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Drugs targeting parasite lysosomes.  


Lysosomes were first described as vacuolar structures containing various hydrolytic enzymes at acidic pH. Subsequent studies revealed that the lysosome/vacuolar system is complex and composed of distinct membrane-enclosed vesicles including endosomes, primary and mature lysosomes, autophagic vesicles, residual bodies, multivesicular bodies, and digestive lysosomes. Lysosomes express a battery of hydrolytic enzymes including proteases, acid phosphatases, glycosidases, and lipases. Parasitic protozoa also possess complex intracellular lysosomes/endosomes/vesicles involved in digestion, transport and recycling of molecules similar to those of mammalian cells. Unique characteristics are ascribed to lysosomes of different parasites and may even differ between parasite stages. Transport of hydrolases and proteins to parasite lysosomes is directed either from the Golgi complex via endosomal vesicles or from endocytic vesicles originated in the cell surface. Inhibition of lysosomal proteases demonstrated that different proteolytic machineries catabolize distinct classes of proteins, and this selectivity may be exploited for the development of effective antiparasitic drugs. This review describes lysosomal molecules that are either validated or potential drug targets for Chagas' disease, sleeping sickness, leishmaniasis, toxoplasmosis, malaria, amebiasis, and giardiasis. PMID:18473838

Doyle, Patricia S; Sajid, Mohamed; O'Brien, Theresa; Dubois, Kelly; Engel, Juan C; Mackey, Zachary B; Reed, Sharon



Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors  

PubMed Central

Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML. PMID:23202731

Sukhai, Mahadeo A.; Prabha, Swayam; Hurren, Rose; Rutledge, Angela C.; Lee, Anna Y.; Sriskanthadevan, Shrivani; Sun, Hong; Wang, Xiaoming; Skrtic, Marko; Seneviratne, Ayesh; Cusimano, Maria; Jhas, Bozhena; Gronda, Marcela; MacLean, Neil; Cho, Eunice E.; Spagnuolo, Paul A.; Sharmeen, Sumaiya; Gebbia, Marinella; Urbanus, Malene; Eppert, Kolja; Dissanayake, Dilan; Jonet, Alexia; Dassonville-Klimpt, Alexandra; Li, Xiaoming; Datti, Alessandro; Ohashi, Pamela S.; Wrana, Jeff; Rogers, Ian; Sonnet, Pascal; Ellis, William Y.; Corey, Seth J.; Eaves, Connie; Minden, Mark D.; Wang, Jean C.Y.; Dick, John E.; Nislow, Corey; Giaever, Guri; Schimmer, Aaron D.



Intracellular drug distribution-based targeting: Exploiting lysosomes to enhance the selectivity of drugs towards cancer cells  

E-print Network

.......................................... 23 1.7. References .............................................................................................................. 27 Chapter 2: The prevalence and mechanism of defective acidification in cancer Cells... of V-ATPase subunit V1E1 ................................................ 75 3.2.6. Western blotting ...................................................................................... 76 3.2.7. Lysosome pH determination...

Ndolo, Rosemary A.



Lysosomal pH Rise during Heat Shock Monitored by a Lysosome-Targeting Near-Infrared Ratiometric Fluorescent Probe.  


Heat stroke is a life-threatening condition, featuring a high body temperature and malfunction of many organ systems. The relationship between heat shock and lysosomes is poorly understood, mainly because of the lack of a suitable research approach. Herein, by incorporating morpholine into a stable hemicyanine skeleton, we develop a new lysosome-targeting near-infrared ratiometric pH?probe. In combination with fluorescence imaging, we show for the first time that the lysosomal pH?value increases but never decreases during heat shock, which might result from lysosomal membrane permeabilization. We also demonstrate that this lysosomal pH?rise is irreversible in living cells. Moreover, the probe is easy to synthesize, and shows superior overall analytical performance as compared to the existing commercial ones. This enhanced performance may enable it to be widely used in more lysosomal models of living cells and in further revealing the mechanisms underlying heat-related pathology. PMID:25154475

Wan, Qiongqiong; Chen, Suming; Shi, Wen; Li, Lihong; Ma, Huimin



High Resolution Crystal Structure of Human ?-Glucuronidase Reveals Structural Basis of Lysosome Targeting  

PubMed Central

Human ?-glucuronidase (GUS) cleaves ?-D-glucuronic acid residues from the non-reducing termini of glycosaminoglycan and its deficiency leads to mucopolysaccharidosis type VII (MPSVII). Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases. The structure revealed several new details including a new glycan chain at Asn272, in addition to that previously observed at Asn173, and coordination of the glycan chain at Asn173 with Lys197 of the lysosomal targeting motif which is essential for phosphotransferase recognition. Analysis of the high resolution structure not only provided new insights into the structural basis for lysosomal targeting but showed significant differences between human GUS, which is medically important in its own right, and E. coli GUS, which can be selectively inhibited in the human gut to prevent prodrug activation and is also widely used as a reporter gene by plant biologists. Despite these differences, both human and E. coli GUS share a high structure homology in all three domains with most of the glycosyl hydrolases, suggesting that they all evolved from a common ancestral gene. PMID:24260279

Hassan, Md. Imtaiyaz; Waheed, Abdul; Grubb, Jeffery H.; Klei, Herbert E.; Korolev, Sergey; Sly, William S.



Generation and characterization of a lysosomally targeted, genetically encoded Ca(2+)-sensor.  


Distinct spatiotemporal Ca2+ signalling events regulate fundamental aspects of eukaryotic cell physiology. Complex Ca2+ signals can be driven by release of Ca2+ from intracellular organelles that sequester Ca2+ such as the ER (endoplasmic reticulum) or through the opening of Ca2+-permeable channels in the plasma membrane and influx of extracellular Ca2+. Late endocytic pathway compartments including late-endosomes and lysosomes have recently been observed to sequester Ca2+ to levels comparable with those found within the ER lumen. These organelles harbour ligand-gated Ca2+-release channels and evidence indicates that they can operate as Ca2+-signalling platforms. Lysosomes sequester Ca2+ to a greater extent than any other endocytic compartment, and signalling from this organelle has been postulated to provide 'trigger' release events that can subsequently elicit more extensive Ca2+ signals from stores including the ER. In order to investigate lysosomal-specific Ca2+ signalling a simple method for measuring lysosomal Ca2+ release is essential. In the present study we describe the generation and characterization of a genetically encoded, lysosomally targeted, cameleon sensor which is capable of registering specific Ca2+ release in response to extracellular agonists and intracellular second messengers. This probe represents a novel tool that will permit detailed investigations examining the impact of lysosomal Ca2+ handling on cellular physiology. PMID:23098255

McCue, Hannah V; Wardyn, Joanna D; Burgoyne, Robert D; Haynes, Lee P



Generation and characterization of a lysosomally targeted, genetically encoded Ca2+-sensor  

PubMed Central

Distinct spatiotemporal Ca2+ signalling events regulate fundamental aspects of eukaryotic cell physiology. Complex Ca2+ signals can be driven by release of Ca2+ from intracellular organelles that sequester Ca2+ such as the ER (endoplasmic reticulum) or through the opening of Ca2+-permeable channels in the plasma membrane and influx of extracellular Ca2+. Late endocytic pathway compartments including late-endosomes and lysosomes have recently been observed to sequester Ca2+ to levels comparable with those found within the ER lumen. These organelles harbour ligand-gated Ca2+-release channels and evidence indicates that they can operate as Ca2+-signalling platforms. Lysosomes sequester Ca2+ to a greater extent than any other endocytic compartment, and signalling from this organelle has been postulated to provide ‘trigger’ release events that can subsequently elicit more extensive Ca2+ signals from stores including the ER. In order to investigate lysosomal-specific Ca2+ signalling a simple method for measuring lysosomal Ca2+ release is essential. In the present study we describe the generation and characterization of a genetically encoded, lysosomally targeted, cameleon sensor which is capable of registering specific Ca2+ release in response to extracellular agonists and intracellular second messengers. This probe represents a novel tool that will permit detailed investigations examining the impact of lysosomal Ca2+ handling on cellular physiology. PMID:23098255

McCue, Hannah V.; Wardyn, Joanna D.; Burgoyne, Robert D.; Haynes, Lee P.



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

SciTech Connect

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.

Haylett, T.; Thilo, L.



IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome  

PubMed Central

Expansion of the polyglutamine repeat within the protein Huntingtin (Htt) causes Huntington's disease, a neurodegenerative disease associated with aging and the accumulation of mutant Htt in diseased neurons. Understanding the mechanisms that influence Htt cellular degradation may target treatments designed to activate mutant Htt clearance pathways. We find that Htt is phosphorylated by the inflammatory kinase IKK, enhancing its normal clearance by the proteasome and lysosome. Phosphorylation of Htt regulates additional post-translational modifications, including Htt ubiquitination, SUMOylation, and acetylation, and increases Htt nuclear localization, cleavage, and clearance mediated by lysosomal-associated membrane protein 2A and Hsc70. We propose that IKK activates mutant Htt clearance until an age-related loss of proteasome/lysosome function promotes accumulation of toxic post-translationally modified mutant Htt. Thus, IKK activation may modulate mutant Htt neurotoxicity depending on the cell's ability to degrade the modified species. PMID:20026656

Thompson, Leslie Michels; Aiken, Charity T.; Kaltenbach, Linda S.; Agrawal, Namita; Illes, Katalin; Khoshnan, Ali; Martinez-Vincente, Marta; Arrasate, Montserrat; O'Rourke, Jacqueline Gire; Khashwji, Hasan; Lukacsovich, Tamas; Zhu, Ya-Zhen; Lau, Alice L.; Massey, Ashish; Hayden, Michael R.; Zeitlin, Scott O.; Finkbeiner, Steven; Green, Kim N.; LaFerla, Frank M.; Bates, Gillian; Huang, Lan; Patterson, Paul H.; Lo, Donald C.; Cuervo, Ana Maria; Marsh, J. Lawrence



Impairment of Lysosomal Activity as a Therapeutic Modality Targeting Cancer Stem Cells of Embryonal Rhabdomyosarcoma Cell Line RD  

PubMed Central

Rhabdomyosarcoma is the most frequent soft tissue sarcoma in children and adolescents, with a high rate of relapse that dramatically affects the clinical outcome. Multiagent chemotherapy, in combination with surgery and/or radiation therapy, is the treatment of choice. However, the relapse rate is disappointingly high and identification of new therapeutic tools is urgently needed. Under this respect, the selective block of key features of cancer stem cells (CSC) appears particularly promising. In this study, we isolated rhabdomyosarcoma CSC with stem-like features (high expression of NANOG and OCT3/4, self-renewal ability, multipotency). Rhabdomyosarcoma CSC showed higher invasive ability and a reduced cytotoxicity to doxorubicin in comparison to native cells, through a mechanism unrelated to the classical multidrug resistance process. This was dependent on a high level of lysosome acidity mediated by a high expression of vacuolar ATPase (V-ATPase). Since it was not associated with other paediatric cancers, like Ewing’s sarcoma and neuroblastoma, V-ATPase higher expression in CSC was rhabdomyosarcoma specific. Inhibition of lysosomal acidification by the V-ATPase inhibitor omeprazole, or by specific siRNA silencing, significantly enhanced doxorubicin cytoxicity. Unexpectedly, lysosomal targeting also blocked cell growth and reduced the invasive potential of rhabdomyosarcoma CSC, even at very low doses of omeprazole (10 and 50 µM, respectively). Based on these observations, we propose lysosome acidity as a valuable target to enhance chemosensitivity of rhabdomyosarcoma CSC, and suggest the use of anti-V-ATPase agents in combination with standard regimens as a promising tool for the eradication of minimal residual disease or the prevention of metastatic disease. PMID:25329465

Salerno, Manuela; Avnet, Sofia; Bonuccelli, Gloria; Hosogi, Shigekuni; Granchi, Donatella; Baldini, Nicola



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


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

Umlauf, Benjamin J; Mercedes, Julia S; Chung, Chin-Ying; Brown, Kathlynn C



Hepatitis C Virus Targets DC-SIGN and L-SIGN To Escape Lysosomal Degradation  

PubMed Central

Hepatitis C virus (HCV) is a major health problem. However, the mechanism of hepatocyte infection is largely unknown. We demonstrate that the dendritic cell (DC)-specific C-type lectin DC-SIGN and its liver-expressed homologue L-SIGN/DC-SIGNR are important receptors for HCV envelope glycoproteins E1 and E2. Mutagenesis analyses demonstrated that both HCV E1 and E2 bind the same binding site on DC-SIGN as the pathogens human immunodeficiency virus type 1 (HIV-1) and mycobacteria, which is distinct from the cellular ligand ICAM-3. HCV virus-like particles are efficiently captured and internalized by DCs through binding of DC-SIGN. Antibodies against DC-SIGN specifically block HCV capture by both immature and mature DCs, demonstrating that DC-SIGN is the major receptor on DCs. Interestingly, internalized HCV virus-like particles were targeted to nonlysosomal compartments within immature DCs, where they are protected from lysosomal degradation in a manner similar to that demonstrated for HIV-1. Lewis X antigen, another ligand of DC-SIGN, was internalized to lysosomes, demonstrating that the internalization pathway of DC-SIGN-captured ligands may depend on the structure of the ligand. Our results suggest that HCV may target DC-SIGN to “hide” within DCs and facilitate viral dissemination. L-SIGN, expressed by THP-1 cells, internalized HCV particles into similar nonlysosomal compartments, suggesting that L-SIGN on liver sinusoidal endothelial cells may capture HCV from blood and transmit it to hepatocytes, the primary target for HCV. We therefore conclude that both DCs and liver sinusoidal endothelial cells may act as reservoirs for HCV and that the C-type lectins DC-SIGN and L-SIGN, as important HCV receptors, may represent a molecular target for clinical intervention in HCV infection. PMID:15254204

Ludwig, Irene S.; Lekkerkerker, Annemarie N.; Depla, Erik; Bosman, Fons; Musters, Rene J. P.; Depraetere, Stany; van Kooyk, Yvette; Geijtenbeek, Teunis B. H.



The role of lysosomes in the selective concentration of mineral elements. A microanalytical study.  


The role of the lysosome during the intracellular concentration of diverse mineral elements has been evidenced by the electron probe X-ray microanalysis (EPMA). This highly sensitive technique allows an in situ chemical analysis of any chemical element with an atomic number greater than 11, present in ultra-thin tissue sections. Therefore, it has been demonstrated by using this EPMA that 21 out of the 92 elements of the periodic table, once injected in a soluble form, were selectively concentrated within lysosomes of several types of mammalian cells. Amongst these 21 elements, 15 are concentrated and precipitated in an insoluble from in association with phosphorus whereas the other 6 are precipitated in association with sulphur. Amongst the 15 elements which precipitate with phosphorus in lysosomes, there are: 3 group IIIB elements of the periodic system, (aluminium, gallium and indium); the rare-earth elements (cerium, gadolinium, lanthanum, thulium and samarium); 2 group IVA elements (hafnium and zirconium), two actinides (uranium and thorium) and elements such as chromium and niobium. The 6 elements which precipitate with sulphur comprise the 3 group VIII elements of the classification (nickel, palladium, platinum) and the 3 group IB elements (copper, silver and gold). The mechanisms responsible for this selective concentration involve enzymatic processes and predominantly acid phosphatases for elements precipitating as phosphates and arylsulfatases for elements precipitating with sulphur. PMID:8793193

Berry, J P



Mr 46,000 mannose 6-phosphate specific receptor: its role in targeting of lysosomal enzymes.  

PubMed Central

Antibodies that block the ligand binding site of the cation-dependent mannose 6-phosphate specific receptor (Mr 46,000 MPR) were used to probe the function of the receptor in transport of lysosomal enzymes. Addition of the antibodies to the medium of Morris hepatoma 7777 cells, which express only the Mr 46,000 MPR, resulted in a decreased intracellular retention and increased secretion of newly synthesized lysosomal enzymes. In fibroblasts and HepG2 cells that express the cation-independent mannose 6-phosphate specific receptor (Mr 215,000 MPR) in addition to the Mr 46,000 MPR, antibodies against the Mr 46,000 MPR inhibited the intracellular retention of newly synthesized lysosomal enzymes only when added to the medium together with antibodies against the Mr 215,000 MPR. Morris hepatoma (M.H.) 7777 did not endocytose lysosomal enzymes, while U937 monocytes, which express both types of MPR, internalized lysosomal enzymes. The uptake was inhibited by antibodies against the Mr 215,000 MPR, but not by antibodies against the Mr 46,000 MPR. These observations suggest that Mr 46,000 MPR mediates transport of endogenous but not endocytosis of exogenous lysosomal enzymes. Internalization of receptor antibodies indicated that the failure to mediate endocytosis of lysosomal enzymes is due to an inability of surface Mr 46,000 MPR to bind ligands rather than its exclusion from the plasma membrane or from internalization. Images Fig. 1. Fig. 2. PMID:2960521

Stein, M; Zijderhand-Bleekemolen, J E; Geuze, H; Hasilik, A; von Figura, K



CDTI target selection criteria  

NASA Technical Reports Server (NTRS)

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.

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



TPC Proteins Are Phosphoinositide-activated Sodium-selective Ion Channels in Endosomes and Lysosomes  

PubMed Central

Summary Mammalian Two-Pore Channels (TPC1, 2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P2, and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na+, not K+, as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes, and may explain the specificity of PI(3,5)P2 in regulating the fusogenic potential of intracellular organelles. PMID:23063126

Wang, Xiang; Zhang, Xiaoli; Dong, Xian-ping; Samie, Mohammad; Li, Xinran; Cheng, Xiping; Goschka, Andrew; Shen, Dongbiao; Zhou, Yandong; Harlow, Janice; Zhu, Michael X.; Clapham, David E.; Ren, Dejian; Xu, Haoxing



Distinctive inhibition of the lysosomal targeting of lysozyme and cathepsin D by drugs affecting pH gradients and protein kinase C.  

PubMed Central

Morphological and biochemical evidence indicates that in several cell types, lysozyme is found in both lysosomes and the medium. Here we report that in calcitriol-treated human promonocytes U937, in which approx. two-thirds of the synthesized lysozyme is secreted, most of the intracellular lysozyme co-localizes with cathepsin D in lysosomal organelles. In the presence of NH4Cl the lysosomal targeting of procathepsin D, but not that of lysozyme, is inhibited. In the presence of 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA; 'TPA'), the lysosomal packaging of lysozyme is almost completely inhibited, while that of procathepsin D is only partially so. However, the inhibition of the lysosomal targeting of procathepsin D by NH4Cl and 4 beta-PMA is additive. The targeting of lysozyme is partially inhibited in the presence of R-59022, an inhibitor of diacylglycerol kinase, whereas it is not affected by 4 alpha-phorbol 12-myristate 13-acetate, an isomer of 4 beta-PMA that does not activate protein kinase C. It is concluded that in U937 cells both carbohydrate-dependent and -independent recognition contributes to the lysosomal targeting of soluble proteins. We suggest that the carbohydrate-independent traffic of proteins to lysosomal compartments is controlled by a signalling pathway involving protein kinase C. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8093011

Radons, J; Biewusch, U; Grässel, S; Geuze, H J; Hasilik, A



MHC II in dendritic cells is targeted to lysosomes or T cell-induced exosomes via distinct multivesicular body pathways.  


Dendritic cells (DCs) express major histocompatibility complex class II (MHC II) to present peptide antigens to T cells. In immature DCs, which bear low cell surface levels of MHC II, peptide-loaded MHC II is ubiquitinated. Ubiquitination drives the endocytosis and sorting of MHC II to the luminal vesicles of multivesicular bodies (MVBs) for lysosomal degradation. Ubiquitination of MHC II is abrogated in activated DCs, resulting in an increased cell surface expression. We here provide evidence for an alternative MVB sorting mechanism for MHC II in antigen-loaded DCs, which is triggered by cognately interacting antigen-specific CD4+ T cells. At these conditions, DCs generate MVBs with MHC II and CD9 carrying luminal vesicles that are secreted as exosomes and transferred to the interacting T cells. Sorting of MHC II into exosomes was, in contrast to lysosomal targeting, independent of MHC II ubiquitination but rather correlated with its incorporation into CD9 containing detergent-resistant membranes. Together, these data indicate two distinct MVB pathways: one for lysosomal targeting and the other for exosome secretion. PMID:19682328

Buschow, Sonja I; Nolte-'t Hoen, Esther N M; van Niel, Guillaume; Pols, Maaike S; ten Broeke, Toine; Lauwen, Marjolein; Ossendorp, Ferry; Melief, Cornelis J M; Raposo, Graça; Wubbolts, Richard; Wauben, Marca H M; Stoorvogel, Willem



Lysosomal storage disorders: The cellular impact of lysosomal dysfunction  

PubMed Central

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



Mutations in the Lysosomal Enzyme-Targeting Pathway and Persistent Stuttering  

PubMed Central

BACKGROUND Stuttering is a disorder of unknown cause characterized by repetitions, prolongations, and interruptions in the flow of speech. Genetic factors have been implicated in this disorder, and previous studies of stuttering have identified linkage to markers on chromosome 12. METHODS We analyzed the chromosome 12q23.3 genomic region in consanguineous Pakistani families, some members of which had nonsyndromic stuttering and in unrelated case and control subjects from Pakistan and North America. RESULTS We identified a missense mutation in the N-acetylglucosamine-1-phosphate transferase gene (GNPTAB), which encodes the alpha and beta catalytic subunits of GlcNAc-phosphotransferase (GNPT [EC]), that was associated with stuttering in a large, consanguineous Pakistani family. This mutation occurred in the affected members of approximately 10% of Pakistani families studied, but it occurred only once in 192 chromosomes from unaffected, unrelated Pakistani control subjects and was not observed in 552 chromosomes from unaffected, unrelated North American control subjects. This and three other mutations in GNPTAB occurred in unrelated subjects with stuttering but not in control subjects. We also identified three mutations in the GNPTG gene, which encodes the gamma subunit of GNPT, in affected subjects of Asian and European descent but not in control subjects. Furthermore, we identified three mutations in the NAGPA gene, which encodes the so-called uncovering enzyme, in other affected subjects but not in control subjects. These genes encode enzymes that generate the mannose-6-phosphate signal, which directs a diverse group of hydrolases to the lysosome. Deficits in this system are associated with the mucolipidoses, rare lysosomal storage disorders that are most commonly associated with bone, connective tissue, and neurologic symptoms. CONCLUSIONS Susceptibility to nonsyndromic stuttering is associated with variations in genes governing lysosomal metabolism. PMID:20147709

Kang, Changsoo; Riazuddin, Sheikh; Mundorff, Jennifer; Krasnewich, Donna; Friedman, Penelope; Mullikin, James C.; Drayna, Dennis



Interaction of HIV-1 Nef Protein with the Host Protein Alix Promotes Lysosomal Targeting of CD4 Receptor.  


Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef. PMID:25118280

Amorim, Nathaly A; da Silva, Eulália M L; de Castro, Rodrigo O; da Silva-Januário, Mara E; Mendonça, Luiza M; Bonifacino, Juan S; da Costa, Luciana J; daSilva, Luis L P



Inhibition of Glycogen Synthase Kinase-3 Ameliorates ?-Amyloid Pathology and Restores Lysosomal Acidification and Mammalian Target of Rapamycin Activity in the Alzheimer Disease Mouse Model  

PubMed Central

Accumulation of ?-amyloid (A?) deposits is a primary pathological feature of Alzheimer disease that is correlated with neurotoxicity and cognitive decline. The role of glycogen synthase kinase-3 (GSK-3) in Alzheimer disease pathogenesis has been debated. To study the role of GSK-3 in A? pathology, we used 5XFAD mice co-expressing mutated amyloid precursor protein and presenilin-1 that develop massive cerebral A? loads. Both GSK-3 isozymes (?/?) were hyperactive in this model. Nasal treatment of 5XFAD mice with a novel substrate competitive GSK-3 inhibitor, L803-mts, reduced A? deposits and ameliorated cognitive deficits. Analyses of 5XFAD hemi-brain samples indicated that L803-mts restored the activity of mammalian target of rapamycin (mTOR) and inhibited autophagy. Lysosomal acidification was impaired in the 5XFAD brains as indicated by reduced cathepsin D activity and decreased N-glycoyslation of the vacuolar ATPase subunit V0a1, a modification required for lysosomal acidification. Treatment with L803-mts restored lysosomal acidification in 5XFAD brains. Studies in SH-SY5Y cells confirmed that GSK-3? and GSK-3? impair lysosomal acidification and that treatment with L803-mts enhanced the acidic lysosomal pool as demonstrated in LysoTracker Red-stained cells. Furthermore, L803-mts restored impaired lysosomal acidification caused by dysfunctional presenilin-1. We provide evidence that mTOR is a target activated by GSK-3 but inhibited by impaired lysosomal acidification and elevation in amyloid precursor protein/A? loads. Taken together, our data indicate that GSK-3 is a player in A? pathology. Inhibition of GSK-3 restores lysosomal acidification that in turn enables clearance of A? burdens and reactivation of mTOR. These changes facilitate amelioration in cognitive function. PMID:23155049

Avrahami, Limor; Farfara, Dorit; Shaham-Kol, Maya; Vassar, Robert; Frenkel, Dan; Eldar-Finkelman, Hagit



Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum  

PubMed Central

Summary Most intracellular Ca2+ signals result from opening of Ca2+ channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca2+ into mitochondria. Ca2+ channels in lysosomes contribute to endo-lysosomal trafficking and Ca2+ signalling, but the role of lysosomal Ca2+ uptake in Ca2+ signalling is unexplored. Inhibition of lysosomal Ca2+ uptake by dissipating the H+ gradient (using bafilomycin A1), perforating lysosomal membranes (using glycyl-L-phenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca2+ signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation. Bafilomycin A1 amplified the Ca2+ signals evoked by photolysis of caged Ins(1,4,5)P3 or by inhibition of ER Ca2+ pumps, and it slowed recovery from them. Ca2+ signals evoked by store-operated Ca2+ entry were unaffected by bafilomycin A1. Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca2+ released by Ins(1,4,5)P3 receptors. PMID:23097044

Lopez-Sanjurjo, Cristina I.; Tovey, Stephen C.; Prole, David L.; Taylor, Colin W.



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

PubMed Central

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



Lysosome-dependent p300/FOXP3 degradation and limits Treg cell functions and enhances targeted therapy against cancers  

PubMed Central

p300 is one of several acetyltransferases that regulate FOXP3 acetylation and functions. Our recent studies have defined a complex set of histone acetyltransferase interactions which can lead to enhanced or repressed changes in FOXP3 function. We have explored the use of a natural p300 inhibitor, Garcinol, as a tool to understand mechanisms by which p300 regulates FOXP3 acetylation. In the presence of Garcinol, p300 appears to become disassociated from the FOXP3 complex and undergoes lysosome-dependent degradation. As a consequence of p300's physical absence, FOXP3 becomes less acetylated and eventually degraded, a process that cannot be rescued by the proteasome inhibitor MG132. p300 plays a complex role in FOXP3 acetylation, as it could also acetylate a subset of four Lys residues that repressively regulate total FOXP3 acetylation. Garcinol acts as a degradation device to reduce the suppressive activity of regulatory T cells (Treg) and to enhance the in vivo anti-tumor activity of a targeted therapeutic anti-p185her2/neu (ERBB2) antibody in MMTV-neu transgenics implanted with neu transformed breast tumor cells. Our studies provide the rationale for molecules that disrupt p300 stability to limit Treg functions in targeted therapies for cancers. PMID:23644046

Du, Taofeng; Nagai, Yasuhiro; Xiao, Yan; Greene, Mark I.; Zhang, Hongtao



The biogenesis of lysosomes and lysosome-related organelles.  


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

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



Imaging of lysosomal pH changes with a fluorescent sensor containing a novel lysosome-locating group.  


Rlyso, a highly selective and sensitive pH sensor, can stain lysosomes with a novel lysosome-locating group, methylcarbitol. Rlyso was successfully used to detect lysosomal pH changes during apoptosis or induced by chloroquine while avoiding the "alkalizing effect" on lysosomes of current lysosomal probes with nitrogen-containing sidechains. PMID:23114612

Zhu, Hao; Fan, Jiangli; Xu, Qunli; Li, Honglin; Wang, Jingyun; Gao, Pan; Peng, Xiaojun



Deletion of an endosomal/lysosomal targeting signal promotes the secretion of Alzheimer's disease amyloid precursor protein (APP).  


Alzheimer's disease amyloid precursor protein (APP) generates a beta-amyloid protein (A beta) that is a main component of the senile plaques found in the brains of Alzheimer's disease patients. APP is thought to undergo proteolysis via two different pathways, the amyloidogenic pathway which produces A beta, and the non-amyloidogenic pathway which releases a large N-terminal fragment into the medium. The proteases that mediate these processes remain unidentified. The physiological function of APP is not clear yet. Therefore, the cytoplasmic region of APP has attracted much interest, because this region is highly conserved among species, and members of the amyloid precursor-like protein (APLP) family. Several potentially functional sequences exist in the region, including signal sequences for protein sorting and a G0-protein binding sequence. We constructed two mutants, 695 deltaNPTY and 695 deltaGYEN. They lack potential endosome/lysosome targeting signals, NPTY and GY, in the cytoplasmic domain of APP695, respectively. The mutant APPs had longer half-lives and were secreted more easily into the medium than the wild type, suggesting that these sequences are important for the secretion and metabolism of APP. PMID:9133629

Ono, Y; Kinouchi, T; Sorimachi, H; Ishiura, S; Suzuki, K



Lysosomal adaptation: how the lysosome responds to external cues.  


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

Settembre, Carmine; Ballabio, Andrea



Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors  

PubMed Central

Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell. In this study we have examined the intracellular trafficking route of S. typhimurium within epithelial cells. Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments approximately 15 min after bacterial internalization. This process was completed approximately 75 min later and did not require microtubules. Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV. Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (approximately 30%) of SCV. Uptake experiments with fluid endocytic tracers such as fluorescein- dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in approximately 75% of lgp- containing vesicles in uninfected cells, while only approximately 15% of SCV contained small amounts of the tracer during the same uptake period. SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with approximately 30% of SCV having detectable amounts of HRP at 6 h after infection. These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and are therefore functionally separated from the endocytic route. Moreover, the unusual intracellular trafficking route of S. typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella- infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes. PMID:7698996



Immature and mature species of the human Prostacyclin Receptor are ubiquitinated and targeted to the 26S proteasomal or lysosomal degradation pathways, respectively  

PubMed Central

Background The human prostacyclin receptor (hIP) undergoes agonist-induced phosphorylation, desensitisation and internalisation and may be recycled to the plasma membrane or targeted for degradation by, as yet, unknown mechanism(s). Results Herein it was sought to investigate the turnover of the hIP under basal conditions and in response to cicaprost stimulation. It was established that the hIP is subject to low-level basal degradation but, following agonist stimulation, degradation is substantially enhanced. Inhibition of the lysosomal pathway prevented basal and agonist-induced degradation of the mature species of the hIP (46-66 kDa). Conversely, inhibition of the proteasomal pathway had no effect on levels of the mature hIP but led to time-dependent accumulation of four newly synthesised immature species (38-44 kDa). It was established that both the mature and immature species of the hIP may be polyubiquitinated and this modification may be required for lysosomal sorting of the mature, internalised receptors and for degradation of the immature receptors by the 26S proteasomes through the ER-associated degradation (ERAD) process, respectively. Moreover, these data substantially advance knowledge of the factors regulating processing and maturation of the hIP, a complex receptor subject to multiple post-translational modifications including N-glycosylation, phosphorylation, isoprenylation, palmitoylation, in addition to polyubiquitination, as determined herein. Conclusion These findings indicate that the hIP is post-translationally modified by ubiquitination, which targets the immature species to the 26S proteasomal degradation pathway and the mature species to the lysosomal degradation pathway. PMID:19781057

Donnellan, Peter D; Kinsella, B Therese



Lysosomal dysfunction causes neurodegeneration in mucolipidosis II 'knock-in' mice  

PubMed Central

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 ?-l-fucosidase, ?-hexosaminidase, ?-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 neurodegeneration. These lysosomal proteins might be a potential target for a valid therapeutic approach for mucolipidosis II disease. PMID:22961545

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



Lipids and lysosomes.  


Lysosomes are cytoplasmic organelles delimited by a single membrane and filled with a variety of hydrolytic enzymes active at acidic pH and collectively capable to degrade the vast majority of macromolecules entering lysosomes via endocytosis, phagocytosis or autophagy. In this review, we describe the lipid composition and the dynamic properties of lysosomal membrane, the main delivery pathways of lipids to lysosomes and their catabolism inside lysosomes. Then, we present the consequences of a lipid accumulation as seen in various lysosomal storage diseases on lysosomal functions. Finally, we discuss about the possible involvement of lysosomes in lipotoxicity. PMID:22978393

Hamer, Isabelle; Van Beersel, Guillaume; Arnould, Thierry; Jadot, Michel



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

NASA Technical Reports Server (NTRS)

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.

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



Inhibition of Glioma Cell Lysosome Exocytosis Inhibits Glioma Invasion  

PubMed Central

Cancer cells invade by secreting enzymes that degrade the extracellular matrix and these are sequestered in lysosomal vesicles. In this study, the effects of the selective lysosome lysing drug GPN and the lysosome exocytosis inhibitor vacuolin-1 on lysosome exocytosis were studied to determine their effect on glioma cell migration and invasion. Both GPN and vacuolin-1 evidently inhibited migration and invasion in transwell experiments and scratch experiments. There are more lysosomes located on the cell membrane of glioma cells than of astrocytes. GPN decreased the lysosome number on the cell membrane. We found that rab27A was expressed in glioma cells, and colocalized with cathepsin D in lysosome. RNAi-Rab27A inhibited lysosome cathepsin D exocytosis and glioma cell invasion in an in vitro assay. Inhibition of cathepsin D inhibited glioma cell migration. The data suggest that the inhibition of lysosome exocytosis from glioma cells plays an important modulatory role in their migration and invasion. PMID:23029308

Zhu, Keqing



Structural Implications for Selective Targeting of PARPs  

PubMed Central

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

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



Localization of Atypical Protein Kinase C Isoforms into Lysosome-Targeted Endosomes through Interaction with p62  

Microsoft Academic Search

An increasing number of independent studies indicate that the atypical protein kinase C (PKC) isoforms (aPKCs) are critically involved in the control of cell proliferation and survival. The aPKCs are targets of important lipid mediators such as ceramide and the products of the PI 3-kinase. In addition, the aPKCs have been shown to interact with Ras and with two novel




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

SciTech Connect

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.

Gonzalez-Noriega, Alfonso [Department of Cell Biology and Physiology, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, PO Box 70228, 04510 Mexico, D.F. (Mexico)]. E-mail:; Ortega Cuellar, Daniel D. [Department of Cell Biology and Physiology, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, PO Box 70228, 04510 Mexico, D.F. (Mexico); Michalak, Colette [Department of Cell Biology and Physiology, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, PO Box 70228, 04510 Mexico, D.F. (Mexico)



Degradation of low density lipoprotein cholesterol esters by lysosomal lipase in vitro. Effect of core physical state and basis of species selectivity.  


The effect of the physical state of low density lipoprotein (LDL) core and the selectivity of the degradation of LDL cholesterol esters (CEs) by the lysosomal acid lipase (LAL) in vitro were investigated. The physical state of LDL was modulated by varying temperature or the triglyceride content of the core. Normal LDL showed an abrupt increase of CE hydrolysis at 24 degrees C and another deviation occurred close to 36 degrees C. 1H-NMR measurements showed that these temperatures coincide with the onset and end temperatures of the LDL core lipid transition, respectively. Enrichment of LDL with triglycerides abolished the abrupt changes both in the CE hydrolysis and in the physical state of LDL lipids. These findings show that there is a correlation between the physical state of LDL lipids and the rate of LAL-mediated hydrolysis of the CEs in the particle. The relative rates of hydrolysis of different CE species were also compared. With native LDL, increasing the length of a saturated acyl chain from 14 to 20 carbons reduced the rate of degradation of CE modestly, while increasing acyl chain unsaturation increased the rate of degradation markedly. However, cholesterol oleate was hydrolyzed more slowly than cholesterol stearate. Essentially the same order of hydrolytic susceptibility was observed when the CE species were incorporated into triglyceride-enriched LDL, reconstituted high density lipoprotein particles or in detergent/phospholipid micelles. These results indicate that the selective hydrolysis of CE species in LDL is determined mainly by the ease with which the CE molecule can emerge from the surface layer reach the active site of LAL. Slower degradation of the more saturated CEs by LAL could lead, under certain conditions, to their accumulation in lysosomes and eventually, to cell death, lysis and deposition of crystalline, poorly mobilizable lipids to the arterial intima. PMID:9461252

Lusa, S; Somerharju, P



Selection criteria for targets of asteroseismic campaigns  

E-print Network

Various dedicated satellite projects are underway or in advanced stages of planning to perform high-precision, long duration time series photometry of stars, with the purpose of using the frequencies of stellar oscillations to put new constraints on the internal structure of stars. It is known (cf. Brown, et al. 1994) that the effectiveness of oscillation frequencies in constraining stellar model parameters is significantly higher if classical parameters such as effective temperature, and luminosity are known with high precision. In order to optimize asteroseismic campaigns it is therefore useful to select targets from among candidates for which good spectroscopic and astrometric data already exists. This paper presents selection criteria, as well as redeterminations of stellar luminosity and reddening for stars satisfying these criteria.

Frank P. Pijpers



Nonresonant and Resonant Frequency-Selectable Induction-Heating Targets  

E-print Network

This paper examines a scheme for developing frequency-selectable induction-heating targets for stimulating temperature-sensitive polymer gels. The phrase “frequency selectable” implies that each target has a frequency at ...

Rodriguez, John I.


[Lysosomes and apoptosis].  


In 1955, Christian de Duve and his coworkers from the School of Medicine in Louvain, Belgium, named a group of cytoplasmic formations surrounded by lipoprotein membrane and containing acid hydrolase enzymes as lysosomes. Biochemical and cytochemical studies showed lysosomes to be found in animal and vegetable eukaryotic cells. Later on, lysosomes were found to be involved in the dynamics of lysosomal system, which consists of a number of various cytoplasmic formations such as primary and secondary lysosomes, endosomes, autophagosomes and postlysosomes. These formations are inter-connected by the mechanism of membrane integration, and in some instances by the cell membrane. Lysosomal system is involved in numerous physiological processes such as degradation of endogenous and exogenous macromolecules (proteins, lipids, polysaccharides and nucleic acids), cytoplasmic formations (mitochondria, peroxisomes, Golgi complex) that have performed their functions, tissue regression (post-lactation mammary gland), hormone secretion regulation (proinsulin to insulin), etc. On the other hand, lysosomal system is also involved in a number of pathologic processes like inflammation, allergic reactions, ischemia, hypoxia, as well as in lysosomal diseases (thesaurismoses), e.g., type II glycogenosis, fucosidosis, mucolipidosis III, etc. In 1974, Christian de Duve introduced the term lysosomotropism, denoting entry of the pharmacologically active, toxic and carcinogenic substances in the lysosomal system irrespective of their chemical nature and mechanism of input. Lysosomotropic substances may act in two ways: (1) causing impairments in the intralysosomal area, with toxic manifestations; and (2) modifying their membrane properties by increasing or decreasing membrane permeability, or by inducing labilizing or stabilizing effects. Damage to the lysosomal system occurs in the late stage of necrosis, while destabilization of lysosomal formations has been recorded in primary processes during apoptosis. PMID:19999542

Viki?-Topi?, Drazen; Carevi?, Olga



Functional Analysis of Lysosomes During Mouse Preimplantation Embryo Development  

PubMed Central

Abstract Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos. PMID:23080372

TSUKAMOTO, Satoshi; HARA, Taichi; YAMAMOTO, Atsushi; OHTA, Yuki; WADA, Ayako; ISHIDA, Yuka; KITO, Seiji; NISHIKAWA, Tetsu; MINAMI, Naojiro; SATO, Ken; KOKUBO, Toshiaki



Escape: A Target Selection Technique Using Visually-cued Gestures  

E-print Network

Escape: A Target Selection Technique Using Visually-cued Gestures Koji Yatani1 , Kurt Partridge2 the selection speed problem through a new target selection technique called Escape. In Escape, targets to twelve pixels wide, Escape performs at a similar error rate and at least 30% faster than Shift

Toronto, University of


Selective targeting of microglia by quantum dots  

PubMed Central

Background Microglia, the resident immune cells of the brain, have been implicated in brain injury and various neurological disorders. However, their precise roles in different pathophysiological situations remain enigmatic and may range from detrimental to protective. Targeting the delivery of biologically active compounds to microglia could help elucidate these roles and facilitate the therapeutic modulation of microglial functions in neurological diseases. Methods Here we employ primary cell cultures and stereotaxic injections into mouse brain to investigate the cell type specific localization of semiconductor quantum dots (QDs) in vitro and in vivo. Two potential receptors for QDs are identified using pharmacological inhibitors and neutralizing antibodies. Results In mixed primary cortical cultures, QDs were selectively taken up by microglia; this uptake was decreased by inhibitors of clathrin-dependent endocytosis, implicating the endosomal pathway as the major route of entry for QDs into microglia. Furthermore, inhibiting mannose receptors and macrophage scavenger receptors blocked the uptake of QDs by microglia, indicating that QD uptake occurs through microglia-specific receptor endocytosis. When injected into the brain, QDs were taken up primarily by microglia and with high efficiency. In primary cortical cultures, QDs conjugated to the toxin saporin depleted microglia in mixed primary cortical cultures, protecting neurons in these cultures against amyloid beta-induced neurotoxicity. Conclusions These findings demonstrate that QDs can be used to specifically label and modulate microglia in primary cortical cultures and in brain and may allow for the selective delivery of therapeutic agents to these cells. PMID:22272874



Lysosomotropic agents: impact on lysosomal membrane permeabilization and cell death.  


Lysosomes are acidic organelles essential for degradation, signalling and cell homoeostasis. In addition, they play a key role in cell death. Permeabilization of the lysosomal membrane and release of hydrolytic enzymes to the cytosol accompanies apoptosis signalling in several systems. The regulatory mechanism of lysosomal stability is, however, poorly understood. Lipophilic or amphiphilic compounds with a basic moiety will become protonated and trapped within lysosomes, and such lysosomotropic behaviour is also found in many pharmacological drugs. The natural sphingolipid sphingosine exhibits lysosomotropic detergent ability and is an endogenous candidate for controlling lysosomal membrane permeabilization. The lysosomotropic properties of certain detergents might be of use in lysosome-targeting anticancer drugs and drug delivery system in the future. The present review summarizes the current knowledge on the targeting and permeabilizing properties of lysosomotropic detergents from a cellular and physicochemical perspective. PMID:25233432

Villamil Giraldo, Ana M; Appelqvist, Hanna; Ederth, Thomas; Ollinger, Karin



The lysosomal membrane complex. Focal point of primary steroid hormone action  

PubMed Central

At short intervals after the intravenous administration of oestradiol-17?, diethylstilboestrol, testosterone or saline control solution to ovariectomized rats, highly purified lysosome samples were prepared in substantial yield from preputial glands, sex accessory organs rich in these organelles. The preparations were essentially devoid of mitochondrial contamination. Exposure in vivo to doses of these hormones varying from 0.1 to 5?g/100g body wt. provoked dose-dependent labilization of the lysosomal membrane surface, as evidenced by significantly diminished structural latency of several characteristic acid hydrolases, including acid phosphatase, ?-glucuronidase and acid ribonuclease II, when such preparations were subsequently challenged in vitro with autolytic conditions, detergent or mechanical stress. Enhanced lytic susceptibility induced by hormone pretreatment was occasionally detectable in the initial preparation without further provocative stimuli in vitro. Comparable results were obtained with the corresponding fractions of uterus, despite the more limited concentration of lysosomes in this steroidal target organ. By the present criteria oestradiol-17? was essentially inert, even in a dose 25 times that effective for its active ?-epimer (<0.1?g/100g body wt.). Pretreatment with diethylstilboestrol exerted substantial membrane-destabilizing influence in preputial-gland lysosome samples from orchidectomized rats. Moreover, administration of testosterone to gonadectomized animals resulted in essentially equivalent dose-dependent augmentation of lysosomal enzyme release in preputial-gland preparations of either sex. The membrane stability of lysosome-enriched preparations from uterus, on the other hand, was unaffected by testosterone pretreatment. The sensitivity, specificity and selectivity of the lysosomal response to sex steroids provide evidence for the physiological significance of this phenomenon as a general mechanism for mediation of secondary biochemical transformations in the hormone-stimulated target cell. PMID:5126905

Szego, Clara M.; Seeler, Barbara J.; Steadman, Rosemarie A.; Hill, Diane F.; Kimura, Arthur K.; Roberts, James A.



Identification of Sites of Mannose 6Phosphorylation on Lysosomal Proteins  

Microsoft Academic Search

Most newly synthesized soluble lysosomal proteins con- tain mannose 6-phosphate (Man-6-P), a specific carbohy- drate modification that is recognized by Man-6-P recep- tors (MPRs) that direct targeting to the lysosome. A number of proteomic studies have focused on lysosomal proteins, exploiting the fact that Man-6-P-containing forms can be purified by affinity chromatography on im- mobilized MPRs. These studies have identified

David E. Sleat; Haiyan Zheng; Meiqian Qian; Peter Lobel



Evaluating the targets of selection during character displacement.  


