Experimental approaches to identify cellular G-quadruplex structures and functions.

PubMed

Di Antonio, Marco; Rodriguez, Raphaël; Balasubramanian, Shankar

2012-05-01

Guanine-rich nucleic acids can fold into non-canonical DNA secondary structures called G-quadruplexes. The formation of these structures can interfere with the biology that is crucial to sustain cellular homeostases and metabolism via mechanisms that include transcription, translation, splicing, telomere maintenance and DNA recombination. Thus, due to their implication in several biological processes and possible role promoting genomic instability, G-quadruplex forming sequences have emerged as potential therapeutic targets. There has been a growing interest in the development of synthetic molecules and biomolecules for sensing G-quadruplex structures in cellular DNA. In this review, we summarise and discuss recent methods developed for cellular imaging of G-quadruplexes, and the application of experimental genomic approaches to detect G-quadruplexes throughout genomic DNA. In particular, we will discuss the use of engineered small molecules and natural proteins to enable pull-down, ChIP-Seq, ChIP-chip and fluorescence imaging of G-quadruplex structures in cellular DNA. Copyright © 2012 Elsevier Inc. All rights reserved.

Dual peptide conjugation strategy for improved cellular uptake and mitochondria targeting.

PubMed

Lin, Ran; Zhang, Pengcheng; Cheetham, Andrew G; Walston, Jeremy; Abadir, Peter; Cui, Honggang

2015-01-21

Mitochondria are critical regulators of cellular function and survival. Delivery of therapeutic and diagnostic agents into mitochondria is a challenging task in modern pharmacology because the molecule to be delivered needs to first overcome the cell membrane barrier and then be able to actively target the intracellular organelle. Current strategy of conjugating either a cell penetrating peptide (CPP) or a subcellular targeting sequence to the molecule of interest only has limited success. We report here a dual peptide conjugation strategy to achieve effective delivery of a non-membrane-penetrating dye 5-carboxyfluorescein (5-FAM) into mitochondria through the incorporation of both a mitochondrial targeting sequence (MTS) and a CPP into one conjugated molecule. Notably, circular dichroism studies reveal that the combined use of α-helix and PPII-like secondary structures has an unexpected, synergistic contribution to the internalization of the conjugate. Our results suggest that although the use of positively charged MTS peptide allows for improved targeting of mitochondria, with MTS alone it showed poor cellular uptake. With further covalent linkage of the MTS-5-FAM conjugate to a CPP sequence (R8), the dually conjugated molecule was found to show both improved cellular uptake and effective mitochondria targeting. We believe these results offer important insight into the rational design of peptide conjugates for intracellular delivery.

Mechanism for the Cellular Uptake of Targeted Gold Nanorods of Defined Aspect Ratios.

PubMed

Yang, Hongrong; Chen, Zhong; Zhang, Lei; Yung, Wing-Yin; Leung, Ken Cham-Fai; Chan, Ho Yin Edwin; Choi, Chung Hang Jonathan

2016-10-01

Biomedical applications of non-spherical nanoparticles such as photothermal therapy and molecular imaging require their efficient intracellular delivery, yet reported details on their interactions with the cell remain inconsistent. Here, the effects of nanoparticle geometry and receptor targeting on the cellular uptake and intracellular trafficking are systematically explored by using C166 (mouse endothelial) cells and gold nanoparticles of four different aspect ratios (ARs) from 1 to 7. When coated with poly(ethylene glycol) strands, the cellular uptake of untargeted nanoparticles monotonically decreases with AR. Next, gold nanoparticles are functionalized with DNA oligonucleotides to target Class A scavenger receptors expressed by C166 cells. Intriguingly, cellular uptake is maximized at a particular AR: shorter nanorods (AR = 2) enter C166 cells more than nanospheres (AR = 1) and longer nanorods (AR = 4 or 7). Strikingly, long targeted nanorods align to the cell membrane in a near-parallel manner followed by rotating by ≈90° to enter the cell via a caveolae-mediated pathway. Upon cellular entry, targeted nanorods of all ARs predominantly traffic to the late endosome without progressing to the lysosome. The studies yield important materials design rules for drug delivery carriers based on targeted, anisotropic nanoparticles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets.

PubMed

Krastev, Dragomir B; Pettitt, Stephen J; Campbell, James; Song, Feifei; Tanos, Barbara E; Stoynov, Stoyno S; Ashworth, Alan; Lord, Christopher J

2018-05-22

Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor "tagged" cells in a genetic screen we carry out a large-scale identification of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites.

Targeting cancer by binding iron: Dissecting cellular signaling pathways

PubMed Central

Lui, Goldie Y.L.; Kovacevic, Zaklina; Richardson, Vera; Merlot, Angelica M.; Kalinowski, Danuta S.; Richardson, Des R.

2015-01-01

Newer and more potent therapies are urgently needed to effectively treat advanced cancers that have developed resistance and metastasized. One such strategy is to target cancer cell iron metabolism, which is altered compared to normal cells and may facilitate their rapid proliferation. This is supported by studies reporting the anti-neoplastic activities of the clinically available iron chelators, desferrioxamine and deferasirox. More recently, ligands of the di-2-pyridylketone thiosemicarbazone (DpT) class have demonstrated potent and selective anti-proliferative activity across multiple cancer-types in vivo, fueling studies aimed at dissecting their molecular mechanisms of action. In the past five years alone, significant advances have been made in understanding how chelators not only modulate cellular iron metabolism, but also multiple signaling pathways implicated in tumor progression and metastasis. Herein, we discuss recent research on the targeting of iron in cancer cells, with a focus on the novel and potent DpT ligands. Several key studies have revealed that iron chelation can target the AKT, ERK, JNK, p38, STAT3, TGF-β, Wnt and autophagic pathways to subsequently inhibit cellular proliferation, the epithelial-mesenchymal transition (EMT) and metastasis. These developments emphasize that these novel therapies could be utilized clinically to effectively target cancer. PMID:26125440

A computational approach to identify cellular heterogeneity and tissue-specific gene regulatory networks.

PubMed

Jambusaria, Ankit; Klomp, Jeff; Hong, Zhigang; Rafii, Shahin; Dai, Yang; Malik, Asrar B; Rehman, Jalees

2018-06-07

The heterogeneity of cells across tissue types represents a major challenge for studying biological mechanisms as well as for therapeutic targeting of distinct tissues. Computational prediction of tissue-specific gene regulatory networks may provide important insights into the mechanisms underlying the cellular heterogeneity of cells in distinct organs and tissues. Using three pathway analysis techniques, gene set enrichment analysis (GSEA), parametric analysis of gene set enrichment (PGSEA), alongside our novel model (HeteroPath), which assesses heterogeneously upregulated and downregulated genes within the context of pathways, we generated distinct tissue-specific gene regulatory networks. We analyzed gene expression data derived from freshly isolated heart, brain, and lung endothelial cells and populations of neurons in the hippocampus, cingulate cortex, and amygdala. In both datasets, we found that HeteroPath segregated the distinct cellular populations by identifying regulatory pathways that were not identified by GSEA or PGSEA. Using simulated datasets, HeteroPath demonstrated robustness that was comparable to what was seen using existing gene set enrichment methods. Furthermore, we generated tissue-specific gene regulatory networks involved in vascular heterogeneity and neuronal heterogeneity by performing motif enrichment of the heterogeneous genes identified by HeteroPath and linking the enriched motifs to regulatory transcription factors in the ENCODE database. HeteroPath assesses contextual bidirectional gene expression within pathways and thus allows for transcriptomic assessment of cellular heterogeneity. Unraveling tissue-specific heterogeneity of gene expression can lead to a better understanding of the molecular underpinnings of tissue-specific phenotypes.

In-situ Click Reaction Coupled with Quantitative Proteomics for Identifying Protein Targets of Catechol Estrogens.

PubMed

Liang, Huei-Chen; Liu, Yi-Chen; Chen, Hsin; Ku, Ming Chun; Do, Quynh-Trang; Wang, Chih-Yen; Tzeng, Shun-Fen; Chen, Shu-Hui

2018-06-13

Catechol estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in-situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography-mass spectrometry (LC-MS). A total of 334 liver proteins were repeatedly identified (n  2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 non-specific binders (keratins) had D/H < 1. These results were confirmed using spiked covalent control (strong binder) and noncovalent control (weak binder), as well as in vitro testing of cytochrome c (D/H = 5.9) which showed covalent conjugation with CEs. Many identified strong binders, such as glutathione transferase, catechol-O-methyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and cytochrome c, are involved in cellular redox processes or detoxification activities. CE conjugation was shown to suppress the superoxide oxidase activity of cytochrome c, suggesting that CEs modification may alter the redox action of cellular proteins. Due to structural similarity and inert alkyne group, EE2 probe is very likely to capture protein targets of CEs in general. Thus, this strategy can be adopted to explore the biological impact of CEs modification in living systems.

Mechanism-based Proteomic Screening Identifies Targets of Thioredoxin-like Proteins*

PubMed Central

Nakao, Lia S.; Everley, Robert A.; Marino, Stefano M.; Lo, Sze M.; de Souza, Luiz E.; Gygi, Steven P.; Gladyshev, Vadim N.

2015-01-01

Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes. PMID:25561728

In vivo Labeling of Constellations of Functionally Identified Neurons for Targeted in vitro Recordings

PubMed Central

Lien, Anthony D.; Scanziani, Massimo

2011-01-01

Relating the functional properties of neurons in an intact organism with their cellular and synaptic characteristics is necessary for a mechanistic understanding of brain function. However, while the functional properties of cortical neurons (e.g., tuning to sensory stimuli) are necessarily determined in vivo, detailed cellular and synaptic analysis relies on in vitro techniques. Here we describe an approach that combines in vivo calcium imaging (for functional characterization) with photo-activation of fluorescent proteins (for neuron labeling), thereby allowing targeted in vitro recording of multiple neurons with known functional properties. We expressed photo-activatable GFP rendered non-diffusible through fusion with a histone protein (H2B–PAGFP) in the mouse visual cortex to rapidly photo-label constellations of neurons in vivo at cellular and sub-cellular resolution using two-photon excitation. This photo-labeling method was compatible with two-photon calcium imaging of neuronal responses to visual stimuli, allowing us to label constellations of neurons with specific functional properties. Photo-labeled neurons were easily identified in vitro in acute brain slices and could be targeted for whole-cell recording. We also demonstrate that in vitro and in vivo image stacks of the same photo-labeled neurons could be registered to one another, allowing the exact in vivo response properties of individual neurons recorded in vitro to be known. The ability to perform in vitro recordings from neurons with known functional properties opens up exciting new possibilities for dissecting the cellular, synaptic, and circuit mechanisms that underlie neuronal function in vivo. PMID:22144948

Determination of the accuracy for targeted irradiations of cellular substructures at SNAKE

NASA Astrophysics Data System (ADS)

Siebenwirth, C.; Greubel, C.; Drexler, S. E.; Girst, S.; Reindl, J.; Walsh, D. W. M.; Dollinger, G.; Friedl, A. A.; Schmid, T. E.; Drexler, G. A.

2015-04-01

In the last 10 years the ion microbeam SNAKE, installed at the Munich 14 MV tandem accelerator, has been successfully used for radiobiological experiments by utilizing pattern irradiation without targeting single cells. Now for targeted irradiation of cellular substructures a precise irradiation device was added to the live cell irradiation setup at SNAKE. It combines a sub-micrometer single ion irradiation facility with a high resolution optical fluorescence microscope. Most systematic errors can be reduced or avoided by using the same light path in the microscope for beam spot verification as well as for and target recognition. In addition online observation of the induced cellular responses is possible. The optical microscope and the beam delivering system are controlled by an in-house developed software which integrates the open-source image analysis software, CellProfiler, for semi-automatic target recognition. In this work the targeting accuracy was determined by irradiation of a cross pattern with 55 MeV carbon ions on nucleoli in U2OS and HeLa cells stably expressing a GFP-tagged repair protein MDC1. For target recognition, nuclei were stained with Draq5 and nucleoli were stained with Syto80 or Syto83. The damage response was determined by live-cell imaging of MDC1-GFP accumulation directly after irradiation. No systematic displacement and a random distribution of about 0.7 μm (SD) in x-direction and 0.8 μm (SD) in y-direction were observed. An independent analysis after immunofluorescence staining of the DNA damage marker yH2AX yielded similar results. With this performance a target with a size similar to that of nucleoli (i.e. a diameter of about 3 μm) is hit with a probability of more than 80%, which enables the investigation of the radiation response of cellular subcompartments after targeted ion irradiation in the future.

Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

PubMed Central

Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

2015-01-01

ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

Activity-based proteome profiling of potential cellular targets of Orlistat--an FDA-approved drug with anti-tumor activities.

PubMed

Yang, Peng-Yu; Liu, Kai; Ngai, Mun Hong; Lear, Martin J; Wenk, Markus R; Yao, Shao Q

2010-01-20

Orlistat, or tetrahydrolipstatin (THL), is an FDA-approved antiobesity drug with potential antitumor activities. Cellular off-targets and potential side effects of Orlistat in cancer therapies, however, have not been extensively explored thus far. In this study, we report the total of synthesis of THL-like protein-reactive probes, in which extremely conservative modifications (i.e., an alkyne handle) were introduced in the parental THL structure to maintain the native biological properties of Orlistat, while providing the necessary functionality for target identification via the bio-orthogonal click chemistry. With these natural productlike, cell-permeable probes, we were able to demonstrate, for the first time, this chemical proteomic approach is suitable for the identification of previously unknown cellular targets of Orlistat. In addition to the expected fatty acid synthase (FAS), we identified a total of eight new targets, some of which were further validated by experiments including Western blotting, recombinant protein expression, and site-directed mutagenesis. Our findings have important implications in the consideration of Orlistat as a potential anticancer drug at its early stages of development for cancer therapy. Our strategy should be broadly useful for off-target identification against quite a number of existing drugs and/or candidates, which are also covalent modifiers of their biological targets.

Identifying MicroRNAs and Transcript Targets in Jatropha Seeds

PubMed Central

Galli, Vanessa; Guzman, Frank; de Oliveira, Luiz F. V.; Loss-Morais, Guilherme; Körbes, Ana P.; Silva, Sérgio D. A.; Margis-Pinheiro, Márcia M. A. N.; Margis, Rogério

2014-01-01

MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs) as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs. PMID:24551031

Cellular and extracellular miRNAs are blood-compartment-specific diagnostic targets in sepsis.

PubMed

Reithmair, Marlene; Buschmann, Dominik; Märte, Melanie; Kirchner, Benedikt; Hagl, Daniel; Kaufmann, Ines; Pfob, Martina; Chouker, Alexander; Steinlein, Ortrud K; Pfaffl, Michael W; Schelling, Gustav

2017-10-01

Septic shock is a common medical condition with a mortality approaching 50% where early diagnosis and treatment are of particular importance for patient survival. Novel biomarkers that serve as prompt indicators of sepsis are urgently needed. High-throughput technologies assessing circulating microRNAs represent an important tool for biomarker identification, but the blood-compartment specificity of these miRNAs has not yet been investigated. We characterized miRNA profiles from serum exosomes, total serum and blood cells (leukocytes, erythrocytes, platelets) of sepsis patients by next-generation sequencing and RT-qPCR (n = 3 × 22) and established differences in miRNA expression between blood compartments. In silico analysis was used to identify compartment-specific signalling functions of differentially regulated miRNAs in sepsis-relevant pathways. In septic shock, a total of 77 and 103 miRNAs were down- and up-regulated, respectively. A majority of these regulated miRNAs (14 in serum, 32 in exosomes and 73 in blood cells) had not been previously associated with sepsis. We found a distinctly compartment-specific regulation of miRNAs between sepsis patients and healthy volunteers. Blood cellular miR-199b-5p was identified as a potential early indicator for sepsis and septic shock. miR-125b-5p and miR-26b-5p were uniquely regulated in exosomes and serum, respectively, while one miRNA (miR-27b-3p) was present in all three compartments. The expression of sepsis-associated miRNAs is compartment-specific. Exosome-derived miRNAs contribute significant information regarding sepsis diagnosis and survival prediction and could serve as newly identified targets for the development of novel sepsis biomarkers. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin.

PubMed

Rink, Jonathan S; Yang, Shuo; Cen, Osman; Taxter, Tim; McMahon, Kaylin M; Misener, Sol; Behdad, Amir; Longnecker, Richard; Gordon, Leo I; Thaxton, C Shad

2017-11-06

Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused on diffuse large B-cell lymphoma (DLBCL) as a model because there is differential cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol uptake through the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). As we previously reported, GC DLBCL are exquisitely sensitive to HDL NP as monotherapy, while ABC DLBCL are less sensitive. Herein, we report that enhanced BCR signaling and resultant de novo cholesterol synthesis in ABC DLBCL drastically reduces the ability of HDL NPs to reduce cellular cholesterol and induce cell death. Therefore, we combined HDL NP with the BCR signaling inhibitor ibrutinib and the SYK inhibitor R406. By targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis, we achieved cellular cholesterol reduction and induced apoptosis in otherwise resistant ABC DLBCL cell lines. These results in lymphoma demonstrate that reduction of cellular cholesterol is a powerful mechanism to induce apoptosis. Cells rich in cholesterol require HDL NP therapy to reduce uptake and molecularly targeted agents that inhibit upstream pathways that stimulate de novo cholesterol synthesis, thus, providing a new paradigm for rationally targeting cholesterol metabolism as therapy for cancer.

Global analysis of bacterial transcription factors to predict cellular target processes.

PubMed

Doerks, Tobias; Andrade, Miguel A; Lathe, Warren; von Mering, Christian; Bork, Peer

2004-03-01

Whole-genome sequences are now available for >100 bacterial species, giving unprecedented power to comparative genomics approaches. We have applied genome-context methods to predict target processes that are regulated by transcription factors (TFs). Of 128 orthologous groups of proteins annotated as TFs, to date, 36 are functionally uncharacterized; in our analysis we predict a probable cellular target process or biochemical pathway for half of these functionally uncharacterized TFs.

Neutrophil degranulation and immunosuppression in patients with GBM: restoration of cellular immune function by targeting arginase I.

PubMed

Sippel, Trisha R; White, Jason; Nag, Kamalika; Tsvankin, Vadim; Klaassen, Marci; Kleinschmidt-DeMasters, B K; Waziri, Allen

2011-11-15

The source of glioblastoma (GBM)-associated immunosuppression remains multifactorial. We sought to clarify and therapeutically target myeloid cell-derived peripheral immunosuppression in patients with GBM. Direct ex vivo T-cell function, serum Arginase I (ArgI) levels, and circulating myeloid lineage populations were compared between patients with GBM and normal donors or patients with other intracranial tumors. Immunofunctional assays were conducted using bulk and sorted cell populations to explore the potential transfer of myeloid cell-mediated immunosuppression and to identify a potential mechanism for these effects. ArgI-mediated immunosuppression was therapeutically targeted in vitro through pharmacologic inhibition or arginine supplementation. We identified a significantly expanded population of circulating, degranulated neutrophils associated with elevated levels of serum ArgI and decreased T-cell CD3ζ expression within peripheral blood from patients with GBM. Sorted CD11b(+) cells from patients with GBM were found to markedly suppress normal donor T-cell function in coculture, and media harvested from mitogen-stimulated GBM peripheral blood mononuclear cell (PBMC) or GBM-associated mixed lymphoid reactions showed ArgI levels that were significantly higher than controls. Critically, T-cell suppression in both settings could be completely reversed through pharmacologic ArgI inhibition or with arginine supplementation. These data indicate that peripheral cellular immunosuppression in patients with GBM is associated with neutrophil degranulation and elevated levels of circulating ArgI, and that T-cell function can be restored in these individuals by targeting ArgI. These data identify a novel pathway of GBM-mediated suppression of cellular immunity and offer a potential therapeutic window for improving antitumor immunity in affected patients.

Identifying Cellular and Molecular Mechanisms for Magnetosensation

PubMed Central

Clites, Benjamin L.; Pierce, Jonathan T.

2017-01-01

Diverse animals ranging from worms and insects to birds and turtles perf orm impressive journeys using the magnetic field of the earth as a cue. Although major cellular and molecular mechanisms for sensing mechanical and chemical cues have been elucidated over the past three decades, the mechanisms that animals use to sense magnetic fields remain largely mysterious. Here we survey progress on the search for magnetosensory neurons and magnetosensitive molecules important for animal behaviors. Emphasis is placed on magnetosensation in insects and birds, as well as on the magnetosensitive neuron pair AFD in the nematode Caenorhabditis elegans. We also review conventional criteria used to define animal magnetoreceptors and suggest how approaches used to identify receptors for other sensory modalities may be adapted for magnetoreceptors. Finally, we discuss prospects for under-utilized and novel approaches to identify the elusive magnetoreceptors in animals. PMID:28772099

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

PubMed

Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

2017-07-17

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

The identification of cellular targets of 17β estradiol using a lytic (T7) cDNA phage display approach.

PubMed

Van Dorst, Bieke; Mehta, Jaytry; Rouah-Martin, Elsa; De Coen, Wim; Blust, Ronny; Robbens, Johan

2011-02-01

To unravel the mechanism of action of chemical compounds, it is crucial to know their cellular targets. A novel in vitro tool that can be used as a fast, simple and cost effective alternative is cDNA phage display. This tool is used in our study to select cellular targets of 17β estradiol (E2). It was possible to select two potential cellular targets of E2 out of the T7 Select™ Human Breast cDNA phage library. The selected cellular targets, autophagy/beclin-1 regulator 1 (beclin 1) and ATP synthase F(0) subunit 6 (ATP6) have so far been unknown as binding proteins of E2. To confirm the E2 binding properties of these selected proteins, surface plasmon resonance (SPR) was used. With SPR the K(d) values were determined to be 0.178±0.031 and 0.401±0.142 nM for the ATP6 phage and beclin 1 phage, respectively. These K(d) values in the low nM range verify that the selected cellular proteins are indeed binding proteins for E2. The selection and identification of these two potential cellular targets of E2, can enhance our current understanding of its mechanism of action. This illustrates the potential of lytic (T7) cDNA phage display in toxicology, to provide important information about cellular targets of chemical compounds. Copyright © 2010 Elsevier Ltd. All rights reserved.

The developing oligodendrocyte: key cellular target in brain injury in the premature infant

PubMed Central

Volpe, Joseph J.; Kinney, Hannah C.; Jensen, Frances, E.; Rosenberg, Paul A.

2011-01-01

Brain injury in the premature infant, a problem of enormous importance, is associated with a high risk of neurodevelopmental disability. The major type of injury involves cerebral white matter and the principal cellular target is the developing oligodendrocyte. The specific phase of the oligodendroglial lineage affected has been defined from study of both human brain and experimental models. This premyelinating cell (pre-OL) is vulnerable because of a series of maturation-dependent events. The pathogenesis of pre-OL injury relates to operation of two upstream mechanisms, hypoxia-ischemia and systemic infection/inflammation, both of which are common occurrences in premature infants. The focus of this review and of our research over the past 15-20 years has been the cellular and molecular bases for the maturation-dependent vulnerability of the pre-OL to the action of the two upstream mechanisms. Three downstream mechanisms have been identified, i.e., microglial activation, excitotoxicity and free radical attack. The work in both experimental models and human brain has identified a remarkable confluence of maturation-dependent factors that render the pre-OL so exquisitely vulnerable to these downstream mechanisms. Most importantly, elucidation of these factors has led to delineation of a series of potential therapeutic interventions, which in experimental models show marked protective properties. The critical next step, i.e., clinical trials in the living infant, is now on the horizon. PMID:21382469

Recent Advances in Superparamagnetic Iron Oxide Nanoparticles for Cellular Imaging and Targeted Therapy Research

PubMed Central

Wang, Yi-Xiang J.; Xuan, Shouhu; Port, Marc; Idee, Jean-Marc

2013-01-01

Advances of nanotechnology have led to the development of nanomaterials with both potential diagnostic and therapeutic applications. Among them, superparamagnetic iron oxide (SPIO) nanoparticles have received particular attention. Over the past decade, various SPIOs with unique physicochemical and biological properties have been designed by modifying the particle structure, size and coating. This article reviews the recent advances in preparing SPIOs with novel properties, the way these physicochemical properties of SPIOs influence their interaction with cells, and the development of SPIOs in liver and lymph nodes magnetic resonance imaging (MRI) contrast. Cellular uptake of SPIO can be exploited in a variety of potential clinical applications, including stem cell and inflammation cell tracking and intra-cellular drug delivery to cancerous cells which offers higher intra-cellular concentration. When SPIOs are used as carrier vehicle, additional advantages can be achieved including magnetic targeting and hyperthermia options, as well as monitoring with MRI. Other potential applications of SPIO include magnetofection and gene delivery, targeted retention of labeled stem cells, sentinel lymph nodes mapping, and magnetic force targeting and cell orientation for tissue engineering. PMID:23621536

Cellular Plasticity-Targeted Therapy in Head and Neck Cancers.

