Ben; Paech, Kerstin; Rau, Markus Michael; Seitz, Stella; Weller, Jochen

    2016-06-01

    We showcase machine learning (ML) inspired target selection algorithms to determine which of all potential targets should be selected first for spectroscopic follow-up. Efficient target selection can improve the ML redshift uncertainties as calculated on an independent sample, while requiring less targets to be observed. We compare seven different ML targeting algorithms with the Sloan Digital Sky Survey (SDSS) target order, and with a random targeting algorithm. The ML inspired algorithms are constructed iteratively by estimating which of the remaining target galaxies will be most difficult for the ML methods to accurately estimate redshifts using the previously observed data. This is performed by predicting the expected redshift error and redshift offset (or bias) of all of the remaining target galaxies. We find that the predicted values of bias and error are accurate to better than 10-30 per cent of the true values, even with only limited training sample sizes. We construct a hypothetical follow-up survey and find that some of the ML targeting algorithms are able to obtain the same redshift predictive power with 2-3 times less observing time, as compared to that of the SDSS, or random, target selection algorithms. The reduction in the required follow-up resources could allow for a change to the follow-up strategy, for example by obtaining deeper spectroscopy, which could improve ML redshift estimates for deeper test data.

  2. PRD125, a potent and selective inhibitor of sterol O-acyltransferase 2 markedly reduces hepatic cholesteryl ester accumulation and improves liver function in lysosomal acid lipase-deficient mice.

    PubMed

    Lopez, Adam M; Chuang, Jen-Chieh; Posey, Kenneth S; Ohshiro, Taichi; Tomoda, Hiroshi; Rudel, Lawrence L; Turley, Stephen D

    2015-11-01

    In most organs, the bulk of cholesterol is unesterified, although nearly all possess a varying capability of esterifying cholesterol through the action of either sterol O-acyltransferase (SOAT) 1 or, in the case of hepatocytes and enterocytes, SOAT2. Esterified cholesterol (EC) carried in plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to whether pharmacological inhibition of SOAT2 might reduce tissue EC accretion in CESD. When weaned at 21 days, Lal(-/-) mice, of either gender, had a whole liver cholesterol content that was 12- to 13-fold more than that of matching Lal(+/+) littermates (23 versus 1.8 mg, respectively). In Lal(-/-) males given the selective SOAT2 inhibitor PRD125 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl-1,7,11-trideacetylpyripyropene A in their diet (∼10 mg/day per kg body weight) from 21 to 53 days, whole liver cholesterol content was 48.6 versus 153.7 mg in untreated 53-day-old Lal(-/-) mice. This difference reflected a 59% reduction in hepatic EC concentration (mg/g), combined with a 28% fall in liver mass. The treated mice also showed a 63% reduction in plasma alanine aminotransferase activity, in parallel with decisive falls in hepatic mRNA expression levels for multiple proteins that reflect macrophage presence and inflammation. These data implicate SOAT2 as a potential target in CESD management. PMID:26283692

  3. Vasopressors During Sepsis: Selection and Targets.

    PubMed

    Gelinas, Jean P; Russell, James A

    2016-06-01

    Clinicians have greatly improved care for septic shock. Urgent resuscitation using intravenous fluids and vasopressors as well as rapid administration of broad spectrum antibiotics are probably the most basic and universally accepted interventions. Various trials have compared different types of vasopressors, associations of vasopressors and inotropes, and pressure targets. End goal-directed therapy algorithms are designed to optimize oxygen delivery by use of fluids, vasopressors, inotropes, and blood products. Patients who have a poor response to resuscitation and patients with known severe ventricular dysfunction might merit advanced hemodynamic monitoring. This review examines important vasopressor and septic shock trials. PMID:27229642

  4. Sexual selection targets cetacean pelvic bones.

    PubMed

    Dines, James P; Otárola-Castillo, Erik; Ralph, Peter; Alas, Jesse; Daley, Timothy; Smith, Andrew D; Dean, Matthew D

    2014-11-01

    Male genitalia evolve rapidly, probably as a result of sexual selection. Whether this pattern extends to the internal infrastructure that influences genital movements remains unknown. Cetaceans (whales and dolphins) offer a unique opportunity to test this hypothesis: since evolving from land-dwelling ancestors, they lost external hind limbs and evolved a highly reduced pelvis that seems to serve no other function except to anchor muscles that maneuver the penis. Here, we create a novel morphometric pipeline to analyze the size and shape evolution of pelvic bones from 130 individuals (29 species) in the context of inferred mating system. We present two main findings: (1) males from species with relatively intense sexual selection (inferred by relative testes size) tend to evolve larger penises and pelvic bones compared to their body length, and (2) pelvic bone shape has diverged more in species pairs that have diverged in inferred mating system. Neither pattern was observed in the anterior-most pair of vertebral ribs, which served as a negative control. This study provides evidence that sexual selection can affect internal anatomy that controls male genitalia. These important functions may explain why cetacean pelvic bones have not been lost through evolutionary time. PMID:25186496

  5. Sexual selection targets cetacean pelvic bones

    PubMed Central

    Dines, J. P.; Otárola-Castillo, E.; Ralph, P.; Alas, J.; Daley, T.; Smith, A. D.; Dean, M. D.

    2014-01-01

    Male genitalia evolve rapidly, probably as a result of sexual selection. Whether this pattern extends to the internal infrastructure that influences genital movements remains unknown. Cetaceans (whales and dolphins) offer a unique opportunity to test this hypothesis: since evolving from land-dwelling ancestors, they lost external hind limbs and evolved a highly reduced pelvis which seems to serve no other function except to anchor muscles that maneuver the penis. Here we create a novel morphometric pipeline to analyze the size and shape evolution of pelvic bones from 130 individuals (29 species) in the context of inferred mating system. We present two main findings: 1) males from species with relatively intense sexual selection (inferred by relative testes size) have evolved relatively large penises and pelvic bones compared to their body size, and 2) pelvic bone shape diverges more quickly in species pairs that have diverged in inferred mating system. Neither pattern was observed in the anterior-most pair of vertebral ribs, which served as a negative control. This study provides evidence that sexual selection can affect internal anatomy that controls male genitalia. These important functions may explain why cetacean pelvic bones have not been lost through evolutionary time. PMID:25186496

  6. Target selection bias transfers across different response actions.

    PubMed

    Moher, Jeff; Song, Joo-Hyun

    2014-06-01

    Target selection is biased by recent experience. For example, a selected target feature may be stored in memory and bias selection on future trials, such that objects matching that feature are "primed" for selection. In the present study, we examined the role of action history in selection biases. Participants searched for a uniquely colored object. Pretrial cues indicated whether participants should respond with a keypress or a reach movement. If the representation of the feature that biases selection is critically bound with its associated action, we would expect priming effects to be restricted to cases where both the response mode and target color are repeated. However, we found that responses to the target were faster when the target color was repeated, even when the response switched from a reach to a keypress, or vice versa. Priming effects were even observed after "no-go" trials in which a response was withheld, and priming effects transferred across response modes when eye movement recordings ensured that participants did not saccade to the target. These results demonstrate that target features are represented in memory separately from their associated actions and can bias selection on subsequent trials even when a different mode of action output is required. PMID:24490945

  7. Computational approaches to selecting and optimising targets for structural biology.

    PubMed

    Overton, Ian M; Barton, Geoffrey J

    2011-09-01

    Selection of protein targets for study is central to structural biology and may be influenced by numerous factors. A key aim is to maximise returns for effort invested by identifying proteins with the balance of biophysical properties that are conducive to success at all stages (e.g. solubility, crystallisation) in the route towards a high resolution structural model. Selected targets can be optimised through construct design (e.g. to minimise protein disorder), switching to a homologous protein, and selection of experimental methodology (e.g. choice of expression system) to prime for efficient progress through the structural proteomics pipeline. Here we discuss computational techniques in target selection and optimisation, with more detailed focus on tools developed within the Scottish Structural Proteomics Facility (SSPF); namely XANNpred, ParCrys, OB-Score (target selection) and TarO (target optimisation). TarO runs a large number of algorithms, searching for homologues and annotating the pool of possible alternative targets. This pool of putative homologues is presented in a ranked, tabulated format and results are also visualised as an automatically generated and annotated multiple sequence alignment. The target selection algorithms each predict the propensity of a selected protein target to progress through the experimental stages leading to diffracting crystals. This single predictor approach has advantages for target selection, when compared with an approach using two or more predictors that each predict for success at a single experimental stage. The tools described here helped SSPF achieve a high (21%) success rate in progressing cloned targets to diffraction-quality crystals. PMID:21906678

  8. Lysosomal Lipid Storage Diseases

    PubMed Central

    Schulze, Heike; Sandhoff, Konrad

    2011-01-01

    Lysosomal lipid storage diseases, or lipidoses, are inherited metabolic disorders in which typically lipids accumulate in cells and tissues. Complex lipids, such as glycosphingolipids, are constitutively degraded within the endolysosomal system by soluble hydrolytic enzymes with the help of lipid binding proteins in a sequential manner. Because of a functionally impaired hydrolase or auxiliary protein, their lipid substrates cannot be degraded, accumulate in the lysosome, and slowly spread to other intracellular membranes. In Niemann-Pick type C disease, cholesterol transport is impaired and unesterified cholesterol accumulates in the late endosome. In most lysosomal lipid storage diseases, the accumulation of one or few lipids leads to the coprecipitation of other hydrophobic substances in the endolysosomal system, such as lipids and proteins, causing a “traffic jam.” This can impair lysosomal function, such as delivery of nutrients through the endolysosomal system, leading to a state of cellular starvation. Therapeutic approaches are currently restricted to mild forms of diseases with significant residual catabolic activities and without brain involvement. PMID:21502308

  9. Computational design of nanoparticle drug delivery systems for selective targeting

    NASA Astrophysics Data System (ADS)

    Duncan, Gregg A.; Bevan, Michael A.

    2015-09-01

    Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ρR, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ρL. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting diseased cells and tissues.Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ρR, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ρL. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting

  10. Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells

    PubMed Central

    Liu, Ji; Lu, Wennan; Guha, Sonia; Baltazar, Gabriel C.; Coffey, Erin E.; Laties, Alan M.; Rubenstein, Ronald C.; Reenstra, William W.

    2012-01-01

    The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTRinh-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4−/− mouse model of recessive Stargardt's disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization. PMID:22572847

  11. UVA Causes Dual Inactivation of Cathepsin B and L Underlying Lysosomal Dysfunction in Human Dermal Fibroblasts

    PubMed Central

    Lamore, Sarah D.; Wondrak, Georg T.

    2013-01-01

    Cutaneous exposure to chronic solar UVA-radiation is a causative factor in photocarcinogenesis and photoaging. Recently, we have identified the thiol-dependent cysteine-protease cathepsin B as a novel UVA-target undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction in fibroblasts. In this study, we examined UVA effects on a wider range of cathepsins and explored the occurrence of UVA-induced cathepsin inactivation in other cultured skin cell types. In dermal fibroblasts, chronic exposure to non-cytotoxic doses of UVA caused pronounced inactivation of the lysosomal cysteine-proteases cathepsin B and L, effects not observed in primary keratinocytes and occurring only to a minor extent in primary melanocytes. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring an established set of protein markers (including LAMP1, LC3-II, and p62) and cell ultrastructural changes detected by electron microscopy, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not display ‘UVA-mimetic’ effects failing to reproduce the UVA-induced phenotype. Taken together, our data demonstrate that chronic UVA inhibits both cathepsin B and L enzymatic activity and that dual inactivation of both enzymes is a causative factor underlying UVA-induced impairment of lysosomal function in dermal fibroblasts. PMID:23603447

  12. N-Pyridineium-2-yl Darrow Red analogue: unique near-infrared lysosome-biomarker for the detection of cancer cells.

    PubMed

    He, Dan-Dan; Liu, Wu; Sun, Ru; Fan, Chen; Xu, Yu-Jie; Ge, Jian-Feng

    2015-02-01

    The lysosome-targetable OFF-ON type pH sensor that does not emit at pH = 4.0 is adopted for the selective detection of cancer cells, and the acidity difference of lysosomes in cancer and normal cells is verified. Three pH probes based on Darrow Red derivatives were designed and prepared that were demonstrated to be lysosome-specific biomarkers with inducible emission at 580-850 nm by the comparable in cellular imaging assays using HeLa, KB, and V79 cells. Of these, a pyridineium-2-yl Darrow Red analogue with a pKa of 2.4 was found to be a lysosome tracker for cancer cells, it is a unique pH sensor for the optical identification and distinction of cancer cells from normal cells and has potential application as a fluorescent biomaker of cancer cells in in vitro assays. PMID:25569205

  13. Target selection biases from recent experience transfer across effectors.

    PubMed

    Moher, Jeff; Song, Joo-Hyun

    2016-02-01

    Target selection is often biased by an observer's recent experiences. However, not much is known about whether these selection biases influence behavior across different effectors. For example, does looking at a red object make it easier to subsequently reach towards another red object? In the current study, we asked observers to find the uniquely colored target object on each trial. Randomly intermixed pre-trial cues indicated the mode of action: either an eye movement or a visually guided reach movement to the target. In Experiment 1, we found that priming of popout, reflected in faster responses following repetition of the target color on consecutive trials, occurred regardless of whether the effector was repeated from the previous trial or not. In Experiment 2, we examined whether an inhibitory selection bias away from a feature could transfer across effectors. While priming of popout reflects both enhancement of the repeated target features and suppression of the repeated distractor features, the distractor previewing effect isolates a purely inhibitory component of target selection in which a previewed color is presented in a homogenous display and subsequently inhibited. Much like priming of popout, intertrial suppression biases in the distractor previewing effect transferred across effectors. Together, these results suggest that biases for target selection driven by recent trial history transfer across effectors. This indicates that representations in memory that bias attention towards or away from specific features are largely independent from their associated actions. PMID:26563393

  14. TDP-43 loss of function increases TFEB activity and blocks autophagosome-lysosome fusion.

    PubMed

    Xia, Qin; Wang, Hongfeng; Hao, Zongbing; Fu, Cheng; Hu, Qingsong; Gao, Feng; Ren, Haigang; Chen, Dong; Han, Junhai; Ying, Zheng; Wang, Guanghui

    2016-01-18

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA-binding protein 43 (TDP-43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP-43 is a multi-functional protein involved in RNA processing and a large number of TDP-43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP-43-linked neurodegeneration remain elusive. In this study, we found that loss of TDP-43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy-lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP-43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP-43-depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP-43-mediated neurodegeneration. PMID:26702100

  15. Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin

    PubMed Central

    Zhou, Xiaolai; Sun, Lirong; Bastos de Oliveira, Francisco; Qi, Xiaoyang; Brown, William J.; Smolka, Marcus B.; Sun, Ying

    2015-01-01

    Mutations in the progranulin (PGRN) gene have been linked to two distinct neurodegenerative diseases, frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL). Accumulating evidence suggests a critical role of PGRN in lysosomes. However, how PGRN is trafficked to lysosomes is still not clear. Here we report a novel pathway for lysosomal delivery of PGRN. We found that prosaposin (PSAP) interacts with PGRN and facilitates its lysosomal targeting in both biosynthetic and endocytic pathways via the cation-independent mannose 6-phosphate receptor and low density lipoprotein receptor-related protein 1. PSAP deficiency in mice leads to severe PGRN trafficking defects and a drastic increase in serum PGRN levels. We further showed that this PSAP pathway is independent of, but complementary to, the previously identified PGRN lysosomal trafficking mediated by sortilin. Collectively, our results provide new understanding on PGRN trafficking and shed light on the molecular mechanisms behind FTLD and NCL caused by PGRN mutations. PMID:26370502

  16. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing.

    PubMed

    Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

    2016-03-01

    The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

  17. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

    PubMed Central

    Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

    2016-01-01

    The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

  18. Evolving Strategies for Target Selection for Antibody-Drug Conjugates.

    PubMed

    Damelin, Marc; Zhong, Wenyan; Myers, Jeremy; Sapra, Puja

    2015-11-01

    Antibody-drug conjugates (ADCs) represent a promising modality for the treatment of cancer. The therapeutic strategy is to deliver a potent drug preferentially to the tumor and not normal tissues by attaching the drug to an antibody that recognizes a tumor antigen. The selection of antigen targets is critical to enabling a therapeutic window for the ADC and has proven to be surprisingly complex. We surveyed the tumor and normal tissue expression profiles of the targets of ADCs currently in clinical development. Our analysis demonstrates a surprisingly broad range of expression profiles and the inability to formalize any optimal parameters for an ADC target. In this context, we discuss additional considerations for ADC target selection, including interdependencies among biophysical properties of the drug, biological functions of the target and strategies for clinical development. The TPBG (5T4) oncofetal antigen and the anti-TPBG ADC A1-mcMMAF are highlighted to demonstrate the relevance of the target's biological function. Emerging platform technologies and novel biological insights are expanding ADC target space and transforming strategies for target selection. PMID:25585957

  19. A SIMPLE LIKELIHOOD METHOD FOR QUASAR TARGET SELECTION

    SciTech Connect

    Kirkpatrick, Jessica A.; Schlegel, David J.; Ross, Nicholas P.; Myers, Adam D.; Hennawi, Joseph F.; Sheldon, Erin S.; Schneider, Donald P.; Weaver, Benjamin A.

    2011-12-20

    We present a new method for quasar target selection using photometric fluxes and a Bayesian probabilistic approach. For our purposes, we target quasars using Sloan Digital Sky Survey (SDSS) photometry to a magnitude limit of g = 22. The efficiency and completeness of this technique are measured using the Baryon Oscillation Spectroscopic Survey (BOSS) data taken in 2010. This technique was used for the uniformly selected (CORE) sample of targets in BOSS year-one spectroscopy to be realized in the ninth SDSS data release. When targeting at a density of 40 objects deg{sup -2} (the BOSS quasar targeting density), the efficiency of this technique in recovering z > 2.2 quasars is 40%. The completeness compared to all quasars identified in BOSS data is 65%. This paper also describes possible extensions and improvements for this technique.

  20. Target Selection for the LBTI Exozodi Key Science Program

    NASA Astrophysics Data System (ADS)

    Weinberger, Alycia J.; Bryden, Geoff; Kennedy, Grant M.; Roberge, Aki; Defrère, Denis; Hinz, Philip M.; Millan-Gabet, Rafael; Rieke, George; Bailey, Vanessa P.; Danchi, William C.; Haniff, Chris; Mennesson, Bertrand; Serabyn, Eugene; Skemer, Andrew J.; Stapelfeldt, Karl R.; Wyatt, Mark C.

    2015-02-01

    The Hunt for Observable Signatures of Terrestrial planetary Systems (HOSTS) on the Large Binocular Telescope Interferometer will survey nearby stars for faint emission arising from ~300 K dust (exozodiacal dust), and aims to determine the exozodiacal dust luminosity function. HOSTS results will enable planning for future space telescopes aimed at direct spectroscopy of habitable zone terrestrial planets, as well as greater understanding of the evolution of exozodiacal disks and planetary systems. We lay out here the considerations that lead to the final HOSTS target list. Our target selection strategy maximizes the ability of the survey to constrain the exozodi luminosity function by selecting a combination of stars selected for suitability as targets of future missions and as sensitive exozodi probes. With a survey of approximately 50 stars, we show that HOSTS can enable an understanding of the statistical distribution of warm dust around various types of stars and is robust to the effects of varying levels of survey sensitivity induced by weather conditions.

  1. Distinct Lysosomal Network Protein Profiles in Parkinsonian Syndrome Cerebrospinal Fluid

    PubMed Central

    Boman, Andrea; Svensson, Samuel; Boxer, Adam; Rojas, Julio C.; Seeley, William W.; Karydas, Anna; Miller, Bruce; Kågedal, Katarina; Svenningsson, Per

    2016-01-01

    Background: Clinical diagnosis of parkinsonian syndromes like Parkinson’s disease (PD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) is hampered by overlapping symptomatology and lack of diagnostic biomarkers, and definitive diagnosis is only possible post-mortem. Objective: Since impaired protein degradation plays an important role in many neurodegenerative disorders, we hypothesized that profiles of select lysosomal network proteins in cerebrospinal fluid could be differentially expressed in these parkinsonian syndromes. Methods: Cerebrospinal fluid samples were collected from PD patients (n = 18), clinically diagnosed 4-repeat tauopathy patients; corticobasal syndrome (CBS) (n = 3) and PSP (n = 8); and pathologically diagnosed PSP (n = 8) and CBD patients (n = 7). Each patient set was compared to its appropriate control group consisting of age and gender matched individuals. Select lysosomal network protein levels were detected via Western blotting. Factor analysis was used to test the diagnostic sensitivity, specificity and accuracy of the select lysosomal network protein expression profiles. Results: PD, CBD and PSP were markedly different in their cerebrospinal fluid lysosomal network protein profiles. Lysosomal-associated membrane proteins 1 and 2 were significantly decreased in PD; early endosomal antigen 1 was decreased and lysozyme increased in PSP; and lysosomal-associated membrane proteins 1 and 2, microtubule-associated protein 1 light chain 3 and lysozyme were increased in CBD. A panel of lysosomal-associated membrane protein 2, lysozyme and microtubule-associated protein 1 light chain discriminated between controls, PD and 4-repeat tauopathies. Conclusions: This study offers proof of concept that select lysosomal network proteins are differentially expressed in cerebrospinal fluid of Parkinson’s disease, corticobasal syndrome and progressive supranuclear palsy. Lysosomal network protein analysis

  2. Mesoscale Nanoparticles Selectively Target the Renal Proximal Tubule Epithelium

    PubMed Central

    Williams, Ryan M.; Shah, Janki; Ng, Brandon D.; Minton, Denise R.; Gudas, Lorraine J.; Park, Christopher Y.; Heller, Daniel A.

    2015-01-01

    We synthesized “mesoscale” nanoparticles, approximately 400 nm in diameter, which unexpectedly localized selectively in renal proximal tubules and up to 7 times more efficiently in the kidney than other organs. Although nanoparticles typically localize in the liver and spleen, modulating their size and opsonization potential allowed for stable targeting of the kidneys through a new proposed uptake mechanism. Applying this kidney targeting strategy, we anticipate use in the treatment of renal disease and the study of renal physiology. PMID:25811353

  3. Newborn screening for lysosomal storage disorders.

    PubMed

    Nakamura, Kimitoshi; Hattori, Kiyoko; Endo, Fumio

    2011-02-15

    Lysosomes are intracellular organelles containing acid hydrolases that degrade biological macromolecules. Lysosomal storage disorders (LSDs) are caused by absent activity of one or more of these enzymes due to mutations of genes encoding lysosomal hydrolases or enzymes that process, target, and transport these enzymes. The specific signs and symptoms of each LSD derive from the type of material accumulated within the lysosome, the site (organ) of accumulation and the response of the body (sometimes in the form of an inflammatory or immune response) to the accumulated material. Interest for inclusion of these disorders in newborn screening programs derives from the availability of effective therapy in the form of enzyme replacement or substrate reduction therapy and bone marrow transplant that may improve long-term outcome especially if started prior to irreversible organ damage. Based on the availability of therapy and suitable screening methods, Gaucher disease, Fabry disease, Pompe disease, mucopolysaccharidosis I and II, Niemann-Pick disease, and Krabbe disease are candidates for newborn screening. Pilot newborn screening projects have been performed for some of these conditions that indicate the feasibility of this approach. This review will provide insight into these screening strategies and discuss their advantages and limitations. © 2011 Wiley-Liss, Inc. PMID:21312327

  4. Target Selection and Determination of Function in Structural Genomics

    PubMed Central

    Watson, James D.; Todd, Annabel E.; Bray, James; Laskowski, Roman A.; Edwards, Aled; Joachimiak, Andrzej; Orengo, Christine A.; Thornton, Janet M.

    2011-01-01

    Summary The first crucial step in any structural genomics project is the selection and prioritization of target proteins for structure determination. There may be a number of selection criteria to be satisfied, including that the proteins have novel folds, that they be representatives of large families for which no structure is known, and so on. The better the selection at this stage, the greater is the value of the structures obtained at the end of the experimental process. This value can be further enhanced once the protein structures have been solved if the functions of the given proteins can also be determined. Here we describe the methods used at either end of the experimental process: firstly, sensitive sequence comparison techniques for selecting a high-quality list of target proteins, and secondly the various computational methods that can be applied to the eventual 3D structures to determine the most likely biochemical function of the proteins in question. PMID:12880206

  5. Feature Extraction and Selection Strategies for Automated Target Recognition

    NASA Technical Reports Server (NTRS)

    Greene, W. Nicholas; Zhang, Yuhan; Lu, Thomas T.; Chao, Tien-Hsin

    2010-01-01

    Several feature extraction and selection methods for an existing automatic target recognition (ATR) system using JPLs Grayscale Optical Correlator (GOC) and Optimal Trade-Off Maximum Average Correlation Height (OT-MACH) filter were tested using MATLAB. The ATR system is composed of three stages: a cursory region of-interest (ROI) search using the GOC and OT-MACH filter, a feature extraction and selection stage, and a final classification stage. Feature extraction and selection concerns transforming potential target data into more useful forms as well as selecting important subsets of that data which may aide in detection and classification. The strategies tested were built around two popular extraction methods: Principal Component Analysis (PCA) and Independent Component Analysis (ICA). Performance was measured based on the classification accuracy and free-response receiver operating characteristic (FROC) output of a support vector machine(SVM) and a neural net (NN) classifier.

  6. Lysosomal Storage Diseases.

    PubMed

    Kaye, Edward M.

    2001-05-01

    Lysosomal storage disorders (LSDs), over 40 different diseases, are now considered treatable disorders. Only a few short years ago, Lysosomal storage disorders were seen as interesting neurodegenerative disorders without any potential for treatment. Effective treatment strategies such as bone marrow transplantation (BMT), enzyme replacement therapy (ERT), and glycolipid synthesis inhibition have been developed in the last 20 years and continue to be researched and evaluated. Bone marrow transplantation began approximately 15 years ago and has shown benefit for some of the lysosomal storage disorders. In order to be effective, the transplant must be performed early in the course of the disease, before the development of irreversible neurologic damage. Diseases such as Hurler appear to respond to BMT, however, improvement in bone disease is much less vigorous than responses in other organs. Krabbe disease responds if the transplant is performed before irreversible signs of neurologic damage appear. Metachromatic leukodystrophy may respond if the transplant can be performed early enough although peripheral nerve findings appear to progress. Other diseases, eg, GM1- and GM2-gangliosidoses do not appear to be altered by BMT. Despite its high cost, ERT has been very effective treatment for type I (non-neuronopathic) Gaucher disease. Enzyme replacement therapy for other LSDs, including ERT for Fabry and Pompe diseases, which are planned to be imminently introduced, and other enzymes such as for Morquio and Hunter diseases that are in the study phases, may be marketed in the very near future. Glycolipid inhibitors, such as N-butyldeoxynijirimycin (OGS-918), have been effective in reducing the liver and spleen volume in type I Gaucher disease. These oral inhibitors may prove to be important adjuncts to ERT and provide the advantage of being able to cross the blood/brain barrier, which limits enzyme access to brain. Currently, clinical studies are being conducted on patients

  7. Kinetics of lysosomal storage of indigestible matter.

    PubMed Central

    Hurley, J; Alward, J

    1975-01-01

    In lysosomal storage diseases and in accumulation of lipofusion in the lysosomes there is a gradual eroding of the lysosomal system due to overloading the lysosomes by molecules which cannot be digested or expelled. The kinetics of this accumulation is examined for tissue cultures in terms of the cell growth rate, lysosomal production rate, and of generation of the indigestible element. PMID:1125388

  8. Selective screening for lysosomal storage diseases with dried blood spots collected on filter paper in 4,700 high-risk colombian subjects.

    PubMed

    Uribe, Alfredo; Giugliani, Roberto

    2013-01-01

    Lysosomal storage disorders (LSDs) are a very heterogeneous group of hereditary disorders. The diagnostic process usually involves complex sampling, processing, testing, and validation procedures, performed by specialized laboratories only, which causes great limitations in reaching a diagnosis for patients affected by these diseases.There are few studies about LSDs in Colombia. The diagnostic limitations often make medical practitioners disregard the possibility of these disorders while diagnosing their patients. The current study documents the results of a 7-year screening in high-risk patients, aimed to detect LSDs using dried blood spots (DBS) collected on filter paper, with a micromethodology that facilitates diagnosis even with a large number of samples.The activities of α-galactosidase A, α glucosidase, α-L-iduronidase, arylsulfatase B, β-galactosidase, β-glucosidase, total hexosaminidase, iduronate sulfatase, and chitotriosidase were analyzed in high-risk patients for lysosomal disease. The catalytic activity was evaluated with fluorometric micromethods using artificial substrates marked with 4-methylumbelliferone.The reference values for a control population were established for the enzymes listed above, and 242 patients were found to have an enzyme deficiency, guiding to the following diagnoses: Fabry disease (n = 31), Pompe disease (n = 16), Hurler Syndrome (n = 15), Maroteaux-Lamy Syndrome (n = 34), GM1 Gangliosidosis (n = 10), Morquio B (n = 1), Gaucher disease (n = 101), Sandhoff disease (n = 1), Mucolipidosis (n = 2), and Hunter Syndrome (n = 31). In conclusion, this protocol provides a comprehensive diagnostic approach which could be carried out in Colombia and made it available to medical services spread around the country, enabling the identification of a large number of patients affected by LSDs, which could potentially benefit from the therapeutic tools already available for many of these diseases. PMID:23609959

  9. Motor cortex guides selection of predictable movement targets

    PubMed Central

    Woodgate, Philip J.W.; Strauss, Soeren; Sami, Saber A.; Heinke, Dietmar

    2016-01-01

    The present paper asks whether the motor cortex contributes to prediction-based guidance of target selection. This question was inspired by recent evidence that suggests (i) recurrent connections from the motor system into the attentional system may extract movement-relevant perceptual information and (ii) that the motor cortex cannot only generate predictions of the sensory consequences of movements but may also operate as predictor of perceptual events in general. To test this idea we employed a choice reaching task requiring participants to rapidly reach and touch a predictable or unpredictable colour target. Motor cortex activity was modulated via transcranial direct current stimulation (tDCS). In Experiment 1 target colour repetitions were predictable. Under such conditions anodal tDCS facilitated selection versus sham and cathodal tDCS. This improvement was apparent for trajectory curvature but not movement initiation. Conversely, where no predictability of colour was embedded reach performance was unaffected by tDCS. Finally, the results of a key-press experiment suggested that motor cortex involvement is restricted to tasks where the predictable target colour is movement-relevant. The outcomes are interpreted as evidence that the motor system contributes to the top-down guidance of selective attention to movement targets. PMID:25835319

  10. Neuroinflammatory paradigms in lysosomal storage diseases

    PubMed Central

    Bosch, Megan E.; Kielian, Tammy

    2015-01-01

    Lysosomal storage diseases (LSDs) include approximately 70 distinct disorders that collectively account for 14% of all inherited metabolic diseases. LSDs are caused by mutations in various enzymes/proteins that disrupt lysosomal function, which impairs macromolecule degradation following endosome-lysosome and phagosome-lysosome fusion and autophagy, ultimately disrupting cellular homeostasis. LSDs are pathologically typified by lysosomal inclusions composed of a heterogeneous mixture of various proteins and lipids that can be found throughout the body. However, in many cases the CNS is dramatically affected, which may result from heightened neuronal vulnerability based on their post-mitotic state. Besides intrinsic neuronal defects, another emerging factor common to many LSDs is neuroinflammation, which may negatively impact neuronal survival and contribute to neurodegeneration. Microglial and astrocyte activation is a hallmark of many LSDs that affect the CNS, which often precedes and predicts regions where eventual neuron loss will occur. However, the timing, intensity, and duration of neuroinflammation may ultimately dictate the impact on CNS homeostasis. For example, a transient inflammatory response following CNS insult/injury can be neuroprotective, as glial cells attempt to remove the insult and provide trophic support to neurons. However, chronic inflammation, as seen in several LSDs, can promote neurodegeneration by creating a neurotoxic environment due to elevated levels of cytokines, chemokines, and pro-apoptotic molecules. Although neuroinflammation has been reported in several LSDs, the cellular basis and mechanisms responsible for eliciting neuroinflammatory pathways are just beginning to be defined. This review highlights the role of neuroinflammation in select LSDs and its potential contribution to neuron loss. PMID:26578874

  11. Integrative analysis to select cancer candidate biomarkers to targeted validation.

    PubMed

    Kawahara, Rebeca; Meirelles, Gabriela V; Heberle, Henry; Domingues, Romênia R; Granato, Daniela C; Yokoo, Sami; Canevarolo, Rafael R; Winck, Flavia V; Ribeiro, Ana Carolina P; Brandão, Thaís Bianca; Filgueiras, Paulo R; Cruz, Karen S P; Barbuto, José Alexandre; Poppi, Ronei J; Minghim, Rosane; Telles, Guilherme P; Fonseca, Felipe Paiva; Fox, Jay W; Santos-Silva, Alan R; Coletta, Ricardo D; Sherman, Nicholas E; Paes Leme, Adriana F

    2015-12-22

    Targeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differentially abundant between the tumor classes. We further tested the strength of the pipeline in selecting candidate biomarkers by immunoblotting, human tissue microarrays, label-free targeted MS and functional experiments. In conclusion, the proposed integrative analysis was able to pre-qualify and prioritize candidate biomarkers from discovery-based proteomics to targeted MS. PMID:26540631

  12. Integrative analysis to select cancer candidate biomarkers to targeted validation

    PubMed Central

    Heberle, Henry; Domingues, Romênia R.; Granato, Daniela C.; Yokoo, Sami; Canevarolo, Rafael R.; Winck, Flavia V.; Ribeiro, Ana Carolina P.; Brandão, Thaís Bianca; Filgueiras, Paulo R.; Cruz, Karen S. P.; Barbuto, José Alexandre; Poppi, Ronei J.; Minghim, Rosane; Telles, Guilherme P.; Fonseca, Felipe Paiva; Fox, Jay W.; Santos-Silva, Alan R.; Coletta, Ricardo D.; Sherman, Nicholas E.; Paes Leme, Adriana F.

    2015-01-01

    Targeted proteomics has flourished as the method of choice for prospecting for and validating potential candidate biomarkers in many diseases. However, challenges still remain due to the lack of standardized routines that can prioritize a limited number of proteins to be further validated in human samples. To help researchers identify candidate biomarkers that best characterize their samples under study, a well-designed integrative analysis pipeline, comprising MS-based discovery, feature selection methods, clustering techniques, bioinformatic analyses and targeted approaches was performed using discovery-based proteomic data from the secretomes of three classes of human cell lines (carcinoma, melanoma and non-cancerous). Three feature selection algorithms, namely, Beta-binomial, Nearest Shrunken Centroids (NSC), and Support Vector Machine-Recursive Features Elimination (SVM-RFE), indicated a panel of 137 candidate biomarkers for carcinoma and 271 for melanoma, which were differentially abundant between the tumor classes. We further tested the strength of the pipeline in selecting candidate biomarkers by immunoblotting, human tissue microarrays, label-free targeted MS and functional experiments. In conclusion, the proposed integrative analysis was able to pre-qualify and prioritize candidate biomarkers from discovery-based proteomics to targeted MS. PMID:26540631

  13. The HYPER-MUCHFUSS project—target selection and analysis

    NASA Astrophysics Data System (ADS)

    Tillich, A.; Geier, S.; Heber, U.; Hirsch, H.; Maxted, P. F. L.; Marsh, T.; Gänsicke, B.; Napiwotzki, R.; Østensen, R.; Scholz, R.-D.

    2010-10-01

    The HYPER-MUCHFUSS project targets a population of high velocity subluminous B stars to discover either close binaries with massive unseen companions or hyper-velocity stars. Our starting point is the enormous database of SDSS. We preselected sdO/B candidates by colour and classified them by visual inspection of their spectra. We measured the radial velocity from the coadded SDSS spectra, which serves as first epoch measurement. Stars with high Galactic rest-frame velocities were selected and second epoch observations were obtained starting in 2007 at several sites. For the brighter targets we also included the SDSS individual spectra as additional information. In the course of our survey we observed 88 out of 265 stars from our target list. We discovered 39 HVS candidates as well as 49 close binaries. In addition we analysed all single spectra of sdBs from SDSS and found 120 close binaries. For the targets with constant RVs we performed a proper motion analysis with the highest possible accuracy from the available digitised photographic plates. Together with the analysed spectra and the calculation of the spectroscopic distance, we calculated complete trajectories and deduced the origins of these stars. Targets with high RV variability on short timescales were selected for follow-up. Numerical simulations based on the period and companion mass distribution of the known sdB binary sample were carried out to optimise the target selection and single out candidate binaries with massive companions. The follow-up campaign using WHT/ISIS and CAHA-3.5m/TWIN started in 2009.

  14. Target Selection for the SDSS-III MARVELS Survey

    NASA Astrophysics Data System (ADS)

    Paegert, Martin; Stassun, Keivan G.; De Lee, Nathan; Pepper, Joshua; Fleming, Scott W.; Sivarani, Thirupathi; Mahadevan, Suvrath; Mack, Claude E., III; Dhital, Saurav; Hebb, Leslie; Ge, Jian

    2015-06-01

    We present the target selection process for the Multi-object APO Radial Velocity Exoplanets Large-area Survey (MARVELS), which is part of the Sloan Digital Sky Survey (SDSS) III. MARVELS is a medium-resolution (R ∼ 11,000) multi-fiber spectrograph capable of obtaining radial velocities for 60 objects at a time in order to find brown dwarfs and giant planets. The survey was configured to target dwarf stars with effective temperatures approximately between 4500 and 6250 K. For the first 2 years MARVELS relied on low-resolution spectroscopic pre-observations to estimate the effective temperature and log (g) for candidate stars and then selected suitable dwarf stars from this pool. Ultimately, the pre-observation spectra proved ineffective at filtering out giant stars; many giants were incorrectly classified as dwarfs, resulting in a giant contamination rate of ∼30% for the first phase of the MARVELS survey. Thereafter, the survey instead applied a reduced proper motion cut to eliminate giants and used the Infrared Flux Method to estimate effective temperatures, using only extant photmetric and proper-motion catalog information. The target selection method introduced here may be useful for other surveys that need to rely on extant catalog data for selection of specific stellar populations.

  15. Highly selective luminescent nanostructures for mitochondrial imaging and targeting.

    PubMed

    Fanizza, E; Iacobazzi, R M; Laquintana, V; Valente, G; Caliandro, G; Striccoli, M; Agostiano, A; Cutrignelli, A; Lopedota, A; Curri, M L; Franco, M; Depalo, N; Denora, N

    2016-02-14

    Here a luminescent hybrid nanostructure based on functionalized quantum dots (QDs) is used as a fluorescent imaging agent able to target selectively mitochondria thanks to the molecular recognition of the translocator protein (TSPO). The selective targeting of such an 18 kDa protein mainly located in the outer mitochondrial membrane and overexpressed in several pathological states including neurodegenerative diseases and cancers may provide valuable information for the early diagnosis and therapy of human disorders. In particular, the rational design of amino functionalized luminescent silica coated QD nanoparticles (QD@SiO2 NPs) provides a versatile nanoplatform to anchor a potent and selective TSPO ligand, characterized by a 2-phenyl-imidazo[1,2-a]pyridine acetamide structure along with a derivatizable carboxylic end group, useful to conjugate the TSPO ligand and achieve TSPO-QD@SiO2 NPs by means of a covalent amide bond. The colloidal stability and optical properties of the proposed nanomaterials are comprehensively investigated and their potential as mitochondrial imaging agents is fully assessed. Sub-cellular fractionation, together with confocal laser scanning fluorescence microscopy and co-localization analysis of targeted TSPO-QD@SiO2 NPs in C6 glioma cells overexpressing the TSPO, proves the great potential of these multifunctional nanosystems as in vitro selective mitochondrial imaging agents. PMID:26763470

  16. Acoustic gaze adjustments during active target selection in echolocating porpoises.

    PubMed

    Wisniewska, Danuta Maria; Johnson, Mark; Beedholm, Kristian; Wahlberg, Magnus; Madsen, Peter Teglberg

    2012-12-15

    Visually dominant animals use gaze adjustments to organize perceptual inputs for cognitive processing. Thereby they manage the massive sensory load from complex and noisy scenes. Echolocation, as an active sensory system, may provide more opportunities to control such information flow by adjusting the properties of the sound source. However, most studies of toothed whale echolocation have involved stationed animals in static auditory scenes for which dynamic information control is unnecessary. To mimic conditions in the wild, we designed an experiment with captive, free-swimming harbor porpoises tasked with discriminating between two hydrophone-equipped targets and closing in on the selected target; this allowed us to gain insight into how porpoises adjust their acoustic gaze in a multi-target dynamic scene. By means of synchronized cameras, an acoustic tag and on-target hydrophone recordings we demonstrate that porpoises employ both beam direction control and range-dependent changes in output levels and pulse intervals to accommodate their changing spatial relationship with objects of immediate interest. We further show that, when switching attention to another target, porpoises can set their depth of gaze accurately for the new target location. In combination, these observations imply that porpoises exert precise vocal-motor control that is tied to spatial perception akin to visual accommodation. Finally, we demonstrate that at short target ranges porpoises narrow their depth of gaze dramatically by adjusting their output so as to focus on a single target. This suggests that echolocating porpoises switch from a deliberative mode of sensorimotor operation to a reactive mode when they are close to a target. PMID:23175527

  17. TMEPAI increases lysosome stability and promotes autophagy.

    PubMed

    Luo, Shenheng; Yang, Meng; Lv, Dan; Jing, Lei; Li, Yuyin; Liu, Zhenxing; Diao, Aipo

    2016-07-01

    Autophagy is emerging as a critical response of normal and cancer cells to environmental changes and plays an important role in cell metabolism and maintenance of damaged organelles. Transmembrane prostate androgen-induced protein (TMEPAI) is a pro-tumorigenic factor with high expression in tumor cells. In this study, we showed that depletion of TMEPAI leads to lysosomal labilization and inhibits autophagy. Further study showed that the inhibition of autophagy induced by the depletion of TMEPAI is involved in regulation of Beclin-1. Depletion of TMEPAI increases the sensitivity of cancer cells to chemotherapeutic drugs. Our study reveals the role of TMEPAI in promoting lysosome stability and autophagy, which might be used as a target for cancer chemotherapeutic treatment. PMID:27163528

  18. Identification and Characterization of Pharmacological Chaperones to Correct Enzyme Deficiencies in Lysosomal Storage Disorders

    PubMed Central

    Khanna, Richie; Powe, Allan C.; Boyd, Robert; Lee, Gary; Flanagan, John J.; Benjamin, Elfrida R.

    2011-01-01

    Abstract Many human diseases result from mutations in specific genes. Once translated, the resulting aberrant proteins may be functionally competent and produced at near-normal levels. However, because of the mutations, the proteins are recognized by the quality control system of the endoplasmic reticulum and are not processed or trafficked correctly, ultimately leading to cellular dysfunction and disease. Pharmacological chaperones (PCs) are small molecules designed to mitigate this problem by selectively binding and stabilizing their target protein, thus reducing premature degradation, facilitating intracellular trafficking, and increasing cellular activity. Partial or complete restoration of normal function by PCs has been shown for numerous types of mutant proteins, including secreted proteins, transcription factors, ion channels, G protein-coupled receptors, and, importantly, lysosomal enzymes. Collectively, lysosomal storage disorders (LSDs) result from genetic mutations in the genes that encode specific lysosomal enzymes, leading to a deficiency in essential enzymatic activity and cellular accumulation of the respective substrate. To date, over 50 different LSDs have been identified, several of which are treated clinically with enzyme replacement therapy or substrate reduction therapy, although insufficiently in some cases. Importantly, a wide range of in vitro assays are now available to measure mutant lysosomal enzyme interaction with and stabilization by PCs, as well as subsequent increases in cellular enzyme levels and function. The application of these assays to the identification and characterization of candidate PCs for mutant lysosomal enzymes will be discussed in this review. In addition, considerations for the successful in vivo use and development of PCs to treat LSDs will be discussed. PMID:21612550

  19. Lysosome-associated miniSOG as a photosensitizer for mammalian cells.

    PubMed

    Ryumina, Alina P; Serebrovskaya, Ekaterina O; Staroverov, Dmitry B; Zlobovskaya, Olga A; Shcheglov, Alexander S; Lukyanov, Sergey A; Lukyanov, Konstantin A

    2016-01-01

    Genetically encoded photosensitizers represent a promising optogenetic tool for the induction of light-controlled oxidative stress strictly localized to a selected intracellular compartment. Here we tested the phototoxic effects of the flavin-containing phototoxic protein miniSOG targeted to the cytoplasmic surfaces of late endosomes and lysosomes by fusion with Rab7. In HeLa Kyoto cells stably expressing miniSOG-Rab7, we demonstrated a high level of cell death upon blue-light illumination. Pepstatin A completely abolished phototoxicity of miniSOG-Rab7, showing a key role for cathepsin D in this model. Using a far-red fluorescence sensor for caspase-3, we observed caspase-3 activation during miniSOG-Rab7-mediated cell death. We conclude that upon illumination, miniSOG-Rab7 induces lysosomal membrane permeabilization (LMP) and leakage of cathepsins into the cytosol, resulting in caspase-dependent apoptosis. PMID:27528074

  20. TM7SF1 (GPR137B): a novel lysosome integral membrane protein.

    PubMed

    Gao, Jialin; Xia, Libin; Lu, Meiqing; Zhang, Binhua; Chen, Yueping; Xu, Rang; Wang, Lizhuo

    2012-09-01

    In the previous proteomic study of human placenta, transmembrane 7 superfamily member 1 (TM7SF1) was found enriched in lysosome compartments. TM7SF1 encodes a 399-amino acid protein with a calculated molecular mass of 45 kDa. Bioinformatic analysis of its amino acid sequence showed that it is a multipass transmembrane protein containing a potential dileucine-based lysosomal targeting signal and four putative N-glycosylation sites. By percoll-gradient centrifugation and further subfraction ways, the lysosomal solute and membrane compartments were isolated respectively. Immunoblotting analysis indicated that TM7SF1 was co-fractioned with lysosome associated membrane protein 2 (LAMP2), which was only detected in lysosomal membrane compartments whereas not detected in the solute compartments. Using specific anti-TM7SF1 antibody and double-immunofluorescence with lysosome membrane protein LAMP1 and Lyso-Tracker Red, the colocalisations of endogenous TM7SF1 with lysosome and late endosome markers were demonstrated. All of this indicated that TM7SF1 is an integral lysosome membrane protein. Rat ortholog of TM7SF1 was found to be strongly expressed in heart, liver, kidney and brain while not or low detected in other tissues. In summary, TM7SF1 was a lysosomal integral membrane protein that shows tissue-specific expression. As a G-protein-coupled receptor in lysosome membrane, TM7SF1 was predicted function as signal transduction across lysosome membrane. PMID:22729905

  1. Target-selective tilt aftereffect during texture learning.

    PubMed

    Pinchuk-Yacobi, Noga; Harris, Hila; Sagi, Dov

    2016-07-01

    Sensory adaptation and perceptual learning are two forms of plasticity in the visual system, with some potential overlapping neural mechanisms and functional benefits. However, they have been largely considered in isolation. Here we examined whether extensive perceptual training with oriented textures (texture discrimination task, TDT) induces adaptation tilt aftereffects (TAE). Texture elements were oriented lines at -22.5° (target) and 22.5° (background). Observers were trained in 5 daily sessions on the TDT, with 800-1000trials/session. Thresholds increased within the daily sessions, showing within-session performance deterioration, but decreased between days, showing learning. To evaluate TAE, perceived vertical (0°) was measured prior to and after each daily session using a single line element. The results showed a TAE of ∼1.5° at retinal locations consistently stimulated by the target, but none at locations consistently stimulated by the background texture. Retinal locations equally stimulated by target and background elements showed a significant TAE (∼0.7°), in a direction expected by target-driven sensory adaptation. Moreover, these locations showed increasing TAE persistence with training. Additional experiments with a modified target, in order to have balanced stimulation around the vertical direction in all target locations, confirmed the locality of the task-dependent TAE. The present results support a strong link between perceptual learning and local orientation-selective adaptation leading to TAE; the latter was shown here to be task and experience dependent. PMID:27359043

  2. PDT: loss of autophagic cytoprotection after lysosomal photodamage

    NASA Astrophysics Data System (ADS)

    Kessel, David; Price, Michael

    2012-02-01

    Photodynamic therapy is known to evoke both autophagy and apoptosis. Apoptosis is an irreversible death pathway while autophagy can serve a cytoprotective function. In this study, we examined two photosensitizing agents that target lysosomes, although they differ in the reactive oxygen species (ROS) formed during irradiation. With both agents, the 'shoulder' on the PDT dose-response curve was substantially attenuated, consistent with loss of a cytoprotective pathway. In contrast, this 'shoulder' is commonly observed when PDT targets mitochondria or the ER. We propose that lysosomal targets may offer the possibility of promoting PDT efficacy by eliminating a potentially protective pathway.

  3. Selective targeting of Mycobacterium smegmatis with trehalose-functionalized nanoparticles†

    PubMed Central

    Jayawardana, Kalana W.; Jayawardena, H. Surangi N.; Wijesundera, Samurdhi A.; De Zoysa, Thareendra; Sundhoro, Madanodaya

    2015-01-01

    Silica and iron oxide nanoparticles with sizes ranging from 6 to 40 nm were functionalized with trehalose. The trehalose-conjugated nanoparticles showed strong interactions with Mycobacterium smegmatis (M. smegmatis) and minimal interactions with macrophage (RAW 264.7) or A549 cells. In addition, trehalose-conjugated silica nanoparticles selectively interacted with M. smegmatis on M. smegmatis-treated A549 cells, demonstrating high potential of trehalose in developing targeted therapy for treating mycobacterial infection. PMID:26121049

  4. TARGET SELECTION FOR THE LBTI EXOZODI KEY SCIENCE PROGRAM

    SciTech Connect

    Weinberger, Alycia J.; Bryden, Geoff; Mennesson, Bertrand; Serabyn, Eugene; Kennedy, Grant M.; Wyatt, Mark C.; Roberge, Aki; Danchi, William C.; Stapelfeldt, Karl R.; Defrère, Denis; Hinz, Philip M.; Rieke, George; Bailey, Vanessa P.; Skemer, Andrew J.; Millan-Gabet, Rafael; Haniff, Chris

    2015-02-01

    The Hunt for Observable Signatures of Terrestrial planetary Systems (HOSTS) on the Large Binocular Telescope Interferometer will survey nearby stars for faint emission arising from ∼300 K dust (exozodiacal dust), and aims to determine the exozodiacal dust luminosity function. HOSTS results will enable planning for future space telescopes aimed at direct spectroscopy of habitable zone terrestrial planets, as well as greater understanding of the evolution of exozodiacal disks and planetary systems. We lay out here the considerations that lead to the final HOSTS target list. Our target selection strategy maximizes the ability of the survey to constrain the exozodi luminosity function by selecting a combination of stars selected for suitability as targets of future missions and as sensitive exozodi probes. With a survey of approximately 50 stars, we show that HOSTS can enable an understanding of the statistical distribution of warm dust around various types of stars and is robust to the effects of varying levels of survey sensitivity induced by weather conditions.