Ecological character displacement occurs when competition imposes divergent selection on interacting species, causing divergence in traits associated with resource use. Generally, divergence is assumed to occur when selection acts on the same, continuously varying trait in both species. However, selection might target multiple traits, and even closely related heterospecifics involved in character displacement might differ in selective targets. We investigated the targets of selection in a species of spadefoot toad, Spea multiplicata, during experimentally imposed competition with a congener, S. bombifrons. When examining traits separately, we found significant selection acting on multiple resource-acquisition traits. Yet, controlling for the independent effects of these traits in a multiple regression revealed that direct selection on a single trait might have contributed toward indirect selection on other correlated traits. Moreover, although we found evidence for plasticity in most traits, competition with S. bombifrons imposed selection on morphology and not on plasticity. Additional experiments suggest that the selective targets during character displacement might differ between the two species involved in this one instance of character displacement. Identifying the targets of competitively mediated selection is crucial, because whether and how character displacement ultimately unfolds depends on the nature of these targets and correlations among them. PMID:21967434

Martin, Ryan A; Pfennig, David W



Linked Target Selection for Saccadic and Smooth Pursuit Eye Movements  

Microsoft Academic Search

In natural situations, motor activity must often choose a single target when multiple distractors are present. The present paper asks how primate smooth pursuit eye movements choose tar- gets, by analysis of a natural target-selection task. Monkeys tracked two targets that started 1.5° eccentric and moved in different directions (up, right, down, and left) toward the posi- tion of fixation.

Justin L. Gardner; Stephen G. Lisberger



General lysosomal hydrolysis can process prorenin accurately.  


Renin, an aspartyl protease that catalyzes the rate-limiting step of the renin-angiotensin system, is first synthesized as an inactive precursor, prorenin. Prorenin is activated by the proteolytic removal of an amino terminal prosegment in the dense granules of the juxtaglomerular (JG) cells of the kidney by one or more proteases whose identity is uncertain but commonly referred to as the prorenin-processing enzyme (PPE). Because several extrarenal tissues secrete only prorenin, we tested the hypothesis that the unique ability of JG cells to produce active renin might be explained by the existence of a PPE whose expression is restricted to JG cells. We found that inducing renin production by the mouse kidney by up to 20-fold was not associated with the concomitant induction of candidate PPEs. Because the renin-containing granules of JG cells also contain several lysosomal hydrolases, we engineered mouse Ren1 prorenin to be targeted to the classical vesicular lysosomes of cultured HEK-293 cells, where it was accurately processed and stored. Furthermore, we found that HEK cell lysosomes hydrolyzed any artificial extensions placed on the protein and that active renin was extraordinarily resistant to proteolytic degradation. Altogether, our results demonstrate that accurate processing of prorenin is not restricted to JG cells but can occur in classical vesicular lysosomes of heterologous cells. The implication is that renin production may not require a specific PPE but rather can be achieved by general hydrolysis in the lysosome-like granules of JG cells. PMID:24965790

Xa, Lucie K; Lacombe, Marie-Josée; Mercure, Chantal; Lazure, Claude; Reudelhuber, Timothy L



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

PubMed Central

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

Lamore, Sarah D.; Wondrak, Georg T.



Automated target selection for DrivenShape  

Microsoft Academic Search

DrivenShape is a data-driven deformation that uses pre-computed data (a.k.a. targets) to approximate the effects of a computationally expensive cloth simulation [Kim and Vendrovsky 2008]. Rather than computing a true, accurate solution, DrivenShape produces a quick approximation that satisfies an acceptable margin of error, where error is defined as a difference in appearance or shape.

Gene S. Lee; Walt Disney; Animation Studios



Predictive saccade target selection in superior colliculus during visual search.  


Searching for a visual object naturally involves sequences of gaze fixations, during which the current foveal image is analyzed and the next object to inspect is selected as a saccade target. Fixation durations during such sequences are short, suggesting that saccades may be concurrently processed. Therefore, the selection of the next saccade target may occur before the current saccade target is acquired. To test this hypothesis, we trained four female rhesus monkeys (Macaca mulatta) to perform a multiple-fixation visual conjunction search task. We simultaneously recorded the activity of sensorimotor neurons in the midbrain superior colliculus (SC) in two monkeys. In this task, monkeys made multiple fixations before foveating the target. Fixation durations were significantly shorter than the latency of the initial responses to the search display, with approximately one-quarter being shorter than the shortest response latencies. The time at which SC sensorimotor activity discriminated the target from distracters occurred significantly earlier for the selection of subsequent fixations than for the selection of the first fixation. Target selection during subsequent fixations occurred even before the visual afferent delay in more than half of the neuronal sample, suggesting that the process of selection can encompass at least two future saccade targets. This predictive selection was present even when differences in saccade latencies were taken into account. Altogether, these findings demonstrate how neural representations on the visual salience map are processed in parallel, thus facilitating visual search. PMID:24741054

Shen, Kelly; Paré, Martin



Selecting asteroids for a targeted spectroscopic survey  

E-print Network

Asteroid spectroscopy reflects surface mineralogy. There are few thousand asteroids whose surfaces have been observed spectrally. Determining the surface properties of those objects is important for many practical and scientific applications, such as for example developing impact deflection strategies or studying history and evolution of the Solar System and planet formation. The aim of this study is to develop a pre-selection method that can be utilized in searching for asteroids of any taxonomic complex. The method could then be utilized im multiple applications such as searching for the missing V-types or looking for primitive asteroids. We used the Bayes Naive Classifier combined with observations obtained in the course of the Sloan Digital Sky Survey and the Wide-field Infrared Survey Explorer surveys as well as a database of asteroid phase curves for asteroids with known taxonomic type. Using the new classification method we have selected a number of possible V-type candidates. Some of the candidates we...

Oszkiewicz, D A; Tomov, T; Birlan, M; Geier, S; Penttilä, A; Poli?ska, M



Lysosomal Storage Disease: Revealing Lysosomal Function and Physiology  

NSDL National Science Digital Library

The discovery over five decades ago of the lysosome, as a degradative organelle and its dysfunction in lysosomal storage disorder patients, was both insightful and simple in concept. Here, we review some of the history and pathophysiology of lysosomal storage disorders to show how they have impacted on our knowledge of lysosomal biology. Although a significant amount of information has been accrued on the molecular genetics and biochemistry of lysosomal storage disorders, we still do not fully understand the mechanistic link between the storage material and disease pathogenesis. However, the accumulation of undegraded substrate(s) can disrupt other lysosomal degradation processes, vesicular traffic, and lysosomal biogenesis to evoke the diverse pathophysiology that is evident in this complex set of disorders.

Emma J. Parkinson-Lawrence (South Australian Pathology Services); Tetyana Shandala (South Australian Pathology Services); Mark Prodoehl (South Australian Pathology Services); Revecca Plew (South Australian Pathology Services); Glenn N. Borlace (South Australian Pathology Services); Doug A. Brooks (South Australian Pathology Services)



Sexual selection targets cetacean pelvic bones.  


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

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



Selectively targeting pain in the trigeminal system.  


We tested whether it is possible to selectively block pain signals in the orofacial area by delivering the permanently charged lidocaine derivative QX-314 into nociceptors via TPRV1 channels. We examined the effects of co-applied QX-314 and capsaicin on nociceptive, proprioceptive, and motor function in the rat trigeminal system. QX-314 alone failed to block voltage-gated sodium channel currents (I(Na)) and action potentials (APs) in trigeminal ganglion (TG) neurons. However, co-application of QX-314 and capsaicin blocked I(Na) and APs in TRPV1-positive TG and dental nociceptive neurons, but not in TRPV1-negative TG neurons or in small neurons from TRPV1 knock-out mice. Immunohistochemistry revealed that TRPV1 is not expressed by trigeminal motor and trigeminal mesencephalic neurons. Capsaicin had no effect on rat trigeminal motor and proprioceptive mesencephalic neurons and therefore should not allow QX-314 to enter these cells. Co-application of QX-314 and capsaicin inhibited the jaw-opening reflex evoked by noxious electrical stimulation of the tooth pulp when applied to a sensory but not a motor nerve, and produced long-lasting analgesia in the orofacial area. These data show that selective block of pain signals can be achieved by co-application of QX-314 with TRPV1 agonists. This approach has potential utility in the trigeminal system for treating dental and facial pain. PMID:20236764

Kim, Hyun Yeong; Kim, Kihwan; Li, Hai Ying; Chung, Gehoon; Park, Chul-Kyu; Kim, Joong Soo; Jung, Sung Jun; Lee, Min Kyung; Ahn, Dong Kuk; Hwang, Se Jin; Kang, Youngnam; Binshtok, Alexander M; Bean, Bruce P; Woolf, Clifford J; Oh, Seog Bae



Target selection and current status of structural genomics for the  

E-print Network

33 Target selection and current status of structural genomics for the completed microbial genomes 3.2 Structural status of completed microbial genomes in the PDB................ 3.3 Metabolic pathways as targets for structural genomics.......................... 3.3.1 Glycolytic pathway

Babu, M. Madan


Predicting selective drug targets in cancer through metabolic networks  

E-print Network

Predicting selective drug targets in cancer through metabolic networks Ori Folger1 , Livnat Jerby1 for cellular proliferation in cancer cell lines. The model predicts 52 cytostatic drug targets, of which 40 of Computer Science, Tel Aviv University, Tel Aviv, Israel, 2 Cancer Research UK, The Beatson Institute

Ruppin, Eytan


Quasar Target Selection Fiber E ciency Heidi Newberg Brian Yanny  

E-print Network

Quasar Target Selection Fiber E ciency Heidi Newberg Brian Yanny February 7, 1997 Abstract We-redshift quasars. With this plan, we expect 54% of the targets to be QSOs. The North Galactic Cap is divided will nd about 17,000 additional quasars in the southern strips, and maybe a few more at very high redshift

Varela, Carlos


Selecting asteroids for a targeted spectroscopic survey  

NASA Astrophysics Data System (ADS)

Context. Asteroid spectroscopy reflects surface mineralogy. There are a few thousand asteroids whose surfaces have been observed spectrally. Determining their surface properties is important for many practical and scientific applications, such as developing impact deflection strategies or studying the history and evolution of the solar system and planet formation. Aims: The aim of this study is to develop a preselection method that can be used to search for asteroids of any taxonomic complex. The method could then be utilized in multiple applications, such as searching for the missing V-types or looking for primitive asteroids. Methods: We used the Bayes Naive Classifier combined with observations obtained in the course of the Sloan Digital Sky Survey and the Wide-field Infrared Survey Explorer surveys, as well as a database of asteroid phase curves for asteroids with a known taxonomic type. With this new classification method, we selected a number of possible V-type candidates. Some of the candidates were then spectrally observed at the Nordic Optical Telescope and South African Large Telescope. Results: We developed and tested the new preselection method. We found three asteroids in the mid-to-outer main belt that probably have differentiated types. Near-infrared observations are still required to confirm this discovery. As in other studies we found that V-type candidates cluster around the Vesta family and are rare in the mid-to-outer main belt. Conclusions: The new method shows that even largely explored large databases when combined could still be exploited further in, for example, solving the missing dunite problem. Tables 6 and A.1 are only available at the CDS via anonymous ftp to ( or via

Oszkiewicz, D. A.; Kwiatkowski, T.; Tomov, T.; Birlan, M.; Geier, S.; Penttilä, A.; Poli?ska, M.



Lysosomal calcium homeostasis defects, not proton pump defects, cause endo-lysosomal dysfunction in PSEN-deficient cells  

PubMed Central

Presenilin (PSEN) deficiency is accompanied by accumulation of endosomes and autophagosomes, likely caused by impaired endo-lysosomal fusion. Recently, Lee et al. (2010. Cell. doi: attributed this phenomenon to PSEN1 enabling the transport of mature V0a1 subunits of the vacuolar ATPase (V-ATPase) to lysosomes. In their view, PSEN1 mediates the N-glycosylation of V0a1 in the endoplasmic reticulum (ER); consequently, PSEN deficiency prevents V0a1 glycosylation, compromising the delivery of unglycosylated V0a1 to lysosomes, ultimately impairing V-ATPase function and lysosomal acidification. We show here that N-glycosylation is not a prerequisite for proper targeting and function of this V-ATPase subunit both in vitro and in vivo in Drosophila melanogaster. We conclude that endo-lysosomal dysfunction in PSEN?/? cells is not a consequence of failed N-glycosylation of V0a1, or compromised lysosomal acidification. Instead, lysosomal calcium storage/release is significantly altered in PSEN?/? cells and neurons, thus providing an alternative hypothesis that accounts for the impaired lysosomal fusion capacity and accumulation of endomembranes that accompanies PSEN deficiency. PMID:22753898

Coen, Katrijn; Flannagan, Ronald S.; Baron, Szilvia; Carraro-Lacroix, Luciene R.; Wang, Dong; Vermeire, Wendy; Michiels, Christine; Munck, Sebastian; Baert, Veerle; Sugita, Shuzo; Wuytack, Frank; Hiesinger, Peter Robin; Grinstein, Sergio




SciTech Connect

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.

Kirkpatrick, Jessica A. [Department of Physics, University of California, Berkeley, CA 94720 (United States); Schlegel, David J.; Ross, Nicholas P. [Lawrence Berkeley National Laboratory, 1 Cyclotron Rd, Berkeley, CA 92420 (United States); Myers, Adam D. [Department of Astronomy, University of Illinois, 1002 West Green Street, Urbana, IL 61801 (United States); Hennawi, Joseph F. [Max-Planck-Institut fuer Astronomie, Koenigstuhl 17, 69117 Heidelberg (Germany); Sheldon, Erin S. [Brookhaven National Laboratory, Physics Department, Mail Stop 510A, Upton, NY 11973-5000 (United States); Schneider, Donald P. [Department of Astronomy and Astrophysics, Pennsylvania State University, University Park, PA 16802 (United States); Weaver, Benjamin A., E-mail: [Center for Cosmology and Particle Physics, New York University, NY 10003 (United States)



Lysosomal Storage Disease: Revealing Lysosomal Function and Physiology - Figure 3  

NSDL National Science Digital Library

This figure shows the morphology of storage compartments commonly observed lysosomal storage disorders: (A) floccular-granular storage, (B) lipid whorls, (C) zebra bodies, and (D) autophagic vacuoles.

Emma J. Parkinson-Lawrence (South Australian Pathology Services); Tetyana Shandala (South Australian Pathology Services); Mark Prodoehl (South Australian Pathology Services); Revecca Plew (South Australian Pathology Services); Glenn N. Borlace (South Australian Pathology Services); Doug A. Brooks (South Australian Pathology Services)



Enhancing lysosome biogenesis attenuates BNIP3-induced cardiomyocyte death  

PubMed Central

Hypoxia-inducible pro-death protein BNIP3 (BCL-2/adenovirus E1B 19-kDa interacting protein 3), provokes mitochondrial permeabilization causing cardiomyocyte death in ischemia-reperfusion injury. Inhibition of autophagy accelerates BNIP3-induced cell death, by preventing removal of damaged mitochondria. We tested the hypothesis that stimulating autophagy will attenuate BNIP3-induced cardiomyocyte death. Neonatal rat cardiac myocytes (NRCMs) were adenovirally transduced with BNIP3 (or LacZ as control; at multiplicity of infection = 100); and autophagy was stimulated with rapamycin (100 nM). Cell death was assessed at 48 h. BNIP3 expression increased autophagosome abundance 8-fold and caused a 3.6-fold increase in cardiomyocyte death as compared with control. Rapamycin treatment of BNIP3-expressing cells led to further increase in autophagosome number without affecting cell death. BNIP3 expression led to accumulation of autophagosome-bound LC3-II and p62, and an increase in autophagosomes, but not autolysosomes (assessed with dual fluorescent mCherry-GFP-LC3 expression). BNIP3, but not the transmembrane deletion variant, interacted with LC3 and colocalized with mitochondria and lysosomes. However, BNIP3 did not target to lysosomes by subcellular fractionation, provoke lysosome permeabilization or alter lysosome pH. Rather, BNIP3-induced autophagy caused a decline in lysosome numbers with decreased expression of the lysosomal protein LAMP-1, indicating lysosome consumption and consequent autophagosome accumulation. Forced expression of transcription factor EB (TFEB) in BNIP3-expressing cells increased lysosome numbers, decreased autophagosomes and increased autolysosomes, prevented p62 accumulation, removed depolarized mitochondria and attenuated BNIP3-induced death. We conclude that BNIP3 expression induced autophagosome accumulation with lysosome consumption in cardiomyocytes. Forced expression of TFEB, a lysosomal biogenesis factor, restored autophagosome processing and attenuated BNIP3-induced cell death. PMID:22302006

Ma, Xiucui; Godar, Rebecca J.; Liu, Haiyan; Diwan, Abhinav



Endocannabinoids Prevent ?-Amyloid-mediated Lysosomal Destabilization in Cultured Neurons*  

PubMed Central

Neuronal cell loss underlies the pathological decline in cognition and memory associated with Alzheimer disease (AD). Recently, targeting the endocannabinoid system in AD has emerged as a promising new approach to treatment. Studies have identified neuroprotective roles for endocannabinoids against key pathological events in the AD brain, including cell death by apoptosis. Elucidation of the apoptotic pathway evoked by ?-amyloid (A?) is thus important for the development of therapeutic strategies that can thwart A? toxicity and preserve cell viability. We have previously reported that lysosomal membrane permeabilization plays a distinct role in the apoptotic pathway initiated by A?. In the present study, we provide evidence that the endocannabinoid system can stabilize lysosomes against A?-induced permeabilization and in turn sustain cell survival. We report that endocannabinoids stabilize lysosomes by preventing the A?-induced up-regulation of the tumor suppressor protein, p53, and its interaction with the lysosomal membrane. We also provide evidence that intracellular cannabinoid type 1 receptors play a role in stabilizing lysosomes against A? toxicity and thus highlight the functionality of these receptors. Given the deleterious effect of lysosomal membrane permeabilization on cell viability, stabilization of lysosomes with endocannabinoids may represent a novel mechanism by which these lipid modulators confer neuroprotection. PMID:20923768

Noonan, Janis; Tanveer, Riffat; Klompas, Allan; Gowran, Aoife; McKiernan, Joanne; Campbell, Veronica A.



A targeted multi-enzyme mechanism for selective microtubule  

E-print Network

1 A targeted multi-enzyme mechanism for selective microtubule polyglutamylation Juliette van Dijk: posttranslational modification, tubulin, motility, polyglutamylase, TTLL Running Title: The multi-enzyme mechanism of polyglutamylation Summary Polyglutamylases are enzymes that form polyglutamate side chains of variable lengths

Paris-Sud XI, Université de


Selective Tumor Cell Targeting Using Low-Affinity, Multivalent Interactions  

E-print Network

Selective Tumor Cell Targeting Using Low-Affinity, Multivalent Interactions Coby B. Carlson interaction between a cell-binding agent (e.g., monoclonal antibody or fragment thereof) and a tumor- associated antigen to direct a cytotoxic moiety selec- tively to the tumor (2). Despite the potential

Kiessling, Laura


Target Selection for Pursuit and Saccadic Eye Movements in Humans  

E-print Network

Target Selection for Pursuit and Saccadic Eye Movements in Humans R. J. Krauzlis Salk Institute for Biological Studies A. Z. Zivotofsky and F. A. Miles National Eye Institute Abstract Eye movements were that guide these two types of eye move- ments. The differences in the details of the effects on pursuit

Krauzlis, Richard J.


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

PubMed Central

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

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



Autophagy in lysosomal storage disorders  

PubMed Central

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

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



Immature and mature species of the human Prostacyclin Receptor are ubiquitinated and targeted to the 26S proteasomal or lysosomal degradation pathways, respectively  

Microsoft Academic Search

BACKGROUND: The human prostacyclin receptor (hIP) undergoes agonist-induced phosphorylation, desensitisation and internalisation and may be recycled to the plasma membrane or targeted for degradation by, as yet, unknown mechanism(s). RESULTS: Herein it was sought to investigate the turnover of the hIP under basal conditions and in response to cicaprost stimulation. It was established that the hIP is subject to low-level

Peter D Donnellan; B Therese Kinsella



Multi-class target recognition based on adaptive feature selection  

NASA Astrophysics Data System (ADS)

In this paper, a new approach of multi-class target recognition is proposed for remote sensing image analysis. A multiclass feature model is built, which is based on sharing features among classes. In order to make the recognition process efficient, we adopted the idea of adaptive feature selection. In each layer of the integrated feature model, the most salient and stable feature are selected first, and then the less ones. Experiments demonstrated the approach proposed is efficient in computation and is adaptive to scene variation.

Wang, Yuehuan; Yao, Wei; Song, Yunfeng; Sang, Nong; Zhang, Tianxu



Lysosomal Storage Disease: Revealing Lysosomal Function and Physiology - Figure 2  

NSDL National Science Digital Library

This figure is a timeline of discoveries in lysosomal storage disorders and their impact on cell biology, including endocytic processes and the subsequent development of enzyme replacement therapy (ERT).

Emma J. Parkinson-Lawrence (South Australian Pathology Services); Tetyana Shandala (South Australian Pathology Services); Mark Prodoehl (South Australian Pathology Services); Revecca Plew (South Australian Pathology Services); Glenn N. Borlace (South Australian Pathology Services); Doug A. Brooks (South Australian Pathology Services)



Target Selection and Imaging Requirements for JWST Fine Phasing  

NASA Technical Reports Server (NTRS)

To achieve and maintain the fine alignment of its segmented primary mirror the James Webb Space Telescope (JWST) plans to use focus-diverse wavefront sensing (WFS) techniques with science camera imagery. The optical requirements for JWST are such that the error contribution from the WFS itself must be limited 10nm rms over all the controllable degrees of freedom of the telescope. In this paper, we will explore the requirements on the target selection and imaging requirements necessary to achieve the desired level of WFS accuracy. Using Monte Carlo simulations we explore the WFS error as a function of wavefront aberrations level, defocus-diversity level, optical bandwidth and imaging signal-to-noise ratio to establish the key imaging requirements. By taking into account practical integration time limits along with the distribution of the defocused point-spread functions, we establish the bright and faint star magnitude limits suitable for WFS target selection.

Green, Joseph J.; Dean, Bruce H.; Ohara, Catherine M.; Zhang, Yan



In vivo selection of tumor-targeting RNA motifs  

PubMed Central

In an effort to target the in vivo context of tumor-specific moieties, a large library of nuclease-resistant RNA oligonucleotides was screened in tumor-bearing mice to identify candidate molecules with the ability to localize to hepatic colon cancer metastases. One of the selected molecules is an RNA aptamer that binds to protein p68, an RNA helicase that has been shown to be upregulated in colorectal cancer. PMID:19946274

Mi, Jing; Liu, Yingmiao; Rabbani, Zahid N.; Yang, Zhongguang; Urban, Johannes H; Sullenger, Bruce A.; Clary, Bryan M.



Glucosamine-Bound Near-Infrared Fluorescent Probes with Lysosomal Specificity for Breast Tumor Imaging1  

PubMed Central

Noninvasive imaging of lysosomes will be useful 1) to elucidate the role of lysosomal parameters in cancer, 2) to diagnose malignant lesions, and 3) to evaluate future lysosome-targeted anticancer therapies. Lysosome-specific labeling of glucosamine-bound near-infrared (NIR) fluorescent probes, IR-1 and IR-2, but not control probe IR-15 without the glucosamine moiety, was observed by fluorescence microscopy in human breast epithelial cell lines. Lysosome labeling and tumor specificity of these NIR probes were investigated by dynamic optical imaging and immunofluorescence staining in human breast tumor xenografts. IR-1 and IR-2 demonstrated faster lysosome labeling rates in highly aggressive MDA-MB-231 and MDA-MB-435 cells compared with less aggressive MCF-7 and nontumorigenic MCF-12A cells. IR-1 and IR-2, but not IR-15, accumulated in human MDA-MB-231, MDA-MB-435, and MCF-7 breast tumor xenografts in vivo. IR-2 demonstrated the highest maximum fluorescence and tumor/normal tissue ratios in all tumor models. Specific lysosome labeling from IR-2 in vivo was validated by colocalization of the NIR fluorescence with CD63 immunofluorescence in tumor sections. IR-1 and IR-2 demonstrated high lysosome-labeling ability and breast tumor-targeting specificity in vitro and in vivo. They are promising for diagnosing malignant lesions and may provide a means for evaluating and monitoring future lysosome-targeted anticancer therapies. PMID:18392136

Li, Cong; Greenwood, Tiffany R; Glunde, Kristine



Target Inhibition Networks: Predicting Selective Combinations of Druggable Targets to Block Cancer Survival Pathways  

PubMed Central

A recent trend in drug development is to identify drug combinations or multi-target agents that effectively modify multiple nodes of disease-associated networks. Such polypharmacological effects may reduce the risk of emerging drug resistance by means of attacking the disease networks through synergistic and synthetic lethal interactions. However, due to the exponentially increasing number of potential drug and target combinations, systematic approaches are needed for prioritizing the most potent multi-target alternatives on a global network level. We took a functional systems pharmacology approach toward the identification of selective target combinations for specific cancer cells by combining large-scale screening data on drug treatment efficacies and drug-target binding affinities. Our model-based prediction approach, named TIMMA, takes advantage of the polypharmacological effects of drugs and infers combinatorial drug efficacies through system-level target inhibition networks. Case studies in MCF-7 and MDA-MB-231 breast cancer and BxPC-3 pancreatic cancer cells demonstrated how the target inhibition modeling allows systematic exploration of functional interactions between drugs and their targets to maximally inhibit multiple survival pathways in a given cancer type. The TIMMA prediction results were experimentally validated by means of systematic siRNA-mediated silencing of the selected targets and their pairwise combinations, showing increased ability to identify not only such druggable kinase targets that are essential for cancer survival either individually or in combination, but also synergistic interactions indicative of non-additive drug efficacies. These system-level analyses were enabled by a novel model construction method utilizing maximization and minimization rules, as well as a model selection algorithm based on sequential forward floating search. Compared with an existing computational solution, TIMMA showed both enhanced prediction accuracies in cross validation as well as significant reduction in computation times. Such cost-effective computational-experimental design strategies have the potential to greatly speed-up the drug testing efforts by prioritizing those interventions and interactions warranting further study in individual cancer cases. PMID:24068907

Tang, Jing; Karhinen, Leena; Xu, Tao; Szwajda, Agnieszka; Yadav, Bhagwan; Wennerberg, Krister; Aittokallio, Tero



Neuropathic Lysosomal Storage Disorders  

PubMed Central

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

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



Context-dependent sequential effects of target selection for action  

PubMed Central

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

Moher, Jeff; Song, Joo-Hyun



Dynamic interactions between visual working memory and saccade target selection.  


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

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




SciTech Connect

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.

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



Clearance of lysosomal glycogen accumulation by Transcription factor EB (TFEB) in muscle cells from lysosomal alpha-glucosidase deficient mice  

PubMed Central

A recently proposed therapeutic approach for lysosomal storage disorders (LSDs) relies upon the ability of transcription factor EB (TFEB) to stimulate autophagy and induce lysosomal exocytosis leading to cellular clearance. This approach is particularly attractive in Glycogen Storage Disease type II (a severe metabolic myopathy and a paradigm for LSDs, also called Pompe disease) as the currently available therapy, replacement of the missing enzyme acid alpha-glucosidase, fails to reverse skeletal muscle pathology. Pompe disease is characterized by both lysosomal abnormality and dysfunctional autophagy. Here we show that TFEB is a viable therapeutic target in Pompe disease: overexpression of TFEB in a newly established conditionally immortalized skeletal muscle cell model reduced glycogen load and lysosomal size; and in the muscle fibers of GFP-LC3 Pompe disease mouse model significantly increased the motility of lysosomes in the fibers, and stimulated the fusion between lysosomes and autophagosomes under stress. Hence, modulation of TFEB activity holds promise for the development of a better therapy. In addition, the newly developed mouse and cell models have many potential applications such as large-scale drug screening for Pompe disease. PMID:23416076

Li, Hoi Ming; Feeney, Erin; Li, Lishu; Zare, Hossein; Puertollano, Rosa; Raben, Nina



Anti-inflammatory drugs selectively target sporangium development in Mucor.  


It is known that acetylsalicylic acid, an anti-inflammatory and anti-mitochondrial drug, targets structure development and functions of yeasts depending on elevated levels of mitochondrial activity. Using antibody probes, we previously reported that sporangia of Mucor circinelloides also contain increased mitochondrial activity, yielding high levels of 3-hydroxyoxylipins. This was, however, not found in Mortierella alpina (subgenus Mortierella). In this study we report that acetylsalicylic acid (aspirin) also targets sporangium development of Mucor circinelloides selectively, while hyphae with lower levels of mitochondrial activity are more resistant. Similar results were obtained when the anti-inflammatory compounds benzoic acid, ibuprofen, indomethacin, and salicylic acid were tested. The anti-inflammatory drugs exerted similar effects on this dimorphic fungus as found under oxygen-limited conditions. Interestingly, sporangium development of Mortierella alpina was found not to be selectively targeted by these drugs. Mortierella alpina, which could not exhibit dimorphic growth under oxygen-limited conditions, was also more sensitive to the anti-inflammatory drugs when compared with Mucor circinelloides. These results prompt further research to assess the applicability of these antimitochondrial antifungals to protect plants and animals against Mucor infections. PMID:20029531

Leeuw, Ntsoaki J; Swart, Chantel W; Ncango, Desmond M; Kriel, Wilmarie M; Pohl, Carolina H; van Wyk, Pieter W J; Kock, Johan L F



Lysosomal photodamage induces cell death via mitochondrial apoptotic pathway  

NASA Astrophysics Data System (ADS)

Lysosomal photosensitizers have been used in photodynamic therapy (PDT). Combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. The lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, we selected N-aspartyl chlorin e6 (NPe6), an effective photosensitizer which preferentially accumulates in lysosomes, to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells treated by NPe6-PDT was studied using real-time single-cell analysis. In this study, the fluorescence probes Cyto c-GFP and DsRed-Mit were used to detect the spatial and temporal changes of cytochrome c in real-time in sub-cell level; the Rhodamine 123 dyes were used to monitor the changes of mitochondrial membrane potential. The results showed that, after PDT treatment,the mitochondrial membrane potential decreased, and cytochrome c released from mitochondria; The caspase-3 was activated obviously. These results suggested that lysosomal photodamage activates mitochondrial apoptotic pathway to induce cell death.

Liu, Lei; Wang, Xian-wang; Li, Hui




SciTech Connect

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.

Batalha, Natalie M. [Department of Physics and Astronomy, San Jose State University, San Jose, CA 95192 (United States); Borucki, William J.; Koch, David G.; Bryson, Stephen T.; Haas, Michael R. [NASA Ames Research Center, Moffett Field, CA (United States); Brown, Timothy M. [Las Cumbres Observatory Global Observatory Telescope Network, Goleta, CA 93117 (United States); Caldwell, Douglas A. [SETI Institute, Mountain View, CA 94043 (United States); Hall, Jennifer R. [Orbital Sciences Corporation/NASA Ames Research Center, Moffett Field, CA 94035 (United States); Gilliland, Ronald L. [STScI, Baltimore, MD 21218 (United States); Latham, David W.; Meibom, Soren [Harvard-Smithsonian, CfA, Cambridge, MA 02138 (United States); Monet, David G. [U.S. Naval Observatory, Flagstaff, AZ 86001 (United States)], E-mail:



Wavelength band selection method for multispectral target detection  

NASA Astrophysics Data System (ADS)

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.

Karlholm, Jorgen; Renhorn, Ingmar



Wavelength band selection method for multispectral target detection.  


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

Karlholm, Jörgen; Renhorn, Ingmar



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

PubMed Central

Background Alzheimer’s disease (AD) is characterized by cerebral deposition of ?-amyloid peptide (A?). A? is produced by sequential cleavage of the Amyloid Precursor Protein (APP) by ?- and ?-secretases. Many studies have demonstrated that the internalization of APP from the cell surface can regulate A? production, although the exact organelle in which A? is produced remains contentious. A number of recent studies suggest that intracellular trafficking also plays a role in regulating A? 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 ?-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 ?-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 the lysosome) using siRNA, we are able to reduce this lysosomal transport. Blocking lysosomal transport of APP reduces A? production by more than a third. Conclusion These data suggests that AP-3 mediates rapid delivery of APP to lysosomes, and that the lysosome is a likely site of A? production. PMID:25085554



Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and iron-replete cells.  


Ferritin is a cytosolic protein that stores excess iron, thereby protecting cells from iron toxicity. Ferritin-stored iron is believed to be utilized when cells become iron deficient; however, the mechanisms underlying the extraction of iron from ferritin have yet to be fully elucidated. Here, we demonstrate that ferritin is degraded in the lysosome under iron-depleted conditions and that the acidic environment of the lysosome is crucial for iron extraction from ferritin and utilization by cells. Ferritin was targeted for degradation in the lysosome even under iron-replete conditions in primary cells; however, the mechanisms underlying lysosomal targeting of ferritin were distinct under depleted and replete conditions. In iron-depleted cells, ferritin was targeted to the lysosome via a mechanism that involved autophagy. In contrast, lysosomal targeting of ferritin in iron-replete cells did not involve autophagy. The autophagy-independent pathway of ferritin delivery to lysosomes was deficient in several cancer-derived cells, and cancer-derived cell lines are more resistant to iron toxicity than primary cells. Collectively, these results suggest that ferritin trafficking may be differentially regulated by cell type and that loss of ferritin delivery to the lysosome under iron-replete conditions may be related to oncogenic cellular transformation. PMID:21444722

Asano, Takeshi; Komatsu, Masaaki; Yamaguchi-Iwai, Yuko; Ishikawa, Fuyuki; Mizushima, Noboru; Iwai, Kazuhiro



The SAMI Galaxy Survey: instrument specification and target selection  

E-print Network

The SAMI Galaxy Survey will observe 3400 galaxies with the Sydney-AAO Multi-object Integral-field spectrograph (SAMI) on the Anglo-Australian Telescope (AAT) in a 3-year 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 hours, and cover a total of 144 square degrees (in GAMA-I). Higher density environments are also included with the addition of eight clusters. The clusters have spectroscopy from 2dFGRS and SDSS and photometry in regions covered by the Slo...

Bryant, J J; 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; Lewis, G; Liske, J; Lopez-Sanchez, A 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



The nutrient-responsive transcription factor TFE3 promotes autophagy, lysosomal biogenesis, and clearance of cellular debris.  


The discovery of a gene network regulating lysosomal biogenesis and its transcriptional regulator transcription factor EB (TFEB) revealed that cells monitor lysosomal function and respond to degradation requirements and environmental cues. 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 guanosine triphosphatases (GTPases) and exhibited mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1)-dependent phosphorylation. Phosphorylated TFE3 was retained in the cytosol through its interaction with the cytosolic chaperone 14-3-3. After 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

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



Selectively Targeting Prostate Cancer with Antiandrogen Equipped Histone Deacetylase Inhibitors  

PubMed Central

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

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



Mucolipin controls lysosome exocytosis in Dictyostelium.  


Mucolipidosis type IV is a poorly understood lysosomal storage disease caused by alterations in the mucolipin lysosomal Ca(2+) channel. In this study, we generated mucolipin-knockout Dictyostelium cells, and observed that lysosome exocytosis was markedly increased in these cells compared with wild-type cells. In addition, mucolipin-knockout cells were more resistant to Ca(2+) deprivation, and the Ca(2+) concentration in their secretory lysosomes was decreased, suggesting that mucolipin transfers Ca(2+) ions from the cytosol to the lumen of secretory lysosomes. We speculate that mucolipin attenuates the fusogenic effect of local cytosolic increases in Ca(2+) by dissipating them into the lumen of lysosomal compartments. PMID:22357942

Lima, Wanessa C; Leuba, Florence; Soldati, Thierry; Cosson, Pierre



Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics  

PubMed Central

Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters—horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)—were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates—saporin-HRP, Alexasaporin and saporin-KQ-RTA—were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of Alexasaporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10–1000 nM. PMID:24859158

Gilabert-Oriol, Roger; Thakur, Mayank; von Mallinckrodt, Benedicta; Bhargava, Cheenu; Wiesner, Burkhard; Eichhorst, Jenny; Melzig, Matthias F.; Fuchs, Hendrik; Weng, Alexander



Lysosomal degradation of membrane lipids.  


The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes. PMID:19836391

Kolter, Thomas; Sandhoff, Konrad



Neuronal lysosomal enzyme replacement using fragment C of tetanus toxin.  

PubMed Central

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 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. Images PMID:1532255

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



Subtype-selective targeting of voltage-gated sodium channels  

PubMed Central

Voltage-gated sodium channels are key to the initiation and propagation of action potentials in electrically excitable cells. Molecular characterization has shown there to be nine functional members of the family, with a high degree of sequence homology between the channels. This homology translates into similar biophysical and pharmacological properties. Confidence in some of the channels as drug targets has been boosted by the discovery of human mutations in the genes encoding a number of them, which give rise to clinical conditions commensurate with the changes predicted from the altered channel biophysics. As a result, they have received much attention for their therapeutic potential. Sodium channels represent well-precedented drug targets as antidysrhythmics, anticonvulsants and local anaesthetics provide good clinical efficacy, driven through pharmacology at these channels. However, electrophysiological characterization of clinically useful compounds in recombinant expression systems shows them to be weak, with poor selectivity between channel types. This has led to the search for subtype-selective modulators, which offer the promise of treatments with improved clinical efficacy and better toleration. Despite developments in high-throughput electrophysiology platforms, this has proven very challenging. Structural biology is beginning to offer us a greater understanding of the three-dimensional structure of voltage-gated ion channels, bringing with it the opportunity to do real structure-based drug design in the future. This discipline is still in its infancy, but developments with the expression and purification of prokaryotic sodium channels offer the promise of structure-based drug design in the not too distant future. PMID:19845672

England, Steve; de Groot, Marcel J



Motor neuron target selectivity and survival after prolonged axotomy  

PubMed Central

Purpose After a cut peripheral nerve is repaired, motor neurons usually regenerate across the lesion site, however they often enter an inappropriate Schwann cell tube and may be directed to an inappropriate target organ such as skin, resulting in continued loss of function. In fact, only about 10% of adults who receive a peripheral nerve repair display full functional recovery. The reasons for this are many and complex, however one aspect is whether the motor neuron has undergone a prolonged period of axotomy prior to nerve repair. Previous studies have suggested a deleterious effect of prolonged axotomy. Methods We examined the influence of prolonged axotomy on target selectivity using a cross-reinnervation model of rat obturator motor neurons regrowing into the distal femoral nerve, with its normal bifurcating pathways to muscle and skin. Results Surprisingly, we found that a prolonged period of axotomy resulted in an increase in motor neuron regeneration accuracy. In addition, we found that regeneration accuracy could be increased even further by a simple surgical manipulation of the distal terminal nerve pathway to skin. Conclusions These results suggest that under certain conditions prolonged axotomy may not be detrimental to the final accuracy of motor neuron regeneration and highlight that a simple manipulation of terminal nerve pathways may be one approach to increase such regeneration accuracy. PMID:23648674

Robinson, Grant A.; Madison, Roger D.



Signaling from lysosomes to mitochondria sensitizes cancer cells to photodynamic treatment  

NASA Astrophysics Data System (ADS)

Previously, we showed that photosensitizers that localize to lysosomes are more effective in killing cancer cells than ones directed to mitochondria after photodynamic treatment (PDT). The photosensitizer, phthalocyanine 4 (Pc 4), localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. However, analogues of Pc 4 (e.g., Pc 181) that primarily target lysosomes still produce mitochondria-mediated cell death, although the time course is slower compared to Pc 4-PDT. In A431 epidermoid carcinoma cells, these new analogues preferentially localized in lysosomes were highly efficient in inducing apoptotic cell death. To assess further how lysosomes contribute to PDT, we monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the acidic vacuolar proton pump that collapses the pH gradient of the lysosomal/endosomal compartment. Bafilomycin by itself was not toxic but greatly enhanced Pc 4-PDT-induced mitochondrial depolarization and cell killing. Both depolarization and cell killing were substantially prevented by iron chelators. The fact that Pc 4-PDT plus bafilomycin treatment did not induce lysosomal membrane damage prior to mitochondrial depolarization suggests that bafilomycin instead induced release of redox active iron from lysosomes into the cytosol that further translocated into mitochondria, where iron-mediated free radical formation occurred. In conclusion, agents that disturb lysosomal function could potentially be used as adjuvants with mitochondrion-targeted photosensitizers to enhance phototoxicity.

Hung, Hsin-I.; Quiogue, Geraldine; Lemasters, John J.; Nieminen, Anna-Liisa



Protective Effects of Positive Lysosomal Modulation in Alzheimer's Disease Transgenic Mouse Models  

PubMed Central

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid ? peptide (A?) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including A?1–42. Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of A? pathology. Systemic PADK injections in APPSwInd and APPswe/PS1?E9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both A? immunostaining as well as A?x-42 sandwich ELISA measures in APPSwInd mice of 10–11 months. More extensive A? deposition in 20-22-month APPswe/PS1?E9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic A?1–38 occurs as A?1–42 levels decrease in the mouse models, indicating that PADK treatment leads to A? truncation. Associated with A? clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces A?1–42 accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of A? pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders. PMID:21695208

Butler, David; Hwang, Jeannie; Estick, Candice; Nishiyama, Akiko; Kumar, Saranya Santhosh; Baveghems, Clive; Young-Oxendine, Hollie B.; Wisniewski, Meagan L.; Charalambides, Ana; Bahr, Ben A.



Introduction Lysosomes are one of the major degradative compartments of  

E-print Network

known mannose 6-phosphate receptors (MPRs) in the trans-Golgi network (TGN) for transport onward to lysosomes. This crucial recognition event directly depends on the acquisition of mannose 6-phosphate (M6P in the Golgi. The first step, the addition of an N-acetyl glucosamine phosphate to selected mannose residues

Gordon, Geoffrey J.


Exocytosis of polymorphonuclear leukocyte lysosomal contents induced by dental plaque.  

PubMed Central

Rabbit polymorphonuclear leukocytes were incubated with a sonically treated suspension of pooled dental plaque to determine if the plaque would induce release of lysosomal enzymes from the polymorphonuclear leukocytes. Cells incubated with plaque at 37 degrees C released significantly greater amounts of the lysosomal enzymes, beta-glucuronidase and lysozyme, than did cells incubated with plaque at 0 degrees C or without plaque at 37 degrees C. This response was both dose and time dependent. Release of the cytoplasmic enzyme lactate dehydrogenase was minimal, and there were no significant differences in lactate dehydrogenase release between cells at 0 and 37 degrees C, or without plaque. These results indicate that dental plaque can induce the selective release of lysosomal enzymes, which could be involved in the periodontal injury produced by dental plaque. PMID:561032

White, R R; Montgomery, E H



DNA Aptamers Selectively Target Leishmania infantum H2A Protein  

PubMed Central

Parasites of the genus Leishmania produce leishmaniasis which affects millions people around the world. Understanding the molecular characteristics of the parasite can increase the knowledge about the mechanisms underlying disease development and progression. Thus, the study of the molecular features of histones has been considered of particular interest because Leishmania does not condense the chromatin during mitosis and, consequently, a different role for these proteins in the biology of the parasite can be expected. Furthermore, the sequence divergences in the amino and in the carboxy-terminal domains of the kinetoplastid core histones convert them in potential diagnostic and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania histones is essential for the progress of this kind of study. Two aptamers which specifically recognize Leishmania infantum H2A histone were cloned from a previously obtained ssDNA enriched population. These aptamers were sequenced and subjected to an in silico analysis. ELONA, slot blot and Western blot were performed to establish aptamer affinity and specificity for LiH2A histone and ELONA assays using peptides corresponding to overlapped sequences of LiH2A were made mapping the aptamers:LiH2A interaction. As “proofs of concept”, aptamers were used to determine the number of parasites in an ELONA platform and to purify LiH2A from complex mixtures. The aptamers showed different secondary structures among them; however, both of them were able to recognize the same peptides located in a side of the protein. In addition, we demonstrate that these aptamers are useful for LiH2A identification and also may be of potential application as diagnostic system and as a laboratory tool with purification purpose. PMID:24205340

Martin, M. Elena; Garcia-Hernandez, Marta; Garcia-Recio, Eva M.; Gomez-Chacon, Geronimo F.; Sanchez-Lopez, Marta; Gonzalez, Victor M.



Reprogramming of lysosomal gene expression by interleukin-4 and Stat6  

PubMed Central

Background Lysosomes play important roles in multiple aspects of physiology, but the problem of how the transcription of lysosomal genes is coordinated remains incompletely understood. The goal of this study was to illuminate the physiological contexts in which lysosomal genes are coordinately regulated and to identify transcription factors involved in this control. Results As transcription factors and their target genes are often co-regulated, we performed meta-analyses of array-based expression data to identify regulators whose mRNA profiles are highly correlated with those of a core set of lysosomal genes. Among the ~50 transcription factors that rank highest by this measure, 65% are involved in differentiation or development, and 22% have been implicated in interferon signaling. The most strongly correlated candidate was Stat6, a factor commonly activated by interleukin-4 (IL-4) or IL-13. Publicly available chromatin immunoprecipitation (ChIP) data from alternatively activated mouse macrophages show that lysosomal genes are overrepresented among Stat6-bound targets. Quantification of RNA from wild-type and Stat6-deficient cells indicates that Stat6 promotes the expression of over 100 lysosomal genes, including hydrolases, subunits of the vacuolar H+ ATPase and trafficking factors. While IL-4 inhibits and activates different sets of lysosomal genes, Stat6 mediates only the activating effects of IL-4, by promoting increased expression and by neutralizing undefined inhibitory signals induced by IL-4. Conclusions The current data establish Stat6 as a broadly acting regulator of lysosomal gene expression in mouse macrophages. Other regulators whose expression correlates with lysosomal genes suggest that lysosome function is frequently re-programmed during differentiation, development and interferon signaling. PMID:24314139



Degradation of Alzheimer's amyloid fibrils by microglia requires delivery of ClC-7 to lysosomes  

PubMed Central

Incomplete lysosomal acidification in microglia inhibits the degradation of fibrillar forms of Alzheimer's amyloid ? peptide (fA?). Here we show that in primary microglia a chloride transporter, ClC-7, is not delivered efficiently to lysosomes, causing incomplete lysosomal acidification. ClC-7 protein is synthesized by microglia but it is mistargeted and appears to be degraded by an endoplasmic reticulum–associated degradation pathway. Activation of microglia with macrophage colony-stimulating factor induces trafficking of ClC-7 to lysosomes, leading to lysosomal acidification and increased fA? degradation. ClC-7 associates with another protein, Ostm1, which plays an important role in its correct lysosomal targeting. Expression of both ClC-7 and Ostm1 is increased in activated microglia, which can account for the increased delivery of ClC-7 to lysosomes. Our findings suggest a novel mechanism of lysosomal pH regulation in activated microglia that is required for fA? degradation. PMID:21441306

Majumdar, Amitabha; Capetillo-Zarate, Estibaliz; Cruz, Dana; Gouras, Gunnar K.; Maxfield, Frederick R.