PubMed

Shang, W; Zhang, Q; Huang, Y; Shanti, R; Alawi, F; Le, A; Jiang, C

2018-06-01

Head and neck cancer is one of the most frequent human malignancies worldwide, with a high rate of recurrence and metastasis. Head and neck squamous cell carcinoma (HNSCC) is cellularly and molecularly heterogeneous, with subsets of undifferentiated cancer cells exhibiting stem cell-like properties, called cancer stem cells (CSCs). Epithelial-mesenchymal transition, gene mutation, and epigenetic modification are associated with the formation of cellular plasticity of tumor cells in HNSCC, contributing to the acquisition of invasive, recurrent, and metastatic properties and therapeutic resistance. Tumor microenvironment (TME) plays a supportive role in the initiation, progression, and metastasis of head and neck cancer. Stromal fibroblasts, vasculature, immune cells, cytokines, and hypoxia constitute the main components of TME in HNSCC, which contributes not only to the acquisition of CSC properties but also to the recurrence and therapeutic resistance of the malignancies. In this review, we discuss the potential mechanisms underlying the development of cellular plasticity, especially the emergence of CSCs, in HNSCC. We also highlight recent studies implicating the complex interplays among TME components, plastic CSCs, tumorigenesis, recurrence, and therapeutic resistance of HNSCC. Finally, we summarize the treatment modalities of HNSCC and reinforce the novel concept of therapeutic targeting CSCs in HNSCC.

Cellular and molecular mechanisms of HIV-1 integration targeting.

PubMed

Engelman, Alan N; Singh, Parmit K

2018-07-01

Integration is central to HIV-1 replication and helps mold the reservoir of cells that persists in AIDS patients. HIV-1 interacts with specific cellular factors to target integration to interior regions of transcriptionally active genes within gene-dense regions of chromatin. The viral capsid interacts with several proteins that are additionally implicated in virus nuclear import, including cleavage and polyadenylation specificity factor 6, to suppress integration into heterochromatin. The viral integrase protein interacts with transcriptional co-activator lens epithelium-derived growth factor p75 to principally position integration within gene bodies. The integrase additionally senses target DNA distortion and nucleotide sequence to help fine-tune the specific phosphodiester bonds that are cleaved at integration sites. Research into virus-host interactions that underlie HIV-1 integration targeting has aided the development of a novel class of integrase inhibitors and may help to improve the safety of viral-based gene therapy vectors.

Elucidation of the Ebola virus VP24 cellular interactome and disruption of virus biology through targeted inhibition of host-cell protein function.

PubMed

García-Dorival, Isabel; Wu, Weining; Dowall, Stuart; Armstrong, Stuart; Touzelet, Olivier; Wastling, Jonathan; Barr, John N; Matthews, David; Carroll, Miles; Hewson, Roger; Hiscox, Julian A

2014-11-07

Viral pathogenesis in the infected cell is a balance between antiviral responses and subversion of host-cell processes. Many viral proteins specifically interact with host-cell proteins to promote virus biology. Understanding these interactions can lead to knowledge gains about infection and provide potential targets for antiviral therapy. One such virus is Ebola, which has profound consequences for human health and causes viral hemorrhagic fever where case fatality rates can approach 90%. The Ebola virus VP24 protein plays a critical role in the evasion of the host immune response and is likely to interact with multiple cellular proteins. To map these interactions and better understand the potential functions of VP24, label-free quantitative proteomics was used to identify cellular proteins that had a high probability of forming the VP24 cellular interactome. Several known interactions were confirmed, thus placing confidence in the technique, but new interactions were also discovered including one with ATP1A1, which is involved in osmoregulation and cell signaling. Disrupting the activity of ATP1A1 in Ebola-virus-infected cells with a small molecule inhibitor resulted in a decrease in progeny virus, thus illustrating how quantitative proteomics can be used to identify potential therapeutic targets.

Cellular function of neuropathy target esterase in lysophosphatidylcholine action

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

Vose, Sarah C.; Center for Children's Environmental Health Research, School of Public Health, University of California, Berkeley, CA 94720; Fujioka, Kazutoshi

2008-11-01

Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC ormore » phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography-mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action.« less

Molecular and cellular alterations in Down syndrome: toward the identification of targets for therapeutics.

PubMed

Créau, Nicole

2012-01-01

Down syndrome is a complex disease that has challenged molecular and cellular research for more than 50 years. Understanding the molecular bases of morphological, cellular, and functional alterations resulting from the presence of an additional complete chromosome 21 would aid in targeting specific genes and pathways for rescuing some phenotypes. Recently, progress has been made by characterization of brain alterations in mouse models of Down syndrome. This review will highlight the main molecular and cellular findings recently described for these models, particularly with respect to their relationship to Down syndrome phenotypes.

Study Identifies New Lymphoma Treatment Target

Cancer.gov

NCI researchers have identified new therapeutic targets for diffuse large B-cell lymphoma. Drugs that hit these targets are under clinical development and the researchers hope to begin testing them in clinical trials of patients with DLBCL.

Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues

PubMed Central

Tulloch, Lindsay B.; Menzies, Stefanie K.; Fraser, Andrew L.; Gould, Eoin R.; King, Elizabeth F.; Zacharova, Marija K.; Florence, Gordon J.

2017-01-01

Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or

Enhanced cellular transport and drug targeting using dendritic nanostructures

NASA Astrophysics Data System (ADS)

Kannan, R. M.; Kolhe, Parag; Kannan, Sujatha; Lieh-Lai, Mary

2003-03-01

Dendrimers and hyperbranched polymers possess highly branched architectures, with a large number of controllable, tailorable, peripheral' functionalities. Since the surface chemistry of these materials can be modified with relative ease, these materials have tremendous potential in targeted drug delivery. The large density of end groups can also be tailored to create enhanced affinity to targeted cells, and can also encapsulate drugs and deliver them in a controlled manner. We are developing tailor-modified dendritic systems for drug delivery. Synthesis, drug/ligand conjugation, in vitro cellular and in vivo drug delivery, and the targeting efficiency to the cell are being studied systematically using a wide variety of experimental tools. Results on PAMAM dendrimers and polyol hyperbranched polymers suggest that: (1) These materials complex/encapsulate a large number of drug molecules and release them at tailorable rates; (2) The drug-dendrimer complex is transported very rapidly through a A549 lung epithelial cancel cell line, compared to free drug, perhaps by endocytosis. The ability of the drug-dendrimer-ligand complexes to target specific asthma and cancer cells is currently being explored using in vitro and in vivo animal models.

Antifungal activity of redox-active benzaldehydes that target cellular antioxidation

USDA-ARS?s Scientific Manuscript database

Many pathogenic fungi are becoming resistant to currently available drugs. Disruption of cellular antioxidation systems should be an effective method for control of fungal pathogens. Such disruption can be achieved with redox-active compounds. The aim of this study was to identify benzaldehydes that...

A genome-wide approach identifies distinct but overlapping subsets of cellular mRNAs associated with Staufen1- and Staufen2-containing ribonucleoprotein complexes

PubMed Central

Furic, Luc; Maher-Laporte, Marjolaine; DesGroseillers, Luc

2008-01-01

Messenger RNAs are associated with multiple RNA-binding proteins to form ribonucleoprotein (mRNP) complexes. These proteins are important regulators of the fate of their target mRNAs. In human cells, Staufen1 and Staufen2 proteins, coded by two different genes, are double-stranded RNA-binding proteins involved in several cellular functions including mRNA localization, translation, and decay. Although 51% identical, these proteins are nevertheless found in different RNA particles. In addition, differential splicing events generate Staufen2 isoforms that only differ at their N-terminal extremities. In this paper, we used a genome-wide approach to identify and compare the mRNA targets of mammalian Staufen proteins. The mRNA content of Staufen mRNPs was identified by probing DNA microarrays with probes derived from mRNAs isolated from immunopurified Staufen-containing complexes following transfection of HEK293T cells with Stau155-HA, Stau259-HA, or Stau262-HA expressors. Our results indicate that 7% and 11% of the cellular RNAs expressed in HEK293T cells are found in Stau1- and in Stau2-containing mRNPs, respectively. A comparison of Stau1- and Stau2-containing mRNAs identifies a relatively low percentage of common mRNAs; the percentage of common mRNAs highly increases when mRNAs in Stau259-HA- and Stau262-containing mRNPs are compared. There is a predominance of mRNAs involved in cell metabolism, transport, transcription, regulation of cell processes, and catalytic activity. All these subsets of mRNAs are mostly distinct from those associated with FMRP or IMP, although some mRNAs overlap. Consistent with a model of post-transcriptionnal gene regulation, our results show that Stau1- and Stau2-mRNPs associate with distinct but overlapping sets of cellular mRNAs. PMID:18094122

A genome-wide approach identifies distinct but overlapping subsets of cellular mRNAs associated with Staufen1- and Staufen2-containing ribonucleoprotein complexes.

PubMed

Furic, Luc; Maher-Laporte, Marjolaine; DesGroseillers, Luc

2008-02-01

Messenger RNAs are associated with multiple RNA-binding proteins to form ribonucleoprotein (mRNP) complexes. These proteins are important regulators of the fate of their target mRNAs. In human cells, Staufen1 and Staufen2 proteins, coded by two different genes, are double-stranded RNA-binding proteins involved in several cellular functions including mRNA localization, translation, and decay. Although 51% identical, these proteins are nevertheless found in different RNA particles. In addition, differential splicing events generate Staufen2 isoforms that only differ at their N-terminal extremities. In this paper, we used a genome-wide approach to identify and compare the mRNA targets of mammalian Staufen proteins. The mRNA content of Staufen mRNPs was identified by probing DNA microarrays with probes derived from mRNAs isolated from immunopurified Staufen-containing complexes following transfection of HEK293T cells with Stau1(55)-HA, Stau2(59)-HA, or Stau2(62)-HA expressors. Our results indicate that 7% and 11% of the cellular RNAs expressed in HEK293T cells are found in Stau1- and in Stau2-containing mRNPs, respectively. A comparison of Stau1- and Stau2-containing mRNAs identifies a relatively low percentage of common mRNAs; the percentage of common mRNAs highly increases when mRNAs in Stau2(59)-HA- and Stau2(62)-containing mRNPs are compared. There is a predominance of mRNAs involved in cell metabolism, transport, transcription, regulation of cell processes, and catalytic activity. All these subsets of mRNAs are mostly distinct from those associated with FMRP or IMP, although some mRNAs overlap. Consistent with a model of post-transcriptional gene regulation, our results show that Stau1- and Stau2-mRNPs associate with distinct but overlapping sets of cellular mRNAs.

Cellular communication and “non-targeted effects”: Modelling approaches

NASA Astrophysics Data System (ADS)

Ballarini, Francesca; Facoetti, Angelica; Mariotti, Luca; Nano, Rosanna; Ottolenghi, Andrea

2009-10-01

During the last decade, a large number of experimental studies on the so-called "non-targeted effects", in particular bystander effects, outlined that cellular communication plays a significant role in the pathways leading to radiobiological damage. Although it is known that two main types of cellular communication (i.e. via gap junctions and/or molecular messengers diffusing in the extra-cellular environment, such as cytokines, NO etc.) play a major role, it is of utmost importance to better understand the underlying mechanisms, and how such mechanisms can be modulated by ionizing radiation. Though the "final" goal is of course to elucidate the in vivo scenario, in the meanwhile also in vitro studies can provide useful insights. In the present paper we will discuss key issues on the mechanisms underlying non-targeted effects and cell communication, for which theoretical models and simulation codes can be of great help. In this framework, we will present in detail three literature models, as well as an approach under development at the University of Pavia. More specifically, we will first focus on a version of the "State-Vector Model" including bystander-induced apoptosis of initiated cells, which was successfully fitted to in vitro data on neoplastic transformation supporting the hypothesis of a protective bystander effect mediated by apoptosis. The second analyzed model, focusing on the kinetics of bystander effects in 3D tissues, was successfully fitted to data on bystander damage in an artificial 3D skin system, indicating a signal range of the order of 0.7-1 mm. A third model for bystander effect, taking into account of spatial location, cell killing and repopulation, showed dose-response curves increasing approximately linearly at low dose rates but quickly flattening out for higher dose rates, also predicting an effect augmentation following dose fractionation. Concerning the Pavia approach, which can model the release, diffusion and depletion/degradation of

Rejuvenating cellular respiration for optimizing respiratory function: targeting mitochondria.