  5. Selective Mitochondrial Targeting Exerts Anxiolytic Effects In Vivo.

    PubMed

    Nussbaumer, Markus; Asara, John M; Teplytska, Larysa; Murphy, Michael P; Logan, Angela; Turck, Christoph W; Filiou, Michaela D

    2016-06-01

    Current treatment strategies for anxiety disorders are predominantly symptom-based. However, a third of anxiety patients remain unresponsive to anxiolytics highlighting the need for more effective, mechanism-based therapeutic approaches. We have previously compared high vs low anxiety mice and identified changes in mitochondrial pathways, including oxidative phosphorylation and oxidative stress. In this work, we show that selective pharmacological targeting of these mitochondrial pathways exerts anxiolytic effects in vivo. We treated high anxiety-related behavior (HAB) mice with MitoQ, an antioxidant that selectively targets mitochondria. MitoQ administration resulted in decreased anxiety-related behavior in HAB mice. This anxiolytic effect was specific for high anxiety as MitoQ treatment did not affect the anxiety phenotype of C57BL/6N and DBA/2J mouse strains. We furthermore investigated the molecular underpinnings of the MitoQ-driven anxiolytic effect and found that MitoQ treatment alters the brain metabolome and that the response to MitoQ treatment is characterized by distinct molecular signatures. These results indicate that a mechanism-driven approach based on selective mitochondrial targeting has the potential to attenuate the high anxiety phenotype in vivo, thus paving the way for translational implementation as long-term MitoQ administration is well-tolerated with no reported side effects in mice and humans. PMID:26567514

  6. Autophagy in lysosomal storage disorders

    PubMed Central

    Lieberman, Andrew P.; Puertollano, Rosa; Raben, Nina; Slaugenhaupt, Susan; Walkley, Steven U.; Ballabio, Andrea

    2012-01-01

    Lysosomes are ubiquitous intracellular organelles that have an acidic internal pH, and play crucial roles in cellular clearance. Numerous functions depend on normal lysosomes, including the turnover of cellular constituents, cholesterol homeostasis, downregulation of surface receptors, inactivation of pathogenic organisms, repair of the plasma membrane and bone remodeling. Lysosomal storage disorders (LSDs) are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. As a consequence, many tissues and organ systems are affected, including brain, viscera, bone and cartilage. The progressive nature of phenotype development is one of the hallmarks of LSDs. In recent years biochemical and cell biology studies of LSDs have revealed an ample spectrum of abnormalities in a variety of cellular functions. These include defects in signaling pathways, calcium homeostasis, lipid biosynthesis and degradation and intracellular trafficking. Lysosomes also play a fundamental role in the autophagic pathway by fusing with autophagosomes and digesting their content. Considering the highly integrated function of lysosomes and autophagosomes it was reasonable to expect that lysosomal storage in LSDs would have an impact upon autophagy. The goal of this review is to provide readers with an overview of recent findings that have been obtained through analysis of the autophagic pathway in several types of LSDs, supporting the idea that LSDs could be seen primarily as “autophagy disorders.” PMID:22647656

  7. A Deterministic Approach to Active Debris Removal Target Selection

    NASA Astrophysics Data System (ADS)

    Lidtke, A.; Lewis, H.; Armellin, R.

    2014-09-01

    Many decisions, with widespread economic, political and legal consequences, are being considered based on space debris simulations that show that Active Debris Removal (ADR) may be necessary as the concerns about the sustainability of spaceflight are increasing. The debris environment predictions are based on low-accuracy ephemerides and propagators. This raises doubts about the accuracy of those prognoses themselves but also the potential ADR target-lists that are produced. Target selection is considered highly important as removal of many objects will increase the overall mission cost. Selecting the most-likely candidates as soon as possible would be desirable as it would enable accurate mission design and allow thorough evaluation of in-orbit validations, which are likely to occur in the near-future, before any large investments are made and implementations realized. One of the primary factors that should be used in ADR target selection is the accumulated collision probability of every object. A conjunction detection algorithm, based on the smart sieve method, has been developed. Another algorithm is then applied to the found conjunctions to compute the maximum and true probabilities of collisions taking place. The entire framework has been verified against the Conjunction Analysis Tools in AGIs Systems Toolkit and relative probability error smaller than 1.5% has been achieved in the final maximum collision probability. Two target-lists are produced based on the ranking of the objects according to the probability they will take part in any collision over the simulated time window. These probabilities are computed using the maximum probability approach, that is time-invariant, and estimates of the true collision probability that were computed with covariance information. The top-priority targets are compared, and the impacts of the data accuracy and its decay are highlighted. General conclusions regarding the importance of Space Surveillance and Tracking for the

  8. The SAMI Galaxy Survey: instrument specification and target selection

    NASA Astrophysics Data System (ADS)

    Bryant, J. J.; Owers, M. S.; Robotham, A. S. G.; Croom, S. M.; Driver, S. P.; Drinkwater, M. J.; Lorente, N. P. F.; Cortese, L.; Scott, N.; Colless, M.; Schaefer, A.; Taylor, E. N.; Konstantopoulos, I. S.; Allen, J. T.; Baldry, I.; Barnes, L.; Bauer, A. E.; Bland-Hawthorn, J.; Bloom, J. V.; Brooks, A. M.; Brough, S.; Cecil, G.; Couch, W.; Croton, D.; Davies, R.; Ellis, S.; Fogarty, L. M. R.; Foster, C.; Glazebrook, K.; Goodwin, M.; Green, A.; Gunawardhana, M. L.; Hampton, E.; Ho, I.-T.; Hopkins, A. M.; Kewley, L.; Lawrence, J. S.; Leon-Saval, S. G.; Leslie, S.; McElroy, R.; Lewis, G.; Liske, J.; López-Sánchez, Á. R.; Mahajan, S.; Medling, A. M.; Metcalfe, N.; Meyer, M.; Mould, J.; Obreschkow, D.; O'Toole, S.; Pracy, M.; Richards, S. N.; Shanks, T.; Sharp, R.; Sweet, S. M.; Thomas, A. D.; Tonini, C.; Walcher, C. J.

    2015-03-01

    The SAMI Galaxy Survey will observe 3400 galaxies with the Sydney-AAO Multi-object Integral-field spectrograph (SAMI) on the Anglo-Australian Telescope in a 3-yr survey which began in 2013. We present the throughput of the SAMI system, the science basis and specifications for the target selection, the survey observation plan and the combined properties of the selected galaxies. The survey includes four volume-limited galaxy samples based on cuts in a proxy for stellar mass, along with low-stellar-mass dwarf galaxies all selected from the Galaxy And Mass Assembly (GAMA) survey. The GAMA regions were selected because of the vast array of ancillary data available, including ultraviolet through to radio bands. These fields are on the celestial equator at 9, 12 and 14.5 h, and cover a total of 144 deg2 (in GAMA-I). Higher density environments are also included with the addition of eight clusters. The clusters have spectroscopy from 2-degree Field Galaxy Redshift Survey (2dFGRS) and Sloan Digital Sky Survey (SDSS) and photometry in regions covered by the SDSS and/or VLT Survey Telescope/ATLAS. The aim is to cover a broad range in stellar mass and environment, and therefore the primary survey targets cover redshifts 0.004 < z < 0.095, magnitudes rpet < 19.4, stellar masses 107-1012 M⊙, and environments from isolated field galaxies through groups to clusters of ˜1015 M⊙.

  9. Targets of Balancing Selection in the Human Genome

    PubMed Central

    Hubisz, Melissa J.; Indap, Amit; Torgerson, Dara G.; Degenhardt, Jeremiah D.; Boyko, Adam R.; Gutenkunst, Ryan N.; White, Thomas J.; Green, Eric D.; Bustamante, Carlos D.; Clark, Andrew G.; Nielsen, Rasmus

    2009-01-01

    Balancing selection is potentially an important biological force for maintaining advantageous genetic diversity in populations, including variation that is responsible for long-term adaptation to the environment. By serving as a means to maintain genetic variation, it may be particularly relevant to maintaining phenotypic variation in natural populations. Nevertheless, its prevalence and specific targets in the human genome remain largely unknown. We have analyzed the patterns of diversity and divergence of 13,400 genes in two human populations using an unbiased single-nucleotide polymorphism data set, a genome-wide approach, and a method that incorporates demography in neutrality tests. We identified an unbiased catalog of genes with signatures of long-term balancing selection, which includes immunity genes as well as genes encoding keratins and membrane channels; the catalog also shows enrichment in functional categories involved in cellular structure. Patterns are mostly concordant in the two populations, with a small fraction of genes showing population-specific signatures of selection. Power considerations indicate that our findings represent a subset of all targets in the genome, suggesting that although balancing selection may not have an obvious impact on a large proportion of human genes, it is a key force affecting the evolution of a number of genes in humans. PMID:19713326

  10. Selection of molecular targets for drug development against trypanosomatids.

    PubMed

    Smirlis, Despina; Soares, Milena Botelho Pereira

    2014-01-01

    Trypanosomatid parasites are a group of flagellated protozoa that includes the genera Leishmania and Trypanosoma, which are the causative agents of diseases (leishmaniases, sleeping sickness and Chagas disease) that cause considerable morbidity and mortality, affecting more than 27 million people worldwide. Today no effective vaccines for the prevention of these diseases exist, whereas current chemotherapy is ineffective, mainly due to toxic side effects of current drugs and to the emergence of drug resistance and lack of cost effectiveness. For these reasons, rational drug design and the search of good candidate drug targets is of prime importance. The search for drug targets requires a multidisciplinary approach. To this end, the completion of the genome project of many trypanosomatid species gives a vast amount of new information that can be exploited for the identification of good drug candidates with a prediction of "druggability" and divergence from mammalian host proteins. In addition, an important aspect in the search for good drug targets is the "target identification" and evaluation in a biological pathway, as well as the essentiality of the gene in the mammalian stage of the parasite, which is provided by basic research and genetic and proteomic approaches. In this chapter we will discuss how these bioinformatic tools and experimental evaluations can be integrated for the selection of candidate drug targets, and give examples of metabolic and signaling pathways in the parasitic protozoa that can be exploited for rational drug design. PMID:24264240

  11. A novel inhibitor of vacuolar ATPase, FR167356, which can discriminate between osteoclast vacuolar ATPase and lysosomal vacuolar ATPase

    PubMed Central

    Niikura, Kazuaki; Takano, Mikiko; Sawada, Masae

    2004-01-01

    Vacuolar ATPase (V-ATPase) has been proposed as a drug target in lytic bone diseases. Studies of bafilomycin derivatives suggest that the key issue regarding the therapeutic usefulness of V-ATPase inhibitors is selective inhibition of osteoclast V-ATPase. Previous efforts to develop therapeutic inhibitors of osteoclast V-ATPase have been frustrated by a lack of synthetically tractable and biologically selective leads. Therefore, we tried to find novel potent and specific V-ATPase inhibitors, which have new structural features and inhibition selectivity, from random screening using osteoclast microsomes. Finally, a novel V-ATPase inhibitor, FR167356, was obtained through chemical modification of a parental hit compound. FR167356 inhibited not only H+ transport activity of osteoclast V-ATPase but also H+ extrusion from cytoplasm of osteoclasts, which depends on the V-ATPase activity. As expected, FR167356 remarkably inhibited bone resorption in vitro. FR167356 also showed inhibitory effects on other V-ATPases, renal brush border V-ATPase, macrophage microsome V-ATPase and lysosomal V-ATPase. However, FR167356 was approximately seven-fold less potent in inhibiting lysosomal V-ATPase compared to osteoclast V-ATPase. Moreover, LDL metabolism in cells, which depends on acidification of lysosome, was blocked merely at higher concentration than bone resorption, suggesting that FR167356 inhibits V-ATPase of osteoclast ruffled border membrane still more selectively than lysosome at the cellular level. These results from the experiments seem to indicate that osteoclast V-ATPase may be different from lysosomal V-ATPase in respect of their structure. FR167356 had a novel chemical structural feature as well as inhibitory characteristics distinctly different from any previously known V-ATPase inhibitor family. Therefore, FR167356 is thought to be a useful tool for estimating the essential characteristics of V-ATPase inhibitors for drug development. PMID:15148249

  12. Context-dependent sequential effects of target selection for action

    PubMed Central

    Moher, Jeff; Song, Joo-Hyun

    2012-01-01

    Humans exhibit variation in behavior from moment to moment even when performing a simple, repetitive task. Errors are typically followed by cautious responses, minimizing subsequent distractor interference. However, less is known about how variation in the execution of an ultimately correct response affects subsequent behavior. We asked participants to reach toward a uniquely colored target presented among distractors and created two categories to describe participants' responses in correct trials based on analyses of movement trajectories; partial errors referred to trials in which observers initially selected a nontarget for action before redirecting the movement and accurately pointing to the target, and direct movements referred to trials in which the target was directly selected for action. We found that latency to initiate a hand movement was shorter in trials following partial errors compared to trials following direct movements. Furthermore, when the target and distractor colors were repeated, movement time and reach movement curvature toward distractors were greater following partial errors compared to direct movements. Finally, when the colors were repeated, partial errors were more frequent than direct movements following partial-error trials, and direct movements were more frequent following direct-movement trials. The dependence of these latter effects on repeated-task context indicates the involvement of higher-level cognitive mechanisms in an integrated attention-action system in which execution of a partial-error or direct-movement response affects memory representations that bias performance in subsequent trials. Altogether, these results demonstrate that whether a nontarget is selected for action or not has a measurable impact on subsequent behavior. PMID:23847303

  13. X-linked Angelman-like syndrome caused by Slc9a6 knockout in mice exhibits evidence of endosomal-lysosomal dysfunction.

    PubMed

    Strømme, Petter; Dobrenis, Kostantin; Sillitoe, Roy V; Gulinello, Maria; Ali, Nafeeza F; Davidson, Cristin; Micsenyi, Matthew C; Stephney, Gloria; Ellevog, Linda; Klungland, Arne; Walkley, Steven U

    2011-11-01

    Mutations in solute carrier family 9 isoform 6 on chromosome Xq26.3 encoding sodium-hydrogen exchanger 6, a protein mainly expressed in early and recycling endosomes are known to cause a complex and slowly progressive degenerative human neurological disease. Three resulting phenotypes have so far been reported: an X-linked Angelman syndrome-like condition, Christianson syndrome and corticobasal degeneration with tau deposition, with each characterized by severe intellectual disability, epilepsy, autistic behaviour and ataxia. Hypothesizing that a sodium-hydrogen exchanger 6 deficiency would most likely disrupt the endosomal-lysosomal system of neurons, we examined Slc9a6 knockout mice with tissue staining and related techniques commonly used to study lysosomal storage disorders. As a result, we found that sodium-hydrogen exchanger 6 depletion leads to abnormal accumulation of GM2 ganglioside and unesterified cholesterol within late endosomes and lysosomes of neurons in selective brain regions, most notably the basolateral nuclei of the amygdala, the CA3 and CA4 regions and dentate gyrus of the hippocampus and some areas of cerebral cortex. In these select neuronal populations, histochemical staining for β-hexosaminidase activity, a lysosomal enzyme involved in the degradation of GM2 ganglioside, was undetectable. Neuroaxonal dystrophy similar to that observed in lysosomal disease was observed in the cerebellum and was accompanied by a marked and progressive loss of Purkinje cells, particularly in those lacking the expression of Zebrin II. On behavioural testing, Slc9a6 knockout mice displayed a discrete clinical phenotype attributable to motor hyperactivity and cerebellar dysfunction. Importantly, these findings show that sodium-hydrogen exchanger 6 loss of function in the Slc9a6-targeted mouse model leads to compromise of endosomal-lysosomal function similar to lysosomal disease and to conspicuous neuronal abnormalities in specific brain regions, which in concert

  14. Highly selective luminescent nanostructures for mitochondrial imaging and targeting

    NASA Astrophysics Data System (ADS)

    Fanizza, E.; Iacobazzi, R. M.; Laquintana, V.; Valente, G.; Caliandro, G.; Striccoli, M.; Agostiano, A.; Cutrignelli, A.; Lopedota, A.; Curri, M. L.; Franco, M.; Depalo, N.; Denora, N.

    2016-02-01

    Here a luminescent hybrid nanostructure based on functionalized quantum dots (QDs) is used as a fluorescent imaging agent able to target selectively mitochondria thanks to the molecular recognition of the translocator protein (TSPO). The selective targeting of such an 18 kDa protein mainly located in the outer mitochondrial membrane and overexpressed in several pathological states including neurodegenerative diseases and cancers may provide valuable information for the early diagnosis and therapy of human disorders. In particular, the rational design of amino functionalized luminescent silica coated QD nanoparticles (QD@SiO2 NPs) provides a versatile nanoplatform to anchor a potent and selective TSPO ligand, characterized by a 2-phenyl-imidazo[1,2-a]pyridine acetamide structure along with a derivatizable carboxylic end group, useful to conjugate the TSPO ligand and achieve TSPO-QD@SiO2 NPs by means of a covalent amide bond. The colloidal stability and optical properties of the proposed nanomaterials are comprehensively investigated and their potential as mitochondrial imaging agents is fully assessed. Sub-cellular fractionation, together with confocal laser scanning fluorescence microscopy and co-localization analysis of targeted TSPO-QD@SiO2 NPs in C6 glioma cells overexpressing the TSPO, proves the great potential of these multifunctional nanosystems as in vitro selective mitochondrial imaging agents.Here a luminescent hybrid nanostructure based on functionalized quantum dots (QDs) is used as a fluorescent imaging agent able to target selectively mitochondria thanks to the molecular recognition of the translocator protein (TSPO). The selective targeting of such an 18 kDa protein mainly located in the outer mitochondrial membrane and overexpressed in several pathological states including neurodegenerative diseases and cancers may provide valuable information for the early diagnosis and therapy of human disorders. In particular, the rational design of amino

  15. Dynamic interactions between visual working memory and saccade target selection.

    PubMed

    Schneegans, Sebastian; Spencer, John P; Schöner, Gregor; Hwang, Seongmin; Hollingworth, Andrew

    2014-01-01

    Recent psychophysical experiments have shown that working memory for visual surface features interacts with saccadic motor planning, even in tasks where the saccade target is unambiguously specified by spatial cues. Specifically, a match between a memorized color and the color of either the designated target or a distractor stimulus influences saccade target selection, saccade amplitudes, and latencies in a systematic fashion. To elucidate these effects, we present a dynamic neural field model in combination with new experimental data. The model captures the neural processes underlying visual perception, working memory, and saccade planning relevant to the psychophysical experiment. It consists of a low-level visual sensory representation that interacts with two separate pathways: a spatial pathway implementing spatial attention and saccade generation, and a surface feature pathway implementing color working memory and feature attention. Due to bidirectional coupling between visual working memory and feature attention in the model, the working memory content can indirectly exert an effect on perceptual processing in the low-level sensory representation. This in turn biases saccadic movement planning in the spatial pathway, allowing the model to quantitatively reproduce the observed interaction effects. The continuous coupling between representations in the model also implies that modulation should be bidirectional, and model simulations provide specific predictions for complementary effects of saccade target selection on visual working memory. These predictions were empirically confirmed in a new experiment: Memory for a sample color was biased toward the color of a task-irrelevant saccade target object, demonstrating the bidirectional coupling between visual working memory and perceptual processing. PMID:25228628

  16. Dynamic interactions between visual working memory and saccade target selection

    PubMed Central

    Schneegans, Sebastian; Spencer, John P.; Schöner, Gregor; Hwang, Seongmin; Hollingworth, Andrew

    2014-01-01

    Recent psychophysical experiments have shown that working memory for visual surface features interacts with saccadic motor planning, even in tasks where the saccade target is unambiguously specified by spatial cues. Specifically, a match between a memorized color and the color of either the designated target or a distractor stimulus influences saccade target selection, saccade amplitudes, and latencies in a systematic fashion. To elucidate these effects, we present a dynamic neural field model in combination with new experimental data. The model captures the neural processes underlying visual perception, working memory, and saccade planning relevant to the psychophysical experiment. It consists of a low-level visual sensory representation that interacts with two separate pathways: a spatial pathway implementing spatial attention and saccade generation, and a surface feature pathway implementing color working memory and feature attention. Due to bidirectional coupling between visual working memory and feature attention in the model, the working memory content can indirectly exert an effect on perceptual processing in the low-level sensory representation. This in turn biases saccadic movement planning in the spatial pathway, allowing the model to quantitatively reproduce the observed interaction effects. The continuous coupling between representations in the model also implies that modulation should be bidirectional, and model simulations provide specific predictions for complementary effects of saccade target selection on visual working memory. These predictions were empirically confirmed in a new experiment: Memory for a sample color was biased toward the color of a task-irrelevant saccade target object, demonstrating the bidirectional coupling between visual working memory and perceptual processing. PMID:25228628

  17. Development of hematin conjugated PLGA nanoparticle for selective cancer targeting.

    PubMed

    Amin, Md Lutful; Kim, Dami; Kim, SeJin

    2016-08-25

    Targeted nanomedicine for cancer therapy has gained widespread popularity and is being extensively explored. Porphyrins have intrinsic tumor localizing ability and have been studied for photodynamic therapy. However, they have not been used as cancer targeting agents for nanomedicines. In this study, PLGA nanoparticles were formulated and an iron-containing blood porphyrin, hematin was conjugated to the surface of the nanoparticles to investigate selectivity towards cancer cell and cellular internalization. Hematin was previously shown to facilitate growth and proliferation of cancer cells. PLGA nanoparticles were characterized by FE-SEM, AFM, DLS, and Zeta potential analyzer. The conjugation of hematin was confirmed by FTIR. HeLa cells were used to study tumor selectivity and uptake. Hematin conjugated particles (ζ potential: -15.19mV) showed higher affinity towards the cancer cells than the control particles. The result indicated that the particles were internalized by heme carrier protein-1. Together these data suggest that hematin is a promising cancer targeting material for nanotherapeutics. PMID:27260086

  18. THINK OUTSIDE THE COLOR BOX: PROBABILISTIC TARGET SELECTION AND THE SDSS-XDQSOQUASAR TARGETING CATALOG

    SciTech Connect

    BOVY, J.; Sheldon, E.; Hennawi, J.F.; Hogg, D.W.; Myers, A.D.; et al.

    2011-03-10

    We present the SDSS-XDQSO quasar targeting catalog for efficient flux-based quasar target selection down to the faint limit of the Sloan Digital Sky Survey (SDSS) catalog, even at medium redshifts (2.5 {approx}< z {approx}< 3) where the stellar contamination is significant. We build models of the distributions of stars and quasars in flux space down to the flux limit by applying the extreme-deconvolution method to estimate the underlying density. We convolve this density with the flux uncertainties when evaluating the probability that an object is a quasar. This approach results in a targeting algorithm that is more principled, more efficient, and faster than other similar methods. We apply the algorithm to derive low-redshift (z < 2.2), medium-redshift (2.2 {le} z {le} 3.5), and high-redshift (z > 3.5) quasar probabilities for all 160,904,060 point sources with dereddened i-band magnitude between 17.75 and 22.45 mag in the 14,555 deg{sup 2} of imaging from SDSS Data Release 8. The catalog can be used to define a uniformly selected and efficient low- or medium-redshift quasar survey, such as that needed for the SDSS-III's Baryon Oscillation Spectroscopic Survey project. We show that the XDQSO technique performs as well as the current best photometric quasar-selection technique at low redshift, and outperforms all other flux-based methods for selecting the medium-redshift quasars of our primary interest. We make code to reproduce the XDQSO quasar target selection publicly available.

  19. Neuropathic Lysosomal Storage Disorders

    PubMed Central

    Pastores, Gregory M.; Maegawa, Gustavo H.B.

    2014-01-01

    The lysosomal storage disorders (LSDs) are a clinically heterogeneous group of inborn errors of metabolism, associated with the accumulation of incompletely degraded macromolecules within several cellular sites. Affected individuals present with a broad range of clinical problems, including hepatosplenomegaly and skeletal dysplasia. Onset of symptoms may range from birth to adulthood. The majority are associated with neurological features, including developmental delay, behavioral/psychiatric disturbances, seizures, acroparesthesia, motor weakness, cerebrovascular ischemic events and extra-pyramidal signs. It should be noted that later-onset forms are often misdiagnosed as symptoms, which might include psychiatric manifestations, are slowly progressive and may precede other neurologic or systemic features. Inheritance is primarily autosomal recessive. For all subtypes, diagnosis can be confirmed using a combination of biochemical and/or molecular assays. In a few LSDs, treatment with either hematopoietic stem cell transplantation, enzyme replacement or substrate reduction therapy is available. Genetic counseling is important, so patients and their families can be informed of reproductive risks, disease prognosis and therapeutic options. Investigations of disease mechanisms are providing insights into potential therapeutic approaches. Symptomatic care, which remains the mainstay for most subtypes, can lead to significant improvement in quality of life. PMID:24176423

  20. SELECTION, PRIORITIZATION, AND CHARACTERISTICS OF KEPLER TARGET STARS

    SciTech Connect

    Batalha, Natalie M.; Borucki, William J.; Koch, David G.; Bryson, Stephen T.; Haas, Michael R.; Brown, Timothy M.; Caldwell, Douglas A.; Hall, Jennifer R.; Latham, David W.; Meibom, Soren; Monet, David G.

    2010-04-20

    The Kepler Mission began its 3.5 year photometric monitoring campaign in 2009 May on a select group of approximately 150,000 stars. The stars were chosen from the {approx} half million in the field of view that are brighter than 16th magnitude. The selection criteria are quantitative metrics designed to optimize the scientific yield of the mission with regard to the detection of Earth-size planets in the habitable zone. This yields more than 90,000 G-type stars on or close to the main sequence, >20, 000 of which are brighter than 14th magnitude. At the temperature extremes, the sample includes approximately 3000 M-type dwarfs and a small sample of O- and B-type MS stars (<200). The small numbers of giants are included in the sample: {approx}5000 stars with surface gravities log(g) < 3.5. We present a brief summary of the selection process and the stellar populations it yields in terms of surface gravity, effective temperature, and apparent magnitude. In addition to the primary, statistically derived target set, several ancillary target lists were manually generated to enhance the science of the mission, examples being: known eclipsing binaries, open cluster members, and high proper motion stars.

  1. Engineering novel cell surface chemistry for selective tumor cell targeting

    SciTech Connect

    Bertozzi, C.R. |

    1997-12-31

    A common feature of many different cancers is the high expression level of the two monosaccharides sialic acid and fucose within the context of cell-surface associated glycoconjugates. A correlation has been made between hypersialylation and/or hyperfucosylation and the highly metastatic phenotype. Thus, a targeting strategy based on sialic acid or fucose expression would be a powerful tool for the development of new cancer cell-selective therapies and diagnostic agents. We have discovered that ketone groups can be incorporated metabolically into cell-surface associated sialic acids. The ketone is can be covalently ligated with hydrazide functionalized proteins or small molecules under physiological conditions. Thus, we have discovered a mechanism to selectively target hydrazide conjugates to highly sialylated cells such as cancer cells. Applications of this technology to the generation of novel cancer cell-selective toxins and MRI contrast reagents will be discussed, in addition to progress towards the use of cell surface fucose residues as vehicles for ketone expression.

  2. Selective targeting of the stress chaperome as a therapeutic strategy

    PubMed Central

    Taldone, Tony; Ochiana, Stefan O.; Patel, Pallav D.; Chiosis, Gabriela

    2014-01-01

    Normal cellular function is maintained by coordinated proteome machinery that performs a vast array of activities. Helping the proteome in such roles is the chaperome, a network of molecular chaperones and folding enzymes. The stressed cell contains, at any time, a complex mixture of chaperome complexes; a majority performs “housekeeping functions” similarly to non-stressed, normal cells, but a finely-tuned fraction buffers the proteome altered by chronic stress. The stress chaperome is epigenetically distinct from its normal, housekeeping counterpart, providing a basis for its selective targeting by small molecules. Here we discuss development of chaperome inhibitors, and how agents targeting chaperome members in stressed cells are in fact being directed towards chaperome complexes and their effect is therefore determined by their ability to sample and engage such complexes. A new approach is needed to target and implement chaperome modulators in the investigation of diseases, and we propose that the classical thinking in drug discovery needs adjustment when developing chaperome-targeting drugs. PMID:25262919

  3. Autophagosome-lysosome fusion triggers a lysosomal response mediated by TLR9 and controlled by OCRL.

    PubMed

    De Leo, Maria Giovanna; Staiano, Leopoldo; Vicinanza, Mariella; Luciani, Alessandro; Carissimo, Annamaria; Mutarelli, Margherita; Di Campli, Antonella; Polishchuk, Elena; Di Tullio, Giuseppe; Morra, Valentina; Levtchenko, Elena; Oltrabella, Francesca; Starborg, Tobias; Santoro, Michele; di Bernardo, Diego; Devuyst, Olivier; Lowe, Martin; Medina, Diego L; Ballabio, Andrea; De Matteis, Maria Antonietta

    2016-08-01

    Phosphoinositides (PtdIns) control fundamental cell processes, and inherited defects of PtdIns kinases or phosphatases cause severe human diseases, including Lowe syndrome due to mutations in OCRL, which encodes a PtdIns(4,5)P2 5-phosphatase. Here we unveil a lysosomal response to the arrival of autophagosomal cargo in which OCRL plays a key part. We identify mitochondrial DNA and TLR9 as the cargo and the receptor that triggers and mediates, respectively, this response. This lysosome-cargo response is required to sustain the autophagic flux and involves a local increase in PtdIns(4,5)P2 that is confined in space and time by OCRL. Depleting or inhibiting OCRL leads to an accumulation of lysosomal PtdIns(4,5)P2, an inhibitor of the calcium channel mucolipin-1 that controls autophagosome-lysosome fusion. Hence, autophagosomes accumulate in OCRL-depleted cells and in the kidneys of Lowe syndrome patients. Importantly, boosting the activity of mucolipin-1 with selective agonists restores the autophagic flux in cells from Lowe syndrome patients. PMID:27398910

  4. Selective follicular targeting by modification of the particle sizes.

    PubMed

    Patzelt, Alexa; Richter, Heike; Knorr, Fanny; Schäfer, Ulrich; Lehr, Claus-Michael; Dähne, Lars; Sterry, Wolfram; Lademann, Juergen

    2011-02-28

    Hair follicles represent interesting target sites for topically applied substances such as topical vaccinations or agents used in the field of regenerative medicine. In recent years, it could be shown that particles penetrate very effectively into the hair follicles. In the present study, the influence of particle size on the follicular penetration depths was examined. The penetration depths of two different types of particles sized 122 to 1000 nm were determined in vitro on porcine skin. The results revealed that the particles of medium size (643 and 646 nm, respectively) penetrated deeper into the porcine hair follicles than smaller or larger particles. It was concluded that by varying the particle size, different sites within the porcine hair follicle can be targeted selectively. For the human terminal hair follicle, the situation can be expected to be similar due to a similar size ratio of the hair follicles. PMID:21087645

  5. Wavelength band selection method for multispectral target detection.

    PubMed

    Karlholm, Jörgen; Renhorn, Ingmar

    2002-11-10

    A framework is proposed for the selection of wavelength bands for multispectral sensors by use of hyperspectral reference data. Using the results from the detection theory we derive a cost function that is minimized by a set of spectral bands optimal in terms of detection performance for discrimination between a class of small rare targets and clutter with known spectral distribution. The method may be used, e.g., in the design of multispectral infrared search and track and electro-optical missile warning sensors, where a low false-alarm rate and a high-detection probability for detection of small targets against a clutter background are of critical importance, but the required high frame rate prevents the use of hyperspectral sensors. PMID:12440532

  6. Target Search & Selection for the DI/EPOXI Spacecraft

    NASA Technical Reports Server (NTRS)

    Grebow, Daniel J.; Bhaskaran, Shyam; Chesley, Steven R.

    2012-01-01

    Upon completion of the Hartley 2 flyby in November 2010, the Deep Impact (DI) spacecraft resided in a solar orbit without possibility for gravity assist with any large body. Conservative estimates of remaining fuel were enough to provide only an 18 m/s impulse on the spacecraft. We present our method and results of our systematic scan of potential small body encounters for DI, and our criteria to narrow the selection to the asteroid 2002 GT as the target flyby body. The mission profile has two deterministic maneuvers to achieve the encounter, the first of which executed on November 25, 2011.

  7. Target Search and Selection for the DI/EPOXI Spacecraft

    NASA Technical Reports Server (NTRS)

    Grebow, Daniel J.; Bhaskaran, Shyam; Chesley, Steven R.

    2012-01-01

    Upon completion of the Hartley 2 flyby in November 2010, the Deep Impact (DI) spacecraft resided in a solar orbit without possibility for gravity assist with any large body. Conservative estimates of remaining fuel were enough to provide only an 18 m/s impulse on the spacecraft. We present our method and results of our systematic scan of potential small body encounters for DI, and our criteria to narrow the selection to the asteroid 2002 GT as the target flyby body. The mission profile has two deterministic maneuvers to achieve the encounter, the first of which executed on November 25, 2011.

  8. Oncotripsy: Targeting cancer cells selectively via resonant harmonic excitation

    NASA Astrophysics Data System (ADS)

    Heyden, S.; Ortiz, M.

    2016-07-01

    We investigate a method of selectively targeting cancer cells by means of ultrasound harmonic excitation at their resonance frequency, which we refer to as oncotripsy. The geometric model of the cells takes into account the cytoplasm, nucleus and nucleolus, as well as the plasma membrane and nuclear envelope. Material properties are varied within a pathophysiologically-relevant range. A first modal analysis reveals the existence of a spectral gap between the natural frequencies and, most importantly, resonant growth rates of healthy and cancerous cells. The results of the modal analysis are verified by simulating the fully-nonlinear transient response of healthy and cancerous cells at resonance. The fully nonlinear analysis confirms that cancerous cells can be selectively taken to lysis by the application of carefully tuned ultrasound harmonic excitation while simultaneously leaving healthy cells intact.

  9. Distinct cathepsins control necrotic cell death mediated by pyroptosis inducers and lysosome-destabilizing agents.

    PubMed

    Brojatsch, Jürgen; Lima, Heriberto; Palliser, Deborah; Jacobson, Lee S; Muehlbauer, Stefan M; Furtado, Raquel; Goldman, David L; Lisanti, Michael P; Chandran, Kartik

    2015-01-01

    Necrotic cell death triggers a range of biological responses including a strong adaptive immune response, yet we know little about the cellular pathways that control necrotic cell death. Inhibitor studies suggest that proteases, and in particular cathepsins, drive necrotic cell death. The cathepsin B-selective inhibitor CA-074-Me blocks all forms of programmed necrosis by an unknown mechanism. We found that cathepsin B deficiency does not prevent induction of pyroptosis and lysosome-mediated necrosis suggesting that CA-074-Me blocks necrotic cell death by targeting cathepsins other than cathepsin B. A single cathepsin, cathepsin C, drives necrotic cell death mediated by the lysosome-destabilizing agent Leu-Leu-OMe (LLOMe). Here we present evidence that cathepsin C-deficiency and CA-074-Me block LLOMe killing in a distinct and cell type-specific fashion. Cathepsin C-deficiency and CA-074-Me block LLOMe killing of all myeloid cells, except for neutrophils. Cathepsin C-deficiency, but not CA-074-Me, blocks LLOMe killing of neutrophils suggesting that CA-074-Me does not target cathepsin C directly, consistent with inhibitor studies using recombinant cathepsin C. Unlike other cathepsins, cathepsin C lacks endoproteolytic activity, and requires activation by other lysosomal proteases, such as cathepsin D. Consistent with this theory, we found that lysosomotropic agents and cathepsin D downregulation by siRNA block LLOMe-mediated necrosis. Our findings indicate that a proteolytic cascade, involving cathepsins C and D, controls LLOMe-mediated necrosis. In contrast, cathepsins C and D were not required for pyroptotic cell death suggesting that distinct cathepsins control pyroptosis and lysosome-mediated necrosis. PMID:25830414

  10. Measuring Cysteine Cathepsin Activity to Detect Lysosomal Membrane Permeabilization.

    PubMed

    Repnik, Urška; Česen, Maruša Hafner; Turk, Boris

    2016-01-01

    During lysosomal membrane permeabilization (LMP), lysosomal lumenal contents can be released into the cytosol. Small molecules are more likely to be released, and cysteine cathepsins, with mature forms possessing a mass of 25-30 kDa, are among the smallest lumenal lysosomal enzymes. In addition, specific substrates for cysteine cathepsins are available to investigators, and therefore the measurement of the cathepsin activity as a hallmark of LMP works well. Here, we present a protocol for measuring the activity of these enzymes after selective plasma membrane permeabilization with a low concentration of digitonin and after total cell membrane lysis with a high concentration of digitonin. A fluorogenic substrate can be added either directly to the well with lysed cells to show LMP or to the cell-free extract to show that the lysosomal membrane has been sufficiently destabilized to allow the translocation of lysosomal enzymes. Although the content of lysosomal cysteine cathepsins differs between cell lines, this method has general applicability, is sensitive, and has high throughput. The presented protocol shows how to measure cysteine cathepsin activity in the presence of lysed cells and also in cell-free extracts. Depending on the aim of the study, one or both types of measurements can be performed. PMID:27140915

  11. Visual target detection paradigm for the study of selective attention.

    PubMed

    Tabert, M H; Chokron, S; Tang, C Y; Wei, T; Brickman, A M; Buchsbaum, M S

    2000-11-01

    The current protocol can be used to examine selective attention. It has been used to acquire behavioral performance data in neurologically healthy normal control subjects and schizophrenic patients. A modified version, also described here, has been used to acquire functional neuroimaging data in normal subjects using positron emission tomography. Subject response accuracy and reaction times are recorded while subjects detect visual stimuli in either hemifield (left vs. right of a fixation point) or along the vertical meridian (above or below fixation). The lateralized presentation of stimuli permits the study of hemispheric specialization for selective attentional processes. Attentional load is manipulated by presenting larger-sized target stimuli alone (i.e., the letter 'O') or smaller-sized target stimuli surrounded by flanking letters. This protocol report includes a description of subject exclusion criteria, procedural details, relevant experimental conditions and variables, suggestions for data analysis, expected results, and a discussion of the protocol's significance for attentional research along with suggestions for future research. PMID:11086266

  12. Selectively Targeting Prostate Cancer with Antiandrogen Equipped Histone Deacetylase Inhibitors

    PubMed Central

    Gryder, Berkley E.; Akbashev, Michelle J.; Rood, Michael K.; Raftery, Eric D.; Meyers, Warren M.; Dillard, Paulette; Khan, Shafiq; Oyelere, Adegboyega K.

    2013-01-01

    Diverse cellular processes relevant to cancer progression are regulated by the acetylation status of proteins. Among such processes is chromatin remodeling via histone proteins, controlled by opposing histone deacetylase (HDAC) and histone acetyltransferase (HAT) enzymes. Histone deacetylase inhibitors (HDACi) show great promise in preclinical cancer models, but clinical trials treating solid tumors have failed to improve patient survival. This is due in part to an inability of HDACi to effectively accumulate in cancerous cells. To address this problem we designed HDACi with secondary pharmacophores to facilitate selective accumulation in malignant cells. We present the first example of HDACi compounds targeted to prostate tumors by equipping them with the additional ability to bind the androgen receptor (AR) with non-steroidal antiandrogen moieties. Leads among these new dual-acting molecules bind to the AR and halt AR transcriptional activity at lower concentrations than clinical antiandrogens. They inhibit key isoforms of HDAC with low nanomolar potency. Fluorescent microscopy reveals varying degrees of AR nuclear localization in response to these compounds that correlates with their HDAC activity. These biological properties translate into potent anticancer activity against hormone dependent (AR+) LNCaP and to a lesser extent against hormone independent (AR−) DU145 prostate cancer, while having greatly reduced toxicity in non-cancerous cells. This illustrates that engaging multiple biological targets with a single chemical probe can achieve both potent and cell-type selective responses. PMID:24004176

  13. Targeting prion-like protein doppel selectively suppresses tumor angiogenesis

    PubMed Central

    Al-Hilal, Taslim A.; Chung, Seung Woo; Choi, Jeong Uk; Kim, Seong Who; Kim, Sang Yoon; Ahsan, Fakhrul; Kim, In-San

    2016-01-01

    Controlled and site-specific regulation of growth factor signaling remains a major challenge for current antiangiogenic therapies, as these antiangiogenic agents target normal vasculature as well tumor vasculature. In this article, we identified the prion-like protein doppel as a potential therapeutic target for tumor angiogenesis. We investigated the interactions between doppel and VEGFR2 and evaluated whether blocking the doppel/VEGFR2 axis suppresses the process of angiogenesis. We discovered that tumor endothelial cells (TECs), but not normal ECs, express doppel; tumors from patients and mouse xenografts expressed doppel in their vasculatures. Induced doppel overexpression in ECs enhanced vascularization, whereas doppel constitutively colocalized and complexed with VEGFR2 in TECs. Doppel inhibition depleted VEGFR2 from the cell membrane, subsequently inducing the internalization and degradation of VEGFR2 and thereby attenuating VEGFR2 signaling. We also synthesized an orally active glycosaminoglycan (LHbisD4) that specifically binds with doppel. We determined that LHbisD4 concentrates over the tumor site and that genetic loss of doppel in TECs decreases LHbisD4 binding and targeting both in vitro and in vivo. Moreover, LHbisD4 eliminated VEGFR2 from the cell membrane, prevented VEGF binding in TECs, and suppressed tumor growth. Together, our results demonstrate that blocking doppel can control VEGF signaling in TECs and selectively inhibit tumor angiogenesis. PMID:26950422

  14. Landslide susceptibility mapping in three selected target zones in Afghanistan

    NASA Astrophysics Data System (ADS)

    Schwanghart, Wolfgang; Seegers, Joe; Zeilinger, Gerold

    2015-04-01

    In May 2014, a large and mobile landslide destroyed the village Ab Barek, a village in Badakshan Province, Afghanistan. The landslide caused several hundred fatalities and once again demonstrated the vulnerability of Afghanistan's population to extreme natural events following more than 30 years of civil war and violent conflict. Increasing the capacity of Afghanistan's population by strengthening the disaster preparedness and management of responsible government authorities and institutions is thus a major component of international cooperation and development strategies. Afghanistan is characterized by high relief and widely varying rock types that largely determine the spatial distribution as well as emplacement modes of mass movements. The major aim of our study is to characterize this variability by conducting a landslide susceptibility analysis in three selected target zones: Greater Kabul Area, Badakhshan Province and Takhar Province. We expand on an existing landslide database by mapping landforms diagnostic for landslides (e.g. head scarps, normal faults and tension cracks), and historical landslide scars and landslide deposits by visual interpretation of high-resolution satellite imagery. We conduct magnitude frequency analysis within subregional physiogeographic classes based on geological maps, climatological and topographic data to identify regional parameters influencing landslide magnitude and frequency. In addition, we prepare a landslide susceptibility map for each area using the Weight-of-Evidence model. Preliminary results show that the three selected target zones vastly differ in modes of landsliding. Low magnitude but frequent rockfall events are a major hazard in the Greater Kabul Area threatening buildings and infrastructure encroaching steep terrain in the city's outskirts. Mass movements in loess covered areas of Badakshan are characterized by medium to large magnitudes. This spatial variability of characteristic landslide magnitudes and

  15. The Amyloid Precursor Protein is rapidly transported from the Golgi apparatus to the lysosome and where it is processed into beta-amyloid

    PubMed Central

    2014-01-01

    Background Alzheimer’s disease (AD) is characterized by cerebral deposition of β-amyloid peptide (Aβ). Aβ is produced by sequential cleavage of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Many studies have demonstrated that the internalization of APP from the cell surface can regulate Aβ production, although the exact organelle in which Aβ is produced remains contentious. A number of recent studies suggest that intracellular trafficking also plays a role in regulating Aβ production, but these pathways are relatively under-studied. The goal of this study was to elucidate the intracellular trafficking of APP, and to examine the site of intracellular APP processing. Results We have tagged APP on its C-terminal cytoplasmic tail with photoactivatable Green Fluorescent Protein (paGFP). By photoactivating APP-paGFP in the Golgi, using the Golgi marker Galactosyltranferase fused to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to follow a population of nascent APP molecules from the Golgi to downstream compartments identified with compartment markers tagged with red fluorescent protein (mRFP or mCherry); including rab5 (early endosomes) rab9 (late endosomes) and LAMP1 (lysosomes). Because γ-cleavage of APP releases the cytoplasmic tail of APP including the photoactivated GFP, resulting in loss of fluorescence, we are able to visualize the cleavage of APP in these compartments. Using APP-paGFP, we show that APP is rapidly trafficked from the Golgi apparatus to the lysosome; where it is rapidly cleared. Chloroquine and the highly selective γ-secretase inhibitor, L685, 458, cause the accumulation of APP in lysosomes implying that APP is being cleaved by secretases in the lysosome. The Swedish mutation dramatically increases the rate of lysosomal APP processing, which is also inhibited by chloroquine and L685, 458. By knocking down adaptor protein 3 (AP-3; a heterotetrameric protein complex required for trafficking many proteins to

  16. Disruption of chaperone-mediated autophagy-dependent degradation of MEF2A by oxidative stress-induced lysosome destabilization

    PubMed Central

    Zhang, Li; Sun, Yang; Fei, Mingjian; Tan, Cheng; Wu, Jing; Zheng, Jie; Tang, Jiqing; Sun, Wei; Lv, Zhaoliang; Bao, Jiandong; Xu, Qiang; Yu, Huixin

    2014-01-01

    Oxidative stress has been implicated in both normal aging and various neurodegenerative disorders and it may be a major cause of neuronal death. Chaperone-mediated autophagy (CMA) targets selective cytoplasmic proteins for degradation by lysosomes and protects neurons against various extracellular stimuli including oxidative stress. MEF2A (myocyte enhancer factor 2A), a key transcription factor, protects primary neurons from oxidative stress-induced cell damage. However, the precise mechanisms of how the protein stability and the transcriptional activity of MEF2A are regulated under oxidative stress remain unknown. In this study, we report that MEF2A is physiologically degraded through the CMA pathway. In pathological conditions, mild oxidative stress (200 μM H2O2) enhances the degradation of MEF2A as well as its activity, whereas excessive oxidative stress (> 400 μM H2O2) disrupts its degradation process and leads to the accumulation of nonfunctional MEF2A. Under excessive oxidative stress, an N-terminal HDAC4 (histone deacetylase 4) cleavage product (HDAC4-NT), is significantly induced by lysosomal serine proteases released from ruptured lysosomes in a PRKACA (protein kinase, cAMP-dependent, catalytic, α)-independent manner. The production of HDAC4-NT, as a MEF2 repressor, may account for the reduced DNA-binding and transcriptional activity of MEF2A. Our work provides reliable evidence for the first time that MEF2A is targeted to lysosomes for CMA degradation; oxidative stress-induced lysosome destabilization leads to the disruption of MEF2A degradation as well as the dysregulation of its function. These findings may shed light on the underlying mechanisms of pathogenic processes of neuronal damage in various neurodegenerative-related diseases. PMID:24879151

  17. Targeted erythropoietin selectively stimulates red blood cell expansion in vivo.

    PubMed

    Burrill, Devin R; Vernet, Andyna; Collins, James J; Silver, Pamela A; Way, Jeffrey C

    2016-05-10

    The design of cell-targeted protein therapeutics can be informed by natural protein-protein interactions that use cooperative physical contacts to achieve cell type specificity. Here we applied this approach in vivo to the anemia drug erythropoietin (EPO), to direct its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) precursors and prevent interaction with EPO-Rs on nonerythroid cells, such as platelets. Our engineered EPO molecule was mutated to weaken its affinity for EPO-R, but its avidity for RBC precursors was rescued via tethering to an antibody fragment that specifically binds the human RBC marker glycophorin A (huGYPA). We systematically tested the impact of these engineering steps on in vivo markers of efficacy, side effects, and pharmacokinetics. huGYPA transgenic mice dosed with targeted EPO exhibited elevated RBC levels, with only minimal platelet effects. This in vivo selectivity depended on the weakening EPO mutation, fusion to the RBC-specific antibody, and expression of huGYPA. The terminal plasma half-life of targeted EPO was ∼28.3 h in transgenic mice vs. ∼15.5 h in nontransgenic mice, indicating that huGYPA on mature RBCs acted as a significant drug sink but did not inhibit efficacy. In a therapeutic context, our targeting approach may allow higher restorative doses of EPO without platelet-mediated side effects, and also may improve drug pharmacokinetics. These results demonstrate how rational drug design can improve in vivo specificity, with potential application to diverse protein therapeutics. PMID:27114509

  18. Mitochondrial genomes are retained by selective constraints on protein targeting

    PubMed Central

    Björkholm, Patrik; Harish, Ajith; Hagström, Erik; Ernst, Andreas M.; Andersson, Siv G. E.

    2015-01-01

    Mitochondria are energy-producing organelles in eukaryotic cells considered to be of bacterial origin. The mitochondrial genome has evolved under selection for minimization of gene content, yet it is not known why not all mitochondrial genes have been transferred to the nuclear genome. Here, we predict that hydrophobic membrane proteins encoded by the mitochondrial genomes would be recognized by the signal recognition particle and targeted to the endoplasmic reticulum if they were nuclear-encoded and translated in the cytoplasm. Expression of the mitochondrially encoded proteins Cytochrome oxidase subunit 1, Apocytochrome b, and ATP synthase subunit 6 in the cytoplasm of HeLa cells confirms export to the endoplasmic reticulum. To examine the extent to which the mitochondrial proteome is driven by selective constraints within the eukaryotic cell, we investigated the occurrence of mitochondrial protein domains in bacteria and eukaryotes. The accessory protein domains of the oxidative phosphorylation system are unique to mitochondria, indicating the evolution of new protein folds. Most of the identified domains in the accessory proteins of the ribosome are also found in eukaryotic proteins of other functions and locations. Overall, one-third of the protein domains identified in mitochondrial proteins are only rarely found in bacteria. We conclude that the mitochondrial genome has been maintained to ensure the correct localization of highly hydrophobic membrane proteins. Taken together, the results suggest that selective constraints on the eukaryotic cell have played a major role in modulating the evolution of the mitochondrial genome and proteome. PMID:26195779

  19. Mitochondrial genomes are retained by selective constraints on protein targeting.

    PubMed

    Björkholm, Patrik; Harish, Ajith; Hagström, Erik; Ernst, Andreas M; Andersson, Siv G E

    2015-08-18

    Mitochondria are energy-producing organelles in eukaryotic cells considered to be of bacterial origin. The mitochondrial genome has evolved under selection for minimization of gene content, yet it is not known why not all mitochondrial genes have been transferred to the nuclear genome. Here, we predict that hydrophobic membrane proteins encoded by the mitochondrial genomes would be recognized by the signal recognition particle and targeted to the endoplasmic reticulum if they were nuclear-encoded and translated in the cytoplasm. Expression of the mitochondrially encoded proteins Cytochrome oxidase subunit 1, Apocytochrome b, and ATP synthase subunit 6 in the cytoplasm of HeLa cells confirms export to the endoplasmic reticulum. To examine the extent to which the mitochondrial proteome is driven by selective constraints within the eukaryotic cell, we investigated the occurrence of mitochondrial protein domains in bacteria and eukaryotes. The accessory protein domains of the oxidative phosphorylation system are unique to mitochondria, indicating the evolution of new protein folds. Most of the identified domains in the accessory proteins of the ribosome are also found in eukaryotic proteins of other functions and locations. Overall, one-third of the protein domains identified in mitochondrial proteins are only rarely found in bacteria. We conclude that the mitochondrial genome has been maintained to ensure the correct localization of highly hydrophobic membrane proteins. Taken together, the results suggest that selective constraints on the eukaryotic cell have played a major role in modulating the evolution of the mitochondrial genome and proteome. PMID:26195779

  20. Saccades and shifting receptive fields: anticipating consequences or selecting targets?

    PubMed Central

    Zirnsak, Marc; Moore, Tirin

    2014-01-01

    Saccadic eye movements cause frequent and substantial displacements of the retinal image, but those displacements go unnoticed. It has been widely assumed that this perceived stability emerges from the shifting of visual receptive fields from their current, presaccadic locations to their future, postsaccadic locations in anticipation of the retinal consequences of saccades. Although evidence consistent with this anticipatory remapping has accumulated over the years, more recent work suggests an alternative view. In this opinion article, we examine the evidence of presaccadic receptive field shifts and their relationship to the perceptual changes that accompany saccades. We argue that both reflect the selection of targets for saccades rather than the anticipation of a displaced retinal image. PMID:25455690

  1. Cancer Immunotherapy: Selected Targets and Small-Molecule Modulators.

    PubMed

    Weinmann, Hilmar

    2016-03-01

    There is a significant amount of excitement in the scientific community around cancer immunotherapy, as this approach has renewed hope for many cancer patients owing to some recent successes in the clinic. Currently available immuno-oncology therapeutics under clinical development and on the market are mostly biologics (antibodies, proteins, engineered cells, and oncolytic viruses). However, modulation of the immune system with small molecules offers several advantages that may be complementary and potentially synergistic to the use of large biologicals. Therefore, the discovery and development of novel small-molecule modulators is a rapidly growing research area for medicinal chemists working in cancer immunotherapy. This review provides a brief introduction into recent trends related to selected targets and pathways for cancer immunotherapy and their small-molecule pharmacological modulators. PMID:26836578

  2. Selective Cell Targeting with Light-Absorbing Microparticles and Nanoparticles

    PubMed Central

    Pitsillides, Costas M.; Joe, Edwin K.; Wei, Xunbin; Anderson, R. Rox; Lin, Charles P.