Glycoproteins of the lysosomal membrane  

Microsoft Academic Search

Three glycoprotein antigens (120, 100, and 80 kD) were detected by mono- and\\/ or polyclonal antibodies generated by immunization with highly purified rat liver lysosomal membranes. All of the antigens were judged to be integral membrane proteins based on the binding of Triton X-114. By immunofluorescence on normal rat kidney cells, a mouse mono- clonal antibody to the 120-kD antigen




Lysosomal localization and mechanism of uptake of Nile blue photosensitizers in tumor cells.  


Nile blue derivatives have been shown to be potentially effective photosensitizers for photodynamic therapy of malignant tumors. Results of a previous study suggested that the high accumulation of these dyes in cells may be the result of dye aggregation, partition in membrane lipids, and/or sequestration in subcellular organelles. In this report, results of studies are presented from an investigation of the subcellular localization and mechanism of accumulation of these dyes in cells in vitro. A video-enhanced fluorescence microscopy was used, and a punctate pattern of fluorescence was seen, most of which was localized in the perinuclear region with extracellular dye concentrations between 1 to 100 nM. These particles resembled characteristic particles identified by standard lysosomal dyes. At higher dye concentrations (1 microM or above), fluorescence in the perinuclear region was too intense to resolve into discrete cellular structures, while fluorescence in other cellular structures including mitochondria and cytomembranes was visible. At even higher dye concentrations (10-100 microM), Nile blue derivatives were seen with a light microscope as blue particles, the size and location of which resembled the punctate fluorescence described above. Results which further suggest that the lysosome is the main site of dye localization include (a) histochemical staining of dye-loaded cells with the lysosomal marker enzyme acid phosphatase, which showed similar localization of the enzyme-staining and dye-containing particles, (b) phototreatment of dye-loaded cells which obliterated the majority of the acid phosphatase-stained particles, and (c) treatments with agents affecting the membrane pH gradient reduced the uptake and enhanced the efflux of dyes, while agents that alter cellular membrane potentials had no effect on dye accumulation. The uptake of the dyes was partially inhibited by inhibitors of oxidative phosphorylation indicating that at least part of the process is energy dependent. These findings, together with previous results showing that the cellular uptake of these dyes is highly concentrative and proportional to the extracellular dye concentration over a wide range, are consistent with the hypothesis that the dyes are mainly localized in the lysosomes via an ion-trapping mechanism. Results of the present study also suggest that the lysosomes may be an intracellular target for photodynamic killing of tumor cells mediated by Nile blue photosensitizers and that lysosomotropic photosensitization may be a strategy for effective and selective destruction of tumor cells. PMID:2021950

Lin, C W; Shulok, J R; Kirley, S D; Cincotta, L; Foley, J W



Pharmacological chaperone therapy for lysosomal storage diseases.  


Pharmacological chaperone therapy is an emerging approach to treat lysosomal storage diseases. Small-molecule chaperones interact with mutant enzymes, favor their correct conformation and enhance their stability. This approach shows significant advantages when compared with existing therapies, particularly in terms of the bioavailability of drugs, oral administration and positive impact on the quality of patients' lives. On the other hand, future research in this field must confront important challenges. The identification of novel chaperones is indispensable to expanding the number of patients amenable to this treatment and to optimize therapeutic efficacy. It is important to develop new allosteric drugs, to address the risk of inhibiting target enzymes. Future research must also be directed towards the exploitation of synergies between chaperone treatment and other therapeutic approaches. PMID:25068986

Parenti, Giancarlo; Moracci, Marco; Fecarotta, Simona; Andria, Generoso



Fading Characteristics of Panchromatic Radar Backscatter from Selected Agricultural Targets  

Microsoft Academic Search

An experiment was performed to determine the fading characteristics of backscattered radar signals from four agricultural targets at 9 GHz. The targets included two different row crops (corn and soybeans), a continuous canopy (alfalfa) and bare ground. After a short review of the statistics of Rayleigh fading backscatter, the data processing method and the results of the experiment are analyzed.

Thomas F. Bush; Fawwaz Uloby



Fading characteristics of panchromatic radar backscatter from selected agricultural targets  

NASA Technical Reports Server (NTRS)

An experiment was performed to empirically determine the fading characteristics of backscattered radar signals from four agricultural targets at 9 GHz. After a short review of the statistics of Rayleigh fading backscatter, the data processing method and results of the data are analyzed. Comparison with theory shows adequate agreement with the experimental results, provided of course, the targets are modeled in a correct manner.

Bush, T. F.; Ulaby, F. T.



Genome-level identification of targets of Hox protein Ultrabithorax in Drosophila: novel mechanisms for target selection  

PubMed Central

Hox proteins are transcription factors and key regulators of segmental identity along the anterior posterior axis across all bilaterian animals. Despite decades of research, the mechanisms by which Hox proteins select and regulate their targets remain elusive. We have carried out whole-genome ChIP-chip experiments to identify direct targets of Hox protein Ultrabithorax (Ubx) during haltere development in Drosophila. Direct targets identified include upstream regulators or cofactors of Ubx. Homothorax, a cofactor of Ubx during embryonic development, is one such target and is required for normal specification of haltere. Although Ubx bound sequences are conserved amongst various insect genomes, no consensus Ubx-specific motif was detected. Surprisingly, binding motifs for certain transcription factors that function either upstream or downstream to Ubx are enriched in these sequences suggesting complex regulatory loops governing Ubx function. Our data supports the hypothesis that specificity during Hox target selection is achieved by associating with other transcription factors. PMID:22355720

Agrawal, Pavan; Habib, Farhat; Yelagandula, Ramesh; Shashidhara, L. S.



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

PubMed Central

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

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



A cation counterflux supports lysosomal acidification  

PubMed Central

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

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



Competition between color and luminance for target selection in smooth pursuit and saccadic  

E-print Network

Competition between color and luminance for target selection in smooth pursuit and saccadic eye of color and luminance for smooth pursuit and saccadic eye movements was investigated using a target selection paradigm. In two experiments, stimuli were varied along the dimensions color and luminance

Carrasco, Marisa


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

ERIC Educational Resources Information Center

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…

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



Chaperone-mediated autophagy: a unique way to enter the lysosome world  

PubMed Central

All cellular proteins undergo continuous synthesis and degradation. This permanent renewal is necessary to maintain a functional proteome and to allow for rapid changes in levels of specific proteins with regulatory purposes. Although for a long time lysosomes were considered unable to contribute to the selective degradation of individual proteins, the discovery of chaperone-mediated autophagy (CMA) changed this notion. Here, we review the characteristics that set CMA apart from other types of lysosomal degradation and the subset of molecules that confer cells the capability to identify individual cytosolic proteins and direct them across the lysosomal membrane for degradation. PMID:22748206

Kaushik, Susmita; Cuervo, Ana Maria



Enrichment and analysis of secretory lysosomes from lymphocyte populations  

PubMed Central

Background In specialized cells, such as mast cells, macrophages, T lymphocytes and Natural Killer cells in the immune system and for instance melanocytes in the skin, secretory lysosomes (SL) have evolved as bifunctional organelles that combine degradative and secretory properties. Mutations in lysosomal storage, transport or sorting molecules are associated with severe immunodeficiencies, autoimmunity and (partial) albinism. In order to analyze the function and content of secretory lysosomes in different cell populations, an efficient enrichment of these organelles is mandatory. Results Based on a combination of differential and density gradient centrifugation steps, we provide a protocol to enrich intact SL from expanded hematopoietic cells, here T lymphocytes and Natural Killer cells. Individual fractions were initially characterized by Western blotting using antibodies against an array of marker proteins for intracellular compartments. As indicated by the presence of LAMP-3 (CD63) and FasL (CD178), we obtained a selective enrichment of SL in one of the resulting organelle fractions. The robustness and reproducibility of the applied separation protocol was examined by a high-resolution proteome analysis of individual SL preparations of different donors by 2D difference gel electrophoresis (2D-DIGE). Conclusion The provided protocol is readily applicable to enrich and isolate intact secretory vesicles from individual cell populations. It can be used to compare SL of normal and transformed cell lines or primary cell populations from healthy donors and patients with lysosomal storage or transport diseases, or from corresponding mutant mice. A subsequent proteome analysis allows the characterization of molecules involved in lysosomal maturation and cytotoxic effector function at high-resolution. PMID:19640298

Schmidt, Hendrik; Gelhaus, Christoph; Lucius, Ralph; Nebendahl, Melanie; Leippe, Matthias; Janssen, Ottmar



Target Selection and Deselection at the Berkeley Structural Genomics Center  

E-print Network

a near- complete structural complement of proteins in the minimal organisms Mycoplasma genitalium and M coverage of the M. pneumoniae and M. genitalium proteomes. The number of Mycoplasma proteins for which TargetDB and PEPCdb. Proteins 2006;62:356­370. © 2005 Wiley-Liss, Inc. Key words: Mycoplasma pneumoniae


Parasite neuropeptide biology: Seeding rational drug target selection?  

PubMed Central

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

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



Target Selection for Pursuit and Saccadic Eye Movements in Humans  

Microsoft Academic Search

Eye movements were recorded from three subjects as they initiated tracking of a small circle (target) moving leftward or rightward, above or below the horizontal meridian, either alone or in the presence of a small square (distractor) moving leftward or rightward on the other side of the horizontal meridian. At the start of each trial, subjects were provided with either

R. J. Krauzlis; A. Z. Zivotofsky; F. A. Miles



Target track extraction in high false density environments using multiple hypothetical frame selection MLPDA  

NASA Astrophysics Data System (ADS)

MLPDA (Maximum Likelihood Probabilistic Data Association) has drawn attention as an effective target track extraction algorithm in high false density environments. In this algorithm, the target track is estimated as the maximum likelihood state vector, by using multiple observation frames that include the target signal and many false signals. The track is confirmed whether it is the true target or not, by comparing its likelihood with a given track confirmation threshold. However, when the target signals are lost at several frames, the conventional MLPDA deteriorates the track estimation accuracy due to false signals in frames without the target signal. In this paper, we propose multiple hypothetical frame selection MLPDA, which can extract the target track under the situation where the target signals are lost in several frames. Specifically, a batch of stored frames is first selected for track extraction. If the track is not confirmed, our algorithm offers multiple frame selection hypotheses where some frames are assumed to be the frames without the target signal and the other frames include the target signal. The track is extracted under these hypotheses, respectively, and the most likely hypothesis is accepted. If all hypotheses are rejected, our proposed method generates hypotheses that increase the number of frames without the target signal, and verifies them again. Furthermore, the hypotheses that have likelihoods above a given threshold are retained in order to modify the wrong frame selection later. Simulation results show the validity of our proposed method.

Mori, Masanori; Matsuzaki, Takashi; Kameda, Hiroshi; Umezawa, Toru



Regional Personalized Electrodes to Select Transcranial Current Stimulation Target  

PubMed Central

Rationale: Personalizing transcranial stimulations promises to enhance beneficial effects for individual patients. Objective: To stimulate specific cortical regions by developing a procedure to bend and position custom shaped electrodes; to probe the effects on cortical excitability produced when the properly customized electrode is targeting different cortical areas. Method: An ad hoc neuronavigation procedure was developed to accurately shape and place the personalized electrodes on the basis of individual brain magnetic resonance images (MRI) on bilateral primary motor (M1) and somatosensory (S1) cortices. The transcranial alternating current stimulation (tACS) protocol published by Feurra et al. (2011b) was used to test the effects on cortical excitability of the personalized electrode when targeting S1 or M1. Results: Neuronal excitability as evaluated by tACS was different when targeting M1 or S1, with the General Estimating Equation model indicating a clear tCS Effect (p?targeted by tCS properly shaping and positioning the stimulating electrode. Significance: Through multimodal brain investigations continuous efforts in understanding the neuronal changes related to specific neurological or psychiatric diseases become more relevant as our ability to build the compensating interventions improves. An important step forward on this path is the ability to target the specific cortical area of interest, as shown in the present pilot work. PMID:23626529

Tecchio, Franca; Cancelli, A.; Cottone, C.; Tomasevic, L.; Devigus, B.; Zito, G.; Ercolani, Matilde; Carducci, F.



Monitoring Autophagy in Lysosomal Storage Disorders  

PubMed Central

Lysosomes are the final destination of the autophagic pathway. It is in the acidic milieu of the lysosomes that autophagic cargo is metabolized and recycled. One would expect that diseases with primary lysosomal defects would be among the first systems in which autophagy would be studied. In reality, this is not the case. Lysosomal storage diseases, a group of more than 60 diverse inherited disorders, have only recently become a focus of autophagic research. Studies of these clinically severe conditions promise not only to clarify pathogenic mechanisms, but also to expand our knowledge of autophagy itself. In this chapter, we will describe the lysosomal storage diseases in which autophagy has been explored, and present the approaches used to evaluate this essential cellular pathway. PMID:19216919

Raben, Nina; Shea, Lauren; Hill, Victoria; Plotz, Paul




PubMed Central

In order to study mechanisms underlying selective enzyme release from human leukocytes during phagocytosis, the effects were studied of compounds which affect microtubule integrity or the accumulation of cyclic nucleotides. Human leukocytes selectively extrude lysosomal enzymes (?-glucuronidase) from viable cells during phagocytosis of zymosan or immune complexes, or upon encounter with immune complexes dispersed along a non-phagocytosable surface such as a millipore filter. In each circumstance, lysosomal enzyme release was reduced by previous treatment of cells with pharmacological doses of drugs which disrupt microtubules (e.g. 10-3–10-5 M colchicine) or with agents which affect accumulation of adenosine 3'5'-monophosphate (cAMP) (e.g. 10-3 M cyclic nucleotides and 2.8 x 10-4–2.8 x 10-6 M prostaglandin E (PGE) and A (PGA) compounds). Preincubation of cells with 5 µg/ml cytochalasin B resulted in complete inhibition of zymosan ingestion, but not of adherence of zymosan particles to plasma membranes or selective enzyme release. In this system, in which enzyme release was independent of particle uptake, preincubation of cells with colchicine, vinblastine, dibutyryl cAMP, or PGE1 also reduced extrusion of lysosomal enzymes. When cell suspensions were incubated with membrane-lytic crystals of monosodium urate (MSU), cytoplasmic as well as lysosomal enzymes were released with subsequent death of the cells. However, enzyme release followed phagocytosis of crystals (as measured by enhanced C-1 oxidation of glucose) and was due to "perforation from within" of the lysosomal membrane, rather than lysis by crystals of the plasma membrane. Enzyme release after MSU ingestion was also reduced when cells were treated with pharmacological doses of the test agents. When cells were killed by Triton X-100, acting on the plasma membrane, C-1 oxidation of glucose was abolished and enzyme release could not be inhibited pharmacologically. These observations suggest that lysosomal enzyme release from human phagocytes can be an active process which accompanies plasma membrane stimulation, is independent of cell death, and may be controlled by cyclic nucleotides and agents which affect microtubules. PMID:4125373

Zurier, Robert B.; Hoffstein, Sylvia; Weissmann, Gerald



Podocytes Degrade Endocytosed Albumin Primarily in Lysosomes  

PubMed Central

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 glomerulosclerosis in albuminuric diseases. Modifiers of lysosomal activity may have therapeutic potential in slowing the progression of glomerulosclerosis by enhancing the ability of podocytes to process and degrade albumin. PMID:24924335

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



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


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

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



Visualization and selective chemical targeting of RNA G-quadruplex structures in the cytoplasm of  

E-print Network

the selectivity and application of stabilizing ligands that target G-quadruplexes within a cellular context. I n on the potential for ligand-binding selectivity, and a crystal structure of TERRA bound to an acridine ligand the possible development of small- molecule ligands that are selective for RNA rather than for DNA G

Cambridge, University of


Tunneling nanotubes mediate rescue of prematurely senescent endothelial cells by endothelial progenitors: exchange of lysosomal pool  

PubMed Central

Although therapeutic effect of adoptive transfer of endothelial progenitor cells (EPC) has been well-substantiated, the actual engraftment is relatively low compared to a robust functional improvement of vasculopathy. Cellular mechanisms governing this action remain elusive. A recently discovered cell-cell communication via tunneling nanotube (TNT) formation is capable of transferring mitochondria and lysosomes between the cells – “organellar diakinesis”. Based on the previous demonstration of lysosomal dysfunction in endothelial cells exposed to AGE-modified collagen I, we inquired whether TNT mechanism may be involved in EPC-mediated repair of stressed endothelial cells. Here we demonstrate that EPC selectively and multiplicatively establish TNT communication with stressed endothelia. The guidance cues for the selectivity are provided by exofacially exposed phosphatidylserine moieties. Lysosomal transfer is associated with the preservation of lysosomal pH gradient, functionally reconstituting lysosomal pool of stressed cells and improving endothelial cell viability, reducing premature senescence and apoptosis. In vivo, adoptive transfer of EPC to streptozotocin-diabetic mice results in a TNT-dependent reduction of senescent endothelial cells and correction of endothelium-dependent vasorelaxation. Collectively, these data establish a selective multiplicative effect of TNT between EPC and stressed endothelia, reconstitution of the lysosomal pool, and improved viability and function of stressed endothelia. PMID:21705809

Yasuda, Kaoru; Khandare, Anupama; Burianovskyy, Leonid; Maruyama, Shoichi; Zhang, Frank; Nasjletti, Alberto



Signal Transduction and Molecular Targets of Selected Flavonoids  

PubMed Central

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

Bode, Ann M.



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

SciTech Connect

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-deficient mice.

Zeng, Jibin; Racicott, Jesse [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada)] [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada); Morales, Carlos R., E-mail: [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada)



A statistical approach to selecting and confirming validation targets in -omics experiments  

E-print Network

A statistical approach to selecting and confirming validation targets in -omics experiments Jeffrey of labor over manual validation of each significant result. 1 #12;Conclusions: For high-throughput -omics

Leek, Jeffrey


Auditory Stream Segregation Improves Infants' Selective Attention to Target Tones Amid Distracters  

ERIC Educational Resources Information Center

This study examined the role of auditory stream segregation in the selective attention to target tones in infancy. Using a task adapted from Bregman and Rudnicky's 1975 study and implemented in a conditioned head-turn procedure, infant and adult listeners had to discriminate the temporal order of 2,200 and 2,400 Hz target tones presented alone,…

Smith, Nicholas A.; Trainor, Laurel J.



Target Tracking with Online Feature Selection in FLIR Imagery Vijay Venkataraman, Guoliang Fan and Xin Fan  

E-print Network

Target Tracking with Online Feature Selection in FLIR Imagery Vijay Venkataraman, Guoliang Fan and Xin Fan School of Electrical and Computer Engineering Oklahoma State University, Stillwater, OK 74078 {vijay.venkataraman,,} Abstract We present a particle filter-based target

Fan, Guoliang


Item Selection Using an Average Growth Approximation of Target Information Functions.  

ERIC Educational Resources Information Center

Derivations of several item selection algorithms for use in fitting test items to target information functions (IFs) are described. These algorithms, which use an average growth approximation of target IFs, were tested by generating six test forms and were found to provide reliable fit. (SLD)

Luecht, Richard M.; Hirsch, Thomas M.



Rhodacyanine Derivative Selectively Targets Cancer Cells and Overcomes Tamoxifen Resistance  

PubMed Central

MKT-077, a rhodacyanine dye, was shown to produce cancer specific cell death. However, complications prevented the use of this compound beyond clinical trials. Here we describe YM-1, a derivative of MKT-077. We found that YM-1 was more cytotoxic and localized differently than MKT-077. YM-1 demonstrated this cytotoxicity across multiple cancer cell lines. This toxicity was limited to cancer cell lines; immortalized cell models were unaffected. Brief applications of YM-1 were found to be non-toxic. Brief treatment with YM-1 restored tamoxifen sensitivity to a refractory tamoxifen-resistant MCF7 cell model. This effect is potentially due to altered estrogen receptor alpha phosphorylation, an outcome precipitated by selective reductions in Akt levels (Akt/PKB). Thus, modifications to the rhodocyanine scaffold could potentially be made to improve efficacy and pharmacokinetic properties. Moreover, the impact on tamoxifen sensitivity could be a new utility for this compound family. PMID:22563386

Koren, John; Miyata, Yoshinari; Kiray, Janine; O'Leary, John C.; Nguyen, Lana; Guo, Jianping; Blair, Laura J.; Li, Xiokai; Jinwal, Umesh K.; Cheng, Jin Q.; Gestwicki, Jason E.; Dickey, Chad A.



New strategies for the treatment of lysosomal storage diseases (review).  


The lysosomal storage diseases (LSDs) are a group of inherited metabolic disorders caused by the deficiency of any of the lysosomal functions, in most cases of lysosomal hydrolases. LSDs are typically characterized by storage of a variety of substrates in multiple tissues and organs and by the variable association of unusual clinical manifestations that are often responsible for physical and neurological handicaps. During the past two decades, research in the field of LSDs has made marked progress, particularly with the development of a variety of innovative therapeutic approaches. These include several strategies aimed at increasing the residual activity of the missing enzyme, such as hematopoietic stem cell transplantation, enzyme replacement therapy, pharmacological chaperone therapy and gene therapy. An alternative approach is based on reducing the synthesis of the stored substrate. More recently, the improved knowledge on LSD pathophysiology has indicated additional targets of therapy. The recent progress made in the treatment of LSDs represents a good model that may be extended to other genetic disorders. PMID:23165354

Parenti, Giancarlo; Pignata, Claudio; Vajro, Pietro; Salerno, Mariacarolina



Microgravity induced selective lesions in immunosignaling: Upstream targets in lymphocytes  

NASA Astrophysics Data System (ADS)

Microgravity is a novel milieu for cells where re-ordering of forces induces different responses. Human lymphocytes undergo a suppression of activation and locomotion in space and modeled microgravity. Based on recovery of activation and locomotion with the phorbol ester PMA, the lesion induced by microgravity is presumed up- stream of the level of PKC signaling. Lymphocytes cultured in ground-based microgravity analog conditions display depressed calcium independent PKC isoforms. Upstream signaling molecules such as Phospholipase C gamma were not sufficiently activated in modeled microgravity. Immunoblotting revealed LAT, which is an adaptor protein crucial for Phospholipase C gamma recruitment in T cell activation, was down regulated in lymphocytes cultured at 72 and 96 hours in modeled microgravity. Also, ZAP 70 kinase, which is a LAT activator, down- regulated (>2 fold) at 96 hours modeled microgravity culture. Microarray analysis of lymphocytes cultured in 1g and modeled microgravity revealed significant down- regulation in upstream T cell activation molecules such as Diacylglycerol kinase, serine/threonine kinases, and tyrosine kinases. All up-stream targets in T cell activation are negatively affected in microgravity. Optimal immune function is critical in the ISS era where long term space travel is inevitable. Elucidation of the key mechanisms affected by microgravity lays the foundation for development of treatments that can counter these deleterious effects.

Sundaresan, A.; Pellis, N.


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

PubMed Central

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

Goschy, Harriet; Muller, Hermann Joseph



Polarized secretion of lysosomal enzymes: co-distribution of cation- independent mannose-6-phosphate receptors and lysosomal enzymes along the osteoclast exocytic pathway  

PubMed Central

The osteoclast is a polarized cell which secretes large amounts of newly synthesized lysosomal enzymes into an apical extracellular lacuna where bone resorption takes place. Using immunocytochemical techniques, we have localized the cation-independent mannose-6-phosphate (Man6P) receptor and lysosomal enzymes in this cell type in order to determine the expression and distribution of this receptor and its ligands. The results demonstrate that the osteoclast expresses large amounts of immunoreactive cation-independent Man6P receptors, despite the fact that most of the lysosomal enzymes it synthesizes are secreted. The lysosomal enzymes and the receptors are co-distributed along the exocytic pathway, i.e., the endoplasmic reticulum, including the perinuclear envelope, the Golgi stacks as well as numerous small transport vesicles that appear to fuse with the ruffled border membrane. Within the Golgi complex, the receptors and lysosomal enzymes were found distributed in two predominant patterns; (a) in all the cisternae, from cis to trans, or (b) predominantly in cis- and trans- Golgi cisternae, with the middle Golgi cisternae being unstained or depleted in antigen. This pattern suggests that enzymes and receptors traverse the Golgi from cis to trans and preferentially accumulate in cis- and in trans-cisternae. This study therefore suggests that, in the osteoclast, Man6P receptors are involved in the vectorial transport and targeting of newly synthesized lysosomal enzymes, presumably via a constitutive pathway, to the apical membrane where they are secreted into the bone-resorbing compartment. This mechanism could insure polarized secretion of lysosomal enzymes into the bone-resorbing lacuna. PMID:2968345



Lysosomes integrate metabolic-inflammatory cross-talk in primary macrophage inflammasome activation.  


Macrophage dysfunction and inflammasome activation have been implicated in the pathogenesis of diabetes and its complications. Prolonged inflammation and impaired healing are hallmarks of the diabetic response to tissue injury, and excessive inflammasome activation has been associated in these phenotypes. However, the mechanisms that regulate the inflammasome in response to lipid metabolic and inflammatory stress are incompletely understood. We have shown previously that IL-1? secretion is induced in primary macrophages exposed to the dietary saturated fatty acid palmitate in combination with LPS. In this study, we sought to unravel the mechanisms underlying the activation of this lipotoxic inflammasome. We demonstrate that palmitate-loaded primary macrophages challenged with LPS activate the NLRP3 inflammasome through a mechanism that involves the lysosome. Interestingly, the lysosome was involved in both the regulation of pro-IL-1? levels and its subsequent cleavage/release. The lysosomal protease cathepsin B was required for IL-1? release but not pro-IL-1? production. In contrast, disrupting lysosomal calcium regulation decreased IL-1? release by reducing pro-IL-1? levels. The calcium pathway involved the calcium-activated phosphatase calcineurin, which stabilized IL-1? mRNA. Our findings provide evidence that the lysosome plays a key role in both the priming and assembly phases of the lipostoxic inflammasome. These findings have potential relevance to the hyperinflammatory phenotypes observed in diabetics during tissue damage or infection and identify lysosomes and calcineurin as potential therapeutic targets. PMID:24532802

Weber, Kassandra; Schilling, Joel D



Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis  

PubMed Central

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

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.



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


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

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



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


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

Ingemann, Linda; Kirkegaard, Thomas



Possible pathways for lysosomal enzyme delivery  

PubMed Central

Immunogold double-labeling and ultrathin cryosections were used to compare the subcellular distribution of albumin, mannose 6-phosphate receptor (MPR), galactosyltransferase, and the lysosomal enzymes cathepsin D, beta-hexosaminidase, and alpha-glucosidase in Hep G2 cells. MPR and lysosomal enzymes were found throughout the stack of Golgi cisternae and in a trans-Golgi reticulum (TGR) of smooth-surfaced tubules with coated buds and vesicles. The trans-Golgi orientation of TGR was ascertained by the co-localization with galactosyltransferase. MPR was particularly abundant in TGR and CURL, the compartment of uncoupling receptors and ligands. Both TGR and CURL also contained lysosomal enzymes, but endogenous albumin was detected in TGR only. The coated buds on TGR tubules contained MPR, lysosomal enzymes, as well as albumin. MPR and lysosomal enzymes were also found in coated pits of the plasma membrane. CURL tubules seemed to give rise to smooth vesicles, often of the multivesicular body type. In CURL, the enzymes were found in the lumina of the smooth vesicles while MPR prevailed in the tubules. These observations suggest a role of CURL in transport of lysosomal enzymes to lysosomes. When the cells were treated with the lysosomotropic amine primaquine, binding of anti-MPR to the cells in culture was reduced by half. Immunocytochemistry showed that MPR accumulated in TGR, especially in coated buds. Since these buds contain endogenous albumin and lysosomal enzymes also, these data suggest that coated vesicles originating from TGR provide for a secretory route in Hep G2 cells and that this pathway is followed by the MPR system as well. PMID:2933416



Lysosomal Enzyme Activities in Experimental Granulomatous Inflammation  

Microsoft Academic Search

Foreign-body (dextran beads) and hypersensitivity (antigen-coupled agarose beads) lung granulomas were induced in BALB\\/c mice by the intratracheal injection of beads. Large granulomas developed, which reached peak intensity within 3 days and declined in size thereafter. Aqueous extracts of both granulomas contained high levels of lysosomal enzymes N-acetyl-?-D-glucosaminidase (NAG) and lysozyme. Lysosomal enzyme activities in the extracts correlated with granuloma

Shuji Kaga; Kazuo Kobayashi; Noriko Yamagata; Hiroko T. Takeuchi; Kazue Yoshida; Tomoko Matsuda; Kazuko Nakatani; Tsuyoshi Kasama; Keita Kasahara; Terumi Takahashi



Selective targeting of radiation-resistant tumor-initiating cells  

PubMed Central

Tumor-initiating cells (TICs) have been shown both experimentally and clinically to be resistant to radiation and chemotherapy, potentially resulting in residual disease that can lead to recurrence. In this study, we demonstrate that TICs isolated from p53 null mouse mammary tumors repair DNA damage following in vivo ionizing radiation more efficiently than the bulk of the tumor cells. Down-regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was observed both in fluorescence activated cell sorting (FACS)-isolated TICs as compared to non-TICs and in TIC-enriched mammospheres as compared to primary tumor cells depleted of TICs. This effect was accompanied by increased Akt signaling, as well as by the direct activation of the canonical Wnt/?-catenin signaling pathway specifically within the TIC subpopulation by phosphorylation of ?-catenin on serine 552. Using limiting dilution transplantation performed on p53 null tumor cells transduced with Wnt reporter lentivirus, we demonstrated that FACS sorting of cells expressing TOP-eGFP resulted in a marked enrichment for TICs. Furthermore, FACS analysis demonstrated that cells with active Wnt signaling overlapped with the TIC subpopulation characterized previously using cell surface markers. Finally, pharmacological inhibition of the Akt pathway in both mammospheres and syngeneic mice bearing tumors was shown to inhibit canonical Wnt signaling as well as the repair of DNA damage selectively in TICs, sensitizing them to ionizing radiation treatment. Thus, these results suggest that pretreatment with Akt inhibitors before ionizing radiation treatment may be of potential therapeutic benefit to patients. PMID:20133717

Zhang, Mei; Atkinson, Rachel L.; Rosen, Jeffrey M.



Selection of aptamers targeting the sialic acid receptor of hemagglutinin by epitope-specific SELEX.  


A new SELEX scheme is proposed for the selection of aptamers targeting a specific epitope of a native protein. Anti-sialic acid receptor (SAR) aptamers that inhibit H1 hemagglutination at a low picomole dose are selected accordingly. PMID:24964092

Lao, Yeh-Hsing; Chiang, Hui-Yu; Yang, Deng-Kai; Peck, Konan; Chen, Lin-Chi



Common Risk Alleles for Inflammatory Diseases Are Targets of Recent Positive Selection  

E-print Network

.16,17 Similarly, it was shown that the positively selected celiac disease (MIM 212750) risk variantARTICLE Common Risk Alleles for Inflammatory Diseases Are Targets of Recent Positive Selection identified hundreds of loci harboring genetic variation influencing inflammatory- disease susceptibility

Raychaudhuri, Soumya


Aptamers Selected for Higher-Affinity Binding Are Not More Specific for the Target Ligand  

E-print Network

Aptamers Selected for Higher-Affinity Binding Are Not More Specific for the Target Ligand James M is required to specify the structures of the higher-affinity aptamers. We are interested in understanding how the more complex aptamers achieve higher affinities for the ligand. In vitro selection produces structural

Heller, Eric


Lysosome-associated protein transmembrane 4 alpha (LAPTM4 alpha) requires two tandemly arranged tyrosine-based signals for sorting to lysosomes.  

PubMed Central

Lysosome-associated protein transmembrane 4 alpha (LAPTM4 alpha) and homologues comprise a family of conserved proteins, which are found in mammals, insects and nematodes. LAPTM4 alpha functions to regulate the intracellular compartmentalization of amphipathic solutes and possibly the sensitivity of cells toward anthracyclines, antibiotics, ionophores, nucleobases and organic cations. This is similar to the multidrug-resistance phenotype exhibited by cells synthesizing high levels of P-glycoprotein. Accordingly, it is possible that LAPTM4 alpha may be a suitable target for development of novel chemotherapeutic agents. LAPTM4 alpha contains four putative membrane-spanning domains and a 55 amino acid C-terminal region that faces the cytoplasm. Localization of LAPTM4 alpha to endosomes and lysosomes appears to be tightly controlled as transient high-level expression of LAPTM4 alpha in cultured cells resulted in no detectable protein on the cell surface. Mutagenic analysis of the C-terminus of LAPTM4 alpha indicated that two tandomly arranged tyrosine-containing motifs in the cytoplasmic domain are required for efficient localization of LAPTM4 alpha to vesicles containing the lysosomal marker lysosomal glycoprotein 120. Although a number of membrane proteins that localize to endosomes/lysosomes contain more than one independently functioning sorting signal, to our knowledge, LAPTM4 alpha is the first example of a membrane protein that requires two tandemly arranged tyrosine-based sorting signals for efficient localization in these compartments. PMID:11980562

Hogue, Douglas L; Nash, Colin; Ling, Victor; Hobman, Tom C



Priming of pop-out modulates attentional target selection in visual search: behavioural and electrophysiological evidence.  


Previous behavioural studies have shown that the repetition of target or distractor features across trials speeds pop-out visual search. We obtained behavioural and event-related brain potential (ERP) measures in two experiments where participants searched for a colour singleton target among homogeneously coloured distractors. An ERP marker of spatially selective attention (N2pc component) was delayed when either target or distractor colours were swapped across successive trials, demonstrating that intertrial feature priming systematically affects the onset of focal-attentional target processing. Results support the hypothesis that priming of pop-out effects are primarily generated at early perceptual/attentional stages of visual processing. PMID:19895829

Eimer, Martin; Kiss, Monika; Cheung, Theodore



Lysosomes and ?-synuclein form a dangerous duet leading to neuronal cell death  

PubMed Central

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). ?-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 ?-synuclein, and for the removal of old or damaged organelles, such as mitochondria. Interestingly, PD-linked ?-synuclein mutants and dopamine-modified wild-type ?-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, ?-synuclein accumulation, and neurotoxicity. In this review, we emphasize the pathophysiological mechanisms connecting ?-synuclein and lysosomal dysfunction in neuronal cell death. PMID:25177278

Bourdenx, Mathieu; Bezard, Erwan; Dehay, Benjamin



Signaling from lysosomes enhances mitochondria-mediated photodynamic therapy in cancer cells  

NASA Astrophysics Data System (ADS)

In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in singlet oxygen formation and cell death. Assessed by confocal microscopy, the photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. A Pc 4 derivative, Pc 181, accumulates into lysosomes. In comparison to Pc 4, Pc 181 was a more effective photosensitizer promoting killing cancer cells after PDT. The mode of cell death after Pc 181-PDT is predominantly apoptosis, and pancaspase and caspase-3 inhibitors prevent onset of the cell death. To assess further how lysosomes contribute to PDT, we monitored cell killing of A431cells after PDT in the presence and absence of bafilomycin, an inhibitor of the acidic vacuolar proton pump that collapses the pH gradient of the lysosomal/endosomal compartment. Bafilomycin by itself did not induce toxicity but greatly enhanced Pc 4-PDT-induced cell killing. In comparison to Pc 4, less enhancement of cell killing by bafilomycin occurred after Pc 181-PDT at photosensitizer doses producing equivalent cell killing in the absence of bafilomycin. These results indicate that lysosomal disruption can augment PDT with Pc 4, which targets predominantly mitochondria, but less so after PDT with Pc 181, since Pc 181 already targets lysosomes.

Quiogue, Geraldine; Saggu, Shalini; Hung, Hsin-I.; Kenney, Malcolm E.; Oleinick, Nancy L.; Lemasters, John J.; Nieminen, Anna-Liisa



Transgenic gene knock-outs: functional genomics and therapeutic target selection.  


The completion of the first draft of the human genome presents both a tremendous opportunity and enormous challenge to the pharmaceutical industry since the whole community, with few exceptions, will soon have access to the same pool of candidate gene sequences from which to select future therapeutic targets. The commercial imperative to select and pursue therapeutically relevant genes from within the overall content of the genome will be particularly intense for those gene families that currently represent the chemically tractable or 'drugable' gene targets. As a consequence the emphasis within exploratory research has shifted towards the evaluation and adoption of technology platforms that can add additional value to the gene selection process, either through functional studies or direct/indirect measures of disease alignment e.g., genetics, differential gene expression, proteomics, tissue distribution, comparative species data etc. The selection of biological targets for the development of potential new medicines relies, in part, on the quality of the in vivo biological data that correlates a particular molecular target with the underlying pathophysiology of a disease. Within the pharmaceutical industry, studies employing transgenic animals and, in particular, animals with specific gene deletions are playing an increasingly important role in the therapeutic target gene selection, drug candidate selection and product development phases of the overall drug discovery process. The potential of phenotypic information from gene knock-outs to contribute to a high-throughput target selection/validation strategy has hitherto been limited by the resources required to rapidly generate and characterise a large number of knock-out transgenics in a timely fashion. The offerings of several companies that provide an opportunity to overcome these hurdles, albeit at a cost, are assessed with respect to the strategic business needs of the pharmaceutical industry. PMID:11257927

Harris, S; Foord, S M



Autophagy-receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome  

PubMed Central

Autophagy targets pathogens, damaged organelles and protein aggregates for lysosomal degradation. These ubiquitinated cargoes are recognised by specific autophagy receptors, which recruit LC3-positive membranes to form autophagosomes. Subsequently, autophagosomes fuse with endosomes and lysosomes, thus facilitating degradation of their content, however, the machinery that targets and mediates fusion of these organelles with autophagosomes remains to be established. Here we demonstrate that myosin VI, in concert with its adaptor proteins NDP52, optineurin, T6BP and Tom1, plays a crucial role in autophagy. We identify Tom1 as a myosin VI binding partner on endosomes and demonstrate that their loss reduces autophagosomal delivery of endocytic cargo and causes a block in autophagosome-lysosome fusion. We propose that myosin VI delivers endosomal membranes containing Tom1 to autophagosomes by docking to NDP52, T6BP and optineurin thereby promoting autophagosome maturation and thus driving fusion with lysosomes. PMID:23023224

Tumbarello, David A.; Waxse, Bennett J.; Arden, Susan D.; Bright, Nicholas A.; Kendrick-Jones, John; Buss, Folma



Comparison of the Cancer Gene Targeting and Biochemical Selectivities of All Targeted Kinase Inhibitors Approved for Clinical Use  

PubMed Central

The anti-proliferative activities of all twenty-five targeted kinase inhibitor drugs that are in clinical use were measured in two large assay panels: (1) a panel of proliferation assays of forty-four human cancer cell lines from diverse tumour tissue origins; and (2) a panel of more than 300 kinase enzyme activity assays. This study provides a head-on comparison of all kinase inhibitor drugs in use (status Nov. 2013), and for six of these drugs, the first kinome profiling data in the public domain. Correlation of drug activities with cancer gene mutations revealed novel drug sensitivity markers, suggesting that cancers dependent on mutant CTNNB1 will respond to trametinib and other MEK inhibitors, and cancers dependent on SMAD4 to small molecule EGFR inhibitor drugs. Comparison of cellular targeting efficacies reveals the most targeted inhibitors for EGFR, ABL1 and BRAF(V600E)-driven cell growth, and demonstrates that the best targeted agents combine high biochemical potency with good selectivity. For ABL1 inhibitors, we computationally deduce optimized kinase profiles for use in a next generation of drugs. Our study shows the power of combining biochemical and cellular profiling data in the evaluation of kinase inhibitor drug action. PMID:24651269

Uitdehaag, Joost C. M.; de Roos, Jeroen A. D. M.; van Doornmalen, Antoon M.; Prinsen, Martine B. W.; de Man, Jos; Tanizawa, Yoshinori; Kawase, Yusuke; Yoshino, Kohichiro; Buijsman, Rogier C.; Zaman, Guido J. R.



The effect of mean luminance on the size selectivity of identified target interneurons in the dragonfly  

Microsoft Academic Search

1.By penetrating axons in the ventral nerve cord of the dragonfly, Aeshna umbrosa, we measured the intracellular responses of target-selective visual interneurons to movement of black square ‘targets’ ranging from 1° to 32° visual angle at several levels of mean background luminance.2.Neuronal responses, measured both in number of spikes and in the magnitude of integrated postsynaptic potentials, showed a preference

Robert M. Olberg; Robert B. Pinter



Computing visual target distinctness through selective filtering, statistical features, and visual patterns  

NASA Astrophysics Data System (ADS)

This paper presents three computational visual distinctness measures, computed from image representational models based on selective filtering, statistical features, and visual patterns, respectively. They are applied to quantify the visual distinctness of targets in complex natural scenes. The measure that applies a simple decision rule to the distances between segregated visual patterns is shown (1) to predict human observer performance in search and detection tasks on complex natural imagery, and (2) to correlate strongly with visual target distinctness estimated by human observers.

Fdez-Vidal, Xose R.; Toet, Alexander; Garcia, J. A.; Fdez-Valdivia, J.



Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT  

Microsoft Academic Search

BackgroundThe stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT.Methodology\\/Principal FindingsIn vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half

Patrice Dubreuil; Sébastien Letard; Marco Ciufolini; Laurent Gros; Martine Humbert; Nathalie Castéran; Laurence Borge; Bérengère Hajem; Anne Lermet; Wolfgang Sippl; Edwige Voisset; Michel Arock; Christian Auclair; Phillip S. Leventhal; Colin D. Mansfield; Alain Moussy; Olivier Hermine



Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle  

Microsoft Academic Search

Potent sequence selective gene inhibition by siRNA 'targeted' therapeutics promises the ultimate level of specificity, but siRNA therapeutics is hindered by poor intracellular uptake, limited blood stability and non-specific immune stimulation. To address these problems, ligand-targeted, sterically stabil- ized nanoparticles have been adapted for siRNA. Self-assembling nanoparticles with siRNA were constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp

Raymond M. Schiffelers; Aslam Ansari; Jun Xu; Qin Zhou; Qingquan Tang; Gert Storm; Grietje Molema; Patrick Y. Lu; Puthupparampil V. Scaria; Martin C. Woodle



[Medicinal therapy for lysosomal storage diseases].  


Lysosomes contain several dozen different enzymes, mostly acid hydrolases. Materials not digested due to deficient lysosomal enzymes are usually large cellular molecules, which are deposited within the cells. The strategy for medicinal therapy of lysosomal storages disease may be to develop the activators of enzymes, to promote coenzyme and cofactor supplementation and to eliminate undegraded materials from blood into urine. In the last several decades, many trials for these strategies has been done. Cysteamine for cystinosis and penicillamine for Wilson's disease has proved useful in treating these patients. Recently, DMSO has been proved to be an activator of acid sphingomyelinase and to accelerate the intracellular mobilization of LDL-derived cholesterol. We treated patients with Niemann-Pick disease type C by oral administration of DMSO, resulting in some clinical benefits such as decreased size of hepatosplenomegaly, and lesser frequency of seizures with improved EEG. However, the progressive clinical course has not been changed although it appeared to slow down. New activators of lysosomal enzymes should be developed for medicinal therapy of lysosomal storage diseases. PMID:8577061

Sakuragawa, N



Software for selecting the most informative sets of genomic loci for multi-target microbial typing  

PubMed Central

Background High-throughput sequencing can identify numerous potential genomic targets for microbial strain typing, but identification of the most informative combinations requires the use of computational screening tools. This paper describes novel software – Automated Selection of Typing Target Subsets (AuSeTTS) - that allows intelligent selection of optimal targets for pathogen strain typing. The objective of this software is to maximise both discriminatory power, using Simpson’s index of diversity (D), and concordance with existing typing methods, using the adjusted Wallace coefficient (AW). The program interrogates molecular typing results for panels of isolates, based on large target sets, and iteratively examines each target, one-by-one, to determine the most informative subset. Results AuSeTTS was evaluated using three target sets: 51 binary targets (13 toxin genes, 16 phage-related loci and 22 SCCmec elements), used for multilocus typing of 153 methicillin-resistant Staphylococcus aureus (MRSA) isolates; 17 MLVA loci in 502 Streptococcus pneumoniae isolates from the MLVA database ( and 12 MLST loci for 98 Cryptococcus spp. isolates. The maximum D for MRSA, 0.984, was achieved with a subset of 20 targets and a D value of 0.954 with 7 targets. Twelve targets predicted MLST with a maximum AW of 0.9994. All 17 S. pneumoniae MLVA targets were required to achieve maximum D of 0.997, but 4 targets reached D of 0.990. Twelve targets predicted pneumococcal serotype with a maximum AW of 0.899 and 9 predicted MLST with maximum AW of 0.963. Eight of the 12 MLST loci were sufficient to achieve the maximum D of 0.963 for Cryptococcus spp. Conclusions Computerised analysis with AuSeTTS allows rapid selection of the most discriminatory targets for incorporation into typing schemes. Output of the program is presented in both tabular and graphical formats and the software is available for free download from PMID:23635100



Spectroscopic Target Selection in the Sloan Digital Sky Survey: The Quasar Sample  

Microsoft Academic Search

We describe the algorithm for selecting quasar candidates for optical spectroscopy in the Sloan Digital Sky Survey. Quasar candidates are selected via their nonstellar colors in ugriz broadband photometry and by matching unresolved sources to the FIRST radio catalogs. The automated algorithm is sensitive to quasars at all redshifts lower than z~5.8. Extended sources are also targeted as low-redshift quasar

Xiaohui Fan; Heidi Jo Newberg; Michael A. Strauss; Daniel E. Vanden Berk; Donald P. Schneider; Brian Yanny; Adam Boucher; Scott Burles; Joshua A. Frieman; James E. Gunn; Patrick B. Hall; Zeljko Ivezic; Stephen Kent; Jon Loveday; Robert H. Lupton; Constance M. Rockosi; David J. Schlegel; Chris Stoughton; Mark SubbaRao; Donald G. York



Effects of ethanol, acetaldehyde and cholesteryl esters on pancreatic lysosomes.  