PubMed

Agrawal, Anurag; Mabalirajan, Ulaganathan

2016-01-15

Altered bioenergetics with increased mitochondrial reactive oxygen species production and degradation of epithelial function are key aspects of pathogenesis in asthma and chronic obstructive pulmonary disease (COPD). This motif is not unique to obstructive airway disease, reported in related airway diseases such as bronchopulmonary dysplasia and parenchymal diseases such as pulmonary fibrosis. Similarly, mitochondrial dysfunction in vascular endothelium or skeletal muscles contributes to the development of pulmonary hypertension and systemic manifestations of lung disease. In experimental models of COPD or asthma, the use of mitochondria-targeted antioxidants, such as MitoQ, has substantially improved mitochondrial health and restored respiratory function. Modulation of noncoding RNA or protein regulators of mitochondrial biogenesis, dynamics, or degradation has been found to be effective in models of fibrosis, emphysema, asthma, and pulmonary hypertension. Transfer of healthy mitochondria to epithelial cells has been associated with remarkable therapeutic efficacy in models of acute lung injury and asthma. Together, these form a 3R model--repair, reprogramming, and replacement--for mitochondria-targeted therapies in lung disease. This review highlights the key role of mitochondrial function in lung health and disease, with a focus on asthma and COPD, and provides an overview of mitochondria-targeted strategies for rejuvenating cellular respiration and optimizing respiratory function in lung diseases. Copyright © 2016 the American Physiological Society.

The targeting of plant cellular systems by injected type III effector proteins.

PubMed

Lewis, Jennifer D; Guttman, David S; Desveaux, Darrell

2009-12-01

The battle between phytopathogenic bacteria and their plant hosts has revealed a diverse suite of strategies and mechanisms employed by the pathogen or the host to gain the higher ground. Pathogens continually evolve tactics to acquire host resources and dampen host defences. Hosts must evolve surveillance and defence systems that are sensitive enough to rapidly respond to a diverse range of pathogens, while reducing costly and damaging inappropriate misexpression. The primary virulence mechanism employed by many bacteria is the type III secretion system, which secretes and translocates effector proteins directly into the cells of their plant hosts. Effectors have diverse enzymatic functions and can target specific components of plant systems. While these effectors should favour bacterial fitness, the host may be able to thwart infection by recognizing the activity or presence of these foreign molecules and initiating retaliatory immune measures. We review the diverse host cellular systems exploited by bacterial effectors, with particular focus on plant proteins directly targeted by effectors. Effector-host interactions reveal different stages of the battle between pathogen and host, as well as the diverse molecular strategies employed by bacterial pathogens to hijack eukaryotic cellular systems.

Targeted cellular ablation based on the morphology of malignant cells

NASA Astrophysics Data System (ADS)

Ivey, Jill W.; Latouche, Eduardo L.; Sano, Michael B.; Rossmeisl, John H.; Davalos, Rafael V.; Verbridge, Scott S.

2015-11-01

Treatment of glioblastoma multiforme (GBM) is especially challenging due to a shortage of methods to preferentially target diffuse infiltrative cells, and therapy-resistant glioma stem cell populations. Here we report a physical treatment method based on electrical disruption of cells, whose action depends strongly on cellular morphology. Interestingly, numerical modeling suggests that while outer lipid bilayer disruption induced by long pulses (~100 μs) is enhanced for larger cells, short pulses (~1 μs) preferentially result in high fields within the cell interior, which scale in magnitude with nucleus size. Because enlarged nuclei represent a reliable indicator of malignancy, this suggested a means of preferentially targeting malignant cells. While we demonstrate killing of both normal and malignant cells using pulsed electric fields (PEFs) to treat spontaneous canine GBM, we proposed that properly tuned PEFs might provide targeted ablation based on nuclear size. Using 3D hydrogel models of normal and malignant brain tissues, which permit high-resolution interrogation during treatment testing, we confirmed that PEFs could be tuned to preferentially kill cancerous cells. Finally, we estimated the nuclear envelope electric potential disruption needed for cell death from PEFs. Our results may be useful in safely targeting the therapy-resistant cell niches that cause recurrence of GBM tumors.

Targeted cellular ablation based on the morphology of malignant cells

PubMed Central

Ivey, Jill W.; Latouche, Eduardo L.; Sano, Michael B.; Rossmeisl, John H.; Davalos, Rafael V.; Verbridge, Scott S.

2015-01-01

Treatment of glioblastoma multiforme (GBM) is especially challenging due to a shortage of methods to preferentially target diffuse infiltrative cells, and therapy-resistant glioma stem cell populations. Here we report a physical treatment method based on electrical disruption of cells, whose action depends strongly on cellular morphology. Interestingly, numerical modeling suggests that while outer lipid bilayer disruption induced by long pulses (~100 μs) is enhanced for larger cells, short pulses (~1 μs) preferentially result in high fields within the cell interior, which scale in magnitude with nucleus size. Because enlarged nuclei represent a reliable indicator of malignancy, this suggested a means of preferentially targeting malignant cells. While we demonstrate killing of both normal and malignant cells using pulsed electric fields (PEFs) to treat spontaneous canine GBM, we proposed that properly tuned PEFs might provide targeted ablation based on nuclear size. Using 3D hydrogel models of normal and malignant brain tissues, which permit high-resolution interrogation during treatment testing, we confirmed that PEFs could be tuned to preferentially kill cancerous cells. Finally, we estimated the nuclear envelope electric potential disruption needed for cell death from PEFs. Our results may be useful in safely targeting the therapy-resistant cell niches that cause recurrence of GBM tumors. PMID:26596248

Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus

PubMed Central

Lipovsky, Alex; Popa, Andreea; Pimienta, Genaro; Wyler, Michael; Bhan, Ashima; Kuruvilla, Leena; Guie, Marie-Aude; Poffenberger, Adrian C.; Nelson, Christian D. S.; Atwood, Walter J.; DiMaio, Daniel

2013-01-01

Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed. PMID:23569269

[Cellular uptake of TPS-L-carnitine synthesised as transporter-based renal targeting prodrug].

PubMed

Li, Li; Zhu, Di; Sun, Xun

2012-11-01

To synthesize transporter-based renal targeting prodrug TPS-L-Carnitine and to determine its cellular uptake in vitro. Triptolide (TP) was conjugated with L-carnitine using succinate as the linker to form TPS-L-Carnitine, which could be specifically recognized by OCTN2, a cationic transporter with high affinity to L-Carnitine and is highly expressed on the apical membrane of renal proximal tubule cells. Cellular uptake assays of the prodrug and its parent drug were performed on HK-2 cells, a human proximal tubule cell line, in different temperature, concentration and in the presence of competitive inhibitors. TPS-L-Carnitine was taken up into HK-2 cells in a saturable and temperature- and concentration-dependent manner. The uptake process could be inhibited by the competitive inhibitors. The uptake of TPS-L-Carnitine was significantly higher than that of TP at 37 degrees C in the same drug concentration. TPS-L-Carnitine was taken through endocytosis mediated by transporter. TPS-L-Carnitine provides a good renal targeting property and lays the foundation for further studies in vivo.

Modeling the mechanics of cancer: effect of changes in cellular and extra-cellular mechanical properties.

PubMed

Katira, Parag; Bonnecaze, Roger T; Zaman, Muhammad H

2013-01-01

Malignant transformation, though primarily driven by genetic mutations in cells, is also accompanied by specific changes in cellular and extra-cellular mechanical properties such as stiffness and adhesivity. As the transformed cells grow into tumors, they interact with their surroundings via physical contacts and the application of forces. These forces can lead to changes in the mechanical regulation of cell fate based on the mechanical properties of the cells and their surrounding environment. A comprehensive understanding of cancer progression requires the study of how specific changes in mechanical properties influences collective cell behavior during tumor growth and metastasis. Here we review some key results from computational models describing the effect of changes in cellular and extra-cellular mechanical properties and identify mechanistic pathways for cancer progression that can be targeted for the prediction, treatment, and prevention of cancer.

Modified Cross-Linking, Ligation, and Sequencing of Hybrids (qCLASH) Identifies Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Targets in Endothelial Cells.

PubMed

Gay, Lauren A; Sethuraman, Sunantha; Thomas, Merin; Turner, Peter C; Renne, Rolf

2018-04-15

Kaposi's sarcoma (KS) tumors are derived from endothelial cells and express Kaposi's sarcoma-associated herpesvirus (KSHV) microRNAs (miRNAs). Although miRNA targets have been identified in B cell lymphoma-derived cells and epithelial cells, little has been done to characterize the KSHV miRNA targetome in endothelial cells. A recent innovation in the identification of miRNA targetomes, cross-linking, ligation, and sequencing of hybrids (CLASH), unambiguously identifies miRNAs and their targets by ligating the two species while both species are still bound within the RNA-induced silencing complex (RISC). We developed a streamlined quick CLASH (qCLASH) protocol that requires a lower cell input than the original method and therefore has the potential to be used on patient biopsy samples. Additionally, we developed a fast-growing, KSHV-negative endothelial cell line derived from telomerase-immortalized vein endothelial long-term culture (TIVE-LTC) cells. qCLASH was performed on uninfected cells and cells infected with either wild-type KSHV or a mutant virus lacking miR-K12-11/11*. More than 1,400 cellular targets of KSHV miRNAs were identified. Many of the targets identified by qCLASH lacked a canonical seed sequence match. Additionally, most target regions in mRNAs originated from the coding DNA sequence (CDS) rather than the 3' untranslated region (UTR). This set of genes includes some that were previously identified in B cells and some new genes that warrant further study. Pathway analysis of endothelial cell targets showed enrichment in cell cycle control, apoptosis, and glycolysis pathways, among others. Characterization of these new targets and the functional consequences of their repression will be important in furthering our understanding of the role of KSHV miRNAs in oncogenesis. IMPORTANCE KS lesions consist of endothelial cells latently infected with KSHV. Cells that make up these lesions express KSHV miRNAs. Identification of the targets of KSHV miRNAs will

Quantitative phase-digital holographic microscopy: a new imaging modality to identify original cellular biomarkers of diseases

NASA Astrophysics Data System (ADS)

Marquet, P.; Rothenfusser, K.; Rappaz, B.; Depeursinge, C.; Jourdain, P.; Magistretti, P. J.

2016-03-01

Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.

Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs.

PubMed

Chen, Shu-Chuan; Jeng, King-Song; Lai, Michael M C

2017-10-15

Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5' untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target. IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of

Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs

PubMed Central

Chen, Shu-Chuan; Jeng, King-Song

2017-01-01

ABSTRACT Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5′ untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target. IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the

High-Risk Human Papillomaviral Oncogenes E6 and E7 Target Key Cellular Pathways to Achieve Oncogenesis.

PubMed

Yeo-Teh, Nicole S L; Ito, Yoshiaki; Jha, Sudhakar

2018-06-08

Infection with high-risk human papillomavirus (HPV) has been linked to several human cancers, the most prominent of which is cervical cancer. The integration of the viral genome into the host genome is one of the manners in which the viral oncogenes E6 and E7 achieve persistent expression. The most well-studied cellular targets of the viral oncogenes E6 and E7 are p53 and pRb, respectively. However, recent research has demonstrated the ability of these two viral factors to target many more cellular factors, including proteins which regulate epigenetic marks and splicing changes in the cell. These have the ability to exert a global change, which eventually culminates to uncontrolled proliferation and carcinogenesis.

Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma

PubMed Central

Kodama, Takahiro; Newberg, Justin Y.; Kodama, Michiko; Rangel, Roberto; Yoshihara, Kosuke; Tien, Jean C.; Parsons, Pamela H.; Wu, Hao; Finegold, Milton J.; Copeland, Neal G.; Jenkins, Nancy A.

2016-01-01

Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets. PMID:27247392

Systematic approach identifies RHOA as a potential biomarker therapeutic target for Asian gastric cancer.