    2003-01-01

    We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond time-resolved microscopy and by thermal modeling. The extent of light-induced damage was investigated by cell lethality, by cell membrane permeability, and by protein inactivation. Strong particle size dependence was found for these interactions. A technique based on light to target endogenous particles is already being exploited to treat pigmented cells in dermatology and ophthalmology. With exogenous particles, phamacokinetics and biodistribution studies are needed before the method can be evaluated against photodynamic therapy for cancer treatment. However, particles are unique, unlike photosensitizers, in that they can remain stable and inert in cells for extended periods. Thus they may be particularly useful for prelabeling cells in engineered tissue before implantation. Subsequent irradiation with laser pulses will allow control of the implanted cells (inactivation or modulation) in a noninvasive manner. PMID:12770906

  3. CD133, Selectively Targeting the Root of Cancer

    PubMed Central

    Schmohl, Jörg U.; Vallera, Daniel A.

    2016-01-01

    Cancer stem cells (CSC) are capable of promoting tumor initiation and self-renewal, two important hallmarks of carcinoma formation. This population comprises a small percentage of the tumor mass and is highly resistant to chemotherapy, causing the most difficult problem in the field of cancer research, drug refractory relapse. Many CSC markers have been reported. One of the most promising and perhaps least ubiquitous is CD133, a membrane-bound pentaspan glycoprotein that is frequently expressed on CSC. There is evidence that directly targeting CD133 with biological drugs might be the most effective way to eliminate CSC. We have investigated two entirely unrelated, but highly effective approaches for selectively targeting CD133. The first involves using a special anti-CD133 single chain variable fragment (scFv) to deliver a catalytic toxin. The second utilizes this same scFv to deliver components of the immune system. In this review, we discuss the development and current status of these CD133 associated biological agents. Together, they show exceptional promise by specific and efficient CSC elimination. PMID:27240402

  4. MESSI: metabolic engineering target selection and best strain identification tool

    PubMed Central

    Kang, Kang; Li, Jun; Lim, Boon Leong; Panagiotou, Gianni

    2015-01-01

    Metabolic engineering and synthetic biology are synergistically related fields for manipulating target pathways and designing microorganisms that can act as chemical factories. Saccharomyces cerevisiae’s ideal bioprocessing traits make yeast a very attractive chemical factory for production of fuels, pharmaceuticals, nutraceuticals as well as a wide range of chemicals. However, future attempts of engineering S. cerevisiae’s metabolism using synthetic biology need to move towards more integrative models that incorporate the high connectivity of metabolic pathways and regulatory processes and the interactions in genetic elements across those pathways and processes. To contribute in this direction, we have developed Metabolic Engineering target Selection and best Strain Identification tool (MESSI), a web server for predicting efficient chassis and regulatory components for yeast bio-based production. The server provides an integrative platform for users to analyse ready-to-use public high-throughput metabolomic data, which are transformed to metabolic pathway activities for identifying the most efficient S. cerevisiae strain for the production of a compound of interest. As input MESSI accepts metabolite KEGG IDs or pathway names. MESSI outputs a ranked list of S. cerevisiae strains based on aggregation algorithms. Furthermore, through a genome-wide association study of the metabolic pathway activities with the strains’ natural variation, MESSI prioritizes genes and small variants as potential regulatory points and promising metabolic engineering targets. Users can choose various parameters in the whole process such as (i) weight and expectation of each metabolic pathway activity in the final ranking of the strains, (ii) Weighted AddScore Fuse or Weighted Borda Fuse aggregation algorithm, (iii) type of variants to be included, (iv) variant sets in different biological levels. Database URL: http://sbb.hku.hk/MESSI/ PMID:26255308

  5. Targeting the actin cytoskeleton: selective antitumor action via trapping PKCɛ.

    PubMed

    Foerster, F; Braig, S; Moser, C; Kubisch, R; Busse, J; Wagner, E; Schmoeckel, E; Mayr, D; Schmitt, S; Huettel, S; Zischka, H; Mueller, R; Vollmar, A M

    2014-01-01

    Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as

  6. Targeting the actin cytoskeleton: selective antitumor action via trapping PKCɛ

    PubMed Central

    Foerster, F; Braig, S; Moser, C; Kubisch, R; Busse, J; Wagner, E; Schmoeckel, E; Mayr, D; Schmitt, S; Huettel, S; Zischka, H; Mueller, R; Vollmar, A M

    2014-01-01

    Targeting the actin cytoskeleton (CSK) of cancer cells offers a valuable strategy in cancer therapy. There are a number of natural compounds that interfere with the actin CSK, but the mode of their cytotoxic action and, moreover, their tumor-specific mechanisms are quite elusive. We used the myxobacterial compound Chondramide as a tool to first elucidate the mechanisms of cytotoxicity of actin targeting in breast cancer cells (MCF7, MDA-MB-231). Chondramide inhibits cellular actin filament dynamics shown by a fluorescence-based analysis (fluorescence recovery after photobleaching (FRAP)) and leads to apoptosis characterized by phosphatidylserine exposure, release of cytochrome C from mitochondria and finally activation of caspases. Chondramide enhances the occurrence of mitochondrial permeability transition (MPT) by affecting known MPT modulators: Hexokinase II bound to the voltage-dependent anion channel (VDAC) translocated from the outer mitochondrial membrane to the cytosol and the proapoptotic protein Bad were recruited to the mitochondria. Importantly, protein kinase C-ɛ (PKCɛ), a prosurvival kinase possessing an actin-binding site and known to regulate the hexokinase/VDAC interaction as well as Bad phosphorylation was identified as the link between actin CSK and apoptosis induction. PKCɛ, which was found overexpressed in breast cancer cells, accumulated in actin bundles induced by Chondramide and lost its activity. Our second goal was to characterize the potential tumor-specific action of actin-binding agents. As the nontumor breast epithelial cell line MCF-10A in fact shows resistance to Chondramide-induced apoptosis and notably express low level of PKCɛ, we suggest that trapping PKCɛ via Chondramide-induced actin hyperpolymerization displays tumor cell specificity. Our work provides a link between targeting the ubiquitously occurring actin CSK and selective inhibition of pro-tumorigenic PKCɛ, thus setting the stage for actin-stabilizing agents as

  7. Neuronal lysosomal enzyme replacement using fragment C of tetanus toxin.

    PubMed

    Dobrenis, K; Joseph, A; Rattazzi, M C

    1992-03-15

    Development of a strategy for efficient delivery of exogenous enzyme to neuronal lysosomes is essential to achieve enzyme replacement in neurodegenerative lysosomal storage diseases. We tested whether effective lysosomal targeting of the human enzyme beta-N-acetylhexosaminidase A (Hex A; beta-N-acetyl-D-hexosaminide N-acetylhexosaminohydrolase, EC 3.2.1.52) can be obtained by coupling it via disulfide linkage to the atoxic fragment C of tetanus toxin (TTC) that is bound avidly by neuronal membrane. TTC-Hex A conjugation resulted in neuronal surface binding and enhanced endocytosis of enzyme as observed in immunofluorescence studies with rat brain cultures. In immunoelectrophoretic quantitative uptake studies, rat neuronal cell cultures contained 16- and 40-fold greater amounts of enzyme after incubation with TTC-Hex A than with nonderivatized Hex A. In cerebral cortex cell cultures from a feline model of human GM2 gangliosidosis (Tay-Sachs and Sandhoff diseases), binding and uptake patterns of the enzymes were similar to those in the rat brain cell cultures. After exposure to extracellular concentrations of enzyme attainable in vivo, lysosomal storage of immunodetectable GM2 ganglioside was virtually eliminated in neurons exposed to TTC-Hex A, whereas a minimal effect was observed with Hex A. These findings demonstrate the usefulness of TTC adducts for effective neuronal lysosomal enzyme replacement. PMID:1532255

  8. Human recombinant lysosomal enzymes produced in microorganisms.

    PubMed

    Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A

    2015-01-01

    Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. PMID:26071627

  9. The Nutrient-Responsive Transcription Factor TFE3, Promotes Autophagy, Lysosomal Biogenesis, and Clearance of Cellular Debris

    PubMed Central

    Martina, José A.; Diab, Heba I.; Lishu, Li; Jeong-A, Lim; Patange, Simona; Raben, Nina; Puertollano, Rosa

    2015-01-01

    The discovery of a gene network regulating lysosomal biogenesis and its transcriptional regulator TFEB revealed that cells monitor lysosomal function and respond to degradation requirements and environmental cues. Here, we report the identification of transcription factor E3 (TFE3) as another regulator of lysosomal homeostasis that induced expression of genes encoding proteins involved in autophagy and lysosomal biogenesis in ARPE-19 cells in response to starvation and lysosomal stress. We found that in nutrient-replete cells, TFE3 was recruited to lysosomes through interaction with active Rag GTPases and exhibited mTORC1-dependent phosphorylation. Phosphorylated TFE3 was retained in the cytosol through its interaction with the cytosolic chaperone 14-3-3. Following starvation, TFE3 rapidly translocated to the nucleus and bound to the CLEAR elements present in the promoter region of many lysosomal genes, thereby inducing lysosomal biogenesis. Depletion of endogenous TFE3 entirely abolished the response of ARPE-19 cells to starvation, suggesting that TFE3 plays a critical role in nutrient sensing and regulation of energy metabolism. Furthermore, overexpression of TFE3 triggered lysosomal exocytosis and resulted in efficient cellular clearance in a cellular model of a lysosomal storage disorder, Pompe disease, thus identifying TFE3 as a potential therapeutic target for the treatment of lysosomal disorders. PMID:24448649

  10. Human hair follicle: reservoir function and selective targeting.

    PubMed

    Blume-Peytavi, U; Vogt, A

    2011-10-01

    Penetration of topically applied compounds may occur via the stratum corneum, skin appendages and hair follicles. The follicular infundibulum increases the surface area, disrupts the epidermal barrier towards the lower parts of the follicle, and serves as a reservoir. Topical delivery of active compounds to specific targets within the skin, especially to distinct hair follicle compartments or cell populations, may help to treat local inflammatory reactions selectively, with reduced systemic side-effects. Various in vitro and in vivo methods exist for studying the hair follicle structure and follicular penetration pathways. These include cyanoacrylate skin surface stripping, confocal microscopy and cyanoacrylate scalp follicle biopsy. The complex anatomical structure as well as the cyclical activity of the hair follicle must be taken into consideration when designing delivery systems. In addition, delivery into and retention inside the infundibular reservoir are controlled by, for example, molecule or particle size, their polarity and the type of preparation. Preferred penetration depth and storage time must also be considered. Particles with release mechanisms should be preferred; however, the release of drugs from nanoparticles still requires further investigations. PMID:21919898

  11. Molecular mechanisms and proposed targets for selected anticancer gold compounds.

    PubMed

    Casini, Angela; Messori, Luigi

    2011-01-01

    Nowadays, gold compounds constitute a family of very promising experimental agents for cancer treatment. Indeed, several gold(I) and gold(III) compounds were shown to manifest outstanding antiproliferative properties in vitro against selected human tumor cell lines and some of them performed remarkably well even in tumor models in vivo. Notably, the peculiar chemical properties of the gold centre impart innovative pharmacological profiles to gold-based metallodrugs most likely in relation to novel molecular mechanisms. The precise mechanisms through which cytotoxic gold compounds produce their biological effects are still largely unknown. Within this frame, the major aim of this review is to define the possible modes of action and the most probable biomolecular targets for a few representative gold compounds on which extensive biochemical and cellular data have been gathered. In particular, we will focus on auranofin and analogues, on gold(III) porphyrins and gold(III) dithiocarbamates. For these three families markedly distinct molecular mechanisms were recently invoked: a direct mitochondrial mechanism involving thioredoxin reductase inhibition in the case of the gold(I) complexes, the influence on some apoptotic proteins--i.e. MAPKs and Bcl-2--for gold(III) porphyrins, and the proteasome inhibition for gold(III) dithiocarbamates. In a few cases the distinct mechanisms may overlap. The general perspectives for the development of new gold compounds as effective anticancer agents with innovative modes of action are critically discussed. PMID:22039866

  12. M-CSF inhibition selectively targets pathological angiogenesis and lymphangiogenesis.

    PubMed

    Kubota, Yoshiaki; Takubo, Keiyo; Shimizu, Takatsune; Ohno, Hiroaki; Kishi, Kazuo; Shibuya, Masabumi; Saya, Hideyuki; Suda, Toshio

    2009-05-11

    Antiangiogenic therapy for the treatment of cancer and other neovascular diseases is desired to be selective for pathological angiogenesis and lymphangiogenesis. Macrophage colony-stimulating factor (M-CSF), a cytokine required for the differentiation of monocyte lineage cells, promotes the formation of high-density vessel networks in tumors and therefore possesses therapeutic potential as an M-CSF inhibitor. However, the physiological role of M-CSF in vascular and lymphatic development, as well as the precise mechanisms underlying the antiangiogenic effects of M-CSF inhibition, remains unclear. Moreover, therapeutic potential of M-CSF inhibition in other neovascular diseases has not yet been evaluated. We used osteopetrotic (op/op) mice to demonstrate that M-CSF deficiency reduces the abundance of LYVE-1(+) and LYVE1(-) macrophages, resulting in defects in vascular and lymphatic development. In ischemic retinopathy, M-CSF was required for pathological neovascularization but was not required for the recovery of normal vasculature. In mouse osteosarcoma, M-CSF inhibition effectively suppressed tumor angiogenesis and lymphangiogenesis, and it disorganized extracellular matrices. In contrast to VEGF blockade, interruption of M-CSF inhibition did not promote rapid vascular regrowth. Continuous M-CSF inhibition did not affect healthy vascular and lymphatic systems outside tumors. These results suggest that M-CSF-targeted therapy is an ideal strategy for treating ocular neovascular diseases and cancer. PMID:19398755

  13. Saccade-target selection of dyslexic children when reading Chinese.

    PubMed

    Pan, Jinger; Yan, Ming; Laubrock, Jochen; Shu, Hua; Kliegl, Reinhold

    2014-04-01

    This study investigates the eye movements of dyslexic children and their age-matched controls when reading Chinese. Dyslexic children exhibited more and longer fixations than age-matched control children, and an increase of word length resulted in a greater increase in the number of fixations and gaze durations for the dyslexic than for the control readers. The report focuses on the finding that there was a significant difference between the two groups in the fixation landing position as a function of word length in single-fixation cases, while there was no such difference in the initial fixation of multi-fixation cases. We also found that both groups had longer incoming saccade amplitudes while the launch sites were closer to the word in single fixation cases than in multi-fixation cases. Our results suggest that dyslexic children's inefficient lexical processing, in combination with the absence of orthographic word boundaries in Chinese, leads them to select saccade targets at the beginning of words conservatively. These findings provide further evidence for parafoveal word segmentation during reading of Chinese sentences. PMID:24508073

  14. Effects of Mode of Target Task Selection on Learning about Plants in a Mobile Learning Environment: Effortful Manual Selection versus Effortless QR-Code Selection

    ERIC Educational Resources Information Center

    Gao, Yuan; Liu, Tzu-Chien; Paas, Fred

    2016-01-01

    This study compared the effects of effortless selection of target plants using quick respond (QR) code technology to effortful manual search and selection of target plants on learning about plants in a mobile device supported learning environment. In addition, it was investigated whether the effectiveness of the 2 selection methods was…

  15. How targets select activation or repression in response to Wnt.

    PubMed

    Murgan, Sabrina; Bertrand, Vincent

    2015-01-01

    In metazoans, the Wnt signaling pathway plays a key role in the regulation of binary decisions during development. During this process different sets of target genes are activated in cells where the Wnt pathway is active (classic target genes) versus cells where the pathway is inactive (opposite target genes). While the mechanism of transcriptional activation is well understood for classic target genes, how opposite target genes are activated in the absence of Wnt remains poorly characterized. Here we discuss how the key transcriptional mediator of the Wnt pathway, the TCF family member POP-1, regulates opposite target genes during C. elegans development. We examine recent findings suggesting that the direction of the transcriptional output (activation or repression) can be determined by the way TCF is recruited and physically interacts with its target gene. PMID:27123368

  16. Target product selection - where can Molecular Pharming make the difference?

    PubMed

    Paul, Mathew J; Teh, Audrey Y H; Twyman, Richard M; Ma, Julian K-C

    2013-01-01

    Four major developments have taken place in the world of Molecular Pharming recently. In the USA, the DARPA initiative challenged plant biotechnology companies to develop strategies for the large-scale manufacture of influenza vaccines, resulting in a successful Phase I clinical trial; in Europe the Pharma-Planta academic consortium gained regulatory approval for a plant-derived monoclonal antibody and completed a first-in-human phase I clinical trial; the Dutch pharmaceutical company Synthon acquired the assets of Biolex Therapeutics, an established Molecular Pharming company with several clinical candidates produced in their proprietary LEX system based on aquatic plants; and finally, the Israeli biotechnology company Protalix Biotherapeutics won FDA approval for the commercial release of a recombinant form of the enzyme glucocerebrosidase produced in carrot cells, the first plant biotechnology-derived biopharmaceutical in the world approved for the market. Commercial momentum is gathering pace with additional candidates now undergoing or awaiting approval for phase III clinical trials. Filling the product pipeline is vital to establish commercial sustainability, and the selection of appropriate target products for Molecular Pharming will be a critical factor. An interesting feature of the four stories outlined above is that they span the use of very different platform technologies addressing different types of molecules which aim to satisfy distinct market demands. In each case, Molecular Pharming was an economically and technically suitable approach, but this decisionmaking process is not necessarily straightforward. Although the various technologies available to Molecular Pharming are broad ranging and flexible, competing technologies are better established, so there needs to be a compelling reason to move into plants. It is most unlikely that plant biotechnology will be the answer for the whole biologics field. In this article, we discuss the current plant

  17. [Gene therapy in lysosomal diseases].

    PubMed

    Moullier, P; Salvetti, A; Bohl, D; Danos, O; Heard, J M

    1996-01-01

    The study of the mechanisms of secretion and recapture of lysosomal enzymes has lead to the proposal of a treatment of lysosomal diseases by enzyme replacement. Autologous implants of genetically modified cells which secrete enzymes ensure systemic distribution of the lacking enzyme. A procedure which permits reimplantation of genetically modified fibroblasts is described. The stable secretion of human glucuronidase by autologous fibroblasts was thus obtained in animal species. This approach should by applicable to the treatment of Hurler's syndrome by obtaining the production and distribution of alpha-L-iduronidase in patients lacking this enzyme by retroviral transfer of the human alpha-L-iduronidase gene to cultured fibroblasts and by preparation of implants. PMID:8881268

  18. Lysosomes and the plasma membrane

    PubMed Central

    Andrews, Norma W.

    2002-01-01

    Studies of the cell invasion mechanism of the parasite Trypanosoma cruzi led to a series of novel findings, which revealed a previously unsuspected ability of conventional lysosomes to fuse with the plasma membrane. This regulated exocytic process, previously regarded mostly as a specialization of certain cell types, was recently shown to play an important role in the mechanism by which cells reseal their plasma membrane after injury. PMID:12147679

  19. Gene therapy for lysosomal disorders.

    PubMed

    Naffakh, N; Bohl, D; Salvetti, A; Moullier, P; Danos, O; Heard, J M

    1994-01-01

    Genetic defects of lysosomal hydrolases result in severe storage diseases and treatments based on enzyme replacement have been proposed. In mice lacking beta-glucuronidase, which develop a disease homologous to human mucopolysaccharidosis type VII (MPS VII, sly syndrome), we have used autologous implants of genetically-modified cells for the continuous in vivo production of the enzyme. A retroviral vector containing the human beta-glucuronidase cDNA under the control of the mouse phosphoglycerate kinase promoter was used to infect primary skin fibroblasts, bone marrow cells, or myoblasts from mutant MPS VII animals. The fibroblasts were embedded into collagen lattices and reimplanted into the peritoneal cavity of recipient MPS VII mice. All animals, when analysed 10 to 155 days later, expressed beta-glucuronidase from the vascularised neo-organs that developed after implantation, and accumulated the enzyme in their tissues. A complete disappearance of the lysosomal storage lesions was observed in their liver and spleen. This procedure has been scaled up for long term lysosomal enzyme delivery in dogs. The bone marrow cells were used for partial hematopoietic reconstruction of sublethally irradiated MPS VII mice. Five months after gene transfer, animals in which under 5% of genetically-modified hematopoietic cells were detected in the spleen showed a drastic reduction of lysosomal storage lesions in the liver and spleen. Genetically-modified myoblasts were transplanted into injured muscles, where they participated in the regeneration of a significant proportion of muscle fibers. Enzyme secretion and liver uptake were observed for at least one month.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8177709

  20. Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells.

    PubMed

    Groth-Pedersen, Line; Aits, Sonja; Corcelle-Termeau, Elisabeth; Petersen, Nikolaj H T; Nylandsted, Jesper; Jäättelä, Marja

    2012-01-01

    Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 siRNAs was preceded by lysosomal membrane permeabilization, and all identified siRNAs induced several changes in the endo-lysosomal compartment, i.e. increased lysosomal volume (KIF11, KIF20A, KIF25, MYO1G, MYH1), increased cysteine cathepsin activity (KIF20A, KIF25), altered lysosomal localization (KIF25, MYH1, TPM2), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide or cisplatin. Similarly to KIF11 siRNA, the KIF11 inhibitor monastrol induced lysosomal membrane permeabilization and sensitized several cancer cell lines to siramesine. While KIF11 inhibitors are under clinical development as mitotic blockers, our data reveal a new function for KIF11 in controlling lysosomal stability and introduce six other molecular motors as putative cancer drug targets. PMID:23071517

  1. Lysosomal and autophagic reactions as predictive indicators of environmental impact in aquatic animals.

    PubMed

    Moore, Michael N; Allen, J Icarus; McVeigh, Allan; Shaw, Jenny

    2006-01-01

    The lysosomal-autophagic system appears to be a common target for many environmental pollutants as lysosomes accumulate many toxic metals and organic xenobiotics, which perturb normal function and damage the lysosomal membrane. In fact, lysosomal membrane integrity or stability appears to be an effective generic indicator of cellular well-being in eukaryotes: in bivalve molluscs and fish, stability is correlated with many toxicological responses and pathological reactions. Prognostic use of adverse lysosomal and autophagic reactions to environmental pollutants has been explored in relation to predicting cellular dysfunction and health in marine mussels, which are extensively used as sensitive bioindicators in monitoring ecosystem health. Derivation of explanatory frameworks for prediction of pollutant impact on health is a major goal; and we have developed a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells and tissues. This model has also complemented the creation of a cell-based computational model for molluscan hepatopancreatic cells that simulates lysosomal, autophagic and other cellular reactions to pollutants. Experimental and simulated results have also indicated that nutritional deprivation-induced autophagy has a protective function against toxic effects mediated by reactive oxygen species (ROS). Finally, coupled measurement of lysosomal-autophagic reactions and modelling is proposed as a practical toolbox for predicting toxic environmental risk. PMID:16874099

  2. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins.

    PubMed

    McEwan, David G; Popovic, Doris; Gubas, Andrea; Terawaki, Seigo; Suzuki, Hironori; Stadel, Daniela; Coxon, Fraser P; Miranda de Stegmann, Diana; Bhogaraju, Sagar; Maddi, Karthik; Kirchof, Anja; Gatti, Evelina; Helfrich, Miep H; Wakatsuki, Soichi; Behrends, Christian; Pierre, Philippe; Dikic, Ivan

    2015-01-01

    The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways. PMID:25498145

  3. Lysosomal ATP imaging in living cells by a water-soluble cationic polythiophene derivative.

    PubMed

    Huang, Bing-Huan; Geng, Zhi-Rong; Ma, Xiao-Yan; Zhang, Cui; Zhang, Zhi-Yang; Wang, Zhi-Lin

    2016-09-15

    Lysosomes in astrocytes and microglia can release ATP as the signaling molecule for the cells through ca(2+)-dependent exocytosis in response to various stimuli. At present, fluorescent probes that can detect ATP in lysosomes have not been reported. In this work, we have developed a new water-soluble cationic polythiophene derivative that can be specifically localized in lysosomes and can be utilized as a fluorescent probe to sense ATP in cells. PEMTEI exhibits high selectivity and sensitivity to ATP at physiological pH values and the detection limit of ATP is as low as 10(-11)M. The probe has low cytotoxicity, good permeability and high photostability in living cells and has been applied successfully to real-time monitoring of the change in concentrations of ATP in lysosomes though fluorescence microscopy. We also demonstrated that lysosomes in Hela cells can release ATP through Ca(2+)-dependent exocytosis in response to drug stimuli. PMID:27131993

  4. Newborn screening for neuropathic lysosomal storage disorders.

    PubMed

    Hwu, Wuh-Liang; Chien, Yin-Hsiu; Lee, Ni-Chung

    2010-08-01

    Interest in newborn screening (NBS) for lysosomal storage disorders (LSDs) has increased significantly due to newly developed enzyme replacement therapy (ERT), the need for early diagnosis, and advances in technical developments. Since the central nervous system cannot be treated by ERT, neuronopathic LSDs are generally not the primary target of NBS. An exception is Krabbe disease, in which hematopoietic stem cell transplantation before the onset of symptoms has benefits. However, NBS for LSD relies on measuring enzyme activities, so the most severely affected individuals (usually patients with neuronopathic subtypes) will be detected together with patients with less severe disease. In the near future, NBS is likely to be developed for diseases such as Gaucher, Niemann-Pick A/B, and certain mucopolysaccharidoses. The ability to predict phenotypes (neuronopathic or not) by enzyme activity and genotyping will therefore be critical for adequate patient management. This article reviews the status of LSD screening and issues concerning detection of neuronopathic LSDs by screening. PMID:20532820

  5. Controlling nematodes in dairy calves using targeted selective treatments.

    PubMed

    O'Shaughnessy, J; Earley, B; Mee, J F; Doherty, M L; Crosson, P; Barrett, D; de Waal, T

    2015-04-30

    With increasing concerns of anthelmintic resistance in cattle nematode populations worldwide, there is a need to explore alternative approaches to nematode control. One alternative approach is the use of targeted selective treatments (TST) where only individual animals are treated instead of the entire group. This study reports the findings of a TST approach in dairy calves conducted over their first grazing season (FGS) to control both gastrointestinal nematode and lungworm challenge. Ninety-six calves with an initial mean (s.d.) age and live weight of 130 (28.3) days and 120 (23.6)kg, respectively, were randomised by breed, age and live weight to one of two treatments; Control (n=24; ×2) and TST (n=24; ×2). Control calves were treated three times at pasture with ivermectin by subcutaneous injection. Individual calves in the TST group were treated at pasture with ivermectin when one of the following thresholds was met: (1) positive for lungworm larvae using the modified Baermann technique or (2) positive or negative for lungworm larvae using the modified Baermann technique with plasma pepsinogen concentration (PP) ≥ two international units of tyrosine/litre and faecal egg count (FEC) ≥ 200 strongyle eggs per gram of faeces. Calves were rotationally grazed from July 3rd 2012 (day 0) to November 2nd 2012 (day 122) when calves were housed. Calves were weighed and sampled (blood and faecal) every three weeks. There was an effect of treatment and time on both FEC [treatment (P=0.023), time (P<0.001)] and PP [treatment (P=0.002), time (P<0.001)]. Both FEC and PP were higher in TST calves. There was a 50% reduction in anthelmintic use in TST calves compared to control calves. Clinical signs of lungworm infection, confirmed by the modified Baermann technique, were evident in TST calves on days 62 and 63 of the study. The average daily live weight gain for control and TST calves was 0.50 (0.02)kg day(-1) and 0.47 (0.03)kg day(-1), respectively (P=0.41). Thus

  6. Impaired lysosomal cobalamin transport in Alzheimer's disease.

    PubMed

    Zhao, Hua; Li, Hongyun; Ruberu, Kalani; Garner, Brett

    2015-01-01

    Cobalamin (vitamin B12) is required for erythrocyte formation and DNA synthesis and it plays a crucial role in maintaining neurological function. As a coenzyme for methionine synthase and methylmalonyl-CoA mutase, cobalamin utilization depends on its efficient transit through the intracellular lysosomal compartment and subsequent delivery to the cytosol and mitochondria. Lysosomal function deteriorates in Alzheimer's disease (AD). Lysosomal acidification is defective in AD and lysosomal proteolysis is disrupted by AD-related presenilin 1 mutation. In this study, we propose that AD related lysosomal dysfunction may impair lysosomal cobalamin transport. The experiments use in vitro and in vivo models of AD to define how lysosomal dysfunction directly affects cobalamin utilization. SH-SY5Y-AβPP mutant cells were treated with a proteasome inhibitor to induce lysosomal amyloid-β accumulation. We metabolically labeled these cells with [57Co] cobalamin and isolated purified lysosomes, mitochondria, and cytosol fractions. The results indicated that proteasome inhibition was associated with lysosomal amyloid-β accumulation and a doubling of lysosomal [57Co] cobalamin levels. We also used AβPPxPS1 transgenic AD mice that were intraperitoneally injected with [57Co] cobalamin. The amount of [57Co] cobalamin in the major organs of these mice was measured and the subcellular [57Co] cobalamin distribution in the brain was assessed. The results demonstrated that lysosomal [57Co] cobalamin level was significantly increased by 56% in the AβPPxPS1 AD mouse brains as compared to wild type control mice. Together these data provide evidence that lysosomal cobalamin may be impaired in AD in association with amyloid-β accumulation. PMID:25125476

  7. Leaving the lysosome behind: novel developments in autophagy inhibition

    PubMed Central

    Solitro, Abigail R; MacKeigan, Jeffrey P

    2016-01-01

    The search for a single silver bullet for the treatment of cancer has now been overshadowed by the identification of multiple therapeutic targets unique to each malignancy and even to each patient. In recent years, autophagy has emerged as one such therapeutic target. In response to both therapeutic and oncogenic stress, cancer cells upregulate and demonstrate an increased dependence upon this intracellular recycling process. Particularly in malignancies that currently lack targeted therapeutic options, autophagy inhibitors are the next hopeful prospects for the treatment of this disease. In this review, we discuss the rapid evolution of autophagy inhibitors from early lysosomotropic agents to next-generation lysosome-targeted drugs and beyond. PMID:26689099

  8. Epidermal Growth Factor Receptor-Targeted Photosensitizer Selectively Inhibits EGFR Signaling and Induces Targeted Phototoxicity In Ovarian Cancer Cells

    PubMed Central

    Abu-Yousif, Adnan O.; Moor, Anne C. E.; Zheng, Xiang; Savellano, Mark D.; Yu, Weiping; Selbo, Pål K.; Hasan, Tayyaba

    2012-01-01

    Targeted photosensitizer delivery to EGFR expressing cells was achieved in the present study using a high purity, targeted photoimmunoconjugate (PIC). When the PDT agent, benzoporphyin monoacid ring A (BPD) was coupled to an EGFR-targeting antibody (cetuximab), we observed altered cellular localization and selective phototoxicity of EGFR-positive cells, but no phototoxicity of EGFR-negative cells. Cetuximab in the PIC formulation blocked EGF-induced activation of the EGFR and downstream signaling pathways. Our results suggest that photoimmunotargeting is a useful dual strategy for the selective destruction of cancer cells and also exerts the receptor-blocking biological function of the antibody. PMID:22266098

  9. Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes multidrug resistance by a novel mechanism involving the hijacking of lysosomal P-glycoprotein (Pgp).

    PubMed

    Jansson, Patric J; Yamagishi, Tetsuo; Arvind, Akanksha; Seebacher, Nicole; Gutierrez, Elaine; Stacy, Alexandra; Maleki, Sanaz; Sharp, Danae; Sahni, Sumit; Richardson, Des R

    2015-04-10

    Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2',7'-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport activity

  10. Di-2-pyridylketone 4,4-Dimethyl-3-thiosemicarbazone (Dp44mT) Overcomes Multidrug Resistance by a Novel Mechanism Involving the Hijacking of Lysosomal P-Glycoprotein (Pgp)*

    PubMed Central

    Jansson, Patric J.; Yamagishi, Tetsuo; Arvind, Akanksha; Seebacher, Nicole; Gutierrez, Elaine; Stacy, Alexandra; Maleki, Sanaz; Sharp, Danae; Sahni, Sumit; Richardson, Des R.

    2015-01-01

    Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2′,7′-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport

  11. Glucose Modulation Induces Lysosome Formation and Increases Lysosomotropic Drug Sequestration via the P-Glycoprotein Drug Transporter.

    PubMed

    Seebacher, Nicole A; Lane, Darius J R; Jansson, Patric J; Richardson, Des R

    2016-02-19

    Pgp is functional on the plasma membrane and lysosomal membrane. Lysosomal-Pgp can pump substrates into the organelle, thereby trapping certain chemotherapeutics (e.g. doxorubicin; DOX). This mechanism serves as a "safe house" to protect cells against cytotoxic drugs. Interestingly, in contrast to DOX, lysosomal sequestration of the novel anti-tumor agent and P-glycoprotein (Pgp) substrate, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), induces lysosomal membrane permeabilization. This mechanism of lysosomal-Pgp utilization enhances cytotoxicity to multidrug-resistant cells. Consequently, Dp44mT has greater anti-tumor activity in drug-resistant relative to non-Pgp-expressing tumors. Interestingly, stressors in the tumor microenvironment trigger endocytosis for cell signaling to assist cell survival. Hence, this investigation examined how glucose variation-induced stress regulated early endosome and lysosome formation via endocytosis of the plasma membrane. Furthermore, the impact of glucose variation-induced stress on resistance to DOX was compared with Dp44mT and its structurally related analogue, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). These studies showed that glucose variation-induced stress-stimulated formation of early endosomes and lysosomes. In fact, through the process of fluid-phase endocytosis, Pgp was redistributed from the plasma membrane to the lysosomal membrane via early endosome formation. This lysosomal-Pgp actively transported the Pgp substrate, DOX, into the lysosome where it became trapped as a result of protonation at pH 5. Due to increased lysosomal DOX trapping, Pgp-expressing cells became more resistant to DOX. In contrast, cytotoxicity of Dp44mT and DpC was potentiated due to more lysosomes containing functional Pgp under glucose-induced stress. These thiosemicarbazones increased lysosomal membrane permeabilization and cell death. This mechanism has critical implications for drug-targeting in

  12. Chelation of lysosomal iron protects against ionizing radiation.

    PubMed

    Berndt, Carsten; Kurz, Tino; Selenius, Markus; Fernandes, Aristi P; Edgren, Margareta R; Brunk, Ulf T

    2010-12-01

    Ionizing radiation causes DNA damage and consequent apoptosis, mainly due to the production of hydroxyl radicals (HO•) that follows radiolytic splitting of water. However, superoxide (O2•-) and H2O2 also form and induce oxidative stress with resulting LMP (lysosomal membrane permeabilization) arising from iron-catalysed oxidative events. The latter will contribute significantly to radiation-induced cell death and its degree largely depends on the quantities of lysosomal redox-active iron present as a consequence of autophagy and endocytosis of iron-rich compounds. Therefore radiation sensitivity might be depressed by lysosome-targeted iron chelators. In the present study, we have shown that cells in culture are significantly protected from ionizing radiation damage if initially exposed to the lipophilic iron chelator SIH (salicylaldehyde isonicotinoyl hydrazone), and that this effect is based on SIH-dependent lysosomal stabilization against oxidative stress. According to its dose-response-modifying effect, SIH is a most powerful radioprotector and a promising candidate for clinical application, mainly to reduce the radiation sensitivity of normal tissue. We propose, as an example, that inhalation of SIH before each irradiation session by patients undergoing treatment for lung malignancies would protect normally aerated lung tissue against life-threatening pulmonary fibrosis, whereas the sensitivity of malignant lung tumours, which usually are non-aerated, will not be affected by inhaled SIH. PMID:20846118

  13. A cation counterflux supports lysosomal acidification

    PubMed Central

    Steinberg, Benjamin E.; Huynh, Kassidy K.; Brodovitch, Alexandre; Jabs, Sabrina; Stauber, Tobias; Jentsch, Thomas J.

    2010-01-01

    The profound luminal acidification essential for the degradative function of lysosomes requires a counter-ion flux to dissipate an opposing voltage that would prohibit proton accumulation. It has generally been assumed that a parallel anion influx is the main or only counter-ion transport that enables acidification. Indeed, defective anion conductance has been suggested as the mechanism underlying attenuated lysosome acidification in cells deficient in CFTR or ClC-7. To assess the individual contribution of counter-ions to acidification, we devised means of reversibly and separately permeabilizing the plasma and lysosomal membranes to dialyze the cytosol and lysosome lumen in intact cells, while ratiometrically monitoring lysosomal pH. Replacement of cytosolic Cl− with impermeant anions did not significantly alter proton pumping, while the presence of permeant cations in the lysosomal lumen supported acidification. Accordingly, the lysosomes were found to acidify to the same pH in both CFTR- and ClC-7–deficient cells. We conclude that cations, in addition to chloride, can support lysosomal acidification and defects in lysosomal anion conductance cannot explain the impaired microbicidal capacity of CF phagocytes. PMID:20566682

  14. Analysis of lysosomal membrane proteins exposed to melanin in HeLa cells

    PubMed Central

    2016-01-01

    Objectives There have been developed to use targeting ability for antimicrobial, anticancerous, gene therapy and cosmetics through analysis of various membrane proteins isolated from cell organelles. Methods It was examined about the lysosomal membrane protein extracted from lysosome isolated from HeLa cell treated by 100 ppm melanin for 24 hours in order to find associated with targeting ability to melanin using by 2-dimensional electrophoresis. Results The result showed 14 up-regulated (1.5-fold) and 13 down-regulated (2.0-fold) spots in relation to melanin exposure. Conclusions It has been found that lysosomal membrane proteins are associated with melanin to decolorize and quantity through cellular activation of lysosome. PMID:27158002

  15. Thiadiazole Carbamates: Potent Inhibitors of Lysosomal Acid Lipase and Potential Niemann-Pick Type C Disease Therapeuticsa

    PubMed Central

    Rosenbaum, Anton I.; Cosner, Casey C.; Mariani, Christopher J.; Maxfield, Frederick R.; Wiest, Olaf; Helquist, Paul

    2010-01-01

    Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized at the cellular level by abnormal accumulation of cholesterol and other lipids in lysosomal storage organelles. Lysosomal acid lipase (LAL) has been recently identified as a potential therapeutic target for NPC. LAL can be specifically inhibited by a variety of 3,4-disubstituted thiadiazole carbamates. An efficient synthesis of the C(3) oxygenated/C(4) aminated analogues has been developed that furnishes the products in high yields and high degrees of purity. Common intermediates can also be used for the synthesis of the C(3) carbon substituted derivatives. Herein we tested various thiadiazole carbamates, amides, esters, and ketones for inhibition of LAL. In addition, we tested a diverse selection of commercially available non-thiadiazole carbamates. Our studies show that, among the compounds examined herein, only thiadiazole carbamates are effective inhibitors of LAL. We present a mechanism for LAL inhibition by these compounds whereby LAL transiently carbamoylates the enzyme similarly to previously described inhibition of acetylcholinesterase by rivastigmine and other carbamates as well as acylation of various lipases by orlistat. PMID:20557099

  16. Evaluating Gaze-Based Interface Tools to Facilitate Point-and-Select Tasks with Small Targets

    ERIC Educational Resources Information Center

    Skovsgaard, Henrik; Mateo, Julio C.; Hansen, John Paulin

    2011-01-01

    Gaze interaction affords hands-free control of computers. Pointing to and selecting small targets using gaze alone is difficult because of the limited accuracy of gaze pointing. This is the first experimental comparison of gaze-based interface tools for small-target (e.g. less than 12 x 12 pixels) point-and-select tasks. We conducted two…

  17. Parasite neuropeptide biology: Seeding rational drug target selection?

    PubMed Central

    McVeigh, Paul; Atkinson, Louise; Marks, Nikki J.; Mousley, Angela; Dalzell, Johnathan J.; Sluder, Ann; Hammerland, Lance; Maule, Aaron G.

    2011-01-01

    The rationale for identifying drug targets within helminth neuromuscular signalling systems is based on the premise that adequate nerve and muscle function is essential for many of the key behavioural determinants of helminth parasitism, including sensory perception/host location, invasion, locomotion/orientation, attachment, feeding and reproduction. This premise is validated by the tendency of current anthelmintics to act on classical neurotransmitter-gated ion channels present on helminth nerve and/or muscle, yielding therapeutic endpoints associated with paralysis and/or death. Supplementary to classical neurotransmitters, helminth nervous systems are peptide-rich and encompass associated biosynthetic and signal transduction components – putative drug targets that remain to be exploited by anthelmintic chemotherapy. At this time, no neuropeptide system-targeting lead compounds have been reported, and given that our basic knowledge of neuropeptide biology in parasitic helminths remains inadequate, the short-term prospects for such drugs remain poor. Here, we review current knowledge of neuropeptide signalling in Nematoda and Platyhelminthes, and highlight a suite of 19 protein families that yield deleterious phenotypes in helminth reverse genetics screens. We suggest that orthologues of some of these peptidergic signalling components represent appealing therapeutic targets in parasitic helminths. PMID:24533265

  18. Targeting extracellular pyrophosphates underpins the high selectivity of nisin.

    PubMed

    Bonev, Boyan B; Breukink, Eefjan; Swiezewska, E; De Kruijff, Ben; Watts, Anthony

    2004-12-01

    The spread of infectious diseases and the increase in antibiotic resistance represent a life-threatening global development that calls for new approaches to control microorganisms. Of all potential targets, the essential and unique pathway of bacterial cell wall synthesis, targeted by the first known antibiotic penicillin, remains a perfect candidate for the development of new antibiotics. Here we show that the lantibiotic nisin exercises its antibacterial action by targeting peptidoglycan intermediates' extracellular pyrophosphate, unique to bacterial cell wall precursors. We show that nisin sequesters cell wall precursors found in the outer leaflet of bacterial plasma membranes, Lipid II and undecaprenyl pyrophosphate, into stable complexes. We propose a model of antibacterial action for nisin in which the terminal amino group of Ile1 targets the pyrophosphate groups of the bacterial cell wall precursors, where it docks via a hydrogen bond. The pyrophosphate moiety, a highly conserved chemical group different from the L-Lys-D-Ala-D-Ala docking motif for vancomycin, has no biochemical analogs with comparable properties and is unlikely to be susceptible to bacterial adaptations akin to those responsible for resistance to penicillins and vancomycin. PMID:15576489

  19. Apolipoprotein L-I Promotes Trypanosome Lysis by Forming Pores in Lysosomal Membranes

    NASA Astrophysics Data System (ADS)

    Pérez-Morga, David; Vanhollebeke, Benoit; Paturiaux-Hanocq, Françoise; Nolan, Derek P.; Lins, Laurence; Homblé, Fabrice; Vanhamme, Luc; Tebabi, Patricia; Pays, Annette; Poelvoorde, Philippe; Jacquet, Alain; Brasseur, Robert; Pays, Etienne

    2005-07-01

    Apolipoprotein L-I is the trypanolytic factor of human serum. Here we show that this protein contains a membrane pore-forming domain functionally similar to that of bacterial colicins, flanked by a membrane-addressing domain. In lipid bilayer membranes, apolipoprotein L-I formed anion channels. In Trypanosoma brucei, apolipoprotein L-I was targeted to the lysosomal membrane and triggered depolarization of this membrane, continuous influx of chloride, and subsequent osmotic swelling of the lysosome until the trypanosome lysed.

  20. Genome-wide polymorphisms show unexpected targets of natural selection.

    PubMed

    Pespeni, Melissa H; Garfield, David A; Manier, Mollie K; Palumbi, Stephen R

    2012-04-01

    Natural selection can act on all the expressed genes of an individual, leaving signatures of genetic differentiation or diversity at many loci across the genome. New power to assay these genome-wide effects of selection comes from associating multi-locus patterns of polymorphism with gene expression and function. Here, we performed one of the first genome-wide surveys in a marine species, comparing purple sea urchins, Strongylocentrotus purpuratus, from two distant locations along the species' wide latitudinal range. We examined 9112 polymorphic loci from upstream non-coding and coding regions of genes for signatures of selection with respect to gene function and tissue- and ontogenetic gene expression. We found that genetic differentiation (F(ST)) varied significantly across functional gene classes. The strongest enrichment occurred in the upstream regions of E3 ligase genes, enzymes known to regulate protein abundance during development and environmental stress. We found enrichment for high heterozygosity in genes directly involved in immune response, particularly NALP genes, which mediate pro-inflammatory signals during bacterial infection. We also found higher heterozygosity in immune genes in the southern population, where disease incidence and pathogen diversity are greater. Similar to the major histocompatibility complex in mammals, balancing selection may enhance genetic diversity in the innate immune system genes of this invertebrate. Overall, our results show that how genome-wide polymorphism data coupled with growing databases on gene function and expression can combine to detect otherwise hidden signals of selection in natural populations. PMID:21993504

  1. A Two-Photon Fluorescent Probe for Lysosomal Thiols in Live Cells and Tissues

    PubMed Central

    Fan, Jiangli; Han, Zhichao; Kang, Yao; Peng, Xiaojun

    2016-01-01

    Lysosome-specific fluorescent probes are exclusive to elucidate the functions of lysosomal thiols. Moreover, two-photon microscopy offers advantages of less phototoxicity, better three dimensional spatial localization, deeper penetration depth and lower self-absorption. However, such fluorescent probes for thiols are still rare. In this work, an efficient two-photon fluorophore 1,8-naphthalimide-based probe conjugating a 2,4-dinitrobenzenesulfonyl chloride and morpholine was designed and synthesized, which exhibited high selectivity and sensitivity towards lysosomal thiols by turn-on fluorescence method quantitatively and was successfully applied to the imaging of thiols in live cells and tissues by two-photon microscopy. PMID:26794434

  2. A Two-Photon Fluorescent Probe for Lysosomal Thiols in Live Cells and Tissues

    NASA Astrophysics Data System (ADS)

    Fan, Jiangli; Han, Zhichao; Kang, Yao; Peng, Xiaojun

    2016-01-01

    Lysosome-specific fluorescent probes are exclusive to elucidate the functions of lysosomal thiols. Moreover, two-photon microscopy offers advantages of less phototoxicity, better three dimensional spatial localization, deeper penetration depth and lower self-absorption. However, such fluorescent probes for thiols are still rare. In this work, an efficient two-photon fluorophore 1,8-naphthalimide-based probe conjugating a 2,4-dinitrobenzenesulfonyl chloride and morpholine was designed and synthesized, which exhibited high selectivity and sensitivity towards lysosomal thiols by turn-on fluorescence method quantitatively and was successfully applied to the imaging of thiols in live cells and tissues by two-photon microscopy.