PubMed Central

Recent studies indicate that altered lysosomal function may be involved in the early stages of pancreatic injury. Chronic consumption of ethanol increases rat pancreatic lysosomal fragility. The aim of this study is to determine whether the lysosomal fragility observed after chronic ethanol consumption is mediated by ethanol per se, its oxidative metabolite acetaldehyde or cholesteryl esters (substances which accumulate in the pancreas after ethanol consumption). Pancreatic lysosomes from chow fed rats were incubated for 30 minutes at 37 degrees C with ethanol, acetaldehyde or phosphatidylcholine vesicles containing cholesteryl oleate. Lysosomal stability was then assessed by determination of: (a) Latency--that is, the per cent increase in lysosomal enzyme activity after addition of Triton X-100 and (b) Supernatant activity--that is, the proportion of lysosomal enzyme remaining in the supernatant after resedimentation of lysosomes. Acid phosphatase, N-acetyl glucosaminidase, beta-glucuronidase and cathepsin B were assayed as lysosomal marker enzymes. Lysosomes incubated with homogenising medium alone or equivalent volumes of phosphatidylcholine vesicles without cholesteryl oleate were used as controls. Cholesteryl oleate at concentrations of 15 and 20 mM increased pancreatic lysosomal fragility as shown by decreased latency and increased supernatant enzyme. In contrast, ethanol (150 mM) and acetaldehyde (5 mM) had no effect on lysosomal stability in vitro. These results suggest that increased pancreatic lysosomal fragility observed with ethanol may be mediated by cholesteryl ester accumulation rather than by ethanol or acetaldehyde. PMID:1398235

Wilson, J S; Apte, M V; Thomas, M C; Haber, P S; Pirola, R C



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

PubMed Central

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

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



Geometric basis-vector selection methods and subpixel target detection as applied to hyperspectral imagery  

Microsoft Academic Search

In this paper, we compare three basis-vector selection methods as applied to subpixel target detection. This is a continuation of previous research in which a similar comparison was performed based on an AVIRIS image. Our goal is to find out to what extent our previous observations apply more broadly to other images, more specifically, a HYDICE image used in this

Peter Bajorski; Emmett J. Ientilucci



Priming of popout: III. A short-term implicit memory system beneficial for rapid target selection  

E-print Network

Priming of popout: III. A short-term implicit memory system beneficial for rapid target selection­8 trials, and is cumulative. Here we establish PoP as an example of short-term implicit memory by showing be more transient implicit memory systems, analogous to better known short-term explicit memory systems

Nakayama, Ken


Efficacy and Selectivity of Phosphodiesterase-Targeted Drugs in Inhibiting Photoreceptor Phosphodiesterase  

E-print Network

-linked immunoassay. RESULTS. Most PDE5-selective inhibitors were excellent PDE6 inhibitors. Vardenafil, a potent PDE5 and potently target PDE5, such as sildenafil (Viagra; Pfizer, New York, NY), vardenafil (Levitra; Bayer.12 Tadalafil and vardenafil, two other ap- proved drugs, show lesser effects on visual function.13

Cote, Rick H.


A solid-phase affinity labeling method for target-selective isolation and modification of proteins.  


Solid-phase affinity labeling of a target protein, peanut agglutinin (PNA), with the specifically designed chemical tool 1 selectively and effectively furnished the labeled PNA. Furthermore, this method was applicable to native human carbonic anhydrase II in red blood cell lysate using the chemical tool 2 without the need for tedious manipulations. PMID:25360454

Kuwahara, Daichi; Hasumi, Takahiro; Kaneko, Hajime; Unno, Madoka; Takahashi, Daisuke; Toshima, Kazunobu



Audience Selection for On-line Brand Advertising: Privacy-friendly Social Network Targeting  

E-print Network

Audience Selection for On-line Brand Advertising: Privacy-friendly Social Network Targeting Foster describes and evaluates privacy-friendly methods for extracting quasi-social networks from browser behavior-generated content, privacy This work was conducted while the authors were at Media6 . Foster Provost thanks NEC

Provost, Foster


Metformin Selectively Targets Cancer Stem Cells, and Acts Together with Chemotherapy to Block Tumor Growth  

E-print Network

Metformin Selectively Targets Cancer Stem Cells, and Acts Together with Chemotherapy to Block Tumor, particularly in combination with chemotherapy. Here, we show that low doses of metformin, a standard drug different types of breast cancer. The combination of metformin and a well-defined chemotherapeutic agent


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

ERIC Educational Resources Information Center

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…

Li, Xingshan; Shen, Wei



Orientation saliency without visual cortex and target selection in archer fish  

E-print Network

with the water (16, 17). Recent studies have demonstrated that these fish can accurately shoot flying insectsOrientation saliency without visual cortex and target selection in archer fish Alik Mokeicheva visual cortex. In behavioral experiments with the archer fish, a proficient hunter with remark- able

ben-Shahar, Ohad


Lysosomal responses to nutritional and contaminant stress in mussel hepatopancreatic digestive cells: a modelling study.  


The lysosomal system occupies a central and crucial role in cellular food degradation (intracellular digestion), toxic responses and internal turnover (autophagy) of the hepatopancreatic digestive cell of the blue mussel Mytilus edulis. Understanding the dynamic response of this system requires factors affecting performance, conceived as a function of the throughput, degradative efficiency and lysosomal membrane stability, to be defined and quantified. A previous carbon/nitrogen flux model has been augmented by separately identifying lysosomal 'target' material (autophagocytosed or endocytosed proteins, carbohydrates and lipids) and 'internal' material (digestive enzymes and lipid membrane components). Additionally, the whole cell's energetic costs for maintaining lysosomal pH and production of these internal components have been incorporated, as has the potentially harmful effect of generation of lipofuscin on the transitory and semi-permanent lysosomal constituents. Inclusion of the three classes of nutrient organic compounds at the whole cell level allows for greater range in the simulated response, including deamination of amino acids to provide molecules as a source of energy, as well as controlling nitrogen and carbon concentrations in the cytosol. Coupled with a more functional framework of pollutant driven reactive oxygen species (ROS) production and antioxidant defence, the separate and combined effects of three stressors (nutritional quality, nutrient quantity and a polycyclic aromatic hydrocarbon [PAH-phenanthrene]) on the digestive cell are simulated and compare favourably with real data. PMID:16730788

McVeigh, Allan; Moore, Michael; Allen, J Icarus; Dyke, Phil



The Ubiquitin–Proteasome System and the Autophagic–Lysosomal System in Alzheimer Disease  

PubMed Central

As neurons age, their survival depends on eliminating a growing burden of damaged, potentially toxic proteins and organelles—a capability that declines owing to aging and disease factors. Here, we review the two proteolytic systems principally responsible for protein quality control in neurons and their important contributions to Alzheimer disease pathogenesis. In the first section, the discovery of paired helical filament ubiquitination is described as a backdrop for discussing the importance of the ubiquitin–proteasome system in Alzheimer disease. In the second section, we review the prominent involvement of the lysosomal system beginning with pathological endosomal–lysosomal activation and signaling at the very earliest stages of Alzheimer disease followed by the progressive failure of autophagy. These abnormalities, which result in part from Alzheimer-related genes acting directly on these lysosomal pathways, contribute to the development of each of the Alzheimer neuropathological hallmarks and represent a promising therapeutic target. PMID:22908190

Ihara, Yasuo; Morishima-Kawashima, Maho; Nixon, Ralph



Lysosomal abnormalities in hereditary spastic paraplegia types SPG15 and SPG11  

PubMed Central

Objective Hereditary spastic paraplegias (HSPs) are among the most genetically diverse inherited neurological disorders, with over 70 disease loci identified (SPG1-71) to date. SPG15 and SPG11 are clinically similar, autosomal recessive disorders characterized by progressive spastic paraplegia along with thin corpus callosum, white matter abnormalities, cognitive impairment, and ophthalmologic abnormalities. Furthermore, both have been linked to early-onset parkinsonism. Methods We describe two new cases of SPG15 and investigate cellular changes in SPG15 and SPG11 patient-derived fibroblasts, seeking to identify shared pathogenic themes. Cells were evaluated for any abnormalities in cell division, DNA repair, endoplasmic reticulum, endosomes, and lysosomes. Results Fibroblasts prepared from patients with SPG15 have selective enlargement of LAMP1-positive structures, and they consistently exhibited abnormal lysosomal storage by electron microscopy. A similar enlargement of LAMP1-positive structures was also observed in cells from multiple SPG11 patients, though prominent abnormal lysosomal storage was not evident. The stabilities of the SPG15 protein spastizin/ZFYVE26 and the SPG11 protein spatacsin were interdependent. Interpretation Emerging studies implicating these two proteins in interactions with the late endosomal/lysosomal adaptor protein complex AP-5 are consistent with shared abnormalities in lysosomes, supporting a converging mechanism for these two disorders. Recent work with Zfyve26?/? mice revealed a similar phenotype to human SPG15, and cells in these mice had endolysosomal abnormalities. SPG15 and SPG11 are particularly notable among HSPs because they can also present with juvenile parkinsonism, and this lysosomal trafficking or storage defect may be relevant for other forms of parkinsonism associated with lysosomal dysfunction. PMID:24999486

Renvoise, Benoit; Chang, Jaerak; Singh, Rajat; Yonekawa, Sayuri; FitzGibbon, Edmond J; Mankodi, Ami; Vanderver, Adeline; Schindler, Alice B; Toro, Camilo; Gahl, William A; Mahuran, Don J; Blackstone, Craig; Pierson, Tyler Mark



Time-dependent effects of hydrophobic amine-containing drugs on lysosome structure and biogenesis in cultured human fibroblasts.  


Many weakly basic amine-containing drugs are known to be extensively sequestered in acidic lysosomes by an ion trapping-type mechanism. The entrapment of drugs in lysosomes has been shown to influence drug activity, cancer cell selectivity, and pharmacokinetics and can cause the hyperaccumulation of various lipids associated with lysosomes. In this work, we have investigated the prolonged time-dependent effects of drugs on lysosomal properties. We have evaluated two amine-containing drugs with intermediate (propranolol) and high (halofantrine) relative degrees of lipophilicity. Interestingly, the cellular accumulation kinetics of these drugs exhibited a biphasic characteristic at therapeutically relevant exposure levels with an initial apparent steady-state occurring at 2 days followed by a second stage of enhanced accumulation. We provide evidence that this secondary drug accumulation coincides with the nuclear localization of transcription factor EB, a master regulator of lysosome biogenesis, and the appearance of an increased number of smaller and lipid-laden lysosomes. Collectively, these results show that hydrophobic lysosomotropic drugs can induce their own cellular accumulation in a time-dependent fashion and that this is associated with an expanded lysosomal volume. These results have important therapeutic implications and may help to explain sources of variability in drug pharmacokinetic distribution and elimination properties observed in vivo. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3287-3296, 2014. PMID:25042198

Logan, Randall; Kong, Alex C; Krise, Jeffrey P



Autonomous target dependent waveband selection for tracking in performance-driven hyperspectral sensing  

NASA Astrophysics Data System (ADS)

Performance-driven sensing is a promising new concept that relies on sensing, processing, and exploiting only the most "decision-relevant" sets of target data for the purpose of reducing requirements on data collection, processing, and communications. An example of a device supporting such a concept is a MEMS-based single pixel Fabry-Perot spectrometer being developed at the Rochester Institute of Technology, which can record selected wavelengths on a per-pixel basis throughout an image. This paper presents an autonomous target-dependent waveband selection approach for performance-driven sensing with an adaptive hyperspectral imaging sensor. Given a target that is to be tracked, a subset of wavebands is estimated from locally recorded hyperspectral data that provides optimal target detectability against local background. The waveband selection algorithm relies on finding a subset of bands that provides maximum separation between a target histogram and local background histogram constructed from the respective bands. To illustrate the concept, we perform a simulation study for vehicle tracking in a set of synthetic DIRSIG rendered HSI images. The simulations demonstrate improved vehicle tracking accuracy when using the adaptively-selected subset of wavebands for tracking by histogram matching compared to performing tracking by histogram matching with regular (fixed) color bands. We extend the framework to a dynamic concept where the waveband subset is updated over time as a function of position estimation accuracy and discuss the full integration of the Feature-Aided Tracking (FAT) component derived from the selected wavebands within a Multiple Hypothesis Tracking (MHT) framework.

Gadaleta, Sabino M.; Kerekes, John P.; Tarplee, Kyle M.



The Adaptive Hybrid Cursor: A Pressure-Based Target Selection Technique for Pen-Based User Interfaces  

Microsoft Academic Search

We present the Adaptive Hybrid Cursor, a novel target acquisition technique for pen-based interfaces. To assist a user in a target selection task, this technique automatically adapts the size of the cursor and\\/or its contexts (the target size and the selection background) based on pen pressure input. We systematically evaluated the new technique with various 2D target acquisition tasks. The

Xiangshi Ren; Jibin Yin; Shengdong Zhao; Yang Li



Peptide nucleic acids targeting ?-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells.  


In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine ?-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies. PMID:25405921

Montagner, Giulia; Gemmo, Chiara; Fabbri, Enrica; Manicardi, Alex; Accardo, Igea; Bianchi, Nicoletta; Finotti, Alessia; Breveglieri, Giulia; Salvatori, Francesca; Borgatti, Monica; Lampronti, Ilaria; Bresciani, Alberto; Altamura, Sergio; Corradini, Roberto; Gambari, Roberto



Reduction of Nanoparticle Avidity Enhances the Selectivity of Vascular Targeting and PET Detection of Pulmonary Inflammation  

PubMed Central

Targeting nanoparticles (NPs) loaded with drugs and probes to precise locations in the body may improve the treatment and detection of many diseases. Generally, to achieve targeting, affinity ligands are introduced on the surface of NPs that can bind to molecules present on the cell of interest. Optimization of ligand density is a critical parameter in controlling NP binding to target cells and a higher ligand density is not always the most effective. In this study, we investigated how NP avidity affects targeting to the pulmonary vasculature, using NPs targeted to ICAM-1. This cell adhesion molecule is expressed by quiescent endothelium at modest levels and is upregulated in a variety of pathological settings. NP avidity was controlled by ligand density, with the expected result that higher avidity NPs demonstrated greater pulmonary uptake than lower avidity NPs in both naïve and pathological mice. However, in comparison with high avidity NPs, low avidity NPs exhibited several-fold higher selectivity of targeting to pathological endothelium. This finding was translated into a PET imaging platform that was more effective in detecting pulmonary vascular inflammation using low avidity NPs. Furthermore, computational modeling revealed that elevated expression of ICAM-1 on the endothelium is critical for multivalent anchoring of NPs with low avidity, while high avidity NPs anchor effectively to both quiescent and activated endothelium. These results provide a paradigm that can be used to optimize NP targeting by manipulating ligand density, and may find biomedical utility for increasing detection of pathological vasculature. PMID:23383962

Zern, Blaine J.; Chacko, Ann-Marie; Liu, Jin; Greineder, Colin F.; Blankemeyer, Eric R.; Radhakrishnan, Ravi; Muzykantov, Vladimir



Gene Transfer Strategies for Correction of Lysosomal Storage Disorders  

Microsoft Academic Search

Lysosomal storage diseases (LSDs) represent a large group of monogenic disorders of metabolism, which affect approximately 1 in 5,000 live births. LSDs result from a single or multiple deficiency of specific lysosomal hydrolases, the enzymes responsible for the luminal catabolization of macromolecular substrates. The consequent accumulation of undigested metabolites in lysosomes leads to polysystemic dysfunction, including progressive neurologic deterioration, mental

Alessandra d’Azzo



A RANKL-PKC?-TFEB signaling cascade is necessary for lysosomal biogenesis in osteoclasts  

PubMed Central

Bone resorption by osteoclasts requires a large number of lysosomes that release proteases in the resorption lacuna. Whether lysosomal biogenesis is a consequence of the action of transcriptional regulators of osteoclast differentiation or is under the control of a different and specific transcriptional pathway remains unknown. We show here, through cell-based assays and cell-specific gene deletion experiments in mice, that the osteoclast differentiation factor RANKL promotes lysosomal biogenesis once osteoclasts are differentiated through the selective activation of TFEB, a member of the MITF/TFE family of transcription factors. This occurs following PKC? phosphorylation of TFEB on three serine residues located in its last 15 amino acids. This post-translational modification stabilizes and increases the activity of this transcription factor. Supporting these biochemical observations, mice lacking in osteoclasts—either TFEB or PKC?—show decreased lysosomal gene expression and increased bone mass. Altogether, these results uncover a RANKL-dependent signaling pathway taking place in differentiated osteoclasts and culminating in the activation of TFEB to enhance lysosomal biogenesis—a necessary step for proper bone resorption. PMID:23599343

Ferron, Mathieu; Settembre, Carmine; Shimazu, Junko; Lacombe, Julie; Kato, Shigeaki; Rawlings, David J.; Ballabio, Andrea; Karsenty, Gerard



Selection Strategy to Generate Aptamer Pairs that Bind to Distinct Sites on Protein Targets  

PubMed Central

Many analytical techniques benefit greatly from the use of affinity reagent pairs, wherein each reagent recognizes a discrete binding site on a target. For example, antibody pairs have been widely used to dramatically increase the specificity of enzyme linked immunosorbent assays (ELISA). Nucleic acid-based aptamers offer many advantageous features relative to protein-based affinity reagents, including well-established chemical synthesis, thermostability and low production cost. However, the generation of suitable aptamer pairs has posed a significant challenge, and few such pairs have been reported to date. To address this important challenge, we present Multivalent Aptamer Isolation SELEX (MAI-SELEX), a technique designed for the efficient selection of aptamer pairs. In contrast to conventional selection methods, our method utilizes two selection modules to generate separate aptamer pools that recognize distinct binding sites on a single target. Using MAI-SELEX, we have isolated two groups of 2?-fluoro-modified RNA aptamers that specifically recognize the ?V or ?3 subunits of integrin ?V?3. These aptamers exhibit low nanomolar affinities for their targets, with minimal cross-reactivity to other closely related integrin homologs. Moreover, we show that these aptamer pairs do not interfere with each other’s binding, and effectively detect the target even in complex mixtures such as undiluted serum. PMID:22624874

Gong, Qiang; Wang, Jinpeng; Ahmad, Kareem M.; Csordas, Andrew; Zhou, Jiehua; Nie, Jeff; Stewart, Ron; Thomson, James A.; Rossi, John J.; Soh, H. Tom



Selective whole genome amplification for resequencing target microbial species from complex natural samples.  


Population genomic analyses have demonstrated power to address major questions in evolutionary and molecular microbiology. Collecting populations of genomes is hindered in many microbial species by the absence of a cost effective and practical method to collect ample quantities of sufficiently pure genomic DNA for next-generation sequencing. Here we present a simple method to amplify genomes of a target microbial species present in a complex, natural sample. The selective whole genome amplification (SWGA) technique amplifies target genomes using nucleotide sequence motifs that are common in the target microbe genome, but rare in the background genomes, to prime the highly processive phi29 polymerase. SWGA thus selectively amplifies the target genome from samples in which it originally represented a minor fraction of the total DNA. The post-SWGA samples are enriched in target genomic DNA, which are ideal for population resequencing. We demonstrate the efficacy of SWGA using both laboratory-prepared mixtures of cultured microbes as well as a natural host-microbe association. Targeted amplification of Borrelia burgdorferi mixed with Escherichia coli at genome ratios of 1:2000 resulted in >10(5)-fold amplification of the target genomes with <6.7-fold amplification of the background. SWGA-treated genomic extracts from Wolbachia pipientis-infected Drosophila melanogaster resulted in up to 70% of high-throughput resequencing reads mapping to the W. pipientis genome. By contrast, 2-9% of sequencing reads were derived from W. pipientis without prior amplification. The SWGA technique results in high sequencing coverage at a fraction of the sequencing effort, thus allowing population genomic studies at affordable costs. PMID:25096321

Leichty, Aaron R; Brisson, Dustin



Monitoring lysosomal activity in nanoparticle-treated cells.  


Certain nanoparticles have been shown to accumulate within lysosome and hence may cause lysosomal pathologies such as phospholipidosis, lysosomal overload, and autophagy. This chapter describes a method for evaluation of lysosomal activity in porcine kidney cells (LLC-PK1) after exposure to nanoparticles. This method uses the accumulation of a cationic fluorescent dye (LysoTracker Red) in acidic cellular compartments as an indicator of total lysosome content. The lysotracker signal is normalized to the signal from a thiol-reactive dye which is proportional to the total number of viable cells. PMID:21116970

Neun, Barry W; Stern, Stephan T



DNA tetraplex-binding drugs: structure-selective targeting is critical for antitumour telomerase inhibition.  


Four-stranded tetraplex ("G-quadruplex") DNA represents a new paradigm for the design of DNA-interactive antitumour drugs, as the formed DNA-drug complexes have been suggested to interfere with critical telomerase function. The unique structural features presented by tetraplex over duplex DNA have stimulated the design of small ligand molecules able to selectively promote the formation and/or stabilisation of such higher-order DNA structures. Current developments in tetraplex-targeted telomerase inhibitors, and importantly their DNA structural selectivity, are explored. PMID:12369989

Perry, P J; Jenkins, T C



Lorcaserin and pimavanserin: emerging selectivity of serotonin receptor subtype-targeted drugs  

PubMed Central

Serotonin (5-hydroxytryptamine, or 5-HT) receptors mediate a plethora of physiological phenomena in the brain and the periphery. Additionally, serotonergic dysfunction has been implicated in nearly every neuropsychiatric disorder. The effects of serotonin are mediated by fourteen GPCRs. Both the therapeutic actions and side effects of commonly prescribed drugs are frequently due to nonspecific actions on various 5-HT receptor subtypes. For more than 20 years, the search for clinically efficacious drugs that selectively target 5-HT receptor subtypes has been only occasionally successful. This review provides an overview of 5-HT receptor pharmacology and discusses two recent 5-HT receptor subtype–selective drugs, lorcaserin and pimavanserin, which target the 5HT2C and 5HT2A receptors and provide new treatments for obesity and Parkinson’s disease psychosis, respectively. PMID:24292660

Meltzer, Herbert Y.; Roth, Bryan L.



Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed  

NASA Astrophysics Data System (ADS)

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.

Serebrovskaya, Ekaterina O.; Ryumina, Alina P.; Boulina, Maria E.; Shirmanova, Marina V.; Zagaynova, Elena V.; Bogdanova, Ekaterina A.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.



Diverse Actions and Target-Site Selectivity of Neonicotinoids: Structural Insights  

PubMed Central

The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids. In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO2 group of imidacloprid and 2) neonicotinoid-unique stacking and CH-? bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non-? nAChR subunits. PMID:19321668

Matsuda, Kazuhiko; Kanaoka, Satoshi; Akamatsu, Miki; Sattelle, David B.



Mitochondrial permeability transition pore as a selective target for anti-cancer therapy  

PubMed Central

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

Suh, Dong H.; Kim, Mi-Kyung; Kim, Hee S.; Chung, Hyun H.; Song, Yong S.



Mechanisms of Dendritic Cell Lysosomal Killing of Cryptococcus  

NASA Astrophysics Data System (ADS)

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.

Hole, Camaron R.; Bui, Hoang; Wormley, Floyd L.; Wozniak, Karen L.



Target Region Selection Is a Critical Determinant of Community Fingerprints Generated by 16S Pyrosequencing  

Microsoft Academic Search

Pyrosequencing of 16S rRNA genes allows for in-depth characterization of complex microbial communities. Although it is known that primer selection can influence the profile of a community generated by sequencing, the extent and severity of this bias on deep-sequencing methodologies is not well elucidated. We tested the hypothesis that the hypervariable region targeted for sequencing and primer degeneracy play important

Purnima S. Kumar; Michael R. Brooker; Scot E. Dowd; Terry Camerlengo; Jonathan H. Badger



Adverse selection in a community-based health insurance scheme in rural Africa: Implications for introducing targeted subsidies  

PubMed Central

Background Although most community-based health insurance (CBHI) schemes are voluntary, problem of adverse selection is hardly studied. Evidence on the impact of targeted subsidies on adverse selection is completely missing. This paper investigates adverse selection in a CBHI scheme in Burkina Faso. First, we studied the change in adverse selection over a period of 4?years. Second, we studied the effect of targeted subsidies on adverse selection. Methods The study area, covering 41 villages and 1 town, was divided into 33 clusters and CBHI was randomly offered to these clusters during 2004–06. In 2007, premium subsidies were offered to the poor households. The data was collected by a household panel survey 2004–2007 from randomly selected households in these 33 clusters (n?=?6795). We applied fixed effect models. Results We found weak evidence of adverse selection before the implementation of subsidies. Adverse selection significantly increased the next year and targeted subsidies largely explained this increase. Conclusions Adverse selection is an important concern for any voluntary health insurance scheme. Targeted subsidies are often used as a tool to pursue the vision of universal coverage. At the same time targeted subsidies are also associated with increased adverse selection as found in this study. Therefore, it’s essential that targeted subsidies for poor (or other high-risk groups) must be accompanied with a sound plan to bridge the financial gap due to adverse selection so that these schemes can continue to serve these populations. PMID:22741549



Lysosomal network proteins as potential novel CSF biomarkers for Alzheimer's disease.  


The success of future intervention strategies for Alzheimer's disease (AD) will likely rely on the development of treatments starting early in the disease course, before irreversible brain damage occurs. The pre-symptomatic stage of AD occurs at least one decade before the clinical onset, highlighting the need for validated biomarkers that reflect this early period. Reliable biomarkers for AD are also needed in research and clinics for diagnosis, patient stratification, clinical trials, monitoring of disease progression and the development of new treatments. Changes in the lysosomal network, i.e., the endosomal, lysosomal and autophagy systems, are among the first alterations observed in an AD brain. In this study, we performed a targeted search for lysosomal network proteins in human cerebrospinal fluid (CSF). Thirty-four proteins were investigated, and six of them, early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were significantly increased in the CSF from AD patients compared with neurological controls. These results were confirmed in a validation cohort of CSF samples, and patients with no neurochemical evidence of AD, apart from increased total-tau, were found to have EEA1 levels corresponding to the increased total-tau levels. These findings indicate that increased levels of LAMP-1, LAMP-2, LC3, Rab3 and Rab7 in the CSF might be specific for AD, and increased EEA1 levels may be a sign of general neurodegeneration. These six lysosomal network proteins are potential AD biomarkers and may be used to investigate lysosomal involvement in AD pathogenesis. PMID:24101586

Armstrong, Andrea; Mattsson, Niklas; Appelqvist, Hanna; Janefjord, Camilla; Sandin, Linnea; Agholme, Lotta; Olsson, Bob; Svensson, Samuel; Blennow, Kaj; Zetterberg, Henrik; Kågedal, Katarina



Intersurgeon Variability in the Selection of Anterior and Posterior Commissures and Its Potential Effects on Target Localization  

Microsoft Academic Search

Background: This study reports the intersurgeon variability in manual selection of the anterior and posterior commissures (AC and PC). The study also investigates the effect of this variability on the localization of targets like the subthalamic nucleus, ventralis intermedius nucleus and globus pallidus internus. The additional effect of variation in the selection of the mid-plane on target localization is also

Srivatsan Pallavaram; Hong Yu; John Spooner; Pierre-François D’Haese; Bobby Bodenheimer; Peter E. Konrad; Benoit M. Dawant



The role of target elevation in prey selection by tiger beetles (Carabidae: Cicindela spp.).  


The elevation of objects in the visual field has long been recognized as a potential distance cue, but it has been demonstrated to a reasonable extent in only four species: humans, frogs, fiddler crabs and backswimmers. Many tiger beetles hunt in flat, sandy areas, and their eyes show "flat-world" adaptations, such as an extended visual streak of higher acuity that corresponds to the horizon. They are therefore possible candidates for the use of elevation as a cue for distance. We tested this empirically and with simulation. In a behavioral prey selection paradigm, in which starved beetles were presented moving prey-targets having different size, speed and elevation, the beetles showed a strong preference for large targets when these were low in the visual field and a weaker preference for small targets when these were near the horizon. Striking of targets above the horizon was reduced compared to sub-horizontal targets, and lacked the size-elevation interaction. We simulated these empirical results with a model that converted elevation to distance, and used distance to estimate the absolute size of the targets. Simulated strike probability was then determined by the similarity between this absolute size and an independently confirmed preferred prey size. The results of the simulation model matched the empirical data as well as the best statistical model of the behavioral results. While some aspects of the model, and the beetles' behavior, differ from the strict geometry of the "elevation hypothesis", our results nevertheless indicate that tiger beetles use elevation to estimate distance to prey, and that it is therefore one of the determinants of prey selection. PMID:17050844

Layne, John E; Chen, P W; Gilbert, Cole



Chlorin e6 Conjugated Interleukin-6 Receptor Aptamers Selectively Kill Target Cells Upon Irradiation  

PubMed Central

Photodynamic therapy (PDT) uses the therapeutic properties of light in combination with certain chemicals, called photosensitizers, to successfully treat brain, breast, prostate, and skin cancers. To improve PDT, current research focuses on the development of photosensitizers to specifically target cancer cells. In the past few years, aptamers have been developed to directly deliver cargo molecules into target cells. We conjugated the photosensitizer chlorin e6 (ce6) with a human interleukin-6 receptor (IL-6R) binding RNA aptamer, AIR-3A yielding AIR-3A-ce6 for application in high efficient PDT. AIR-3A-ce6 was rapidly and specifically internalized by IL-6R presenting (IL-6R+) cells. Upon light irradiation, targeted cells were selectively killed, while free ce6 did not show any toxic effect. Cells lacking the IL-6R were also not affected by AIR-3A-ce6. With this approach, we improved the target specificity of ce6-mediated PDT. In the future, other tumor-specific aptamers might be used to selectively localize photosensitizers into cells of interest and improve the efficacy and specificity of PDT in cancer and other diseases. PMID:24481022

Kruspe, Sven; Meyer, Cindy; Hahn, Ulrich



Neurologic Abnormalities in Mouse Models of the Lysosomal Storage Disorders Mucolipidosis II and Mucolipidosis III ?  

PubMed Central

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an ?2?2?2 hexameric enzyme that catalyzes the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. Mutations in the ?/? subunit precursor gene cause the severe lysosomal storage disorder mucolipidosis II (ML II) or the more moderate mucolipidosis III alpha/beta (ML III ?/?), while mutations in the ? subunit gene cause the mildest disorder, mucolipidosis III gamma (ML III ?). Here we report neurologic consequences of mouse models of ML II and ML III ?. The ML II mice have a total loss of acid hydrolase phosphorylation, which results in depletion of acid hydrolases in mesenchymal-derived cells. The ML III ? mice retain partial phosphorylation. However, in both cases, total brain extracts have normal or near normal activity of many acid hydrolases reflecting mannose 6-phosphate-independent lysosomal targeting pathways. While behavioral deficits occur in both models, the onset of these changes occurs sooner and the severity is greater in the ML II mice. The ML II mice undergo progressive neurodegeneration with neuronal loss, astrocytosis, microgliosis and Purkinje cell depletion which was evident at 4 months whereas ML III ? mice have only mild to moderate astrocytosis and microgliosis at 12 months. Both models accumulate the ganglioside GM2, but only ML II mice accumulate fucosylated glycans. We conclude that in spite of active mannose 6-phosphate-independent targeting pathways in the brain, there are cell types that require at least partial phosphorylation function to avoid lysosomal dysfunction and the associated neurodegeneration and behavioral impairments. PMID:25314316

Idol, Rachel A.; Wozniak, David F.; Fujiwara, Hideji; Yuede, Carla M.; Ory, Daniel S.; Kornfeld, Stuart; Vogel, Peter



Targeting hunter distribution based on host resource selection and kill sites to manage disease risk  

PubMed Central

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 selection and kill sites as a promising tool for applying selective hunting to the management of transmissible diseases in a game species. PMID:24324876

Dugal, Cherie J; van Beest, Floris M; Vander Wal, Eric; Brook, Ryan K



Targeting hunter distribution based on host resource selection and kill sites to manage disease risk.  


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 selection and kill sites as a promising tool for applying selective hunting to the management of transmissible diseases in a game species. PMID:24324876

Dugal, Cherie J; van Beest, Floris M; Vander Wal, Eric; Brook, Ryan K



Target Selection and Deselection at the Berkeley StructuralGenomics Center  

SciTech Connect

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 percentage of accurately modeled proteinswould rise from 50 percent (243 of 486) to 58 percent (283 of 486).Sequences and data on experimental progress on our targets are availablein the public databases Target DB and PEPCdb.

Chandonia, John-Marc; Kim, Sung-Hou; Brenner, Steven E.




SciTech Connect

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 these data, and we analyze the spectra obtained during the first year. During this year, 11,263 new z > 2.20 quasars were spectroscopically confirmed by BOSS, roughly double the number of previously known quasars with z > 2.20. Our current algorithms select an average of 15 z > 2.20 quasars deg{sup -2} from 40 targets deg{sup -2} using single-epoch SDSS imaging. Multi-epoch optical data and data at other wavelengths can further improve the efficiency and completeness of BOSS QTS.

Ross, Nicholas P.; Kirkpatrick, Jessica A.; Carithers, William C.; Ho, Shirley [Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720 (United States); Myers, Adam D. [Department of Astronomy, MC-221, University of Illinois, 1002 West Green Street, Urbana, IL 61801 (United States); Sheldon, Erin S. [Brookhaven National Laboratory, Blgd 510, Upton, NY 11375 (United States); Yeche, Christophe; Aubourg, Eric [CEA, Centre de Saclay, IRFU, 91191 Gif-sur-Yvette (France); Strauss, Michael A.; Lee, Khee-Gan [Department of Astrophysical Sciences, Princeton University, Princeton, NJ 08544 (United States); Bovy, Jo; Blanton, Michael R.; Hogg, David W. [Center for Cosmology and Particle Physics, New York University, 4 Washington Place, New York, NY 10003 (United States); Richards, Gordon T. [Department of Physics, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104 (United States); Brandt, W. N. [Department of Astronomy and Astrophysics, The Pennsylvania State University, 525 Davey Laboratory, University Park, PA 16802 (United States); Croft, Rupert A. C. [Bruce and Astrid McWilliams Center for Cosmology, Carnegie Mellon University, Pittsburgh, PA 15213 (United States); Da Silva, Robert [Department of Astronomy and Astrophysics, University of California, Santa Cruz, Santa Cruz, CA 95064 (United States); Dawson, Kyle [Department of Physics and Astronomy, University of Utah, UT (United States); Eisenstein, Daniel J. [Steward Observatory, 933 North Cherry Avenue, Tucson, AZ 85721 (United States); Hennawi, Joseph F., E-mail: [Max-Planck-Institut fuer Astronomie, Konigstuhl 17, 69117 Heidelberg (Germany); and others



The lytic granules of natural killer cells are dual-function organelles combining secretory and pre-lysosomal compartments  

PubMed Central

Cytolytic lymphocytes contain specialized lytic granules whose secretion during cell-mediated cytolysis results in target cell death. Using serial section EM of RNK-16, a natural killer cell line, we show that there are structurally distinct types of granules. Each type is composed of varying proportions of a dense core domain and a multivesicular cortical domain. The dense core domains contain secretory proteins thought to play a role in cytolysis, including cytolysin and chondroitin sulfate proteoglycan. In contrast, the multivesicular domains contain lysosomal proteins, including acid phosphatase, alpha-glucosidase, cathepsin D, and LGP-120. In addition to their protein content, the lytic granules have other properties in common with lysosomes. The multivesicular regions of the granules have an acidic pH, comparable to that of endosomes and lysosomes. The granules take up exogenous cationized ferritin with lysosome-like kinetics, and this uptake is blocked by weak bases and low temperature. The multivesicular domains of the granules are rich in the 270-kD mannose-6-phosphate receptor, a marker which is absent from mature lysosomes but present in earlier endocytic compartments. Thus, the natural killer granules represent an unusual dual-function organelle, where a regulated secretory compartment, the dense core, is contained within a pre-lysosomal compartment, the multivesicular domain. PMID:2277062



Targeted delivery of photosensitizers: efficacy and selectivity issues revealed by multifunctional ORMOSIL nanovectors in cellular systems  

NASA Astrophysics Data System (ADS)

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 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. Electronic supplementary information (ESI) available: Experimental procedures and additional characterization of nanoparticles. See DOI: 10.1039/c3nr00402c

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



Patient selection and targeted treatment in the management of platinum-resistant ovarian cancer  

PubMed Central

Ovarian cancer (OC) has the highest mortality rate of any gynecologic cancer, and patients generally have a poor prognosis due to high chemotherapy resistance and late stage disease diagnosis. Platinum-resistant OC can be treated with cytotoxic chemotherapy such as paclitaxel, topotecan, pegylated liposomal doxorubicin, and gemcitabine, but many patients eventually relapse upon treatment. Fortunately, there are currently a number of targeted therapies in development for these patients who have shown promising results in recent clinical trials. These treatments often target the vascular endothelial growth factor pathway (eg, bevacizumab and aflibercept), DNA repair mechanisms (eg, iniparib and olaparib), or they are directed against folate related pathways (eg, pemetrexed, farletuzumab, and vintafolide). As many targeted therapies are only effective in a subset of patients, there is an increasing need for the identification of response predictive biomarkers. Selecting the right patients through biomarker screening will help tailor therapy to patients and decrease superfluous treatment to those who are biomarker negative; this approach should lead to improved clinical results and decreased toxicities. In this review the current targeted therapies used for treating platinum-resistant OC are discussed. Furthermore, use of prognostic and response predictive biomarkers to define OC patient populations that may benefit from specific targeted therapies is also highlighted. PMID:24109193

Leamon, Christopher P; Lovejoy, Chandra D; Nguyen, Binh



Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing.  


RNA interference (RNAi)-mediated gene silencing has become a valuable tool for functional studies, reverse genomics, and drug discoveries. One major challenge of using RNAi is to identify the most effective short interfering RNAs (siRNAs) sites of a given gene. Although several published bioinformatic prediction models have proven useful, the process to select and validate optimal siRNA sites for a given gene remains empirical and laborious. Here, we developed a fluorescence-based selection system using a retroviral vector backbone, namely pSOS, which was based on the premise that candidate siRNAs would knockdown the chimeric transcript between GFP and target gene. The expression of siRNA was driven by the opposing convergent H1 and U6 promoters. This configuration simplifies the cloning of duplex siRNA oligonucleotide cassettes. We demonstrated that GFP signal reduction was closely correlated with siRNA knockdown efficiency of human beta-catenin, as well as with the inhibition of beta-catenin/Tcf4 signaling activity. The pSOS should not only facilitate the selection and validation of candidate siRNA sites, but also provide efficient delivery tools of siRNAs via viral vectors in mammalian cells. Thus, the pSOS system represents an efficient and user-friendly strategy to select and validate siRNA target sites. PMID:17449199

Luo, Qing; Kang, Quan; Song, Wen-Xin; Luu, Hue H; Luo, Xiaoji; An, Naili; Luo, Jinyong; Deng, Zhong-Liang; Jiang, Wei; Yin, Hong; Chen, Jin; Sharff, Katie A; Tang, Ni; Bennett, Erwin; Haydon, Rex C; He, Tong-Chuan



Newborn screening for lysosomal storage disorders.  


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

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



Lysosomal dysfunction results in altered energy balance.  


The mucopolysaccharidosis (MPS) type VII mouse was originally described as the adipose storage deficiency mouse because of its extreme lean phenotype of unknown etiology. Here, we show that adipose storage deficiency and lower leptin levels are common to five different lysosomal storage diseases (LSDs): MPSI, MPSIIIB, MPSVII, Niemann-Pick type A/B, and infantile neuronal ceroid lipofuscinosis. Elevated circulating pro-inflammatory proteins (VCAM1 and MCP1) were found in multiple LSDs. Multiple anti-inflammatory strategies (dexamethasone, MCP1 deficiency, M3 expression) failed to alter adiposity in LSD animals. All of the models had normal or greater caloric intake and lower to normal metabolic rate, fasting plasma glucose, non-esterified fatty acids, cholesterol, and triglycerides. Triglycerides were lower in the livers of MPSI mice, and the trend was lower in the muscle. Lipid absorption and processing in MPSI mice were indistinguishable from those in normal mice following oral gavage of olive oil. The increased lean mass of MPSI and MPSIIIB mice suggests a shift in adipose triglycerides to lysosomal storage. In agreement, MPSI livers had a similar total caloric content but reduced caloric density, indicating a shift in energy from lipids to proteins/carbohydrates (lysosomal storage). Enzyme replacement therapy normalized the caloric density within 48 h without reducing total caloric content. This was due to an increase in lipids. Recycling of stored material is likely reduced or nonexistent. Therefore, to maintain homeostasis, energy is likely diverted to synthesis at the expense of typical energy storage depots. Thus, these diseases will serve as important tools in studying the role of lysosome function in metabolism and obesity. PMID:17911106

Woloszynek, Josh C; Coleman, Trey; Semenkovich, Clay F; Sands, Mark S



Lysosomal and mitochondrial permeabilization mediates zinc(II) cationic phthalocyanine phototoxicity.  


In order to find a novel photosensitizer to be used in photodynamic therapy for cancer treatment, we have previously showed that the cationic zinc(II) phthalocyanine named Pc13, the sulfur-linked dye 2,9(10),16(17),23(24)-tetrakis[(2-trimethylammonium) ethylsulfanyl]phthalocyaninatozinc(II) tetraiodide, exerts a selective phototoxic effect on human nasopharynx KB carcinoma cells and induces an apoptotic response characterized by an increase in the activity of caspase-3. Since the activation of an apoptotic pathway by chemotherapeutic agents contributes to the elimination of malignant cells, in this study we investigated the molecular mechanisms underlying the antitumor action of Pc13. We found that after light exposure, Pc13 induced the production of reactive oxygen species (ROS), which are mediating the resultant cytotoxic action on KB cells. ROS led to an early permeabilization of lysosomal membranes as demonstrated by the reduction of lysosome fluorescence with acridine orange and the release of lysosomal proteases to cytosol. Treatment with antioxidants inhibited ROS generation, preserved the integrity of lysosomal membrane and increased cell proliferation in a concentration-dependent manner. Lysosome disruption was followed by mitochondrial depolarization, cytosolic release of cytochrome C and caspases activation. Although no change in the total amount of Bax was observed, the translocation of Bax from cytosol to mitochondria, the cleavage of the pro-apoptotic protein Bid, together with the decrease of the anti-apoptotic proteins Bcl-XL and Bcl-2 indicated the involvement of Bcl-2 family proteins in the induction of the mitochondrial pathway. It was also demonstrated that cathepsin D, but not caspase-8, contributed to Bid cleavage. In conclusion, Pc13-induced cell photodamage is triggered by ROS generation and activation of the mitochondrial apoptotic pathway through the release of lysosomal proteases. In addition, our results also indicated that Pc13 induced a caspase-dependent apoptotic response, being activation of caspase-8, -9 and -3 the result of a post-mitochondrial event. PMID:23994488

Marino, Julieta; García Vior, María C; Furmento, Verónica A; Blank, Viviana C; Awruch, Josefina; Roguin, Leonor P



Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes  

PubMed Central

Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways. PMID:25337829

Lopez Sanjurjo, Cristina I.; Tovey, Stephen C.; Taylor, Colin W.



Dried blood spots for the enzymatic diagnosis of lysosomal storage diseases in dogs and cats  

PubMed Central

Background In people lysosomal storage diseases (LSD) can be diagnosed by assaying enzyme activities in dried blood spots (DBS). Objective The aim of this study was to evaluate the feasibility of using DBS samples from dogs and cats to measure lysosomal enzymatic activities and diagnose LSD. Methods Drops of fresh whole blood collected in EDTA from dogs and cats with known or suspected LSD and from clinically healthy dogs and cats were placed on neonatal screening cards, dried, and mailed to the Metabolic Laboratory, University Children’s Hospital, Frankfurt, Germany. Activities of selected lysosomal enzymes were measured using fluorescent substrates in a 2-mm diameter disk (~2.6 ?L blood) punched from the DBS. Results were expressed as nmol substrate hydrolyzed per mL of blood per minute or hour. Results Reference values were established for several lysosomal enzyme activities in DBS from dogs and cats; for most enzymes, activities were higher than those published for human samples. Activities of ?-glucuronidase, N-acetylglucosamine-4-sulfatase (arylsulfatase B), ?-mannosidase, ?-galactosidase, ?-fucosidase, and hexosaminidase A were measureable in DBS from healthy cats and dogs; ?-iduronidase activity was measureable only in cats. In samples from animals with LSD, markedly reduced activity of a specific enzyme was found. In contrast, in samples from cats affected with mucolipidosis II activities of lysosomal enzymes were markedly increased. Conclusions Measurement of lysosomal enzyme activities in DBS provides an inexpensive, simple, and convenient method to screen animals for suspected LSD and requires only a small sample volume. For diseases in which the relevant enzyme activity can be measured in DBS, a specific diagnosis can be made. PMID:23121383

Sewell, Adrian C.; Haskins, Mark E.; Giger, Urs



Theater targets plume edge extraction and hardbody aimpoint selection using morphological image processing  

NASA Astrophysics Data System (ADS)

(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.

Paiva, Clifford A.



In vivo phage display selection of an ovarian cancer targeting peptide for SPECT/CT imaging.  