PubMed

Chang, Hae Ryung; Nam, Seungyoon; Lee, Jinhyuk; Kim, Jin-Hee; Jung, Hae Rim; Park, Hee Seo; Park, Sungjin; Ahn, Young Zoo; Huh, Iksoo; Balch, Curt; Ku, Ja-Lok; Powis, Garth; Park, Taesung; Jeong, Jin-Hyun; Kim, Yon Hui

2016-12-06

Gastric cancer (GC) is a highly heterogeneous disease, in dire need of specific, biomarker-driven cancer therapies. While the accumulation of cancer "Big Data" has propelled the search for novel molecular targets for GC, its specific subpathway and cellular functions vary from patient to patient. In particular, mutations in the small GTPase gene RHOA have been identified in recent genome-wide sequencing of GC tumors. Moreover, protein overexpression of RHOA was reported in Chinese populations, while RHOA mutations were found in Caucasian GC tumors. To develop evidence-based precision medicine for heterogeneous cancers, we established a systematic approach to integrate transcriptomic and genomic data. Predicted signaling subpathways were then laboratory-validated both in vitro and in vivo, resulting in the identification of new candidate therapeutic targets. Here, we show: i) differences in RHOA expression patterns, and its pathway activity, between Asian and Caucasian GC tumors; ii) in vitro and in vivo perturbed RHOA expression inhibits GC cell growth in high RHOA-expressing cell lines; iii) inverse correlation between RHOA and RHOB expression; and iv) an innovative small molecule design strategy for RHOA inhibitors. In summary, RHOA, and its oncogenic signaling pathway, represent a strong biomarker-driven therapeutic target for Asian GC. This comprehensive strategy represents a promising approach for the development of "hit" compounds.

Label-Free Raman Microspectral Analysis for Comparison of Cellular Uptake and Distribution between Non-Targeted and EGFR-Targeted Biodegradable Polymeric Nanoparticles

PubMed Central

Chernenko, Tatyana; Buyukozturk, Fulden; Miljkovic, Milos; Carrier, Rebecca; Diem, Max; Amiji, Mansoor

2013-01-01

Active targeted delivery of nanoparticle-encapsulated agents to tumor cells in vivo is expected to enhance therapeutic effect with significantly less non-specific toxicity. Active targeting is based on surface modification of nanoparticles with ligands that bind with extracellular targets and enhance payload delivery in the cells. In this study, we have used label-free Raman micro-spectral analysis and kinetic modeling to study cellular interactions and intracellular delivery of C6-ceramide using a non-targeted and an epidermal growth factor receptor (EGFR) targeted biodegradable polymeric nano-delivery systems, in EGFR-expressing human ovarian adenocarcinoma (SKOV3) cells. The results show that EGFR peptide-modified nanoparticles were rapidly internalized in SKOV3 cells leading to significant intracellular accumulation as compared to non-specific uptake by the non-targeted nanoparticles. Raman micro-spectral analysis enables visualization and quantification of the carrier system, drug-load, and responses of the biological systems interrogated, without exogenous staining and labeling procedures. PMID:24298430

Cellular Factors Targeting APCs to Modulate Adaptive T Cell Immunity

PubMed Central

Do, Jeongsu; Min, Booki

2014-01-01

The fate of adaptive T cell immunity is determined by multiple cellular and molecular factors, among which the cytokine milieu plays the most important role in this process. Depending on the cytokines present during the initial T cell activation, T cells become effector cells that produce different effector molecules and execute adaptive immune functions. Studies thus far have primarily focused on defining how these factors control T cell differentiation by targeting T cells themselves. However, other non-T cells, particularly APCs, also express receptors for the factors and are capable of responding to them. In this review, we will discuss how APCs, by responding to those cytokines, influence T cell differentiation and adaptive immunity. PMID:25126585

Polymeric nanocomposites loaded with fluoridated hydroxyapatite Ln3+ (Ln = Eu or Tb)/iron oxide for magnetic targeted cellular imaging

PubMed Central

Pan, Jie; Liu, Wei-Jiao; Hua, Chao; Wang, Li-Li; Wan, Dong; Gong, Jun-Bo

2015-01-01

Objective To fabricate polymeric nanocomposites with excellent photoluminescence, magnetic properties, and stability in aqueous solutions, in order to improve specificity and sensitivity of cellular imaging under a magnetic field. Methods Fluoridated Ln3+-doped HAP (Ln3+-HAP) NPs and iron oxides (IOs) can be encapsulated with biocompatible polymers via a modified solvent exaction/evaporation technique to prepare polymeric nanocomposites with fluoridated Ln3+-HAP/iron oxide. The nanocomposites were characterized for surface morphology, fluorescence spectra, magnetic properties and in vitro cytotoxicity. Magnetic targeted cellular imaging of such nanocomposites was also evaluated with confocal laser scanning microscope using A549 cells with or without magnetic field. Results The fabricated nanocomposites showed good stability and excellent luminescent properties, as well as low in vitro cytotoxicity, indicating that the nanocomposites are suitable for biological applications. Nanocomposites under magnetic field achieved much higher cellular uptake via an energy-dependent pathway than those without magnetic field. Conclusion The nanocomposites fabricated in this study will be a promising tool for magnetic targeted cellular imaging with improved specificity and enhanced selection. PMID:26487962

Unambiguous Identification of β-Tubulin as the Direct Cellular Target Responsible for the Cytotoxicity of Chalcone by Photoaffinity Labeling.

PubMed

Zhou, Bo; Yu, Xingxin; Zhuang, Chunlin; Villalta, Peter; Lin, Yong; Lu, Junxuan; Xing, Chengguo

2016-07-05

Chalcone is a simple and potentially privileged structure in medicinal chemistry with a diverse repertoire of biological activities, among which cytotoxicity is of particular interest. The sharp structure-activity relationship (SAR) for chalcone's cytotoxicity suggests structure-specific target interactions. Despite the numerous putative targets proposed, evidence for direct target interactions in cells is unavailable. In this study, guided by the sharp cytotoxic SAR, we developed a cytotoxic chalcone-based photoaffinity labeling (PAL) probe, (E)-3-(3-azidophenyl)-1-[3,5-dimethoxy-4-(prop-2-yn-1-yloxy)phenyl]-2-methylprop-2-en-1-one (C95; IC50 : 0.38±0.01 μm), along with two structurally similar non-cytotoxic probes. These probes were used to search for the direct cellular target responsible for chalcone's cytotoxicity through intact cell-based PAL experiments, in which β-tubulin was identified to specifically interact with the cytotoxic probe (i.e., C95) but not the non-cytotoxic probes. A set of phenotypical and biochemical assays further reinforced β-tubulin as the cytotoxic target of chalcones. Peptide mass quantitation by mass spectrometric analysis revealed one peptide potentially labeled by C95, providing information on chalcone's binding site on β-tubulin. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Yeast Reporter Assay to Identify Cellular Components of Ricin Toxin A Chain Trafficking.

PubMed

Becker, Björn; Schnöder, Tina; Schmitt, Manfred J

2016-12-06

RTA, the catalytic A-subunit of the ribosome inactivating A/B toxin ricin, inhibits eukaryotic protein biosynthesis by depurination of 28S rRNA. Although cell surface binding of ricin holotoxin is mainly mediated through its B-subunit (RTB), sole application of RTA is also toxic, albeit to a significantly lower extent, suggesting alternative pathways for toxin uptake and transport. Since ricin toxin trafficking in mammalian cells is still not fully understood, we developed a GFP-based reporter assay in yeast that allows rapid identification of cellular components required for RTA uptake and subsequent transport through a target cell. We hereby show that Ypt6p, Sft2p and GARP-complex components play an important role in RTA transport, while neither the retromer complex nor COPIB vesicles are part of the transport machinery. Analyses of yeast knock-out mutants with chromosomal deletion in genes whose products regulate ADP-ribosylation factor GTPases (Arf-GTPases) and/or retrograde Golgi-to-ER (endoplasmic reticulum) transport identified Sso1p, Snc1p, Rer1p, Sec22p, Erv46p, Gea1p and Glo3p as novel components in RTA transport, suggesting the developed reporter assay as a powerful tool to dissect the multistep processes of host cell intoxication in yeast.

Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming

PubMed Central

McClellan, Michael J.; Wood, C. David; Ojeniyi, Opeoluwa; Cooper, Tim J.; Kanhere, Aditi; Arvey, Aaron; Webb, Helen M.; Palermo, Richard D.; Harth-Hertle, Marie L.; Kempkes, Bettina; Jenner, Richard G.; West, Michelle J.

2013-01-01

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of

Systematic approach identifies RHOA as a potential biomarker therapeutic target for Asian gastric cancer

PubMed Central

Jung, Hae Rim; Park, Hee Seo; Park, Sungjin; Ahn, Young Zoo; Huh, Iksoo; Balch, Curt; Ku, Ja-Lok; Powis, Garth; Park, Taesung; Jeong, Jin-Hyun; Kim, Yon Hui

2016-01-01

Gastric cancer (GC) is a highly heterogeneous disease, in dire need of specific, biomarker-driven cancer therapies. While the accumulation of cancer “Big Data” has propelled the search for novel molecular targets for GC, its specific subpathway and cellular functions vary from patient to patient. In particular, mutations in the small GTPase gene RHOA have been identified in recent genome-wide sequencing of GC tumors. Moreover, protein overexpression of RHOA was reported in Chinese populations, while RHOA mutations were found in Caucasian GC tumors. To develop evidence-based precision medicine for heterogeneous cancers, we established a systematic approach to integrate transcriptomic and genomic data. Predicted signaling subpathways were then laboratory-validated both in vitro and in vivo, resulting in the identification of new candidate therapeutic targets. Here, we show: i) differences in RHOA expression patterns, and its pathway activity, between Asian and Caucasian GC tumors; ii) in vitro and in vivo perturbed RHOA expression inhibits GC cell growth in high RHOA-expressing cell lines; iii) inverse correlation between RHOA and RHOB expression; and iv) an innovative small molecule design strategy for RHOA inhibitors. In summary, RHOA, and its oncogenic signaling pathway, represent a strong biomarker-driven therapeutic target for Asian GC. This comprehensive strategy represents a promising approach for the development of “hit” compounds. PMID:27806312

RchyOptimyx: Cellular Hierarchy Optimization for Flow Cytometry

PubMed Central

Aghaeepour, Nima; Jalali, Adrin; O’Neill, Kieran; Chattopadhyay, Pratip K.; Roederer, Mario; Hoos, Holger H.; Brinkman, Ryan R.

2013-01-01

Analysis of high-dimensional flow cytometry datasets can reveal novel cell populations with poorly understood biology. Following discovery, characterization of these populations in terms of the critical markers involved is an important step, as this can help to both better understand the biology of these populations and aid in designing simpler marker panels to identify them on simpler instruments and with fewer reagents (i.e., in resource poor or highly regulated clinical settings). However, current tools to design panels based on the biological characteristics of the target cell populations work exclusively based on technical parameters (e.g., instrument configurations, spectral overlap, and reagent availability). To address this shortcoming, we developed RchyOptimyx (cellular hieraRCHY OPTIMization), a computational tool that constructs cellular hierarchies by combining automated gating with dynamic programming and graph theory to provide the best gating strategies to identify a target population to a desired level of purity or correlation with a clinical outcome, using the simplest possible marker panels. RchyOptimyx can assess and graphically present the trade-offs between marker choice and population specificity in high-dimensional flow or mass cytometry datasets. We present three proof-of-concept use cases for RchyOptimyx that involve 1) designing a panel of surface markers for identification of rare populations that are primarily characterized using their intracellular signature; 2) simplifying the gating strategy for identification of a target cell population; 3) identification of a non-redundant marker set to identify a target cell population. PMID:23044634

A novel mouse model identifies cooperating mutations and therapeutic targets critical for chronic myeloid leukemia progression

PubMed Central

Giotopoulos, George; van der Weyden, Louise; Osaki, Hikari; Rust, Alistair G.; Gallipoli, Paolo; Meduri, Eshwar; Horton, Sarah J.; Chan, Wai-In; Foster, Donna; Prinjha, Rab K.; Pimanda, John E.; Tenen, Daniel G.; Vassiliou, George S.; Koschmieder, Steffen; Adams, David J.