  3. How does the genetic assassin select its neuronal target?

    PubMed

    Stevens, James C; Fisher, Elizabeth M C; Mead, Simon

    2011-04-01

    Through many different routes of analysis, including human familial studies and animal models, we are identifying an increasing number of genes that are causative for human neurodegenerative disease and are now in a position for many such disorders to dissect the molecular pathology that gives rise to neuronal death. Yet a paradox remains: The majority of the genes identified cause neurodegeneration in specific neuronal subtypes, but the genes themselves are ubiquitously expressed. Furthermore, the different mutations in the same gene may cause quite different types of neurodegeneration. Something in our understanding of neurodegenerative disease is clearly missing, and we refer to this as the phenomenon of "neuronal targeting." Here we discuss possible explanations for neuronal targeting, why specific neuronal subtypes are vulnerable to specific mutations in ubiquitously expressed genes. PMID:21373885

  4. The safety of ONRAB® in select non-target wildlife.

    PubMed

    Fry, Tricia L; Vandalen, Kaci K; Duncan, Colleen; Vercauteren, Kurt

    2013-08-20

    ONRAB(®) is a recombinant human adenovirus type 5 (HAd5) with the rabies glycoprotein gene incorporated into its genome. ONRAB(®) has been used in Canada as an oral rabies vaccine in target wildlife species such as: red fox (Vulpes vulpes), raccoon (Procyon lotor), and striped skunk (Mepthis mephitis). We evaluated the safety of ONRAB(®) in non-target wildlife species likely to contact the vaccine baits during oral rabies vaccine campaigns in the United States. We investigated the effects of oral inoculation of high titer ONRAB(®), approximately ten times the dose given to target species, in wood rats (Neotoma spp.), eastern cottontail rabbits (Sylvilagus floridanus), Virginia opossums (Didelphis virginiana), eastern wild turkeys (Meleagris gallopavo silvestri), and fox squirrels (Sciurus niger). We performed real-time polymerase chain reaction (PCR) on fecal swabs, oral swabs, and tissues, including lung, liver, kidney, small intestine, large intestine, and when appropriate nasal turbinates, to detect ONRAB(®) DNA from inoculated animals. By seven days post-inoculation, turkeys, opossums, and cottontails had all stopped shedding ONRAB(®) DNA. One wood rat and one fox squirrel still had detectable levels of ONRAB(®) DNA in fecal swabs 14 days post-inoculation. Real-time PCR analysis of the tissues revealed some ONRAB(®) DNA persisting in certain tissues; however, there were no significant gross or histologic lesions associated with ONRAB(®) in any of the species studied. Our results suggest that many non-target species are not likely to be impacted by the distribution of ONRAB(®) as part of oral rabies vaccination programs in the United States. PMID:23831321

  5. Target selection by natural and redesigned PUF proteins.

    PubMed

    Porter, Douglas F; Koh, Yvonne Y; VanVeller, Brett; Raines, Ronald T; Wickens, Marvin

    2015-12-29

    Pumilio/fem-3 mRNA binding factor (PUF) proteins bind RNA with sequence specificity and modularity, and have become exemplary scaffolds in the reengineering of new RNA specificities. Here, we report the in vivo RNA binding sites of wild-type (WT) and reengineered forms of the PUF protein Saccharomyces cerevisiae Puf2p across the transcriptome. Puf2p defines an ancient protein family present throughout fungi, with divergent and distinctive PUF RNA binding domains, RNA-recognition motifs (RRMs), and prion regions. We identify sites in RNA bound to Puf2p in vivo by using two forms of UV cross-linking followed by immunopurification. The protein specifically binds more than 1,000 mRNAs, which contain multiple iterations of UAAU-binding elements. Regions outside the PUF domain, including the RRM, enhance discrimination among targets. Compensatory mutants reveal that one Puf2p molecule binds one UAAU sequence, and align the protein with the RNA site. Based on this architecture, we redesign Puf2p to bind UAAG and identify the targets of this reengineered PUF in vivo. The mutant protein finds its target site in 1,800 RNAs and yields a novel RNA network with a dramatic redistribution of binding elements. The mutant protein exhibits even greater RNA specificity than wild type. The redesigned protein decreases the abundance of RNAs in its redesigned network. These results suggest that reengineering using the PUF scaffold redirects and can even enhance specificity in vivo. PMID:26668354

  6. Selective imaging of adherent targeted ultrasound contrast agents

    PubMed Central

    Zhao, S; Kruse, D E; Ferrara, K W; Dayton, P A

    2007-01-01

    The goal of ultrasonic molecular imaging is the detection of targeted contrast agents bound to receptors on endothelial cells. We propose imaging methods that can distinguish adherent microbubbles from tissue and from freely circulating microbubbles, each of which would otherwise obscure signal from molecularly targeted adherent agents. The methods are based on a harmonic signal model of the returned echoes over a train of pulses. The first method utilizes an ‘image–push–image’ pulse sequence where adhesion of contrast agents is rapidly promoted by acoustic radiation force and the presence of adherent agents is detected by the signal change due to targeted microbubble adhesion. The second method rejects tissue echoes using a spectral high-pass filter. Free agent signal is suppressed by a pulse-to-pulse low-pass filter in both methods. An overlay of the adherent and/or flowing contrast agents on B-mode images can be readily created for anatomical reference. Contrast-to-tissue ratios from adherent microbubbles exceeding 30 dB and 20 dB were achieved for the two methods proposed, respectively. The performance of these algorithms is compared, emphasizing the significance and potential applications in ultrasonic molecular imaging. PMID:17404455

  7. The unfolded protein response selectively targets active smoothened mutants.

    PubMed

    Marada, Suresh; Stewart, Daniel P; Bodeen, William J; Han, Young-Goo; Ogden, Stacey K

    2013-06-01

    The Hedgehog signaling pathway, an essential regulator of developmental patterning, has been implicated in playing causative and survival roles in a range of human cancers. The signal-transducing component of the pathway, Smoothened, has revealed itself to be an efficacious therapeutic target in combating oncogenic signaling. However, therapeutic challenges remain in cases where tumors acquire resistance to Smoothened antagonists, and also in cases where signaling is driven by active Smoothened mutants that exhibit reduced sensitivity to these compounds. We previously demonstrated that active Smoothened mutants are subjected to prolonged endoplasmic reticulum (ER) retention, likely due to their mutations triggering conformation shifts that are detected by ER quality control. We attempted to exploit this biology and demonstrate that deregulated Hedgehog signaling driven by active Smoothened mutants is specifically attenuated by ER stressors that induce the unfolded protein response (UPR). Upon UPR induction, active Smoothened mutants are targeted by ER-associated degradation, resulting in attenuation of inappropriate pathway activity. Accordingly, we found that the UPR agonist thapsigargin attenuated mutant Smoothened-induced phenotypes in vivo in Drosophila melanogaster. Wild-type Smoothened and physiological Hedgehog patterning were not affected, suggesting that UPR modulation may provide a novel therapeutic window to be evaluated for targeting active Smoothened mutants in disease. PMID:23572559

  8. Deacetylation of TFEB promotes fibrillar Aβ degradation by upregulating lysosomal biogenesis in microglia.

    PubMed

    Bao, Jintao; Zheng, Liangjun; Zhang, Qi; Li, Xinya; Zhang, Xuefei; Li, Zeyang; Bai, Xue; Zhang, Zhong; Huo, Wei; Zhao, Xuyang; Shang, Shujiang; Wang, Qingsong; Zhang, Chen; Ji, Jianguo

    2016-06-01

    Microglia play a pivotal role in clearance of Aβ by degrading them in lysosomes, countering amyloid plaque pathogenesis in Alzheimer's disease (AD). Recent evidence suggests that lysosomal dysfunction leads to insufficient elimination of toxic protein aggregates. We tested whether enhancing lysosomal function with transcription factor EB (TFEB), an essential regulator modulating lysosomal pathways, would promote Aβ clearance in microglia. Here we show that microglial expression of TFEB facilitates fibrillar Aβ (fAβ) degradation and reduces deposited amyloid plaques, which are further enhanced by deacetylation of TFEB. Using mass spectrometry analysis, we firstly confirmed acetylation as a previously unreported modification of TFEB and found that SIRT1 directly interacted with and deacetylated TFEB at lysine residue 116. Subsequently, SIRT1 overexpression enhanced lysosomal function and fAβ degradation by upregulating transcriptional levels of TFEB downstream targets, which could be inhibited when TFEB was knocked down. Furthermore, overexpression of deacetylated TFEB at K116R mutant in microglia accelerated intracellular fAβ degradation by stimulating lysosomal biogenesis and greatly reduced the deposited amyloid plaques in the brain slices of APP/PS1 transgenic mice. Our findings reveal that deacetylation of TFEB could regulate lysosomal biogenesis and fAβ degradation, making microglial activation of TFEB a possible strategy for attenuating amyloid plaque deposition in AD. PMID:27209302

  9. Multiple Domains of GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal Enzymes.

    PubMed

    van Meel, Eline; Lee, Wang-Sik; Liu, Lin; Qian, Yi; Doray, Balraj; Kornfeld, Stuart

    2016-04-01

    The Golgi enzyme UDP-GlcNAc:lysosomal enzymeN-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2β2γ2hexamer, mediates the initial step in the addition of the mannose 6-phosphate targeting signal on newly synthesized lysosomal enzymes. This tag serves to direct the lysosomal enzymes to lysosomes. A key property of GlcNAc-1-phosphotransferase is its unique ability to distinguish the 60 or so lysosomal enzymes from the numerous non-lysosomal glycoproteins with identical Asn-linked glycans. In this study, we demonstrate that the two Notch repeat modules and the DNA methyltransferase-associated protein interaction domain of the α subunit are key components of this recognition process. Importantly, different combinations of these domains are involved in binding to individual lysosomal enzymes. This study also identifies the γ-binding site on the α subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the γ subunit is required for optimal phosphorylation. These findings serve to explain how GlcNAc-1-phosphotransferase recognizes a large number of proteins that lack a common structural motif. PMID:26833567

  10. Non-esterified Cholesterol Content of Lysosomes Modulates Susceptibility to Oxidant-induced Permeabilization

    PubMed Central

    Reiners, John J.; Kleinman, Miriam; Kessel, David; Mathieu, Patricia A.; Caruso, Joseph A.

    2010-01-01

    Reactive oxygen species (ROS) can induce lysosomal membrane permeabilization (LMP). Photoirradiation of murine hepatoma 1c1c7 cultures preloaded with the photosensitizer NPe6 generates singlet oxygen within acidic organelles, and causes LMP and the activation of procaspases. Treatment with the cationic amphiphilic drugs (CADs) U18666A, imipramine, and clozapine stimulated the accumulation of filipin-stainable non-esterified cholesterol/sterols in late endosomes/lysosomes, but not in mitochondria. Concentration-response studies demonstrated an inverse relationship between lysosomal non-esterified cholesterol/sterol contents and susceptibility to NPe6 photoirradiation-induced intracellular membrane oxidation, LMP, and activation of procaspases-9 and -3. Similarly, the kinetics of restoration of NPe6 photoirradiation-induced LMP paralleled the losses of lysosomal cholesterol that occurred upon replating U18666A-treated cultures in CAD-free medium. Consistent with the oxidation of lysosomal cholesterol, filipin staining in U18666A-treated cultures progressively decreased with increasing photoirradiating light dose. U18666A also suppressed the inductions of LMP and procaspase activation by exogenously added hydrogen peroxide. However, neither U18666A nor imipramine suppressed the induction of apoptosis by agents that did not directly induce LMP. These studies indicate that lysosomal non-esterified cholesterol/sterol content modulates susceptibility to ROS-induced LMP, and possibly does so by being an alternative target for oxidants and lowering the probability of damage to other lysosomal membrane lipids and/or proteins. PMID:21074609

  11. Signal Transduction and Molecular Targets of Selected Flavonoids

    PubMed Central

    Bode, Ann M.

    2013-01-01

    Abstract Significance: Diet exerts a major influence on the risk for developing cancer and heart disease. Food factors such as flavonoids are alleged to protect cells from premature aging and disease by shielding DNA, proteins, and lipids from oxidative damage. Recent Advances: Our work has focused on clarifying the effects of dietary components on cancer cell proliferation and tumor growth, discovering mechanisms to explain the effects, and identifying the specific molecular targets of these compounds. Our strategy for identifying specific molecular targets of phytochemicals involves the use of supercomputer technology combined with protein crystallography, molecular biology, and experimental laboratory verification. Critical Issues: One of the greatest challenges for scientists is to reduce the accumulation of distortion and half truths reported in the popular media regarding the health benefits of certain foods or food supplements. The use of these is not new, but interest has increased dramatically because of perceived health benefits that are presumably acquired without unpleasant side effects. Flavonoids are touted to exert many beneficial effects in vitro. However, whether they can produce these effects in vivo is disputed. Future Directions: The World Health Organization indicates that one third of all cancer deaths are preventable and that diet is closely linked to prevention. Based on this idea and epidemiological findings, attention has centered on dietary phytochemicals as an effective intervention in cancer development. However, an unequivocal link between diet and cancer has not been established. Thus, identifying cancer preventive dietary agents with specific molecular targets is essential to move forward toward successful cancer prevention. Antioxid. Redox Signal. 19, 163–180. PMID:23458437

  12. Leveraging Big Data to Transform Target Selection and Drug Discovery

    PubMed Central

    Chen, B; Butte, AJ

    2016-01-01

    The advances of genomics, sequencing, and high throughput technologies have led to the creation of large volumes of diverse datasets for drug discovery. Analyzing these datasets to better understand disease and discover new drugs is becoming more common. Recent open data initiatives in basic and clinical research have dramatically increased the types of data available to the public. The past few years have witnessed successful use of big data in many sectors across the whole drug discovery pipeline. In this review, we will highlight the state of the art in leveraging big data to identify new targets, drug indications, and drug response biomarkers in this era of precision medicine. PMID:26659699

  13. Targeting Mitochondria with Avocatin B Induces Selective Leukemia Cell Death.

    PubMed

    Lee, Eric A; Angka, Leonard; Rota, Sarah-Grace; Hanlon, Thomas; Mitchell, Andrew; Hurren, Rose; Wang, Xiao Ming; Gronda, Marcela; Boyaci, Ezel; Bojko, Barbara; Minden, Mark; Sriskanthadevan, Shrivani; Datti, Alessandro; Wrana, Jeffery L; Edginton, Andrea; Pawliszyn, Janusz; Joseph, Jamie W; Quadrilatero, Joe; Schimmer, Aaron D; Spagnuolo, Paul A

    2015-06-15

    Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function. PMID:26077472

  14. A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation.

    PubMed

    Li, Xinran; Rydzewski, Nicholas; Hider, Ahmad; Zhang, Xiaoli; Yang, Junsheng; Wang, Wuyang; Gao, Qiong; Cheng, Xiping; Xu, Haoxing

    2016-04-01

    To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca(2+) channel TRPML1 cause lysosomal storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca(2+)-dependent centripetal movement of lysosomes towards the perinuclear region (where autophagosomes accumulate) following autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca(2+) sensor that associates physically with the minus-end-directed dynactin-dynein motor, while PtdIns(3,5)P(2), a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P(2)-TRPML1-ALG-2-dynein signalling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely to be caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Ca(2+) release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation. PMID:26950892

  15. Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content

    PubMed Central

    Appelqvist, Hanna; Sandin, Linnea; Björnström, Karin; Saftig, Paul; Garner, Brett; Öllinger, Karin; Kågedal, Katarina

    2012-01-01

    Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis. PMID:23166840

  16. Lysosomal Acid Phosphatase Biosynthesis and Dysfunction: A Mini Review Focused on Lysosomal Enzyme Dysfunction in Brain.

    PubMed

    Ashtari, N; Jiao, X; Rahimi-Balaei, M; Amiri, S; Mehr, S E; Yeganeh, B; Marzban, H

    2016-01-01

    Lysosomes are membrane-bound organelles that are responsible for degrading and recycling macromolecules. Lysosomal dysfunction occurs in enzymatic and non-enzymatic deficiencies, which result in abnormal accumulation of materials. Although lysosomal storage disorders affect different organs, the central nervous system is the most vulnerable. Evidence shows the role of lysosomal dysfunction in different neurodegenerative diseases, such as Niemann-Pick Type C disease, juvenile neuronal ceroid lipofuscinosis, Alzheimer's disease and Parkinson's disease. Lysosomal enzymes such as lysosomal acid phosphatase 2 (Acp2) play a critical role in mannose-6-phosphate removal and Acp2 controls molecular and cellular functions in the brain during development and adulthood. Acp2 is essential in cerebellar development, and mutations in this gene cause severe cerebellar neurodevelopmental and neurodegenerative disorders. In this mini-review, we highlight lysosomal dysfunctions in the pathogenesis of neurodevelopmental and/or neurodegenerative diseases with special attention to Acp2 dysfunction. PMID:27132795

  17. Loss of Mitochondrial Function Impairs Lysosomes.

    PubMed

    Demers-Lamarche, Julie; Guillebaud, Gérald; Tlili, Mouna; Todkar, Kiran; Bélanger, Noémie; Grondin, Martine; Nguyen, Angela P; Michel, Jennifer; Germain, Marc

    2016-05-01

    Alterations in mitochondrial function, as observed in neurodegenerative diseases, lead to disrupted energy metabolism and production of damaging reactive oxygen species. Here, we demonstrate that mitochondrial dysfunction also disrupts the structure and function of lysosomes, the main degradation and recycling organelle. Specifically, inhibition of mitochondrial function, following deletion of the mitochondrial protein AIF, OPA1, or PINK1, as well as chemical inhibition of the electron transport chain, impaired lysosomal activity and caused the appearance of large lysosomal vacuoles. Importantly, our results show that lysosomal impairment is dependent on reactive oxygen species. Given that alterations in both mitochondrial function and lysosomal activity are key features of neurodegenerative diseases, this work provides important insights into the etiology of neurodegenerative diseases. PMID:26987902

  18. Computational selection of antibody-drug conjugate targets for breast cancer

    PubMed Central

    Fauteux, François; Hill, Jennifer J.; Jaramillo, Maria L.; Pan, Youlian; Phan, Sieu; Famili, Fazel; O'Connor-McCourt, Maureen

    2016-01-01

    The selection of therapeutic targets is a critical aspect of antibody-drug conjugate research and development. In this study, we applied computational methods to select candidate targets overexpressed in three major breast cancer subtypes as compared with a range of vital organs and tissues. Microarray data corresponding to over 8,000 tissue samples were collected from the public domain. Breast cancer samples were classified into molecular subtypes using an iterative ensemble approach combining six classification algorithms and three feature selection techniques, including a novel kernel density-based method. This feature selection method was used in conjunction with differential expression and subcellular localization information to assemble a primary list of targets. A total of 50 cell membrane targets were identified, including one target for which an antibody-drug conjugate is in clinical use, and six targets for which antibody-drug conjugates are in clinical trials for the treatment of breast cancer and other solid tumors. In addition, 50 extracellular proteins were identified as potential targets for non-internalizing strategies and alternative modalities. Candidate targets linked with the epithelial-to-mesenchymal transition were identified by analyzing differential gene expression in epithelial and mesenchymal tumor-derived cell lines. Overall, these results show that mining human gene expression data has the power to select and prioritize breast cancer antibody-drug conjugate targets, and the potential to lead to new and more effective cancer therapeutics. PMID:26700623

  19. Solid tumor therapy by selectively targeting stromal endothelial cells.

    PubMed

    Liu, Shihui; Liu, Jie; Ma, Qian; Cao, Liu; Fattah, Rasem J; Yu, Zuxi; Bugge, Thomas H; Finkel, Toren; Leppla, Stephen H

    2016-07-12

    Engineered tumor-targeted anthrax lethal toxin proteins have been shown to strongly suppress growth of solid tumors in mice. These toxins work through the native toxin receptors tumor endothelium marker-8 and capillary morphogenesis protein-2 (CMG2), which, in other contexts, have been described as markers of tumor endothelium. We found that neither receptor is required for tumor growth. We further demonstrate that tumor cells, which are resistant to the toxin when grown in vitro, become highly sensitive when implanted in mice. Using a range of tissue-specific loss-of-function and gain-of-function genetic models, we determined that this in vivo toxin sensitivity requires CMG2 expression on host-derived tumor endothelial cells. Notably, engineered toxins were shown to suppress the proliferation of isolated tumor endothelial cells. Finally, we demonstrate that administering an immunosuppressive regimen allows animals to receive multiple toxin dosages and thereby produces a strong and durable antitumor effect. The ability to give repeated doses of toxins, coupled with the specific targeting of tumor endothelial cells, suggests that our strategy should be efficacious for a wide range of solid tumors. PMID:27357689

  20. Disulfiram-induced cytotoxicity and endo-lysosomal sequestration of zinc in breast cancer cells

    PubMed Central

    Wiggins, Helen L.; Wymant, Jennifer M.; Solfa, Francesca; Hiscox, Stephen E.; Taylor, Kathryn M.; Westwell, Andrew D.; Jones, Arwyn T.

    2015-01-01

    Disulfiram, a clinically used alcohol-deterrent has gained prominence as a potential anti-cancer agent due to its impact on copper-dependent processes. Few studies have investigated zinc effects on disulfiram action, despite it having high affinity for this metal. Here we studied the cytotoxic effects of disulfiram in breast cancer cells, and its relationship with both intra and extracellular zinc. MCF-7 and BT474 cancer cell lines gave a striking time-dependent biphasic cytotoxic response between 0.01 and 10 μM disulfiram. Co-incubation of disulfiram with low-level zinc removed this effect, suggesting that availability of extracellular zinc significantly influences disulfiram efficacy. Live-cell confocal microscopy using fluorescent endocytic probes and the zinc dye Fluozin-3 revealed that disulfiram selectively and rapidly increased zinc levels in endo-lysosomes. Disulfiram also caused spatial disorganization of late endosomes and lysosomes, suggesting they are novel targets for this drug. This relationship between disulfiram toxicity and ionophore activity was consolidated via synthesis of a new disulfiram analog and overall we demonstrate a novel mechanism of disulfiram-cytotoxicity with significant clinical implications for future use as a cancer therapeutic. PMID:25557293

  1. Podocytes degrade endocytosed albumin primarily in lysosomes.

    PubMed

    Carson, John M; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and

  2. Podocytes Degrade Endocytosed Albumin Primarily in Lysosomes

    PubMed Central

    Carson, John M.; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B.; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and

  3. Recent advances in gene therapy for lysosomal storage disorders

    PubMed Central

    Rastall, David PW; Amalfitano, Andrea

    2015-01-01

    Lysosomal storage disorders (LSDs) are a group of genetic diseases that result in metabolic derangements of the lysosome. Most LSDs are due to the genetic absence of a single catabolic enzyme, causing accumulation of the enzyme’s substrate within the lysosome. Over time, tissue-specific substrate accumulations result in a spectrum of symptoms and disabilities that vary by LSD. LSDs are promising targets for gene therapy because delivery of a single gene into a small percentage of the appropriate target cells may be sufficient to impact the clinical course of the disease. Recently, there have been several significant advancements in the potential for gene therapy of these disorders, including the first human trials. Future clinical trials will build upon these initial attempts, with an improved understanding of immune system responses to gene therapy, the obstacle that the blood–brain barrier poses for neuropathic LSDs, as well other biological barriers that, when overcome, may facilitate gene therapy for LSDs. In this manuscript, we will highlight the recent innovations in gene therapy for LSDs and discuss the clinical limitations that remain to be overcome, with the goal of fostering an understanding and further development of this important field. PMID:26170711

  4. Lysosomal and vacuolar sorting: not so different after all!

    PubMed

    de Marcos Lousa, Carine; Denecke, Jurgen

    2016-06-15

    Soluble hydrolases represent the main proteins of lysosomes and vacuoles and are essential to sustain the lytic properties of these organelles typical for the eukaryotic organisms. The sorting of these proteins from ER residents and secreted proteins is controlled by highly specific receptors to avoid mislocalization and subsequent cellular damage. After binding their soluble cargo in the early stage of the secretory pathway, receptors rely on their own sorting signals to reach their target organelles for ligand delivery, and to recycle back for a new round of cargo recognition. Although signals in cargo and receptor molecules have been studied in human, yeast and plant model systems, common denominators and specific examples of diversification have not been systematically explored. This review aims to fill this niche by comparing the structure and the function of lysosomal/vacuolar sorting receptors (VSRs) from these three organisms. PMID:27284057

  5. Insights into the molecular basis of a bispecific antibody's target selectivity

    PubMed Central

    Mazor, Yariv; Hansen, Anna; Yang, Chunning; Chowdhury, Partha S; Wang, Jihong; Stephens, Geoffrey; Wu, Herren; Dall’Acqua, William F

    2015-01-01

    Bispecific antibodies constitute a valuable class of therapeutics owing to their ability to bind 2 distinct targets. Dual targeting is thought to enhance biological efficacy, limit escape mechanisms, and increase target selectivity via a strong avidity effect mediated by concurrent binding to both antigens on the surface of the same cell. However, factors that regulate the extent of target selectivity are not well understood. We show that dual targeting alone is not sufficient to promote efficient target selectivity, and report the substantial roles played by the affinity of the individual arms, overall avidity and valence. More particularly, various monovalent bispecific IgGs composed of an anti-CD70 moiety paired with variants of the anti-CD4 mAb ibalizumab were tested for preferential binding and selective depletion of CD4+/CD70+ T cells over cells expressing only one of the target antigens that resulted from antibody dependent cell-mediated cytotoxicity. Variants exhibiting reduced CD4 affinity showed a greater degree of target selectivity, while the overall efficacy of the bispecific molecule was not affected. PMID:25730144

  6. Rapid screening of endonuclease target site preference using a modified bacterial two-plasmid selection.

    PubMed

    Wolfs, Jason M; Kleinstiver, Benjamin P; Edgell, David R

    2014-01-01

    Homing endonucleases and other site-specific endonucleases have potential applications in genome editing, yet efficient targeting requires a thorough understanding of DNA-sequence specificity. Here, we describe a modified two-plasmid genetic selection in Escherichia coli that allows rapid profiling of nucleotide substitutions within a target site of given endonucleases. The selection utilizes a toxic plasmid (pTox) that encodes a DNA gyrase toxin in addition to the endonuclease target site. Cleavage of the toxic plasmid by an endonuclease expressed from a second plasmid (pEndo) facilitates growth under selective conditions. The modified protocol utilizes competent cells harboring the endonuclease expression plasmid into which target site plasmids are transformed. Replica plating on nonselective and selective media plates identifies cleavable and non-cleavable targets. Thus, a library of randomized target sites, or many individual target sites, can be analyzed using a single transformation. Both cleavable and non-cleavable targets can be analyzed by DNA sequencing to gain information about nucleotide preference in the endonuclease's target site. PMID:24510263

  7. Epigenetic Editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes

    PubMed Central

    de Groote, Marloes L.; Verschure, Pernette J.; Rots, Marianne G.

    2012-01-01

    Despite significant advances made in epigenetic research in recent decades, many questions remain unresolved, especially concerning cause and consequence of epigenetic marks with respect to gene expression modulation (GEM). Technologies allowing the targeting of epigenetic enzymes to predetermined DNA sequences are uniquely suited to answer such questions and could provide potent (bio)medical tools. Toward the goal of gene-specific GEM by overwriting epigenetic marks (Epigenetic Editing, EGE), instructive epigenetic marks need to be identified and their writers/erasers should then be fused to gene-specific DNA binding domains. The appropriate epigenetic mark(s) to change in order to efficiently modulate gene expression might have to be validated for any given chromatin context and should be (mitotically) stable. Various insights in such issues have been obtained by sequence-specific targeting of epigenetic enzymes, as is presented in this review. Features of such studies provide critical aspects for further improving EGE. An example of this is the direct effect of the edited mark versus the indirect effect of recruited secondary proteins by targeting epigenetic enzymes (or their domains). Proof-of-concept of expression modulation of an endogenous target gene is emerging from the few EGE studies reported. Apart from its promise in correcting disease-associated epi-mutations, EGE represents a powerful tool to address fundamental epigenetic questions. PMID:23002135

  8. Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases

    PubMed Central

    Oh, Doo-Byoung

    2015-01-01

    Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy. [BMB Reports 2015; 48(8): 438-444] PMID:25999178

  9. Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases.

    PubMed

    Oh, Doo-Byoung

    2015-08-01

    Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy. PMID:25999178

  10. Nanoparticles restore lysosomal acidification defects: Implications for Parkinson and other lysosomal-related diseases.

    PubMed

    Bourdenx, Mathieu; Daniel, Jonathan; Genin, Emilie; Soria, Federico N; Blanchard-Desce, Mireille; Bezard, Erwan; Dehay, Benjamin

    2016-03-01

    Lysosomal impairment causes lysosomal storage disorders (LSD) and is involved in pathogenesis of neurodegenerative diseases, notably Parkinson disease (PD). Strategies enhancing or restoring lysosomal-mediated degradation thus appear as tantalizing disease-modifying therapeutics. Here we demonstrate that poly(DL-lactide-co-glycolide) (PLGA) acidic nanoparticles (aNP) restore impaired lysosomal function in a series of toxin and genetic cellular models of PD, i.e. ATP13A2-mutant or depleted cells or glucocerebrosidase (GBA)-mutant cells, as well as in a genetic model of lysosomal-related myopathy. We show that PLGA-aNP are transported to the lysosome within 24 h, lower lysosomal pH and rescue chloroquine (CQ)-induced toxicity. Re-acidification of defective lysosomes following PLGA-aNP treatment restores lysosomal function in different pathological contexts. Finally, our results show that PLGA-aNP may be detected after intracerebral injection in neurons and attenuate PD-related neurodegeneration in vivo by mechanisms involving a rescue of compromised lysosomes. PMID:26761717

  11. Initial basalt target site selection evaluation for the Mars penetrator drop test

    NASA Technical Reports Server (NTRS)

    Bunch, T. E.; Quaide, W. L.; Polkowski, G.

    1976-01-01

    Potential basalt target sites for an air drop penetrator test were described and the criteria involved in site selection were discussed. A summary of the background field geology and recommendations for optimum sites are also presented.

  12. Target-selective degradation of proteins by porphyrins upon visible photo-irradiation.

    PubMed

    Tanimoto, Shuho; Matsumura, Shuichi; Toshima, Kazunobu

    2008-08-21

    A porphyrin derivative effectively and selectively degraded the target transcription factor, human estrogen receptor-alpha (hER-alpha), upon visible light irradiation, in the absence of additives and under neutral conditions. PMID:18665297

  13. Salience-Based Selection: Attentional Capture by Distractors Less Salient Than the Target

    PubMed Central

    Goschy, Harriet; Müller, Hermann Joseph

    2013-01-01

    Current accounts of attentional capture predict the most salient stimulus to be invariably selected first. However, existing salience and visual search models assume noise in the map computation or selection process. Consequently, they predict the first selection to be stochastically dependent on salience, implying that attention could even be captured first by the second most salient (instead of the most salient) stimulus in the field. Yet, capture by less salient distractors has not been reported and salience-based selection accounts claim that the distractor has to be more salient in order to capture attention. We tested this prediction using an empirical and modeling approach of the visual search distractor paradigm. For the empirical part, we manipulated salience of target and distractor parametrically and measured reaction time interference when a distractor was present compared to absent. Reaction time interference was strongly correlated with distractor salience relative to the target. Moreover, even distractors less salient than the target captured attention, as measured by reaction time interference and oculomotor capture. In the modeling part, we simulated first selection in the distractor paradigm using behavioral measures of salience and considering the time course of selection including noise. We were able to replicate the result pattern we obtained in the empirical part. We conclude that each salience value follows a specific selection time distribution and attentional capture occurs when the selection time distributions of target and distractor overlap. Hence, selection is stochastic in nature and attentional capture occurs with a certain probability depending on relative salience. PMID:23382820

  14. Evolutionary dynamics and population control during in vitro selection and amplification with multiple targets.

    PubMed Central

    Shi, Hua; Fan, Xiaochun; Ni, Zhuoyu; Lis, John T

    2002-01-01

    Iterative cycles of in vitro selection and amplification allow rare functional nucleic acid molecules, aptamers, to be isolated from large sequence pools. Here we present an analysis of the progression of a selection experiment that simultaneously yielded two families of RNA aptamers against two disparate targets: the intended target protein (B52/SRp55) and the partitioning matrix. We tracked the sequence abundance and binding activity to reveal the enrichment of the aptamers through successive generations of selected pools. The two aptamer families showed distinct trajectories of evolution, as did members within a single family. We also developed a method to control the relative abundance of an aptamer family in selected pools. This method, involving specific ribonuclease digestion, can be used to reduce the background selection for aptamers that bind the matrix. Additionally, it can be used to isolate a full spectrum of aptamers in a sequential and exhaustive manner for all the different targets in a mixture. PMID:12458799

  15. Joint Skewness and Its Application in Unsupervised Band Selection for Small Target Detection

    NASA Astrophysics Data System (ADS)

    Geng, Xiurui; Sun, Kang; Ji, Luyan; Tang, Hairong; Zhao, Yongchao

    2015-04-01

    Few band selection methods are specially designed for small target detection. It is well known that the information of small targets is most likely contained in non-Gaussian bands, where small targets are more easily separated from the background. On the other hand, correlation of band set also plays an important role in the small target detection. When the selected bands are highly correlated, it will be unbeneficial for the subsequent detection. However, the existing non-Gaussianity-based band selection methods have not taken the correlation of bands into account, which generally result in high correlation of obtained bands. In this paper, combining the third-order (third-order tensor) and second-order (correlation) statistics of bands, we define a new concept, named joint skewness, for multivariate data. Moreover, we also propose an easy-to-implement approach to estimate this index based on high-order singular value decomposition (HOSVD). Based on the definition of joint skewness, we present an unsupervised band selection for small target detection for hyperspectral data, named joint skewness band selection (JSBS). The evaluation results demonstrate that the bands selected by JSBS are very effective in terms of small target detection.

  16. Joint Skewness and Its Application in Unsupervised Band Selection for Small Target Detection

    PubMed Central

    Geng, Xiurui; Sun, Kang; Ji, Luyan; Tang, Hairong; Zhao, Yongchao

    2015-01-01

    Few band selection methods are specially designed for small target detection. It is well known that the information of small targets is most likely contained in non-Gaussian bands, where small targets are more easily separated from the background. On the other hand, correlation of band set also plays an important role in the small target detection. When the selected bands are highly correlated, it will be unbeneficial for the subsequent detection. However, the existing non-Gaussianity-based band selection methods have not taken the correlation of bands into account, which generally result in high correlation of obtained bands. In this paper, combining the third-order (third-order tensor) and second-order (correlation) statistics of bands, we define a new concept, named joint skewness, for multivariate data. Moreover, we also propose an easy-to-implement approach to estimate this index based on high-order singular value decomposition (HOSVD). Based on the definition of joint skewness, we present an unsupervised band selection for small target detection for hyperspectral data, named joint skewness band selection (JSBS). The evaluation results demonstrate that the bands selected by JSBS are very effective in terms of small target detection. PMID:25873018

  17. The lh3 Glycosyltransferase Directs Target-Selective Peripheral Nerve Regeneration.

    PubMed

    Isaacman-Beck, Jesse; Schneider, Valerie; Franzini-Armstrong, Clara; Granato, Michael

    2015-11-18

    Functional PNS regeneration requires injured axons to return to their original synaptic targets, yet the mechanisms underlying target-selective regeneration have remained elusive. Using live-cell imaging in zebrafish we find that regenerating motor axons exhibit a strong preference for their original muscle territory and that axons probe both correct and incorrect trajectories extensively before selecting their original path. We show that this process requires the glycosyltransferase lh3 and that post-injury expression of lh3 in Schwann cells is sufficient to restore target-selective regeneration. Moreover, we demonstrate that Schwann cells neighboring the transection site express the lh3 substrate collagen4a5 and that during regeneration collagen4a5 destabilizes axons probing inappropriate trajectories to ensure target-selective regeneration, possibly through the axonal repellant slit1a. Our results demonstrate that selective ECM components match subpopulations of regenerating axons with their original targets and reveal a previously unappreciated mechanism that conveys synaptic target selection to regenerating axons in vivo. VIDEO ABSTRACT. PMID:26549330

  18. Non-targeted evaluation of selectivity of water-compatible class selective adsorbents for the analysis of steroids in wastewater.

    PubMed

    Kopperi, Matias; Riekkola, Marja-Liisa

    2016-05-12

    Selective adsorbents for solid-phase extraction are needed to meet the low concentration requirements of new environmental quality standard directives, especially for the analysis of estrogens in wastewater. In this work, bulk polymerization procedures were first optimized for the synthesis of non-imprinted polymers (NIP) with low non-specific adsorption of nonpolar compounds in aqueous environments. Water-compatible molecularly imprinted polymers (MIP) were then synthetized by increasing the selectivity of the polymer towards steroids with a testosterone template (average imprinting factor > 10). In addition, the affinity of synthetized entrapped β-cyclodextrin-epichlorohydrin polymers (ECD) towards steroids was clarified. The polymers were applied to the extraction of spiked wastewater effluent samples and their performance compared to commercially available adsorbents. The selectivity of the studied adsorbents was evaluated utilizing liquid chromatography ‒ mass spectrometry as well as comprehensive two-dimensional gas chromatography ‒ time-of-flight mass spectrometry. Affinity between adsorbents and steroids as well as matrix removal potential were measured with targeted methodologies, and two novel non-targeted methodologies were proposed to quantitatively measure adsorbent selectivity by utilizing chemometrics. Semi-quantitative selectivity was measured from the ratio of peak areas between steroidal and other compounds. Semi-qualitative selectivity was calculated from the ratio between the number of tentatively identified steroidal and other compounds. The synthetized polymers provided good matrix removal potential (ion suppression 15-30%) and semi-qualitative selectivity (∼4 units) compared to the commercial adsorbents (ion suppression 45-80%, selectivity < 3 units). Simple non-targeted approaches provided a novel method of quantifying the selectivity of extraction. PMID:27114222

  19. The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes

    SciTech Connect

    Zeng, Jibin; Racicott, Jesse; Morales, Carlos R.

    2009-11-01

    Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM{sub 2}AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin

  20. Robust Ground Target Detection by SAR and IR Sensor Fusion Using Adaboost-Based Feature Selection.

    PubMed

    Kim, Sungho; Song, Woo-Jin; Kim, So-Hyun

    2016-01-01

    Long-range ground targets are difficult to detect in a noisy cluttered environment using either synthetic aperture radar (SAR) images or infrared (IR) images. SAR-based detectors can provide a high detection rate with a high false alarm rate to background scatter noise. IR-based approaches can detect hot targets but are affected strongly by the weather conditions. This paper proposes a novel target detection method by decision-level SAR and IR fusion using an Adaboost-based machine learning scheme to achieve a high detection rate and low false alarm rate. The proposed method consists of individual detection, registration, and fusion architecture. This paper presents a single framework of a SAR and IR target detection method using modified Boolean map visual theory (modBMVT) and feature-selection based fusion. Previous methods applied different algorithms to detect SAR and IR targets because of the different physical image characteristics. One method that is optimized for IR target detection produces unsuccessful results in SAR target detection. This study examined the image characteristics and proposed a unified SAR and IR target detection method by inserting a median local average filter (MLAF, pre-filter) and an asymmetric morphological closing filter (AMCF, post-filter) into the BMVT. The original BMVT was optimized to detect small infrared targets. The proposed modBMVT can remove the thermal and scatter noise by the MLAF and detect extended targets by attaching the AMCF after the BMVT. Heterogeneous SAR and IR images were registered automatically using the proposed RANdom SAmple Region Consensus (RANSARC)-based homography optimization after a brute-force correspondence search using the detected target centers and regions. The final targets were detected by feature-selection based sensor fusion using Adaboost. The proposed method showed good SAR and IR target detection performance through feature selection-based decision fusion on a synthetic database generated

  1. Robust Ground Target Detection by SAR and IR Sensor Fusion Using Adaboost-Based Feature Selection

    PubMed Central

    Kim, Sungho; Song, Woo-Jin; Kim, So-Hyun

    2016-01-01

    Long-range ground targets are difficult to detect in a noisy cluttered environment using either synthetic aperture radar (SAR) images or infrared (IR) images. SAR-based detectors can provide a high detection rate with a high false alarm rate to background scatter noise. IR-based approaches can detect hot targets but are affected strongly by the weather conditions. This paper proposes a novel target detection method by decision-level SAR and IR fusion using an Adaboost-based machine learning scheme to achieve a high detection rate and low false alarm rate. The proposed method consists of individual detection, registration, and fusion architecture. This paper presents a single framework of a SAR and IR target detection method using modified Boolean map visual theory (modBMVT) and feature-selection based fusion. Previous methods applied different algorithms to detect SAR and IR targets because of the different physical image characteristics. One method that is optimized for IR target detection produces unsuccessful results in SAR target detection. This study examined the image characteristics and proposed a unified SAR and IR target detection method by inserting a median local average filter (MLAF, pre-filter) and an asymmetric morphological closing filter (AMCF, post-filter) into the BMVT. The original BMVT was optimized to detect small infrared targets. The proposed modBMVT can remove the thermal and scatter noise by the MLAF and detect extended targets by attaching the AMCF after the BMVT. Heterogeneous SAR and IR images were registered automatically using the proposed RANdom SAmple Region Consensus (RANSARC)-based homography optimization after a brute-force correspondence search using the detected target centers and regions. The final targets were detected by feature-selection based sensor fusion using Adaboost. The proposed method showed good SAR and IR target detection performance through feature selection-based decision fusion on a synthetic database generated

  2. Targeting autophagy in neurodegenerative diseases.

    PubMed

    Vidal, René L; Matus, Soledad; Bargsted, Leslie; Hetz, Claudio

    2014-11-01

    The most prevalent neurodegenerative disorders involve protein misfolding and the aggregation of specific proteins. Autophagy is becoming an attractive target to treat neurodegenerative disorders through the selective degradation of abnormally folded proteins by the lysosomal pathway. However, accumulating evidence indicates that autophagy impairment at different regulatory steps may contribute to the neurodegenerative process. Thus, a complex scenario is emerging where autophagy may play a dual role in neurodegenerative diseases by causing the downstream effect of promoting the degradation of misfolded proteins and an upstream effect where its deregulation perturbs global proteostasis, contributing to disease progression. Challenges in the future development of therapeutic strategies to target the autophagy pathway are discussed. PMID:25270767

  3. Neuraminidase of Influenza A Virus Binds Lysosome-Associated Membrane Proteins Directly and Induces Lysosome Rupture

    PubMed Central

    Ju, Xiangwu; Yan, Yiwu; Liu, Qiang; Li, Ning; Sheng, Miaomiao; Zhang, Lifang; Li, Xiao; Liang, Zhu; Huang, Fengming; Liu, Kangtai; Zhao, Yan; Zhang, Yanxu; Zou, Zhen; Du, Jianchao; Zhong, Ying; Zhou, Huandi; Yang, Peng; Lu, Huijun; Tian, Mingyao; Li, Dangsheng; Zhang, Jianming

    2015-01-01

    ABSTRACT As a recycling center, lysosomes are filled with numerous acid hydrolase enzymes that break down waste materials and invading pathogens. Recently, lysosomal cell death has been defined as “lysosomal membrane permeabilization and the consequent leakage of lysosome contents into cytosol.” Here, we show that the neuraminidase (NA) of H5N1 influenza A virus markedly deglycosylates and degrades lysosome-associated membrane proteins (LAMPs; the most abundant membrane proteins of lysosome), which induces lysosomal rupture, and finally leads to cell death of alveolar epithelial carcinoma A549 cells and human tracheal epithelial cells. The NA inhibitors peramivir and zanamivir could effectively block the deglycosylation of LAMPs, inhibit the virus cell entry, and prevent cell death induced by the H5N1 influenza virus. The NA of seasonal H1N1 virus, however, does not share these characteristics. Our findings not only reveal a novel role of NA in the early stage of the H5N1 influenza virus life cycle but also elucidate the molecular mechanism of lysosomal rupture crucial for influenza virus induced cell death. IMPORTANCE The integrity of lysosomes is vital for maintaining cell homeostasis, cellular defense and clearance of invading pathogens. This study shows that the H5N1 influenza virus could induce lysosomal rupture through deglycosylating lysosome-associated membrane proteins (LAMPs) mediated by the neuraminidase activity of NA protein. NA inhibitors such as peramivir and zanamivir could inhibit the deglycosylation of LAMPs and protect lysosomes, which also further interferes with the H5N1 influenza virus infection at early stage of life cycle. This work is significant because it presents new concepts for NA's function, as well as for influenza inhibitors' mechanism of action, and could partially explain the high mortality and high viral load after H5N1 virus infection in human beings and why NA inhibitors have more potent therapeutic effects for lethal avian

  4. Concept for On-Board Safe Landing Target Selection and Landing for the Mars 2020 Mission

    NASA Astrophysics Data System (ADS)

    Brugarolas, P.; Chen, A.; Johnson, A.; Casoliva, J.; Singh, G.; Stehura, A.; Way, D.; Dutta, S.

    2014-06-01

    We present a concept for a potential enhancement to Mars 2020 to enable landing on hazardous landing sites. It adds to MSL-EDL the capability to select and divert to a safe site through on-board terrain relative localization and target selection.

  5. Cancer Cell-Selective In Vivo Near Infrared Photoimmunotherapy Targeting Specific Membrane Molecules

    PubMed Central

    Mitsunaga, Makoto; Ogawa, Mikako; Kosaka, Nobuyuki; Rosenblum, Lauren T.; Choyke, Peter L; Kobayashi, Hisataka

    2011-01-01

    Three major modes of cancer therapies, surgery, radiation and chemotherapy, have been the mainstay of modern oncologic therapy. To minimize side effects, molecular targeted cancer therapies including armed antibody therapy have been developed with limited success. In this study, we developed a new type of molecular targeted cancer therapy, photoimmunotherapy (PIT), employing a target-specific photosensitizer based on a near infrared (NIR) phthalocyanine dye, IR700, conjugated to monoclonal antibodies (MAb) targeting epidermal growth factor receptors (EGFR). Cell death was induced immediately only upon irradiating, MAb-IR700 bound, target cells with NIR light. In vivo tumor shrinkage after irradiation with NIR light was observed only in target EGFR-expressing cells. The MAb-IR700 conjugates were most effective when bound to the cell membrane, producing no phototoxicity when not bound, suggesting a different mechanism for PIT compared with conventional photodynamic therapies. Target selective PIT enables treatment of cancer based on MAb binding on the cell membrane. PMID:22057348

  6. PPARα in lysosomal biogenesis: A perspective

    PubMed Central

    Ghosh, Arunava; Pahan, Kalipada

    2016-01-01

    Lysosomes are membrane-bound vesicles containing hydrolytic enzymes, ubiquitously present in all eukaryotic cells. Classically considered to be central to the cellular waste management machinery, recent studies revealed the role of lysosomes in a wide array of cellular processes like, degradation, cellular development, programmed cell death, secretion, plasma membrane repair, nutritional responses, and lipid metabolism. We recently studied the regulation of TFEB, considered to be the master regulator of lysosomal biogenesis, by activation of peroxisomal proliferator activated receptor α (PPARα), one of the key regulators of lipid metabolism. In this article, we discuss how the recent finding could be put in to perspective with the previous findings that relate lysosomal biogenesis to lipid metabolism, and comment on the possibility of a bi-directional interplay between these two distinct cellular processes upon activation of PPARα. PMID:26621249

  7. Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

    PubMed Central

    Polishchuk, Elena V.; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; van IJzendoorn, Sven C.D.; Chan, Jefferson; Chang, Christopher J.; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, Roman S.

    2014-01-01

    Summary Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease. PMID:24909901

  8. Wilson disease protein ATP7B utilizes lysosomal exocytosis to maintain copper homeostasis.

    PubMed

    Polishchuk, Elena V; Concilli, Mafalda; Iacobacci, Simona; Chesi, Giancarlo; Pastore, Nunzia; Piccolo, Pasquale; Paladino, Simona; Baldantoni, Daniela; van IJzendoorn, Sven C D; Chan, Jefferson; Chang, Christopher J; Amoresano, Angela; Pane, Francesca; Pucci, Piero; Tarallo, Antonietta; Parenti, Giancarlo; Brunetti-Pierri, Nicola; Settembre, Carmine; Ballabio, Andrea; Polishchuk, Roman S

    2014-06-23

    Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease. PMID:24909901

  9. Lysosomal storage diseases and the heat shock response: convergences and therapeutic opportunities

    PubMed Central

    Ingemann, Linda; Kirkegaard, Thomas

    2014-01-01

    Lysosomes play a vital role in the maintenance of cellular homeostasis through the recycling of cell constituents, a key metabolic function which is highly dependent on the correct function of the lysosomal hydrolases and membrane proteins, as well as correct membrane lipid stoichiometry and composition. The critical role of lysosomal functionality is evident from the severity of the diseases in which the primary lesion is a genetically defined loss-of-function of lysosomal hydrolases or membrane proteins. This group of diseases, known as lysosomal storage diseases (LSDs), number more than 50 and are associated with severe neurodegeneration, systemic disease, and early death, with only a handful of the diseases having a therapeutic option. Another key homeostatic system is the metabolic stress response or heat shock response (HSR), which is induced in response to a number of physiological and pathological stresses, such as protein misfolding and aggregation, endoplasmic reticulum stress, oxidative stress, nutrient deprivation, elevated temperature, viral infections, and various acute traumas. Importantly, the HSR and its cardinal members of the heat shock protein 70 family has been shown to protect against a number of degenerative diseases, including severe diseases of the nervous system. The cytoprotective actions of the HSR also include processes involving the lysosomal system, such as cell death, autophagy, and protection against lysosomal membrane permeabilization, and have shown promise in a number of LSDs. This review seeks to describe the emerging understanding of the interplay between these two essential metabolic systems, the lysosomes and the HSR, with a particular focus on their potential as a therapeutic target for LSDs. PMID:24837749

  10. The BH3 Mimetic Obatoclax Accumulates in Lysosomes and Causes Their Alkalinization.

    PubMed

    Stamelos, Vasileios A; Fisher, Natalie; Bamrah, Harnoor; Voisey, Carolyn; Price, Joshua C; Farrell, William E; Redman, Charles W; Richardson, Alan

    2016-01-01

    Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive preclinical and clinical evaluation as a cancer therapeutic. Despite this, it is clear that obatoclax has additional pharmacological effects that contribute to its cytotoxic activity. It has been claimed that obatoclax, either alone or in combination with other molecularly targeted therapeutics, induces an autophagic form of cell death. In addition, obatoclax has been shown to inhibit lysosomal function, but the mechanism of this has not been elucidated. We have evaluated the mechanism of action of obatoclax in eight ovarian cancer cell lines. Consistent with its function as a BH3 mimetic, obatoclax induced apoptosis in three cell lines. However, in the remaining cell lines another form of cell death was evident because caspase activation and PARP cleavage were not observed. Obatoclax also failed to show synergy with carboplatin and paclitaxel, chemotherapeutic agents which we have previously shown to be synergistic with authentic Bcl-2 family antagonists. Obatoclax induced a profound accumulation of LC-3 but knockdown of Atg-5 or beclin had only minor effects on the activity of obatoclax in cell growth assays suggesting that the inhibition of lysosomal function rather than stimulation of autophagy may play a more prominent role in these cells. To evaluate how obatoclax inhibits lysosomal function, confocal microscopy studies were conducted which demonstrated that obatoclax, which contains two basic pyrrole groups, accumulates in lysosomes. Studies using pH sensitive dyes demonstrated that obatoclax induced lysosomal alkalinization. Furthermore, obatoclax was synergistic in cell growth/survival assays with bafilomycin and chloroquine, two other drugs which cause lysosomal alkalinization. These studies explain, for the first time, how obatoclax inhibits lysosomal function and suggest that lysosomal

  11. The BH3 Mimetic Obatoclax Accumulates in Lysosomes and Causes Their Alkalinization

    PubMed Central

    Stamelos, Vasileios A.; Fisher, Natalie; Bamrah, Harnoor; Voisey, Carolyn; Price, Joshua C.; Farrell, William E.; Redman, Charles W.; Richardson, Alan

    2016-01-01

    Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive preclinical and clinical evaluation as a cancer therapeutic. Despite this, it is clear that obatoclax has additional pharmacological effects that contribute to its cytotoxic activity. It has been claimed that obatoclax, either alone or in combination with other molecularly targeted therapeutics, induces an autophagic form of cell death. In addition, obatoclax has been shown to inhibit lysosomal function, but the mechanism of this has not been elucidated. We have evaluated the mechanism of action of obatoclax in eight ovarian cancer cell lines. Consistent with its function as a BH3 mimetic, obatoclax induced apoptosis in three cell lines. However, in the remaining cell lines another form of cell death was evident because caspase activation and PARP cleavage were not observed. Obatoclax also failed to show synergy with carboplatin and paclitaxel, chemotherapeutic agents which we have previously shown to be synergistic with authentic Bcl-2 family antagonists. Obatoclax induced a profound accumulation of LC-3 but knockdown of Atg-5 or beclin had only minor effects on the activity of obatoclax in cell growth assays suggesting that the inhibition of lysosomal function rather than stimulation of autophagy may play a more prominent role in these cells. To evaluate how obatoclax inhibits lysosomal function, confocal microscopy studies were conducted which demonstrated that obatoclax, which contains two basic pyrrole groups, accumulates in lysosomes. Studies using pH sensitive dyes demonstrated that obatoclax induced lysosomal alkalinization. Furthermore, obatoclax was synergistic in cell growth/survival assays with bafilomycin and chloroquine, two other drugs which cause lysosomal alkalinization. These studies explain, for the first time, how obatoclax inhibits lysosomal function and suggest that lysosomal

  12. Lysosomal destabilization in p53-induced apoptosis

    PubMed Central

    Yuan, Xi-Ming; Li, Wei; Dalen, Helge; Lotem, Joseph; Kama, Rachel; Sachs, Leo; Brunk, Ulf T.