The often fatal outcome of ovarian cancer (OC) is related to inadequate detection methods, which may be overcome by development of nuclear imaging agents. Cancer targeting peptides have been identified using in vivo bacteriophage (phage) display technology; however, the majority of these ligands target tumor vasculature. To overcome this problem, a two-tier phage display method was employed to select an ovarian cancer targeting peptide with good pharmacokinetic and imaging properties. A fUSE5 15-amino acid peptide library was screened against xenografted human OC SKOV-3 tumors in mice, which was followed by selection against enriched SKOV-3 cells. The selected peptide RSLWSDFYASASRGP (J18) was synthesized with a GSG-spacer and a 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) chelator and radiolabeled with (111)In. SKOV-3 xenografted mice were used to evaluate the biodistribution and single photon emission computed tomography (SPECT) imaging capabilities of the radiolabeled peptide. Competitive binding experiments using (111)In-DOTA-GSG-J18 indicated that the peptide displayed a half maximal inhibitory concentration (IC50) value of 10.5 ± 1.1 ?M. Biodistribution studies revealed that tumor uptake was 1.63 ± 0.68, 0.60 ± 0.32, 0.31 ± 0.12 and 0.10 ± 0.02% injected dose/g at 30 min, 1 h, 2 h and 4 h post-injection of (111)In-DOTA-GSG-J18, respectively. SPECT/CT imaging demonstrated good tumor uptake and minimal background binding. This study demonstrated successful utilization of a two-tier phage display selection process to identify an ovarian cancer avid peptide with excellent SPECT/CT imaging capabilities. PMID:25250205

Soendergaard, Mette; Newton-Northup, Jessica R; Deutscher, Susan L



In vivo phage display selection of an ovarian cancer targeting peptide for SPECT/CT imaging  

PubMed Central

The often fatal outcome of ovarian cancer (OC) is related to inadequate detection methods, which may be overcome by development of nuclear imaging agents. Cancer targeting peptides have been identified using in vivo bacteriophage (phage) display technology; however, the majority of these ligands target tumor vasculature. To overcome this problem, a two-tier phage display method was employed to select an ovarian cancer targeting peptide with good pharmacokinetic and imaging properties. A fUSE5 15-amino acid peptide library was screened against xenografted human OC SKOV-3 tumors in mice, which was followed by selection against enriched SKOV-3 cells. The selected peptide RSLWSDFYASASRGP (J18) was synthesized with a GSG-spacer and a 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) chelator and radiolabeled with 111In. SKOV-3 xenografted mice were used to evaluate the biodistribution and single photon emission computed tomography (SPECT) imaging capabilities of the radiolabeled peptide. Competitive binding experiments using 111In-DOTA-GSG-J18 indicated that the peptide displayed a half maximal inhibitory concentration (IC50) value of 10.5 ± 1.1 ?M. Biodistribution studies revealed that tumor uptake was 1.63 ± 0.68, 0.60 ± 0.32, 0.31 ± 0.12 and 0.10 ± 0.02% injected dose/g at 30 min, 1 h, 2 h and 4 h post-injection of 111In-DOTA-GSG-J18, respectively. SPECT/CT imaging demonstrated good tumor uptake and minimal background binding. This study demonstrated successful utilization of a two-tier phage display selection process to identify an ovarian cancer avid peptide with excellent SPECT/CT imaging capabilities.

Soendergaard, Mette; Newton-Northup, Jessica R; Deutscher, Susan L



Target Selection for the LBTI Hunt for Observable Signatures of Terrestrial Planetary Systems  

NASA Astrophysics Data System (ADS)

The Hunt for Observable Signatures of Terrestrial planetary Systems (HOSTS) on the Large Binocular Telescope Interferometer (LBTI) will survey nearby stars for faint exozodiacal dust (exozodi). This warm circumstellar dust, analogous to the interplanetary dust found in the vicinity of the Earth in our own system, is produced in comet breakups and asteroid collisions. Exozodi will be the major source of astrophysical noise for a future space telescope aimed at direct imaging and spectroscopy of habitable zone terrestrial planets (exo-Earths). About 20% of nearby field stars have cold dust coming from planetesimals at large distances from the stars (Eiroa et al. 2013). Much less is known about exozodi; current detection limits for individual stars are at best ~ 500 times our solar system's level (aka. 500 zodi). LBTI-HOSTS will be the first survey capable of measuring exozodi at the 10 zodi level (3s). Detections of warm dust will also reveal new information about planetary system architectures and evolution. We describe the target star selection by the LBTI Science Team to satisfy the goals of the HOSTS survey - to inform mission design and target selection for a future exo-Earth mission. We are interested in both 1) actual stars likely to be observed by such a mission and 2) stars whose observation will enable sensible extrapolations for stars that cannot be observed with LBTI. We integrated two approaches to generate the HOSTS target list. The mission-driven approach concentrates on F, G, and K-type stars that are the best targets for future direct observations of exo-Earths, thereby providing model-independent "ground truth" dust observations. However, not every potential target of a future exo-Earth mission can be observed with LBTI. The sensitivity-driven approach selects targets based on maximizing the exozodi sensitivity that can be achieved, without consideration of exo-Earth mission constraints. This naturally chooses more luminous stars (A and early F-type stars). In both cases, all stars are close enough to Earth such that their habitable zones are resolvable by LBTI and bright enough at N-band (10 µm) to provide excellent sensitivity. We also discuss observational and astrophysical motivations for excluding binaries of certain separations.

Roberge, A.; Weinberger, A.; Kennedy, G.; Defrère, D.; LBTI Instrument; Science Teams



Gold nanorods for target selective SPECT/CT imaging and photothermal therapy in vivo  

PubMed Central

The development of theranostic agents with high detection sensitivity and antitumor efficacy at low concentration is a challenging task for target selective imaging and therapy of cancers. In this study, folate-conjugated and radioactive-iodine-labeled gold nanorods (GNRs) were designed and synthesized for target selective SPECT/CT imaging and subsequent thermal ablation of folate-receptor-overexpressing cancers. Both (ortho-pyridyl) disulfide-poly(ethylene glycol)-folate and a short peptide, H2N-Tyr-Asn-Asn-Leu-Ala-Cys-OH, were conjugated on the surface of the GNRs through thiol chemistry. The tyrosine in the peptide sequence was introduced for radioactive-iodine labeling through an iodine-tyrosine interaction. The labeling efficiency of radioactive iodine was more than 99%. Radiochemical stability tests on iodine-125-labeled GNRs in human serum showed that 91% of the iodine-125 remained intact on the GNRs after incubation for 24 h. In vitro and in vivo results in this study confirmed the potential utility of folate-conjugated and iodine-125-labeled GNRs as smart theranostic agents. This type of platform may also be useful for the targeted SPECT/CT imaging and photothermal therapy of inflammatory diseases such as atherosclerosis and arthritis, in which folate-receptor-overexpressing macrophages play pivotal roles. PMID:23256055

Jang, Boseung; Park, Seonhwa; Kang, Se Hun; Kim, Joa Kyum; Kim, Seok-Ki; Kim, In-Hoo; Choi, Yongdoo



Selection Sampling from Large Data Sets for Targeted Inference in Mixture Modeling  

PubMed Central

One of the challenges in using Markov chain Monte Carlo for model analysis in studies with very large datasets is the need to scan through the whole data at each iteration of the sampler, which can be computationally prohibitive. Several approaches have been developed to address this, typically drawing computationally manageable subsamples of the data. Here we consider the specific case where most of the data from a mixture model provides little or no information about the parameters of interest, and we aim to select subsamples such that the information extracted is most relevant. The motivating application arises in flow cytometry, where several measurements from a vast number of cells are available. Interest lies in identifying specific rare cell subtypes and characterizing them according to their corresponding markers. We present a Markov chain Monte Carlo approach where an initial subsample of the full dataset is used to guide selection sampling of a further set of observations targeted at a scientifically interesting, low probability region. We define a Sequential Monte Carlo strategy in which the targeted subsample is augmented sequentially as estimates improve, and introduce a stopping rule for determining the size of the targeted subsample. An example from flow cytometry illustrates the ability of the approach to increase the resolution of inferences for rare cell subtypes. PMID:20865145

Manolopoulou, Ioanna; Chan, Cliburn; West, Mike



PITPs as Targets for Selectively Interfering With Phosphoinositide Signaling in Cells  

PubMed Central

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

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.



The anticancer effect of cytotoxin 1 from Naja atra Cantor venom is mediated by a lysosomal cell death pathway involving lysosomal membrane permeabilization and cathepsin B release.  


The cytotoxin family of cobra venom proteins, also called cardiotoxins, can activate both necrotic and apoptotic cell death pathways in cancer cells. Cytotoxin 1 (CTX1)from Naja atra Cantor venom is a 60 amino acid, 6698 Da protein with as yet untested anticancer efficacy and cell selectivity. We tested the toxicity of CTX1 on a number of cancer cell lines (MCF-7, P388, K562, and H22) and on one normal human cell line (16HBE). The rank order of cytotoxicity was MCF-7 > P388 ? K562 >H22 ? 16HBE, indicating that the effect of CTX1 on certain cancer cell types was relatively selective.Treatment with CTX1 greatly prolonged the survival of P388 ascites tumors bearing KM mice compared to cyclophosphamide treatment. Cell viability, apoptosis, and lysosomal permeability assays all demonstrated that CTX1 induced dose- and time-dependent cell death, with most cells exhibiting the morphological and biochemical features of late apoptosis and necrosis. Mitochondrial membrane potential was lost in CTX1-treated P388 cells. In addition, CTX1 induced an increase in both lysosomal membrane permeability and cathepsin B protease activity. These analyses reveal that CTX1 possesses significant and selective anticancer activity, likely by inducing programmed cell death through mitochondrial and/or lysosomal pathways. PMID:23711147

Wu, Minyan; Ming, Wei; Tang, Ya; Zhou, Shengming; Kong, Tianhan; Dong, Weihua



Lysosomal NEU1 deficiency affects Amyloid Precursor Protein levels and amyloid-? secretion via deregulated lysosomal exocytosis  

PubMed Central

Alzheimer’s disease (AD) belongs to a category of adult neurodegenerative conditions which are associated with intracellular and extracellular accumulation of neurotoxic protein aggregates. Understanding how these aggregates are formed, secreted and propagated by neurons has been the subject of intensive research, but so far no preventive or curative therapy for AD is available and clinical trials have been largely unsuccessful. Here we show that deficiency of the lysosomal sialidase NEU1 leads to the spontaneous occurrence of an AD-like amyloidogenic process in mice. This involves two consecutive events linked to NEU1 loss-of-function – accumulation and amyloidogenic processing of an oversialylated amyloid precursor protein in lysosomes, and extracellular release of A?-peptides by excessive lysosomal exocytosis. Furthermore, cerebral injection of NEU1 in an established AD mouse model substantially reduces ?-amyloid plaques. Our findings identify an additional pathway for the secretion of A? and define NEU1 as a potential therapeutic molecule for AD. PMID:24225533

Annunziata, Ida; Patterson, Annette; Helton, Danielle; Hu, Huimin; Moshiach, Simon; Gomero, Elida; Nixon, Ralph; d'Azzo, Alessandra



Lysosomal NEU1 deficiency affects amyloid precursor protein levels and amyloid-? secretion via deregulated lysosomal exocytosis.  


Alzheimer's disease (AD) belongs to a category of adult neurodegenerative conditions, which are associated with intracellular and extracellular accumulation of neurotoxic protein aggregates. Understanding how these aggregates are formed, secreted and propagated by neurons has been the subject of intensive research, but so far no preventive or curative therapy for AD is available, and clinical trials have been largely unsuccessful. Here we show that deficiency of the lysosomal sialidase NEU1 leads to the spontaneous occurrence of an AD-like amyloidogenic process in mice. This involves two consecutive events linked to NEU1 loss-of-function--accumulation and amyloidogenic processing of an oversialylated amyloid precursor protein in lysosomes, and extracellular release of A? peptides by excessive lysosomal exocytosis. Furthermore, cerebral injection of NEU1 in an established AD mouse model substantially reduces ?-amyloid plaques. Our findings identify an additional pathway for the secretion of A? and define NEU1 as a potential therapeutic molecule for AD. PMID:24225533

Annunziata, Ida; Patterson, Annette; Helton, Danielle; Hu, Huimin; Moshiach, Simon; Gomero, Elida; Nixon, Ralph; d'Azzo, Alessandra



The transcription factor NRSF contributes to epileptogenesis by selective repression of a subset of target genes.  


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. PMID:25117540

McClelland, Shawn; Brennan, Gary P; Dubé, Celine; Rajpara, Seeta; Iyer, Shruti; Richichi, Cristina; Bernard, Christophe; Baram, Tallie Z



Trihydroxamate Siderophore-Fluoroquinolone Conjugates are Selective Sideromycin Antibiotics that Target Staphylococcus aureus  

PubMed Central

Siderophores are multidentate iron(III) chelators used by bacteria for iron assimilation. Sideromycins, also called siderophore-antibiotic conjugates, are a unique subset of siderophores that enter bacterial cells via siderophore uptake pathways and deliver the toxic antibiotic in a ‘Trojan Horse’ fashion. Sideromycins represent a novel antibiotic delivery technology with untapped potential for developing sophisticated microbe-selective antibacterial agents that limit the emergence of bacterial resistance. The chemical synthesis of a series of mono-, bis-, and trihydroxamate sideromycins are described here along with their biological evaluation in antibacterial susceptibility assays. The linear hydroxamate siderophores used for the sideromycins in this study were derived from the ferrioxamine family and inspired by the naturally occurring salmycin sideromycins. The antibacterial agents used were a ?-lactam carbacepholosporin, Lorabid®, and a fluoroquinolone, ciprofloxacin, chosen for the different locations of their biological targets, the periplasm (extracellular) and the cytoplasm (intracellular). The linear hydroxamate-based sideromycins were selectively toxic towards Gram-positive bacteria, especially Staphylococcus aureus SG511 (MIC = 1.0 µM for the trihydroxamate-fluoroquinolone sideromycin). Siderophore-sideromycin competition assays demonstrated that only the fluoroquinolone sideromycins required membrane transport to reach their cytoplasmic biological target and that a trihydroxamate siderophore backbone was required for protein-mediated active transport of the sideromycins into S. aureus cells via siderophore uptake pathways. This work represents a comprehensive study of linear hydroxamate sideromycins and teaches how to build effective hydroxamate-based sideromycins as Gram-positive selective antibiotic agents. PMID:23350642

Wencewicz, Timothy A.; Long, Timothy E.; Mollmann, Ute; Miller, Marvin J.



Correlation between OFF and ON channels underlies dark target selectivity in an insect visual system.  


In both vertebrates and invertebrates, evidence supports separation of luminance increments and decrements (ON and OFF channels) in early stages of visual processing (Hartline, 1938; Joesch et al., 2010); however, less is known about how these parallel pathways are recombined to encode form and motion. In Drosophila, genetic knockdown of inputs to putative ON and OFF pathways and direct recording from downstream neurons in the wide-field motion pathway reveal that local elementary motion detectors exist in pairs that separately correlate contrast polarity channels, ON with ON and OFF with OFF (Joesch et al., 2013). However, behavioral responses to reverse-phi motion of discrete features reveal additional correlations of the opposite signs (Clark et al., 2011). We here present intracellular recordings from feature detecting neurons in the dragonfly that provide direct physiological evidence for the correlation of OFF and ON pathways. These neurons show clear polarity selectivity for feature contrast, responding strongly to targets that are darker than the background and only weakly to dark contrasting edges. These dark target responses are much stronger than the linear combination of responses to ON and OFF edges. We compare these data with output from elementary motion detector-based models (Eichner et al., 2011; Clark et al., 2011), with and without stages of strong center-surround antagonism. Our data support an alternative elementary small target motion detector model, which derives dark target selectivity from the correlation of a delayed OFF with an un-delayed ON signal at each individual visual processing unit (Wiederman et al., 2008, 2009). PMID:23926274

Wiederman, Steven D; Shoemaker, Patrick A; O'Carroll, David C



A gene with major phenotypic effects as a target for selection vs. homogenizing gene flow.  


Genes with major phenotypic effects facilitate quantifying the contribution of genetic vs. plastic effects to adaptive divergence. A classical example is Ectodysplasin (Eda), the major gene controlling lateral plate phenotype in three-spined stickleback. Completely plated marine stickleback populations evolved repeatedly towards low-plated freshwater populations, representing a prime example of parallel evolution by natural selection. However, many populations remain polymorphic for lateral plate number. Possible explanations for this polymorphism include relaxation of selection, disruptive selection or a balance between divergent selection and gene flow. We investigated 15 polymorphic stickleback populations from brackish and freshwater habitats in coastal North-western Europe. At each site, we tracked changes in allele frequency at the Eda gene between subadults in fall, adults in spring and juveniles in summer. Eda genotypes were also compared for body size and reproductive investment. We observed a fitness advantage for the Eda allele for the low morph in freshwater and for the allele for the complete morph in brackish water. Despite these results, the differentiation at the Eda gene was poorly correlated with habitat characteristics. Neutral population structure was the best predictor of spatial variation in lateral plate number, suggestive of a substantial effect of gene flow. A meta-analysis revealed that the signature of selection at Eda was weak compared to similar studies in stickleback. We conclude that a balance between divergent selection and gene flow can maintain stickleback populations polymorphic for lateral plate number and that ecologically relevant genes may not always contribute much to local adaptation, even when targeted by selection. PMID:24192132

Raeymaekers, Joost A M; Konijnendijk, Nellie; Larmuseau, Maarten H D; Hellemans, Bart; De Meester, Luc; Volckaert, Filip A M



Selection of target mutation in rat gastrointestinal tract E. coli by minute dosage of enrofloxacin  

PubMed Central

It has been suggested that bacterial resistance is selected within a mutation selection window of antibiotics. More recent studies showed that even extremely low concentration of antibiotic could select resistant bacteria in vitro. Yet little is known about the exact antibiotic concentration range that can effectively select for resistant organisms in animal gastrointestinal (GI) tract. In this study, the effect of different dosages of enrofloxacin on resistance and mutation development in rat GI tract E. coli was investigated by determining the number of resistant E. coli recoverable from rat fecal samples. Our data showed that high dose antibiotic treatment could effectively eliminate E. coli with single gyrA mutation in the early course of treatment, yet the eradication effects diminished upon prolonged treatment. Therapeutic and sub-therapeutic dose (1/10 and 1/100 of therapeutic doses) of enrofloxacin could effectively select for mutation in GI tract E. coli at the later course of enrofloxacin treatment and during the cessation periods. Surprisingly, very low dose of enrofloxacin (1/1000 therapeutic dose) could also select for mutation in GI tract E. coli at the later course of enrofloxacin treatment, only with slightly lower efficiency. No enrofloxacin-resistant E. coli could be selected at all test levels of enrofloxacin during long term treatment and the strength of antibiotic treatment does not alter the overall level of E. coli in rat GI tract. This study demonstrated that long term antibiotic treatment seems to be the major trigger for the development of target mutations in GI tract E. coli, which provided insight into the rational use of antibiotics in animal husbandry. PMID:25237308

Lin, Dachuan; Chen, Kaichao; Li, Ruichao; Liu, Lizhang; Guo, Jiubiao; Yao, Wen; Chen, Sheng



Targeted insertion of foreign genes into the tobacco plastid genome without physical linkage to the selectable marker gene  

SciTech Connect

To determine whether targeted DNA insertion into the tobacco plastid genome can be obtained without physical linkage to a selectable marker gene, we carried out biolistic transformation of chloroplasts in tobacco leaf segments with a 1:1 mix of two independently targeted antibiotic resistance genes. Plastid transformants were selected by spectinomycin resistance due to expression of an integrated aadA gene. Integration of the unselected kanamycin resistance (kan) gene into the same plastid genome was established by Southern probing in {approx}20% of the spectinomycin-selected clones. Efficient cotransformation will facilitate targeted plastid genome modification without physical linkage to a marker gene. 26 refs., 5 figs., 1 tab.

Carrer, H.; Maliga, P. [State Univ. of New Jersey, Piscataway, NJ (United States)] [State Univ. of New Jersey, Piscataway, NJ (United States)



Plasma chitotriosidase activity in children with lysosomal storage disorders.  


Chitotriosidase (ChT) is an enzyme that is selectively activated in tissue macrophage. This property of ChT makes it a potential marker for many disease process and prognostication. Present study has been carried out to know the significance of ChT as a screening marker in lysosomal storage disorders (LSDs) where tissue macrophage activation is commonly observed due to accumulation of substrate in various organs of the body. Study comprises of 20 healthy children in the age range of 10 days to 5 yrs and 56 children in the age range of 2.5 months to 13 yrs with regression of milestones, skeletal dysplasia, neuroregression and hepatosplenomegaly were selected for plasma ChT who had confirmed LSDs as carried out by specific lysosomal enzyme study from the leukocytes or fibroblasts. Plasma ChT was 55.21 +/- 20.81 nmol/ml/hr in twenty healthy age matched controls. Plasma ChT level was 42.88 to 79.78 nmol/ml/hr in thirteen of 56 (23.21%) children with LSDs like Morquio-B, Pompe, Metachromatic leucodystrophy (MLD), Sandhoff and Niemann-Pick disease type C (NPD-C). While in 43 (76.78%) children it was in the range of 213.74 to 23,511.40 nmol/ml/hr. who had LSDs like Morquio-B, Sly syndrome, MLD, GM2 Gangliosidosis, NPD-A/B and Gaucher disease (GD). Marked elevated ChT (4,000 to 23,511 nmol/ml/hr) was observed in all cases of GD (n=7) and NDP-A/B. It can be concluded from the present study that moderately raised activity of ChT can be utilized as a positive predictive test for certain LSD's. Those with marked elevated ChT have confirmed GD or NPD-A/B making it a strong screening marker for this group of diseases. PMID:19936666

Sheth, Jayesh J; Sheth, Frenny J; Oza, Nrupesh J; Gambhir, Prakash S; Dave, Usha P; Shah, Raju C



The gaia survey contribution to EChO target selection and characterization  

NASA Astrophysics Data System (ADS)

The scientific output of the proposed EChO mission (in terms of spectroscopic characterization of the atmospheres of transiting extrasolar planets) will be maximized by a careful selection of targets and by a detailed characterization of the main physical parameters (such as masses and radii) of both the planets and their stellar hosts. To achieve this aim, the availability of high-quality data from other space-borne and ground-based programs will play a crucial role. Here we identify and discuss the elements of the Gaia catalogue that will be of utmost relevance for the selection and characterization of transiting planet systems to be observed by the proposed EChO mission.

Sozzetti, Alessandro; Damasso, Mario



The RNA Polymerase II Trigger Loop Functions in Substrate Selection and is Directly Targeted by ?-amanitin  

PubMed Central

Summary Structural, biochemical and genetic studies have led to proposals that a mobile element of multi-subunit RNA polymerases, the Trigger Loop (TL), plays a critical role in catalysis and can be targeted by antibiotic inhibitors. Here we present evidence that the Saccharomyces cerevisiae RNA Polymerase II (Pol II) TL participates in substrate selection. Amino acid substitutions within the Pol II TL preferentially alter substrate usage and enzyme fidelity, as does inhibition of transcription by ?-amanitin. Finally, substitution of His1085 in the TL specifically renders Pol II highly resistant to ?-amanitin, indicating a functional interaction between His1085 and ?-amanitin that is supported by re-refinement of an ?-amanitin-Pol II crystal structure. We propose that ?-amanitin inhibited Pol II elongation, which is slow and exhibits reduced substrate selectivity, results from direct ?-amanitin interference with the TL. PMID:18538653

Kaplan, Craig D.; Larsson, Karl-Magnus; Kornberg, Roger D.



The diffusion properties of ion implanted species in selected target materials  

SciTech Connect

Experiments important to the future success of the Holifield Radioactive Ion Beam Facility (HRIBF) are in progress at the Oak Ridge National Laboratory which are designed to select the most appropriate target material for generating a particular radioactive ion beam (RIB). The 25-MV HHIRF 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 {open_quotes}on-line{close_quotes} at the UNISOR facility. The intensity versus time of implanted species, which diffuse from the high-temperature target material ({approximately}1700{degrees}C) and are ionized in the FEBIAD ion source, is used to determine release times for a particular projectile/target material combination. From such release data, diffusion coefficients can be derived by fitting the theoretical results obtained by computational solution of Fick`s second equation to experimental data. The diffusion coefficient can be used subsequently to predict the release properties of the particular element from the same material in other target geometries and at other temperatures, provided that the activation energy is also known. Diffusion coefficients for Cl 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. Brief descriptions of the experimental apparatus and procedures utilized in the present experiments and plans for future related experiments are presented.

Alton, G.D. [Oak Ridge National Lab., TN (United States); Dellwo, J.; Carter, H.K.; Kormicki, J.; Bartolo, G. di; Batchelder, J.C.; Breitenbach, J.; Chediak, J.A.; Jentoff-Nilsen, K.; Ichikawa, S.



Linking Albinism and Immunity: The Secrets of Secretory Lysosomes  

NSDL National Science Digital Library

Lysosomes are membrane-bound organelles that are found in all mammalian cells and contain hydrolases and lipases required for protein and membrane degradation. In many cells of the immune system, lysosomes also contain secretory proteins that can be released by regulated exocytosis in response to an external stimulus, providing different cell types with a wide range of effector functions. Melanosomes also use a lysosome-related organelle to secrete melanin for pigmentation. Links between albinism and immunity in patients have uncovered a number of key proteins required for lysosomal secretion and have revealed a versatile secretory mechanism that can be fine-tuned by distinct interactions in different cell types.

Jane Stinchcombe (Sir William Dunn School of Pathology;); Giovanna Bossi (Sir William Dunn School of Pathology;); Gillian Griffiths (Sir William Dunn School of Pathology;)



Selection, characterization, and biosensing application of high affinity congener-specific microcystin-targeting aptamers.  


The efficiency of current microcystin detection methods has been hampered by the low detection limits required in drinking water and that routine detection is restricted to a few of the congeners with high degree of undesired cross-reactivity. Here, we report the development of novel microcystin-targeting molecules and their application in microcystin detection. We have selected DNA aptamers from a diverse random library that exhibit high affinity and specificity to microcystin-LR, -YR, and -LA. We obtained aptamers that bind to all chosen congeners with high affinity with K(D) ranging from 28 to 60 nM. More importantly, we also obtained aptamers that are selective among the different congeners, with selectivity from 3-folds difference in binding affinity to total discrimination (K(D) of 50 nM versus nonspecific binding). Electrochemical aptasensors constructed with the selected aptamers were able to achieve sensitive and congener-specific microcystin detection with detection limit as low as 10 pM. PMID:22958101

Ng, Andy; Chinnappan, Raja; Eissa, Shimaa; Liu, Hechun; Tlili, Chaker; Zourob, Mohammed



Clinical neurogenetics: neuropathic lysosomal storage disorders.  


The lysosomal storage disorders 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. Most are associated with neurologic features. Later-onset forms are often misdiagnosed as symptoms, which might include psychiatric manifestations, are slowly progressive, and may precede other neurologic or systemic features. Symptomatic care, which remains the mainstay for most subtypes, can lead to significant improvement in quality of life. PMID:24176423

Pastores, Gregory M; Maegawa, Gustavo H B



Selective targeting of liver cancer with the endothelial marker CD146  

PubMed Central

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

Thomann, Stefan; Longerich, Thomas; Bazhin, Alexandr V.; Mier, Walter; Schemmer, Peter; Ryschich, Eduard



Phosphatidylserine-selective targeting and anticancer effects of SapC-DOPS nanovesicles on brain tumors  

PubMed Central

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

Blanco, Victor M.; Chu, Zhengtao; Vallabhapurapu, Subrahmanya D.; Sulaiman, Mahaboob K.; Kendler, Ady; Rixe, Olivier; Warnick, Ronald E.; Franco, Robert S.; Qi, Xiaoyang



Multifunctional envelope-type nano device for controlled intracellular trafficking and selective targeting in vivo.  


Nanomedicine is expected to be a basic technology for using nucleic acids as a drug, in which treating the cause of diseases represent the ultimate therapy. However, a sophisticated delivery system is required for efficient delivery of RNA/DNA, since these compounds need precise control of intracellular trafficking as well as biodistribution. Here we report on the use of a multifunctional envelope-type nano device (MEND) which is capable of intracellular trafficking such as endosomal escape, delivery to mitochondria, as well as active targeting to selective tissues/cells in vivo. In this review, we focused on the controlled intracellular trafficking of antigens for advanced immunotherapy, and then introduced a mitochondrial delivery system as an organelle targeting system for unmet medical needs. We also provide a successful in vivo delivery of siRNA to the liver based on a newly designed pH-responsive cationic lipid. Finally we will discuss an important role of an active targeting system using a peptide ligand to adipose vasculature. These progresses in drug delivery system will break through the barriers exist in our body, tissues and cells and open a window for future Nanomedicine. PMID:24794902

Kajimoto, Kazuaki; Sato, Yusuke; Nakamura, Takashi; Yamada, Yuma; Harashima, Hideyoshi



Positive-negative selection gene targeting with the diphtheria toxin A-chain gene in mouse embryonic stem cells  

Microsoft Academic Search

The diphtheria toxin A-chain gene was used in a positive-negative selection gene targeting vector to alter the CD4 gene which is transcriptionally silent in mouse embryonic stem cells. Expression of the toxin gene was driven by a constitutively active enhancer, yet the targeting construct exhibited only minimal transient toxicity while enriching for targeted clones 9- to 29-fold. Germline transmissiion of

James W. McCarrick; Jane R. Parnes; Rho H. Seong; Davor Solter; Barbara B. Knowles



Dynamics of saccade target selection: Race model analysis of double step and search step saccade production in human and macaque  

Microsoft Academic Search

We investigated how saccade target selection by humans and macaque monkeys reacts to unexpected changes of the image. This was explored using double step and search step tasks in which a target, presented alone or as a singleton in a visual search array, steps to a different location on infrequent, random trials. We report that human and macaque monkey performance

C. R. Camalier; A. Gotler; A. Murthy; K. G. Thompson; G. D. Logan; T. J. Palmeri; J. D. Schall



Parallel in Vivo and in Vitro Selection Using Phage Display Identifies Protease-dependent Tumor-targeting Peptides*S  

E-print Network

-targeting Peptides*S Received for publication,April 26, 2010 Published, JBC Papers in Press,May 11, 2010, DOI 10 activatable cell-penetrating peptides (ACPPs) that target contrast agents to in vivo sites of matrix of tumor versus normal tissue. Selected sequences were synthesized as fluorescently labeled peptides

Tsien, Roger Y.


Practical considerations for dose selection in pediatric patients to ensure target exposure requirements.  


Pediatric dosing recommendations are often not based on allometry, despite recognition that metabolic processes in mammals scale to the ¾ power. This report reviews the allometric size model for clearance and its implications for defining doses for children while considering practical limitations. Fondaparinux exposures in children were predicted using allometric and mg/kg dosing. Additional simulations further refined the dose based on the predicted Cmax, target exposure range, complexity of the dosing regimen, and previous exposure/response data. The percent reduction of the adult dose of an oral lozenge fixed-dose formulation which would predict similar exposures in children and adults was recommended based on simulations. Allometric dosing predicted a consistent fondaparinux exposure across the weight range. Size-optimized mg/kg dosing, which partially approximates the allometric relationship, allows for consistent fondaparinux exposures (i.e., 0.12 mg/kg ?35 kg or 0.1 mg/kg >35 kg). Simulations of the oral lozenge formulation demonstrated rapidly changing clearance in children less than 6 years prohibiting practical dosing recommendations for satisfying all conventional exposure metrics (Cmax and AUC) in this age group. In children between 13 and 18 or 6 and 13 years, a 8.6% and 54% reduction in dose would maintain target exposures but dose reductions of 12.5% or 62.5% were ultimately recommended as deemed manufacturable. Dose selection in children should consider the known and/or predicted covariate relationships which affect exposure. Presented examples applied the allometric model in dose selection with the goal of PK bridging and considered practical limitations in dose selection. PMID:24841797

Barbour, April M; Fossler, Michael J; Barrett, Jeffrey



Bezielle Selectively Targets Mitochondria of Cancer Cells to Inhibit Glycolysis and OXPHOS  

PubMed Central

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

Chen, Vivian; Staub, Richard E.; Fong, Sylvia; Tagliaferri, Mary; Cohen, Isaac; Shtivelman, Emma



Targeting gene expression selectively in cancer cells by using the progression-elevated gene-3 promoter  

PubMed Central

One impediment to effective cancer-specific gene therapy is the rarity of regulatory sequences targeting gene expression selectively in tumor cells. Although many tissue-specific promoters are recognized, few cancer-selective gene promoters are available. Progression-elevated gene-3 (PEG-3) is a rodent gene identified by subtraction hybridization that displays elevated expression as a function of transformation by diversely acting oncogenes, DNA damage, and cancer cell progression. The promoter of PEG-3, PEG-Prom, displays robust expression in a broad spectrum of human cancer cell lines with marginal expression in normal cellular counterparts. Whereas GFP expression, when under the control of a CMV promoter, is detected in both normal and cancer cells, when GFP is expressed under the control of the PEG-Prom, cancer-selective expression is evident. Mutational analysis identifies the AP-1 and PEA-3 transcription factors as primary mediators of selective, cancer-specific expression of the PEG-Prom. Synthesis of apoptosis-inducing genes, under the control of the CMV promoter, inhibits the growth of both normal and cancer cells, whereas PEG-Prom-mediated expression of these genes kills only cancer cells and spares normal cells. The efficacy of the PEG-Prom as part of a cancer gene therapeutic regimen is further documented by in vivo experiments in which PEG-Prom-controlled expression of an apoptosis-inducing gene completely inhibited prostate cancer xenograft growth in nude mice. These compelling observations indicate that the PEG-Prom, with its cancer-specific expression, provides a means of selectively delivering genes to cancer cells, thereby providing a crucial component in developing effective cancer gene therapies. PMID:15647352

Su, Zhao-Zhong; Sarkar, Devanand; Emdad, Luni; Duigou, Gregory J.; Young, Charles S. H.; Ware, Joy; Randolph, Aaron; Valerie, Kristoffer; Fisher, Paul B.



Animal models for lysosomal storage disorders.  


The lysosomal storage disorders (LSD) represent a heterogeneous group of inherited diseases characterized by the accumulation of non-metabolized macromolecules (by-products of cellular turnover) in different tissues and organs. LSDs primarily develop as a consequence of a deficiency in a lysosomal hydrolase or its co-factor. The majority of these enzymes are glycosidases and sulfatases, which in normal conditions participate in degradation of glycoconjugates: glycoproteins, glycosaminoproteoglycans, and glycolipids. Significant insights have been gained from studies of animal models, both in understanding mechanisms of disease and in establishing proof of therapeutic concept. These studies have led to the introduction of therapy for certain LSD subtypes, primarily by enzyme replacement or substrate reduction therapy. Animal models have been useful in elucidating molecular changes, particularly prior to onset of symptoms. On the other hand, it should be noted certain animal (mouse) models may have the underlying biochemical defect, but not show the course of disease observed in human patients. There is interest in examining therapeutic options in the larger spontaneous animal models that may more closely mimic the brain size and pathology of humans. This review will highlight lessons learned from studies of animal models of disease, drawing primarily from publications in 2011-2012. PMID:24010835

Pastores, G M; Torres, P A; Zeng, B-J



Production impact of a targeted selective treatment system based on liveweight gain in a commercial flock.  


The sustainability of sheep production is hindered by anthelmintic resistance. Options to slow down or prevent resistance have been widely studied but their application in the field is still limited. In this study, the practical application and effect of a targeted selective treatment (TST) approach for the treatment of parasitic gastroenteritis was investigated in lambs (n?=?385) over a 2 year period. At 14-day intervals during the grazing season, liveweight, breech soiling and anthelmintic treatments were individually recorded. Selection of lambs for anthelmintic treatment in the TST group was based on pre-calculated individual growth rates, with a matched cohort routinely treated (RT) with anthelmintic drug every 6 weeks. The adoption of a TST approach had no negative effect on the liveweight gains of the lambs, time to finishing or breech soiling measures compared to RT lambs; however a 50% decrease in anthelmintic treatment was observed in the TST group. The time to implement this system averaged 2?min per lamb. It is concluded that the TST could be suitable for commercial sheep farms, in association with automated weighing systems, potentially reducing selection for anthelmintic resistance, while having no negative effect on production. PMID:24685103

Busin, V; Kenyon, F; Parkin, T; McBean, D; Laing, N; Sargison, N D; Ellis, K



Target dependence of orientation and direction selectivity of corticocortical projection neurons in the mouse V1  

PubMed Central

Higher order visual areas that receive input from the primary visual cortex (V1) are specialized for the processing of distinct features of visual information. However, it is still incompletely understood how this functional specialization is acquired. Here we used in vivo two photon calcium imaging in the mouse visual cortex to investigate whether this functional distinction exists at as early as the level of projections from V1 to two higher order visual areas, AL and LM. Specifically, we examined whether sharpness of orientation and direction selectivity and optimal spatial and temporal frequency of projection neurons from V1 to higher order visual areas match with that of target areas. We found that the V1 input to higher order visual areas were indeed functionally distinct: AL preferentially received inputs from V1 that were more orientation and direction selective and tuned for lower spatial frequency compared to projection of V1 to LM, consistent with functional differences between AL and LM. The present findings suggest that selective projections from V1 to higher order visual areas initiates parallel processing of sensory information in the visual cortical network. PMID:24068987

Matsui, Teppei; Ohki, Kenichi



Hyperspectral band selection based on the aggregation of proximity measures for automated target detection  

NASA Astrophysics Data System (ADS)

Band selection is an important unsolved challenge in hyperspectral image processing that has been used for dimensionality reduction and classification improvement. To date, numerous researchers have investigated the unsupervised selection of band groups using measures such as correlation and Kullback-Leibler divergence. However, no clear winner has emerged across data sets and detection tasks. Herein, we investigate the utility of aggregating different proximity measures for band group selection. Specifically, we employ the Choquet integral with respect to different measures (capacities) as it is able to yield a variety of aggregation functions like t-norms, t-conorms and averaging operators. We explore the utility of aggregation in the context of single band, single band group, band group dimensionality reduction and multiple band group combinations in conjunction with support vector machine (SVM) based classification. Our preliminary experiments indicate there is value in aggregating different proximity measures. In some instances an intersection operator works well while in other cases a union operator is best. As may be expected, this can, and does vary per detection task. We also see that depending on the difficulty of the target detection problem, different aggregation, band grouping and combination strategies prevail. Advantages of our approach include; flexibility, the aggregation operator can be learned, and the method can default to a single proximity measure if needed and result, in the worst case, in no performance loss. Experiments are performed on three hyperspectral benchmark data sets to demonstrate the applicability of the proposed concepts.

Ball, John E.; Anderson, Derek T.; Samiappan, Sathish



Selective occlusion of tumor blood vessels by targeted delivery of an antibody-photosensitizer conjugate.  


The irregular vasculature and high interstitial pressure of solid tumors hinder the delivery of cytotoxic agents to cancer cells. As a consequence, the doses of chemotherapy necessary to achieve complete tumor eradication are associated with unacceptably high toxicities. The selective thrombosis of tumor blood vessels has been postulated as an alternative avenue for combating cancer, depriving tumors of nutrients and oxygen and causing an avalanche of tumor cell deaths. The human antibody L19, specific to the EDB domain of fibronectin, a marker of angiogenesis, is capable of selective in vivo localization around tumor blood vessels and is thus a suitable agent for delivering toxic payloads to the tumor neovasculature. Here we show that a chemical conjugate of the L19 antibody with the photosensitizer bis(triethanolamine)Sn(IV) chlorin e(6), after intravenous injection and irradiation with red light, caused an arrest of tumor growth in mice with subcutaneous tumors. By contrast, a photosensitizer conjugate obtained with an antibody of identical pharmacokinetic properties but irrelevant specificity did not exhibit a significant therapeutic effect. These results confirm that vascular targeting strategies, aimed at the selective occlusion/disruption of tumor blood vessels, have a significant anticancer therapeutic potential and encourage the use of antibody-photosensitizer conjugates for the therapy of superficial tumors and possibly other angiogenesis-related pathologies. PMID:16217760

Fabbrini, Monica; Trachsel, Eveline; Soldani, Patrizia; Bindi, Stefano; Alessi, Patrizia; Bracci, Luisa; Kosmehl, Hartwig; Zardi, Luciano; Neri, Dario; Neri, Paolo



Comparison of endoplasmic reticulum targeted and non-targeted cytoplasmic GFP as a selectable marker in citrus protoplast transformation  

Microsoft Academic Search

In order to improve transformation efficiency in citrus two green fluorescent protein (GFP) constructions, an endoplasmic reticulum targeted (GFP-ER) and cytoplasmic targeted (Cy-GFP), were used to study expression and stability of GFP in transgenic plants. Results from citrus protoplast-GFP transformation showed that GFP-ER had brighter fluorescence than the Cy-GFP. Although both of these constructs gave transient expression at the protoplast

Ahmad A. Omar; Jude W. Grosser



Drug-induced lysosomal storage of sulfated glycosaminoglycans. Studies on the underlying structure-activity relationships.  


Some immunomodulatory drugs have previously been shown to induce lysosomal storage of sulfated glycosaminoglycans (sGAG) in intact organisms and cultured cells. These compounds consist of a planary aromatic ring system and two symmetric side chains each carrying a protonizable nitrogen. The purpose of this study was to test a larger collection of such compounds for their potencies to induce lysosomal storage of sGAG in cultured fibroblasts of rat cornea. The cells were exposed (72 h) to various compounds differing with respect to the aromatic ring system or the side chains. Lysosomal sGAG-storage was demonstrated by selective cytochemical staining with cuprolinic blue. The threshold concentration, i.e., the concentration necessary to induce cuprolinic blue-positive cytoplasmic inclusions in at least 1% of the cells, was determined for each compound. The threshold concentrations were distributed over a range of 0.3-30 microM. It should be emphasized that the threshold concentration of a given compound is not a constant, but depends on the volume of cell culture medium per surface area of cell monolayer, since the lysosomal accumulation lowers the initial drug concentration in the medium. If the ratio of medium volume:cell monolayer surface is increased as compared with standard cell culture conditions, the threshold concentration will be lowered. The compounds were ranked according to their threshold concentrations as determined under standard conditions. The following conclusions can be drawn from the ranking: the type of the central aromatic ring system and the distance between the ring system and the protonizable nitrogen atoms of the side chains influence the potency to induce lysosomal sGAG-storage. Regarding the ring system, the potency decreases as follows: acridine approximately anthrachinone > fenfluorenone approximately fenfluorene > xanthenone; xanthene > dibenzofuran approximately dibenzothiophene. In intact organisms, these structure-activity relationships may be superimposed by drug metabolism and pharmacokinetic factors. PMID:8303713

Handrock, K; Lüllmann-Rauch, R; Vogt, R D



Lysosome dysfunction in the pathogenesis of kidney diseases.  


The lysosome, an organelle central to macromolecule degradation and recycling, plays a pivotal role in normal cell processes, ranging from autophagy to redox regulation. Not surprisingly, lysosomes are an integral part of the renal epithelial molecular machinery that facilitates normal renal physiology. Two inherited diseases that manifest as kidney dysfunction are Fabry's disease and cystinosis, each of which is caused by a primary biochemical defect at the lysosome resulting from loss-of-function mutations in genes that encode lysosomal proteins. The functions of the lysosomes in the kidney and how lysosomal dysfunction might contribute to Fabry's disease and cystinosis are discussed. Unlike most other pediatric renal diseases, therapies are available for Fabry's disease and cystinosis, but require early diagnosis. Recent analysis of ceroid neuronal lipofuscinosis type 3 (Cln3) null mice, a mouse model of lysosomal disease that is primarily associated with neurological deficits, revealed renal functional abnormalities. As current and future therapeutics increase the life-span of those suffering from diseases like neuronal ceroid lipofuscinosis, it remains a distinct possibility that many more lysosomal disorders that primarily manifest as infant and juvenile neurodegenerative diseases may also include renal disease phenotypes. PMID:24217784

Surendran, Kameswaran; Vitiello, Seasson P; Pearce, David A



Expanding Newborn Screening for Lysosomal Disorders: Opportunities and Challenges  

ERIC Educational Resources Information Center

Newborn screening (NBS), since its implementation in the 1960s, has traditionally been successful in reducing mortality and disability in children with a range of different conditions. Lysosomal storage disorders (LSD) are a heterogeneous group of inherited metabolic diseases that result from lysosomal dysfunction. Based on available treatment and…

Waggoner, Darrel J.; Tan, Christopher A.



Linking Albinism and Immunity: The Secrets of Secretory Lysosomes  

Microsoft Academic Search

Lysosomes are membrane-bound organelles that are found in all mammalian cells and contain hydrolases and lipases required for protein and membrane degradation. In many cells of the immune system, lysosomes also contain secretory proteins that can be released by regulated exocytosis in response to an external stimulus, providing different cell types with a wide range of effector functions. Melanosomes also

Jane Stinchcombe; Giovanna Bossi; Gillian M. Griffiths



Stability of antibody-conjugated gold nanoparticles in the endo-lysosomal nanoenvironment: Implications for non-invasive radiofrequency-based cancer therapy  

PubMed Central

The use of non-invasive radiofrequency (RF) electric fields as an energy source for thermal activation of nanoparticles within cancer cells could be a valuable addition to the emerging field of nano-mediated cancer therapies. Based on investigations of cell death through hyperthermia, and offering the ability for total body penetration by RF fields, this technique is thought to compliment and possibly out-perform existing nano-heat-treatments that utilize alternative heat production via optical or magnetic stimuli. However, it remains a challenge to understand fully the complex RF-nanoparticle-intracellular interactions before full system optimization can be engineered. Herein we have shown that liver cancer cells can selectively internalize antibody-conjugated gold nanoparticles (AuNPs) through receptor-mediated endocytosis, with the nanoparticles predominantly accumulating and aggregating within cytoplasmic endo-lysosomes. After exposure to an external RF field, non-aggregated AuNPs absorbed and dissipated energy as heat causing thermal damage to the targeted cancer cells. We also observed that RF absorption and heat dissipation is dependent on solubility of AuNPs in the colloid, which is pH dependent. Furthermore, by modulating endo-lysosomal pH it is possible to prevent intracellular AuNP aggregation and enhance thermal cytotoxicity in hepatocellular cancer cells. PMID:22349096

Raoof, Mustafa; Corr, Stuart J.; Kaluarachchi, Warna D.; Massey, Katheryn L.; Briggs, Katrina; Zhu, Cihui; Cheney, Matthew A.; Wilson, Lon J.; Curley, Steven A.