2015-01-01

The introduction of highly selective ABL-tyrosine kinase inhibitors (TKIs) has revolutionized therapy for chronic myeloid leukemia (CML). However, TKIs are only efficacious in the chronic phase of the disease and effective therapies for TKI-refractory CML, or after progression to blast crisis (BC), are lacking. Whereas the chronic phase of CML is dependent on BCR-ABL, additional mutations are required for progression to BC. However, the identity of these mutations and the pathways they affect are poorly understood, hampering our ability to identify therapeutic targets and improve outcomes. Here, we describe a novel mouse model that allows identification of mechanisms of BC progression in an unbiased and tractable manner, using transposon-based insertional mutagenesis on the background of chronic phase CML. Our BC model is the first to faithfully recapitulate the phenotype, cellular and molecular biology of human CML progression. We report a heterogeneous and unique pattern of insertions identifying known and novel candidate genes and demonstrate that these pathways drive disease progression and provide potential targets for novel therapeutic strategies. Our model greatly informs the biology of CML progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease. PMID:26304963

Cellular pH regulators: potentially promising molecular targets for cancer chemotherapy.

PubMed

Izumi, Hiroto; Torigoe, Takayuki; Ishiguchi, Hiroshi; Uramoto, Hidetaka; Yoshida, Yoichiro; Tanabe, Mizuho; Ise, Tomoko; Murakami, Tadashi; Yoshida, Takeshi; Nomoto, Minoru; Kohno, Kimitoshi

2003-12-01

One of the major obstacles to the successful treatment of cancer is the complex biology of solid tumour development. Although regulation of intracellular pH has been shown to be critically important for many cellular functions, pH regulation has not been fully investigated in the field of cancer. It has, however, been shown that cellular pH is crucial for biological functions such as cell proliferation, invasion and metastasis, drug resistance and apoptosis. Hypoxic conditions are often observed during the development of solid tumours and lead to intracellular and extracellular acidosis. Cellular acidosis has been shown to be a trigger in the early phase of apoptosis and leads to activation of endonucleases inducing DNA fragmentation. To avoid intracellular acidification under such conditions, pH regulators are thought to be up-regulated in tumour cells. Four major types of pH regulator have been identified: the proton pump, the sodium-proton exchanger family (NHE), the bicarbonate transporter family (BCT) and the monocarboxylate transporter family (MCT). Here, we describe the structure and function of pH regulators expressed in tumour tissue. Understanding pH regulation in tumour cells may provide new ways of inducing tumour-specific apoptosis, thus aiding cancer chemotherapy.

Identifying transcription factor functions and targets by phenotypic activation

PubMed Central

Chua, Gordon; Morris, Quaid D.; Sopko, Richelle; Robinson, Mark D.; Ryan, Owen; Chan, Esther T.; Frey, Brendan J.; Andrews, Brenda J.; Boone, Charles; Hughes, Timothy R.

2006-01-01

Mapping transcriptional regulatory networks is difficult because many transcription factors (TFs) are activated only under specific conditions. We describe a generic strategy for identifying genes and pathways induced by individual TFs that does not require knowledge of their normal activation cues. Microarray analysis of 55 yeast TFs that caused a growth phenotype when overexpressed showed that the majority caused increased transcript levels of genes in specific physiological categories, suggesting a mechanism for growth inhibition. Induced genes typically included established targets and genes with consensus promoter motifs, if known, indicating that these data are useful for identifying potential new target genes and binding sites. We identified the sequence 5′-TCACGCAA as a binding sequence for Hms1p, a TF that positively regulates pseudohyphal growth and previously had no known motif. The general strategy outlined here presents a straightforward approach to discovery of TF activities and mapping targets that could be adapted to any organism with transgenic technology. PMID:16880382

Silver nanoparticles induced alterations in multiple cellular targets, which are critical for drug susceptibilities and pathogenicity in fungal pathogen (Candida albicans)

PubMed Central

Radhakrishnan, Venkatraman Srinivasan; Reddy Mudiam, Mohana Krishna; Kumar, Manish; Dwivedi, Surya Prakash; Singh, Surinder Pal; Prasad, Tulika

2018-01-01

Purpose A significant increase in the incidence of fungal infections and drug resistance has been observed in the past decades due to limited availability of broad-spectrum antifungal drugs. Nanomedicines have shown significant antimicrobial potential against various drug-resistant microbes. Silver nanoparticles (AgNps) are known for their antimicrobial properties and lower host toxicity; however, for clinical applications, evaluation of their impact at cellular and molecular levels is essential. The present study aims to understand the cellular and molecular mechanisms of AgNp-induced toxicity in a common fungal pathogen, Candida albicans. Methods AgNps were synthesized by chemical reduction method and characterized using UV–visible spectroscopy, X-ray powder diffraction, transmission electron microscopy, scanning electron microscopy–energy dispersive X-ray spectroscopy, energy dispersive X-ray fluorescence, and zeta potential. The anti-Candida activity of AgNps was assessed by broth microdilution and spot assays. Effects of AgNps on cellular and molecular targets were assessed by monitoring the intracellular reactive oxygen species (ROS) production in the absence and presence of natural antioxidant, changes in surface morphology, cellular ultrastructure, membrane microenvironment, membrane fluidity, membrane ergosterol, and fatty acids. Results Spherical AgNps (10–30 nm) showed minimum inhibitory concentration (minimum concentration required to inhibit the growth of 90% of organisms) at 40 μg/mL. Our results demonstrated that AgNps induced dose-dependent intracellular ROS which exerted antifungal effects; however, even scavenging ROS by antioxidant could not offer protection from AgNp mediated killing. Treatment with AgNps altered surface morphology, cellular ultrastructure, membrane microenvironment, membrane fluidity, ergosterol content, and fatty acid composition, especially oleic acid. Conclusion To summarize, AgNps affected multiple cellular targets

Viral Activation of Cellular Metabolism

PubMed Central

Sanchez, Erica L.; Lagunoff, Michael

2015-01-01

To ensure optimal environments for their replication and spread, viruses have evolved to alter many host cell pathways. In the last decade, metabolomic studies have shown that eukaryotic viruses induce large-scale alterations in host cellular metabolism. Most viruses examined to date induce aerobic glycolysis also known as the Warburg effect. Many viruses tested also induce fatty acid synthesis as well as glutaminolysis. These modifications of carbon source utilization by infected cells can increase available energy for virus replication and virion production, provide specific cellular substrates for virus particles and create viral replication niches while increasing infected cell survival. Each virus species also likely requires unique metabolic changes for successful spread and recent research has identified additional virus-specific metabolic changes induced by many virus species. A better understanding of the metabolic alterations required for each virus may lead to novel therapeutic approaches through targeted inhibition of specific cellular metabolic pathways. PMID:25812764

Dendritic cells are early cellular targets of Listeria monocytogenes after intestinal delivery and are involved in bacterial spread in the host.

PubMed

Pron, B; Boumaila, C; Jaubert, F; Berche, P; Milon, G; Geissmann, F; Gaillard, J L

2001-05-01

We studied the sequence of cellular events leading to the dissemination of Listeria monocytogenes from the gut to draining mesenteric lymph nodes (MLNs) by confocal microscopy of immunostained tissue sections from a rat ligated ileal loop system. OX-62-positive cells beneath the epithelial lining of Peyer's patches (PPs) were the first Listeria targets identified after intestinal inoculation. These cells had other features typical of dendritic cells (DCs): they were large, pleiomorphic and major histocompatibility complex class II(hi). Listeria were detected by microscopy in draining MLNs as early as 6 h after inoculation. Some 80-90% of bacteria were located in the deep paracortical regions, and 100% of the bacteria were present in OX-62-positive cells. Most infected cells contained more than five bacteria each, suggesting that they had arrived already loaded with bacteria. At later stages, the bacteria in these areas were mostly present in ED1-positive mononuclear phagocytes. These cells were also infected by an actA mutant defective in cell-to-cell spreading. This suggests that Listeria are transported by DCs from PPs to the deep paracortical regions of draining MLNs and are then transmitted to other cell populations by mechanisms independent of ActA. Another pathway of dissemination to MLNs was identified, probably involving free Listeria and leading to the infection of ED3-positive mononuclear phagocytes in the subcapsular sinus and adjacent paracortical areas. This study provides evidence that DCs are major cellular targets of L. monocytogenes in PPs and that DCs may be involved in the early dissemination of this pathogen. DCs were not sites of active bacterial replication, making these cells ideal vectors of infection.

Rodent Models of Experimental Endometriosis: Identifying Mechanisms of Disease and Therapeutic Targets

PubMed Central

Bruner-Tran, Kaylon L.; Mokshagundam, Shilpa; Herington, Jennifer L.; Ding, Tianbing; Osteen, Kevin G.

2018-01-01

Background: Although it has been more than a century since endometriosis was initially described in the literature, understanding the etiology and natural history of the disease has been challenging. However, the broad utility of murine and rat models of experimental endometriosis has enabled the elucidation of a number of potentially targetable processes which may otherwise promote this disease. Objective: To review a variety of studies utilizing rodent models of endometriosis to illustrate their utility in examining mechanisms associated with development and progression of this disease. Results: Use of rodent models of endometriosis has provided a much broader understanding of the risk factors for the initial development of endometriosis, the cellular pathology of the disease and the identification of potential therapeutic targets. Conclusion: Although there are limitations with any animal model, the variety of experimental endometriosis models that have been developed has enabled investigation into numerous aspects of this disease. Thanks to these models, our under-standing of the early processes of disease development, the role of steroid responsiveness, inflammatory processes and the peritoneal environment has been advanced. More recent models have begun to shed light on how epigenetic alterations con-tribute to the molecular basis of this disease as well as the multiple comorbidities which plague many patients. Continued de-velopments of animal models which aid in unraveling the mechanisms of endometriosis development provide the best oppor-tunity to identify therapeutic strategies to prevent or regress this enigmatic disease.

Cellular target of streptomycin in the internal ear.

PubMed

Meza, G; López, I; Paredes, M A; Peñaloza, Y; Poblano, A

1989-01-01

The cellular target of streptomycin (STP) was investigated by analyzing the activity of glutamate decarboxylase (GAD) or choline acetyltransferase (ChAT) enzymes of synthesis of GABA and acetylcholine (Ach), respectively, [supposedly located in hair cells (GAD) or efferent terminals (ChAT)] in control and in 50 day-STP-treated colored guinea pig vestibular homogenates. Vestibular and auditory function were assessed by measuring postrotatory nystagmus response (PNR) and auditory brainstem evoked potentials (ABP). Morphological changes were followed by light and electron microscopy. STP-treated animals exhibited a GAD decrease of 83.6% with respect to controls whereas ChAT did not suffer any change. Assessment of PNR and ABP showed that STP affected only the former since animals lost it between the 20th and the 30th day of treatment, whereas ABP was not modified. Morphological experiments detected vestibular hair cell deterioration as the only cell type affected by STP. These results confirm the predilection of STP to affect vestibular function by damage to hair cells and show that this effect can be followed by measurement of GAD and ChAT in the vestibule as markers for hair cells and efferent terminals, respectively.