    2002-01-01

    The tumor suppressor wild-type p53 can induce apoptosis. M1-t-p53 myeloid leukemic cells have a temperature-sensitive p53 protein that changes its conformation to wild-type p53 after transfer from 37°C to 32°C. We have now found that these cells showed an early lysosomal rupture after transfer to 32°C. Mitochondrial damage, including decreased membrane potential and release of cytochrome c, and the appearance of apoptotic cells occurred later. Lysosomal rupture, mitochondrial damage, and apoptosis were all inhibited by the cytokine IL-6. Some other compounds can also inhibit apoptosis induced by p53. The protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited the decrease in mitochondrial membrane potential and cytochrome c release, the Ca2+-ATPase inhibitor thapsigargin inhibited only cytochrome c release, and the antioxidant butylated hydroxyanisole inhibited only the decrease in mitochondrial membrane potential. In contrast to IL-6, these other compounds that inhibited some of the later occurring mitochondrial damage did not inhibit the earlier p53-induced lysosomal damage. The results indicate that apoptosis is induced by p53 through a lysosomal-mitochondrial pathway that is initiated by lysosomal destabilization, and that this pathway can be dissected by using different apoptosis inhibitors. These findings on the induction of p53-induced lysosomal destabilization can also help to formulate new therapies for diseases with apoptotic disorders. PMID:11959917

  13. Lysosomal Storage Disorders in the Newborn

    PubMed Central

    Staretz-Chacham, Orna; Lang, Tess C.; LaMarca, Mary E.; Krasnewich, Donna; Sidransky, Ellen

    2009-01-01

    Lysosomal storage disorders are rare inborn errors of metabolism, with a combined incidence of 1 in 1500 to 7000 live births. These relatively rare disorders are seldom considered when evaluating a sick newborn. A significant number of the >50 different lysosomal storage disorders, however, do manifest in the neonatal period and should be part of the differential diagnosis of several perinatal phenotypes. We review the earliest clinical features, diagnostic tests, and treatment options for lysosomal storage disorders that can present in the newborn. Although many of the lysosomal storage disorders are characterized by a range in phenotypes, the focus of this review is on the specific symptoms and clinical findings that present in the perinatal period, including neurologic, respiratory, endocrine, and cardiovascular manifestations, dysmorphic features, hepatosplenomegaly, skin or ocular involvement, and hydrops fetalis/congenital ascites. A greater awareness of these features may help to reduce misdiagnosis and promote the early detection of lysosomal storage disorders. Implementing therapy at the earliest stage possible is crucial for several of the lysosomal storage disorders; hence, an early appreciation of these disorders by physicians who treat newborns is essential. PMID:19336380

  14. Activator of G-Protein Signaling 3-Induced Lysosomal Biogenesis Limits Macrophage Intracellular Bacterial Infection.

    PubMed

    Vural, Ali; Al-Khodor, Souhaila; Cheung, Gordon Y C; Shi, Chong-Shan; Srinivasan, Lalitha; McQuiston, Travis J; Hwang, Il-Young; Yeh, Anthony J; Blumer, Joe B; Briken, Volker; Williamson, Peter R; Otto, Michael; Fraser, Iain D C; Kehrl, John H

    2016-01-15

    Many intracellular pathogens cause disease by subverting macrophage innate immune defense mechanisms. Intracellular pathogens actively avoid delivery to or directly target lysosomes, the major intracellular degradative organelle. In this article, we demonstrate that activator of G-protein signaling 3 (AGS3), an LPS-inducible protein in macrophages, affects both lysosomal biogenesis and activity. AGS3 binds the Gi family of G proteins via its G-protein regulatory (GoLoco) motif, stabilizing the Gα subunit in its GDP-bound conformation. Elevated AGS3 levels in macrophages limited the activity of the mammalian target of rapamycin pathway, a sensor of cellular nutritional status. This triggered the nuclear translocation of transcription factor EB, a known activator of lysosomal gene transcription. In contrast, AGS3-deficient macrophages had increased mammalian target of rapamycin activity, reduced transcription factor EB activity, and a lower lysosomal mass. High levels of AGS3 in macrophages enhanced their resistance to infection by Burkholderia cenocepacia J2315, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus, whereas AGS3-deficient macrophages were more susceptible. We conclude that LPS priming increases AGS3 levels, which enhances lysosomal function and increases the capacity of macrophages to eliminate intracellular pathogens. PMID:26667172

  15. Microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta.

    PubMed Central

    Coleman, R A; Haynes, E B

    1984-01-01

    The placenta plays a major role in transporting lipid to the developing foetus. Since previous studies have suggested that placental lipid transport involves intermediate esterification steps, we investigated selected microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta. Between gestational days 10 and 14, microsomal phosphatidic acid phosphatase specific activity was 6-fold greater than the activity in adult rat liver. Phosphatidic acid phosphatase activity decreased 50% on day 15. Studies employing several different phosphorylated substrates indicated a high degree of substrate specificity. Lysosomal triacylglycerol lipase and cholesterol esterase activities decreased about 50% between days 15 and 18, then rose late in gestation. No changes were observed in the specific activities of fatty acid: CoA ligase, glycerolphosphate acyltransferase, lysophosphatidate acyltransferase, diacylglycerol acyltransferase or diacylglycerol cholinephosphotransferase during the final 12 days of gestation. Kinetic observations (competitive inhibition by alternative substrates, pH-dependence and thermal inactivation) were consistent with the hypothesis that glycerol phosphate and dihydroxyacetone phosphate can be acylated by a single microsomal enzyme in placenta. Except for fatty acid: CoA ligase, the activities of microsomal and lysosomal enzymes of triacylglycerol metabolism were comparable with those in adult rat liver. These observations are consistent with physiological studies suggesting that triacylglycerol synthetic and degradative pathways are very active in rat placenta. PMID:6696738

  16. PLEKHM1: Adapting to life at the lysosome

    PubMed Central

    McEwan, David G; Dikic, Ivan

    2015-01-01

    The endosomal system and autophagy are 2 intertwined pathways that share a number of common protein factors as well as a final destination, the lysosome. Identification of adaptor platforms that can link both pathways are of particular importance, as they serve as common nodes that can coordinate the different trafficking arms of the endolysosomal system. Using a mass spectrometry approach to identify interaction partners of active (GTP-bound) RAB7, the late endosome/lysosome GTPase, and yeast 2-hybrid screening to identify LC3/GABARAP interaction partners we discovered the multivalent adaptor protein PLEKHM1. We discovered a highly conserved LC3-interaction region (LIR) between 2 PH domains of PLEKHM1 that mediated direct binding to all LC3/GABARAP family members. Subsequent mass spectrometry analysis of PLEKHM1 precipitated from cells revealed the HOPS (homotypic fusion and protein sorting) complex as a prominent interaction partner. Functionally, depletion of PLEKHM1, HOPS, or RAB7 results in decreased autophagosome-lysosome fusion. In Plekhm1 knockout (KO) mouse embryonic fibroblasts (MEFs) we observed increased lipidated LC3B, decreased colocalization between LC3B and LAMP1 under amino acid starvation conditions and decreased autolysosome formation. Finally, PLEKHM1 binding to LC3-positive autophagosomes was also essential for selective autophagy pathways, as shown by clearance of puromycin-aggregates, in a PLEKHM1-LIR-dependent manner. Overall, we have identified PLEKHM1 as an endolysosomal adaptor platform that acts as a central hub to integrate endocytic and autophagic pathways at the lysosome. PMID:25905573

  17. Para-toluenesulfonamide induces tongue squamous cell carcinoma cell death through disturbing lysosomal stability.

    PubMed

    Liu, Zhe; Liang, Chenyuan; Zhang, Zhuoyuan; Pan, Jian; Xia, Hui; Zhong, Nanshan; Li, Longjiang

    2015-11-01

    Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors. In the present study, we investigated the inhibitory effects of PTS on tongue squamous cell carcinoma (Tca-8113) and explored the lysosomal and mitochondrial changes after PTS treatment in vitro. High-performance liquid chromatography showed that PTS selectively accumulated in Tca-8113 cells with a relatively low concentration in normal fibroblasts. Next, the effects of PTS on cell viability, invasion, and cell death were determined. PTS significantly inhibited Tca-8113 cells' viability and invasive ability with increased cancer cell death. Flow cytometric analysis and the lactate dehydrogenase release assay showed that PTS induced cancer cell death by activating apoptosis and necrosis simultaneously. Morphological changes, such as cellular shrinkage, nuclear condensation as well as formation of apoptotic body and secondary lysosomes, were observed, indicating that PTS might induce cell death through disturbing lysosomal stability. Lysosomal integrity assay and western blot showed that PTS increased lysosomal membrane permeabilization associated with activation of lysosomal cathepsin B. Finally, PTS was shown to inhibit ATP biosynthesis and induce the release of mitochondrial cytochrome c. Therefore, our findings provide a novel insight into the use of PTS in cancer therapy. PMID:26302210

  18. Lysosomes and α-synuclein form a dangerous duet leading to neuronal cell death

    PubMed Central

    Bourdenx, Mathieu; Bezard, Erwan; Dehay, Benjamin

    2014-01-01

    Neurodegenerative diseases are (i) characterized by a selective neuronal vulnerability to degeneration in specific brain regions; and (ii) likely to be caused by disease-specific protein misfolding. Parkinson’s disease (PD) is characterized by the presence of intraneuronal proteinacious cytoplasmic inclusions, called Lewy Bodies (LB). α-Synuclein, an aggregation prone protein, has been identified as a major protein component of LB and the causative for autosomal dominant PD. Lysosomes are responsible for the clearance of long-lived proteins, such as α-synuclein, and for the removal of old or damaged organelles, such as mitochondria. Interestingly, PD-linked α-synuclein mutants and dopamine-modified wild-type α-synuclein block its own degradation, which result in insufficient clearance, leading to its aggregation and cell toxicity. Moreover, both lysosomes and lysosomal proteases have been found to be involved in the activation of certain cell death pathways. Interestingly, lysosomal alterations are observed in the brains of patients suffering from sporadic PD and also in toxic and genetic rodent models of PD-related neurodegeneration. All these events have unraveled a causal link between lysosomal impairment, α-synuclein accumulation, and neurotoxicity. In this review, we emphasize the pathophysiological mechanisms connecting α-synuclein and lysosomal dysfunction in neuronal cell death. PMID:25177278

  19. Translocation of the ABC transporter ABCD4 from the endoplasmic reticulum to lysosomes requires the escort protein LMBD1.

    PubMed

    Kawaguchi, Kosuke; Okamoto, Takumi; Morita, Masashi; Imanaka, Tsuneo

    2016-01-01

    We previously demonstrated that ABCD4 does not localize to peroxisomes but rather, the endoplasmic reticulum (ER), because it lacks the NH2-terminal hydrophilic region required for peroxisomal targeting. It was recently reported that mutations in ABCD4 result in a failure to release vitamin B12 from lysosomes. A similar phenotype is caused by mutations in LMBRD1, which encodes the lysosomal membrane protein LMBD1. These findings suggested to us that ABCD4 translocated from the ER to lysosomes in association with LMBD1. In this report, it is demonstrated that ABCD4 interacts with LMBD1 and then localizes to lysosomes, and this translocation depends on the lysosomal targeting ability of LMBD1. Furthermore, endogenous ABCD4 was localized to both lysosomes and the ER, and its lysosomal localization was disturbed by knockout of LMBRD1. To the best of our knowledge, this is the first report demonstrating that the subcellular localization of the ABC transporter is determined by its association with an adaptor protein. PMID:27456980

  20. Translocation of the ABC transporter ABCD4 from the endoplasmic reticulum to lysosomes requires the escort protein LMBD1

    PubMed Central

    Kawaguchi, Kosuke; Okamoto, Takumi; Morita, Masashi; Imanaka, Tsuneo

    2016-01-01

    We previously demonstrated that ABCD4 does not localize to peroxisomes but rather, the endoplasmic reticulum (ER), because it lacks the NH2-terminal hydrophilic region required for peroxisomal targeting. It was recently reported that mutations in ABCD4 result in a failure to release vitamin B12 from lysosomes. A similar phenotype is caused by mutations in LMBRD1, which encodes the lysosomal membrane protein LMBD1. These findings suggested to us that ABCD4 translocated from the ER to lysosomes in association with LMBD1. In this report, it is demonstrated that ABCD4 interacts with LMBD1 and then localizes to lysosomes, and this translocation depends on the lysosomal targeting ability of LMBD1. Furthermore, endogenous ABCD4 was localized to both lysosomes and the ER, and its lysosomal localization was disturbed by knockout of LMBRD1. To the best of our knowledge, this is the first report demonstrating that the subcellular localization of the ABC transporter is determined by its association with an adaptor protein. PMID:27456980

  1. Selective focusing through target identification and experimental acoustic signature extraction: Numerical experiments.

    PubMed

    Rodriguez, S; Jacob, X; Gibiat, V

    2016-05-01

    Using transducer arrays and appropriate emission delays allow to focus acoustic waves at a chosen location in a medium. The focusing spatial accuracy depends on the accurate knowledge of its acoustic properties. When those properties are unknown, methods based on the Time-Reversal principle allow accurate focusing. Still, these methods are either intrusive (an active source has to be introduced at the target location first), either blind (the target cannot be selected in the presence of several objects.) The purpose of the present work is to achieve non-invasive accurate focusing on a selected target using inaccurate acoustic properties for the investigated medium. Potential applications are for instance noninvasive surgery based on High Intensity Focused Ultrasound (HIFU). Numerical experiments are presented and demonstrate accurate focusing on a previously designated target located in an unknown heterogeneous medium. PMID:26890791

  2. Phylogeny-driven target selection for large-scale genome-sequencing (and other) projects

    PubMed Central

    Göker, Markus; Klenk, Hans-Peter

    2013-01-01

    Despite the steadily decreasing costs of genome sequencing, prioritizing organisms for sequencing remains important in large-scale projects. Phylogeny-based selection is of interest to identify those organisms whose genomes can be expected to differ most from those that have already been sequenced. Here, we describe a method that infers a phylogenetic scoring independent of which set of organisms has previously been targeted, which is computationally simple and easy to apply in practice. The scoring itself, as well as pre- and post-processing of the data, is illustrated using two real-world examples in which the method has already been applied for selecting targets for genome sequencing. These projects are the JGI CSP Genomic Encyclopedia of Bacteria and Archaea phase I, targeting 1,000 type strains, and, on a smaller-scale, the phylogenomics of the Roseobacter clade. Potential artifacts of the method are discussed and compared to a selection approach based on the taxonomic classification. PMID:23991265

  3. Target selection of classical pulsating variables for space-based photometry

    NASA Astrophysics Data System (ADS)

    Plachy, E.; Molnar, L.; Szabo, R.; Kolenberg, K.; Banyai, E.

    2016-05-01

    In a few years the Kepler and TESS missions will provide ultra- precise photometry for thousands of RR Lyrae and hundreds of Cepheid stars. In the extended Kepler mission all targets are proposed in the Guest Observer (GO) Program, while the TESS space telescope will work with full frame images and a ~15-16th mag brightness limit with the possibility of short cadence measurements for a limited number of pre-selected objects. This paper highlights some details of the enormous and important work of the target selection process made by the members of Working Group 7 (WG#7) of the Kepler and TESS Asteroseismic Science Consortium.

  4. Effect of reactive oxygen species on lysosomal membrane integrity. A study on a lysosomal fraction.

    PubMed

    Zdolsek, J M; Svensson, I

    1993-01-01

    Using a lysosome-enriched "light mitochondrial" fraction of a rat liver homogenate, the effects of the reactive oxygen species hydrogen peroxide, superoxide- and hydroxyl radicals were determined. Alterations in the intralysosomal pH and the release of a lysosomal marker enzyme, N-acetyl-glucosaminidase, were used as indicators of changes in the lysosomal membrane integrity. Lipid peroxidation of the fraction was assayed by TBARS measurement. Neither superoxide radicals, generated by hypoxanthine/xanthine oxidase, nor a bolus dose of hydrogen peroxide (0.5-1.5 mM) induced any lysosomal damage. If, however, Fe(III)ADP was included in the superoxide radical-generating system, lysosomal membrane damage was detected, both as an increase in lysosomal pH and as a release of N-acetyl-glucosaminidase, but only after a lag phase of about 7 min. Lipid peroxidation, on the other hand, proceeded gradually. Lysosomes treated with hydrogen peroxide displayed similar dose-dependent alterations, albeit only if both Fe(III)ADP and the reducing amino acid cysteine were added. In the latter system, however, alterations of the lysosomal membrane stability occurred more rapidly, showing a lag phase of only 2 min. Lipid peroxidation, which proceeded faster and displayed no lag phase, levelled out within 10 min. The results indicate that neither superoxide radicals nor hydrogen peroxide are by themselves damaging to lysosomes. Available catalytically active iron in Fe(II) form, however, allows reactions yielding powerful oxidative species--probably hydroxyl radicals formed via Fenton reactions--to take place inducing peroxidation of the lysosomal membranes resulting in dissipation of the proton-gradient and leakage of their enzyme contents. PMID:8148962

  5. Hybrid EEG and eye movement interface to multi-directional target selection.

    PubMed

    Kim, Minho; Chae, Yongwook; Jo, Sungho

    2013-01-01

    This work addresses the development of a low-cost hybrid interface with eye tracking and brain signals. Eye movement detection is used for search task and EEG-based brain computer interface (BCI) for selection task. Multi-directional target selection experiments with the hybrid interface device were conducted with five subjects to evaluate the proposed hybrid interface scheme. The task asked each user to move a cursor onto a circular target among twelve possible positions and select it. Using the Fitts' law, the interface performance was compared with the computer mouse. With two BCI selection confirmation schemes, the hybrid interface attained 2-2.7 bit/s overall. Based on the results, the potential of the proposed hybrid interface was discussed. PMID:24109799

  6. Targeted Phosphoproteome Analysis Using Selected/Multiple Reaction Monitoring (SRM/MRM).

    PubMed

    Adachi, Jun; Narumi, Ryohei; Tomonaga, Takeshi

    2016-01-01

    Mass spectrometry-based phosphoproteomics has been rapidly spread based on the advancement of mass spectrometry and development of efficient enrichment techniques for phosphorylated proteins or peptides. Non-targeted approach has been employed in most of the studies for phosphoproteome analysis. However, targeted approach using selected/multiple reaction monitoring (SRM/MRM) is an indispensible technique used for the quantitation of known targets especially when we have many samples to quantitate phosphorylation events on proteins in biological or clinical samples. We herein describe the application of a large-scale phosphoproteome analysis and SRM-based quantitation for the systematic discovery and validation of biomarkers. PMID:26700043

  7. Nonstructural Proteins Are Preferential Positive Selection Targets in Zika Virus and Related Flaviviruses.

    PubMed

    Sironi, Manuela; Forni, Diego; Clerici, Mario; Cagliani, Rachele

    2016-09-01

    The Flavivirus genus comprises several human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Although ZIKV usually causes mild symptoms, growing evidence is linking it to congenital birth defects and to increased risk of Guillain-Barré syndrome. ZIKV encodes a polyprotein that is processed to produce three structural and seven nonstructural (NS) proteins. We investigated the evolution of the viral polyprotein in ZIKV and in related flaviviruses (DENV, Spondweni virus, and Kedougou virus). After accounting for saturation issues, alignment uncertainties, and recombination, we found evidence of episodic positive selection on the branch that separates DENV from the other flaviviruses. NS1 emerged as the major selection target, and selected sites were located in immune epitopes or in functionally important protein regions. Three of these sites are located in an NS1 region that interacts with structural proteins and is essential for virion biogenesis. Analysis of the more recent evolutionary history of ZIKV lineages indicated that positive selection acted on NS5 and NS4B, this latter representing the preferential target. All selected sites were located in the N-terminal portion of NS4B, which inhibits interferon response. One of the positively selected sites (26M/I/T/V) in ZIKV also represents a selection target in sylvatic DENV2 isolates, and a nearby residue evolves adaptively in JEV. Two additional positively selected sites are within a protein region that interacts with host (e.g. STING) and viral (i.e. NS1, NS4A) proteins. Notably, mutations in the NS4B region of other flaviviruses modulate neurovirulence and/or neuroinvasiveness. These results suggest that the positively selected sites we identified modulate viral replication and contribute to immune evasion. These sites should be prioritized in future experimental studies. However, analyses herein detected no selective events associated to the spread of the Asian

  8. Multiple factors dictate target selection by Hfq-binding small RNAs

    PubMed Central

    Beisel, Chase L; Updegrove, Taylor B; Janson, Ben J; Storz, Gisela

    2012-01-01

    Hfq-binding small RNAs (sRNAs) in bacteria modulate the stability and translational efficiency of target mRNAs through limited base-pairing interactions. While these sRNAs are known to regulate numerous mRNAs as part of stress responses, what distinguishes targets and non-targets among the mRNAs predicted to base pair with Hfq-binding sRNAs is poorly understood. Using the Hfq-binding sRNA Spot 42 of Escherichia coli as a model, we found that predictions using only the three unstructured regions of Spot 42 substantially improved the identification of previously known and novel Spot 42 targets. Furthermore, increasing the extent of base-pairing in single or multiple base-pairing regions improved the strength of regulation, but only for the unstructured regions of Spot 42. We also found that non-targets predicted to base pair with Spot 42 lacked an Hfq-binding site, folded into a secondary structure that occluded the Spot 42 targeting site, or had overlapping Hfq-binding and targeting sites. By modifying these features, we could impart Spot 42 regulation on non-target mRNAs. Our results thus provide valuable insights into the requirements for target selection by sRNAs. PMID:22388518

  9. Selective attention modulates the effect of target location probability on redundant signal processing.

    PubMed

    Chang, Ting-Yun; Little, Daniel R; Yang, Cheng-Ta

    2016-08-01

    We investigated the decision process underlying the detection of targets at multiple locations. In three experiments using the same observers, target location probability and attentional instructions were manipulated. A redundant-target detection task was conducted in which participants were required to detect a dot presented at one of two locations. When the dot appeared at the two locations with equal frequency (Experiment 1), those participants who were found to have limited to unlimited capacity were shown to adopt a parallel, self-terminating strategy. By contrast, those participants who had supercapacity were shown to process redundant targets in a coactive manner. When targets were presented with unequal probability, two participants adopted a parallel, self-terminating strategy regardless of whether they were informed the target location probability (Experiment 3) or not (Experiment 2). For the remaining two participants, the strategy changed from parallel, self-terminating to serial, self-terminating as a result of the probability instructions. In Experiments 2 and 3, all the participants were of unlimited to limited capacity. Taken together, these results suggest that target location probability differently affects the selection of a decision strategy and highlight the role of controlled attention in selecting a decision strategy. PMID:27188653

  10. Lysosomal Storage Diseases—Regulating Neurodegeneration

    PubMed Central

    Onyenwoke, Rob U.; Brenman, Jay E.

    2015-01-01

    Autophagy is a complex pathway regulated by numerous signaling events that recycles macromolecules and can be perturbed in lysosomal storage diseases (LSDs). The concept of LSDs, which are characterized by aberrant, excessive storage of cellular material in lysosomes, developed following the discovery of an enzyme deficiency as the cause of Pompe disease in 1963. Great strides have since been made in better understanding the biology of LSDs. Defective lysosomal storage typically occurs in many cell types, but the nervous system, including the central nervous system and peripheral nervous system, is particularly vulnerable to LSDs, being affected in two-thirds of LSDs. This review provides a summary of some of the better characterized LSDs and the pathways affected in these disorders. PMID:27081317

  11. Immunomodulatory gene therapy in lysosomal storage disorders

    PubMed Central

    Koeberl, D.D.; Kishnani, P.S.

    2010-01-01

    Significant advances in therapy for lysosomal storage disorders have occurred with an accelerating pace over the past decade. Although enzyme replacement therapy has improved the outcome of lysosomal storage disorders, antibody responses have occurred and sometimes prevented efficacy, especially in cross-reacting immune material negative patients with Pompe disease. Preclinical gene therapy experiments have revealed the relevance of immune responses to long-term efficacy. The choice of regulatory cassette played a critical role in evading humoral and cellular immune responses to gene therapy in knockout mouse models, at least in adult animals. Liver-specific regulatory cassettes prevented antibody formation and enhanced the efficacy of gene therapy. Regulatory T cells prevented transgene directed immune responses, as shown by adoptive transfer of antigen-specific immune tolerance to enzyme therapy. Immunomodulatory gene therapy with a very low vector dose could enhance the efficacy of enzyme therapy in Pompe disease and other lysosomal storage disorders. PMID:19807648

  12. Immunomodulatory gene therapy in lysosomal storage disorders.

    PubMed

    Koeberl, Dwight D; Kishnani, Priya S

    2009-12-01

    Significant advances in therapy for lysosomal storage disorders have occurred with an accelerating pace over the past decade. Although enzyme replacement therapy has improved the outcome of lysosomal storage disorders, antibody responses have occurred and sometimes prevented efficacy, especially in cross-reacting immune material negative patients with Pompe disease. Preclinical gene therapy experiments have revealed the relevance of immune responses to long-term efficacy. The choice of regulatory cassette played a critical role in evading humoral and cellular immune responses to gene therapy in knockout mouse models, at least in adult animals. Liver-specific regulatory cassettes prevented antibody formation and enhanced the efficacy of gene therapy. Regulatory T cells prevented transgene directed immune responses, as shown by adoptive transfer of antigen-specific immune tolerance to enzyme therapy. Immunomodulatory gene therapy with a very low vector dose could enhance the efficacy of enzyme therapy in Pompe disease and other lysosomal storage disorders. PMID:19807648

  13. Selection of DNA nanoparticles with preferential binding to aggregated protein target.

    PubMed

    Ruff, Laura E; Sapre, Ajay A; Plaut, Justin S; De Maere, Elisabeth; Mortier, Charlotte; Nguyen, Valerie; Separa, Kevin; Vandenbogaerde, Sofie; Vandewalle, Laura; Esener, Sadik C; Messmer, Bradley T

    2016-06-01

    High affinity and specificity are considered essential for affinity reagents and molecularly-targeted therapeutics, such as monoclonal antibodies. However, life's own molecular and cellular machinery consists of lower affinity, highly multivalent interactions that are metastable, but easily reversible or displaceable. With this inspiration, we have developed a DNA-based reagent platform that uses massive avidity to achieve stable, but reversible specific recognition of polyvalent targets. We have previously selected these DNA reagents, termed DeNAno, against various cells and now we demonstrate that DeNAno specific for protein targets can also be selected. DeNAno were selected against streptavidin-, rituximab- and bevacizumab-coated beads. Binding was stable for weeks and unaffected by the presence of soluble target proteins, yet readily competed by natural or synthetic ligands of the target proteins. Thus DeNAno particles are a novel biomolecular recognition agent whose orthogonal use of avidity over affinity results in uniquely stable yet reversible binding interactions. PMID:26969734

  14. Near Surface Swimming of Salmonella Typhimurium Explains Target-Site Selection and Cooperative Invasion

    PubMed Central

    Kreibich, Saskia; Vonaesch, Pascale; Andritschke, Daniel; Rout, Samuel; Weidner, Kerstin; Sormaz, Milos; Songhet, Pascal; Horvath, Peter; Chabria, Mamta; Vogel, Viola; Spori, Doris M.; Jenny, Patrick; Hardt, Wolf-Dietrich

    2012-01-01

    Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1.5–3 s in “near surface swimming”. This increased the local pathogen density and facilitated “scanning” of the host surface topology. We observed transient TTSS-1 and fim-independent “stopping” and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria. PMID:22911370

  15. Selection of DNA nanoparticles with preferential binding to aggregated protein target

    PubMed Central

    Ruff, Laura E.; Sapre, Ajay A.; Plaut, Justin S.; De Maere, Elisabeth; Mortier, Charlotte; Nguyen, Valerie; Separa, Kevin; Vandenbogaerde, Sofie; Vandewalle, Laura; Esener, Sadik C.; Messmer, Bradley T.

    2016-01-01

    High affinity and specificity are considered essential for affinity reagents and molecularly-targeted therapeutics, such as monoclonal antibodies. However, life's own molecular and cellular machinery consists of lower affinity, highly multivalent interactions that are metastable, but easily reversible or displaceable. With this inspiration, we have developed a DNA-based reagent platform that uses massive avidity to achieve stable, but reversible specific recognition of polyvalent targets. We have previously selected these DNA reagents, termed DeNAno, against various cells and now we demonstrate that DeNAno specific for protein targets can also be selected. DeNAno were selected against streptavidin-, rituximab- and bevacizumab-coated beads. Binding was stable for weeks and unaffected by the presence of soluble target proteins, yet readily competed by natural or synthetic ligands of the target proteins. Thus DeNAno particles are a novel biomolecular recognition agent whose orthogonal use of avidity over affinity results in uniquely stable yet reversible binding interactions. PMID:26969734

  16. Arylsulfatase K, a Novel Lysosomal Sulfatase*

    PubMed Central

    Wiegmann, Elena Marie; Westendorf, Eva; Kalus, Ina; Pringle, Thomas H.; Lübke, Torben; Dierks, Thomas

    2013-01-01

    The human sulfatase family has 17 members, 13 of which have been characterized biochemically. These enzymes specifically hydrolyze sulfate esters in glycosaminoglycans, sulfolipids, or steroid sulfates, thereby playing key roles in cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked to severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase K (ARSK), was identified bioinformatically through its conserved sulfatase signature sequence directing posttranslational generation of the catalytic formylglycine residue in sulfatases. However, overall sequence identity of ARSK with other human sulfatases is low (18–22%). Here we demonstrate that ARSK indeed shows desulfation activity toward arylsulfate pseudosubstrates. When expressed in human cells, ARSK was detected as a 68-kDa glycoprotein carrying at least four N-glycans of both the complex and high-mannose type. Purified ARSK turned over p-nitrocatechol and p-nitrophenyl sulfate. This activity was dependent on cysteine 80, which was verified to undergo conversion to formylglycine. Kinetic parameters were similar to those of several lysosomal sulfatases involved in degradation of sulfated glycosaminoglycans. An acidic pH optimum (∼4.6) and colocalization with LAMP1 verified lysosomal functioning of ARSK. Further, it carries mannose 6-phosphate, indicating lysosomal sorting via mannose 6-phosphate receptors. ARSK mRNA expression was found in all tissues tested, suggesting a ubiquitous physiological substrate and a so far non-classified lysosomal storage disorder in the case of ARSK deficiency, as shown before for all other lysosomal sulfatases. PMID:23986440

  17. Strategies for the Discovery of Target-Specific or Isoform-Selective Modulators.

    PubMed

    Zhan, Peng; Itoh, Yukihiro; Suzuki, Takayoshi; Liu, Xinyong

    2015-10-01

    Currently, the creation of class- and isoform-selective modulators of biologically important targets is a particularly challenging problem because different isoforms within a protein family often show striking similarity in spatial quaternary structure, especially at the catalytic sites or binding pockets. Therefore, an understanding of both the precise three-dimensional structure of the target protein and the mechanisms of action of modulators is important for developing more effective and selective agents. In this Perspective, we discuss currently available rational design strategies for obtaining class- and isoform-selective inhibitors and we illustrate these strategies with the aid of specific examples from the recent literature. The strategies covered include: (1) target-derived (-dependent) de novo drug discovery methodologies, and (2) follow-on derivatization approaches from initially identified active molecules (hit-to-lead and lead-to-candidate efforts). We also comment on prospects for further development and integration of strategies to achieve target-specific or isoform-selective inhibition. PMID:26086931

  18. Joint Effect of Insertion of Spaces and Word Length in Saccade Target Selection in Chinese Reading

    ERIC Educational Resources Information Center

    Li, Xingshan; Shen, Wei

    2013-01-01

    The present study examined how insertion of spaces before and after a word affects saccade target selection in Chinese reading. We found that inserting spaces in Chinese text changes the eye movement behaviour of Chinese readers. They are less likely to fixate on the character near the space and will try their best to process the entire word with…

  19. Photometric Observations of Selected BRITE Target Stars at Mt. Suhora Observatory

    NASA Astrophysics Data System (ADS)

    Stachowski, G.; Ogloza, W.; Drozdz, M.; Zakrzewski, B.

    2015-07-01

    We present the results of ground-based photometric observations of selected bright stars targeted by the BRITE satellite mission, carried out at Mt. Suhora Observatory using the new small telescope and a CCD camera, with the chip partially covered by a neutral density filter.

  20. The SDSS-IV Extended Baryon Oscillation Spectroscopic Survey: Luminous Red Galaxy Target Selection

    NASA Astrophysics Data System (ADS)

    Prakash, Abhishek; Licquia, Timothy C.; Newman, Jeffrey A.; Ross, Ashley J.; Myers, Adam D.; Dawson, Kyle S.; Kneib, Jean-Paul; Percival, Will J.; Bautista, Julian E.; Comparat, Johan; Tinker, Jeremy L.; Schlegel, David J.; Tojeiro, Rita; Ho, Shirley; Lang, Dustin; Rao, Sandhya M.; McBride, Cameron K.; Ben Zhu, Guangtun; Brownstein, Joel R.; Bailey, Stephen; Bolton, Adam S.; Delubac, Timothée; Mariappan, Vivek; Blanton, Michael R.; Reid, Beth; Schneider, Donald P.; Seo, Hee-Jong; Carnero Rosell, Aurelio; Prada, Francisco

    2016-06-01

    We describe the algorithm used to select the luminous red galaxy (LRG) sample for the extended Baryon Oscillation Spectroscopic Survey (eBOSS) of the Sloan Digital Sky Survey IV (SDSS-IV) using photometric data from both the SDSS and the Wide-field Infrared Survey Explorer. LRG targets are required to meet a set of color selection criteria and have z-band and i-band MODEL magnitudes z < 19.95 and 19.9 < i < 21.8, respectively. Our algorithm selects roughly 50 LRG targets per square degree, the great majority of which lie in the redshift range 0.6 < z < 1.0 (median redshift 0.71). We demonstrate that our methods are highly effective at eliminating stellar contamination and lower-redshift galaxies. We perform a number of tests using spectroscopic data from SDSS-III/BOSS ancillary programs to determine the redshift reliability of our target selection and its ability to meet the science requirements of eBOSS. The SDSS spectra are of high enough signal-to-noise ratio that at least ∼89% of the target sample yields secure redshift measurements. We also present tests of the uniformity and homogeneity of the sample, demonstrating that it should be clean enough for studies of the large-scale structure of the universe at higher redshifts than SDSS-III/BOSS LRGs reached.

  1. TARGET SELECTION FOR THE APACHE POINT OBSERVATORY GALACTIC EVOLUTION EXPERIMENT (APOGEE)

    SciTech Connect

    Zasowski, G.; Johnson, Jennifer A.; Andrews, B.; Epstein, C.; Frinchaboy, P. M.; Jackson, K.; Majewski, S. R.; Chojnowski, S. D.; Skrutskie, M. F.; Beaton, R. L.; Nidever, D. L.; Pinto, H. J. Rocha; Girardi, L.; Cudworth, K. M.; Munn, J.; Blake, C. H.; Covey, K.; Deshpande, R.; Fleming, S. W.; Fabbian, D. [Instituto de Astrofisica de Canarias, Calle Via Lactea s and others

    2013-10-01

    The Apache Point Observatory Galactic Evolution Experiment (APOGEE) is a high-resolution infrared spectroscopic survey spanning all Galactic environments (i.e., bulge, disk, and halo), with the principal goal of constraining dynamical and chemical evolution models of the Milky Way. APOGEE takes advantage of the reduced effects of extinction at infrared wavelengths to observe the inner Galaxy and bulge at an unprecedented level of detail. The survey's broad spatial and wavelength coverage enables users of APOGEE data to address numerous Galactic structure and stellar populations issues. In this paper we describe the APOGEE targeting scheme and document its various target classes to provide the necessary background and reference information to analyze samples of APOGEE data with awareness of the imposed selection criteria and resulting sample properties. APOGEE's primary sample consists of {approx}10{sup 5} red giant stars, selected to minimize observational biases in age and metallicity. We present the methodology and considerations that drive the selection of this sample and evaluate the accuracy, efficiency, and caveats of the selection and sampling algorithms. We also describe additional target classes that contribute to the APOGEE sample, including numerous ancillary science programs, and we outline the targeting data that will be included in the public data releases.

  2. The SDSS-IV Extended Baryon Oscillation Spectroscopic Survey: Luminous Red Galaxy Target Selection

    NASA Astrophysics Data System (ADS)

    Prakash, Abhishek; Licquia, Timothy C.; Newman, Jeffrey A.; Ross, Ashley J.; Myers, Adam D.; Dawson, Kyle S.; Kneib, Jean-Paul; Percival, Will J.; Bautista, Julian E.; Comparat, Johan; Tinker, Jeremy L.; Schlegel, David J.; Tojeiro, Rita; Ho, Shirley; Lang, Dustin; Rao, Sandhya M.; McBride, Cameron K.; Ben Zhu, Guangtun; Brownstein, Joel R.; Bailey, Stephen; Bolton, Adam S.; Delubac, Timothée; Mariappan, Vivek; Blanton, Michael R.; Reid, Beth; Schneider, Donald P.; Seo, Hee-Jong; Carnero Rosell, Aurelio; Prada, Francisco

    2016-06-01

    We describe the algorithm used to select the luminous red galaxy (LRG) sample for the extended Baryon Oscillation Spectroscopic Survey (eBOSS) of the Sloan Digital Sky Survey IV (SDSS-IV) using photometric data from both the SDSS and the Wide-field Infrared Survey Explorer. LRG targets are required to meet a set of color selection criteria and have z-band and i-band MODEL magnitudes z < 19.95 and 19.9 < i < 21.8, respectively. Our algorithm selects roughly 50 LRG targets per square degree, the great majority of which lie in the redshift range 0.6 < z < 1.0 (median redshift 0.71). We demonstrate that our methods are highly effective at eliminating stellar contamination and lower-redshift galaxies. We perform a number of tests using spectroscopic data from SDSS-III/BOSS ancillary programs to determine the redshift reliability of our target selection and its ability to meet the science requirements of eBOSS. The SDSS spectra are of high enough signal-to-noise ratio that at least ˜89% of the target sample yields secure redshift measurements. We also present tests of the uniformity and homogeneity of the sample, demonstrating that it should be clean enough for studies of the large-scale structure of the universe at higher redshifts than SDSS-III/BOSS LRGs reached.

  3. Target Selection for the Arecibo Pisces-Perseus Supercluster Survey (APPSS)

    NASA Astrophysics Data System (ADS)

    Craig, David W.; O'Donoghue, Aileen A.; Haynes, Martha P.; Rosenberg, Jessica L.; Venkatesan, Aparna; Hallenbeck, Gregory L.; Jones, Michael; Koopmann, Rebecca A.; Undergraduate ALFALFA Team

    2016-01-01

    The Arecibo Pisces-Perseus Supercluster Survey (APPSS) is a new large targeted HI survey now underway using Arecibo's L-band Wide receiver system. A major goal is to constrain models of the Pisces Perseus infall, producing 5-σ detections of infall motions ˜500 km s-1. We are targeting sources that are likely to be at the PPS distance, but that are just below the the HI mass detection threshold of the ALFALFA survey. We expect to identify ˜800 objects of mass ˜108—9 M⊙ which will alllow us to constrain the lower mass end of the HI mass function in this infall environment.We have pursued a multi-pronged approach to target selection for this survey. Sources from ALFALFA, SDSS, and the GALEX GCAT single source catalogs were matched and intercompared via multi-band color photometry, surface brightnesses, and appearance in SDSS images. Final target selection based on visual inspection of SDSS images was found to correlate well with a color-selection technique based on GALEX/NUV - SDSS/r. Along with the details of the source selection we will discuss the facilitation and implementation of this process via a multi-institution collaborative website, and early results from the APSS survey.This work has been supported by NSF grant AST-1211005.

  4. Endo-Lysosomal Dysfunction in Human Proximal Tubular Epithelial Cells Deficient for Lysosomal Cystine Transporter Cystinosin

    PubMed Central

    Van Den Heuvel, Lambertus; Pastore, Anna; Dijkman, Henry; De Matteis, Maria Antonietta; Levtchenko, Elena N.

    2015-01-01

    Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC) deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles. PMID:25811383

  5. Molecular beacons with a homo-DNA stem: improving target selectivity

    PubMed Central

    Crey-Desbiolles, Caroline; Ahn, Dae-Ro; Leumann, Christian J.

    2005-01-01

    Molecular beacons (MBs) are stem–loop DNA probes used for identifying and reporting the presence and localization of nucleic acid targets in vitro and in vivo via target-dependent dequenching of fluorescence. A drawback of conventional MB design is present in the stem sequence that is necessary to keep the MBs in a closed conformation in the absence of a target, but that can participate in target binding in the open (target-on) conformation, giving rise to the possibility of false-positive results. In order to circumvent these problems, we designed MBs in which the stem was replaced by an orthogonal DNA analog that does not cross-pair with natural nucleic acids. Homo-DNA seemed to be specially suited, as it forms stable adenine-adenine base pairs of the reversed Hoogsteen type, potentially reducing the number of necessary building blocks for stem design to one. We found that MBs in which the stem part was replaced by homo-adenylate residues can easily be synthesized using conventional automated DNA synthesis. As conventional MBs, such hybrid MBs show cooperative hairpin to coil transitions in the absence of a DNA target, indicating stable homo-DNA base pair formation in the closed conformation. Furthermore, our results show that the homo-adenylate stem is excluded from DNA target binding, which leads to a significant increase in target binding selectivity. PMID:15879349

  6. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

    PubMed Central

    Siklos, Marton; BenAissa, Manel; Thatcher, Gregory R.J.

    2015-01-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy. PMID:26713267

  7. Pathogenic cascades in lysosomal disease-Why so complex?

    PubMed

    Walkley, S U

    2009-04-01

    Lysosomal disease represents a large group of more than 50 clinically recognized conditions resulting from inborn errors of metabolism affecting the organelle known as the lysosome. The lysosome is an integral part of the larger endosomal/lysosomal system, and is closely allied with the ubiquitin-proteosomal and autophagosomal systems, which together comprise essential cell machinery for substrate degradation and recycling, homeostatic control, and signalling. More than two-thirds of lysosomal diseases affect the brain, with neurons appearing particularly vulnerable to lysosomal compromise and showing diverse consequences ranging from specific axonal and dendritic abnormalities to neuron death. While failure of lysosomal function characteristically leads to lysosomal storage, new studies argue that lysosomal diseases may also be appropriately viewed as 'states of deficiency' rather than simply overabundance (storage). Interference with signalling events and salvage processing normally controlled by the endosomal/lysosomal system may represent key mechanisms accounting for the inherent complexity of lysosomal disorders. Analysis of lysosomal disease pathogenesis provides a unique window through which to observe the importance of the greater lysosomal system for normal cell health. PMID:19130290

  8. Targeted Ultrasound-Assisted Cancer-Selective Chemical Labeling and Subsequent Cancer Imaging using Click Chemistry.

    PubMed

    Wang, Hua; Gauthier, Marianne; Kelly, Jamie R; Miller, Rita J; Xu, Ming; O'Brien, William D; Cheng, Jianjun

    2016-04-25

    Metabolic sugar labeling followed by the use of reagent-free click chemistry is an established technique for in vitro cell targeting. However, selective metabolic labeling of the target tissues in vivo remains a challenge to overcome, which has prohibited the use of this technique for targeted in vivo applications. Herein, we report the use of targeted ultrasound pulses to induce the release of tetraacetyl N-azidoacetylmannosamine (Ac4 ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac4 ManAz-loaded MBs showed great stability under physiological conditions, but rapidly collapsed in the presence of tumor-localized ultrasound pulses. The released Ac4 ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 by subsequent click chemistry. We demonstrated for the first time that Ac4 ManAz-loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer-selective labeling and targeted cancer therapies. PMID:27010510

  9. A Fluorescent Indicator for Imaging Lysosomal Zinc(II) with Förster Resonance Energy Transfer (FRET)-Enhanced Photostability and a Narrow Band of Emission

    PubMed Central

    Sreenath, Kesavapillai; Yuan, Zhao; Allen, John R.

    2015-01-01

    We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET-conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)-enhanced emission of 3 and 4 in lysosomes. It was further shown using two-color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino-functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells. PMID:25382395

  10. A new lactoferrin- and iron-dependent lysosomal death pathway is induced by benzo[a]pyrene in hepatic epithelial cells

    SciTech Connect

    Gorria, Morgane; Tekpli, Xavier; Rissel, Mary |; Sergent, Odile; Huc, Laurence |; Landvik, Nina; Fardel, Olivier; Dimanche-Boitrel, Marie-Therese |; Holme, Jorn A.; Lagadic-Gossmann, Dominique |

    2008-04-15

    While lysosomal disruption seems to be a late step of necrosis, a moderate lysosomal destabilization has been suggested to participate early in the apoptotic cascade. The origin of lysosomal dysfunction and its precise role in apoptosis or apoptosis-like process still needs to be clarified, especially upon carcinogen exposure. In this study, we focused on the implication of lysosomes in cell death induced by the prototype carcinogen benzo[a]pyrene (B[a]P; 50 nM) in rat hepatic epithelial F258 cells. We first demonstrated that B[a]P affected lysosomal morphology (increase in size) and pH (alkalinization), and that these changes were involved in caspase-3 activation and cell death. Subsequently, we showed that lysosomal modifications were partly dependent on mitochondrial dysfunction, and that lysosomes together with mitochondria participate in B[a]P-induced oxidative stress. Using two iron chelators (desferrioxamine and deferiprone) and siRNA targeting the lysosomal iron-binding protease lactoferrin, we further demonstrated that both lysosomal iron content and lactoferrin were required for caspase-3 activation and apoptosis-like cell death.

  11. Lysosome biogenesis/scattering increases host cell susceptibility to invasion by Trypanosoma cruzi metacyclic forms and resistance to tissue culture trypomastigotes.

    PubMed

    Cortez, Cristian; Real, Fernando; Yoshida, Nobuko

    2016-05-01

    A fundamental question to be clarified concerning the host cell invasion by Trypanosoma cruzi is whether the insect-borne and mammalian-stage parasites use similar mechanisms for invasion. To address that question, we analysed the cell invasion capacity of metacyclic trypomastigotes (MT) and tissue culture trypomastigotes (TCT) under diverse conditions. Incubation of parasites for 1 h with HeLa cells in nutrient-deprived medium, a condition that triggered lysosome biogenesis and scattering, increased MT invasion and reduced TCT entry into cells. Sucrose-induced lysosome biogenesis increased HeLa cell susceptibility to MT and resistance to TCT. Treatment of cells with rapamycin, which inhibits mammalian target of rapamycin (mTOR), induced perinuclear lysosome accumulation and reduced MT invasion while augmenting TCT invasion. Metacylic trypomastigotes, but not TCT, induced mTOR dephosphorylation and the nuclear translocation of transcription factor EB (TFEB), a mTOR-associated lysosome biogenesis regulator. Lysosome biogenesis/scattering was stimulated upon HeLa cell interaction with MT but not with TCT. Recently, internalized MT, but not TCT, were surrounded by colocalized lysosome marker LAMP2 and mTOR. The recombinant gp82 protein, the MT-specific surface molecule that mediates invasion, induced mTOR dephosphorylation, nuclear TFEB translocation and lysosome biogenesis/scattering. Taken together, our data clearly indicate that MT invasion is mainly lysosome-dependent, whereas TCT entry is predominantly lysosome-independent. PMID:26572924

  12. BORC, a Multisubunit Complex that Regulates Lysosome Positioning

    PubMed Central

    Pu, Jing; Schindler, Christina; Jia, Rui; Jarnik, Michal; Backlund, Peter; Bonifacino, Juan S.

    2016-01-01

    SUMMARY The positioning of lysosomes within the cytoplasm is emerging as a critical determinant of many lysosomal functions. Here we report the identification of a multi-subunit complex named BORC that regulates lysosome positioning. BORC comprises eight subunits, some of which are shared with the BLOC-1 complex involved in the biogenesis of lysosome-related organelles, and the others of which are products of previously uncharacterized open reading frames. BORC associates peripherally with the lysosomal membrane, where it functions to recruit the small GTPase Arl8. This initiates a chain of interactions that promotes the Kinesin-1-dependent movement of lysosomes toward the plus ends of microtubules in the peripheral cytoplasm. Interference with BORC or other components of this pathway results in collapse of the lysosomal population into the pericentriolar region. In turn, this causes reduced cell spreading and migration, highlighting the importance of BORC-dependent centrifugal transport for non-degradative functions of lysosomes. PMID:25898167

  13. Giant Lysosomes as a Chemotherapy Resistance Mechanism in Hepatocellular Carcinoma Cells

    PubMed Central

    Colombo, Federico; Trombetta, Elena; Cetrangolo, Paola; Maggioni, Marco; Razini, Paola; De Santis, Francesca; Torrente, Yvan; Prati, Daniele; Torresani, Erminio; Porretti, Laura

    2014-01-01

    Despite continuous improvements in therapeutic protocols, cancer-related mortality is still one of the main problems facing public health. The main cause of treatment failure is multi-drug resistance (MDR: simultaneous insensitivity to different anti-cancer agents), the underlying molecular and biological mechanisms of which include the activity of ATP binding cassette (ABC) proteins and drug compartmentalisation in cell organelles. We investigated the expression of the main ABC proteins and the role of cytoplasmic vacuoles in the MDR of six hepatocellular carcinoma (HCC) cell lines, and confirmed the accumulation of the yellow anti-cancer drug sunitinib in giant (four lines) and small cytoplasmic vacuoles of lysosomal origin (two lines). ABC expression analyses showed that the main ABC protein harboured by all of the cell lines was PGP, whose expression was not limited to the cell membrane but was also found on lysosomes. MTT assays showed that the cell lines with giant lysosomes were more resistant to sorafenib treatment than those with small lysosomes (p<0.01), and that verapamil incubation can revert this resistance, especially if it is administered after drug pre-incubation. The findings of this study demonstrate the involvement of PGP-positive lysosomes in drug sequestration and MDR in HCC cell lines. The possibility of modulating this mechanism using PGP inhibitors could lead to the development of new targeted strategies to enhance HCC treatment. PMID:25493932

  14. Long-Term Memories Bias Sensitivity and Target Selection in Complex Scenes

    PubMed Central

    Patai, Eva Zita; Doallo, Sonia; Nobre, Anna Christina

    2014-01-01

    In everyday situations we often rely on our memories to find what we are looking for in our cluttered environment. Recently, we developed a new experimental paradigm to investigate how long-term memory (LTM) can guide attention, and showed how the pre-exposure to a complex scene in which a target location had been learned facilitated the detection of the transient appearance of the target at the remembered location (Summerfield, Lepsien, Gitelman, Mesulam, & Nobre, 2006; Summerfield, Rao, Garside, & Nobre, 2011). The present study extends these findings by investigating whether and how LTM can enhance perceptual sensitivity to identify targets occurring within their complex scene context. Behavioral measures showed superior perceptual sensitivity (d′) for targets located in remembered spatial contexts. We used the N2pc event-related potential to test whether LTM modulated the process of selecting the target from its scene context. Surprisingly, in contrast to effects of visual spatial cues or implicit contextual cueing, LTM for target locations significantly attenuated the N2pc potential. We propose that the mechanism by which these explicitly available LTMs facilitate perceptual identification of targets may differ from mechanisms triggered by other types of top-down sources of information. PMID:23016670

  15. Extreme selective sweeps independently targeted the X chromosomes of the great apes

    PubMed Central

    Nam, Kiwoong; Munch, Kasper; Hobolth, Asger; Dutheil, Julien Yann; Veeramah, Krishna R.; Woerner, August E.; Hammer, Michael F.; Mailund, Thomas; Schierup, Mikkel Heide

    2015-01-01

    The unique inheritance pattern of the X chromosome exposes it to natural selection in a way that is different from that of the autosomes, potentially resulting in accelerated evolution. We perform a comparative analysis of X chromosome polymorphism in 10 great ape species, including humans. In most species, we identify striking megabase-wide regions, where nucleotide diversity is less than 20% of the chromosomal average. Such regions are found exclusively on the X chromosome. The regions overlap partially among species, suggesting that the underlying targets are partly shared among species. The regions have higher proportions of singleton SNPs, higher levels of population differentiation, and a higher nonsynonymous-to-synonymous substitution ratio than the rest of the X chromosome. We show that the extent to which diversity is reduced is incompatible with direct selection or the action of background selection and soft selective sweeps alone, and therefore, we suggest that very strong selective sweeps have independently targeted these specific regions in several species. The only genomic feature that we can identify as strongly associated with loss of diversity is the location of testis-expressed ampliconic genes, which also have reduced diversity around them. We hypothesize that these genes may be responsible for selective sweeps in the form of meiotic drive caused by an intragenomic conflict in male meiosis. PMID:25941379

  16. Treatment Strategies that Enhance the Efficacy and Selectivity of Mitochondria-Targeted Anticancer Agents

    PubMed Central

    Modica-Napolitano, Josephine S.; Weissig, Volkmar

    2015-01-01

    Nearly a century has passed since Otto Warburg first observed high rates of aerobic glycolysis in a variety of tumor cell types and suggested that this phenomenon might be due to an impaired mitochondrial respiratory capacity in these cells. Subsequently, much has been written about the role of mitochondria in the initiation and/or progression of various forms of cancer, and the possibility of exploiting differences in mitochondrial structure and function between normal and malignant cells as targets for cancer chemotherapy. A number of mitochondria-targeted compounds have shown efficacy in selective cancer cell killing in pre-clinical and early clinical testing, including those that induce mitochondria permeability transition and apoptosis, metabolic inhibitors, and ROS regulators. To date, however, none has exhibited the standards for high selectivity and efficacy and low toxicity necessary to progress beyond phase III clinical trials and be used as a viable, single modality treatment option for human cancers. This review explores alternative treatment strategies that have been shown to enhance the efficacy and selectivity of mitochondria-targeted anticancer agents in vitro and in vivo, and may yet fulfill the clinical promise of exploiting the mitochondrion as a target for cancer chemotherapy. PMID:26230693

  17. Separate visual signals for saccade initiation during target selection in the primate superior colliculus.

    PubMed

    White, Brian J; Munoz, Douglas P

    2011-02-01

    The primary function of the superior colliculus (SC) is to orient the visual system toward behaviorally relevant stimuli defined by features such as color. However, a longstanding view has held that visual activity in the SC arises exclusively from achromatic pathways. Recently, we reported evidence that the primate SC is highly sensitive to signals originating from chromatic pathways, but these signals are delayed relative to luminance signals (White et al., 2009). Here, we describe a functional consequence of this difference in visual arrival time on the processes leading to target selection and saccade initiation. Two rhesus monkeys performed a simple color-singleton selection task in which stimuli carried a chromatic component only (target and distractors were isoluminant with the background, but differed in chromaticity) or a combined chromatic-achromatic component (36% luminance contrast added equally to all stimuli). Although visual responses were delayed in the chromatic-only relative to the combined chromatic-achromatic condition, SC neurons discriminated the target from distractors at approximately the same time provided stimulus chromaticity was held constant. However, saccades were triggered sooner, and with more errors, with the chromatic-achromatic condition, suggesting that luminance signals associated with these stimuli increased the probability of triggering a saccade before the target color was adequately discriminated. These results suggest that separate mechanisms may independently influence the saccadic command in the SC, one linked to the arrival time of pertinent visual signals, and another linked to the output of the visual selection process. PMID:21289164

  18. Treatment Strategies that Enhance the Efficacy and Selectivity of Mitochondria-Targeted Anticancer Agents.

    PubMed

    Modica-Napolitano, Josephine S; Weissig, Volkmar

    2015-01-01

    Nearly a century has passed since Otto Warburg first observed high rates of aerobic glycolysis in a variety of tumor cell types and suggested that this phenomenon might be due to an impaired mitochondrial respiratory capacity in these cells. Subsequently, much has been written about the role of mitochondria in the initiation and/or progression of various forms of cancer, and the possibility of exploiting differences in mitochondrial structure and function between normal and malignant cells as targets for cancer chemotherapy. A number of mitochondria-targeted compounds have shown efficacy in selective cancer cell killing in pre-clinical and early clinical testing, including those that induce mitochondria permeability transition and apoptosis, metabolic inhibitors, and ROS regulators. To date, however, none has exhibited the standards for high selectivity and efficacy and low toxicity necessary to progress beyond phase III clinical trials and be used as a viable, single modality treatment option for human cancers. This review explores alternative treatment strategies that have been shown to enhance the efficacy and selectivity of mitochondria-targeted anticancer agents in vitro and in vivo, and may yet fulfill the clinical promise of exploiting the mitochondrion as a target for cancer chemotherapy. PMID:26230693

  19. Purification of Lysosomes Using Supraparamagnetic Iron Oxide Nanoparticles (SPIONs).

    PubMed

    Rofe, Adam P; Pryor, Paul R

    2016-04-01

    Lysosomes can be rapidly isolated from tissue culture cells using supraparamagnetic iron oxide particles (SPIONs). In this protocol, colloidal iron dextran (FeDex) particles, a type of SPION, are taken up by cultured mouse macrophage cells via the endocytic pathway. The SPIONs accumulate in lysosomes, the end point of the endocytic pathway, permitting the lysosomes to be isolated magnetically. The purified lysosomes are suitable for in vitro fusion assays or for proteomic analysis. PMID:27037068

  20. Increased lysosomal biogenesis in activated microglia and exacerbated neuronal damage after traumatic brain injury in progranulin-deficient mice.