Cold atmospheric plasma treatment selectively targets head and neck squamous cell carcinoma cells.  


The treatment of locoregional recurrence (LRR) of head and neck squamous cell carcinoma (HNSCC) often requires a combination of surgery, radiation therapy and/or chemotherapy. Survival outcomes are poor and the treatment outcomes are morbid. Cold atmospheric plasma (CAP) is an ionized gas produced at room temperature under laboratory conditions. We have previously demonstrated that treatment with a CAP jet device selectively targets cancer cells using in vitro melanoma and in vivo bladder cancer models. In the present study, we wished to examine CAP selectivity in HNSCC in vitro models, and to explore its potential for use as a minimally invasive surgical approach that allows for specific cancer cell or tumor tissue ablation without affecting the surrounding healthy cells and tissues. Four HNSCC cell lines (JHU-022, JHU-028, JHU-029, SCC25) and 2 normal oral cavity epithelial cell lines (OKF6 and NOKsi) were subjected to cold plasma treatment for durations of 10, 30 and 45 sec, and a helium flow of 20 l/min-1 for 10 sec was used as a positive treatment control. We showed that cold plasma selectively diminished HNSCC cell viability in a dose-response manner, as evidenced by MTT assays; the viability of the OKF6 cells was not affected by the cold plasma. The results of colony formation assays also revealed a cell-specific response to cold plasma application. Western blot analysis did not provide evidence that the cleavage of PARP occurred following cold plasma treatment. In conclusion, our results suggest that cold plasma application selectively impairs HNSCC cell lines through non-apoptotic mechanisms, while having a minimal effect on normal oral cavity epithelial cell lines. PMID:25050490

Guerrero-Preston, Rafael; Ogawa, Takenori; Uemura, Mamoru; Shumulinsky, Gary; Valle, Blanca L; Pirini, Francesca; Ravi, Rajani; Sidransky, David; Keidar, Michael; Trink, Barry



Effective heritability of targets of sex-ratio selection under environmental sex determination.  


Selection is expected to maintain primary sex ratios at an evolutionary equilibrium. In organisms with temperature-dependent sex determination (TSD), targets of sex-ratio selection include the thermal sensitivity of the sex-determining pathway (hereafter, sex determination threshold) and nest-site choice. However, offspring sex may be canalized for nests located in thermally extreme environments; thus, genetic variance for the sex determination threshold is not expressed and is invisible to direct selection. The concept of 'effective heritability' accounts for this dependence and provides a more realistic prediction of the expected evolutionary response to selection in the wild. Past estimates of effective heritability of the sex determination threshold, which were derived from laboratory data, suggested that the potential for the sex determination threshold to evolve in the wild was extremely low. We re-evaluated original estimates of this parameter by analysing field-collected measures of nest temperatures, vegetation cover and clutch sex ratios from nests in a population of painted turtles (Chrysemys picta). We coupled these data with measurements of broad-sense heritability of the sex determination threshold in C. picta, using an experiment that splits clutches of eggs between a constant temperature (i.e. typical laboratory incubation) and a daily fluctuating temperature (i.e. similar to natural nests) with the same mean. We found that (i) the effective heritability of the sex determination threshold appears to have been historically underestimated and the effective heritability of nest-site choice has been overestimated and (ii) significant family-by-incubation treatment interaction exists for sex for C. picta between constant- and fluctuating-temperature regimes. Our results suggest that the thermal sensitivity of the sex-determining pathway may play a larger, more complex role in the microevolution of TSD than traditionally thought. PMID:21261771

McGaugh, S E; Janzen, F J



Possible ATP release through lysosomal exocytosis from primary sensory neurons.  


The adenosine triphosphate (ATP) plays important roles under physiological and pathological conditions such as traumatic brain injury, neuroinflammation and neuropathic pain. In the present study, we set out to study the role of lysosomal vesicles on ATP release from the dorsal root ganglion neurons. We found that the lysosomal vesicles, which contain the quinacrine-positive fluorescence and express the vesicular nucleotide transporter (VNUT), were localized within the soma and growth cone of the cultured dorsal root ganglion neurons. In addition, the number of the quinacrine staining was decreased by application of lysosomal exocytosis activators, and this decrease was suppressed by the metformin and vacuolin-1, which suppressed lysosomal exocytosis. Thus, these findings suggest that ATP release via the lysosomal exocytosis may be one of the pathways for ATP release in response to stimulation. PMID:23237805

Jung, Junyang; Shin, Youn Ho; Konishi, Hiroyuki; Lee, Seo Jin; Kiyama, Hiroshi



New Molecules and Old Drugs as Emerging Approaches to Selectively Target Human Glioblastoma Cancer Stem Cells  

PubMed Central

Despite relevant progress obtained by multimodal treatment, glioblastoma (GBM), the most aggressive primary brain tumor, is still incurable. The most encouraging advancement of GBM drug research derives from the identification of cancer stem cells (CSCs), since these cells appear to represent the determinants of resistance to current standard therapies. The goal of most ongoing studies is to identify drugs able to affect CSCs biology, either inducing selective toxicity or differentiating this tumor cell population into nontumorigenic cells. Moreover, the therapeutic approach for GBM could be improved interfering with chemo- or radioresistance mechanisms, microenvironment signals, and the neoangiogenic process. During the last years, molecular targeted compounds such as sorafenib and old drugs, like metformin, displayed interesting efficacy in preclinical studies towards several tumors, including GBM, preferentially affecting CSC viability. In this review, the latest experimental results, controversies, and prospective application concerning these promising anticancer drugs will be discussed. PMID:24527434

Wurth, Roberto; Barbieri, Federica; Florio, Tullio



Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro  

PubMed Central

Inappropriate signalling through the EGFR and ErbB2/HER2 members of the epidermal growth factor family of receptor tyrosine kinases is well recognised as being causally linked to a variety of cancers. Consequently, monoclonal antibodies specific for these receptors have become increasingly important components of effective treatment strategies for cancer. Increasing evidence suggests that ErbB3 plays a critical role in cancer progression and resistance to therapy. We hypothesised that co-targeting the preferred ErbB2/ErbB3 heterodimer with a bispecific single-chain Fv (bs-scFv) antibody would promote increased targeting selectivity over antibodies specific for a single tumour-associated antigen (TAA). In addition, we hypothesised that targeting this important heterodimer could induce a therapeutic effect. Here, we describe the construction and evaluation of the A5-linker-ML3.9 bs-scFv (ALM), an anti-ErbB3/ErbB2 bs-scFv. The A5-linker-ML3.9 bs-scFv exhibits selective targeting of tumour cells in vitro and in vivo that co-express the two target antigens over tumour cells that express only one target antigen or normal cells that express low levels of both antigens. The A5-linker-ML3.9 bs-scFv also exhibits significantly greater in vivo targeting of ErbB2‘+'/ErbB3‘+' tumours than derivative molecules that contain only one functional arm targeting ErbB2 or ErbB3. Binding of ALM to ErbB2‘+'/ErbB3‘+' cells mediates inhibition of tumour cell growth in vitro by effectively targeting the therapeutic anti-ErbB3 A5 scFv. This suggests both that ALM could provide the basis for an effective therapeutic agent and that engineered antibodies selected to co-target critical functional pairs of TAAs can enhance the targeting specificity and efficacy of antibody-based cancer therapeutics. PMID:18841159

Robinson, M K; Hodge, K M; Horak, E; Sundberg, A L; Russeva, M; Shaller, C C; von Mehren, M; Shchaveleva, I; Simmons, H H; Marks, J D; Adams, G P



Mature Epitope Density - A strategy for target selection based on immunoinformatics and exported prokaryotic proteins  

PubMed Central

Background Current immunological bioinformatic approaches focus on the prediction of allele-specific epitopes capable of triggering immunogenic activity. The prediction of major histocompatibility complex (MHC) class I epitopes is well studied, and various software solutions exist for this purpose. However, currently available tools do not account for the concentration of epitope products in the mature protein product and its relation to the reliability of target selection. Results We developed a computational strategy based on measuring the epitope's concentration in the mature protein, called Mature Epitope Density (MED). Our method, though simple, is capable of identifying promising vaccine targets. Our online software implementation provides a computationally light and reliable analysis of bacterial exoproteins and their potential for vaccines or diagnosis projects against pathogenic organisms. We evaluated our computational approach by using the Mycobacterium tuberculosis (Mtb) H37Rv exoproteome as a gold standard model. A literature search was carried out on 60 out of 553 Mtb's predicted exoproteins, looking for previous experimental evidence concerning their possible antigenicity. Half of the 60 proteins were classified as highest scored by the MED statistic, while the other half were classified as lowest scored. Among the lowest scored proteins, ~13% were confirmed as not related to antigenicity or not contributing to the bacterial pathogenicity, and 70% of the highest scored proteins were confirmed as related. There was no experimental evidence of antigenic or pathogenic contributions for three of the highest MED-scored Mtb proteins. Hence, these three proteins could represent novel putative vaccine and drug targets for Mtb. A web version of MED is publicly available online at Conclusions The software presented here offers a practical and accurate method to identify potential vaccine and diagnosis candidates against pathogenic bacteria by "reading" results from well-established reverse vaccinology software in a novel way, considering the epitope's concentration in the mature portion of the protein. PMID:24564223



Targeting the XIAP/caspase-7 complex selectively kills caspase-3-deficient malignancies  

PubMed Central

Caspase-3 downregulation (CASP3/DR) in tumors frequently confers resistance to cancer therapy and is significantly correlated with a poor prognosis in cancer patients. Because CASP3/DR cancer cells rely heavily on the activity of caspase-7 (CASP7) to initiate apoptosis, inhibition of activated CASP7 (p19/p12-CASP7) by X-linked inhibitor of apoptosis protein (XIAP) is a potential mechanism by which apoptosis is prevented in those cancer cells. Here, we identify the pocket surrounding the Cys246 residue of p19/p12-CASP7 as a target for the development of a protein-protein interaction (PPI) inhibitor of the XIAP:p19/p12-CASP7 complex. Interrupting this PPI directly triggered CASP7-dependent apoptotic signaling that bypassed the activation of the apical caspases and selectively killed CASP3/DR malignancies in vitro and in vivo without adverse side effects in nontumor cells. Importantly, CASP3/DR combined with p19/p12-CASP7 accumulation correlated with the aggressive evolution of clinical malignancies and a poor prognosis in cancer patients. Moreover, targeting of this PPI effectively killed cancer cells with multidrug resistance due to microRNA let-7a-1–mediated CASP3/DR and resensitized cancer cells to chemotherapy-induced apoptosis. These findings not only provide an opportunity to treat CASP3/DR malignancies by targeting the XIAP:p19/p12-CASP7 complex, but also elucidate the molecular mechanism underlying CASP3/DR in cancers. PMID:23979166

Lin, Yuan-Feng; Lai, Tsung-Ching; Chang, Chih-Kang; Chen, Chi-Long; Huang, Ming-Shyan; Yang, Chih-Jen; Liu, Hon-Ge; Dong, Jhih-Jhong; Chou, Yi-An; Teng, Kuo-Hsun; Chen, Shih-Hsun; Tian, Wei-Ting; Jan, Yi-Hua; Hsiao, Michael; Liang, Po-Huang



NuMA is required for the selective induction of p53 target genes.  


The p53 tumor suppressor protein is a transcription factor controlling various outcomes, such as growth arrest and apoptosis, through the regulation of different sets of target genes. The nuclear mitotic apparatus protein (NuMA) plays important roles in spindle pole organization during mitosis and in chromatin regulation in the nucleus during interphase. Although NuMA has been shown to colocalize with several nuclear proteins, including high-mobility-group proteins I and Y and GAS41, the role of NuMA during interphase remains unclear. Here we report that NuMA binds to p53 to modulate p53-mediated transcription. Acute and partial ablation of NuMA attenuates the induction of the proarrested p21 gene following DNA damage, subsequently causing impaired cell cycle arrest. Interestingly, NuMA knockdown had little effect on the induction of the p53-dependent proapoptotic PUMA gene. Furthermore, NuMA is required for the recruitment of cyclin-dependent kinase 8 (Cdk8), a component of the Mediator complex and a promoter of p53-mediated p21 gene function. These data demonstrate that NuMA is critical for the target selectivity of p53-mediated transcription. PMID:23589328

Ohata, Hirokazu; Miyazaki, Makoto; Otomo, Ryo; Matsushima-Hibiya, Yuko; Otsubo, Chihiro; Nagase, Takahiro; Arakawa, Hirofumi; Yokota, Jun; Nakagama, Hitoshi; Taya, Yoichi; Enari, Masato



Searching for life on Mars: selection of molecular targets for ESA's aurora ExoMars mission.  


The European Space Agency's ExoMars mission will seek evidence of organic compounds of biological and non-biological origin at the martian surface. One of the instruments in the Pasteur payload may be a Life Marker Chip that utilizes an immunoassay approach to detect specific organic molecules or classes of molecules. Therefore, it is necessary to define and prioritize specific molecular targets for antibody development. Target compounds have been selected to represent meteoritic input, fossil organic matter, extant (living, recently dead) organic matter, and contamination. Once organic molecules are detected on Mars, further information is likely to derive from the detailed distribution of compounds rather than from single molecular identification. This will include concentration gradients beneath the surface and gradients from generic to specific compounds. The choice of biomarkers is informed by terrestrial biology but is wide ranging, and nonterrestrial biology may be evident from unexpected molecular distributions. One of the most important requirements is to sample where irradiation and oxidation are minimized, either by drilling or by using naturally excavated exposures. Analyzing regolith samples will allow for the search of both extant and fossil biomarkers, but sequential extraction would be required to optimize the analysis of each of these in turn. PMID:17723091

Parnell, John; Cullen, David; Sims, Mark R; Bowden, Stephen; Cockell, Charles S; Court, Richard; Ehrenfreund, Pascale; Gaubert, Francois; Grant, William; Parro, Victor; Rohmer, Michel; Sephton, Mark; Stan-Lotter, Helga; Steele, Andrew; Toporski, Jan; Vago, Jorge



Target selective micelles for bombesin receptors incorporating Au(III)-dithiocarbamato complexes.  


Pure sterically stabilized micelles (SSM) of DSPE-PEG2000, and sterically stabilized mixed micelles (SSMM) containing PC or DOPC phospholipids (5, 10 or 20% mol/mol with respect to DSPE-PEG2000) are developed as delivery systems for the gold based cytotoxic drug Au(III)-dithiocarbamato complex AuL12. In particular, SSMM containing 5% of PC at 5mM of lipid concentration encapsulates 61.0 ?g of AuL12 with a DL% of 1.13. The gold complex remains stable up to 72 h when incorporated in the aggregate, as indicated by UV-vis measurements. Incorporation in micelle composition of a low amount of the peptide derivative MonY-BN-AA1, containing a bombesin peptide analogue does not influence structural parameters of the micelles (diameter around 20 nm) neither the AuL12 loading parameters. Target selective properties of the peptide containing full aggregate on PC-3 cells overexpressing the GRP/bombesin receptors are observed by in vitro cytotoxic studies: a decrease of cell viability, ? 50%, is obtained in cells treated with AuL12-targeted micelles at 10 ?M drug concentration for 48 h with respect to untargeted micelles. PMID:25014371

Ringhieri, Paola; Iannitti, Roberta; Nardon, Chiara; Palumbo, Rosanna; Fregona, Dolores; Morelli, Giancarlo; Accardo, Antonella



Lysosomes and Fas-mediated liver cell death  

PubMed Central

A number of studies, mostly performed ex vivo, suggest that lysosomes are involved in apoptosis as a result of a release of their cathepsins into the cytosol. These enzymes could then contribute to the permeabilization of the outer mitochondrial membrane; they could also activate effector caspases. The present study aims at testing whether the membrane of liver lysosomes is disrupted during Fas-mediated cell death of hepatocytes in vivo, a process implicated in several liver pathologies. Apoptosis was induced by injecting mice with aFas (anti-Fas antibody). The state of lysosomes was assessed by determining the proportion of lysosomal enzymes (?-galactosidase, ?-glucuronidase, cathepsin C and cathepsin B) present in homogenate supernatants, devoid of intact lysosomes, and by analysing the behaviour in differential and isopycnic centrifugation of ?-galactosidase. Apoptosis was monitored by measuring caspase 3 activity (DEVDase) and the release of sulfite cytochrome c reductase, an enzyme located in the mitochondrial intermembrane space. Results show that an injection of 10 ?g of aFas causes a rapid and large increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase. This modifies neither the proportion of unsedimentable lysosomal enzyme in the homogenates nor the behaviour of lysosomes in centrifugation. Experiments performed with a lower dose of aFas (5 ?g) indicate that unsedimentable lysosomal hydrolase activity increases in the homogenate after injection but with a marked delay with respect to the increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase. Comparative experiments ex vivo performed with Jurkat cells show an increase in unsedimentable lysosomal hydrolases, but much later than caspase 3 activation, and a release of dipeptidyl peptidase III and DEVDase into culture medium. It is proposed that the weakening of lysosomes observed after aFas treatment in vivo and ex vivo results from a necrotic process that takes place late after initiation of apoptosis. PMID:17129211

Wattiaux, Robert; Wattiaux-De Coninck, Simone; Thirion, Jacqueline; Gasingirwa, Mane-Christine; Jadot, Michel



A cysteine-specific lysosomal transport system provides a major route for the delivery of thiol to human fibroblast lysosomes: possible role in supporting lysosomal proteolysis  

Microsoft Academic Search

Lysosomes constitute only 4% of the intra- cellular volume of a normal human fibroblast. When human fibroblasts are incubated for 2-5 min with 20 t~M (asS)cystine in Krebs-Ringer phosphate solution at pH 7.4, a minimum of 50-60% of the total radioactiv- ity taken up by the cells is found sequestered into the lysosomal compartment in the form of cysteine. A

Ronald L. Pisoni; Tracy L. Acker; Karen M. Lisowski; Rosemary M. Lemons; Jess G. Thoene



Tumor-targeted delivery of liposome-encapsulated doxorubicin by use of a peptide that selectively binds to irradiated tumors  

PubMed Central

Tumor-targeted drug delivery improves anti-tumor efficacy and reduces systemic toxicity by limiting bioavailability of cytotoxic drugs to within tumors. Targeting reagents, such as peptides or antibodies recognizing molecular targets over-expressed within tumors, have been used to improve liposome-encapsulated drug accumulation within tumors and resulted in enhanced tumor growth control. In this report, we expand the scope of targeting reagents by showing that one peptide, HVGGSSV which was isolated from an in vivo screening of phage-displayed peptide library due to its selective binding within irradiated tumors, enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in enhanced cytotoxicity within tumors. Targeting liposomes (TL) and non-targeting liposomes (nTL) were labeled with Alexa Fluor 750. Biodistribution of the liposomes within tumor-bearing mice was studied with near infrared (NIR) imaging. In the single dose pharmacokinetic study, the liposomal doxorubicin has an extended circulation half life as compared to the free doxorubicin. Targeting liposomes partitioned to the irradiated tumors and improved drug deposition and retention within tumors. The tumor-targeted delivery of doxorubicin improved tumor growth control as indicated with reduced tumor growth rate and tumor cell proliferation, enhanced tumor blood vessel destruction, and increased treatment-associated apoptosis and necrosis of tumor cells. Collectively, the results demonstrated the remarkable capability of the HVGGSSV peptide in radiation-guided drug delivery to tumors. PMID:21075152

Lowery, Amanda; Onishko, Halina; Hallahan, Dennis E.; Han, Zhaozhong



Engineering of Targeted Nanoparticles for Cancer Therapy Using Internalizing Aptamers Isolated by Cell-Uptake Selection  

E-print Network

One of the major challenges in the development of targeted nanoparticles (NPs) for cancer therapy is to discover targeting ligands that allow for differential binding and uptake by the target cancer cells. Using prostate ...

Xiao, Zeyu


BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells  

PubMed Central

Summary Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However, recent studies have shown that relative redox levels in primary tumors can be heterogeneous, suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies, we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First, the majority of functionally-defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed “ROS-low”). Second, ROS-low LSCs aberrantly over-express BCL-2. Third, BCL-2 inhibition reduced oxidative phosphorylation and selectively eradicated quiescent LSCs. Based on these findings, we propose a model wherein the unique physiology of ROS-low LSCs provides an opportunity for selective targeting via disruption of BCL-2-dependent oxidative phosphorylation. PMID:23333149

Lagadinou, Eleni D.; Sach, Alexander; Callahan, Kevin; Rossi, Randall M.; Neering, Sarah J.; Minhajuddin, Mohammad; Ashton, John M.; Pei, Shanshan; Grose, Valerie; O'Dwyer, Kristen M.; Liesveld, Jane L.; Brookes, Paul S.; Becker, Michael W.; Jordan, Craig T.



Cyclodextrin Induces Calcium-Dependent Lysosomal Exocytosis  

PubMed Central

Cyclodextrins (CDs) have long been used to manipulate cellular cholesterol levels both in vitro and in vivo, but their direct effects at a cellular level are not well characterized. Recently, CDs have garnered much interest because of their ability to clear stored cholesterol from Niemann Pick Type C (NPC) cells and markedly prolong the life of NPC1 disease mice. Here, we investigate the hypothesis that treatment with 2-hydroxypropyl- ?-cyclodextrin (HPB-CD) stimulates lysosomal exocytosis in a calcium-enhanced manner. We propose that this exocytosis is the mechanism by which HPB-CD ameliorates the endolysosomal cholesterol storage phenotype in NPC cells. These findings have significant implications for the use of HPB-CD in biochemical assays and data interpretation as well as for their use for the treatment for NPC and other disorders. PMID:21124786

Chen, Fannie W.; Li, Chunlei; Ioannou, Yiannis A.




PubMed Central

The mast cell-rupturing component present in the lysosomes of rabbit exudate PMN neutrophil leukocytes has been identified and some of its physical and chemical properties have been described. The active agent is a low molecular weight (1200 to 2400) polypeptide containing a relatively large proportion of the basic amino acid, arginine. It is thermostable and dialyzable, and does not cause contraction of the isolated guinea pig ileum. The mast cell-rupturing activity of the agent is destroyed by trypsin. A second permeability factor with a larger molecular weight is present in crude extracts of PMN granules. Although this substance does not lyse mast cells, it is capable of evoking delayed permeability responses in rabbit skin. PMID:5926298

Seegers, Winnifred; Janoff, Aaron



Biogenesis of Lysosome-related Organelles Complex-1 Subunit 1 (BLOS1) Interacts with Sorting Nexin 2 and the Endosomal Sorting Complex Required for Transport-I (ESCRT-I) Component TSG101 to Mediate the Sorting of Epidermal Growth Factor Receptor into Endosomal Compartments.  


Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is a component of the molecular machinery required for the biogenesis of specialized organelles and lysosomal targeting of cargoes via the endosomal to lysosomal trafficking pathway. BLOS1, one subunit of BLOC-1, is implicated in lysosomal trafficking of membrane proteins. We found that the degradation and trafficking of epidermal growth factor receptor (EGFR) were delayed in BLOS1 knockdown cells, which were rescued through BLOS1 overexpression. A key feature to the delayed EGFR degradation is the accumulation of endolysosomes in BLOS1 knockdown cells or BLOS1 knock-out mouse embryonic fibroblasts. BLOS1 interacted with SNX2 (a retromer subunit) and TSG101 (an endosomal sorting complex required for transport subunit-I) to mediate EGFR lysosomal trafficking. These results suggest that coordination of the endolysosomal trafficking proteins is important for proper targeting of EGFR to lysosomes. PMID:25183008

Zhang, Aili; He, Xin; Zhang, Ling; Yang, Lin; Woodman, Philip; Li, Wei



A guide to picking the most selective kinase inhibitor tool compounds for pharmacological validation of drug targets  

PubMed Central

To establish the druggability of a target, genetic validation needs to be supplemented with pharmacological validation. Pharmacological studies, especially in the kinase field, are hampered by the fact that many reference inhibitors are not fully selective for one target. Fortunately, the initial trickle of selective inhibitors released in the public domain has steadily swelled into a stream. However, rationally picking the most selective tool compound out of the increasing amounts of available inhibitors has become progressively difficult due to the lack of accurate quantitative descriptors of drug selectivity. A recently published approach, termed ‘selectivity entropy’, is an improved way of expressing selectivity as a single-value parameter and enables rank ordering of inhibitors. We provide a guide to select the best tool compounds for pharmacological validation experiments of candidate drug targets using selectivity entropy. In addition, we recommend which inhibitors to use for studying the biology of the 20 most investigated kinases that are clinically relevant: Abl (ABL1), AKT1, ALK, Aurora A/B, CDKs, MET, CSF1R (FMS), EGFR, FLT3, ERBB2 (HER2), IKBKB (IKK2), JAK2/3, JNK1/2/3 (MAPK8/9/10), MEK1/2, PLK1, PI3Ks, p38? (MAPK14), BRAF, SRC and VEGFR2 (KDR). PMID:22250956

Uitdehaag, Joost CM; Verkaar, Folkert; Alwan, Husam; de Man, Jos; Buijsman, Rogier C; Zaman, Guido JR



Dynamics of saccade target selection: Race model analysis of double step and search step saccade production in human and macaque  

PubMed Central

We investigated how saccade target selection by humans and macaque monkeys reacts to unexpected changes of the image. This was explored using double step and search step tasks in which a target, presented alone or as a singleton in a visual search array, steps to a different location on infrequent, random trials. We report that human and macaque monkey performance are qualitatively indistinguishable. Performance is stochastic with the probability of producing a compensated saccade to the final target location decreasing with the delay of the step. Compensated saccades to the final target location are produced with latencies relative to the step that are comparable to or less than the average latency of saccades on trials with no target step. Noncompensated errors to the initial target location are produced with latencies less than the average latency of saccades on trials with no target step. Noncompensated saccades to the initial target location are followed by corrective saccades to the final target location following an intersaccade interval that decreases with the interval between the target step and the initiation of the noncompensated saccade. We show that this pattern of results cannot be accounted for by a race between two stochastically independent processes producing the saccade to the initial target location and another process producing the saccade to the final target location. However, performance can be accounted for by a race between three stochastically independent processes – a GO process producing the saccade to the initial target location, a STOP process interrupting that GO process, and another GO process producing the saccade to the final target location. Furthermore, if the STOP process and second GO process start at the same time, then the model can account for the incidence and latency of mid-flight corrections and rapid corrective saccades. This model provides a computational account of saccade production when the image changes unexpectedly. PMID:17604806

Camalier, C. R.; Gotler, A.; Murthy, A.; Thompson, K.G.; Logan, G.D.; Palmeri, T.J.; Schall, J.D.



Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity  

PubMed Central

The study of the potential risks associated with the manufacture, use, and disposal of nanoscale materials, and their mechanisms of toxicity, is important for the continued advancement of nanotechnology. Currently, the most widely accepted paradigms of nanomaterial toxicity are oxidative stress and inflammation, but the underlying mechanisms are poorly defined. This review will highlight the significance of autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Most endocytic routes of nanomaterial cell uptake converge upon the lysosome, making the lysosomal compartment the most common intracellular site of nanoparticle sequestration and degradation. In addition to the endo-lysosomal pathway, recent evidence suggests that some nanomaterials can also induce autophagy. Among the many physiological functions, the lysosome, by way of the autophagy (macroautophagy) pathway, degrades intracellular pathogens, and damaged organelles and proteins. Thus, autophagy induction by nanoparticles may be an attempt to degrade what is perceived by the cell as foreign or aberrant. While the autophagy and endo-lysosomal pathways have the potential to influence the disposition of nanomaterials, there is also a growing body of literature suggesting that biopersistent nanomaterials can, in turn, negatively impact these pathways. Indeed, there is ample evidence that biopersistent nanomaterials can cause autophagy and lysosomal dysfunctions resulting in toxicological consequences. PMID:22697169



Development of targeted therapies for Parkinson's disease and related synucleinopathies.  


Therapeutic efforts in neurodegenerative diseases have been very challenging, particularly due to a lack of validated and mechanism-based therapeutic targets and biomarkers. The basic idea underlying the novel therapeutic approaches reviewed here is that by exploring the molecular basis of neurodegeneration in a rare lysosomal disease such as Gaucher's disease (GD), new molecular targets will be identified for therapeutic development in common synucleinopathies. Accumulation of ?-synuclein plays a key role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies, suggesting that improved clearance of ?-synuclein may be of therapeutic benefit. To achieve this goal, it is important to identify specific mechanisms and targets involved in the clearance of ?-synuclein. Recent discovery of clinical, genetic, and pathological linkage between GD and PD offers a unique opportunity to examine lysosomal glucocerebrosidase, an enzyme mutated in GD, for development of targeted therapies in synucleinopathies. While modulation of glucocerebrosidase and glycolipid metabolism offers a viable approach to treating disorders associated with synuclein accumulation, the compounds described to date either lack the ability to penetrate the CNS or have off-target effects that may counteract or limit their capabilities to mediate the desired pharmacological action. However, recent emergence of selective inhibitors of glycosphingolipid biosynthesis and noninhibitory pharmacological chaperones of glycosphingolipid processing enzymes that gain access to the CNS provide a novel approach that may overcome some of the limitations of compounds reported to date. These new strategies may allow for development of targeted treatments for synucleinopathies that affect both children and adults. PMID:24668939

Sybertz, Edmund; Krainc, Dimitri



Estradiol and selective estrogen receptor modulators differentially regulate target genes with estrogen receptors alpha and beta.  


Estrogens and selective estrogen receptor modulators (SERMs) interact with estrogen receptor (ER) alpha and beta to activate or repress gene transcription. To understand how estrogens and SERMs exert tissue-specific effects, we performed microarray analysis to determine whether ERalpha or ERbeta regulate different target genes in response to estrogens and SERMs. We prepared human U2OS osteosarcoma cells that are stably transfected with a tetracycline-inducible vector to express ERalpha or ERbeta. Western blotting, immunohistochemistry, and immunoprecipitation studies confirmed that U2OS-ERalpha cells synthesized only ERalpha and that U2OS-ERbeta cells expressed exclusively ERbeta. U2OS-ERalpha and U2OS-ERbeta cells were treated either with 17beta-estradiol (E2), raloxifene, and tamoxifen for 18 h. Labeled cRNAs were hybridized with U95Av2 GeneChips (Affymetrix). A total of 228, 190, and 236 genes were significantly activated or repressed at least 1.74-fold in U2OS-ERalpha and U2OS-ERbeta cells by E2, raloxifene, and tamoxifen, respectively. Most genes regulated in ERalpha cells in response to E2, raloxifene, and tamoxifen were distinct from those regulated in ERbeta cells. Only 38 of the 228 (17%) genes were regulated by E2 in both U2OS-ERalpha and U2OS-ERbeta cells. Raloxifene and tamoxifen regulated only 27% of the same genes in both the ERalpha and ERbeta cells. A subset of genes involved in bone-related activities regulated by E2, raloxifene, and tamoxifen were also distinct. Our results demonstrate that most genes regulated by ERalpha are distinct from those regulated by ERbeta in response to E2 and SERMs. These results indicate that estrogens and SERMs exert tissue-specific effects by regulating unique sets of targets genes through ERalpha and ERbeta PMID:14699072

Tee, Meng Kian; Rogatsky, Inez; Tzagarakis-Foster, Christina; Cvoro, Aleksandra; An, Jinping; Christy, Robert J; Yamamoto, Keith R; Leitman, Dale C



TBLR1 as an AR coactivator selectively activates AR target genes to inhibit prostate cancer growth  

PubMed Central

Androgen Receptor (AR), a steroid hormone receptor, is critical for prostate cancer growth. However, activation of AR by androgens can also lead to growth suppression and differentiation. Transcriptional cofactors play an important role in this switch between proliferative and anti-proliferative AR target gene programs. TBLR1, a core component of the nuclear receptor corepressor (NCoR) complex, shows both co-repressor and co-activator activities on nuclear receptors, but little is known about its effects on AR and prostate cancer. We characterized TBLR1 as a coactivator of AR in prostate cancer cells and the activation is both phosphorylation and 19S proteosome dependent. We showed that TBLR1 physically interacts with AR and directly occupies the androgen response elements of affected AR target genes in an androgen-dependent manner. TBLR1 is primarily localized in the nucleus in benign prostate cells and nuclear expression is significantly reduced in prostate cancer cells in culture. Similarly, in human tumor samples, the expression of TBLR1 in the nucleus is significantly reduced in the malignant glands compared to the surrounding benign prostatic glands (p<0.005). Stable ectopic expression of nuclear TBLR1 leads to androgen-dependent growth suppression of prostate cancer cells in vitro and in vivo by selective activation of androgen regulated genes associated with differentiation (e.g. KRT18) and growth suppression (e.g. NKX3.1), but not cell proliferation of the prostate. Understanding the molecular switches involved in the transition from AR dependent growth promotion to AR dependent growth suppression will lead to more successful prostate cancer treatments. PMID:24243687

Daniels, Garrett; Li, Yirong; Gellert, Lan Lin; Zhou, Albert; Melamed, Jonathan; Wu, Xinyu; Zhang, Xinming; Zhang, David; Meruelo, Daniel; Logan, Susan K.; Basch, Ross; Lee, Peng



IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors  

PubMed Central

Upregulation of the ERK1 and ERK2 (ERK1/2) MAP kinase (MAPK) cascade occurs in >30% of cancers1, often through mutational activation of receptor tyrosine kinases or other upstream genes, including KRAS and BRAF 2. Efforts to target endogenous MAPKs are challenged by the fact that these kinases are required for viability in mammals3,4. Additionally, the effectiveness of new inhibitors of mutant BRAF has been diminished by acquired tumor resistance through selection for BRAF-independent mechanisms of ERK1/2 induction2,5,6. Furthermore, recently identified ERK1/2-inducing mutations in MEK1 and MEK2 (MEK1/2) MAPK genes in melanoma confer resistance to emerging therapeutic MEK inhibitors, underscoring the challenges facing direct kinase inhibition in cancer7,8. MAPK scaffolds, such as IQ motif–containing GTPase activating protein 1 (IQGAP1)9,10, assemble pathway kinases to affect signal transmission11–13, and disrupting scaffold function therefore offers an orthogonal approach to MAPK cascade inhibition. Consistent with this, we found a requirement for IQGAP1 in RAS-driven tumorigenesis in mouse and human tissue. In addition, the ERK1/2-binding14 IQGAP1 WW domain peptide disrupted IQGAP1-ERK1/2 interactions, inhibited RAS- and RAF-driven tumorigenesis, bypassed acquired resistance to the BRAF inhibitor vemurafenib (PLX-4032) and acted as a systemically deliverable therapeutic to significantly increase the lifespan of tumor-bearing mice. Scaffold-kinase interaction blockade acts by a mechanism distinct from direct kinase inhibition and may be a strategy to target overactive oncogenic kinase cascades in cancer. PMID:23603816

Zehnder, Ashley M; Zhang, Jiajing; Zarnegar, Brian; Sage, Julien; Khavari, Paul A



Neural Control of Visual Search by Frontal Eye Field: Effects of Unexpected Target Displacement on Visual Selection and Saccade Preparation  

PubMed Central

The dynamics of visual selection and saccade preparation by the frontal eye field was investigated in macaque monkeys performing a search-step task combining the classic double-step saccade task with visual search. Reward was earned for producing a saccade to a color singleton. On random trials the target and one distractor swapped locations before the saccade and monkeys were rewarded for shifting gaze to the new singleton location. A race model accounts for the probabilities and latencies of saccades to the initial and final singleton locations and provides a measure of the duration of a covert compensation process—target-step reaction time. When the target stepped out of a movement field, noncompensated saccades to the original location were produced when movement-related activity grew rapidly to a threshold. Compensated saccades to the final location were produced when the growth of the original movement-related activity was interrupted within target-step reaction time and was replaced by activation of other neurons producing the compensated saccade. When the target stepped into a receptive field, visual neurons selected the new target location regardless of the monkeys’ response. When the target stepped out of a receptive field most visual neurons maintained the representation of the original target location, but a minority of visual neurons showed reduced activity. Chronometric analyses of the neural responses to the target step revealed that the modulation of visually responsive neurons and movement-related neurons occurred early enough to shift attention and saccade preparation from the old to the new target location. These findings indicate that visual activity in the frontal eye field signals the location of targets for orienting, whereas movement-related activity instantiates saccade preparation. PMID:19261711

Murthy, Aditya; Ray, Supriya; Shorter, Stephanie M.; Schall, Jeffrey D.; Thompson, Kirk G.



Neural control of visual search by frontal eye field: effects of unexpected target displacement on visual selection and saccade preparation.  


The dynamics of visual selection and saccade preparation by the frontal eye field was investigated in macaque monkeys performing a search-step task combining the classic double-step saccade task with visual search. Reward was earned for producing a saccade to a color singleton. On random trials the target and one distractor swapped locations before the saccade and monkeys were rewarded for shifting gaze to the new singleton location. A race model accounts for the probabilities and latencies of saccades to the initial and final singleton locations and provides a measure of the duration of a covert compensation process-target-step reaction time. When the target stepped out of a movement field, noncompensated saccades to the original location were produced when movement-related activity grew rapidly to a threshold. Compensated saccades to the final location were produced when the growth of the original movement-related activity was interrupted within target-step reaction time and was replaced by activation of other neurons producing the compensated saccade. When the target stepped into a receptive field, visual neurons selected the new target location regardless of the monkeys' response. When the target stepped out of a receptive field most visual neurons maintained the representation of the original target location, but a minority of visual neurons showed reduced activity. Chronometric analyses of the neural responses to the target step revealed that the modulation of visually responsive neurons and movement-related neurons occurred early enough to shift attention and saccade preparation from the old to the new target location. These findings indicate that visual activity in the frontal eye field signals the location of targets for orienting, whereas movement-related activity instantiates saccade preparation. PMID:19261711

Murthy, Aditya; Ray, Supriya; Shorter, Stephanie M; Schall, Jeffrey D; Thompson, Kirk G



In Vitro Attachment of Radioactive Endotoxins to Lysosomes  

PubMed Central

The experiments reported here demonstrate that under certain conditions endotoxin can interact with lysosomes in vitro. After incubation of large granular fraction with 51Cr-labeled antigen under the conditions required for acid hydrolytic activity, radioactivity was associated with the pellet after centrifugation. This effect can be inhibited by preincubation of the large granular fraction with unlabeled homologous or heterologous endotoxins. High resolution autoradiography showed that 14C-labeled endotoxin was predominantly attached to the lysosomes contained in the large granular fraction. The mechanism of this interaction and its relationship to the toxic effect of endotoxins on lysosomes are discussed. Images PMID:4949505

Bona, C.; Chedid, L.; Lamensans, A.



Emerging roles of molecular chaperones and co-chaperones in selective autophagy: focus on BAG proteins  

Microsoft Academic Search

Macroautophagy is a catabolic process by which the cell degrades cytoplasmic components through the lysosomal machinery. While\\u000a initially acknowledged as a rather unspecific bulk degradation process, growing lines of evidence indicate the selectivity\\u000a of macroautophagy pathways in the removal of misfolded or aggregated proteins. How such substrates are recognized and specifically\\u000a targeted to the macroautophagy machinery has become a hotspot

Martin Gamerdinger; Serena Carra; Christian Behl


Proteasome Failure Promotes Positioning of Lysosomes around the Aggresome via Local Block of Microtubule-Dependent Transport  

PubMed Central

Ubiquitinated proteins aggregate upon proteasome failure, and the aggregates are transported to the aggresome. In aggresomes, protein aggregates are actively degraded by the autophagy-lysosome pathway, but why targeting the aggresome promotes degradation of aggregated species is currently unknown. Here we report that the important factor in this process is clustering of lysosomes around the aggresome via a novel mechanism. Proteasome inhibition causes formation of a zone around the centrosome where microtubular transport of lysosomes is suppressed, resulting in their entrapment and accumulation. Microtubule-dependent transport of other organelles, including autophagosomes, mitochondria, and endosomes, is also blocked in this entrapment zone (E-zone), while movement of organelles at the cell periphery remains unaffected. Following the whole-genome small interfering RNA (siRNA) screen for proteins involved in aggresome formation, we defined the pathway that regulates formation of the E-zone, including the Stk11 protein kinase, the Usp9x deubiquitinating enzyme, and their substrate kinase MARK4. Therefore, upon proteasome failure, targeting of aggregated proteins of the aggresome is coordinated with lysosome positioning around this body to facilitate degradation of the abnormal species. PMID:24469403

Zaarur, Nava; Meriin, Anatoli B.; Bejarano, Eloy; Xu, Xiaobin; Gabai, Vladimir L.; Cuervo, Ana Maria



The Intracellular Domain of Sortilin Interacts with Amyloid Precursor Protein and Regulates Its Lysosomal and Lipid Raft Trafficking  

PubMed Central

The processing of Amyloid precursor protein (APP) is multifaceted, comprising of protein transport, internalization and sequential proteolysis. However, the exact mechanism of APP intracellular trafficking and distribution remains unclear. To determine the interaction between sortilin and APP and the effect of sortilin on APP trafficking and processing, we studied the binding site and its function by mapping experiments, colocalization, coimmunoprecipitation and sucrose gradient fractionation. We identified for the first time that sortilin interacts with APP at both N- and C-terminal regions. The sortilin-FLVHRY (residues 787–792) and APP-NPTYKFFE (residues 759–766) motifs are crucial for the C-terminal interaction. We also found that lack of the FLVHRY motif reduces APP lysosomal targeting and increases APP distribution in lipid rafts in co-transfected HEK293 cells. These results are consistent with our in vivo data where sortilin knockout mice showed a decrease of APP lysosomal distribution and an increase of APP in lipid rafts. We further confirmed that overexpression of sortilin-FLVHRY mutants failed to rescue the lysosomal degradation of APP. Thus, our data suggests that sortilin is implicated in APP lysosomal and lipid raft targeting via its carboxyl-terminal F/YXXXXF/Y motif. Our study provides new molecular insights into APP trafficking and processing. PMID:23704887

Yang, Miao; Virassamy, Balaji; Vijayaraj, Swarna Lekha; Lim, Yoon; Saadipour, Khalil; Wang, Yan-Jiang; Han, Yan-Chuang; Zhong, Jin-Hua; Morales, Carlos R.; Zhou, Xin-Fu



Selective Targeting of the Cysteine Proteome by Thioredoxin and Glutathione Redox Systems  

PubMed Central

Thioredoxin (Trx) and GSH are the major thiol antioxidants protecting cells from oxidative stress-induced cytotoxicity. Redox states of Trx and GSH have been used as indicators of oxidative stress. Accumulating studies suggest that Trx and GSH redox systems regulate cell signaling and metabolic pathways differently and independently during diverse stressful conditions. In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx- and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized more than 1.3-fold by ARF, whereas BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than the GSH system, support distinct function of Trx and GSH in cellular redox control, and show for the first time in mammalian cells selective targeting peptidyl Cys and biological pathways due to deficient function of the Trx system. PMID:23946468

Go, Young-Mi; Roede, James R.; Walker, Douglas I.; Duong, Duc M.; Seyfried, Nicholas T.; Orr, Michael; Liang, Yongliang; Pennell, Kurt D.; Jones, Dean P.



Actin-dependent propulsion of endosomes and lysosomes byrecruitment of n-wasp  

SciTech Connect

We examined the spatial and temporal control of actin assembly in living Xenopus eggs. Within minutes of egg activation,dynamic actin-rich comet tails appeared on a subset of cytoplasmic vesicles that were enriched in protein kinase C (PKC), causing the vesicles to move through the cytoplasm. Actin comet tail formation in vivo was stimulated by the PKC activator phorbol myristate acetate (PMA),and this process could be reconstituted in a cell-free system. We used this system to define the characteristics that distinguish vesicles associated with actin comet tails from other vesicles in the extract. We found that the protein, N-WASP, was recruited to the surface of every vesicle associated with an actin comet tail, suggesting that vesicle movement results from actin assembly nucleated by the Arp2/3 complex, the immediate downstream target of N-WASP, The motile vesicles accumulated the dye acridine orange, a marker for endosomes and lysosomes. Furthermore, vesicles associated with actin comet tails had the morphological features of multivesicular endosomes as revealed by electron microscopy. Endosomes and lysosomes from mammalian cells preferentially nucleated actin assembly and moved in the Xenopus egg extract system. These results define endosomes and lysosomes as recruitment sites for the actin nucleation machinery and demonstrate that actin assembly contributes to organelle movement. Conversely, by nucleating actin assembly, intracellular membranes may contribute to the dynamic organization of the actin cytoskeleton.

Taunton J; Rowning BA; Coughlin ML; Wu M; Moon RT; Mitchison TJ; Larabell CA



Inhibitor screening of pharmacological chaperones for lysosomal ?-glucocerebrosidase by capillary electrophoresis.  


Pharmacological chaperones (PCs) represent a promising therapeutic strategy for treatment of lysosomal storage disorders based on enhanced stabilization and trafficking of mutant protein upon orthosteric and/or allosteric binding. Herein, we introduce a simple yet reliable enzyme assay using capillary electrophoresis (CE) for inhibitor screening of PCs that target the lysosomal enzyme, ?-glucocerebrosidase (GCase). The rate of GCase-catalyzed hydrolysis of the synthetic substrate, 4-methylumbelliferyl-?-D: -glucopyranoside was performed using different classes of PCs by CE with UV detection under standardized conditions. The pH and surfactant dependence of inhibitor binding on recombinant GCase activity was also examined. Enzyme inhibition studies were investigated for five putative PCs including isofagomine (IFG), ambroxol, bromhexine, diltiazem, and fluphenazine. IFG was confirmed as a potent competitive inhibitor of recombinant GCase with half-maximal inhibitory concentration (IC(50)) of 47.5?±?0.1 and 4.6?±?1.4 nM at pH 5.2 and pH 7.2, respectively. In contrast, the four other non-carbohydrate amines were demonstrated to function as mixed-type inhibitors with high micromolar activity at neutral pH relative to acidic pH conditions reflective of the lysosome. CE offers a convenient platform for characterization of PCs as a way to accelerate the clinical translation of previously approved drugs for oral treatment of rare genetic disorders, such as Gaucher disease. PMID:21286689

Shanmuganathan, Meera; Britz-McKibbin, Philip



Green tea extract selectively targets nanomechanics of live metastatic cancer cells  

NASA Astrophysics Data System (ADS)

Green tea extract (GTE) is known to be a potential anticancer agent (Yang et al 2009 Nat. Rev. Cancer 9 429-39) with various biological activities (Lu et al 2005 Clin. Cancer Res. 11 1675-83 Yang et al 1998 Carcinogenesis 19 611-6) yet the precise mechanism of action is still unclear. The biomechanical response of GTE treated cells taken directly from patient's body samples was measured using atomic force microscopy (AFM) (Binnig et al 1986 Phys. Rev. Lett. 56 930). We found significant increase in stiffness of GTE treated metastatic tumor cells, with a resulting value similar to untreated normal mesothelial cells, whereas mesothelial cell stiffness after GTE treatment is unchanged. Immunofluorescence analysis showed an increase in cytoskeletal-F-actin in GTE treated tumor cells, suggesting GTE treated tumor cells display mechanical, structural and morphological features similar to normal cells, which appears to be mediated by annexin-I expression, as determined by siRNA analysis of an in vitro cell line model. Our data indicates that GTE selectively targets human metastatic cancer cells but not normal mesothelial cells, a finding that is significantly advantageous compared to conventional chemotherapy agents.