Distinctive Behaviors of Druggable Proteins in Cellular Networks

PubMed Central

Workman, Paul; Al-Lazikani, Bissan

2015-01-01

The interaction environment of a protein in a cellular network is important in defining the role that the protein plays in the system as a whole, and thus its potential suitability as a drug target. Despite the importance of the network environment, it is neglected during target selection for drug discovery. Here, we present the first systematic, comprehensive computational analysis of topological, community and graphical network parameters of the human interactome and identify discriminatory network patterns that strongly distinguish drug targets from the interactome as a whole. Importantly, we identify striking differences in the network behavior of targets of cancer drugs versus targets from other therapeutic areas and explore how they may relate to successful drug combinations to overcome acquired resistance to cancer drugs. We develop, computationally validate and provide the first public domain predictive algorithm for identifying druggable neighborhoods based on network parameters. We also make available full predictions for 13,345 proteins to aid target selection for drug discovery. All target predictions are available through canSAR.icr.ac.uk. Underlying data and tools are available at https://cansar.icr.ac.uk/cansar/publications/druggable_network_neighbourhoods/. PMID:26699810

USP1 deubiquitinase: cellular functions, regulatory mechanisms and emerging potential as target in cancer therapy

PubMed Central

2013-01-01

Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the best-characterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells. PMID:23937906

Functional screening for miRNAs targeting Smad4 identified miR-199a as a negative regulator of TGF-β signalling pathway

PubMed Central

Zhang, Yan; Fan, Kai-Ji; Sun, Qiang; Chen, Ai-Zhong; Shen, Wen-Long; Zhao, Zhi-Hu; Zheng, Xiao-Fei; Yang, Xiao

2012-01-01

The transforming growth factor-β (TGF-β) signalling pathway participates in various biological processes. Dysregulation of Smad4, a central cellular transducer of TGF-β signalling, is implicated in a wide range of human diseases and developmental disorders. However, the mechanisms underlying Smad4 dysregulation are not fully understood. Using a functional screening approach based on luciferase reporter assays, we identified 39 microRNAs (miRNAs) as potential regulators of Smad4 from an expression library of 388 human miRNAs. The screening was supported by bioinformatic analysis, as 24 of 39 identified miRNAs were also predicted to target Smad4. MiR-199a, one of the identified miRNAs, was inversely correlated with Smad4 expression in various human cancer cell lines and gastric cancer tissues, and repressed Smad4 expression and blocked canonical TGF-β transcriptional responses in cell lines. These effects were dependent on the presence of a conserved, but not perfect seed paired, miR-199a-binding site in the Smad4 3′-untranslated region (UTR). Overexpression of miR-199a significantly inhibited the ability of TGF-β to induce gastric cancer cell growth arrest and apoptosis in vitro, and promoted anchorage-independent growth in soft agar, suggesting that miR-199a plays an oncogenic role in human gastric tumourigenesis. In conclusion, our functional screening uncovers multiple miRNAs that regulate the cellular responsiveness to TGF-β signalling and reveals important roles of miR-199a in gastric cancer by directly targeting Smad4. PMID:22821565

Selecting a Targeting Method to Identify BPL Households in India

ERIC Educational Resources Information Center

Alkire, Sabina; Seth, Suman

2013-01-01

This paper proposes how to select a methodology to target multidimensionally poor households, and how to update that targeting exercise periodically. We present this methodology in the context of discussions regarding the selection of a targeting methodology in India. In 1992, 1997, and 2002 the Indian government identified households that are…

UniDrug-target: a computational tool to identify unique drug targets in pathogenic bacteria.

PubMed

Chanumolu, Sree Krishna; Rout, Chittaranjan; Chauhan, Rajinder S

2012-01-01

Targeting conserved proteins of bacteria through antibacterial medications has resulted in both the development of resistant strains and changes to human health by destroying beneficial microbes which eventually become breeding grounds for the evolution of resistances. Despite the availability of more than 800 genomes sequences, 430 pathways, 4743 enzymes, 9257 metabolic reactions and protein (three-dimensional) 3D structures in bacteria, no pathogen-specific computational drug target identification tool has been developed. A web server, UniDrug-Target, which combines bacterial biological information and computational methods to stringently identify pathogen-specific proteins as drug targets, has been designed. Besides predicting pathogen-specific proteins essentiality, chokepoint property, etc., three new algorithms were developed and implemented by using protein sequences, domains, structures, and metabolic reactions for construction of partial metabolic networks (PMNs), determination of conservation in critical residues, and variation analysis of residues forming similar cavities in proteins sequences. First, PMNs are constructed to determine the extent of disturbances in metabolite production by targeting a protein as drug target. Conservation of pathogen-specific protein's critical residues involved in cavity formation and biological function determined at domain-level with low-matching sequences. Last, variation analysis of residues forming similar cavities in proteins sequences from pathogenic versus non-pathogenic bacteria and humans is performed. The server is capable of predicting drug targets for any sequenced pathogenic bacteria having fasta sequences and annotated information. The utility of UniDrug-Target server was demonstrated for Mycobacterium tuberculosis (H37Rv). The UniDrug-Target identified 265 mycobacteria pathogen-specific proteins, including 17 essential proteins which can be potential drug targets. UniDrug-Target is expected to accelerate

Identifying Molecular Targets for PTSD Treatment Using Single Prolonged Stress

DTIC Science & Technology

2015-10-01

1 AWARD NUMBER: W81XWH-13-1-0377 TITLE: Identifying Molecular Targets For PTSD Treatment Using Single Prolonged Stress PRINCIPAL...TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-13-1-0377 Identifying Molecular Targets For PTSD Treatment Using Single Prolonged Stress 5b. GRANT...brain GR and β-AR expression alters glutamatergic and GABAergic function in neural circuits that mediate SPS-induced deficits in extinction retention

Overexpression of FurA in Anabaena sp. PCC 7120 reveals new targets for this regulator involved in photosynthesis, iron uptake and cellular morphology.

PubMed

González, Andrés; Bes, M Teresa; Barja, François; Peleato, M Luisa; Fillat, María F

2010-11-01

Previous genomic analyses of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 have identified three ferric uptake regulator (Fur) homologs with low sequence identities and probably different functions in the cell. FurA is a constitutive protein that shares the highest homology with Fur from heterotrophic bacteria and appears to be essential for in vitro growth. In this study, we have analysed the effects of FurA overexpression on the Anabaena sp. phenotype and investigated which of the observed alterations were directly operated by FurA. Overexpression of the regulator led to changes in cellular morphology, resulting in shorter filaments with rounded cells of different sizes. The furA-overexpressing strain showed a slower photoautotrophic growth and a marked decrease in the oxygen evolution rate. Overexpression of the regulator also decreased both catalase and superoxide dismutase activities, but did not lead to an increase in the levels of intracellular reactive oxygen species. By combining phenotypic studies, reverse transcription-PCR analyses and electrophoretic mobility shift assays, we identified three novel direct targets of FurA, including genes encoding a siderophore outer membrane transporter (schT), bacterial actins (mreBCD) and the PSII reaction center protein D1 (psbA). The affinity of FurA for these novel targets was markedly affected by the absence of divalent metal ions, confirming previous evidence of a critical role for the metal co-repressor in the function of the regulator in vivo. The results unravel new cellular processes modulated by FurA, supporting its role as a global transcriptional regulator in Anabaena sp. PCC 7120.

Single-domain antibody bioconjugated near-IR quantum dots for targeted cellular imaging of pancreatic cancer.

PubMed

Zaman, Md Badruz; Baral, Toya Nath; Jakubek, Zygmunt J; Zhang, Jianbing; Wu, Xiaohua; Lai, Edward; Whitfield, Dennis; Yu, Kui

2011-05-01

Successful targeted imaging of BxPC3 human pancreatic cancer cells is feasible with near-IR CdTeSe/CdS quantum dots (QDs) functionalized with single-domain antibody (sdAb) 2A3. For specific targeting, sdAbs are superior to conventional antibodies, especially in terms of stability, aggregation, and production cost. The bright CdTeSe/CdS QDs were synthesized to emit in the diagnostic window of 650-900 nm with a narrow emission band. 2A3 was derived from llama and is small in size of 13 kDa, but with fully-functional recognition to the target carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a possible biomarker as a therapeutic target of pancreatic cancer. For compelling imaging, optical may be the most sensible among the various imaging modalities, regarding the sensitivity and cost. This first report on sdAb-conjugated near-IR QDs with high signal to background sensitivity for targeted cellular imaging brings insights into the development of optical molecular imaging for early stage cancer diagnosis.

Principles of Unconventional Myosin Function and Targeting

PubMed Central

Hartman, M. Amanda; Finan, Dina; Sivaramakrishnan, Sivaraj; Spudich, James A.

2016-01-01

Unconventional myosins are a superfamily of actin-based motors implicated in diverse cellular processes. In recent years, much progress has been made in describing their biophysical properties, and headway has been made into analyzing their cellular functions. Here, we focus on the principles that guide in vivo motor function and targeting to specific cellular locations. Rather than describe each motor comprehensively, we outline the major themes that emerge from research across the superfamily and use specific examples to illustrate each. In presenting the data in this format, we seek to identify open questions in each field as well as to point out commonalities between them. To advance our understanding of myosins’ roles in vivo, clearly we must identify their cellular cargoes and the protein complexes that regulate motor attachment to fully appreciate their functions on the cellular and developmental levels. PMID:21639800

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

PubMed

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

2013-12-09

Gene vaccines offer attractive rationales for prophylactic as well as therapeutic treatments of type I allergies. DNA and mRNA vaccines have been shown to prevent from allergic sensitization and to counterbalance established allergic immune reactions. Recent advances in gene vaccine manipulation offer additional opportunities for modulation of T helper cell profiles by specific targeting of cellular compartments. DNA vaccines encoding the major birch pollen allergen Bet v 1.0101 were equipped with different leader sequences to shuttle the antigen to lysosomes (LIMP-II), to trigger cellular secretion (hTPA), or to induce proteasomal degradation via forced ubiquitination (ubi). Mice were pre-vaccinated with these constructs and the protective efficacy was tested by subcutaneous Th2-promoting challenges, followed by allergen inhalation. IgG antibody subclass distribution and allergen-specific IgE as well as cytokine profiles from re-stimulated splenocytes and from BALFs were assessed. The cellular composition of BALFs, and lung resistance and compliance were determined. Immunization with all targeting variants protected from allergic sensitization, i.e. IgE induction, airway hyperresponsiveness, lung inflammation, and systemic and local Th2 cytokine expression. Surprisingly, protection did not clearly correlate with the induction of a systemic Th1 cytokine profile, but rather with proliferating CD4+ CD25+ FoxP3+ T regulatory cells in splenocyte cultures. Targeting the allergen to proteasomal or lysosomal degradation severely down-regulated antibody induction after vaccination, while T cell responses remained unaffected. Although secretion of antigen promoted the highest numbers of Th1 cells, this vaccine type was the least efficient in suppressing the establishment of an allergic immune response. This comparative analysis highlights the modulatory effect of antigen targeting on the resulting immune response, with a special emphasis on prophylactic anti-allergy DNA

Antibodies against the mono-methylated arginine-glycine repeat (MMA-RG) of the Epstein-Barr virus nuclear antigen 2 (EBNA2) identify potential cellular proteins targeted in viral transformation.

PubMed

Ayoubian, Hiresh; Fröhlich, Thomas; Pogodski, Dagmar; Flatley, Andrew; Kremmer, Elisabeth; Schepers, Aloys; Feederle, Regina; Arnold, Georg J; Grässer, Friedrich A

2017-08-01

The Epstein-Barr virus is a human herpes virus with oncogenic potential. The virus-encoded nuclear antigen 2 (EBNA2) is a key mediator of viral tumorigenesis. EBNA2 features an arginine-glycine (RG) repeat at amino acids (aa)339-354 that is essential for the transformation of lymphocytes and contains symmetrically (SDMA) and asymmetrically (ADMA) di-methylated arginine residues. The SDMA-modified EBNA2 binds the survival motor neuron protein (SMN), thus mimicking SMD3, a cellular SDMA-containing protein that interacts with SMN. Accordingly, a monoclonal antibody (mAb) specific for the SDMA-modified RG repeat of EBNA2 also binds to SMD3. With the novel mAb 19D4 we now show that EBNA2 contains mono-methylated arginine (MMA) residues within the RG repeat. Using 19D4, we immune-precipitated and analysed by mass spectrometry cellular proteins in EBV-transformed B-cells that feature MMA motifs that are similar to the one in EBNA2. Among the cellular proteins identified, we confirmed by immunoprecipitation and/or Western blot analyses Aly/REF, Coilin, DDX5, FXR1, HNRNPK, LSM4, MRE11, NRIP, nucleolin, PRPF8, RBM26, SMD1 (SNRDP1) and THRAP3 proteins that are either known to contain MMA residues or feature RG repeat sequences that probably serve as methylation substrates. The identified proteins are involved in splicing, tumorigenesis, transcriptional activation, DNA stability and RNA processing or export. Furthermore, we found that several proteins involved in energy metabolism are associated with MMA-modified proteins. Interestingly, the viral EBNA1 protein that features methylated RG repeat motifs also reacted with the antibodies. Our results indicate that the region between aa 34-52 of EBNA1 contains ADMA or SDMA residues, while the region between aa 328-377 mainly contains MMA residues.

Targeting Neutrophilic Inflammation Using Polymersome-Mediated Cellular Delivery.