    PubMed

    Tanaka, Y; Matsuwaki, T; Yamanouchi, K; Nishihara, M

    2013-10-10

    Progranulin (PGRN) is known to play a role in the pathogenesis of neurodegenerative diseases. Recently, it has been demonstrated that patients with the homozygous mutation in the GRN gene present with neuronal ceroid lipofuscinosis, and there is growing evidence that PGRN is related to lysosomal function. In the present study, we investigated the possible role of PGRN in the lysosomes of activated microglia in the cerebral cortex after traumatic brain injury (TBI). We showed that the mouse GRN gene has two possible coordinated lysosomal expression and regulation (CLEAR) sequences that bind to transcription factor EB (TFEB), a master regulator of lysosomal genes. PGRN was colocalized with Lamp1, a lysosomal marker, and Lamp1-positive areas in GRN-deficient (KO) mice were significantly expanded compared with wild-type (WT) mice after TBI. Expression of all the lysosome-related genes examined in KO mice was significantly higher than that in WT mice. The number of activated microglia with TFEB localized to the nucleus was also significantly increased in KO as compared with WT mice. Since the TFEB translocation is regulated by the mammalian target of rapamycin complex 1 (mTORC1) activity in the lysosome, we compared ribosomal S6 kinase 1 (S6K1) phosphorylation that reflects mTORC1 activity. S6K1 phosphorylation in KO mice was significantly lower than that in WT mice. In addition, the number of nissl-positive and fluoro-jade B-positive cells around the injury was significantly decreased and increased, respectively, in KO as compared with WT mice. These results suggest that PGRN localized in the lysosome is involved in the activation of mTORC1, and its deficiency leads to increased TFEB nuclear translocation with a resultant increase in lysosomal biogenesis in activated microglia and exacerbated neuronal damage in the cerebral cortex after TBI. PMID:23830905

  1. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

    PubMed Central

    Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

    2015-01-01

    A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer. PMID:26221730

  2. Engineering Multi-Walled Carbon Nanotube Therapeutic Bionanofluids to Selectively Target Papillary Thyroid Cancer Cells

    PubMed Central

    Paliouras, Miltiadis; Mitmaker, Elliot J.; Trifiro, Mark A.

    2016-01-01

    Background The incidence of papillary thyroid carcinoma (PTC) has risen steadily over the past few decades as well as the recurrence rates. It has been proposed that targeted ablative physical therapy could be a therapeutic modality in thyroid cancer. Targeted bio-affinity functionalized multi-walled carbon nanotubes (BioNanofluid) act locally, to efficiently convert external light energy to heat thereby specifically killing cancer cells. This may represent a promising new cancer therapeutic modality, advancing beyond conventional laser ablation and other nanoparticle approaches. Methods Thyroid Stimulating Hormone Receptor (TSHR) was selected as a target for PTC cells, due to its wide expression. Either TSHR antibodies or Thyrogen or purified TSH (Thyrotropin) were chemically conjugated to our functionalized Bionanofluid. A diode laser system (532 nm) was used to illuminate a PTC cell line for set exposure times. Cell death was assessed using Trypan Blue staining. Results TSHR-targeted BioNanofluids were capable of selectively ablating BCPAP, a TSHR-positive PTC cell line, while not TSHR-null NSC-34 cells. We determined that a 2:1 BCPAP cell:α-TSHR-BioNanofluid conjugate ratio and a 30 second laser exposure killed approximately 60% of the BCPAP cells, while 65% and >70% of cells were ablated using Thyrotropin- and Thyrogen-BioNanofluid conjugates, respectively. Furthermore, minimal non-targeted killing was observed using selective controls. Conclusion A BioNanofluid platform offering a potential therapeutic path for papillary thyroid cancer has been investigated, with our in vitro results suggesting the development of a potent and rapid method of selective cancer cell killing. Therefore, BioNanofluid treatment emphasizes the need for new technology to treat patients with local recurrence and metastatic disease who are currently undergoing either re-operative neck explorations, repeated administration of radioactive iodine and as a last resort external beam

  3. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    PubMed

    Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

    2015-01-01

    A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer. PMID:26221730

  4. Target selection and comparison of mission design for space debris removal by DLR's advanced study group

    NASA Astrophysics Data System (ADS)

    van der Pas, Niels; Lousada, Joao; Terhes, Claudia; Bernabeu, Marc; Bauer, Waldemar

    2014-09-01

    Space debris is a growing problem. Models show that the Kessler syndrome, the exponential growth of debris due to collisions, has become unavoidable unless an active debris removal program is initiated. The debris population in LEO with inclination between 60° and 95° is considered as the most critical zone. In order to stabilize the debris population in orbit, especially in LEO, 5 to 10 objects will need to be removed every year. The unique circumstances of such a mission could require that several objects are removed with a single launch. This will require a mission to rendezvous with a multitude of objects orbiting on different altitudes, inclinations and planes. Removal models have assumed that the top priority targets will be removed first. However this will lead to a suboptimal mission design and increase the ΔV-budget. Since there is a multitude of targets to choose from, the targets can be selected for an optimal mission design. In order to select a group of targets for a removal mission the orbital parameters and political constraints should also be taken into account. Within this paper a number of the target selection criteria are presented. The possible mission targets and their order of retrieval is dependent on the mission architecture. A comparison between several global mission architectures is given. Under consideration are 3 global missions of which a number of parameters are varied. The first mission launches multiple separate deorbit kits. The second launches a mother craft with deorbit kits. The third launches an orbital tug which pulls the debris in a lower orbit, after which a deorbit kit performs the final deorbit burn. A RoM mass and cost comparison is presented. The research described in this paper has been conducted as part of an active debris removal study by the Advanced Study Group (ASG). The ASG is an interdisciplinary student group working at the DLR, analyzing existing technologies and developing new ideas into preliminary

  5. Comparison of five peptide vectors for improved brain delivery of the lysosomal enzyme arylsulfatase A.

    PubMed

    Böckenhoff, Annika; Cramer, Sandra; Wölte, Philipp; Knieling, Simeon; Wohlenberg, Claudia; Gieselmann, Volkmar; Galla, Hans-Joachim; Matzner, Ulrich

    2014-02-26

    Enzyme replacement therapy (ERT) is a treatment option for lysosomal storage disorders (LSDs) caused by deficiencies of soluble lysosomal enzymes. ERT depends on receptor-mediated transport of intravenously injected recombinant enzyme to lysosomes of patient cells. The blood-brain barrier (BBB) prevents efficient transfer of therapeutic polypeptides from the blood to the brain parenchyma and thus hinders effective treatment of LSDs with CNS involvement. We compared the potential of five brain-targeting peptides to promote brain delivery of the lysosomal enzyme arylsulfatase A (ASA). Fusion proteins between ASA and the protein transduction domain of the human immunodeficiency virus TAT protein (Tat), an Angiopep peptide (Ang-2), and the receptor-binding domains of human apolipoprotein B (ApoB) and ApoE (two versions, ApoE-I and ApoE-II) were generated. All ASA fusion proteins were enzymatically active and targeted to lysosomes when added to cultured cells. In contrast to wild-type ASA, which is taken up by mannose-6-phosphate receptors, all chimeric proteins were additionally endocytosed via mannose-6-phosphate-independent routes. For ASA-Ang-2, ASA-ApoE-I, and ASA-ApoE-II, uptake was partially due to the low-density lipoprotein receptor-related protein 1. Transendothelial transfer in a BBB cell culture model was elevated for ASA-ApoB, ASA-ApoE-I, and ASA-ApoE-II. Brain delivery was, however, increased only for ASA-ApoE-II. ApoE-II was also superior to wild-type ASA in reducing lysosomal storage in the CNS of ASA-knock-out mice treated by ERT. Therefore, the ApoE-derived peptide appears useful to treat metachromatic leukodystrophy and possibly other neurological disorders more efficiently. PMID:24573272

  6. A novel bifunctional mitochondria-targeted anticancer agent with high selectivity for cancer cells.

    PubMed

    He, Huan; Li, Dong-Wei; Yang, Li-Yun; Fu, Li; Zhu, Xun-Jin; Wong, Wai-Kwok; Jiang, Feng-Lei; Liu, Yi

    2015-01-01

    Mitochondria have recently emerged as novel targets for cancer therapy due to its important roles in fundamental cellular function. Discovery of new chemotherapeutic agents that allow for simultaneous treatment and visualization of cancer is urgent. Herein, we demonstrate a novel bifunctional mitochondria-targeted anticancer agent (FPB), exhibiting both imaging capability and anticancer activity. It can selectively accumulate in mitochondria and induce cell apoptosis. Notably, it results in much higher toxicity toward cancer cells owing to much higher uptake by cancer cells. These features make it highly attractive in cancer imaging and treatment. PMID:26337336

  7. A novel bifunctional mitochondria-targeted anticancer agent with high selectivity for cancer cells

    PubMed Central

    He, Huan; Li, Dong-Wei; Yang, Li-Yun; Fu, Li; Zhu, Xun-Jin; Wong, Wai-Kwok; Jiang, Feng-Lei; Liu, Yi

    2015-01-01

    Mitochondria have recently emerged as novel targets for cancer therapy due to its important roles in fundamental cellular function. Discovery of new chemotherapeutic agents that allow for simultaneous treatment and visualization of cancer is urgent. Herein, we demonstrate a novel bifunctional mitochondria-targeted anticancer agent (FPB), exhibiting both imaging capability and anticancer activity. It can selectively accumulate in mitochondria and induce cell apoptosis. Notably, it results in much higher toxicity toward cancer cells owing to much higher uptake by cancer cells. These features make it highly attractive in cancer imaging and treatment. PMID:26337336

  8. Selective targeting of melanoma by PEG-masked protein-based multifunctional nanoparticles

    PubMed Central

    Vannucci, Luca; Falvo, Elisabetta; Fornara, Manuela; Di Micco, Patrizio; Benada, Oldrich; Krizan, Jiri; Svoboda, Jan; Hulikova-Capkova, Katarina; Morea, Veronica; Boffi, Alberto; Ceci, Pierpaolo

    2012-01-01

    Background Nanoparticle-based systems are promising for the development of imaging and therapeutic agents. The main advantage of nanoparticles over traditional systems lies in the possibility of loading multiple functionalities onto a single molecule, which are useful for therapeutic and/or diagnostic purposes. These functionalities include targeting moieties which are able to recognize receptors overexpressed by specific cells and tissues. However, targeted delivery of nanoparticles requires an accurate system design. We present here a rationally designed, genetically engineered, and chemically modified protein-based nanoplatform for cell/tissue-specific targeting. Methods Our nanoparticle constructs were based on the heavy chain of the human protein ferritin (HFt), a highly symmetrical assembly of 24 subunits enclosing a hollow cavity. HFt-based nanoparticles were produced using both genetic engineering and chemical functionalization methods to impart several functionalities, ie, the α-melanocyte-stimulating hormone peptide as a melanoma-targeting moiety, stabilizing and HFt-masking polyethylene glycol molecules, rhodamine fluorophores, and magnetic resonance imaging agents. The constructs produced were extensively characterized by a number of physicochemical techniques, and assayed for selective melanoma-targeting in vitro and in vivo. Results Our HFt-based nanoparticle constructs functionalized with the α-melanocyte-stimulating hormone peptide moiety and polyethylene glycol molecules were specifically taken up by melanoma cells but not by other cancer cell types in vitro. Moreover, experiments in melanoma-bearing mice indicate that these constructs have an excellent tumor-targeting profile and a long circulation time in vivo. Conclusion By masking human HFt with polyethylene glycol and targeting it with an α-melanocyte-stimulating hormone peptide, we developed an HFt-based melanoma-targeting nanoplatform for application in melanoma diagnosis and treatment

  9. Reactivation of Lysosomal Ca2+ Efflux Rescues Abnormal Lysosomal Storage in FIG4-Deficient Cells.

    PubMed

    Zou, Jianlong; Hu, Bo; Arpag, Sezgi; Yan, Qing; Hamilton, Audra; Zeng, Yuan-Shan; Vanoye, Carlos G; Li, Jun

    2015-04-29

    Loss of function of FIG4 leads to Charcot-Marie-Tooth disease Type 4J, Yunis-Varon syndrome, or an epilepsy syndrome. FIG4 is a phosphatase with its catalytic specificity toward 5'-phosphate of phosphatidylinositol-3,5-diphosphate (PI3,5P2). However, the loss of FIG4 decreases PI3,5P2 levels likely due to FIG4's dominant effect in scaffolding a PI3,5P2 synthetic protein complex. At the cellular level, all these diseases share similar pathology with abnormal lysosomal storage and neuronal degeneration. Mice with no FIG4 expression (Fig4(-/-)) recapitulate the pathology in humans with FIG4 deficiency. Using a flow cytometry technique that rapidly quantifies lysosome sizes, we detected an impaired lysosomal fission, but normal fusion, in Fig4(-/-) cells. The fission defect was associated with a robust increase of intralysosomal Ca(2+) in Fig4(-/-) cells, including FIG4-deficient neurons. This finding was consistent with a suppressed Ca(2+) efflux of lysosomes because the endogenous ligand of lysosomal Ca(2+) channel TRPML1 is PI3,5P2 that is deficient in Fig4(-/-) cells. We reactivated the TRPML1 channels by application of TRPML1 synthetic ligand, ML-SA1. This treatment reduced the intralysosomal Ca(2+) level and rescued abnormal lysosomal storage in Fig4(-/-) culture cells and ex vivo DRGs. Furthermore, we found that the suppressed Ca(2+) efflux in Fig4(-/-) culture cells and Fig4(-/-) mouse brains profoundly downregulated the expression/activity of dynamin-1, a GTPase known to scissor organelle membranes during fission. This downregulation made dynamin-1 unavailable for lysosomal fission. Together, our study revealed a novel mechanism explaining abnormal lysosomal storage in FIG4 deficiency. Synthetic ligands of the TRPML1 may become a potential therapy against diseases with FIG4 deficiency. PMID:25926456

  10. The use of selection in recovery of transgenic targets for mutation analysis.

    PubMed

    Lundberg, K S; Kretz, P L; Provost, G S; Short, J M

    1993-02-01

    Transgenic animal mutagenesis assays using lambda shuttle vectors have recently been described for isolation and characterization of spontaneous and chemical induced DNA mutations. Extensive information on lambda and E. coli genetics provides a wealth of techniques to allow selection of mutant target genes. Here we describe the modification of an E. coli host which permits two methods for the direct selection of mutant genes. These methods reduce the number of plates needed to be screened for a comparable amount of frequency data by 20-100-fold and thus provide a significant savings of the materials and time required for the screening of mutations. In addition, mutants selected by these approaches described here may alter or broaden the spectrum of mutations that are recoverable. Ultimately, a combination of selective and nonselective techniques may prove valuable for the analysis of mutations produced in vivo in transgenic animals. PMID:7678177

  11. Vacuolar ATPase in Phagosome-Lysosome Fusion

    PubMed Central

    Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S.; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

    2015-01-01

    The vacuolar H+-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. PMID:25903133

  12. Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses

    PubMed Central

    Corrotte, M.; Castro-Gomes, T.; Koushik, A.B.; Andrews, N.W.

    2016-01-01

    Rapid plasma membrane repair is essential to restore cellular homeostasis and improve cell survival after injury. Several mechanisms for plasma membrane repair have been proposed, including formation of an intracellular vesicle patch, reduction of plasma membrane tension, lesion removal by endocytosis, and/or shedding of the wounded membrane. Under all conditions studied to date, plasma membrane repair is strictly dependent on the entry of calcium into cells, from the extracellular medium. Calcium-dependent exocytosis of lysosomes is an important early step in the plasma membrane repair process, and defects in plasma membrane repair have been observed in cells carrying mutations responsible for serious lysosomal diseases, such as Chediak–Higashi (Huynh, Roth, Ward, Kaplan, & Andrews, 2004) and Niemann–Pick Disease type A (Tam et al., 2010). A functional role for release of the lysosomal enzyme acid sphingomyelinase, which generates ceramide on the cell surface and triggers endocytosis, has been described (Corrotte et al., 2013; Tam et al., 2010). Therefore, procedures for measuring the extent of lysosomal fusion with the plasma membrane of wounded cells are important indicators of the cellular repair response. The importance of carefully selecting the methodology for experimental plasma membrane injury, in order not to adversely impact the membrane repair machinery, is becoming increasingly apparent. Here, we describe physiologically relevant methods to induce different types of cellular wounds, and sensitive assays to measure the ability of cells to secrete lysosomes and reseal their plasma membrane. PMID:25665445

  13. Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

    PubMed Central

    Aizawa, Shu; Fujiwara, Yuuki; Contu, Viorica Raluca; Hase, Katsunori; Takahashi, Masayuki; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

    2016-01-01

    ABSTRACT Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy. PMID:27046251

  14. Lysosomal Integral Membrane Protein-2: A New Player in Lysosome-Related Pathology

    PubMed Central

    Gonzalez, Ashley; Valeiras, Mark; Sidransky, Ellen; Tayebi, Nahid

    2014-01-01

    Lysosomes require the presence of many specialized proteins to facilitate their roles in cellular maintenance. One such protein that has proven to be an important player in the lysosomal field is lysosomal integral membrane protein-2 (LIMP-2), encoded by the gene SCARB2. LIMP-2 is required for the normal biogenesis and maintenance of lysosomes and endosomes and has been identified as the specific receptor for glucocerebrosidase, the enzyme deficient in Gaucher disease. Research into LIMP-2 and the SCARB2 gene indicate that it may be a factor contributing to the clinical heterogeneity seen among patients with Gaucher disease. Mutations in SCARB2 have also been identified as the cause of action myoclonus renal failure (AMRF), and in some cases progressive myoclonic epilepsy. A total of 14 disease-causing SCARB2 mutations have been identified to date. The role of LIMP-2 in human pathology has expanded with its identification as a component of the intercalated disc in cardiac muscle and as a receptor for specific enteroviruses, two unanticipated findings that reaffirm the myriad roles of lysosomal proteins. Studies into the full impact of LIMP-2 deficiency and the LIMP2/glucocerebrosidase molecular pathway will lead to a better understanding of disease pathogenesis in Gaucher disease and AMRF, and to new insights into lysosomal processing, trafficking and function. PMID:24389070

  15. Application of support vector machine-based ranking strategies to search for target-selective compounds.

    PubMed

    Wassermann, Anne Mai; Geppert, Hanna; Bajorath, Jürgen

    2011-01-01

    Support vector machine (SVM)-based selectivity searching has recently been introduced to identify compounds in virtual screening libraries that are not only active for a target protein, but also selective for this target over a closely related member of the same protein family. In simulated virtual screening calculations, SVM-based strategies termed preference ranking and one-versus-all ranking were successfully applied to rank a database and enrich high-ranking positions with selective compounds while removing nonselective molecules from high ranks. In contrast to the original SVM approach developed for binary classification, these strategies enable learning from more than two classes, considering that distinguishing between selective, promiscuously active, and inactive compounds gives rise to a three-class prediction problem. In this chapter, we describe the extension of the one-versus-all strategy to four training classes. Furthermore, we present an adaptation of the preference ranking strategy that leads to higher recall of selective compounds than previously investigated approaches and is applicable in situations where the removal of nonselective compounds from high-ranking positions is not required. PMID:20838983

  16. Designing the nanobiointerface of fluorescent nanodiamonds: highly selective targeting of glioma cancer cells

    NASA Astrophysics Data System (ADS)

    Slegerova, Jitka; Hajek, Miroslav; Rehor, Ivan; Sedlak, Frantisek; Stursa, Jan; Hruby, Martin; Cigler, Petr

    2014-12-01

    Core-shell nanoparticles based on fluorescent nanodiamonds coated with a biocompatible N-(2-hydroxypropyl)methacrylamide copolymer shell were developed for background-free near-infrared imaging of cancer cells. The particles showed excellent colloidal stability in buffers and culture media. After conjugation with a cyclic RGD peptide they selectively targeted integrin αvβ3 receptors on glioblastoma cells with high internalization efficacy.Core-shell nanoparticles based on fluorescent nanodiamonds coated with a biocompatible N-(2-hydroxypropyl)methacrylamide copolymer shell were developed for background-free near-infrared imaging of cancer cells. The particles showed excellent colloidal stability in buffers and culture media. After conjugation with a cyclic RGD peptide they selectively targeted integrin αvβ3 receptors on glioblastoma cells with high internalization efficacy. Electronic supplementary information (ESI) available: Materials and methods, colloidal stability studies and cell viability studies. See DOI: 10.1039/c4nr02776k

  17. Impact of high-risk conjunctions on Active Debris Removal target selection

    NASA Astrophysics Data System (ADS)

    Lidtke, Aleksander A.; Lewis, Hugh G.; Armellin, Roberto

    2015-10-01

    Space debris simulations show that if current space launches continue unchanged, spacecraft operations might become difficult in the congested space environment. It has been suggested that Active Debris Removal (ADR) might be necessary in order to prevent such a situation. Selection of objects to be targeted by ADR is considered important because removal of non-relevant objects will unnecessarily increase the cost of ADR. One of the factors to be used in this ADR target selection is the collision probability accumulated by every object. This paper shows the impact of high-probability conjunctions on the collision probability accumulated by individual objects as well as the probability of any collision occurring in orbit. Such conjunctions cannot be predicted far in advance and, consequently, not all the objects that will be involved in such dangerous conjunctions can be removed through ADR. Therefore, a debris remediation method that would address such events at short notice, and thus help prevent likely collisions, is suggested.

  18. Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization.

    PubMed

    Viennet, Thibault; Viegas, Aldino; Kuepper, Arne; Arens, Sabine; Gelev, Vladimir; Petrov, Ognyan; Grossmann, Tom N; Heise, Henrike; Etzkorn, Manuel

    2016-08-26

    Nuclear magnetic resonance (NMR) spectroscopy has the intrinsic capabilities to investigate proteins in native environments. In general, however, NMR relies on non-natural protein purity and concentration to increase the desired signal over the background. We here report on the efficient and specific hyperpolarization of low amounts of a target protein in a large isotope-labeled background by combining dynamic nuclear polarization (DNP) and the selectivity of protein interactions. Using a biradical-labeled ligand, we were able to direct the hyperpolarization to the protein of interest, maintaining comparable signal enhancement with about 400-fold less radicals than conventionally used. We could selectively filter out our target protein directly from crude cell lysate obtained from only 8 mL of fully isotope-enriched cell culture. Our approach offers effective means to study proteins with atomic resolution in increasingly native concentrations and environments. PMID:27351143

  19. Photostick: a method for selective isolation of target cells from culture

    PubMed Central

    Chien, Miao-Ping; Werley, Christopher A.; Farhi, Samouil L.

    2015-01-01

    Sorting of target cells from a heterogeneous pool is technically difficult when the selection criterion is complex, e.g. a dynamic response, a morphological feature, or a combination of multiple parameters. At present, mammalian cell selections are typically performed either via static fluorescence (e.g. fluorescence activated cell sorter), via survival (e.g. antibiotic resistance), or via serial operations (flow cytometry, laser capture microdissection). Here we present a simple protocol for selecting cells based on any static or dynamic property that can be identified by video microscopy and image processing. The “photostick” technique uses a cell-impermeant photochemical crosslinker and digital micromirror array-based patterned illumination to immobilize selected cells on the culture dish. Other cells are washed away with mild protease treatment. The crosslinker also labels the selected cells with a fluorescent dye and a biotin for later identification. The photostick protocol preserves cell viability, permits genetic profiling of selected cells, and can be performed with complex functional selection criteria such as neuronal firing patterns. PMID:25705368

  20. Microfluidics for Drug Discovery and Development: From Target Selection to Product Lifecycle Management

    PubMed Central

    Kang, Lifeng; Chung, Bong Geun; Langer, Robert; Khademhosseini, Ali

    2009-01-01

    Microfluidic technologies’ ability to miniaturize assays and increase experimental throughput have generated significant interest in the drug discovery and development domain. These characteristics make microfluidic systems a potentially valuable tool for many drug discovery and development applications. Here, we review the recent advances of microfluidic devices for drug discovery and development and highlight their applications in different stages of the process, including target selection, lead identification, preclinical tests, clinical trials, chemical synthesis, formulations studies, and product management. PMID:18190858

  1. Microfluidics for drug discovery and development: from target selection to product lifecycle management.

    PubMed

    Kang, Lifeng; Chung, Bong Geun; Langer, Robert; Khademhosseini, Ali

    2008-01-01

    Microfluidic technologies' ability to miniaturize assays and increase experimental throughput have generated significant interest in the drug discovery and development domain. These characteristics make microfluidic systems a potentially valuable tool for many drug discovery and development applications. Here, we review the recent advances of microfluidic devices for drug discovery and development and highlight their applications in different stages of the process, including target selection, lead identification, preclinical tests, clinical trials, chemical synthesis, formulations studies and product management. PMID:18190858

  2. Functionalized paper SERS (P-SERS) substrates for selective targeting of analytes in complex samples

    NASA Astrophysics Data System (ADS)

    Yu, Wei W.; Hoppmann, Eric P.

    2015-05-01

    Surface enhanced Raman spectroscopy (SERS) requires the analyte molecule to be close to the plasmonic surface in order to generate SERS enhancement. This limitation restricts the practical application of SERS to molecules that possess functional groups that interact strongly with gold or silver surfaces. Moreover, the identification of target analytes in a complex sample matrix is made even more difficult when interferents compete with the target for binding to the plasmonic surface, resulting in overlapping spectral signatures. In this work, we report a strategy to functionalize inkjet printed P-SERS substrates by strategically placing supramolecular structures (such as nucleic acid aptamers) onto the gold nanoparticles. This promotes the selective interaction of target molecules with the plasmonic surface, leading to improved sensor performance.

  3. Mitochondrial permeability transition pore as a selective target for anti-cancer therapy

    PubMed Central

    Suh, Dong H.; Kim, Mi-Kyung; Kim, Hee S.; Chung, Hyun H.; Song, Yong S.

    2012-01-01

    Mitochondrial outer membrane permeabilization (MOMP) is the ultimate step in dozens of lethal apoptotic signal transduction pathways which converge on mitochondria. One of the representative systems proposed to be responsible for the MOMP is the mitochondrial permeability transition pore (MPTP). Although the molecular composition of the MPTP is not clearly understood, the MPTP attracts much interest as a promising target for resolving two conundrums regarding cancer treatment: tumor selectivity and resistance to treatment. The regulation of the MPTP is closely related to metabolic reprogramming in cancer cells including mitochondrial alterations. Restoration of deregulated apoptotic machinery in cancer cells by tumor-specific modulation of the MPTP could therefore be a promising anti-cancer strategy. Currently, a number of MPTP-targeting agents are under pre-clinical and clinical studies. Here, we reviewed the structure and regulation of the MPTP as well as the current status of the development of promising MPTP-targeting drugs. PMID:23483560

  4. Spectral selective radio frequency emissions from laser induced breakdown of target materials

    SciTech Connect

    Vinoth Kumar, L.; Manikanta, E.; Leela, Ch.; Prem Kiran, P.

    2014-08-11

    The radio frequency emissions scanned over broad spectral range (30 MHz–1 GHz) from single shot nanosecond (7 ns) and picosecond (30 ps) laser induced breakdown (LIB) of different target materials (atmospheric air, aluminum, and copper) are presented. The dominant emissions from ns-LIB, compared to those from the ps-LIB, indicate the presence and importance of atomic and molecular clusters in the plasma. The dynamics of laser pulse-matter interaction and the properties of the target materials were found to play an important role in determining the plasma parameters which subsequently determine the emissions. Thus, with a particular laser and target material, the emissions were observed to be spectral selective. The radiation detection capability was observed to be relatively higher, when the polarization of the input laser and the antenna is same.

  5. Hydrogen peroxide induces lysosomal protease alterations in PC12 cells.

    PubMed

    Lee, Daniel C; Mason, Ceceile W; Goodman, Carl B; Holder, Maurice S; Kirksey, Otis W; Womble, Tracy A; Severs, Walter B; Palm, Donald E

    2007-09-01

    Alterations in lysosomal proteases have been implicated in many neurodegenerative diseases. The current study demonstrates a concentration-dependent decrease in PC12 cell viability and transient changes in cystatin C (CYSC), cathepsin B (CATB), cathepsin D (CATD) and caspase-3 following exposure to H2O2. Furthermore, activation of CATD occurred following exposure to H2O2 and cysteine protease suppression, while inhibition of CATD with pepstatin A significantly improved cell viability. Additionally, significant PARP cleavage, suggestive of caspase-3-like activity, was observed following H2O2 exposure, while inhibition of caspase-3 significantly increased cell viability compared to H2O2 administration alone. Collectively, our data suggest that H2O2 induced cell death is regulated at least in part by caspase-3 and CATD. Furthermore, cysteine protease suppression increases CATD expression and activity. These studies provide insight for alternate pathways and potential therapeutic targets of cell death associated with oxidative stress and lysosomal protease alterations. PMID:17440810

  6. G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes

    PubMed Central

    Marchese, Adriano; Paing, May M.; Temple, Brenda R.S.; Trejo, JoAnn

    2010-01-01

    The heptahelical G protein–coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine- and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs. PMID:17995450

  7. Targeting hunter distribution based on host resource selection and kill sites to manage disease risk

    PubMed Central

    Dugal, Cherie J; van Beest, Floris M; Vander Wal, Eric; Brook, Ryan K

    2013-01-01

    Endemic and emerging diseases are rarely uniform in their spatial distribution or prevalence among cohorts of wildlife. Spatial models that quantify risk-driven differences in resource selection and hunter mortality of animals at fine spatial scales can assist disease management by identifying high-risk areas and individuals. We used resource selection functions (RSFs) and selection ratios (SRs) to quantify sex- and age-specific resource selection patterns of collared (n = 67) and hunter-killed (n = 796) nonmigratory elk (Cervus canadensis manitobensis) during the hunting season between 2002 and 2012, in southwestern Manitoba, Canada. Distance to protected area was the most important covariate influencing resource selection and hunter-kill sites of elk (AICw = 1.00). Collared adult males (which are most likely to be infected with bovine tuberculosis (Mycobacterium bovis) and chronic wasting disease) rarely selected for sites outside of parks during the hunting season in contrast to adult females and juvenile males. The RSFs showed selection by adult females and juvenile males to be negatively associated with landscape-level forest cover, high road density, and water cover, whereas hunter-kill sites of these cohorts were positively associated with landscape-level forest cover and increasing distance to streams and negatively associated with high road density. Local-level forest was positively associated with collared animal locations and hunter-kill sites; however, selection was stronger for collared juvenile males and hunter-killed adult females. In instances where disease infects a metapopulation and eradication is infeasible, a principle goal of management is to limit the spread of disease among infected animals. We map high-risk areas that are regularly used by potentially infectious hosts but currently underrepresented in the distribution of kill sites. We present a novel application of widely available data to target hunter distribution based on host resource

  8. BioGPS: navigating biological space to predict polypharmacology, off-targeting, and selectivity.

    PubMed

    Siragusa, Lydia; Cross, Simon; Baroni, Massimo; Goracci, Laura; Cruciani, Gabriele

    2015-03-01

    The structural comparison of protein binding sites is increasingly important in drug design; identifying structurally similar sites can be useful for techniques such as drug repurposing, and also in a polypharmacological approach to deliberately affect multiple targets in a disease pathway, or to explain unwanted off-target effects. Once similar sites are identified, identifying local differences can aid in the design of selectivity. Such an approach moves away from the classical "one target one drug" approach and toward a wider systems biology paradigm. Here, we report a semiautomated approach, called BioGPS, that is based on the software FLAP which combines GRID Molecular Interactions Fields (MIFs) and pharmacophoric fingerprints. BioGPS comprises the automatic preparation of protein structure data, identification of binding sites, and subsequent comparison by aligning the sites and directly comparing the MIFs. Chemometric approaches are included to reduce the complexity of the resulting data on large datasets, enabling focus on the most relevant information. Individual site similarities can be analyzed in terms of their Pharmacophoric Interaction Field (PIF) similarity, and importantly the differences in their PIFs can be extracted. Here we describe the BioGPS approach, and demonstrate its applicability to rationalize off-target effects (ERα and SERCA), to classify protein families and explain polypharmacology (ABL1 kinase and NQO2), and to rationalize selectivity between subfamilies (MAP kinases p38α/ERK2 and PPARδ/PPARγ). The examples shown demonstrate a significant validation of the method and illustrate the effectiveness of the approach. PMID:25556939

  9. THE SDSS-III BARYON OSCILLATION SPECTROSCOPIC SURVEY: QUASAR TARGET SELECTION FOR DATA RELEASE NINE

    SciTech Connect

    Ross, Nicholas P.; Kirkpatrick, Jessica A.; Carithers, William C.; Ho, Shirley; Myers, Adam D.; Sheldon, Erin S.; Yeche, Christophe; Aubourg, Eric; Strauss, Michael A.; Lee, Khee-Gan; Bovy, Jo; Blanton, Michael R.; Hogg, David W.; Richards, Gordon T.; Brandt, W. N.; Croft, Rupert A. C.; Da Silva, Robert; Dawson, Kyle; Eisenstein, Daniel J.; Hennawi, Joseph F.; and others

    2012-03-01

    The SDSS-III Baryon Oscillation Spectroscopic Survey (BOSS), a five-year spectroscopic survey of 10,000 deg{sup 2}, achieved first light in late 2009. One of the key goals of BOSS is to measure the signature of baryon acoustic oscillations (BAOs) in the distribution of Ly{alpha} absorption from the spectra of a sample of {approx}150,000 z > 2.2 quasars. Along with measuring the angular diameter distance at z Almost-Equal-To 2.5, BOSS will provide the first direct measurement of the expansion rate of the universe at z > 2. One of the biggest challenges in achieving this goal is an efficient target selection algorithm for quasars in the redshift range 2.2 < z < 3.5, where their colors tend to overlap those of the far more numerous stars. During the first year of the BOSS survey, quasar target selection (QTS) methods were developed and tested to meet the requirement of delivering at least 15 quasars deg{sup -2} in this redshift range, with a goal of 20 out of 40 targets deg{sup -2} allocated to the quasar survey. To achieve these surface densities, the magnitude limit of the quasar targets was set at g {<=} 22.0 or r {<=} 21.85. While detection of the BAO signature in the distribution of Ly{alpha} absorption in quasar spectra does not require a uniform target selection algorithm, many other astrophysical studies do. We have therefore defined a uniformly selected subsample of 20 targets deg{sup -2}, for which the selection efficiency is just over 50% ({approx}10 z > 2.20 quasars deg{sup -2}). This 'CORE' subsample will be fixed for Years Two through Five of the survey. For the remaining 20 targets deg{sup -2}, we will continue to develop improved selection techniques, including the use of additional data sets beyond the Sloan Digital Sky Survey (SDSS) imaging data. In this paper, we describe the evolution and implementation of the BOSS QTS algorithms during the first two years of BOSS operations (through 2011 July), in support of the science investigations based on

  10. Virus Replication, Cytopathology, and Lysosomal Enzyme Response of Mitotic and Interphase Hep-2 Cells Infected with Poliovirus

    PubMed Central

    Bienz, Kurt; Egger, Denise; Wolff, David A.

    1973-01-01

    Mitotic Hep-2 cells, selected by the PEL (colloidal silica) density gradient method and held in mitosis with Colcemid, are readily infected by poliovirus type I (Mahoney). They produce and release the same amount of virus as interphase, random-growing cells. In contrast to interphase cells, mitotic cells show no detectable virus-induced cytopathic effect at the light microscopy level and only slight alterations, consisting of small clusters of vacuoles, at the electron microscopy level. Mitotic cells contain the same total amount of lysosomal enzymes per cell as interphase cells, but they display no redistribution of lysosomal enzymes during the virus infection as interphase cells do. This supports the view that lysosomal enzyme redistribution is associated with the cytopathic effect in poliovirus infection but shows that virus synthesis and release is not dependent on either the cytopathic effect or lysosomal enzyme release. The possible reasons for the lack of cytopathic effect in mitotic cells are discussed. Images PMID:4121707

  11. Target Selection and Deselection at the Berkeley StructuralGenomics Center

    SciTech Connect

    Chandonia, John-Marc; Kim, Sung-Hou; Brenner, Steven E.

    2005-03-22

    At the Berkeley Structural Genomics Center (BSGC), our goalis to obtain a near-complete structural complement of proteins in theminimal organisms Mycoplasma genitalium and M. pneumoniae, two closelyrelated pathogens. Current targets for structure determination have beenselected in six major stages, starting with those predicted to be mosttractable to high throughput study and likely to yield new structuralinformation. We report on the process used to select these proteins, aswell as our target deselection procedure. Target deselection reducesexperimental effort by eliminating targets similar to those recentlysolved by the structural biology community or other centers. We measurethe impact of the 69 structures solved at the BSGC as of July 2004 onstructure prediction coverage of the M. pneumoniae and M. genitaliumproteomes. The number of Mycoplasma proteins for which thefold couldfirst be reliably assigned based on structures solved at the BSGC (24 M.pneumoniae and 21 M. genitalium) is approximately 25 percent of the totalresulting from work at all structural genomics centers and the worldwidestructural biology community (94 M. pneumoniae and 86M. genitalium)during the same period. As the number of structures contributed by theBSGC during that period is less than 1 percent of the total worldwideoutput, the benefits of a focused target selection strategy are apparent.If the structures of all current targets were solved, the percentage ofM. pneumoniae proteins for which folds could be reliably assigned wouldincrease from approximately 57 percent (391 of 687) at present to around80 percent (550 of 687), and the percentage of the proteome that could beaccurately modeled would increase from around 37 percent (254 of 687) toabout 64 percent (438 of 687). In M. genitalium, the percentage of theproteome that could be structurally annotated based on structures of ourremaining targets would rise from 72 percent (348 of 486) to around 76percent (371 of 486), with the

  12. Impairment of lysosomal integrity by B10, a glycosylated derivative of betulinic acid, leads to lysosomal cell death and converts autophagy into a detrimental process

    PubMed Central

    Gonzalez, P; Mader, I; Tchoghandjian, A; Enzenmüller, S; Cristofanon, S; Basit, F; Debatin, K-M; Fulda, S

    2012-01-01

    In this study, we report a novel mechanism of action for a cytotoxic derivative of betulinic acid (BA). B10 is a semi-synthetic glycosylated derivative of BA selected for its enhanced cytotoxic activity. Interestingly, although B10 induces apoptosis, caspase-3 downregulation incompletely prevents B10-induced cell death, Bcl-2 overexpression fails to protect cells and DNA fragmentation rates do not reflect cell death rates in contrast to cytoplasmic membrane permeabilization. These results implicate that apoptotic and non-apoptotic cell death coexist upon B10 treatment. Unexpectedly, we found that B10 induces autophagy and also abrogates the autophagic flux. B10 destabilizes lysosomes as shown by Lysotracker Red staining and by cathepsin Z and B release from lysosomes into the cytoplasm. Consistently, the cathepsin inhibitor Ca074Me significantly decreases B10-induced cell death, further supporting the fact that the release of lysosomal enzymes contributes to B10-triggered cell death. Downregulation of ATG7, ATG5 or BECN1 by RNAi significantly decreases caspase-3 activation, lysosomal permeabilization and cell death. Thus, by concomitant induction of autophagy and inhibition of the autophagic flux, B10 turns autophagy into a cell death mechanism. These findings have important implications for the therapeutic exploitation of BA derivatives, particularly in apoptosis-resistant cancers. PMID:22343715

  13. Implications of structural genomics target selection strategies: Pfam5000, whole genome, and random approaches

    SciTech Connect

    Chandonia, John-Marc; Brenner, Steven E.

    2004-07-14

    The structural genomics project is an international effort to determine the three-dimensional shapes of all important biological macromolecules, with a primary focus on proteins. Target proteins should be selected according to a strategy which is medically and biologically relevant, of good value, and tractable. As an option to consider, we present the Pfam5000 strategy, which involves selecting the 5000 most important families from the Pfam database as sources for targets. We compare the Pfam5000 strategy to several other proposed strategies that would require similar numbers of targets. These include including complete solution of several small to moderately sized bacterial proteomes, partial coverage of the human proteome, and random selection of approximately 5000 targets from sequenced genomes. We measure the impact that successful implementation of these strategies would have upon structural interpretation of the proteins in Swiss-Prot, TrEMBL, and 131 complete proteomes (including 10 of eukaryotes) from the Proteome Analysis database at EBI. Solving the structures of proteins from the 5000 largest Pfam families would allow accurate fold assignment for approximately 68 percent of all prokaryotic proteins (covering 59 percent of residues) and 61 percent of eukaryotic proteins (40 percent of residues). More fine-grained coverage which would allow accurate modeling of these proteins would require an order of magnitude more targets. The Pfam5000 strategy may be modified in several ways, for example to focus on larger families, bacterial sequences, or eukaryotic sequences; as long as secondary consideration is given to large families within Pfam, coverage results vary only slightly. In contrast, focusing structural genomics on a single tractable genome would have only a limited impact in structural knowledge of other proteomes: a significant fraction (about 30-40 percent of the proteins, and 40-60 percent of the residues) of each proteome is classified in small

  14. Targeted delivery of photosensitizers: efficacy and selectivity issues revealed by multifunctional ORMOSIL nanovectors in cellular systems

    NASA Astrophysics Data System (ADS)

    Selvestrel, Francesco; Moret, Francesca; Segat, Daniela; Woodhams, Josephine H.; Fracasso, Giulio; Echevarria, Iria M. Rio; Baù, Luca; Rastrelli, Federico; Compagnin, Chiara; Reddi, Elena; Fedeli, Chiara; Papini, Emanuele; Tavano, Regina; MacKenzie, Alexandra; Bovis, Melissa; Yaghini, Elnaz; MacRobert, Alexander J.; Zanini, Silvia; Boscaini, Anita; Colombatti, Marco; Mancin, Fabrizio

    2013-06-01

    PEGylated and non-PEGylated ORMOSIL nanoparticles prepared by microemulsion condensation of vinyltriethoxy-silane (VTES) were investigated in detail for their micro-structure and ability to deliver photoactive agents. With respect to pure silica nanoparticles, organic modification substantially changes the microstructure and the surface properties. This in turn leads to a modulation of both the photophysical properties of embedded photosensitizers and the interaction of the nanoparticles with biological entities such as serum proteins. The flexibility of the synthetic procedure allows the rapid preparation and screening of multifunctional nanosystems for photodynamic therapy (PDT). Selective targeting of model cancer cells was tested by using folate, an integrin specific RGD peptide and anti-EGFR antibodies. Data suggest the interference of the stealth-conferring layer (PEG) with small targeting agents, but not with bulky antibodies. Moreover, we showed that selective photokilling of tumour cells may be limited even in the case of efficient targeting because of intrinsic transport limitations of active cellular uptake mechanisms or suboptimum localization.PEGylated and non-PEGylated ORMOSIL nanoparticles prepared by microemulsion condensation of vinyltriethoxy-silane (VTES) were investigated in detail for their micro-structure and ability to deliver photoactive agents. With respect to pure silica nanoparticles, organic modification substantially changes the microstructure and the surface properties. This in turn leads to a modulation of both the photophysical properties of embedded photosensitizers and the interaction of the nanoparticles with biological entities such as serum proteins. The flexibility of the synthetic procedure allows the rapid preparation and screening of multifunctional nanosystems for photodynamic therapy (PDT). Selective targeting of model cancer cells was tested by using folate, an integrin specific RGD peptide and anti-EGFR antibodies. Data

  15. Molecular pathologies of and enzyme replacement therapies for lysosomal diseases.

    PubMed

    Sakuraba, Hitoshi; Sawada, Makoto; Matsuzawa, Fumiko; Aikawa, Sei-ichi; Chiba, Yasunori; Jigami, Yoshifumi; Itoh, Kohji

    2006-08-01

    Lysosomal diseases comprise a group of inherited disorders resulting from defects of lysosomal enzymes and their cofactors, and in many of them the nervous system is affected. Recently, enzyme replacement therapy with recombinant lysosomal enzymes has been clinically available for several lysosomal diseases. Such enzyme replacement therapies can improve non-neurological disorders but is not effective for neurological ones. In this review, we discuss the molecular pathologies of lysosomal diseases from the protein structural aspect, current enzyme replacement therapies, and attempts to develop enzyme replacement therapies effective for lysosomal diseases associated with neurological disorders, i.e., production of enzymes, brain-specific delivery and incorporation of lysosomal enzymes into cells. PMID:16918392

  16. Mechanisms of Dendritic Cell Lysosomal Killing of Cryptococcus

    PubMed Central

    Hole, Camaron R.; Bui, Hoang; Wormley, Floyd L.; Wozniak, Karen L.

    2012-01-01

    Cryptococcus neoformans is an opportunistic pulmonary fungal pathogen that disseminates to the CNS causing fatal meningitis in immunocompromised patients. Dendritic cells (DCs) phagocytose C. neoformans following inhalation. Following uptake, cryptococci translocate to the DC lysosomal compartment and are killed by oxidative and non-oxidative mechanisms. DC lysosomal extracts kill cryptococci in vitro; however, the means of antifungal activity remain unknown. Our studies determined non-oxidative antifungal activity by DC lysosomal extract. We examined DC lysosomal killing of cryptococcal strains, anti-fungal activity of purified lysosomal enzymes, and mechanisms of killing against C. neoformans. Results confirmed DC lysosome fungicidal activity against all cryptococcal serotypes. Purified lysosomal enzymes, specifically cathepsin B, inhibited cryptococcal growth. Interestingly, cathepsin B combined with its enzymatic inhibitors led to enhanced cryptococcal killing. Electron microscopy revealed structural changes and ruptured cryptococcal cell walls following treatment. Finally, additional studies demonstrated that osmotic lysis was responsible for cryptococcal death. PMID:23074646

  17. Mechanisms of dendritic cell lysosomal killing of Cryptococcus.

    PubMed

    Hole, Camaron R; Bui, Hoang; Wormley, Floyd L; Wozniak, Karen L

    2012-01-01

    Cryptococcus neoformans is an opportunistic pulmonary fungal pathogen that disseminates to the CNS causing fatal meningitis in immunocompromised patients. Dendritic cells (DCs) phagocytose C. neoformans following inhalation. Following uptake, cryptococci translocate to the DC lysosomal compartment and are killed by oxidative and non-oxidative mechanisms. DC lysosomal extracts kill cryptococci in vitro; however, the means of antifungal activity remain unknown. Our studies determined non-oxidative antifungal activity by DC lysosomal extract. We examined DC lysosomal killing of cryptococcal strains, anti-fungal activity of purified lysosomal enzymes, and mechanisms of killing against C. neoformans. Results confirmed DC lysosome fungicidal activity against all cryptococcal serotypes. Purified lysosomal enzymes, specifically cathepsin B, inhibited cryptococcal growth. Interestingly, cathepsin B combined with its enzymatic inhibitors led to enhanced cryptococcal killing. Electron microscopy revealed structural changes and ruptured cryptococcal cell walls following treatment. Finally, additional studies demonstrated that osmotic lysis was responsible for cryptococcal death. PMID:23074646

  18. Mechanisms of Dendritic Cell Lysosomal Killing of Cryptococcus

    NASA Astrophysics Data System (ADS)

    Hole, Camaron R.; Bui, Hoang; Wormley, Floyd L.; Wozniak, Karen L.