Cross, Sarah E.; Jin, Yu-Sheng; Lu, Qing-Yi; Rao, JianYu; Gimzewski, James K.



Selective BCL-2 Inhibition by ABT-199 Causes On Target Cell Death in Acute Myeloid Leukemia  

PubMed Central

B-cell leukemia/lymphoma 2 (BCL-2) prevents commitment to programmed cell death at the mitochondrion. It remains a challenge to identify those tumors that are best treated by inhibition of BCL-2. Here we demonstrate that acute myeloid leukemia (AML) cell lines, primary patient samples, and murine primary xenografts are very sensitive to treatment with the selective BCL-2 antagonist ABT-199. In primary patient cells, the median IC50 was approximately 10 nM, and cell death occurred within 2 h. Our ex vivo sensitivity results compare favorably with those observed for chronic lymphocytic leukemia (CLL), a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, mitochondrial studies using BH3 profiling demonstrate activity at the mitochondrion that correlates well with cytotoxicity, supporting an on target mitochondrial mechanism of action. Our protein and BH3 profiling studies provide promising tools that can be tested as predictive biomarkers in any clinical trial of ABT-199 in AML. PMID:24346116

Pan, Rongqing; Hogdal, Leah J.; Benito, Juliana M; Bucci, Donna; Han, Lina; Borthakur, Gautam; Cortes, Jorge; DeAngelo, Daniel J.; Debose, LaKeisha; Mu, Hong; Dohner, Hartmut; Gaidzik, Verena I.; Galinsky, Ilene; Golfman, Leonard S.; Haferlach, Torsten; Harutyunyan, Karine G.; Hu, Jianhua; Leverson, Joel D; Marcucci, Guido; Muschen, Markus; Newman, Rachel; Park, Eugene; Ruvolo, Peter P.; Ruvolo, Vivian; Ryan, Jeremy; Schindela, Sonja; Zweidler-McKay, Patrick; Stone, Richard M.; Kantarjian, Hagop; Andreeff, Michael; Konopleva, Marina; Letai, Anthony G.



Estrogen receptor ? can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation  

PubMed Central

Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ER?) receptors. More specifically, ER? represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ER? represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ER? can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells. PMID:23828038

Marques, Maud; Laflamme, Liette; Gaudreau, Luc



Intracellular selection of peptide inhibitors that target disulphide-bridged A?42 oligomers.  


The ?-amyloid (A?) peptide aggregates into a number of soluble and insoluble forms, with soluble oligomers thought to be the primary factor implicated in Alzheimer's disease pathology. As a result, a wide range of potential aggregation inhibitors have been developed. However, in addition to problems with solubility and protease susceptibility, many have inadvertently raised the concentration of these soluble neurotoxic species. Sandberg et al. previously reported a ?-hairpin stabilized variant of A?42 that results from an intramolecular disulphide bridge (A21C/A31C; A?42cc), which generates highly toxic oligomeric species incapable of converting into mature fibrils. Using an intracellular protein-fragment complementation (PCA) approach, we have screened peptide libraries using E. coli that harbor an oxidizing environment to permit cytoplasmic disulphide bond formation. Peptides designed to target either the first or second ?-strand have been demonstrated to bind to A?42cc, lower amyloid cytotoxicity, and confer bacterial cell survival. Peptides have consequently been tested using wild-type A?42 via ThT binding assays, circular dichroism, MTT cytotoxicity assays, fluorescence microscopy, and atomic force microscopy. Results demonstrate that amyloid-PCA selected peptides function by both removing amyloid oligomers as well as inhibiting their formation. These data further support the use of semirational design combined with intracellular PCA methodology to develop A? antagonists as candidates for modification into drugs capable of slowing or even preventing the onset of AD. PMID:24947815

Acerra, Nicola; Kad, Neil M; Cheruvara, Harish; Mason, Jody M



Ken & barbie selectively regulates the expression of a subset of Jak/STAT pathway target genes.  


A limited number of evolutionarily conserved signal transduction pathways are repeatedly reused during development to regulate a wide range of processes. Here we describe a new negative regulator of JAK/STAT signaling and identify a potential mechanism by which the pleiotropy of responses resulting from pathway activation is generated in vivo. As part of a genetic interaction screen, we have identified Ken & Barbie (Ken) , which is an ortholog of the mammalian proto-oncogene BCL6 , as a negative regulator of the JAK/STAT pathway. Ken genetically interacts with the pathway in vivo and recognizes a DNA consensus sequence overlapping that of STAT92E in vitro. Tissue culture-based assays demonstrate the existence of Ken-sensitive and Ken-insensitive STAT92E binding sites, while ectopically expressed Ken is sufficient to downregulate a subset of JAK/STAT pathway target genes in vivo. Finally, we show that endogenous Ken specifically represses JAK/STAT-dependent expression of ventral veins lacking (vvl) in the posterior spiracles. Ken therefore represents a novel regulator of JAK/STAT signaling whose dynamic spatial and temporal expression is capable of selectively modulating the transcriptional repertoire elicited by activated STAT92E in vivo. PMID:16401426

Arbouzova, Natalia I; Bach, Erika A; Zeidler, Martin P



Microfluidic enrichment of a target cell type from a heterogenous suspension by adhesion-based negative selection.  


The enrichment or isolation of a selected type of cells in a heterogeneous suspension is challenging when the surface markers of these cells are not completely known. Here, we present a 3-stage arrangement of peptide-coated microfluidic channels that can recover a small number of adipose-derived stem cells (ADSCs) from a heterogenous suspension by negative selection depletion of the non-target cell types. PMID:19606304

Green, James V; Murthy, Shashi K



MDMA induces cardiac contractile dysfunction through autophagy upregulation and lysosome destabilization in rats.  


The underlying mechanisms of cardiotoxicity of 3,4-methylenedioxymethylamphetamine (MDMA, "ecstasy") abuse are unclear. Autophagy exerts either adaptive or maladaptive effects on cardiac function in various pathological settings, but nothing is known on the role of autophagy in the MDMA cardiotoxicity. Here, we investigated the mechanism through which autophagy may be involved in MDMA-induced cardiac contractile dysfunction. Rats were injected intraperitoneally with MDMA (20mg/kg) or saline. Left ventricular (LV) echocardiography and LV pressure measurement demonstrated reduction of LV systolic contractility 24h after MDMA administration. Western blot analysis showed a time-dependent increase in the levels of microtubule-associated protein light chain 3-II (LC3-II) and cathepsin-D after MDMA administration. Electron microscopy showed the presence of autophagic vacuoles in cardiomyocytes. MDMA upregulated phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) at Thr172, mammalian target of rapamycin (mTOR) at Thr2446, Raptor at Ser792, and Unc51-like kinase (ULK1) at Ser555, suggesting activation of autophagy through the AMPK-mTOR pathway. The effects of autophagic inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) on LC3-II levels indicated that MDMA enhanced autophagosome formation, but attenuated autophagosome clearance. MDMA also induced release of cathepsins into cytosol, and western blotting and electron microscopy showed cardiac troponin I (cTnI) degradation and myofibril damage, respectively. 3-MA, CQ, and a lysosomal inhibitor, E64c, inhibited cTnI proteolysis and improved contractile dysfunction after MDMA administration. In conclusion, MDMA causes lysosome destabilization following activation of the autophagy-lysosomal pathway, through which released lysosomal proteases damage myofibrils and induce LV systolic dysfunction in rat heart. PMID:24491919

Shintani-ishida, Kaori; Saka, Kanju; Yamaguchi, Koji; Hayashida, Makiko; Nagai, Hisashi; Takemura, Genzou; Yoshida, Ken-ichi



Adhesive but not Signaling Activity of Drosophila N-cadherin is Essential for Target Selection of Photoreceptor Afferents  

PubMed Central

Drosophila N-cadherin (CadN) is an evolutionarily conserved, atypical classical cadherin, which has a large complex extracellular domain and a catenin-binding cytoplasmic domain. We have previously shown that CadN regulates target selection of R7 photoreceptor axons. To determine the functional domains of CadN, we conducted a structure-function analysis focusing on its in vitro adhesive activity and in vivo function in R7 growth cones. We found that the cytoplasmic domain of CadN is largely dispensable for the targeting of R7 growth cones, and it is not essential for mediating homophilic interaction in cultured cells. Instead, the cytoplasmic domain of CadN is required for maintaining proper growth cone morphology. Domain swapping with the extracellular domain of CadN2, a related but non-adhesive cadherin, revealed that the CadN extracellular domain is required for both adhesive activity and R7 targeting. Using a target-mosaic system, we generated CadN mutant clones in the optic lobe and examined the target-selection of genetically wild-type R7 growth cones to CadN mutant target neurons. We found that CadN, but neither LAR nor Liprin-?, is required in the medulla neurons for R7 growth cones to select sthe correct medulla layer. Together, these data suggest that CadN mediates homophilic adhesive interactions between R7 growth cones and medulla neurons to regulate layer-specific target selection. PMID:17320070

Yonekura, Shinichi; Xu, Lei; Ting, Chun-Yuan; Lee, Chi-Hon



Regulation of lysosome biogenesis and functions in osteoclasts  

PubMed Central

In order to resorb the mineralized bone extracellular matrix, the osteoclast relies on the generation of a resorption lacuna characterized by the presence of specific proteases and a low pH. Hence, bone resorption by osteoclasts is highly dependent on lysosomes, the organelles specialized in intra- and extracellular material degradation. This is best illustrated by the fact that multiple forms of human osteopetrosis are caused by mutations in genes encoding for lysosomal proteins. Yet, until recently, the molecular mechanisms regulating lysosomal biogenesis and function in osteoclasts were poorly understood. Here we review the latest developments in the study of lysosomal biogenesis and function in osteoclasts with an emphasis on the transcriptional control of these processes. PMID:23966172

Lacombe, Julie; Karsenty, Gerard; Ferron, Mathieu



Genetic Regulation of Caenorhabditis elegans Lysosome Related Organelle Function  

E-print Network

Lysosomes are membrane-bound organelles that contain acid hydrolases that degrade cellular proteins, lipids, nucleic acids, and oligosaccharides, and are important for cellular maintenance and protection against age-related ...

Soukas, Alexander A.


Attention Blinks for Selection, Not Perception or Memory: Reading Sentences and Reporting Targets  

ERIC Educational Resources Information Center

In whole report, a sentence presented sequentially at the rate of about 10 words/s can be recalled accurately, whereas if the task is to report only two target words (e.g., red words), the second target suffers an attentional blink if it appears shortly after the first target. If these two tasks are carried out simultaneously, is there an…

Potter, Mary C.; Wyble, Brad; Olejarczyk, Jennifer



Target selection for saccadic eye movements: prelude activity in the superior colliculus during a direction-discrimination task.  


We investigated the role of the superior colliculus (SC) in saccade target selection while macaque monkeys performed a direction-discrimination task. The monkeys selected one of two possible saccade targets based on the direction of motion in a stochastic random-dot display; the difficulty of the task was varied by adjusting the strength of the motion signal in the display. One of the two saccade targets was positioned within the movement field of the SC neuron under study while the other target was positioned well outside the movement field. Approximately 30% of the neurons in the intermediate and deep layers of the SC discharged target-specific preludes of activity that "predicted" target choices well before execution of the saccadic eye movement. Across the population of neurons, the strength of the motion signal in the display influenced the intensity of this "predictive" prelude activity: SC activity signaled the impending saccade more reliably when the motion signal was strong than when it was weak. The dependence of neural activity on motion strength could not be explained by small variations in the metrics of the saccadic eye movements. Predictive activity was particularly strong in a subpopulation of neurons with directional visual responses that we have described previously. For a subset of SC neurons, therefore, prelude activity reflects the difficulty of the direction discrimination in addition to the target of the impending saccade. These results are consistent with the notion that a restricted network of SC neurons plays a role in the process of saccade target selection. PMID:11698541

Horwitz, G D; Newsome, W T



[Enzyme replacement therapy of lysosomal storage diseases].  


Extraction and purification of an acid ?-glucosidase from human placenta (alglucerase) for the treatment of Gaucher disease, replaced a few years later by a recombinant enzyme (imiglucérase, Cerezyme(®)), has paved the way to the development of enzyme replacement therapies (ERT) for the treatment of lysosomal storage diseases (LSD) among which Fabry disease for which the long-term efficacy of the two currently available preparations (agalsidase alfa, Replagal(®) and Fabrazyme(®)) is still being investigated. Mucopolysaccharidosis (MPS) type I (Hurler and Scheie diseases), II (Hunter syndrome) and VI (Maroteaux-Lamy disease) also benefit from ERT using laronidase (Aldurazyme(®)), idursulfase (Elaprase(®)) and galsulfase (Naglazyme(®)), respectively. ERT reduces the hepatosplenomegaly and improves the physical and respiratory capacities of MPS patients with a globally acceptable safety profile although the possibility of infusion-associated should always be kept in mind. Alglucosidase alpha (Myozyme(®)) improves the cardiomyopathy and life expectancy of infants suffering from Pompe disease and is under evaluation for the treatment of the juvenile and adult forms of the disease. CNS involvement remains a major challenge for many LSD and innovative research and approaches are needed to address the fact that recombinant enzymes do not cross the blood-brain barrier and therefore are not expected to lead to any improvement in CNS damages, except if alternative routes such as intrathecal administration would be developed. Molecular chaperones (e.g. migalastat for Fabry disease) and inhibitors of glucosylceramide synthesis (e.g. eliglustat tartrate for Gaucher disease) are currently under investigation in various clinical trials. PMID:21211680

Germain, D P; Boucly, C; Carlier, R Y; Caudron, E; Charlier, P; Colas, F; Jabbour, F; Martinez, V; Mokhtari, S; Orlikowski, D; Pellegrini, N; Perronne, C; Prigent, H; Rubinsztajn, R; Benistan, K



Lysosome and Phagosome Stability in Lethal Cell Injury  

PubMed Central

In two types of cell injury in a tissue culture system, the possibility was tested that lysosome rupture may be a lethal cellular reaction to injury, and thus an important general cause of irreversibility of damage in injured tissue. Prior labeling of secondary lysosomes with the fluorochrome acridine orange, or with ferritin, was used to trace changes in lysosomes after applying an injury. The metabolic inhibitors iodoacetate and cyanide were used together to block the cell's energy supply, or attachment of antiserum and subsequent complement attack were used to damage the surface membrane, producing rapid loss of cell volume control. Living cells were studied by time-lapse phase-contrast cinemicrography and fluorescence microscopy, and samples were fixed at intervals for electron microscopy. The cytolytic action of complement was lethal to sensitized cells within 2 hours, but results showed that lysosomes did not rupture for approximately 4 hours and in fact did not release the fluorescent dye until after reaching the postmortem necrotic phase of injury. Cells treated with metabolic inhibitors also showed irreversible alterations, while lysosomes remained intact and retained the ferritin marker. The fluorochrome marker, acridine orange, escaped from lysosomes early after metabolic injury, but the significance of this observation is not clear. The results are interpreted as evidence against the concept that lysosome rupture threatens the survival of injured cells. The original suicide bag mechanism of cell damage thus is apparently not operative in the systems studied. Lysosomes appear to be relatively stable organelles which, following injury of the types studied, burst only after cell death, acting then as scavengers which help to clear cellular debris. ImagesFigs 5-7Fig 18Fig 19Fig 20Figs 21-23Fig 8Fig 9Fig 10Fig 11Figs 24-27Fig 12Figs 13 and 14Fig 1Fig 2Fig 3Fig 4Fig 15Fig 16Fig 17 PMID:4340333

Hawkins, Hal K.; Ericsson, Jan L. E.; Biberfeld, Peter; Trump, Benjamin F.



The autophagy/lysosome pathway is impaired in SCA7 patients and SCA7 knock-in mice.  


There is still no treatment for polyglutamine disorders, but clearance of mutant proteins might represent a potential therapeutic strategy. Autophagy, the major pathway for organelle and protein turnover, has been implicated in these diseases. To determine whether the autophagy/lysosome system contributes to the pathogenesis of spinocerebellar ataxia type 7 (SCA7), caused by expansion of a polyglutamine tract in the ataxin-7 protein, we looked for biochemical, histological and transcriptomic abnormalities in components of the autophagy/lysosome pathway in a knock-in mouse model of the disease, postmortem brain and peripheral blood mononuclear cells (PBMC) from patients. In the mouse model, mutant ataxin-7 accumulated in inclusions immunoreactive for the autophagy-associated proteins mTOR, beclin-1, p62 and ubiquitin. Atypical accumulations of the autophagosome/lysosome markers LC3, LAMP-1, LAMP2 and cathepsin-D were also found in the cerebellum of the SCA7 knock-in mice. In patients, abnormal accumulations of autophagy markers were detected in the cerebellum and cerebral cortex of patients, but not in the striatum that is spared in SCA7, suggesting that autophagy might be impaired by the selective accumulation of mutant ataxin-7. In vitro studies demonstrated that the autophagic flux was impaired in cells overexpressing full-length mutant ataxin-7. Interestingly, the expression of the early autophagy-associated gene ATG12 was increased in PBMC from SCA7 patients in correlation with disease severity. These results provide evidence that the autophagy/lysosome pathway is impaired in neurons undergoing degeneration in SCA7. Autophagy/lysosome-associated molecules might, therefore, be useful markers for monitoring the effects of potential therapeutic approaches using modulators of autophagy in SCA7 and other autophagy/lysosome-associated neurodegenerative disorders. PMID:24859968

Alves, Sandro; Cormier-Dequaire, Florence; Marinello, Martina; Marais, Thibaut; Muriel, Marie-Paule; Beaumatin, Florian; Charbonnier-Beaupel, Fanny; Tahiri, Khadija; Seilhean, Danielle; El Hachimi, Khalid; Ruberg, Merle; Stevanin, Giovanni; Barkats, Martine; den Dunnen, Wilfred; Priault, Muriel; Brice, Alexis; Durr, Alexandra; Corvol, Jean-Christophe; Sittler, Annie



Precipitation effects on the selection of suitable non-variant targets intended for atmospheric correction of satellite remotely sensed imagery  

NASA Astrophysics Data System (ADS)

One of the most well-established atmospheric correction methods of satellite imagery is the use of the empirical line method using non-variant targets. Non-variant targets serve as pseudo-invariant targets since their reflectance values are stable across time. A recent adaptation of the empirical line method incorporates the use of ground reflectance measurements of selected non-variant targets. Most of the users are not aware of the existing conditions of the pseudo-invariant targets; i.e., whether they are dry or wet. Any omission of such effects may cause erroneous results; therefore, remote sensing users must be aware of such effects. This study assessed the effects of precipitation on five types of commonly located surfaces, including asphalt, concrete and sand, intended as pseudo-invariant targets for atmospheric correction. Spectroradiometric measurements were taken in wet and dry conditions to obtain the spectral signatures of the targets, from January 2010 to May 2011 (46 campaigns). An atmospheric correction of eleven Landsat TM/ETM + satellite images using the empirical line method was conducted. To identify the effects of precipitation, a comparison was conducted of the atmospheric path radiance component for wet and dry conditions. It was found that precipitation conditions such as rainfall affected the reflectance values of the surfaces, especially sand. Therefore, precipitation conditions need to be considered when using non-variant targets in atmospheric correction methods.

Themistocleous, Kyriacos; Hadjimitsis, Diofantos G.; Retalis, Adrianos; Chrysoulakis, Nektarios; Michaelides, Silas



Abstract--Our sensor selection algorithm targets the problem of global self-localization of multi-sensor mobile  

E-print Network

Abstract--Our sensor selection algorithm targets the problem of global self-localization of multi-sensor mobile robots. The algorithm builds on the probabilistic reasoning using Bayes filters to estimate sensor measurement uncertainty and sensor validity in robot localization. For quantifying measurement uncertainty we

Koschan, Andreas


Targeting adequate thermal stability and fire safety in selecting ionic liquid-based electrolytes for energy storage  

E-print Network

1 Targeting adequate thermal stability and fire safety in selecting ionic liquid-based electrolytes electrolyte solutions with nonvolatile and nonflammable ionic liquids instead of actual carbonate mixtures could be safer. However, few definitions of thermal stability of electrolytes based on ionic liquids

Paris-Sud XI, Université de


Staurosporine tethered peptide ligands that target cAMP-dependent protein kinase (PKA): Optimization and selectivity profiling  

E-print Network

of PKA reported to date; (c) the BL was found to be modular, where attachment of active site targeted Article history: Received 21 June 2009 Revised 22 July 2009 Accepted 23 July 2009 Available online 26 July to provide guidance toward the development of kinase selective reagents while uncovering new allosteric sites

Ghosh, Indraneel


A target-selected Apc-mutant rat kindred enhances the modeling of familial human colon cancer  

E-print Network

A target-selected Apc-mutant rat kindred enhances the modeling of familial human colon cancer James) Progress toward the understanding and management of human colon cancer can be significantly advanced several unique and favorable features for the study of colon cancer. Tumor-bearing Pirc rats can live

Dove, William


Efficacy Trial of a Selective Prevention Program Targeting Both Eating Disorder Symptoms and Unhealthy Weight Gain among Female College Students  

ERIC Educational Resources Information Center

Objective: Evaluate a selective prevention program targeting both eating disorder symptoms and unhealthy weight gain in young women. Method: Female college students at high-risk for these outcomes by virtue of body image concerns (N = 398; M age = 18.4 years, SD = 0.6) were randomized to the Healthy Weight group-based 4-hr prevention program,…

Stice, Eric; Rohde, Paul; Shaw, Heather; Marti, C. Nathan



Reward has a residual impact on target selection in visual search, but not on the suppression of distractors  

Microsoft Academic Search

In the reinforcement learning literature, good outcome following selection of a visual object is thought to bias perception and attention in favour of similar objects in later experience. This impact of reward might be instantiated in two ways: Reward could prime target features or it could act to facilitate suppression of distractors present when reward was received. Here we report

Clayton Hickey; Leonardo Chelazzi; Jan Theeuwes



Transitional B cells are the target of negative selection in the B cell compartment  

PubMed Central

B lymphocytes recognize antigen through membrane-bound antigen- receptors, membrane IgM and IgD (mIgM and mIgD). Binding to foreign antigens initiates a cascade of biochemical events that lead to activation and differentiation. In contrast, binding to self-antigens leads to death or to inactivation. It is commonly believed that the B cells acquire the ability to discriminate between self and nonself in the early phases of development. We report here that immature B cells, which have just emerged from the mIgMneg, B220pos pool, are not deleted upon binding of self-antigen. In vivo, developing B cells become sensitive to tolerance induction in a relatively late window of differentiation, when they are in transition from the immature (HSAbright, B220dull) to the mature (HSAdull, B220bright) stage. In the transitional B cells, early markers of differentiation such as Pgp1 (CD44) and ThB reach the highest level of expression, while the expression of CD23 and mIgD, late markers of differentiation, and expression of class II MHC, progressively increases. Most of the transitional B cells, but only few of the mature and of the immature B cells, express the fas antigen, while mature B cells, but not immature and transitional B cells, express bcl-2 protein. mIgM is present in low amounts in immature B cells, reaches the highest level of expression in transitional B cells and is down-regulated in mature resting B cells, where it is coexpressed with mIgD. The high expression of mIgM, the presence of the fas antigen and the absence of bcl-2 protein is compatible with the high sensitivity of transitional B cells to negative selection. In vitro, immature B cells die rapidly by apoptosis after cross-linking of mIgM. This result, combined with the resistance of immature B cells to elimination in vivo, suggests that early in development the stroma cell microenvironment modulates signals transduced through mIgM. The functional and phenotypic division of IgMpos bone marrow B cells in three compartments not only allows to define the target population of physiological processes like negative selection, but will also be a helpful tool for an accurate description of possible developmental blocks in mutant mice. PMID:7760002



Enzymatic reduction of disulfide bonds in lysosomes: Characterization of a Gamma-interferon-inducible lysosomal thiol reductase (GILT)  

NASA Astrophysics Data System (ADS)

Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter- and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin, but the mechanism of lysosomal reduction is unknown. We describe here a lysosomal thiol reductase optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro. The active site, determined by mutagenesis, consists of a pair of cysteine residues separated by two amino acids, similar to other enzymes of the thioredoxin family. The enzyme is a soluble glycoprotein that is synthesized as a precursor. After delivery into the endosomal/lysosomal system by the mannose 6-phosphate receptor, N- and C-terminal prosequences are removed. The enzyme is expressed constitutively in antigen-presenting cells and induced by IFN-? in other cell types, suggesting a potentially important role in antigen processing.

Arunachalam, Balasubramanian; Phan, Uyen T.; Geuze, Hans J.; Cresswell, Peter



From Target Selection to Post-Stimulation Analysis: Example of an Unconventional Faulted Reservoir  

NASA Astrophysics Data System (ADS)

As the global balance of supply and demand forces the hydrocarbon industry toward unconventional resources, technology- and economics-driven shale oil and gas production is gaining momentum throughout many basins worldwide. Production from such unconventional plays is facilitated by massive hydraulic fracturing treatments aimed at increasing permeability and reactivating natural fractures. Large-scale faulting and fracturing partly control stress distribution, hence stimulation-derived hydraulically-induced fracture systems development. Therefore, careful integrated approaches to target selection, treatment staging, and stimulation methods need to be used to economically maximize ultimate hydrocarbon recovery. We present a case study of a multistage, multilateral stimulation project in the Fort Worth Basin, Texas. Wells had to be drilled within city limits in a commercially developing building area. Well locations and trajectories were determined in and around large-scale faults using 3D surface seismic with throws varying from seven to thirty meters. As a result, three horizontal wells were drilled in the Lower Barnett Shale section, 150 m apart with the central well landed about 25 m shallower than the outside laterals. Surface seismic indicates that the surface locations are on top of a major fault complex with the lateral sections drilling away from the major fault system and through a smaller fault. Modeling of the borehole-based microseismic monitoring options led to the selection of an optimum set of configurations given the operational restrictions faced: monitoring would mainly take place using a horizontal array to be tractored downhole and moved according to the well and stage to be monitored. Wells were completed using a perf-and-plug approach allowing for each stimulation stage to obtain a precise orientation of the various three-component accelerometers of the monitoring array as well as the calibration of the velocity model used to process the microseismic data acquired. Real-time microseismic monitoring allowed (i) to avoid the water-bearing formation below the zone of interest, (ii) to bypass the faulted zone, and (iii) to modify as needed the perforation and stimulation plans. Completion led to an initial gas production of over 3 MMCF/day each. Early decline rates confirm successful completion in avoiding the faulted areas. Initial observations of the slickwater fracturing stimulation treatments for these three wells using an integrated approach involving mechanical modelling calibrated using microseismic data indicate that (i) a long bi-wing-like fracture system initiated prior to being followed by a complex fracture network; thus, explaining the fact that some events are mapped relatively far away from the injection site, (ii) proppant generally settled down in the near wellbore area during the fracture network development due to rapid decrease of fluid flow velocity away from the injection side. Initial b-value results seem to indicate that the target reservoir is naturally fractured and that the influence of a large fault system in the vicinity of the treated zone could be asserted.

LeCalvez, J. H.; Williams, M.; Xu, W.; Stokes, J.; Moros, H.; Maxwell, S. C.; Conners, S.




SciTech Connect

This first paper in a series describes the design of a study testing whether pre-appearance signatures of solar magnetic active regions were detectable using various tools of local helioseismology. The ultimate goal is to understand flux-emergence mechanisms by setting observational constraints on pre-appearance subsurface changes, for comparison with results from simulation efforts. This first paper provides details of the data selection and preparation of the samples, each containing over 100 members, of two populations: regions on the Sun that produced a numbered NOAA active region, and a 'control' sample of areas that did not. The seismology is performed on data from the GONG network; accompanying magnetic data from SOHO/MDI are used for co-temporal analysis of the surface magnetic field. Samples are drawn from 2001-2007, and each target is analyzed for 27.7 hr prior to an objectively determined time of emergence. The results of two analysis approaches are published separately: one based on averages of the seismology- and magnetic-derived signals over the samples, another based on Discriminant Analysis of these signals, for a statistical test of detectable differences between the two populations. We include here descriptions of a new potential-field calculation approach and the algorithm for matching sample distributions over multiple variables. We describe known sources of bias and the approaches used to mitigate them. We also describe unexpected bias sources uncovered during the course of the study and include a discussion of refinements that should be included in future work on this topic.

Leka, K. D.; Barnes, G.; Birch, A. C.; Dunn, T.; Javornik, B.; Braun, D. C. [NorthWest Research Associates, Boulder, CO 80301 (United States)] [NorthWest Research Associates, Boulder, CO 80301 (United States); Gonzalez-Hernandez, I. [National Solar Observatory, Tucson, AZ 85719 (United States)] [National Solar Observatory, Tucson, AZ 85719 (United States)



Selective inhibition of cell-free translation by oligonucleotides targeted to a mRNA hairpin structure.  

PubMed Central

Using an in vitro selection approach we have previously isolated oligodeoxy aptamers that can bind to a DNA hairpin structure without disrupting the double-stranded stem. We report here that these oligomers can bind to the RNA version of this hairpin, mostly through pairing with a designed 6 nt anchor. The part of the aptamer selected against the DNA hairpin did not increase stability of the RNA-aptamer complex. However, it contributed to the binding site for Escherichia coli RNase H, leading to very efficient cleavage of the target RNA. In addition, a 2'- O -methyloligoribonucleotide analogue of one selected sequence selectively blocked in vitro translation of luciferase in wheat germ extract by binding to the hairpin region inserted upstream of the initiation codon of the reporter gene. Therefore, non-complementary oligomers can exhibit antisense properties following hybridization with the target RNA. Our study also suggests that in vitro selection might provide a means to extend the repertoire of sequences that can be targetted by antisense oligonucleotides to structured RNA motifs of biological importance. PMID:9580674

Le Tinevez, R; Mishra, R K; Toulme, J J



Identification and Characterization of Genes Involved in Leishmania Pathogenesis: The Potential for Drug Target Selection  

PubMed Central

Identifying and characterizing Leishmania donovani genes and the proteins they encode for their role in pathogenesis can reveal the value of this approach for finding new drug targets. Effective drug targets are likely to be proteins differentially expressed or required in the amastigote life cycle stage found in the patient. Several examples and their potential for chemotherapeutic disruption are presented. A pathway nearly ubiquitous in living cells targeted by anticancer drugs, the ubiquitin system, is examined. New findings in ubiquitin and ubiquitin-like modifiers in Leishmania show how disruption of those pathways could point to additional drug targets. The programmed cell death pathway, now recognized among protozoan parasites, is reviewed for some of its components and evidence that suggests they could be targeted for antiparasitic drug therapy. Finally, the endoplasmic reticulum quality control system is involved in secretion of many virulence factors. How disruptions in this pathway reduce virulence as evidence for potential drug targets is presented. PMID:22091403

Duncan, Robert; Gannavaram, Sreenivas; Dey, Ranadhir; Debrabant, Alain; Lakhal-Naouar, Ines; Nakhasi, Hira L.



Targeted Multifunctional Nanoparticles cure and image Brain Tumors: Selective MRI Contrast Enhancement and Photodynamic Therapy  

Microsoft Academic Search

Aimed at targeted therapy and imaging of brain tumors, our approach uses targeted, multi-functional nano-particles (NP). A typical nano-particle contains a biologically inert, non-toxic matrix, biodegradable and bio-eliminable over a long time period. It also contains active components, such as fluorescent chemical indicators, photo-sensitizers, MRI contrast enhancement agents and optical imaging dyes. In addition, its surface contains molecular targeting units,

Raoul Kopelman



New biotechnological and nanomedicine strategies for treatment of lysosomal storage disorders  

PubMed Central

This review discusses the multiple bio- and nano-technological strategies developed in the last few decades for treatment of a group of fatal genetic diseases, termed lysosomal storage disorders. Some basic foundation on the biomedical causes and social and clinical relevance of these diseases is provided. Several treatment modalities, from those currently available to novel therapeutic approaches under development, are also discussed; these include gene and cell therapies, substrate reduction therapy, chemical chaperones, enzyme replacement therapy, multifunctional chimeras, targeting strategies, and drug carrier approaches. PMID:20112244

Muro, Silvia



[Enzyme replacement therapies for lysosomal storage disorders.].  


The lysosome was discovered in 1955 by Christian de Duve. The first demonstration by Hers of a link between an enzyme deficiency and a storage disorder (Pompe disease) paved the way for seminal discoveries culminating in the successful treatment of Gaucher disease with beta-glucosidase. Today, enzyme replacement therapy is a reality for Fabry disease, mucopolysaccharidosis type I (MPS I) and mucopolysaccharidosis type VI (MPS VI). In patients with MPS I, laronidase (recombinant human alpha-L-iduronidase) significantly improves respiratory function and physical capacities, reduces hepatomegaly and glycosaminoglycan storage, and has a favorable safety profile. Following positive results from phase I and phase II studies, a randomized, double-blind, multicentre phase III trial of galsulfase (recombinant human arylsulfatase B) was conducted in patients affected with MPS VI. Data from this study confirmed the safety and efficacy of galsulfase which significantly improved the 12-minutes walk test and reduced urinary dermatan sulftate. Recombinant human alpha-glucosidase (rhGAA) is currently in clinical trials for therapy of Pompe disease. Clinical data show that the enzyme efficiently clears glycogen from cardiac muscle and type I skeletal muscle fibers, but not type II fibers. Clinical trials with recombinant human enzymes are ongoing in MPS II and about to begin in Niemann-Pick B disease. Significant challenges remain for enzyme replacement therapies, particularly the treatment of central nervous system disease. Alternative strategies, such as substrate deprivation and enzyme enhancement therapy which employs small molecules as "pharmacological chaperones" to rescue misfolded and/or unstable mutant enzymes that have residual function, are currently being investigated. double dagger. PMID:16324679

Germain, Dominique P



Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target  

NASA Astrophysics Data System (ADS)

Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.



A role for presenilins in autophagy revisited: normal acidification of lysosomes in cells lacking PSEN1 and PSEN2  

PubMed Central

Presenilins (PS1 and PS2) are the catalytic subunits of the ?-secretase complex, and genes encoding mutant PS1 and PS2 variants cause familial forms of Alzheimer’s disease (FAD). Lee et al. (2010) recently reported that loss of PS1 activity lead to impairments in autophagosomal function as a consequence of lysosomal alkalinization, caused by failed maturation of the proton translocating V0a1 subunit of the vacuolar (H+)-ATPase and targeting to the lysosome. We have reexamined these issues in mammalian cells and in brains of mice lacking PS (PScdko) and have been unable to find evidence that the turnover of autophagic substrates, vesicle pH, V0a1 maturation or lysosome function is altered compared to wild-type counterparts. Collectively, our studies fail to document a role for presenilins in regulating cellular autophagosomal function. On the other hand, our transcriptome studies of PScdko mouse brains reveal, for the first time, a role for PS in regulating lysosomal biogenesis. PMID:22723704

Zhang, Xulun; Garbett, Krassimira; Veeraraghavalu, Karthikeyan; Wilburn, Brian; Gilmore, Reid; Mirnics, Karoly; Sisodia, Sangram S.



Lysosomal Localization of TRPML3 Depends on TRPML2 and the Mucolipidosis-associated Protein TRPML1*S  

PubMed Central

Mucolipidosis type IV is an autosomal recessive lysosomal storage disorder characterized by severe neurodegeneration, achlorhydria, and visual impairments such as corneal opacity and strabismus. The disease arises due to mutations in a group 2 transient receptor potential (TRP)-related cation channel, TRPML1. Mammals encode two additional TRPML proteins named TRPML2 and TRPML3. Information regarding the propensity of these proteins to multimerize, their subcellular distribution and mechanisms that regulate their trafficking are limited. Here we demonstrate that TRPMLs interact to form homo- and heteromultimers. Moreover, the presence of either TRPML1 or TRPML2 specifically influences the spatial distribution of TRPML3. TRPML1 and TRPML2 homo-multimers are lysosomal proteins, whereas TRPML3 homomultimers are in the endoplasmic reticulum. However, TRPML3 localizes to lysosomes when coexpressed with either TRPML1 or TRPML2 and is comparably mislocalized when lysosomal targeting of TRPML1 and TRPML2 is disrupted. Conversely, TRPML3 does not cause retention of TRPML1 or TRPML2 in the endoplasmic reticulum. These data demonstrate that there is a hierarchy controlling the subcellular distributions of the TRPMLs such that TRPML1 and TRPML2 dictate the localization of TRPML3 and not vice versa. PMID:16606612

Venkatachalam, Kartik; Hofmann, Thomas; Montell, Craig



Deep sequencing of large library selections allows computational discovery of diverse sets of zinc fingers that bind common targets  

PubMed Central

The Cys2His2 zinc finger (ZF) is the most frequently found sequence-specific DNA-binding domain in eukaryotic proteins. The ZF’s modular protein–DNA interface has also served as a platform for genome engineering applications. Despite decades of intense study, a predictive understanding of the DNA-binding specificities of either natural or engineered ZF domains remains elusive. To help fill this gap, we developed an integrated experimental-computational approach to enrich and recover distinct groups of ZFs that bind common targets. To showcase the power of our approach, we built several large ZF libraries and demonstrated their excellent diversity. As proof of principle, we used one of these ZF libraries to select and recover thousands of ZFs that bind several 3-nt targets of interest. We were then able to computationally cluster these recovered ZFs to reveal several distinct classes of proteins, all recovered from a single selection, to bind the same target. Finally, for each target studied, we confirmed that one or more representative ZFs yield the desired specificity. In sum, the described approach enables comprehensive large-scale selection and characterization of ZF specificities and should be a great aid in furthering our understanding of the ZF domain. PMID:24214968

Persikov, Anton V.; Rowland, Elizabeth F.; Oakes, Benjamin L.; Singh, Mona; Noyes, Marcus B.



Comparing the Selection and Placement of Best Management Practices in Improving Water Quality Using a Multiobjective Optimization and Targeting Method  

PubMed Central

Suites of Best Management Practices (BMPs) are usually selected to be economically and environmentally efficient in reducing nonpoint source (NPS) pollutants from agricultural areas in a watershed. The objective of this research was to compare the selection and placement of BMPs in a pasture-dominated watershed using multiobjective optimization and targeting methods. Two objective functions were used in the optimization process, which minimize pollutant losses and the BMP placement areas. The optimization tool was an integration of a multi-objective genetic algorithm (GA) and a watershed model (Soil and Water Assessment Tool—SWAT). For the targeting method, an optimum BMP option was implemented in critical areas in the watershed that contribute the greatest pollutant losses. A total of 171 BMP combinations, which consist of grazing management, vegetated filter strips (VFS), and poultry litter applications were considered. The results showed that the optimization is less effective when vegetated filter strips (VFS) are not considered, and it requires much longer computation times than the targeting method to search for optimum BMPs. Although the targeting method is effective in selecting and placing an optimum BMP, larger areas are needed for BMP implementation to achieve the same pollutant reductions as the optimization method. PMID:24619160

Chiang, Li-Chi; Chaubey, Indrajeet; Maringanti, Chetan; Huang, Tao



Caging of plumbagin on silver nanoparticles imparts selectivity and sensitivity to plumbagin for targeted cancer cell apoptosis.  


Plumbagin is a nutraceutical with potent anti-cancer activity. However, the therapeutic efficacy of plumbagin is overshadowed by the lack of sensitivity and selectivity towards cancer cells. The present study evaluated the use of nano-biotechnological intervention to cage plumbagin in silver nanoparticles for selective targeting of its biological effects towards cancerous cells. Caging of plumbagin in silver nanoparticles imparted selectivity and sensitivity to plumbagin for selective killing of cancer cells by altering the redox signalling events in the cancer cells. The selectivity and sensitivity of plumbagin towards cancer cells was due to the cumulative expression of the properties of plumbagin and nanoparticles which specifically affected the differential cancer cell microenvironment by altering the pyruvate kinase activity that regulates the ROS challenge in cancerous cells. The positive surface charge of plumbagin caged silver nanoparticles (PCSN) aids in getting them targeted towards anionic cancerous cells due to the exposed terminal carboxyl group of sialic acid residues. Furthermore, we observed that the effective concentration of the drug required to induce apoptosis was brought down to 50% upon caging of plumbagin on silver nanoparticles. We observed no such effect with the individual compound alone. The results indicated that the physico-chemical and biochemical properties of plumbagin significantly changed after conjugation with nanomaterials that facilitated "adding-in" therapeutical values to plumbagin which would otherwise be overshadowed by its lack of sensitivity and selectivity towards cancer cells. PMID:25188862

Duraipandy, N; Lakra, Rachita; Kunnavakkam Vinjimur, Srivatsan; Samanta, Debasis; K, Purna Sai; Kiran, Manikantan Syamala



A Targetable, Injectable Adenoviral Vector for Selective Gene Delivery to Pulmonary Endothelium in Vivo  

Microsoft Academic Search

Adenoviral (Ad) vectors are promising gene therapy vehicles due to their in vivo stability and effi- ciency, but their potential utility is compromised by their restricted tropism. Targeting strategies have been devised to improve the efficacy of these agents, but specific targeting following in vivo systemic administration of vector has not previously been demonstrated. The distinct aim of the current

Paul N. Reynolds; Kurt R. Zinn; Vitaliy D. Gavrilyuk; Irina V. Balyasnikova; Buck E. Rogers; Donald J. Buchsbaum; Ming H. Wang; David J. Miletich; William E. Grizzle; Joanne T. Douglas; Sergei M. Danilov; David T. Curiel



Identified target-selective visual interneurons descending from the dragonfly brain  

Microsoft Academic Search

1.Eight large interneurons descending in the dragonfly (Aeshna umbrosa, Anax junius) ventral nerve cord from the brain to the thoracic ganglia were identified anatomically with intracellular dye injection (Fig. 3). All eight were strictly visual and responded only to movements of small patterns, such as black squares, ‘targets’, moving on a white background.2.The target interneurons all projected from the protocerebrum

Robert M. Olberg



Serial linkage of target selection for orienting and tracking eye movements  

Microsoft Academic Search

Many natural actions require the coordination of two different kinds of movements. How are targets chosen under these circumstances: do central commands instruct different movement systems in parallel, or does the execution of one movement activate a serial chain that automatically chooses targets for the other movement? We examined a natural eye tracking action that consists of orienting saccades and

Stephen G. Lisberger; Justin L. Gardner



Negligible Genetic Diversity of Mycobacterium tuberculosis Host Immune System Protein Targets: Evidence of Limited Selective Pressure  

Microsoft Academic Search

A common theme in medical microbiology is that the amount of amino acid sequence variation in proteins that are targets of the host immune system greatly exceeds that found in metabolic enzymes or other housekeeping proteins. Twenty-four Mycobacterium tuberculosis genes coding for targets of the host immune system were sequenced in 16 strains representing the breadth of genomic diversity in

James M. Musser; Amol Amin; Srinivas Ramaswamy


Radiation-induced cell death: importance of lysosomal destabilization  

PubMed Central

The mechanisms involved in radiation-induced cellular injury and death remain incompletely understood. In addition to the direct formation of highly reactive hydroxyl radicals (HO·) by radiolysis of water, oxidative stress events in the cytoplasm due to formation of H2O2 may also be important. Since the major pool of low-mass redox-active intracellular iron seems to reside within lysosomes, arising from the continuous intralysosomal autophagocytotic degradation of ferruginous materials, formation of H2O2 inside and outside these organelles may cause lysosomal labilization with release to the cytosol of lytic enzymes and low-mass iron. If of limited magnitude, such release may induce ‘reparative autophagocytosis’, causing additional accumulation of redox-active iron within the lysosomal compartment. We have used radio-resistant histiocytic lymphoma (J774) cells to assess the importance of intralysosomal iron and lysosomal rupture in radiation-induced cellular injury. We found that a 40 Gy radiation dose increased the ‘loose’ iron content of the (still viable) cells approx. 5-fold when assayed 24 h later. Cytochemical staining revealed that most redox-active iron was within the lysosomes. The increase of intralysosomal iron was associated with ‘reparative autophagocytosis’, and sensitized cells to lysosomal rupture and consequent apoptotic/necrotic death following a second, much lower dose of radiation (20 Gy) 24 h after the first one. A high-molecular-mass derivative of desferrioxamine, which specifically localizes intralysosomally following endocytic uptake, added to the culture medium before either the first or the second dose of radiation, stabilized lysosomes and largely prevented cell death. These observations may provide a biological rationale for fractionated radiation. PMID:15813701



Superior antitumoral activity of dimerized targeted single-chain TRAIL fusion proteins under retention of tumor selectivity  

PubMed Central

Although targeting of the death receptors (DRs) DR4 and DR5 still appears a suitable antitumoral strategy, the limited clinical responses to recombinant soluble TNF-related apoptosis inducing ligand (TRAIL) necessitate novel reagents with improved apoptotic activity/tumor selectivity. Apoptosis induction by a single-chain TRAIL (scTRAIL) molecule could be enhanced >10-fold by generation of epidermal growth factor receptor (EGFR)-specific scFv-scTRAIL fusion proteins. By forcing dimerization of scFv-scTRAIL based on scFv linker modification, we obtained a targeted scTRAIL composed predominantly of dimers (Db-scTRAIL), exceeding the activity of nontargeted scTRAIL ?100-fold on Huh-7 hepatocellular and Colo205 colon carcinoma cells. Increased activity of Db-scTRAIL was also demonstrated on target-negative cells, suggesting that, in addition to targeting, oligomerization equivalent to an at least dimeric assembly of standard TRAIL per se enhances apoptosis signaling. In the presence of apoptosis sensitizers, such as the proteasomal inhibitor bortezomib, Db-scTRAIL was effective at picomolar concentrations in vitro (EC50 ?2 × 10?12 M). Importantly, in vivo, Db-scTRAIL was well tolerated and displayed superior antitumoral activity in mouse xenograft (Colo205) tumor models. Our results show that both targeting and controlled dimerization of scTRAIL fusion proteins provides a strategy to enforce apoptosis induction, together with retained tumor selectivity and good in vivo tolerance. PMID:22495350

Siegemund, M; Pollak, N; Seifert, O; Wahl, K; Hanak, K; Vogel, A; Nussler, A K; Gottsch, D; Munkel, S; Bantel, H; Kontermann, R E; Pfizenmaier, K



Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation  

PubMed Central

Introduction Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. Methods Bone marrow–derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4+ T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. Results The frequency of PS particle–positive CD11c+/CD11b+ BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4+ T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. Conclusion These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4+ T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles. PMID:25152619

Seydoux, Emilie; Rothen-Rutishauser, Barbara; Nita, Izabela M; Balog, Sandor; Gazdhar, Amiq; Stumbles, Philip A; Petri-Fink, Alke; Blank, Fabian; von Garnier, Christophe



Discovery-2: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria  

PubMed Central

Background Drug resistance to anti-malarial compounds remains a serious problem, with resistance to newer pharmaceuticals developing at an alarming rate. The development of new anti-malarials remains a priority, and the rational selection of putative targets is a key element of this process. Discovery-2 is an update of the original Discovery in silico resource for the rational selection of putative drug target proteins, enabling researchers to obtain information for a protein which may be useful for the selection of putative drug targets, and to perform advanced filtering of proteins encoded by the malaria genome based on a series of molecular properties. Methods An updated in silico resource has been developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein properties used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions. Newly added features include drugability measures from ChEMBL, automated literature relations and links to clinical trial information. Searching by chemical structure is also available. Results The updated functionality of the Discovery-2 resource is presented, together with a detailed case study of the Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (PfSAHH) protein. A short example of a chemical search with pyrimethamine is also illustrated. Conclusion The updated Discovery-2 resource allows researchers to obtain detailed properties of proteins from the malaria genome, which may be of interest in the target selection process, and to perform advanced filtering and selection of proteins based on a relevant range of molecular characteristics. PMID:23537208



Nematode control in spring-born suckler beef calves using targeted selective anthelmintic treatments.  