    2012-10-01

    Cryptococcus neoformans is an opportunistic pulmonary fungal pathogen that disseminates to the CNS causing fatal meningitis in immunocompromised patients. Dendritic cells (DCs) phagocytose C. neoformans following inhalation. Following uptake, cryptococci translocate to the DC lysosomal compartment and are killed by oxidative and non-oxidative mechanisms. DC lysosomal extracts kill cryptococci in vitro; however, the means of antifungal activity remain unknown. Our studies determined non-oxidative antifungal activity by DC lysosomal extract. We examined DC lysosomal killing of cryptococcal strains, anti-fungal activity of purified lysosomal enzymes, and mechanisms of killing against C. neoformans. Results confirmed DC lysosome fungicidal activity against all cryptococcal serotypes. Purified lysosomal enzymes, specifically cathepsin B, inhibited cryptococcal growth. Interestingly, cathepsin B combined with its enzymatic inhibitors led to enhanced cryptococcal killing. Electron microscopy revealed structural changes and ruptured cryptococcal cell walls following treatment. Finally, additional studies demonstrated that osmotic lysis was responsible for cryptococcal death.

  19. Autocrine-Based Selection of Drugs That Target Ion Channels from Combinatorial Venom Peptide Libraries.

    PubMed

    Zhang, Hongkai; Du, Mingjuan; Xie, Jia; Liu, Xiao; Sun, Jingying; Wang, Wei; Xin, Xiu; Possani, Lourival D; Yea, Kyungmoo; Lerner, Richard A

    2016-08-01

    Animal venoms represent a rich source of pharmacologically active peptides that interact with ion channels. However, a challenge to discovering drugs remains because of the slow pace at which venom peptides are discovered and refined. An efficient autocrine-based high-throughput selection system was developed to discover and refine venom peptides that target ion channels. The utility of this system was demonstrated by the discovery of novel Kv1.3 channel blockers from a natural venom peptide library that was formatted for autocrine-based selection. We also engineered a Kv1.3 blocker peptide (ShK) derived from sea anemone to generate a subtype-selective Kv1.3 blocker with a long half-life in vivo. PMID:27197631

  20. Differential sorting of lysosomal enzymes in mannose 6-phosphate receptor-deficient fibroblasts.

    PubMed Central

    Ludwig, T; Munier-Lehmann, H; Bauer, U; Hollinshead, M; Ovitt, C; Lobel, P; Hoflack, B

    1994-01-01

    In higher eukaryotes, the transport of soluble lysosomal enzymes involves the recognition of their mannose 6-phosphate signal by two receptors: the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). It is not known why these two different proteins are present in most cell types. To investigate their relative function in lysosomal enzyme targeting, we created cell lines that lack either or both MPRs. This was accomplished by mating CD-MPR-deficient mice with Thp mice that carry a CI-MPR deleted allele. Fibroblasts prepared from embryos that lack the two receptors exhibit a massive missorting of multiple lysosomal enzymes and accumulate undigested material in their endocytic compartments. Fibroblasts that lack the CI-MPR, like those lacking the CD-MPR, exhibit a milder phenotype and are only partially impaired in sorting. This demonstrates that both receptors are required for efficient intracellular targeting of lysosomal enzymes. More importantly, comparison of the phosphorylated proteins secreted by the different cell types indicates that the two receptors may interact in vivo with different subgroups of hydrolases. This observation may provide a rational explanation for the existence of two distinct mannose 6-phosphate binding proteins in mammalian cells. Images PMID:8062819

  1. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

    NASA Astrophysics Data System (ADS)

    Serebrovskaya, Ekaterina O.; Ryumina, Alina P.; Boulina, Maria E.; Shirmanova, Marina V.; Zagaynova, Elena V.; Bogdanova, Ekaterina A.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

    2014-07-01

    KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

  2. Cancer cell injury by cytotoxins from cobra venom is mediated through lysosomal damage

    PubMed Central

    2005-01-01

    Cytotoxins from cobra venom are known to manifest cytotoxicity in various cell types. It is widely accepted that the plasma membrane is a target of cytotoxins, but the mechanism of their action remains obscure. Using the confocal spectral imaging technique, we show for the first time that cytotoxins from cobra venom penetrate readily into living cancer cells and accumulate markedly in lysosomes. Cytotoxins CT1 and CT2 from Naja oxiana, CT3 from Naja kaouthia and CT1 from Naja haje are demonstrated to possess this property with respect to human lung adenocarcinoma A549 and promyelocytic leukaemia HL60 cells. Immobilized plasma membrane binding accompanies the internalization of CT3 from Naja kaouthia in the HL60 cells, but it is very weak for other cytotoxins. Detectable membrane binding is not a property of any of the cytotoxins tested in A549 cells. The kinetics and concentration-dependence of cytotoxin accumulation in lysosomes correlate well with their cytotoxic effects. On the basis of the results obtained, we propose that lysosomes are a primary target of the lytic action of cytotoxins. Plasma membrane permeabilization seems to be a downstream event relative to lysosome rupture. Direct damage to the plasma membrane may be a complementary mechanism, but its relative contribution to the cytotoxic action depends on the cytotoxin structure and cell type. PMID:15847607

  3. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies

    PubMed Central

    Wang, Xiang; Ding, Jian; Meng, Ling-hua

    2015-01-01

    The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents. PMID:26364801

  4. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies.

    PubMed

    Wang, Xiang; Ding, Jian; Meng, Ling-hua

    2015-10-01

    The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents. PMID:26364801

  5. ATG7 deficiency suppresses apoptosis and cell death induced by lysosomal photodamage

    PubMed Central

    Kessel, David H.; Price, Michael; Reiners, Jr., John J.

    2012-01-01

    Photodynamic therapy (PDT) involves photosensitizing agents that, in the presence of oxygen and light, initiate formation of cytotoxic reactive oxygen species (ROS). PDT commonly induces both apoptosis and autophagy. Previous studies with murine hepatoma 1c1c7 cells indicated that loss of autophagy-related protein 7 (ATG7) inhibited autophagy and enhanced the cytotoxicity of photosensitizers that mediate photodamage to mitochondria or the endoplasmic reticulum. In this study, we examined two photosensitizing agents that target lysosomes: the chlorin NPe6 and the palladium bacteriopheophorbide WST11. Irradiation of wild-type 1c1c7 cultures loaded with either photosensitizer induced apoptosis and autophagy, with a blockage of autophagic flux. An ATG7- or ATG5-deficiency suppressed the induction of autophagy in PDT protocols using either photosensitizer. Whereas ATG5-deficient cells were quantitatively similar to wild-type cultures in their response to NPe6 and WST11 PDT, an ATG7-deficiency suppressed the apoptotic response (as monitored by analyses of chromatin condensation and procaspase-3/7 activation) and increased the LD50 light dose by > 5-fold (as monitored by colony-forming assays). An ATG7-deficiency did not prevent immediate lysosomal photodamage, as indicated by loss of the lysosomal pH gradient. However, unlike wild-type and ATG5-deficient cells, the lysosomes of ATG7-deficient cells recovered this gradient within 4 h of irradiation, and never underwent permeabilization (monitored as release of endocytosed 10-kDa dextran polymers). We propose that the efficacy of lysosomal photosensitizers is in part due to both promotion of autophagic stress and suppression of autophagic prosurvival functions. In addition, an effect of ATG7 unrelated to autophagy appears to modulate lysosomal photodamage. PMID:22889762

  6. Salinomycin induces selective cytotoxicity to MCF-7 mammosphere cells through targeting the Hedgehog signaling pathway.

    PubMed

    Fu, Ying-Zi; Yan, Yuan-Yuan; He, Miao; Xiao, Qing-Huan; Yao, Wei-Fan; Zhao, Lin; Wu, Hui-Zhe; Yu, Zhao-Jin; Zhou, Ming-Yi; Lv, Mu-Tian; Zhang, Shan-Shan; Chen, Jian-Jun; Wei, Min-Jie

    2016-02-01

    Breast cancer stem cells (BCSCs) are believed to be responsible for tumor chemoresistance, recurrence, and metastasis formation. Salinomycin (SAL), a carboxylic polyether ionophore, has been reported to act as a selective breast CSC inhibitor. However, the molecular mechanisms underlying SAL-induced cytotoxicity on BCSCs remain unclear. The Hedgehog (Hh) signaling pathway plays an important role in CSC maintenance and carcinogenesis. Here, we investigated whether SAL induces cytotoxicity on BCSCs through targeting Hh pathway. In the present study, we cultured breast cancer MCF-7 cells in suspension in serum-free medium to obtain breast CSC-enriched MCF-7 mammospheres (MCF-7 MS). MCF-7 MS cells possessed typical BCSC properties, such as CD44+CD24-/low phenotype, high expression of OCT4 (a stem cell marker), increased colony-forming ability, strong migration and invasion capabilities, differentiation potential, and strong tumorigenicity in xenografted mice. SAL exhibited selective cytotoxicity to MCF-7 MS cells relative to MCF-7 cells. The Hh pathway was highly activated in BCSC-enriched MCF-7 MS cells and SAL inhibited Hh signaling activation by downregulating the expression of critical components of the Hh pathway such as PTCH, SMO, Gli1, and Gli2, and subsequently repressing the expression of their essential downstream targets including C-myc, Bcl-2, and Snail (but not cyclin D1). Conversely, Shh-induced Hh signaling activation could largely reverse SAL-mediated inhibitory effects. These findings suggest that SAL-induced selective cytotoxicity against MCF-7 MS cells is associated with the inhibition of Hh signaling activation and the expression of downstream targets and the Hh pathway is an important player and a possible drug target in the pathogenesis of BCSCs. PMID:26718029

  7. Characterization of parasite-specific indels and their proposed relevance for selective anthelminthic drug targeting.

    PubMed

    Wang, Qi; Heizer, Esley; Rosa, Bruce A; Wildman, Scott A; Janetka, James W; Mitreva, Makedonka

    2016-04-01

    Insertions and deletions (indels) are important sequence variants that are considered as phylogenetic markers that reflect evolutionary adaptations in different species. In an effort to systematically study indels specific to the phylum Nematoda and their structural impact on the proteins bearing them, we examined over 340,000 polypeptides from 21 nematode species spanning the phylum, compared them to non-nematodes and identified indels unique to nematode proteins in more than 3000 protein families. Examination of the amino acid composition revealed uneven usage of amino acids for insertions and deletions. The amino acid composition and cost, along with the secondary structure constitution of the indels, were analyzed in the context of their biological pathway associations. Species-specific indels could enable indel-based targeting for drug design in pathogens/parasites. Therefore, we screened the spatial locations of the indels in the parasite's protein 3D structures, determined the location of the indel and identified potential unique drug targeting sites. These indels could be confirmed by RNA-Seq data. Examples are presented illustrating the close proximity of some indels to established small-molecule binding pockets that can potentially facilitate selective targeting to the parasites and bypassing their host, thus reducing or eliminating the toxicity of the potential drugs. This study presents an approach for understanding the adaptation of pathogens/parasites at a molecular level, and outlines a strategy to identify such nematode-selective targets that remain essential to the organism. With further experimental characterization and validation, it opens a possible channel for the development of novel treatments with high target specificity, addressing both host toxicity and resistance concerns. PMID:26829384

  8. Theater targets plume edge extraction and hardbody aimpoint selection using morphological image processing

    NASA Astrophysics Data System (ADS)

    Paiva, Clifford A.

    1997-06-01

    (U) Future successful ballistic missile booster intercepts will require advanced automatic target detection, tracking, classification and identification (ADTCI) image processing techniques. Two such techniques are presented in this classified SECRET paper using the synthetic scene generator model (SSGM) in combination with the advanced systems (AVS) image processing package. Two challenging multispectral cases are treated: (1) missile hardbody occultation by the missile exhaust plume, and (2) variable plume/hardbody system (PHS) gradient intensities generated by missile tumbling due to exiting the sensible atmosphere. The target detection, tracking and edge extraction methods selected for this study include morphological, open-close operations within decision- level fusion for the obscuration case and pixel-level fusion for variable edge intensities. Other investigators have approached this issue on similar image processing techniques. The multispectral (2.69 - 2.95 micrometer SWIR; 4.17 - 4.2, 4.35 - 4.50 micrometer MWIR; and 8.0 - 12.0 micrometer LWIR) target/background imagery includes SWIRM/MWIR boost phase track (with occlusion problem) and LWIR aimpoint selection (with tumbling problem). The two classified missile systems are: (1) a depressed-angle submarine launched ballistic missile (SLBM) and (2) a medium range ballistic missile (MRBM). The results indicate that for 6 degrees of freedom (6 DOF) hardbodies, ATDCI geometrical pattern reference libraries should be optimized to accommodate the extreme variable gradient geometries for tumbling midcourse targets. For boost- phase missile hardbody occultation by missile exhaust plumes, segmentation and feature extraction should be implemented in each bandpass before processing to the ATDCI classifier. This study demonstrates that although the plume/hardbody system edges were extracted, the geometry of the target edge often deviated from symmetry.

  9. Sticky Patches on Lipid Nanoparticles Enable the Selective Targeting and Killing of Untargetable Cancer Cells.

    PubMed

    Sempkowski, Michelle; Zhu, Charles; Menzenski, Monica Zofia; Kevrekidis, Ioannis G; Bruchertseifer, Frank; Morgenstern, Alfred; Sofou, Stavroula

    2016-08-23

    Effective targeting by uniformly functionalized nanoparticles is limited to cancer cells expressing at least two copies of targeted receptors per nanoparticle footprint (approximately ≥2 × 10(5) receptor copies per cell); such a receptor density supports the required multivalent interaction between the neighboring receptors and the ligands from a single nanoparticle. To enable selective targeting below this receptor density, ligands on the surface of lipid vesicles were displayed in clusters that were designed to form at the acidic pH of the tumor interstitium. Vesicles with clustered HER2-targeting peptides within such sticky patches (sticky vesicles) were compared to uniformly functionalized vesicles. On HER2-negative breast cancer cells MDA-MB-231 and MCF7 {expressing (8.3 ± 0.8) × 10(4) and (5.4 ± 0.9) × 10(4) HER2 copies per cell, respectively}, only the sticky vesicles exhibited detectable specific targeting (KD ≈ 49-69 nM); dissociation (0.005-0.009 min(-1)) and endocytosis rates (0.024-0.026 min(-1)) were independent of HER2 expression for these cells. MDA-MB-231 and MCF7 were killed only by sticky vesicles encapsulating doxorubicin (32-40% viability) or α-particle emitter (225)Ac (39-58% viability) and were not affected by uniformly functionalized vesicles (>80% viability). Toxicities on cardiomyocytes and normal breast cells (expressing HER2 at considerably lower but not insignificant levels) were not observed, suggesting the potential of tunable clustered ligand display for the selective killing of cancer cells with low receptor densities. PMID:27468779

  10. Newborn screening for lysosomal storage disorders.

    PubMed

    Matern, Dietrich; Gavrilov, Dimitar; Oglesbee, Devin; Raymond, Kimiyo; Rinaldo, Piero; Tortorelli, Silvia

    2015-04-01

    Every newborn in the U.S. is screened for at least 29 disorders, where evidence suggests that early detection is possible and beneficial. With new or improved treatment options and development of high-throughput screening tests, additional conditions have been proposed for inclusion in newborn screening programs. Among those are several lysosomal storage disorders that have been evaluated in limited pilot studies or that are already included in a few national or international newborn screening programs. These conditions include Pompe disease, Niemann-Pick type A/B disease, Fabry disease, Krabbe disease, Mucopolysaccharidoses types I and II, and Gaucher disease. Here, we review the current state of newborn screening for these lysosomal storage disorders. PMID:25891428

  11. Newborn screening for lysosomal storage disorders.

    PubMed

    Meikle, Peter J; Grasby, Dallas J; Dean, Caroline J; Lang, Debbie L; Bockmann, Michelle; Whittle, Alison M; Fietz, Michael J; Simonsen, Henrik; Fuller, Maria; Brooks, Douglas A; Hopwood, John J

    2006-08-01

    Lysosomal storage disorders (LSD) are chronic progressive diseases that have a devastating impact on the patient and family. Most patients are clinically normal at birth but develop symptoms early in childhood. Despite no curative treatment, a number of therapeutic options are available to improve quality of life. To achieve this, there is a pressing need for newborn screening to identify affected individuals early, before the onset of severe irreversible pathology. We have developed a multiplexed immune-quantification assay of 11 different lysosomal proteins for the identification of individuals with an LSD and evaluated this assay in a retrospective study using blood-spots from; newborns subsequently diagnosed with an LSD (n=19, six different LSD), individuals sampled after diagnosis of an LSD (n=92, 11 different LSD), newborn controls (n=433), and adult controls (n=200). All patients with mucopolysaccharidosis type I (MPS I), MPS II, MPS IIIA, MPS VI, metachromatic leukodystrophy, Niemann-Pick disease type A/B, and multiple sulfatase deficiency could be identified by reduced enzyme levels compared to controls. All mucolipidosis type II/III patients were identified by the elevation of several lysosomal enzymes, above the control range. Most Fabry, Pompe, and Gaucher disease patients were identified from either single protein differences or profiles of multiple protein markers. Newborn screening for multiple LSD is achievable using multiplexed immune-quantification of a panel of lysosomal proteins. With further validation, this method could be readily incorporated into existing screening laboratories and will have a substantial impact on patient management and counseling of families. PMID:16600651

  12. Pathogenic LRRK2 mutations, through increased kinase activity, produce enlarged lysosomes with reduced degradative capacity and increase ATP13A2 expression.

    PubMed

    Henry, Anastasia G; Aghamohammadzadeh, Soheil; Samaroo, Harry; Chen, Yi; Mou, Kewa; Needle, Elie; Hirst, Warren D

    2015-11-01

    Lysosomal dysfunction plays a central role in the pathogenesis of several neurodegenerative disorders, including Parkinson's disease (PD). Several genes linked to genetic forms of PD, including leucine-rich repeat kinase 2 (LRRK2), functionally converge on the lysosomal system. While mutations in LRRK2 are commonly associated with autosomal-dominant PD, the physiological and pathological functions of this kinase remain poorly understood. Here, we demonstrate that LRRK2 regulates lysosome size, number and function in astrocytes, which endogenously express high levels of LRRK2. Expression of LRRK2 G2019S, the most common pathological mutation, produces enlarged lysosomes and diminishes the lysosomal capacity of these cells. Enlarged lysosomes appears to be a common phenotype associated with pathogenic LRRK2 mutations, as we also observed this effect in cells expressing other LRRK2 mutations; R1441C or Y1699C. The lysosomal defects associated with these mutations are dependent on both the catalytic activity of the kinase and autophosphorylation of LRRK2 at serine 1292. Further, we demonstrate that blocking LRRK2's kinase activity, with the potent and selective inhibitor PF-06447475, rescues the observed defects in lysosomal morphology and function. The present study also establishes that G2019S mutation leads to a reduction in lysosomal pH and increased expression of the lysosomal ATPase ATP13A2, a gene linked to a parkinsonian syndrome (Kufor-Rakeb syndrome), in brain samples from mouse and human LRRK2 G2019S carriers. Together, these results demonstrate that PD-associated LRRK2 mutations perturb lysosome function in a kinase-dependent manner, highlighting the therapeutic promise of LRRK2 kinase inhibitors in the treatment of PD. PMID:26251043

  13. Newborn Screening for Lysosomal Storage Diseases

    PubMed Central

    Gelb, Michael H.; Scott, C. Ronald; Turecek, Frantisek

    2015-01-01

    BACKGROUND There is worldwide interest in newborn screening for lysosomal storage diseases because of the development of treatment options that give better results when carried out early in life. Screens with high differentiation between affected and nonaffected individuals are critical because of the large number of potential false positives. CONTENT This review summarizes 3 screening methods: (a) direct assay of enzymatic activities using tandem mass spectrometry or fluorometry, (b) immunocapture-based measurement of lysosomal enzyme abundance, and (c) measurement of biomarkers. Assay performance is compared on the basis of small-scale studies as well as on large-scale pilot studies of mass spectrometric and fluorometric screens. SUMMARY Tandem mass spectrometry and fluorometry techniques for direct assay of lysosomal enzymatic activity in dried blood spots have emerged as the most studied approaches. Comparative mass spectrometry vs fluorometry studies show that the former better differentiates between nonaffected vs affected individuals. This in turn leads to a manageable number of screen positives that can be further evaluated with second-tier methods. PMID:25477536

  14. Strategies for Assaying Lysosomal Membrane Permeabilization.

    PubMed

    Repnik, Urška; Hafner Česen, Maruša; Turk, Boris

    2016-01-01

    Late endosomal organelles have an acidic pH and contain hydrolytic enzymes to degrade cargo delivered either from the extracellular environment by endocytosis or from within the cell itself by autophagy. In the event of lysosomal membrane permeabilization (LMP), the contents of late endosomes and lysosomes can be released into the cytosol and then initiate apoptosis. Compounds that can trigger LMP are therefore candidates for the induction of apoptosis, in particular in anticancer therapy. Alternatively, drug-delivery systems, such as nanoparticles, can have side effects that can include LMP, which has toxic consequences for the cells. To determine when, to what extent, and with what consequences LMP occurs is therefore of paramount importance for the evaluation of new potentially LMP-inducing compounds. In this introduction, we provide an overview of some basic assays for assessing LMP, such as staining with lysosomotropic dyes and measurement of cysteine cathepsin activity, and discuss additional strategies for the detection of the release of endogenous lysosomal molecules or preloaded exogenous tracers into the cytosol. PMID:27250949

  15. Intracellular sphingosine releases calcium from lysosomes

    PubMed Central

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. DOI: http://dx.doi.org/10.7554/eLife.10616.001 PMID:26613410

  16. Intracellular sphingosine releases calcium from lysosomes.

    PubMed

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. PMID:26613410

  17. Sequential Retraction Segregates SGN Processes during Target Selection in the Cochlea

    PubMed Central

    Druckenbrod, Noah R.

    2015-01-01

    A hallmark of the nervous system is the presence of precise patterns of connections between different types of neurons. Many mechanisms can be used to establish specificity, including homophilic adhesion and synaptic refinement, but the range of strategies used across the nervous system remains unclear. To broaden the understanding of how neurons find their targets, we studied the developing murine cochlea, where two classes of spiral ganglion neurons (SGNs), type I and type II, navigate together to the sensory epithelium and then diverge to contact inner hair cells (IHCs) or outer hair cells (OHCs), respectively. Neurons with type I and type II morphologies are apparent before birth, suggesting that target selection might be accomplished by excluding type I processes from the OHC region. However, because type I processes appear to overshoot into type II territory postnatally, specificity may also depend on elimination of inappropriate synapses. To resolve these differences, we analyzed the morphology and dynamic behaviors of individual fibers and their branches as they interact with potential partners. We found that SGN processes continue to be segregated anatomically in the postnatal cochlea. Although type I-like fibers branched locally, few branches contacted OHCs, arguing against synaptic elimination. Instead, time-lapse imaging studies suggest a prominent role for retraction, first positioning processes to the appropriate region and then corralling branches during a subsequent period of exuberant growth and refinement. Thus, sequential stages of retraction can help to achieve target specificity, adding to the list of mechanisms available for sculpting neural circuits. SIGNIFICANCE STATEMENT During development, different types of neurons must form connections with specific synaptic targets, thereby creating the precise wiring diagram necessary for adult function. Although studies have revealed multiple mechanisms for target selection, we still know little about

  18. PITPs as Targets for Selectively Interfering With Phosphoinositide Signaling in Cells

    PubMed Central

    Nile, Aaron H.; Tripathi, Ashutosh; Yuan, Peihua; Mousley, Carl J.; Suresh, Sundari; Wallace, Iain Michael; Shah, Sweety D.; Pohlhaus, Denise Teotico; Temple, Brenda; Nislow, Corey; Giaever, Guri; Tropsha, Alexander; Davis, Ronald W.; St Onge, Robert P.; Bankaitis, Vytas A.

    2013-01-01

    Sec14-like phosphatidylinositol transfer proteins (PITPs) integrate diverse territories of intracellular lipid metabolism with stimulated phosphatidylinositol-4-phosphate production, and are discriminating portals for interrogating phosphoinositide signaling. Yet, neither Sec14-like PITPs, nor PITPs in general, have been exploited as targets for chemical inhibition for such purposes. Herein, we validate the first small molecule inhibitors (SMIs) of the yeast PITP Sec14. These SMIs are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and are effective inhibitors in vitro and in vivo. We further establish Sec14 is the sole essential NPPM target in yeast, that NPPMs exhibit exquisite targeting specificities for Sec14 (relative to related Sec14-like PITPs), propose a mechanism for how NPPMs exert their inhibitory effects, and demonstrate NPPMs exhibit exquisite pathway selectivity in inhibiting phosphoinositide signaling in cells. These data deliver proof-of-concept that PITP-directed SMIs offer new and generally applicable avenues for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies. PMID:24292071

  19. The contribution of the major planet search surveys to EChO target selection

    NASA Astrophysics Data System (ADS)

    Micela, Giuseppina; Bakos, Gáspár Á.; Lopez-Morales, Mercedes; Maxted, Pierre F. L.; Pagano, Isabella; Sozzetti, Alessandro; Wheatley, Peter J.

    2015-12-01

    The EChO core science will be based on a three tier survey, each with increasing sensitivity, in order to study the population of exo-planets from super-Earths to Jupiter-like planets, in the very hot to temperate zones (temperatures of 300 K - 3000 K) of F to M-type host stars. To achieve a meaningful outcome, an accurate selection of the target sample is needed. In this paper we analyse the targets, suitable for EChO observations, expected to result from a sample of present and forthcoming detection surveys. Present day discovered exoplanets are sufficient to provide a large and diverse sample. However we expect that results from ongoing and planned surveys, aimed at identifying new planets, will increase the sample of smaller planets allowing us to optimize the EChO sample selection. The analysis of the expected yields of a representative set of those surveys both from ground and space shows that they will be able to provide a large number of targets, covering an ample range of planetary and stellar parameters, fitting the EChO capabilities.

  20. The transcription factor NRSF contributes to epileptogenesis by selective repression of a subset of target genes

    PubMed Central

    McClelland, Shawn; Brennan, Gary P; Dubé, Celine; Rajpara, Seeta; Iyer, Shruti; Richichi, Cristina; Bernard, Christophe; Baram, Tallie Z

    2014-01-01

    The mechanisms generating epileptic neuronal networks following insults such as severe seizures are unknown. We have previously shown that interfering with the function of the neuron-restrictive silencer factor (NRSF/REST), an important transcription factor that influences neuronal phenotype, attenuated development of this disorder. In this study, we found that epilepsy-provoking seizures increased the low NRSF levels in mature hippocampus several fold yet surprisingly, provoked repression of only a subset (∼10%) of potential NRSF target genes. Accordingly, the repressed gene-set was rescued when NRSF binding to chromatin was blocked. Unexpectedly, genes selectively repressed by NRSF had mid-range binding frequencies to the repressor, a property that rendered them sensitive to moderate fluctuations of NRSF levels. Genes selectively regulated by NRSF during epileptogenesis coded for ion channels, receptors, and other crucial contributors to neuronal function. Thus, dynamic, selective regulation of NRSF target genes may play a role in influencing neuronal properties in pathological and physiological contexts. DOI: http://dx.doi.org/10.7554/eLife.01267.001 PMID:25117540

  1. Evodiamine selectively targets cancer stem-like cells through the p53-p21-Rb pathway.

    PubMed

    Han, Seula; Woo, Jong Kyu; Jung, Yuchae; Jeong, Dawoon; Kang, Minsook; Yoo, Young-Ji; Lee, Hani; Oh, Seung Hyun; Ryu, Jae-Ha; Kim, Woo-Young

    2016-01-22

    In spite of the recent improvements, the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer, a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here, we report on a component of a traditional Chinese medicine, evodiamine, which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulk cancer cells at the G2/M phase, it did not hold CSLC in a specific cell cycle phase but instead, selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly, evodiamine selectively activated p53 and p21 and decreased inactive Rb, the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer. PMID:26713361

  2. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    PubMed

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  3. Aurora B kinase is a potent and selective target in MYCN-driven neuroblastoma

    PubMed Central

    Azorsa, David O.; Ormanoglu, Pinar; Buehler, Eugen; Guha, Rajarshi; Keller, Jonathan M.; Griner, Lesley A. Mathews; Ferrer, Marc; Song, Young K.; Liao, Hongling; Mendoza, Arnulfo; Gryder, Berkley E.; Sindri, Sivasish; He, Jianbin; Wen, Xinyu; Zhang, Shile; Shern, John F.; Yohe, Marielle E.; Taschner-Mandl, Sabine; Shohet, Jason M.; Thomas, Craig J.; Martin, Scott E.; Ambros, Peter F.; Khan, Javed

    2015-01-01

    Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials. PMID:26497213

  4. Selective cellular uptake and induction of apoptosis of cancer-targeted selenium nanoparticles.

    PubMed

    Huang, Yanyu; He, Lizhen; Liu, Wen; Fan, Cundong; Zheng, Wenjie; Wong, Yum-Shing; Chen, Tianfeng

    2013-09-01

    Selenium nanoparticles (SeNPs) have garnered a great deal of attention as potential cancer therapeutic payloads. However, the in vivo targeting drug delivery has been challenging. Herein, we describe the synthesis of tansferrin (Tf)-conjugated SeNPs and its use as a cancer-targeted drug delivery system to achieve enhanced cellular uptake and anticancer efficacy. Tf as targeting ligand significantly enhances the cellular uptake of doxorubicin (DOX)-loaded SeNPs through clathrin-mediated and caveolae/lipid raft-mediated endocytosis in cancer cells overexpressing transferrin receptor, and increases their selectivity between cancer and normal cells. DOX-loaded and Tf-conjugated SeNPs (Tf-SeNPs) exhibits unprecedented enhanced cytotoxicity toward cancer cells through induction of apoptosis with the involvement of intrinsic and extrinsic pathways. Internalized Tf-SeNPs triggers intracellular ROS overproduction, thus activates p53 and MAPKs pathways to promote cell apoptosis. In the nude mice xenograft experiment, Tf-SeNPs significantly inhibits the tumor growth via induction of p53-mediated apoptosis. This cancer-targeted design of SeNPs opens a new path for synergistic treating of cancer with higher efficacy and decreased side effects. PMID:23800743

  5. Host factors in retroviral integration and the selection of integration target sites

    PubMed Central

    Craigie, Robert; Bushman, Frederic D.

    2015-01-01

    In order to replicate, a retrovirus must integrate a DNA copy of the viral RNA genome into a chromosome of the host cell. The study of retroviral integration has advanced considerably in the last few years. Here we focus on host factor interactions and the linked area of integration targeting. Genome-wide screens for cellular factors affecting HIV replication have identified a series of host cell proteins that may mediate subcellular trafficking of integration complexes, nuclear import, and integration target site selection. The cell transcriptional co-activator protein LEDGF/p75 has been identified as a tethering factor important for HIV integration, and recently, BET proteins (Brd2, 4, and 4) have been identified as tethering factors for the gammaretroviruses. A new class of HIV inhibitors has been developed targeting the HIV-1 IN-LEDGF binding site, though surprisingly these inhibitors appear to block assembly late during replication and do not act at the integration step. Going forward, genome-wide studies of HIV-host interactions offer many new starting points to investigate HIV replication and identify potential new inhibitor targets. PMID:26104434

  6. The Role of Microscopy in Understanding Atherosclerotic Lysosomal Lipid Metabolism

    NASA Astrophysics Data System (ADS)

    Gray Jerome, W.; Yancey, Patricia G.

    2003-02-01

    Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.

  7. Localisation of macrophage lysosomal enzyme in experimental toxoplasma retinitis.

    PubMed Central

    Yoshizumi, M O

    1977-01-01

    Rabbit retinal tissue experimentally infected with Toxoplasma gondii was processed for the lysosomal enzyme aryl sulphatase. Abundant lysosomal activity was found in lysosomal bodies of the infected macrophages. There appeared to be a lack of fusion of the lysosomal bodies with the phagosomes containing the organisms. Examination of the majority of macrophage vacuoles containing trophozoites failed to show consistently lead sulphide deposition for aryl sulphatase activity. By light microscopy 83% of 115 macrophage vacuoles containing the trophozoites of T. gondii showed an absence of lysosomal enzyme activity; 7% of the vacuole containing the trophozoites were found to contain lysosomal enzyme activity. In the remaining 10% of the vacuoles containing the trophozoites of T. gondii the presence or absence of lysosomal enzyme activity could not be determined with certainty. The frequent absence of lysosomal enzyme activity within the phagosomes containing T. gondii organisms may be related to the parasite's ability to multiply and encyst in an intracellular locus. The abundant lysosomal enzyme activity in the lysosomal bodies within the cytoplasm of the infected macrophages may contribute to the cellular destruction of surrounding tissues when infected macrophages burst open owing to proliferation of the trophozoites. Images PMID:871468

  8. Candidate targets of balancing selection in the genome of Staphylococcus aureus.

    PubMed

    Thomas, Jonathan C; Godfrey, Paul A; Feldgarden, Michael; Robinson, D Ashley

    2012-04-01

    Signatures of balancing selection can highlight polymorphisms and functions that are important to the long-term fitness of a species. We performed a first genome-wide scan for balancing selection in a bacterial species, Staphylococcus aureus, which is a common cause of serious antimicrobial-resistant infections of humans. Using a sliding window approach, the genomes of 16 strains of S. aureus, including 5 new genome sequences presented here, and 1 outgroup strain of S. epidermidis were scanned for signatures of balancing selection. A total of 195 short windows were investigated based on their extreme values of both Tajima's D (>2.03) and π/K ratios (>0.12) relative to the rest of the genome. To test the unusualness of these windows, an Approximate Bayesian Computation framework was used to select a null demographic model that better accounted for the observed data than did the standard neutral model. A total of 186 windows were demonstrated to be unusual under the null model and, thus, represented candidate loci under balancing selection. These 186 candidate windows were located within 99 candidate genes that were spread across 62 different loci. Nearly all the signal (97.2%) was located within coding sequences; balancing selection on gene regulation apparently occurs through the targeting of global regulators such as agr and gra/aps. The agr locus had some of the strongest signatures of balancing selection, which provides new insight into the causes of diversity at this locus. The list of candidate genes included multiple virulence-associated genes and was significantly enriched for functions in amino acid and inorganic ion transport and metabolism and in defense mechanisms against innate immunity and antimicrobials, highlighting these particular functions as important to the fitness of this pathogen. PMID:22114360

  9. Candidate Targets of Balancing Selection in the Genome of Staphylococcus aureus

    PubMed Central

    Thomas, Jonathan C.; Godfrey, Paul A.; Feldgarden, Michael; Robinson, D. Ashley

    2012-01-01

    Signatures of balancing selection can highlight polymorphisms and functions that are important to the long-term fitness of a species. We performed a first genome-wide scan for balancing selection in a bacterial species, Staphylococcus aureus, which is a common cause of serious antimicrobial-resistant infections of humans. Using a sliding window approach, the genomes of 16 strains of S. aureus, including 5 new genome sequences presented here, and 1 outgroup strain of S. epidermidis were scanned for signatures of balancing selection. A total of 195 short windows were investigated based on their extreme values of both Tajima's D (>2.03) and π/K ratios (>0.12) relative to the rest of the genome. To test the unusualness of these windows, an Approximate Bayesian Computation framework was used to select a null demographic model that better accounted for the observed data than did the standard neutral model. A total of 186 windows were demonstrated to be unusual under the null model and, thus, represented candidate loci under balancing selection. These 186 candidate windows were located within 99 candidate genes that were spread across 62 different loci. Nearly all the signal (97.2%) was located within coding sequences; balancing selection on gene regulation apparently occurs through the targeting of global regulators such as agr and gra/aps. The agr locus had some of the strongest signatures of balancing selection, which provides new insight into the causes of diversity at this locus. The list of candidate genes included multiple virulence-associated genes and was significantly enriched for functions in amino acid and inorganic ion transport and metabolism and in defense mechanisms against innate immunity and antimicrobials, highlighting these particular functions as important to the fitness of this pathogen. PMID:22114360

  10. Selection of Nearby Star Targets for the Subaru Strategic Exploration of Exoplanets and Disks

    NASA Astrophysics Data System (ADS)

    Kandori, R.; Tamura, M.; Morino, J.; Ishii, M.; Suzuki, R.; Hashimoto, J.; Kusakabe, N.; Narita, N.; Sato, B.; Yamada, T.; Enya, K.; Goto, M.; Carson, J.; Thalmann, C.; McElwain, M.; Moro-Martin, A.; Knapp, J.; Turner, E. L.

    2009-08-01

    SEEDS (the Subaru Strategic Exploration of Exoplanets and Disks with Hi-CIAO/AO188) is a strategic five-year campaign of direct imaging surveys of exoplanets/disks using the Subaru telescope equipped with the new adaptive optics system AO188 and our new high-contrast instrument, HiCIAO. The goals of the survey are to address the following key issues in exoplanet/disk sciences: (1) the detection and census of exoplanets; (2) the evolution of protoplanetary and debris disks; and (3) the link between exoplanets and disks. Targets prepared for the SEEDS exoplanet searches are in four categories, including nearby stars. We present our scientific motivations and current status of the SEEDS target selection in the nearby stars category.

  11. Pancratistatin selectively targets cancer cell mitochondria and reduces growth of human colon tumor xenografts.

    PubMed

    Griffin, Carly; Karnik, Aditya; McNulty, James; Pandey, Siyaram

    2011-01-01

    The naturally occurring Amaryllidaceae alkaloid pancratistatin exhibits potent apoptotic activity against a large panel of cancer cells lines and has an insignificant effect on noncancerous cell lines, although with an elusive cellular target. Many current chemotherapeutics induce apoptosis via genotoxic mechanisms and thus have low selectivity. The observed selectivity of pancratistatin for cancer cells promoted us to consider the hypothesis that this alkaloid targets cancer cell mitochondria rather than DNA or its replicative machinery. In this study, we report that pancratistatin decreased mitochondrial membrane potential and induced apoptotic nuclear morphology in p53-mutant (HT-29) and wild-type p53 (HCT116) colorectal carcinoma cell lines, but not in noncancerous colon fibroblast (CCD-18Co) cells. Interestingly, pancratistatin was found to be ineffective against mtDNA-depleted (ρ(0)) cancer cells. Moreover, pancratistatin induced cell death in a manner independent of Bax and caspase activation, and did not alter β-tubulin polymerization rate nor cause double-stranded DNA breaks. For the first time we report the efficacy of pancratistatin in vivo against human colorectal adenocarcinoma xenografts. Intratumor administration of pancratistatin (3 mg/kg) caused significant reduction in the growth of subcutaneous HT-29 tumors in Nu/Nu mice (n = 6), with no apparent toxicity to the liver or kidneys as indicated by histopathologic analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Altogether, this work suggests that pancratistatin may be a novel mitochondria-targeting compound that selectively induces apoptosis in cancer cells and significantly reduces tumor growth. PMID:21220492

  12. Target selection and mass estimation for manned NEO exploration using a baseline mission design

    NASA Astrophysics Data System (ADS)

    Boden, Ralf C.; Hein, Andreas M.; Kawaguchi, Junichiro

    2015-06-01

    In recent years Near-Earth Objects (NEOs) have received an increased amount of interest as a target for human exploration. NEOs offer scientifically interesting targets, and at the same time function as a stepping stone for achieving future Mars missions. The aim of this research is to identify promising targets from the large number of known NEOs that qualify for a manned sample-return mission with a maximum duration of one year. By developing a baseline mission design and a mass estimation model, mission opportunities are evaluated based on on-orbit mass requirements, safety considerations, and the properties of the potential targets. A selection of promising NEOs is presented and the effects of mission requirements and restrictions are discussed. Regarding safety aspects, the use of free-return trajectories provides the lowest on-orbit mass, when compared to an alternative design that uses system redundancies to ensure return of the spacecraft to Earth. It is discovered that, although a number of targets are accessible within the analysed time frame, no NEO offers both easy access and high incentive for its exploration. Under the discussed aspects a first human exploration mission going beyond the vicinity of Earth will require a trade off between targets that provide easy access and those that are of scientific interest. This lack of optimal mission opportunities can be seen in the small number of only 4 NEOs that meet all requirements for a sample-return mission and remain below an on-orbit mass of 500 metric Tons (mT). All of them require a mass between 315 and 492 mT. Even less ideal, smaller asteroids that are better accessible require an on-orbit mass that exceeds the launch capability of future heavy lift vehicles (HLV) such as SLS by at least 30 mT. These mass requirements show that additional efforts are necessary to increase the number of available targets and reduce on-orbit mass requirements through advanced mission architectures. The need for on

  13. The influence of synesthesia on eye movements: no synesthetic pop-out in an oculomotor target selection task.

    PubMed

    Nijboer, Tanja C W; Satris, Gabriela; Van der Stigchel, Stefan

    2011-12-01

    Recent research on grapheme-colour synesthesia has focused on whether visual attention is necessary to induce a synesthetic percept. The current study investigated the influence of synesthesia on overt visual attention during an oculomotor target selection task. Chromatic and achromatic stimuli were presented with one target among distractors (e.g. a '2' (target) among multiple '5's (distractors)). Participants executed an eye movement to the target. Synesthetes and controls showed a comparable target selection performance across conditions and a 'pop-out effect' was only seen in the chromatic condition. As a pop-out effect was absent for the synesthetes in the achromatic condition, a synesthetic element appears not to elicit a synesthetic colour, even when it is the target. The synesthetic percepts are not pre-attentively available to distinguish the synesthetic target from synesthetic distractors when elements are presented in the periphery. Synesthesia appears to require full recognition to bind form and colour. PMID:21531581

  14. The SDSS-IV Extended Baryon Oscillation Spectroscopic Survey: Quasar Target Selection

    NASA Astrophysics Data System (ADS)

    Myers, Adam D.; Palanque-Delabrouille, Nathalie; Prakash, Abhishek; Pâris, Isabelle; Yeche, Christophe; Dawson, Kyle S.; Bovy, Jo; Lang, Dustin; Schlegel, David J.; Newman, Jeffrey A.; Petitjean, Patrick; Kneib, Jean-Paul; Laurent, Pierre; Percival, Will J.; Ross, Ashley J.; Seo, Hee-Jong; Tinker, Jeremy L.; Armengaud, Eric; Brownstein, Joel; Burtin, Etienne; Cai, Zheng; Comparat, Johan; Kasliwal, Mansi; Kulkarni, Shrinivas R.; Laher, Russ; Levitan, David; McBride, Cameron K.; McGreer, Ian D.; Miller, Adam A.; Nugent, Peter; Ofek, Eran; Rossi, Graziano; Ruan, John; Schneider, Donald P.; Sesar, Branimir; Streblyanska, Alina; Surace, Jason

    2015-12-01

    As part of the Sloan Digital Sky Survey (SDSS) IV the extended Baryon Oscillation Spectroscopic Survey (eBOSS) will improve measurements of the cosmological distance scale by applying the Baryon Acoustic Oscillation (BAO) method to quasar samples. eBOSS will adopt two approaches to target quasars over 7500 deg2. First, a “CORE” quasar sample will combine the optical selection in ugriz using a likelihood-based routine called XDQSOz, with a mid-IR-optical color cut. eBOSS CORE selection (to g < 22 or r < 22) should return ˜70 deg-2 quasars at redshifts 0.9 < z < 2.2 and ˜7 deg-2z > 2.1 quasars. Second, a selection based on variability in multi-epoch imaging from the Palomar Transient Factory should recover an additional ˜3-4 deg-2z > 2.1 quasars to g < 22.5. A linear model of how imaging systematics affect target density recovers the angular distribution of eBOSS CORE quasars over 96.7% (76.7%) of the SDSS north (south) Galactic Cap area. The eBOSS CORE quasar sample should thus be sufficiently dense and homogeneous over 0.9 < z < 2.2 to yield the first few-percent-level BAO constraint near \\bar{z}˜ 1.5.eBOSS quasars at z > 2.1 will be used to improve BAO measurements in the Lyα Forest. Beyond its key cosmological goals, eBOSS should be the next-generation quasar survey, comprising >500,000 new quasars and >500,000 uniformly selected spectroscopically confirmed 0.9 < z < 2.2 quasars. At the conclusion of eBOSS, the SDSS will have provided unique spectra for more than 800,000 quasars.

  15. In Vivo Selection Against Human Colorectal Cancer Xenografts Identifies an Aptamer That Targets RNA Helicase Protein DHX9.

    PubMed

    Mi, Jing; Ray, Partha; Liu, Jenny; Kuan, Chien-Tsun; Xu, Jennifer; Hsu, David; Sullenger, Bruce A; White, Rebekah R; Clary, Bryan M

    2016-01-01

    The ability to selectively target disease-related tissues with molecules is critical to the design of effective therapeutic and diagnostic reagents. Recognizing the differences between the in vivo environment and in vitro conditions, we employed an in vivo selection strategy to identify RNA aptamers (targeting motifs) that could localize to tumor in situ. One of the selected molecules is an aptamer that binds to the protein DHX9, an RNA helicase that is known to be upregulated in colorectal cancer. Upon systemic administration, the aptamer preferentially localized to the nucleus of cancer cells in vivo and thus has the potential to be used for targeted delivery. PMID:27115840

  16. Global changes in STAT target selection and transcription regulation upon interferon treatments

    PubMed Central

    Hartman, Stephen E.; Bertone, Paul; Nath, Anjali K.; Royce, Thomas E.; Gerstein, Mark; Weissman, Sherman; Snyder, Michael

    2005-01-01

    The STAT (signal transducer and activator of transcription) proteins play a crucial role in the regulation of gene expression, but their targets and the manner in which they select them remain largely unknown. Using chromatin immunoprecipitation and DNA microarray analysis (ChIP-chip), we have identified the regions of human chromosome 22 bound by STAT1 and STAT2 in interferon-treated cells. Analysis of the genomic loci proximal to these binding sites introduced new candidate STAT1 and STAT2 target genes, several of which are affiliated with proliferation and apoptosis. The genes on chromosome 22 that exhibited interferon-induced up- or down-regulated expression were determined and correlated with the STAT-binding site information, revealing the potential regulatory effects of STAT1 and STAT2 on their target genes. Importantly, the comparison of STAT1-binding sites upon interferon (IFN)-γ and IFN-α treatments revealed dramatic changes in binding locations between the two treatments. The IFN-α induction revealed nonconserved STAT1 occupancy at IFN-γ-induced sites, as well as novel sites of STAT1 binding not evident in IFN-γ-treated cells. Many of these correlated with binding by STAT2, but others were STAT2 independent, suggesting that multiple mechanisms direct STAT1 binding to its targets under different activation conditions. Overall, our results reveal a wealth of new information regarding IFN/STAT-binding targets and also fundamental insights into mechanisms of regulation of gene expression in different cell states. PMID:16319195

  17. Selective pressures for accurate altruism targeting: evidence from digital evolution for difficult-to-test aspects of inclusive fitness theory.

    PubMed

    Clune, Jeff; Goldsby, Heather J; Ofria, Charles; Pennock, Robert T

    2011-03-01

    Inclusive fitness theory predicts that natural selection will favour altruist genes that are more accurate in targeting altruism only to copies of themselves. In this paper, we provide evidence from digital evolution in support of this prediction by competing multiple altruist-targeting mechanisms that vary in their accuracy in determining whether a potential target for altruism carries a copy of the altruist gene. We compete altruism-targeting mechanisms based on (i) kinship (kin targeting), (ii) genetic similarity at a level greater than that expected of kin (similarity targeting), and (iii) perfect knowledge of the presence of an altruist gene (green beard targeting). Natural selection always favoured the most accurate targeting mechanism available. Our investigations also revealed that evolution did not increase the altruism level when all green beard altruists used the same phenotypic marker. The green beard altruism levels stably increased only when mutations that changed the altruism level also changed the marker (e.g. beard colour), such that beard colour reliably indicated the altruism level. For kin- and similarity-targeting mechanisms, we found that evolution was able to stably adjust altruism levels. Our results confirm that natural selection favours altruist genes that are increasingly accurate in targeting altruism to only their copies. Our work also emphasizes that the concept of targeting accuracy must include both the presence of an altruist gene and the level of altruism it produces. PMID:20843843

  18. Selective pressures for accurate altruism targeting: evidence from digital evolution for difficult-to-test aspects of inclusive fitness theory

    PubMed Central

    Clune, Jeff; Goldsby, Heather J.; Ofria, Charles; Pennock, Robert T.

    2011-01-01

    Inclusive fitness theory predicts that natural selection will favour altruist genes that are more accurate in targeting altruism only to copies of themselves. In this paper, we provide evidence from digital evolution in support of this prediction by competing multiple altruist-targeting mechanisms that vary in their accuracy in determining whether a potential target for altruism carries a copy of the altruist gene. We compete altruism-targeting mechanisms based on (i) kinship (kin targeting), (ii) genetic similarity at a level greater than that expected of kin (similarity targeting), and (iii) perfect knowledge of the presence of an altruist gene (green beard targeting). Natural selection always favoured the most accurate targeting mechanism available. Our investigations also revealed that evolution did not increase the altruism level when all green beard altruists used the same phenotypic marker. The green beard altruism levels stably increased only when mutations that changed the altruism level also changed the marker (e.g. beard colour), such that beard colour reliably indicated the altruism level. For kin- and similarity-targeting mechanisms, we found that evolution was able to stably adjust altruism levels. Our results confirm that natural selection favours altruist genes that are increasingly accurate in targeting altruism to only their copies. Our work also emphasizes that the concept of targeting accuracy must include both the presence of an altruist gene and the level of altruism it produces. PMID:20843843

  19. Inhibitor focusing: direct selection of drug targets from proteomes using activity-based probes.

    PubMed

    Nomanbhoy, Tyzoon K; Rosenblum, Jonathan; Aban, Arwin; Burbaum, Jonathan J

    2003-02-01

    In the latter stages of drug discovery and development, assays that establish drug selectivity and toxicity are important when side effects, which are often due to lack of specificity, determine drug candidate viability. There has been no comprehensive or systematic methodology to measure these factors outside of whole-animal assays, and such phenomenological assays generally fail to establish the additional targets of a given small molecule, or the molecular origin of toxicity. Consequently, small-molecule development programs destined for failure often reach advanced stages of testing, and the money and time invested in such programs could be saved if information on selectivity were available early in the process. Here, we present a methodology that utilizes chemical ABPs in combination with small-molecule inhibitors to selectively label small-molecule binding sites in whole proteomic samples. In principle, the ABP and small molecule will compete for similar binding sites, such that the small molecule will protect against modification by the ABP. Thus, after removal of the small molecule, the binding site for the ABP will be revealed, and a second probe can then be used to label the small-molecule binding sites selectively. To demonstrate this experimentally, we mapped the binding sites of the DPP4 inhibitor, IT, in a number of different tissue types. PMID:15090140

  20. Selective activity of deguelin identifies therapeutic targets for androgen receptor-positive breast cancer.

    PubMed

    Robles, Andrew J; Cai, Shengxin; Cichewicz, Robert H; Mooberry, Susan L

    2016-06-01

    Triple-negative breast cancers (TNBC) are aggressive malignancies with no effective targeted therapies. Recent gene expression profiling of these heterogeneous cancers and the classification of cell line models now allows for the identification of compounds with selective activities against molecular subtypes of TNBC. The natural product deguelin was found to have selective activity against MDA-MB-453 and SUM-185PE cell lines, which both model the luminal androgen receptor (LAR) subtype of TNBC. Deguelin potently inhibited proliferation of these cells with GI50 values of 30 and 61 nM, in MDA-MB-453 and SUM-185PE cells, respectively. Deguelin had exceptionally high selectivity, 197 to 566-fold, for these cell lines compared to cell lines representing other TNBC subtypes. Deguelin's mechanisms of action were investigated to determine how it produced these potent and selective effects. Our results show that deguelin has dual activities, inhibiting PI3K/Akt/mTOR signaling, and decreasing androgen receptor levels and nuclear localization. Based on these data, we hypothesized that the combination of the mTOR inhibitor rapamycin and the antiandrogen enzalutamide would have efficacy in LAR models. Rapamycin and enzalutamide showed additive effects in MDA-MB-453 cells, and both drugs had potent antitumor efficacy in a LAR xenograft model. These results suggest that the combination of antiandrogens and mTOR inhibitors might be an effective strategy for the treatment of androgen receptor-expressing TNBC. PMID:27255535

  1. Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

    PubMed Central

    Newton, Phillip T; Vuppalapati, Karuna K; Bouderlique, Thibault; Chagin, Andrei S

    2015-01-01

    Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H+-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A1 or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity. PMID:26259639

  2. Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration.