As anthelmintic resistance is increasingly being reported in cattle worldwide, there is a need to explore alternative approaches to gastrointestinal nematode control in cattle. A novel approach is the use of targeted selective treatments (TST) where only individual animals are treated instead of the entire group. The study objective was to determine if anthelmintic usage could be reduced using a TST-based approach in rotationally grazed first-grazing season suckler beef calves without affecting calf performance. Eighty-eight spring-born suckler beef calves, naïve to anthelmintics, with an initial mean (s.d.) age and live weight of 159 (22.4) days and 221 (42.4) kg, respectively, were used. All calves were vaccinated at pasture against dictyocaulosis at 8 and 12 weeks old. On August 9th 2013 (Week 0), when the trial began, calves were randomised by age, weight, sex, dam breed and sire breed to one of two treatments: (1) standard treatment (positive control) (n=44) and (2) TST (n=44). Samples collected one week prior to the start of the study were used as baseline covariates. Each treatment group was replicated once. All calves in the control groups were treated subcutaneously with levamisole on Week 0 and on Week 6. Individual calves in the TST groups were only eligible for treatment at pasture with the same product if predetermined thresholds were reached [plasma pepsinogen ?2.0 international units of tyrosine/litre and faecal egg count ?200 eggs per gram of faeces]. The trial concluded at housing on Week 13. Data were analysed using repeated measures mixed models ANOVA (PROC MIXED) (SAS 9.3). No calves in the TST groups were treated for gastrointestinal nematodes during the study period as they did not reach pre-determined treatment thresholds. Mean (sem) calf daily live weight gain for control and TST groups was 0.90 (±0.04) and 0.92 (±0.03) kg, respectively (P=0.68). Using an ELISA to detect antibodies to Dictyocaulus viviparus at Week 11, 81% of calves were seropositive. Gastrointestinal nematode challenge in spring-born suckler beef calves under these conditions can potentially be controlled with minimal anthelmintic treatments whilst not significantly impairing calf performance, provided appropriate control measures are taken to prevent dictyocaulosis from occurring. PMID:25085771

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



Synthesis and evaluation of aza-peptidyl inhibitors of the lysosomal asparaginyl endopeptidase, legumain  

PubMed Central

Legumain or asparaginly endopeptidase (AEP) is a lysosomal cysteine protease with a high level of specificity for cleavage of protein substrates after an asparagine residue. It is also capable of cleaving after aspartic acids sites when in the acidic environment of the lysosome. Legumain expression and activity is linked to a number of pathological conditions including cancer, atherosclerosis and inflammation, yet its biological role in these pathologies is not well-understood. Highly potent and selective inhibitors of legumain would not only be valuable for studying the functional roles of legumain in these conditions, but may have therapeutic potential as well. We describe here the design, synthesis and in vitro evaluation of selective legumain inhibitors based on the aza-asparaginyl scaffold. We synthesized a library of aza-peptidyl inhibitors with various non-natural amino acids and different electrophilic warheads, and characterized the kinetic properties of inactivation of legumain. We also synthesized fluorescently labeled inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have second order rate constants of up to 5×104 M?1s?1 and IC50 values as low as 4 nM against recombinant mouse legumain. In addition, the inhibitors are highly selective toward legumain and have little or no cross-reactivity with cathepsins. Overall, we have identified several valuable new inhibitors of legumain that can be used to study legumain function in multiple disease models. PMID:22243962

Lee, Jiyoun; Bogyo, Matthew



Synthesis and evaluation of aza-peptidyl inhibitors of the lysosomal asparaginyl endopeptidase, legumain.  


Legumain or asparaginly endopeptidase (AEP) is a lysosomal cysteine protease with a high level of specificity for cleavage of protein substrates after an asparagine residue. It is also capable of cleaving after aspartic acids sites when in the acidic environment of the lysosome. Legumain expression and activity is linked to a number of pathological conditions including cancer, atherosclerosis and inflammation, yet its biological role in these pathologies is not well-understood. Highly potent and selective inhibitors of legumain would not only be valuable for studying the functional roles of legumain in these conditions, but may have therapeutic potential as well. We describe here the design, synthesis and in vitro evaluation of selective legumain inhibitors based on the aza-asparaginyl scaffold. We synthesized a library of aza-peptidyl inhibitors with various non-natural amino acids and different electrophilic warheads, and characterized the kinetic properties of inactivation of legumain. We also synthesized fluorescently labeled inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have second order rate constants of up to 5 × 10(4)M(-1)s(-1) and IC(50) values as low as 4 nM against recombinant mouse legumain. In addition, the inhibitors are highly selective toward legumain and have little or no cross-reactivity with cathepsins. Overall, we have identified several valuable new inhibitors of legumain that can be used to study legumain function in multiple disease models. PMID:22243962

Lee, Jiyoun; Bogyo, Matthew



Oxidative Stress and Autophagy in the Regulation of Lysosome-Dependent Neuron Death  

PubMed Central

Abstract Lysosomes critically regulate the pH-dependent catabolism of extracellular and intracellular macromolecules delivered from the endocytic/heterophagy and autophagy pathways, respectively. The importance of lysosomes to cell survival is underscored not only by their unique ability effectively to degrade metalloproteins and oxidatively damaged macromolecules, but also by the distinct potential for induction of both caspase-dependent and -independent cell death with a compromise in the integrity of lysosome function. Oxidative stress and free radical damage play a principal role in cell death induced by lysosome dysfunction and may be linked to several upstream and downstream stimuli, including alterations in the autophagy degradation pathway, inhibition of lysosome enzyme function, and lysosome membrane damage. Neurons are sensitive to lysosome dysfunction, and the contribution of oxidative stress and free radical damage to lysosome dysfunction may contribute to the etiology of neurodegenerative disease. This review provides a broad overview of lysosome function and explores the contribution of oxidative stress and autophagy to lysosome dysfunction–induced neuron death. Putative signaling pathways that either induce lysosome dysfunction or result from lysosome dysfunction or both, and the role of oxidative stress, free radical damage, and lysosome dysfunction in pediatric lysosomal storage disorders (neuronal ceroid lipofuscinoses or NCL/Batten disease) and in Alzheimer's disease are emphasized. Antioxid. Redox Signal. 11, 481–496. PMID:18764739

Pivtoraiko, Violetta N.; Stone, Sara L.; Roth, Kevin A.



Gene expression profiling of mucolipidosis type IV fibroblasts reveals deregulation of genes with relevant functions in lysosome physiology.  


Mucolipidosis type IV (MLIV, MIM 252650) is an autosomal recessive lysosomal storage disorder that causes mental and motor retardation as well as visual impairment. The lysosomal storage defect in MLIV is consistent with abnormalities of membrane traffic and organelle dynamics in the late endocytic pathway. MLIV is caused by mutations in the MCOLN1 gene, which codes for mucolipin-1 (MLN1), a member of the large family of transient receptor potential (TRP) cation channels. Although a number of studies have been performed on mucolipin-1, the pathological mechanisms underlying MLIV are not fully understood. To identify genes that characterize pathogenic changes in mucolipidosis type IV, we compared the expression profiles of three MLIV and three normal skin fibroblasts cell lines using oligonucleotide microarrays. Genes that were differentially expressed in patients' cells were identified. 231 genes were up-regulated, and 116 down-regulated. Real-Time RT-PCR performed on selected genes in six independent MLIV fibroblasts cell lines was generally consistent with the microarray findings. This study allowed to evidence the modulation at the transcriptional level of a discrete number of genes relevant in biological processes which are altered in the disease such as endosome/lysosome trafficking, lysosome biogenesis, organelle acidification and lipid metabolism. PMID:18258208

Bozzato, Andrea; Barlati, Sergio; Borsani, Giuseppe



Membrane proteins of dense lysosomes from Chinese hamster ovary cells  

SciTech Connect

In this work membrane proteins from lysosomes were studied in order to gain more information on the biogenesis and intracellular sorting of this class of membrane proteins. Membrane proteins were isolated from a purified population of lysosomes. These proteins were then examined for various co- and post-translational modifications which could serve as potential intracellular sorting signals. Biochemical analysis using marker enzymatic activities detected no plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, mitochondria, or cytosol. Analysis after incorporation of ({sup 3}H)thymidine or ({sup 3}H)uridine detected no nuclei or ribosomes. A fraction containing integral membrane proteins was obtained from the dense lysosomes by extraction with Triton X-114. Twenty-three polypeptides which incorporated both ({sup 35}S)methionine and ({sup 3}H)leucine were detected by SDS PAGE in this membrane fraction, and ranged in molecular weight from 30-130 kDa. After incorporation by cells of various radioactive metabolic precursors, the membrane fraction from dense lysosomes was examined and was found to be enriched in mannose, galactose, fucose, palmitate, myristate, and sulfate, but was depleted in phosphate. The membrane fraction from dense lysosomes was then analyzed by SDS PAGE to determine the apparent molecular weights of modified polypepties.

Chance, S.C.



Changes in lysosomal enzymes in acute experimental liver injury  

PubMed Central

1. An investigation has been made of the changes occurring in lysosomal enzyme activities during the early development of experimentally produced liver injury in the rat. Three enzymes have been studied: acid phosphatase, acid ribonuclease and ?-glucuronidase. Four different methods of inducing liver injury have been used: administration of carbon tetrachloride, thioacetamide, dimethylnitrosamine and the fungal toxin sporidesmin. 2. The majority of the data presented concern alterations produced by carbon tetrachloride. Despite the extensive central necrosis and accompanying fat accumulation which this poison produced in the liver, only small changes in the activity and latency of lysosomal enzymes could be detected. In the early (pre-necrotic) period of injury these changes were insignificant. At a late stage of injury, when extensive centrilobular necrosis was present, there were indications of lysosomal rupture. 3. The results obtained with the other three hepatotoxins were similar to those described for carbon tetrachloride in that no evidence of early lysosomal rupture was obtained during the pre-necrotic period. It is concluded that lysosomes probably play no role in the early development of the four types of liver injury studied but, instead, are involved in later scavenging processes. PMID:5837787

Slater, T. F.; Greenbaum, A. L.



Selection and optimization of gene targets for the metabolic engineering of E. coli  

E-print Network

This thesis is about identifying genetic interventions that improve the performance of targeted pathways in the metabolism of the bacterium Escherichia coli. Three case studies illustrate three disparate approaches to ...

Fischer, Curt R., Ph. D. Massachusetts Institute of Technology



Structure-Based Design of a Potent, Selective, and Brain Penetrating PDE2 Inhibitor with Demonstrated Target Engagement.  


Structure-guided design led to the identification of the novel, potent, and selective phosphodiesterase 2 (PDE2) inhibitor 12. Compound 12 demonstrated a >210-fold selectivity versus PDE10 and PDE11 and was inactive against all other PDE family members up to 10 ?M. In vivo evaluation of 12 provided evidence that it is able to engage the target and to increase cGMP levels in relevant brain regions. Hence, 12 is a valuable tool compound for the better understanding of the role of PDE2 in cognitive impairment and other central nervous system related disorders. PMID:25221665

Buijnsters, Peter; De Angelis, Meri; Langlois, Xavier; Rombouts, Frederik J R; Sanderson, Wendy; Tresadern, Gary; Ritchie, Alison; Trabanco, Andrés A; VanHoof, Greet; Roosbroeck, Yves Van; Andrés, José-Ignacio



Mitochondrial Targeted Coenzyme Q, Superoxide, and Fuel Selectivity in Endothelial Cells  

E-print Network

Background: Previously, we reported that the ‘‘antioxidant’’ compound ‘‘mitoQ’’ (mitochondrial-targeted ubiquinol/ ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I... consequences of loading mitochondria with Coenzyme Q (CoQ10) analogs. In past studies of bovine aortic endothelial (BAE) cell mitochondria, we demonstrated that a mitochondrial targeted CoQ compound termed ‘‘mitoQ’’ (mitoquinone, mitoquinol, or a mixture...

Fink, Brian D.; O'Malley, Yunxia; Dake, Brian L.; Ross, Nicholette C.; Prisinzano, Thomas E.; Sivitz, William I.



A minimally invasive multifunctional nanoscale system for selective targeting, imaging, and NIR photothermal therapy of malignant tumors  

NASA Astrophysics Data System (ADS)

The anti-EGFR antibody, cetuximab, was labeled with IRDye 800CW fluorescent dye and conjugated to gold nanorods (GNR). GNR with aspect ratio of ~ 4 and plasmon resonance peak at ~785 nm were fabricated for use in these experiments. The IRDye:cetuximab:nanorod conjugate treatment with NIR light selectively heated the GNR and was sufficient to treat cancers. Excitation induced fluorescence of the IRDye 800CW enabling real-time imaging. We characterized and optimized the parameters for the conjugation of the GNR to cetuximab to facilitate active targeting of the nanorods to the site of the tumor. This combination of selective targeting, imaging, and photothermal treating of malignant cells is a viable approach for a variety of squamous cell carcinomas.

Green, H. N.; Martyshkin, D. V.; Rosenthal, E. L.; Mirov, S. B.



Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis  

E-print Network

M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely ...

Farhat, Maha R


Cancer-Selective Targeting of the NF-?B Survival Pathway with GADD45?/MKK7 Inhibitors  

PubMed Central

Summary Constitutive NF-?B signaling promotes survival in multiple myeloma (MM) and other cancers; however, current NF-?B-targeting strategies lack cancer cell specificity. Here, we identify the interaction between the NF-?B-regulated antiapoptotic factor GADD45? and the JNK kinase MKK7 as a therapeutic target in MM. Using a drug-discovery strategy, we developed DTP3, a D-tripeptide, which disrupts the GADD45?/MKK7 complex, kills MM cells effectively, and, importantly, lacks toxicity to normal cells. DTP3 has similar anticancer potency to the clinical standard, bortezomib, but more than 100-fold higher cancer cell specificity in vitro. Notably, DTP3 ablates myeloma xenografts in mice with no apparent side effects at the effective doses. Hence, cancer-selective targeting of the NF-?B pathway is possible and, at least for myeloma patients, promises a profound benefit. PMID:25314077

Tornatore, Laura; Sandomenico, Annamaria; Raimondo, Domenico; Low, Caroline; Rocci, Alberto; Tralau-Stewart, Cathy; Capece, Daria; D'Andrea, Daniel; Bua, Marco; Boyle, Eileen; van Duin, Mark; Zoppoli, Pietro; Jaxa-Chamiec, Albert; Thotakura, Anil K.; Dyson, Julian; Walker, Brian A.; Leonardi, Antonio; Chambery, Angela; Driessen, Christoph; Sonneveld, Pieter; Morgan, Gareth; Palumbo, Antonio; Tramontano, Anna; Rahemtulla, Amin; Ruvo, Menotti; Franzoso, Guido



Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection  

Microsoft Academic Search

Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing

Courtney L. Frazier; Joseph San Filippo; Alan M. Lambowitz; David A. Mills



Inhibition of lysosome degradation on autophagosome formation and responses to GMI, an immunomodulatory protein from Ganoderma microsporum  

PubMed Central

BACKGROUND AND PURPOSE Autophagic cell death is considered a self-destructive process that results from large amounts of autophagic flux. In our previous study, GMI, a recombinant fungal immunomodulatory protein cloned from Ganoderma microsporum, induced autophagic cell death in lung cancer cells. The aim of this study was to examine the role of autophagosome accumulation in GMI-mediated cell death. EXPERIMENTAL APPROACH Western blot analysis, flow cytometry and confocal microscopy were used to evaluate the effects of different treatments, including silencing of ATP6V0A1 by use of short hairpin RNAi, on GMI-mediated cell death, lung cancer cell viability and autophagosome accumulation in vitro. KEY RESULTS Lysosome inhibitors bafilomycin-A1 and chloroquine increased GMI-mediated autophagic cell death. GMI and bafilomycin-A1 co-treatment induced the accumulation of large amounts of autophagosomes, but did not significantly induce apoptosis. GMI elicited autophagy through the PKB (Akt)/mammalian target of rapamycin signalling pathway. Silencing of ATP6V0A1, one subunit of vesicular H+-ATPases (V-ATPases) that mediates lysosome acidification, spontaneously induced autophagosome accumulation, but did not affect lysosome acidity. GMI-mediated autophagosome accumulation and cytotoxicity was increased in shATP6V0A1 lung cancer cells. Furthermore, ATP6V0A1 silencing decreased autophagosome and lysosome fusion in GMI-treated CaLu-1/GFP-LC3 lung cancer cells. CONCLUSION AND IMPLICATIONS We demonstrated that autophagosome accumulation induces autophagic cell death in a GMI treatment model, and ATP6V0A1 plays an important role in mediating autophagosome-lysosome fusion. Our findings provide new insights into the mechanisms involved in the induction of autophagic cell death. PMID:22708544

Hsin, I-Lun; Sheu, Gwo-Tarng; Jan, Ming-Shiou; Sun, Hai-Lun; Wu, Tzu-Chin; Chiu, Ling-Yen; Lue, Ko-Huang; Ko, Jiunn-Liang



ATP release through lysosomal exocytosis from peripheral nerves: the effect of lysosomal exocytosis on peripheral nerve degeneration and regeneration after nerve injury.  


Studies have shown that lysosomal activation increases in Schwann cells after nerve injury. Lysosomal activation is thought to promote the engulfment of myelin debris or fragments of injured axons in Schwann cells during Wallerian degeneration. However, a recent interpretation of lysosomal activation proposes a different view of the phenomenon. During Wallerian degeneration, lysosomes become secretory vesicles and are activated for lysosomal exocytosis. The lysosomal exocytosis triggers adenosine 5'-triphosphate (ATP) release from peripheral neurons and Schwann cells during Wallerian degeneration. Exocytosis is involved in demyelination and axonal degradation, which facilitate nerve regeneration following nerve degeneration. At this time, released ATP may affect the communication between cells in peripheral nerves. In this review, our description of the relationship between lysosomal exocytosis and Wallerian degeneration has implications for the understanding of peripheral nerve degenerative diseases and peripheral neuropathies, such as Charcot-Marie-Tooth disease or Guillain-Barré syndrome. PMID:25101301

Jung, Junyang; Jo, Hyun Woo; Kwon, Hyunseob; Jeong, Na Young



The Dendritic Cell Receptor for Endocytosis, DEC205, Can Recycle and Enhance Antigen Presentation via Major Histocompatibility Complex Class II-positive Lysosomal Compartments  

Microsoft Academic Search

Many receptors for endocytosis recycle into and out of cells through early endosomes. We now find in dendritic cells that the DEC-205 multilectin receptor targets late endosomes or lysosomes rich in major his- tocompatibility complex class II (MHC II) products, whereas the homologous macrophage mannose recep- tor (MMR), as expected, is found in more peripheral endosomes. To analyze this finding,

Karsten Mahnke; Ming Guo; Sena Lee; Homero Sepulveda; Suzy L. Swain; Michel Nussenzweig; Ralph M. Steinman



Effects of mitogenic stimulation of lymphocytes on lysosomal enzyme activity.  


Changes in the activities of several lysosomal enzymes were studied during transformation of mouse spleen cells in vitro. The activity of beta-glucuronidase increased during culture in the presence of T or B-cell mitogens, and lymphoblasts contained higher levels of activity than did small, non-transformed lymphocytes. Moreover, lymphoblasts in well-transformed cultures had higher activities than those in poorly-transformed cultures. The activities of other lysosomal enzymes (N-acetyl-beta-glucosaminidase, alpha-mannosidase, beta-glucosidase) also increased during mitogenic stimulation, but each at different rates, although aryl sulphatase was unaffected. Such differences may be of importance when lymphocytes are used for diagnosis of inherited lysosomal deficiency diseases. PMID:6434192

Olsen, I; Dean, M F; Muir, H; Smith, R; Jenne, B M; Hand, C



Selecting target populations for ROPS retrofit programs in Pennsylvania and Vermont.  


Agriculture has the highest injury and fatality rates when compared with other U.S. industries, and tractor overturns remain the leading cause of agricultural fatalities. Rollover protection structures (ROPS) are the only proven devices to protect a tractor operator in the event of an overturn. These devices are 99% effective when used with a seatbelt. Nearly 49% of tractors in the U.S. are not equipped with a ROPS. Interventions such as social marketing, community awareness campaigns, and financial incentives have been directed at encouraging farmers to install ROPS on their unprotected tractors. The purpose of this study was to conduct similar comparisons of ROPS protection and readiness to retrofit in different segments of the Vermont and Pennsylvania farm communities. A telephone survey was used to collect data on ROPS prevalence, farm demographic characteristics, and farmer's stage of change relative to installing ROPS on farm tractors. Our data provide new and unique information on the prevalence of ROPS-equipped tractors relative to commodity, farm size, and a variety of other demographic variables. Extrapolating from these data, the commodities studied account for roughly 162,072 tractors across the two states. Of these, 85,927 (53%) do not have ROPS. Of these unprotected tractors, 77,203 are in Pennsylvania and 8,724 are in Vermont. Our other two research questions dealt with the farmer's stage of change and possible ways to segment this population. The stage of change portion of our work demonstrates that most Pennsylvania and Vermont farmers are not contemplating ROPS retrofitting in the near future. Since no major differences were found in the stage of change, the number of unprotected tractors was examined for each of the commodity groups. In Pennsylvania, 29% of all unprotected tractors were found on cash crop farms. This trend was even more apparent on smaller farms than large farms. This led to the selection of smaller cash crop farms as the target audience for social marketing messages. In contrast, researchers in Vermont found a bimodal distribution of unprotected tractors. Of all the commodity groups surveyed in Vermont, vegetable and cash crop farmers were least likely to have even one protected tractor to use on the farm. Probably the most encouraging finding from this study is that over 85% of Pennsylvania farms and over 87% of Vermont farms surveyed had at least one tractor available that had ROPS protection. Of those farms, 25.5% of the Pennsylvania farms and 46% of the Vermont farms have ROPS on all of their tractors. Both of these findings were greater than the findings from a 2006 survey of New York State farms, which found that 75% of surveyed New York farms have ROPS on at least one tractor and 18% have ROPS on all tractors. Even with these encouraging data, the goal of 100% of tractors with ROPS is far from being met. There are still an estimated 90,000 unprotected tractors on Pennsylvania and Vermont farms, and these farm owners are currently unmotivated to install ROPS. However as demonstrated in New York State, it may be possible to use social marketing that combines persuasive messages and cost-sharing to persuade these farmers that ROPS are indeed important and accessible. PMID:24400422

Yoder, A M; Sorensen, J A; Foster, F; Myers, M; Murphy, D; Cook, G; May, J; Jenkins, P



Strategies for Delivery of Therapeutics into the Central Nervous System for Treatment of Lysosomal Storage Disorders  

PubMed Central

Lysosomal storage disorders (LSDs) are a group of about fifty life-threatening conditions caused by genetic defects affecting lysosomal components. The underscoring molecular deficiency leads to widespread cellular dysfunction through most tissues in the body, including peripheral organs and the central nervous system (CNS). Efforts during the last few decades have rendered a remarkable advance regarding our knowledge, medical awareness, and early detection of these genetic defects, as well as development of several treatment modalities. Clinical and experimental strategies encompassing enzyme replacement, gene and cell therapies, substrate reduction, and chemical chaperones are showing considerable potential in attenuating the peripheral pathology. However, a major drawback has been encountered regarding the suboptimal impact of these approaches on the CNS pathology. Particular anatomical and biochemical constraints of this tissue pose a major obstacle to the delivery of therapeutics into the CNS. Approaches to overcome these obstacles include modalities of local administration, strategies to enhance the blood-CNS permeability, intranasal delivery, use of exosomes, and those exploiting targeting of transporters and transcytosis pathways in the endothelial lining. The later two approaches are being pursued at the time by coupling therapeutic agents to affinity moieties and drug delivery systems capable of targeting these natural transport routes. This approach is particularly promising, as using paths naturally active at this interface may render safe and effective delivery of LSD therapies into the CNS. PMID:24688886

Muro, Silvia



Update on selective treatments targeting neutrophilic inflammation in atherogenesis and atherothrombosis.  


Atherosclerosis is the most common pathological process underlying cardiovascular diseases. Current therapies are largely focused on alleviating hyperlipidaemia and preventing thrombotic complications, but do not completely eliminate risk of suffering recurrent acute ischaemic events. Specifically targeting the inflammatory processes may help to reduce this residual risk of major adverse cardiovascular events in atherosclerotic patients. The involvement of neutrophils in the pathophysiology of atherosclerosis is an emerging field, where evidence for their causal contribution during various stages of atherosclerosis is accumulating. Therefore, the identification of neutrophils as a potential therapeutic target may offer new therapeutic perspective to reduce the current atherosclerotic burden. This narrative review highlights the expanding role of neutrophils in atherogenesis and discusses on the potential treatment targeting neutrophil-related inflammation and associated atherosclerotic plaque vulnerability. PMID:24285257

Gomes Quinderé, Ana Luíza; Benevides, Norma Maria Barros; Carbone, Federico; Mach, François; Vuilleumier, Nicolas; Montecucco, Fabrizio



Common Risk Alleles for Inflammatory Diseases Are Targets of Recent Positive Selection  

PubMed Central

Genome-wide association studies (GWASs) have identified hundreds of loci harboring genetic variation influencing inflammatory-disease susceptibility in humans. It has been hypothesized that present day inflammatory diseases may have arisen, in part, due to pleiotropic effects of host resistance to pathogens over the course of human history, with significant selective pressures acting to increase host resistance to pathogens. The extent to which genetic factors underlying inflammatory-disease susceptibility has been influenced by selective processes can now be quantified more comprehensively than previously possible. To understand the evolutionary forces that have shaped inflammatory-disease susceptibility and to elucidate functional pathways affected by selection, we performed a systems-based analysis to integrate (1) published GWASs for inflammatory diseases, (2) a genome-wide scan for signatures of positive selection in a population of European ancestry, (3) functional genomics data comprised of protein-protein interaction networks, and (4) a genome-wide expression quantitative trait locus (eQTL) mapping study in peripheral blood mononuclear cells (PBMCs). We demonstrate that loci for inflammatory-disease susceptibility are enriched for genomic signatures of recent positive natural selection, with selected loci forming a highly interconnected protein-protein interaction network. Further, we identify 21 loci for inflammatory-disease susceptibility that display signatures of recent positive selection, of which 13 also show evidence of cis-regulatory effects on genes within the associated locus. Thus, our integrated analyses highlight a set of susceptibility loci that might subserve a shared molecular function and has experienced selective pressure over the course of human history; today, these loci play a key role in influencing susceptibility to multiple different inflammatory diseases, in part through alterations of gene expression in immune cells. PMID:23522783

Raj, Towfique; Kuchroo, Manik; Replogle, Joseph M.; Raychaudhuri, Soumya; Stranger, Barbara E.; De Jager, Philip L.



Common risk alleles for inflammatory diseases are targets of recent positive selection.  


Genome-wide association studies (GWASs) have identified hundreds of loci harboring genetic variation influencing inflammatory-disease susceptibility in humans. It has been hypothesized that present day inflammatory diseases may have arisen, in part, due to pleiotropic effects of host resistance to pathogens over the course of human history, with significant selective pressures acting to increase host resistance to pathogens. The extent to which genetic factors underlying inflammatory-disease susceptibility has been influenced by selective processes can now be quantified more comprehensively than previously possible. To understand the evolutionary forces that have shaped inflammatory-disease susceptibility and to elucidate functional pathways affected by selection, we performed a systems-based analysis to integrate (1) published GWASs for inflammatory diseases, (2) a genome-wide scan for signatures of positive selection in a population of European ancestry, (3) functional genomics data comprised of protein-protein interaction networks, and (4) a genome-wide expression quantitative trait locus (eQTL) mapping study in peripheral blood mononuclear cells (PBMCs). We demonstrate that loci for inflammatory-disease susceptibility are enriched for genomic signatures of recent positive natural selection, with selected loci forming a highly interconnected protein-protein interaction network. Further, we identify 21 loci for inflammatory-disease susceptibility that display signatures of recent positive selection, of which 13 also show evidence of cis-regulatory effects on genes within the associated locus. Thus, our integrated analyses highlight a set of susceptibility loci that might subserve a shared molecular function and has experienced selective pressure over the course of human history; today, these loci play a key role in influencing susceptibility to multiple different inflammatory diseases, in part through alterations of gene expression in immune cells. PMID:23522783

Raj, Towfique; Kuchroo, Manik; Replogle, Joseph M; Raychaudhuri, Soumya; Stranger, Barbara E; De Jager, Philip L



Target Selection by the Frontal Cortex during Coordinated Saccadic and Smooth Pursuit Eye Movements  

Microsoft Academic Search

Oculomotor tracking of moving objects is an important component of visually based cognition and planning. Such tracking is achieved by a combination of saccades and smoothpursuit eye movements. In particular, the saccadic and smoothpursuit systems interact to often choose the same target, and to maximize its visibility through time. How do multiple brain regions interact, including frontal cortical areas, to

Krishna Srihasam; Daniel Bullock; Stephen Grossberg



Target Selection by the Frontal Cortex during Coordinated Saccadic and Smooth Pursuit Eye Movements  

Microsoft Academic Search

Oculomotor tracking of moving objects is an important component of visually based cognition and planning. Such tracking is achieved by a combination of saccades and smooth-pursuit eye movements. In particular, the saccadic and smooth-pursuit systems interact to often choose the same target, and to maximize its visibility through time. How do multiple brain regions interact, including frontal cortical areas, to

Krishna Srihasam; Daniel Bullock; Stephen Grossberg



Bull's-Eye!: Selecting Target Schools Can Boost an Organization's Bottom Line.  

ERIC Educational Resources Information Center

Many companies make mistakes when identifying which schools to include in their college relations programs. This article describes which criteria can be used for identifying target schools, and how to deal with the issue of the reduction in numbers of schools due to financial constraints. (ADT)

Gray, Kevin



Insect-selective spider toxins targeting voltage-gated sodium channels  

Microsoft Academic Search

The voltage-gated sodium (Nav) channel is a target for a number of drugs, insecticides and neurotoxins. These bind to at least seven identified neurotoxin binding sites and either block conductance or modulate Nav channel gating. A number of peptide neurotoxins from the venoms of araneomorph and mygalomorph spiders have been isolated and characterized and determined to interact with several of

Graham M. Nicholson



Searching for Life on Mars: Selection of Molecular Targets for ESA's Aurora ExoMars Mission  

Microsoft Academic Search

The European Space Agency's ExoMars mission will seek evidence of organic compounds of biological and non-biological origin at the martian surface. One of the instruments in the Pasteur payload may be a Life Marker Chip that utilizes an immunoassay approach to detect specific organic molecules or classes of molecules. Therefore, it is necessary to define and prioritize specific molecular targets

John Parnell; David Cullen; Mark R. Sims; Stephen Bowden; Charles S. Cockell; Richard Court; Pascale Ehrenfreund; Francois Gaubert; William Grant; Victor Parro; Michel Rohmer; Mark Sephton; Helga Stan-Lotter; Andrew Steele; Jan Toporski; Jorge Vago



Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte  

E-print Network

, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115; and §Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215 Edited by Owen N and validating drug targets and poten- tially for therapy. Lymphocytes and other primary blood cells

Lieberman, Judy


Update on the Pfam5000 Strategy for Selection of Structural Genomics Targets  

E-print Network

structural genomics projects at 9 pilot centers through the Protein Structure Initiative (PSI). The primary-dimensional shapes of all important biological macromolecules, with a primary focus on proteins. Target proteins diversity found in nature [1-5]. In the United States, the National Institutes of Health are supporting


Advanced Feature Selection Methodology for Automatic Target Recognition Dale E. Nelson  

E-print Network

recognition (ATR) is a difficult task. When applied to air-to-air targets using High Range Resolution (HRR) radar, the task becomes even more difficult. Figure 1 shows the typical way the HRR signal is obtained for that time segment. The difficulty of ATR using HRR data lies in the extreme variability in the radar

Starzyk, Janusz A.


Genomic Expression Analyses Reveal Lysosomal, Innate Immunity Proteins, as Disease Correlates in Murine Models of a Lysosomal Storage Disorder  

PubMed Central

Niemann-Pick Type C (NPC) disease is a rare, genetic, lysosomal disorder with progressive neurodegeneration. Poor understanding of the pathophysiology and a lack of blood-based diagnostic markers are major hurdles in the treatment and management of NPC and several additional, neurological lysosomal disorders. To identify disease severity correlates, we undertook whole genome expression profiling of sentinel organs, brain, liver, and spleen of Balb/c Npc1?/? mice relative to Npc1+/? at an asymptomatic stage, as well as early- and late-symptomatic stages. Unexpectedly, we found prominent up regulation of innate immunity genes with age-dependent change in their expression, in all three organs. We shortlisted a set of 12 secretory genes whose expression steadily increased with age in both brain and liver, as potential plasma correlates of neurological and/or liver disease. Ten were innate immune genes with eight ascribed to lysosomes. Several are known to be elevated in diseased organs of murine models of other lysosomal diseases including Gaucher’s disease, Sandhoff disease and MPSIIIB. We validated the top candidate lysozyme, in the plasma of Npc1?/? as well as Balb/c Npc1nmf164 mice (bearing a point mutation closer to human disease mutants) and show its reduction in response to an emerging therapeutic. We further established elevation of innate immunity in Npc1?/? mice through multiple functional assays including inhibition of bacterial infection as well as cellular analysis and immunohistochemistry. These data revealed neutrophil elevation in the Npc1?/? spleen and liver (where large foci were detected proximal to damaged tissue). Together our results yield a set of lysosomal, secretory innate immunity genes that have potential to be developed as pan or specific plasma markers for neurological diseases associated with lysosomal storage and where diagnosis is a major problem. Further, the accumulation of neutrophils in diseased organs (hitherto not associated with NPC) suggests their role in pathophysiology and disease exacerbation. PMID:23094108

Alam, Md. Suhail; Getz, Michelle; Safeukui, Innocent; Yi, Sue; Tamez, Pamela; Shin, Jenny; Velazquez, Peter; Haldar, Kasturi



APOL1 risk variants enhance podocyte necrosis through compromising lysosomal membrane permeability.  


Development of higher rates of nondiabetic glomerulosclerosis (GS) in African Americans has been attributed to two coding sequence variants (G1 and G2) in the APOL1 gene. To date, the cellular function and the role of APOL1 variants (Vs) in GS are still unknown. In this study, we examined the effects of overexpressing wild-type (G0) and kidney disease risk variants (G1 and G2) of APOL1 in human podocytes using a lentivirus expression system. Interestingly, G0 inflicted podocyte injury only at a higher concentration; however, G1 and G2 promoted moderate podocyte injury at lower and higher concentrations. APOL1Vs expressing podocytes displayed diffuse distribution of both Lucifer yellow dye and cathepsin L as manifestations of enhanced lysosomal membrane permeability (LMP). Chloroquine attenuated the APOL1Vs-induced increase in podocyte injury, consistent with targeting lysosomes. The chloride channel blocker DIDS prevented APOL1Vs- induced injury, indicating a role for chloride influx in osmotic swelling of lysosomes. Direct exposure of noninfected podocytes with conditioned media from G1- and G2-expressing podocytes also induced injury, suggesting a contributory role of the secreted component of G1 and G2 as well. Adverse host factors (AHFs) such as hydrogen peroxide, hypoxia, TNF-?, and puromycin aminonucleoside augmented APOL1- and APOL1Vs-induced podocyte injury, while the effect of human immunodeficiency virus (HIV) on podocyte injury was overwhelming under conditions of APOLVs expression. We conclude that G0 and G1 and G2 APOL1 variants have the potential to induce podocyte injury in a manner which is further augmented by AHFs, with HIV infection being especially prominent. PMID:24899058

Lan, Xiqian; Jhaveri, Aakash; Cheng, Kang; Wen, Hongxiu; Saleem, Moin A; Mathieson, Peter W; Mikulak, Joanna; Aviram, Sharon; Malhotra, Ashwani; Skorecki, Karl; Singhal, Pravin C



Wortmannin causes mistargeting of procathepsin D. evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes  

PubMed Central

At present little is known of the biochemical machinery controlling transport of newly synthesized lysosomal hydrolases from the trans- Golgi network (TGN) to endosomes. The demonstration that Vps34p (a protein required for targeting soluble hydrolases to the vacuole in Saccharomyces cerevisiae) is a phosphatidylinositol 3-kinase (PI3-K) suggested the possibility that a homologous enzyme might be involved in the equivalent step in mammalian cells. Using the PI3-K inhibitors wortmannin and LY294002, I provide evidence to support this hypothesis. Treatment of K-562 cells with wortmannin induced secretion of procathepsin D, with half-maximal inhibition of accurate targeting to lysosomes at 10-20 nM. Kinetic analysis indicated that a late Golgi (TGN) step was affected, and that other constitutive vesicular transport events were not. The M6P recognition signal was still generated in the presence of wortmannin suggesting that the drug was directly inhibiting export of the receptor-ligand complex from the TGN, while removal of the drug led to a rapid restoration of accurate sorting. At the concentrations used, wortmannin and LY294002 are presently accepted to be specific inhibitors of PI3-K. I conclude that these data implicate such an enzyme in the trafficking of M6P-receptor- ligand complexes from the TGN towards lysosomes. PMID:7642698



MLL fusion protein-driven AML is selectively inhibited by targeted disruption of the MLL-PAFc interaction  

PubMed Central

MLL rearrangements are common in leukemia and considered an adverse risk factor. Through interactions with the polymerase-associated factor complex (PAFc), mixed lineage leukemia (MLL) fusion proteins activate genes critical for blocking differentiation, such as HOXA9. Here we investigate whether the MLL-PAFc interaction can be exploited therapeutically using both genetic and biochemical approaches. We tested the genetic requirement of the PAFc in acute myeloid leukemia (AML) using a conditional allele of the PAFc subunit, Cdc73. We show that the PAFc is indiscriminately necessary for the proliferation of AML cells through the epigenetic regulation of proleukemogenic target genes, such as MEIS1 and Bcl2. To investigate the therapeutic potential of targeting the MLL-PAFc interaction, we engineered a dominant negative fragment of MLL capable of binding to the PAFc. Disruption of the MLL-PAFc interaction selectively inhibits the proliferation of MLL leukemic cells without affecting cells transformed by an unrelated E2A-HLF fusion protein. Using in vivo hematopoietic reconstitution assays, we demonstrate that disruption of the MLL-PAFc does not alter normal hematopoietic stem cell function. Together, our data show a selective growth inhibition of MLL-associated leukemic cells and tolerance of normal hematopoiesis to disruption of the MLL-PAFc interaction establishing the MLL-PAFc interaction as an attractive therapeutic target. PMID:23900238

Chen, Wei; Jones, Morgan; Granowicz, Eric M.; Maillard, Ivan; Hess, Jay L.



Validity of Teacher Ratings in Selecting Influential Aggressive Adolescents for a Targeted Preventive Intervention  

PubMed Central

This study describes a method for using teacher nominations and ratings to identify socially influential, aggressive middle school students for participation in a targeted violence prevention intervention. The teacher nomination method is compared with peer nominations of aggression and influence to obtain validity evidence. Participants were urban, predominantly African American and Latino sixth-grade students who were involved in a pilot study for a large multi-site violence prevention project. Convergent validity was suggested by the high correlation of teacher ratings of peer influence and peer nominations of social influence. The teacher ratings of influence demonstrated acceptable sensitivity and specificity when predicting peer nominations of influence among the most aggressive children. Results are discussed m terms of the application of teacher nominations and ratings in large trials and full implementation of targeted prevention programs. PMID:16378226

Henry, David B.; Miller-Johnson, Shari; Simon, Thomas R.; Schoeny, Michael E.



Selection of Target Sites for Mobile DNA Integration in the Human Genome  

PubMed Central

DNA sequences from retroviruses, retrotransposons, DNA transposons, and parvoviruses can all become integrated into the human genome. Accumulation of such sequences accounts for at least 40% of our genome today. These integrating elements are also of interest as gene-delivery vectors for human gene therapy. Here we present a comprehensive bioinformatic analysis of integration targeting by HIV, MLV, ASLV, SFV, L1, SB, and AAV. We used a mathematical method which allowed annotation of each base pair in the human genome for its likelihood of hosting an integration event by each type of element, taking advantage of more than 200 types of genomic annotation. This bioinformatic resource documents a wealth of new associations between genomic features and integration targeting. The study also revealed that the length of genomic intervals analyzed strongly affected the conclusions drawn—thus, answering the question “What genomic features affect integration?” requires carefully specifying the length scale of interest. PMID:17166054

Berry, Charles; Hannenhalli, Sridhar; Leipzig, Jeremy; Bushman, Frederic D



On and off-target effects of telomere uncapping G-quadruplex selective ligands based on pentacyclic acridinium salts  

PubMed Central

Quadruplexes DNA are present in telomeric DNA as well as in several cancer-related gene promoters and hence affect gene expression and subsequent biological processes. The conformations of G4 provide selective recognition sites for small molecules and thus these structures have become important drug-design targets for cancer treatment. The DNA G-quadruplex binding pentacyclic acridinium salt RHPS4 (1) has many pharmacological attributes of an ideal telomere-targeting agent but has undesirable off-target liabilities. Notably a cardiovascular effect was evident in a guinea pig model, manifested by a marked and sustained increase in QTcB interval. In accordance with this, significant interaction with the human recombinant ?2 adrenergic receptor, and M1, M2 and M3 muscarinic receptors was observed, together with a high inhibition of the hERG tail current tested in a patch clamp assay. Two related pentacyclic structures, the acetylamines (2) and (3), both show a modest interaction with ?2 adrenergic receptor, and do not significatively inhibit the hERG tail current while demonstrating potent telomere on-target properties comparing closely with 1. Of the two isomers, the 2-acetyl-aminopentacycle (2) more closely mimics the overall biological profile of 1 and this information will be used to guide further synthetic efforts to identify novel variants of this chemotype, to maximize on-target and minimize off-target activities. Consequently, the improvement of toxicological profile of these compounds could therefore lead to the obtainment of suitable molecules for clinical development offering new pharmacological strategies in cancer treatment. PMID:24330541



Target Selection for SETI: 1. A Catalog of Nearby Habitable Stellar Systems  

Microsoft Academic Search

In preparation for the advent of the Allen Telescope Array, the SETI\\u000aInstitute has the need to greatly expand its former list of ~2000 targets\\u000acompiled for Project Phoenix, a search for extraterrestrial technological\\u000asignals. In this paper we present a catalog of stellar systems that are\\u000apotentially habitable to complex life forms (including intelligent life), which\\u000acomprises the largest

Margaret C. Turnbull; Jill C. Tarter



Selective Targeting of p53 Gene Mutational Hotspots in Human Cancers by Etiologically Defined Carcinogens  

Microsoft Academic Search

In lung and liver cancers. pS3 mutations are mostly G:C to T:A transversions. This type of mutation is known to be induced by benzo(a)pyrcne and aflatoxin B, which are associated with the etiology of lung and liver cancers, respectively. Using a novel assay based on DNA polymerase fingerprint analysis, we identified p53 nucleotides targeted by these carcinogens. Thirteen of 14

Alain Puisieux; Susan Lim; John Groopman; Mehmet Ozturk