    PubMed

    Berg, Russell D; Levitte, Steven; O'Sullivan, Mary P; O'Leary, Seónadh M; Cambier, C J; Cameron, James; Takaki, Kevin K; Moens, Cecilia B; Tobin, David M; Keane, Joseph; Ramakrishnan, Lalita

    2016-03-24

    A zebrafish genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in lysosomal cysteine cathepsins that manifests hallmarks of human lysosomal storage diseases. Under homeostatic conditions, mutant macrophages accumulate undigested lysosomal material, which disrupts endocytic recycling and impairs their migration to, and thus engulfment of, dying cells. This causes a buildup of unengulfed cell debris. During mycobacterial infection, macrophages with lysosomal storage cannot migrate toward infected macrophages undergoing apoptosis in the tuberculous granuloma. The unengulfed apoptotic macrophages undergo secondary necrosis, causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal storage similarly impairs migration to newly infecting mycobacteria. This phenotype is recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of their alveolar macrophages exhibit lysosomal accumulations of tobacco smoke particulates and do not migrate to Mycobacterium tuberculosis. The incapacitation of highly microbicidal first-responding macrophages may contribute to smokers' susceptibility to tuberculosis. PMID:27015311

  3. Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration

    PubMed Central

    Berg, Russell D.; Levitte, Steven; O’Sullivan, Mary P.; O’Leary, Seónadh M.; Cambier, C.J.; Cameron, James; Takaki, Kevin K.; Moens, Cecilia B.; Tobin, David M.; Keane, Joseph; Ramakrishnan, Lalita

    2016-01-01

    Summary A zebrafish genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in lysosomal cysteine cathepsins that manifests hallmarks of human lysosomal storage diseases. Under homeostatic conditions, mutant macrophages accumulate undigested lysosomal material, which disrupts endocytic recycling and impairs their migration to, and thus engulfment of, dying cells. This causes a buildup of unengulfed cell debris. During mycobacterial infection, macrophages with lysosomal storage cannot migrate toward infected macrophages undergoing apoptosis in the tuberculous granuloma. The unengulfed apoptotic macrophages undergo secondary necrosis, causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal storage similarly impairs migration to newly infecting mycobacteria. This phenotype is recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of their alveolar macrophages exhibit lysosomal accumulations of tobacco smoke particulates and do not migrate to Mycobacterium tuberculosis. The incapacitation of highly microbicidal first-responding macrophages may contribute to smokers’ susceptibility to tuberculosis. PMID:27015311

  4. Rag GTPases are cardioprotective by regulating lysosomal function

    PubMed Central

    Kim, Young Chul; Mo, Jung-Soon; Jewell, Jenna L.; Russell, Ryan C.; Wu, Xiaohui; Sadoshima, Junichi; Guan, Kun-Liang

    2014-01-01

    The Rag family proteins are Ras-like small GTPases that play a critical role in amino acid-stimulated mTORC1 activation by recruiting mTORC1 to lysosome. Despite progress in the mechanistic understanding of Rag GTPases in mTORC1 activation, little is known about the physiological function of Rag GTPases in vivo. Here, we show that loss of RagA and RagB (RagA/B) in cardiomyocytes results in hypertrophic cardiomyopathy and phenocopies lysosomal storage diseases although mTORC1 activity is not substantially impaired in vivo. We demonstrate that despite upregulation of lysosomal protein expression by constitutive activation of the transcription factor EB (TFEB) in RagA/B knockout mouse embryonic fibroblasts, lysosomal acidification is compromised due to decreased v-ATPase level in the lysosome fraction. Our study uncovers RagA/B GTPases as key regulators of lysosomal function and cardiac protection. PMID:24980141

  5. Sorting Nexin 11 Regulates Lysosomal Degradation of Plasma Membrane TRPV3.

    PubMed

    Li, Caiyue; Ma, Wenbo; Yin, Shikui; Liang, Xin; Shu, Xiaodong; Pei, Duanqing; Egan, Terrance M; Huang, Jufang; Pan, Aihua; Li, Zhiyuan

    2016-05-01

    The trafficking of ion channels to/from the plasma membrane is considered an important mechanism for cellular activity and an interesting approach for disease therapies. The transient receptor potential vanilloid 3 (TRPV3) ion channel is widely expressed in skin keratinocytes, and its trafficking mechanism to/from the plasma membrane is unknown. Here, we report that the vesicular trafficking protein sorting nexin 11 (SNX11) downregulates the level of the TRPV3 plasma membrane protein. Overexpression of SNX11 causes a decrease in the level of TRPV3 current and TRPV3 plasma membrane protein in TRPV3-transfected HEK293T cells. Subcellular localizations and western blots indicate that SNX11 interacts with TRPV3 and targets it to lysosomes for degradation, which is blocked by the lysosomal inhibitors chloroquine and leupeptin. Both TRPV3 and SNX11 are highly expressed in HaCaT cells. We show that TRPV3 agonists-activated Ca(2+) influxes and the level of native TRPV3 total protein in HaCaT cells are decreased by overexpression of SNX11 and increased by knockdown of SNX11. Our findings reveal that SNX11 promotes the trafficking of TRPV3 from the plasma membrane to lysosomes for degradation via protein-protein interactions, which demonstrates a previously unknown function of SNX11 as a regulator of TRPV3 trafficking from the plasma membrane to lysosomes. PMID:26818531

  6. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells

    PubMed Central

    Shim, Su Yeon; Karri, Srinivasarao; Law, Sampson; Schatzl, Hermann M.; Gilch, Sabine

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrPSc) of the cellular prion protein (PrPc). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrPc into PrPSc. Within neurons, PrPSc accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrPSc accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases. PMID:26865414

  7. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells.

    PubMed

    Shim, Su Yeon; Karri, Srinivasarao; Law, Sampson; Schatzl, Hermann M; Gilch, Sabine

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrP(Sc)) of the cellular prion protein (PrP(c)). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrP(c) into PrP(Sc). Within neurons, PrP(Sc) accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrP(Sc) accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases. PMID:26865414

  8. Combination Therapies for Lysosomal Storage Diseases: A Complex Answer to a Simple Problem.

    PubMed

    Macauley, Shannon L

    2016-06-01

    Abstract Lysosomal storage diseases (LSDs) are a group of 40-50 rare monogenic disorders that result in disrupted lysosomal function and subsequent lysosomal pathology. Depending on the protein or enzyme deficiency associated with each disease, LSDs affect an array of organ systems and elicit a complex set of secondary disease mechanisms that make many of these disorders difficult to fully treat. The etiology of most LSDs is known and the innate biology of lysosomal enzymes favors therapeutic intervention, yet most attempts at treating LSDs with enzyme replacement strategies fall short of being curative. Even with the advent of more sophisticated approaches, like substrate reduction therapy, pharmacologic chaperones, gene therapy or stem cell therapy, comprehensive treatments for LSDs have yet to be achieved. Given the limitations with individual therapies, recent research has focused on using a combination approach to treat LSDs. By coupling protein-, cell-, and gene- based therapies with small molecule drugs, researchers have found greater success in eradicating the clinical features of disease. This review seeks to discuss the positive and negatives of singular therapies used to treat LSDs, and discuss how, in combination, studies have demonstrated a more holistic benefit on pathological and functional parameters. By optimizing routes of delivery, therapeutic timing, and targeting secondary disease mechanisms, combination therapy represents the future for LSD treatment. PMID:27491211

  9. Selective targeting of bioengineered platelets to prostate cancer vasculature: new paradigm for therapeutic modalities.

    PubMed

    Montecinos, Viviana P; Morales, Claudio H; Fischer, Thomas H; Burns, Sarah; San Francisco, Ignacio F; Godoy, Alejandro S; Smith, Gary J

    2015-07-01

    Androgen deprivation therapy (ADT) provides palliation for most patients with advanced prostate cancer (CaP); however, greater than 80% subsequently fail ADT. ADT has been indicated to induce an acute but transient destabilization of the prostate vasculature in animal models and humans. Human re-hydrated lyophilized platelets (hRL-P) were investigated as a prototype for therapeutic agents designed to target selectively the tumour-associated vasculature in CaP. The ability of hRL-P to bind the perturbed endothelial cells was tested using thrombin- and ADP-activated human umbilical vein endothelial cells (HUVEC), as well as primary xenografts of human prostate tissue undergoing acute vascular involution in response to ADT. hRL-P adhered to activated HUVEC in a dose-responsive manner. Systemically administered hRL-P, and hRL-P loaded with super-paramagnetic iron oxide (SPIO) nanoparticles, selectively targeted the ADT-damaged human microvasculature in primary xenografts of human prostate tissue. This study demonstrated that hRL-P pre-loaded with chemo-therapeutics or nanoparticles could provide a new paradigm for therapeutic modalities to prevent the rebound/increase in prostate vasculature after ADT, inhibiting the transition to castration-recurrent growth. PMID:25736582

  10. Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics

    SciTech Connect

    Shi, Tujin; Su, Dian; Liu, Tao; Tang, Keqi; Camp, David G.; Qian, Weijun; Smith, Richard D.

    2012-04-01

    Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for e.g., detecting low-abundance biomarkers likely present at the pg/mL to low ng/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in the cells or tissues. Herein we review recent advances in methods and technologies, including front-end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides or their posttranslational modifications (PTMs), as well as advances in MS instrumentation, which have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low-abundance proteins at low to sub- ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed.

  11. Selective targeting of liver cancer with the endothelial marker CD146

    PubMed Central

    Thomann, Stefan; Longerich, Thomas; Bazhin, Alexandr V.; Mier, Walter; Schemmer, Peter; Ryschich, Eduard

    2014-01-01

    Hepatocellular carcinomas are well-vascularized tumors; the endothelial cells in these tumors have a specific phenotype. Our aim was to develop a new approach for tumor-specific drug delivery with monoclonal antibody targeting of endothelial ligands. CD146, a molecule expressed on the endothelial surface of hepatocellular carcinoma, was identified as a promising candidate for targeting. In the present study, endothelial cells immediately captured circulating anti-CD146 (ME-9F1) antibody, while antibody binding in tumors was significantly higher than in hepatic endothelium. Macroscopically, after intravenous injection, there were no differences in the mean accumulation of anti-CD146 antibody in tumor compared to liver tissue, due to a compensating higher blood vessel density in the liver tissue. Additional blockade of nontumoral epitopes and intra-arterial administration, improved selective antibody capture in the tumor microvasculature and largely prevented antibody distribution in the lung and liver. The potential practical use of this approach was demonstrated by imaging of radionuclide-labeled ME-9F1 antibody, which showed excellent tumor-selective uptake. Our results provide a promising principle for the use of endothelial markers for intratumoral drug delivery. Tumor endothelium–based access might offer new opportunities for the imaging and therapy of hepatocellular carcinoma and other liver malignancies. PMID:25238265

  12. Does Angling Technique Selectively Target Fishes Based on Their Behavioural Type?

    PubMed Central

    Wilson, Alexander D. M.; Brownscombe, Jacob W.; Sullivan, Brittany; Jain-Schlaepfer, Sofia; Cooke, Steven J.

    2015-01-01

    Recently, there has been growing recognition that fish harvesting practices can have important impacts on the phenotypic distributions and diversity of natural populations through a phenomenon known as fisheries-induced evolution. Here we experimentally show that two common recreational angling techniques (active crank baits versus passive soft plastics) differentially target wild largemouth bass (Micropterus salmoides) and rock bass (Ambloplites rupestris) based on variation in their behavioural tendencies. Fish were first angled in the wild using both techniques and then brought back to the laboratory and tested for individual-level differences in common estimates of personality (refuge emergence, flight-initiation-distance, latency-to-recapture and with a net, and general activity) in an in-lake experimental arena. We found that different angling techniques appear to selectively target these species based on their boldness (as characterized by refuge emergence, a standard measure of boldness in fishes) but not other assays of personality. We also observed that body size was independently a significant predictor of personality in both species, though this varied between traits and species. Our results suggest a context-dependency for vulnerability to capture relative to behaviour in these fish species. Ascertaining the selective pressures angling practices exert on natural populations is an important area of fisheries research with significant implications for ecology, evolution, and resource management. PMID:26284779

  13. Update on the Pfam5000 Strategy for Selection of StructuralGenomics Targets

    SciTech Connect

    Chandonia, John-Marc; Brenner, Steven E.

    2005-06-27

    Structural Genomics is an international effort to determine the three-dimensional shapes of all important biological macromolecules, with a primary focus on proteins. Target proteins should be selected according to a strategy that is medically and biologically relevant, of good financial value, and tractable. In 2003, we presented the ''Pfam5000'' strategy, which involves selecting the 5,000 most important families from the Pfam database as sources for targets. In this update, we show that although both the Pfam database and the number of sequenced genomes have increased in size, the expected benefits of the Pfam5000 strategy have not changed substantially. Solving the structures of proteins from the 5,000 largest Pfam families would allow accurate fold assignment for approximately 65 percent of all prokaryotic proteins (covering 54 percent of residues) and 63 percent of eukaryotic proteins (42 percent of residues). Fewer than 2,300 of the largest families on this list remain to be solved, making the project feasible in the next five years given the expected throughput to be achieved in the production phase of the Protein Structure Initiative.

  14. Phosphatidylserine-selective targeting and anticancer effects of SapC-DOPS nanovesicles on brain tumors.

    PubMed

    Blanco, Víctor M; Chu, Zhengtao; Vallabhapurapu, Subrahmanya D; Sulaiman, Mahaboob K; Kendler, Ady; Rixe, Olivier; Warnick, Ronald E; Franco, Robert S; Qi, Xiaoyang

    2014-08-30

    Brain tumors, either primary (e.g., glioblastoma multiforme) or secondary (metastatic), remain among the most intractable and fatal of all cancers. We have shown that nanovesicles consisting of Saposin C (SapC) and dioleylphosphatidylserine (DOPS) are able to effectively target and kill cancer cells both in vitro and in vivo. These actions are a consequence of the affinity of SapC-DOPS for phosphatidylserine, an acidic phospholipid abundantly present in the outer membrane of a variety of tumor cells and tumor-associated vasculature. In this study, we first characterize SapC-DOPS bioavailability and antitumor effects on human glioblastoma xenografts, and confirm SapC-DOPS specificity towards phosphatidylserine by showing that glioblastoma targeting is abrogated after in vivo exposure to lactadherin, which binds phosphatidylserine with high affinity. Second, we demonstrate that SapC-DOPS selectively targets brain metastases-forming cancer cells both in vitro, in co-cultures with human astrocytes, and in vivo, in mouse models of brain metastases derived from human breast or lung cancer cells. Third, we demonstrate that SapC-DOPS have cytotoxic activity against metastatic breast cancer cells in vitro, and prolong the survival of mice harboring brain metastases. Taken together, these results support the potential of SapC-DOPS for the diagnosis and therapy of primary and metastatic brain tumors. PMID:25051370

  15. Selection of flowing liquid lead target structural materials for accelerator driven transmutation applications

    SciTech Connect

    Park, J.J.; Buksa, J.J.

    1994-08-01

    The beam entry window and container for a liquid lead spallation target will be exposed to high fluxes of protons and neutrons that are both higher in magnitude and energy than have been experienced in proton accelerators and fission reactors, as well as in a corrosive environment. The structural material of the target should have a good compatibility with liquid lead, a sufficient mechanical strength at elevated temperatures, a good performance under an intense irradiation environment, and a low neutron absorption cross section; these factors have been used to rank the applicability of a wide range of materials for structural containment Nb-1Zr has been selected for use as the structural container for the LANL ABC/ATW molten lead target. Corrosion and mass transfer behavior for various candidate structural materials in liquid lead are reviewed, together with the beneficial effects of inhibitors and various coatings to protect substrate against liquid lead corrosion. Mechanical properties of some candidate materials at elevated temperatures and the property changes resulting from 800 MeV proton irradiation are also reviewed.

  16. Target detection as a tool of selective spray application on trees and weeds in orchards

    NASA Astrophysics Data System (ADS)

    Doruchowski, Grzegorz; Jaeken, Peter; Holownicki, Ryszard

    1999-01-01

    A spectral detection system discriminating the targets for the non target area was tested during spray applications in apple and pear orchards. The objective of the test was to evaluate the accuracy of the system working at different application parameters and to estimate the rate of possible spray savings obtained during applications on the trees of different size and weeds of different density. The system consisted of the spray units equipped with optic sensor and a control unit which could operate up to 16 spray units. Each spray unit had an optic detector and two light sources emitting two beams of light at the wavelengths 670 and 750. The ratio between emitted and reflected light for each wavelength was the basis for discriminating between the presence or the absence of chlorophyll. The information was processed and used to control the electric solenoid valves opening or shutting off the nozzles. The target detection system worked technically properly. It enabled the selective spray application with spray savings adequate to the tree row profile. In intensive apple and pear orchards 16-25 percent reduction of spray volume was obtained. For herbicide applications the detection system discriminated weeds for the bare ground. Both sensitivity of the sensors and weed density had a significant influence on the spray savings. At medium sensitivity, a considerable spray saving amounting 23 percent was obtained only on the plots with very low weed coverage.

  17. Phosphatidylserine-selective targeting and anticancer effects of SapC-DOPS nanovesicles on brain tumors

    PubMed Central

    Blanco, Víctor M.; Chu, Zhengtao; Vallabhapurapu, Subrahmanya D.; Sulaiman, Mahaboob K.; Kendler, Ady; Rixe, Olivier; Warnick, Ronald E.; Franco, Robert S.; Qi, Xiaoyang

    2014-01-01

    Brain tumors, either primary (e.g., glioblastoma multiforme) or secondary (metastatic), remain among the most intractable and fatal of all cancers. We have shown that nanovesicles consisting of Saposin C (SapC) and dioleylphosphatidylserine (DOPS) are able to effectively target and kill cancer cells both in vitro and in vivo. These actions are a consequence of the affinity of SapC-DOPS for phosphatidylserine, an acidic phospholipid abundantly present in the outer membrane of a variety of tumor cells and tumor-associated vasculature. In this study, we first characterize SapC-DOPS bioavailability and antitumor effects on human glioblastoma xenografts, and confirm SapC-DOPS specificity towards phosphatidylserine by showing that glioblastoma targeting is abrogated after in vivo exposure to lactadherin, which binds phosphatidylserine with high affinity. Second, we demonstrate that SapC-DOPS selectively targets brain metastases-forming cancer cells both in vitro, in co-cultures with human astrocytes, and in vivo, in mouse models of brain metastases derived from human breast or lung cancer cells. Third, we demonstrate that SapC-DOPS nanovesicles have cytotoxic activity against metastatic breast cancer cells in vitro, and prolong the survival of mice harboring brain metastases. Taken together, these results support the potential of SapC-DOPS for the diagnosis and therapy of primary and metastatic brain tumors. PMID:25051370

  18. Selective synaptic targeting of the excitatory and inhibitory presynaptic organizers FGF22 and FGF7

    PubMed Central

    Terauchi, Akiko; Timmons, Kendall M.; Kikuma, Koto; Pechmann, Yvonne; Kneussel, Matthias; Umemori, Hisashi

    2015-01-01

    ABSTRACT Specific formation of excitatory and inhibitory synapses is crucial for proper functioning of the brain. Fibroblast growth factor 22 (FGF22) and FGF7 are postsynaptic-cell-derived presynaptic organizers necessary for excitatory and inhibitory presynaptic differentiation, respectively, in the hippocampus. For the establishment of specific synaptic networks, these FGFs must localize to appropriate synaptic locations – FGF22 to excitatory and FGF7 to inhibitory postsynaptic sites. Here, we show that distinct motor and adaptor proteins contribute to intracellular microtubule transport of FGF22 and FGF7. Excitatory synaptic targeting of FGF22 requires the motor proteins KIF3A and KIF17 and the adaptor protein SAP102 (also known as DLG3). By contrast, inhibitory synaptic targeting of FGF7 requires the motor KIF5 and the adaptor gephyrin. Time-lapse imaging shows that FGF22 moves with SAP102, whereas FGF7 moves with gephyrin. These results reveal the basis of selective targeting of the excitatory and inhibitory presynaptic organizers that supports their different synaptogenic functions. Finally, we found that knockdown of SAP102 or PSD95 (also known as DLG4), which impairs the differentiation of excitatory synapses, alters FGF7 localization, suggesting that signals from excitatory synapses might regulate inhibitory synapse formation by controlling the distribution of the inhibitory presynaptic organizer. PMID:25431136

  19. Scientific objectives and selection of targets for the SMART-1 Infrared Spectrometer (SIR)

    USGS Publications Warehouse

    Basilevsky, A.T.; Keller, H.U.; Nathues, A.; Mall, U.; Hiesinger, H.; Rosiek, M.

    2004-01-01

    The European SMART-1 mission to the Moon, primarily a testbed for innovative technologies, was launched in September 2003 and will reach the Moon in 2005. On board are several scientific instruments, including the point-spectrometer SMART-1 Infrared Spectrometer (SIR). Taking into account the capabilities of the SMART-1 mission and the SIR instrument in particular, as well as the open questions in lunar science, a selection of targets for SIR observations has been compiled. SIR can address at least five topics: (1) Surface/regolith processes; (2) Lunar volcanism; (3) Lunar crust structure; (4) Search for spectral signatures of ices at the lunar poles; and (5) Ground truth and study of geometric effects on the spectral shape. For each topic we will discuss specific observation modes, necessary to achieve our scientific goals. The majority of SIR targets will be observed in the nadir-tracking mode. More than 100 targets, which require off-nadir pointing and off-nadir tracking, are planned. It is expected that results of SIR observations will significantly increase our understanding of the Moon. Since the exact arrival date and the orbital parameters of the SMART-1 spacecraft are not known yet, a more detailed planning of the scientific observations will follow in the near future. ?? 2004 Elsevier Ltd. All rights reserved.

  20. Phosphatidic acid osmotically destabilizes lysosomes through increased permeability to K+ and H+.

    PubMed

    Yi, Y-P; Wang, X; Zhang, G; Fu, T-S; Zhang, G-J

    2006-06-01

    Lysosomal destabilization is a critical event not only for the organelle but also for living cells. However, what factors can affect lysosomal stability is not fully studied. In this work, the effects of phosphatidic acid (PA) on the lysosomal integrity were investigated. Through the measurements of lysosomal beta-hexosaminidase free activity, intralysosomal pH, leakage of lysosomal protons and lysosomal latency loss in hypotonic sucrose medium, we established that PA could increase the lysosomal permeability to K+ and H+, and enhance the lysosomal osmotic sensitivity. Treatment of lysosomes with PA promoted entry of K+ into the organelle via K+/H+ exchange, which could produce osmotic stresses and osmotically destabilize the lysosomes. In addition, PA-induced increase in the lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shocks. The results suggest that PA may play a role in the lysosomal destabilization. PMID:16917129

  1. Akt mediated ROS-dependent selective targeting of mutant KRAS tumors.

    PubMed

    Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

    2014-10-01

    Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers. PMID:26461287

  2. Selective target inactivation rather than global metabolic dormancy causes antibiotic tolerance in uropathogens.

    PubMed

    Goneau, Lee W; Yeoh, Nigel S; MacDonald, Kyle W; Cadieux, Peter A; Burton, Jeremy P; Razvi, Hassan; Reid, Gregor

    2014-01-01

    Persister cells represent a multidrug-tolerant (MDT), physiologically distinct subpopulation of bacteria. The ability of these organisms to survive lethal antibiotic doses raises concern over their potential role in chronic disease, such as recurrent urinary tract infection (RUTI). Persistence is believed to be conveyed through global metabolic dormancy, which yields organisms unresponsive to external stimuli. However, recent studies have contested this stance. Here, various antibiotics that target different cellular processes were used to dissect the activity of transcription, translation, and peptidoglycan turnover in persister cells. Differential susceptibility patterns were found in type I and type II persisters, and responses differed between Staphylococcus saprophyticus and Escherichia coli uropathogens. Further, SOS-deficient strains were sensitized to ciprofloxacin, suggesting DNA gyrase activity in persisters and indicating the importance of active DNA repair systems for ciprofloxacin tolerance. These results indicate that global dormancy per se cannot sufficiently account for antibiotic tolerance. Rather, the activity of individual cellular processes dictates multidrug tolerance in an antibiotic-specific fashion. Furthermore, the susceptibility patterns of persisters depended on their mechanisms of onset, with subinhibitory antibiotic pretreatments selectively shutting down cognate targets and increasing the persister fraction against the same agent. Interestingly, antibiotics targeting transcription and translation enhanced persistence against multiple agents indirectly related to these processes. Conducting these assays with uropathogenic E. coli isolated from RUTI patients revealed an enriched persister fraction compared to organisms cleared with standard antibiotic therapy. This finding suggests that persister traits are either selected for during prolonged antibiotic treatment or initially contribute to therapy failure. PMID:24449771

  3. Lysosomal Cholesterol Accumulation Inhibits Subsequent Hydrolysis Of Lipoprotein Cholesteryl Ester

    PubMed Central

    Jerome, W. Gray; Cox, Brian E.; Griffin, Evelyn E.; Ullery, Jody C.

    2010-01-01

    Human macrophages incubated for prolonged periods with mildly oxidized LDL (oxLDL) or cholesteryl ester-rich lipid dispersions (DISP) accumulate free and esterified cholesterol within large, swollen lysosomes similar to those in foam cells of atherosclerosis. The cholesteryl ester (CE) accumulation is, in part, the result of inhibition of lysosomal hydrolysis due to increased lysosomal pH mediated by excessive lysosomal free cholesterol (FC). To determine if the inhibition of hydrolysis was long lived and further define the extent of the lysosomal defect, we incubated THP-1 macrophages with oxLDL or DISP to produce lysosome sterol engorgement and then chased with acetylated LDL (acLDL). Unlike oxLDL or DISP, CE from acLDL normally is hydrolyzed rapidly. Three days of incubation with oxLDL or DISP produced an excess of CE in lipid-engorged lysosomes, indicative of inhibition. After prolonged oxLDL or DISP pretreatment, subsequent hydrolysis of acLDL CE was inhibited. Coincident with the inhibition, the lipid-engorged lysosomes failed to maintain an acidic pH during both the initial pretreatment and subsequent acLDL incubation. This indicates that the alterations in lysosomes were general, long-lived and affected subsequent lipoprotein metabolism. This same phenomenon, occurring within atherosclerotic foam cells, could significantly affect lesion progression. PMID:18312718

  4. A lysosome-centered view of nutrient homeostasis.

    PubMed

    Mony, Vinod K; Benjamin, Shawna; O'Rourke, Eyleen J

    2016-01-01

    Lysosomes are highly acidic cellular organelles traditionally viewed as sacs of enzymes involved in digesting extracellular or intracellular macromolecules for the regeneration of basic building blocks, cellular housekeeping, or pathogen degradation. Bound by a single lipid bilayer, lysosomes receive their substrates by fusing with endosomes or autophagosomes, or through specialized translocation mechanisms such as chaperone-mediated autophagy or microautophagy. Lysosomes degrade their substrates using up to 60 different soluble hydrolases and release their products either to the cytosol through poorly defined exporting and efflux mechanisms or to the extracellular space by fusing with the plasma membrane. However, it is becoming evident that the role of the lysosome in nutrient homeostasis goes beyond the disposal of waste or the recycling of building blocks. The lysosome is emerging as a signaling hub that can integrate and relay external and internal nutritional information to promote cellular and organismal homeostasis, as well as a major contributor to the processing of energy-dense molecules like glycogen and triglycerides. Here we describe the current knowledge of the nutrient signaling pathways governing lysosomal function, the role of the lysosome in nutrient mobilization, and how lysosomes signal other organelles, distant tissues, and even themselves to ensure energy homeostasis in spite of fluctuations in energy intake. At the same time, we highlight the value of genomics approaches to the past and future discoveries of how the lysosome simultaneously executes and controls cellular homeostasis. PMID:27050453

  5. Imaging and imagination: understanding the endo-lysosomal system

    PubMed Central

    van Meel, Eline

    2008-01-01

    Lysosomes are specialized compartments for the degradation of endocytosed and intracellular material and essential regulators of cellular homeostasis. The importance of lysosomes is illustrated by the rapidly growing number of human disorders related to a defect in lysosomal functioning. Here, we review current insights in the mechanisms of lysosome biogenesis and protein sorting within the endo-lysosomal system. We present increasing evidence for the existence of parallel pathways for the delivery of newly synthesized lysosomal proteins directly from the trans-Golgi network (TGN) to the endo-lysosomal system. These pathways are either dependent or independent of mannose 6-phosphate receptors and likely involve multiple exits for lysosomal proteins from the TGN. In addition, we discuss the different endosomal intermediates and subdomains that are involved in sorting of endocytosed cargo. Throughout our review, we highlight some examples in the literature showing how imaging, especially electron microscopy, has made major contributions to our understanding of the endo-lysosomal system today. PMID:18274773

  6. A lysosome-centered view of nutrient homeostasis

    PubMed Central

    Mony, Vinod K.; Benjamin, Shawna; O'Rourke, Eyleen J.

    2016-01-01

    ABSTRACT Lysosomes are highly acidic cellular organelles traditionally viewed as sacs of enzymes involved in digesting extracellular or intracellular macromolecules for the regeneration of basic building blocks, cellular housekeeping, or pathogen degradation. Bound by a single lipid bilayer, lysosomes receive their substrates by fusing with endosomes or autophagosomes, or through specialized translocation mechanisms such as chaperone-mediated autophagy or microautophagy. Lysosomes degrade their substrates using up to 60 different soluble hydrolases and release their products either to the cytosol through poorly defined exporting and efflux mechanisms or to the extracellular space by fusing with the plasma membrane. However, it is becoming evident that the role of the lysosome in nutrient homeostasis goes beyond the disposal of waste or the recycling of building blocks. The lysosome is emerging as a signaling hub that can integrate and relay external and internal nutritional information to promote cellular and organismal homeostasis, as well as a major contributor to the processing of energy-dense molecules like glycogen and triglycerides. Here we describe the current knowledge of the nutrient signaling pathways governing lysosomal function, the role of the lysosome in nutrient mobilization, and how lysosomes signal other organelles, distant tissues, and even themselves to ensure energy homeostasis in spite of fluctuations in energy intake. At the same time, we highlight the value of genomics approaches to the past and future discoveries of how the lysosome simultaneously executes and controls cellular homeostasis. PMID:27050453

  7. FAMACHA©: A potential tool for targeted selective treatment of chronic fasciolosis in sheep.

    PubMed

    Olah, Sophie; van Wyk, Jan A; Wall, Richard; Morgan, Eric R

    2015-09-15

    The liver fluke Fasciola hepatica causes considerable damage to the health, welfare and productivity of ruminants in temperate areas, and its control is challenged by anthelmintic resistance. Targeted selective treatment (TST) is an increasingly established strategy for preserving anthelmintic efficacy in grazing livestock, yet no practical indicators are available to target individuals for treatment against fluke infection. This paper evaluates the FAMACHA(©) system, a colour chart for the non-invasive detection of anaemia in small ruminants, for this purpose. FAMACHA(©) scores were collected from 288 sheep prior to slaughter during the winter period, when fluke infections were largely mature, and condemned livers were recovered and adult flukes extracted. Average FAMACHA(©) score was significantly higher (=paler conjunctivae) in animals whose livers were condemned (3.6, n=62) than in those whose livers were not condemned (2.1). The number of adult flukes recovered ranged from 2 to 485, and was positively correlated with FAMACHA(©) score (r(2)=0.54, p<0.001). Packed cell volume was correlated negatively with both FAMACHA(©) score (n=240, r=0.23, p<0.001) and fluke number (r=0.24, p<0.001). Nematode faecal egg count (FEC) did not correlate with FAMACHA(©) score, and selective treatment of individual sheep with FAMACHA(©) scores above 2 or 3 would have preserved between 27 and 100% of nematodes in refugia on the basis of FEC, depending on group and the threshold used for treatment. FAMACHA(©) holds promise as a tool for selective treatment of sheep against adult F. hepatica, in support of refugia-based control of fluke and nematode infections, and further field evaluation is warranted. PMID:26223154

  8. Target Selection Signals Influence Perceptual Decisions by Modulating the Onset and Rate of Evidence Accumulation.

    PubMed

    Loughnane, Gerard M; Newman, Daniel P; Bellgrove, Mark A; Lalor, Edmund C; Kelly, Simon P; O'Connell, Redmond G

    2016-02-22

    Computational and neurophysiological research has highlighted neural processes that accumulate sensory evidence for perceptual decisions [1]. These processes have been studied in the context of highly simplified perceptual discrimination paradigms in which the physical evidence appears at times and locations that are either entirely predictable or exogenously cued (e.g., by the onset of the stimulus itself). Yet, we are rarely afforded such certainty in everyday life. For example, when driving along a busy motorway, we must continually monitor the movements of surrounding vehicles for events that call for a lane change. In such scenarios, it is unknown which of the continuously present information sources will become relevant or when. Although it is well established that evidence integration provides an effective mechanism for countering the impact of noise [2], the question of how this mechanism is implemented in the face of uncertain evidence onsets has yet to be answered. Here, we show that when monitoring two potential sources of information for evidence occurring unpredictably in both time and space, the human brain employs discrete, early target selection signals that significantly modulate the onset and rate of neural evidence accumulation, and thereby the timing and accuracy of perceptual reports. These selection signals share many of the key characteristics of the N2pc component highlighted in the literature on visual search [3, 4] yet are present even in the absence of distractors and under situations of low temporal and spatial uncertainty. These data provide novel insights into how target selection supports decision making in uncertain environments. PMID:26853360

  9. Bezielle Selectively Targets Mitochondria of Cancer Cells to Inhibit Glycolysis and OXPHOS

    PubMed Central

    Chen, Vivian; Staub, Richard E.; Fong, Sylvia; Tagliaferri, Mary; Cohen, Isaac; Shtivelman, Emma

    2012-01-01

    Bezielle (BZL101) is a candidate oral drug that has shown promising efficacy and excellent safety in the early phase clinical trials for advanced breast cancer. Bezielle is an aqueous extract from the herb Scutellaria barbata. We have reported previously that Bezielle was selectively cytotoxic to cancer cells while sparing non-transformed cells. In tumor, but not in non-transformed cells, Bezielle induced generation of ROS and severe DNA damage followed by hyperactivation of PARP, depletion of the cellular ATP and NAD, and inhibition of glycolysis. We show here that tumor cells' mitochondria are the primary source of reactive oxygen species induced by Bezielle. Treatment with Bezielle induces progressively higher levels of mitochondrial superoxide as well as peroxide-type ROS. Inhibition of mitochondrial respiration prevents generation of both types of ROS and protects cells from Bezielle-induced death. In addition to glycolysis, Bezielle inhibits oxidative phosphorylation in tumor cells and depletes mitochondrial reserve capacity depriving cells of the ability to produce ATP. Tumor cells lacking functional mitochondria maintain glycolytic activity in presence of Bezielle thus supporting the hypothesis that mitochondria are the primary target of Bezielle. The metabolic effects of Bezielle towards normal cells are not significant, in agreement with the low levels of oxidative damage that Bezielle inflicts on them. Bezielle is therefore a drug that selectively targets cancer cell mitochondria, and is distinguished from other such drugs by its ability to induce not only inhibition of OXPHOS but also of glycolysis. This study provides a better understanding of the mechanism of Bezielle's cytotoxicity, and the basis of its selectivity towards cancer cells. PMID:22319564

  10. Lysosome triggered near-infrared fluorescence imaging of cellular trafficking processes in real time.

    PubMed

    Grossi, Marco; Morgunova, Marina; Cheung, Shane; Scholz, Dimitri; Conroy, Emer; Terrile, Marta; Panarella, Angela; Simpson, Jeremy C; Gallagher, William M; O'Shea, Donal F

    2016-01-01

    Bioresponsive NIR-fluorophores offer the possibility for continual visualization of dynamic cellular processes with added potential for direct translation to in vivo imaging. Here we show the design, synthesis and lysosome-responsive emission properties of a new NIR fluorophore. The NIR fluorescent probe design differs from typical amine functionalized lysosomotropic stains with off/on fluorescence switching controlled by a reversible phenol/phenolate interconversion. Emission from the probe is shown to be highly selective for the lysosomes in co-imaging experiments using a HeLa cell line expressing the lysosomal-associated membrane protein 1 fused to green fluorescent protein. The responsive probe is capable of real-time continuous imaging of fundamental cellular processes such as endocytosis, lysosomal trafficking and efflux in 3D and 4D. The advantage of the NIR emission allows for direct translation to in vivo tumour imaging, which is successfully demonstrated using an MDA-MB-231 subcutaneous tumour model. This bioresponsive NIR fluorophore offers significant potential for use in live cellular and in vivo imaging, for which currently there is a deficit of suitable molecular fluorescent tools. PMID:26927507

  11. Lysosome triggered near-infrared fluorescence imaging of cellular trafficking processes in real time

    PubMed Central

    Grossi, Marco; Morgunova, Marina; Cheung, Shane; Scholz, Dimitri; Conroy, Emer; Terrile, Marta; Panarella, Angela; Simpson, Jeremy C.; Gallagher, William M.; O'Shea, Donal F.

    2016-01-01

    Bioresponsive NIR-fluorophores offer the possibility for continual visualization of dynamic cellular processes with added potential for direct translation to in vivo imaging. Here we show the design, synthesis and lysosome-responsive emission properties of a new NIR fluorophore. The NIR fluorescent probe design differs from typical amine functionalized lysosomotropic stains with off/on fluorescence switching controlled by a reversible phenol/phenolate interconversion. Emission from the probe is shown to be highly selective for the lysosomes in co-imaging experiments using a HeLa cell line expressing the lysosomal-associated membrane protein 1 fused to green fluorescent protein. The responsive probe is capable of real-time continuous imaging of fundamental cellular processes such as endocytosis, lysosomal trafficking and efflux in 3D and 4D. The advantage of the NIR emission allows for direct translation to in vivo tumour imaging, which is successfully demonstrated using an MDA-MB-231 subcutaneous tumour model. This bioresponsive NIR fluorophore offers significant potential for use in live cellular and in vivo imaging, for which currently there is a deficit of suitable molecular fluorescent tools. PMID:26927507

  12. Current molecular genetics strategies for the diagnosis of lysosomal storage disorders.

    PubMed

    Giugliani, Roberto; Brusius-Facchin, Ana-Carolina; Pasqualim, Gabriela; Leistner-Segal, Sandra; Riegel, Mariluce; Matte, Ursula

    2016-01-01

    Lysosomal storage disorders (LSDs) are a group of almost 50 monogenic diseases characterized by mutations causing deficiency of lysosomal enzymes or non-enzyme proteins involved in transport across the lysosomal membrane, protein maturation or lysosomal biogenesis. Usually, affected patients are normal at birth and have a progressive and severe disease with high morbidity and reduced life expectancy. The overall incidence of LSDs is usually estimated as 1:5000, but newborn screening studies are indicating that it could be much higher. Specific therapies were already developed for selected LSDs, making the timely and correct diagnosis very important for successful treatment and also for genetic counseling. In most LSD cases the biochemical techniques provide a reliable diagnosis. However, the identification of pathogenic mutations by genetic analysis is being increasingly recommended to provide additional information. In this paper we discuss the conventional methods for genetic analysis used in the LSDs [restriction fragment length polymorphism (RFLP), amplification-refractory mutation system (ARMS), single strand conformation polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC), real-time polymerase chain reaction, high resolution melting (HRM), multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing] and also the newer approaches [massive parallel sequencing, array comparative genomic hybridization (CGH)]. PMID:26567866

  13. Targeted Curing of All Lysogenic Bacteriophage from Streptococcus pyogenes Using a Novel Counter-selection Technique

    PubMed Central

    Euler, Chad W.; Juncosa, Barbara; Ryan, Patricia A.; Deutsch, Douglas R.; McShan, W. Michael; Fischetti, Vincent A.

    2016-01-01

    Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and

  14. Targeted Curing of All Lysogenic Bacteriophage from Streptococcus pyogenes Using a Novel Counter-selection Technique.

    PubMed

    Euler, Chad W; Juncosa, Barbara; Ryan, Patricia A; Deutsch, Douglas R; McShan, W Michael; Fischetti, Vincent A

    2016-01-01

    Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and

  15. Selective inhibition of the kinase DYRK1A by targeting its folding process.

    PubMed

    Kii, Isao; Sumida, Yuto; Goto, Toshiyasu; Sonamoto, Rie; Okuno, Yukiko; Yoshida, Suguru; Kato-Sumida, Tomoe; Koike, Yuka; Abe, Minako; Nonaka, Yosuke; Ikura, Teikichi; Ito, Nobutoshi; Shibuya, Hiroshi; Hosoya, Takamitsu; Hagiwara, Masatoshi

    2016-01-01

    Autophosphorylation of amino-acid residues is part of the folding process of various protein kinases. Conventional chemical screening of mature kinases has missed inhibitors that selectively interfere with the folding process. Here we report a cell-based assay that evaluates inhibition of a kinase at a transitional state during the folding process and identify a folding intermediate-selective inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), which we refer to as FINDY. FINDY suppresses intramolecular autophosphorylation of Ser97 in DYRK1A in cultured cells, leading to its degradation, but does not inhibit substrate phosphorylation catalysed by the mature kinase. FINDY also suppresses Ser97 autophosphorylation of recombinant DYRK1A, suggesting direct inhibition, and shows high selectivity for DYRK1A over other DYRK family members. In addition, FINDY rescues DYRK1A-induced developmental malformations in Xenopus laevis embryos. Our study demonstrates that transitional folding intermediates of protein kinases can be targeted by small molecules, and paves the way for developing novel types of kinase inhibitors. PMID:27102360

  16. Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat.

    PubMed

    Robb, Ellen L; Gawel, Justyna M; Aksentijević, Dunja; Cochemé, Helena M; Stewart, Tessa S; Shchepinova, Maria M; Qiang, He; Prime, Tracy A; Bright, Thomas P; James, Andrew M; Shattock, Michael J; Senn, Hans M; Hartley, Richard C; Murphy, Michael P

    2015-12-01

    Superoxide is the proximal reactive oxygen species (ROS) produced by the mitochondrial respiratory chain and plays a major role in pathological oxidative stress and redox signaling. While there are tools to detect or decrease mitochondrial superoxide, none can rapidly and specifically increase superoxide production within the mitochondrial matrix. This lack impedes progress, making it challenging to assess accurately the roles of mitochondrial superoxide in cells and in vivo. To address this unmet need, we synthesized and characterized a mitochondria-targeted redox cycler, MitoParaquat (MitoPQ) that comprises a triphenylphosphonium lipophilic cation conjugated to the redox cycler paraquat. MitoPQ accumulates selectively in the mitochondrial matrix driven by the membrane potential. Within the matrix, MitoPQ produces superoxide by redox cycling at the flavin site of complex I, selectively increasing superoxide production within mitochondria. MitoPQ increased mitochondrial superoxide in isolated mitochondria and cells in culture ~a thousand-fold more effectively than untargeted paraquat. MitoPQ was also more toxic than paraquat in the isolated perfused heart and in Drosophila in vivo. MitoPQ enables the selective generation of superoxide within mitochondria and is a useful tool to investigate the many roles of mitochondrial superoxide in pathology and redox signaling in cells and in vivo. PMID:26454075

  17. Molecular targeted PDT with selective delivery of ICG Photo-Immunoconjugates (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Wang, Sijia; Hüttmann, Gereon; Hasan, Tayyaba; Rahmanzadeh, Ramtin

    2016-03-01

    Light-induced inhibition of intracellular molecules holds great promise for a selective treatment of cancer and other diseases. Challenges for the targeting of intracellular proteins are the synthesis of effective photoimmuno-conjugates and their functional delivery inside living cells. In earlier studies we have shown, that photodynamic inactivation of the nuclear Ki-67 protein leads to an effective elimination of proliferating tumor cells. Here we show a selective treatment for EGFR and Ki-67 positive cancer cells after light-controlled delivery of indocyanine green (ICG) photo-immunoconjugates. The Ki-67 antibody TuBB-9, which recognizes an active state of the protein, was labeled with different ratios of ICG and encapsulated into immuno-liposomes that selectively deliver the conjugates to EGFR overexpressing cells. To overcome endosomal entrapment of the delivered agents, ovarian carcinoma cells were treated with the photosensitizer benzoporphyrin monoacid derivative (BPD) and irradiated first for endosomal escape of the TuBB-9-ICG constructs. 24 h after irradiation TuBB-9-ICG antibodies showed a relocalization from spots in the cytoplasm to the cell nucleus. A second irradiation of the delivered TuBB-9-ICG led to a significant elimination of cells after Ki-67 inactivation.

  18. Selective inhibition of the kinase DYRK1A by targeting its folding process

    PubMed Central

    Kii, Isao; Sumida, Yuto; Goto, Toshiyasu; Sonamoto, Rie; Okuno, Yukiko; Yoshida, Suguru; Kato-Sumida, Tomoe; Koike, Yuka; Abe, Minako; Nonaka, Yosuke; Ikura, Teikichi; Ito, Nobutoshi; Shibuya, Hiroshi; Hosoya, Takamitsu; Hagiwara, Masatoshi

    2016-01-01

    Autophosphorylation of amino-acid residues is part of the folding process of various protein kinases. Conventional chemical screening of mature kinases has missed inhibitors that selectively interfere with the folding process. Here we report a cell-based assay that evaluates inhibition of a kinase at a transitional state during the folding process and identify a folding intermediate-selective inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), which we refer to as FINDY. FINDY suppresses intramolecular autophosphorylation of Ser97 in DYRK1A in cultured cells, leading to its degradation, but does not inhibit substrate phosphorylation catalysed by the mature kinase. FINDY also suppresses Ser97 autophosphorylation of recombinant DYRK1A, suggesting direct inhibition, and shows high selectivity for DYRK1A over other DYRK family members. In addition, FINDY rescues DYRK1A-induced developmental malformations in Xenopus laevis embryos. Our study demonstrates that transitional folding intermediates of protein kinases can be targeted by small molecules, and paves the way for developing novel types of kinase inhibitors. PMID:27102360

  19. Combinatorial support vector machines approach for virtual screening of selective multi-target serotonin reuptake inhibitors from large compound libraries.

    PubMed

    Shi, Z; Ma, X H; Qin, C; Jia, J; Jiang, Y Y; Tan, C Y; Chen, Y Z

    2012-02-01

    Selective multi-target serotonin reuptake inhibitors enhance antidepressant efficacy. Their discovery can be facilitated by multiple methods, including in silico ones. In this study, we developed and tested an in silico method, combinatorial support vector machines (COMBI-SVMs), for virtual screening (VS) multi-target serotonin reuptake inhibitors of seven target pairs (serotonin transporter paired with noradrenaline transporter, H(3) receptor, 5-HT(1A) receptor, 5-HT(1B) receptor, 5-HT(2C) receptor, melanocortin 4 receptor and neurokinin 1 receptor respectively) from large compound libraries. COMBI-SVMs trained with 917-1951 individual target inhibitors correctly identified 22-83.3% (majority >31.1%) of the 6-216 dual inhibitors collected from literature as independent testing sets. COMBI-SVMs showed moderate to good target selectivity in misclassifying as dual inhibitors 2.2-29.8% (majority <15.4%) of the individual target inhibitors of the same target pair and 0.58-7.1% of the other 6 targets outside the target pair. COMBI-SVMs showed low dual inhibitor false hit rates (0.006-0.056%, 0.042-0.21%, 0.2-4%) in screening 17 million PubChem compounds, 168,000 MDDR compounds, and 7-8181 MDDR compounds similar to the dual inhibitors. Compared with similarity searching, k-NN and PNN methods, COMBI-SVM produced comparable dual inhibitor yields, similar target selectivity, and lower false hit rate in screening 168,000 MDDR compounds. The annotated classes of many COMBI-SVMs identified MDDR virtual hits correlate with the reported effects of their predicted targets. COMBI-SVM is potentially useful for searching selective multi-target agents without explicit knowledge of these agents. PMID:22064367

  20. A precisely substituted benzopyran targets androgen refractory prostate cancer cells through selective modulation of estrogen receptors

    SciTech Connect

    Kumar, Rajeev; Verma, Vikas; Sharma, Vikas; Jain, Ashish; Singh, Vishal; Sarswat, Amit; Maikhuri, Jagdamba P.; Sharma, Vishnu L.; Gupta, Gopal

    2015-03-15

    Dietary consumption of phytoestrogens like genistein has been linked with lower incidence of prostate cancer. The estradiol-like benzopyran core of genistein confers estrogen receptor-β (ER-β) selectivity that imparts weak anti-proliferative activity against prostate cancer cells. DL-2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2H-1-benzopyran (BP), a SERM designed with benzopyran core, targeted androgen independent prostate cancer (PC-3) cells 14-times more potently than genistein, ~ 25% more efficiently than tamoxifen and 6.5-times more actively than ICI-182780, without forfeiting significant specificity in comparison to genistein. BP increased apoptosis (annexin-V and TUNEL labeling), arrested cell cycle, and significantly increased caspase-3 activity along with mRNA expressions of estrogen receptor (ER)-β and FasL (qPCR) in PC-3 cells. In classical ERE-luc reporter assay BP behaved as a potent ER-α antagonist and ER-β agonist. Accordingly, it decreased expression of ER-α target PS2 (P < 0.01) and increased expression of ER-β target TNF-α (P < 0.05) genes in PC-3. ER-β deficient PC-3 (siRNA-transfected) was resistant to apoptotic and anti-proliferative actions of SERMs, including stimulation of FasL expression by BP. BP significantly inhibited phosphorylation of Akt and ERK-1/2, JNK and p38 in PC-3 (immunoblotting), and thus adopted a multi-pathway mechanism to exert a more potent anti-proliferative activity against prostate cancer cells than natural and synthetic SERMs. Its precise ER-subtype specific activity presents a unique lead structure for further optimization. - Highlights: • BP with benzopyran core of genistein was identified for ER-β selective action. • BP was 14-times more potent than genistien in targeting prostate cancer cells. • It behaved as a potent ER-β agonist and ER-α antagonist in gene reporter assays. • BP's anti-proliferative action was inhibited significantly in ER-β deficient cells. • BP — a unique lead structure