PubMed

Robertson, James D; Ward, Jon R; Avila-Olias, Milagros; Battaglia, Giuseppe; Renshaw, Stephen A

2017-05-01

Neutrophils are key effector cells in inflammation and play an important role in neutralizing invading pathogens. During inflammation resolution, neutrophils undergo apoptosis before they are removed by macrophages, but if apoptosis is delayed, neutrophils can cause extensive tissue damage and chronic disease. Promotion of neutrophil apoptosis is a potential therapeutic approach for treating persistent inflammation, yet neutrophils have proven difficult cells to manipulate experimentally. In this study, we deliver therapeutic compounds to neutrophils using biocompatible, nanometer-sized synthetic vesicles, or polymersomes, which are internalized by binding to scavenger receptors and subsequently escape the early endosome through a pH-triggered disassembly mechanism. This allows polymersomes to deliver molecules into the cell cytosol of neutrophils without causing cellular activation. After optimizing polymersome size, we show that polymersomes can deliver the cyclin-dependent kinase inhibitor (R)-roscovitine into human neutrophils to promote apoptosis in vitro. Finally, using a transgenic zebrafish model, we show that encapsulated (R)-roscovitine can speed up inflammation resolution in vivo more efficiently than the free drug. These results show that polymersomes are effective intracellular carriers for drug delivery into neutrophils. This has important consequences for the study of neutrophil biology and the development of neutrophil-targeted therapeutics. Copyright © 2017 The Authors.

A side-effect free method for identifying cancer drug targets.

PubMed

Ashraf, Md Izhar; Ong, Seng-Kai; Mujawar, Shama; Pawar, Shrikant; More, Pallavi; Paul, Somnath; Lahiri, Chandrajit

2018-04-27

Identifying effective drug targets, with little or no side effects, remains an ever challenging task. A potential pitfall of failing to uncover the correct drug targets, due to side effect of pleiotropic genes, might lead the potential drugs to be illicit and withdrawn. Simplifying disease complexity, for the investigation of the mechanistic aspects and identification of effective drug targets, have been done through several approaches of protein interactome analysis. Of these, centrality measures have always gained importance in identifying candidate drug targets. Here, we put forward an integrated method of analysing a complex network of cancer and depict the importance of k-core, functional connectivity and centrality (KFC) for identifying effective drug targets. Essentially, we have extracted the proteins involved in the pathways leading to cancer from the pathway databases which enlist real experimental datasets. The interactions between these proteins were mapped to build an interactome. Integrative analyses of the interactome enabled us to unearth plausible reasons for drugs being rendered withdrawn, thereby giving future scope to pharmaceutical industries to potentially avoid them (e.g. ESR1, HDAC2, F2, PLG, PPARA, RXRA, etc). Based upon our KFC criteria, we have shortlisted ten proteins (GRB2, FYN, PIK3R1, CBL, JAK2, LCK, LYN, SYK, JAK1 and SOCS3) as effective candidates for drug development.

Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal Dominant Polycystic Kidney Disease

DTIC Science & Technology

2016-09-01

AWARD NUMBER: W81XWH-15-1-0420 TITLE: Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal Dominant Polycystic Kidney Disease...Autosomal Dominant Polycystic Kidney Disease 5b. GRANT NUMBER W81XWH-15-1-0420 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Kenneth R. Hallows, MD...polycystic kidney disease (ADPKD) is a common inherited disorder where patients, over the course of decades, develop large fluid filled cysts that

Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2-Monoacylglycerols and N-acyl Amides

PubMed Central

Takacs, Sara M.; Stuart, Jordyn M.; Basnet, Arjun; Raboune, Siham; Widlanski, Theodore S.; Doherty, Patrick; Bradshaw, Heather B.

2013-01-01

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling. PMID:23874457

A genome-wide RNAi screen identifies novel targets of neratinib resistance leading to identification of potential drug resistant genetic markers.

PubMed

Seyhan, Attila A; Varadarajan, Usha; Choe, Sung; Liu, Wei; Ryan, Terence E

2012-04-01

Neratinib (HKI-272) is a small molecule tyrosine kinase inhibitor of the ErbB receptor family currently in Phase III clinical trials. Despite its efficacy, the mechanism of potential cellular resistance to neratinib and genes involved with it remains unknown. We have used a pool-based lentiviral genome-wide functional RNAi screen combined with a lethal dose of neratinib to discover chemoresistant interactions with neratinib. Our screen has identified a collection of genes whose inhibition by RNAi led to neratinib resistance including genes involved in oncogenesis (e.g. RAB33A, RAB6A and BCL2L14), transcription factors (e.g. FOXP4, TFEC, ZNF), cellular ion transport (e.g. CLIC3, TRAPPC2P1, P2RX2), protein ubiquitination (e.g. UBL5), cell cycle (e.g. CCNF), and genes known to interact with breast cancer-associated genes (e.g. CCNF, FOXP4, TFEC, several ZNF factors, GNA13, IGFBP1, PMEPA1, SOX5, RAB33A, RAB6A, FXR1, DDO, TFEC, OLFM2). The identification of novel mediators of cellular resistance to neratinib could lead to the identification of new or neoadjuvant drug targets. Their use as patient or treatment selection biomarkers could make the application of anti-ErbB therapeutics more clinically effective.

Geothermal Target Areas in Colorado as Identified by Remote Sensing Techniques

DOE Data Explorer

Khalid Hussein

2012-02-01

This layer contains the areas identified as targets of potential geothermal activity. The Criteria used to identify the target areas include: hot/warm surface exposures modeled from ASTER/Landsat satellite imagery and geological characteristics, alteration mineral commonly associated with hot springs (clays, Si, and FeOx) modeled from ASTER and Landsat data, Colorado Geological Survey (CGS) known thermal hot springs/wells and heat-flow data points, Colorado deep-seated fault zones, weakened basement identified from isostatic gravity data, and Colorado sedimentary and topographic characteristics.

Approaches to Validate and Manipulate RNA Targets with Small Molecules in Cells.

PubMed

Childs-Disney, Jessica L; Disney, Matthew D

2016-01-01

RNA has become an increasingly important target for therapeutic interventions and for chemical probes that dissect and manipulate its cellular function. Emerging targets include human RNAs that have been shown to directly cause cancer, metabolic disorders, and genetic disease. In this review, we describe various routes to obtain bioactive compounds that target RNA, with a particular emphasis on the development of small molecules. We use these cases to describe approaches that are being developed for target validation, which include target-directed cleavage, classic pull-down experiments, and covalent cross-linking. Thus, tools are available to design small molecules to target RNA and to identify the cellular RNAs that are their targets.

Controllability analysis of the directed human protein interaction network identifies disease genes and drug targets

PubMed Central

Vinayagam, Arunachalam; Gibson, Travis E.; Lee, Ho-Joon; Yilmazel, Bahar; Roesel, Charles; Hu, Yanhui; Kwon, Young; Sharma, Amitabh; Liu, Yang-Yu; Perrimon, Norbert; Barabási, Albert-László

2016-01-01

The protein–protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here, we characterize the structural controllability of a large directed human PPI network comprising 6,339 proteins and 34,813 interactions. This network allows us to classify proteins as “indispensable,” “neutral,” or “dispensable,” which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network’s control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets. PMID:27091990

Target-specific cellular uptake of PLGA nanoparticles coated with poly(L-lysine)-poly(ethylene glycol)-folate conjugate.

PubMed

Kim, Sun Hwa; Jeong, Ji Hoon; Chun, Ki Woo; Park, Tae Gwan

2005-09-13

Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with anionic surface charge were surface coated with cationic di-block copolymer, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL) conjugate, for enhancing their site-specific intracellular delivery against folate receptor overexpressing cancer cells. The PLGA nanoparticles coated with the conjugate were characterized in terms of size, surface charge, and change in surface composition by XPS. By employing the flow cytometry method and confocal image analysis, the extent of cellular uptake was comparatively evaluated under various conditions. PLL-PEG-FOL coated PLGA nanoparticles demonstrated far greater extent of cellular uptake to KB cells, suggesting that they were mainly taken up by folate receptor-mediated endocytosis. The enhanced cellular uptake was also observed even in the presence of serum proteins, possibly due to the densely seeded PEG chains. The PLL-PEG-FOL coated PLGA nanoparticles could be potentially applied for cancer cell targeted delivery of various therapeutic agents.

Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal Dominant Polycystic Kidney Disease

DTIC Science & Technology

2016-09-01

AWARD NUMBER: W81XWH-15-1-0419 TITLE: Cellular Energy Pathways as Novel Targets for the Therapy of Autosomal Dominant Polycystic Kidney Disease...Polycystic Kidney Disease 5b. GRANT NUMBER W81XWH-15-1-0419 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Michael J. Caplan, MD, PhD Kenneth...dominant polycystic kidney disease (ADPKD) is a common inherited disorder where patients, over the course of decades, develop large fluid filled

Deep Sequencing Reveals Direct Targets of Gammaherpesvirus-Induced mRNA Decay and Suggests That Multiple Mechanisms Govern Cellular Transcript Escape

PubMed Central

Clyde, Karen; Glaunsinger, Britt A.

2011-01-01

One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV68), is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5) have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq). Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection. PMID:21573023

Comparing the effects of mitochondrial targeted and localized antioxidants with cellular antioxidants in human skin cells exposed to UVA and hydrogen peroxide.

PubMed

Oyewole, Anne O; Wilmot, Marie-Claire; Fowler, Mark; Birch-Machin, Mark A

2014-01-01

Skin cancer and aging are linked to increased cellular reactive oxygen species (ROS), particularly following exposure to ultraviolet A (UVA) in sunlight. As mitochondria are the main source of cellular ROS, this study compared the protective effects of mitochondria-targeted and -localized antioxidants (MitoQ and tiron, respectively) with cellular antioxidants against oxidative stress-induced [UVA and hydrogen peroxide (H2O2)] mitochondrial DNA (mtDNA) damage in human dermal fibroblasts. With the use of a long quantitative PCR assay, tiron (EC50 10 mM) was found to confer complete (100%) protection (P<0.001) against both UVA- and H2O2-induced mtDNA damage, whereas MitoQ (EC50 750 nM) provided less protection (17 and 32%, respectively; P<0.05). This particular protective effect of tiron was greater than a range of cellular antioxidants investigated. The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway provides cellular protection against oxidative stress. An ELISA assay for the Nrf2 target gene heme oxygenase-1 (HO-1) and studies using Nrf2 small interfering RNA both indicated that tiron's mode of action was Nrf2 independent. The comet assay showed that tiron's protective effect against H2O2-induced nuclear DNA damage was greater than the cellular antioxidants and MitoQ (P<0.001). This study provides a platform to investigate molecules with similar structure to tiron as potent and clinically relevant antioxidants.

Chemical Proteomics Identifies Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1 as the Molecular Target of Quercetin in Its Anti-cancer Effects in PC-3 Cells*

PubMed Central

Ko, Chia-Chen; Chen, Yun-Ju; Chen, Chih-Ta; Liu, Yu-Chih; Cheng, Fong-Chi; Hsu, Kai-Chao; Chow, Lu-Ping

2014-01-01

Quercetin, a flavonoid abundantly present in plants, is widely used as a phytotherapy in prostatitis and prostate cancer. Although quercetin has been reported to have a number of therapeutic effects, the cellular target(s) responsible for its anti-cancer action has not yet been clearly elucidated. Here, employing affinity chromatography and mass spectrometry, we identified heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) as a direct target of quercetin. A specific interaction between quercetin and hnRNPA1 was validated by immunoblotting and in vitro binding experiments. We found that quercetin bound the C-terminal region of hnRNPA1, impairing the ability of hnRNPA1 to shuttle between the nucleus and cytoplasm and ultimately resulting in its cytoplasmic retention. In addition, hnRNPA1 was recruited to stress granules after treatment of cells with quercetin for up to 48 h, and the levels of cIAP1 (cellular inhibitor of apoptosis), an internal ribosome entry site translation-dependent protein, were reduced by hnRNPA1 regulation. This is the first report that anti-cancer effects of quercetin are mediated, in part, by impairing functions of hnRNPA1, insights that were obtained using a chemical proteomics strategy. PMID:24962584

A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast

DTIC Science & Technology

2004-05-01

AD Award Number: DAMD17-03-1-0232 TITLE: A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast PRINCIPAL INVESTIGATOR...Approach to Identify Novel Breast DAMD17-03-1-0232 Cancer Gene Targets in Yeast 6. A UTHOR(S) Craig Bennett, Ph.D. 7. PERFORMING ORGANIZA TION NAME(S...Unlimited 13. ABSTRACT (Maximum 200 Words) We are using the yeast Saccharomyces cerevisiae to identify new cancer gene targets that interact with the

Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

PubMed

Morohashi, Kengo; Sahara, Hiroeki; Watashi, Koichi; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

2011-04-29

Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

Cyclosporin A Associated Helicase-Like Protein Facilitates the Association of Hepatitis C Virus RNA Polymerase with Its Cellular Cyclophilin B

PubMed Central

Sahara, Hiroeki; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

2011-01-01

Background Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Principal Findings Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. Conclusions We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology. PMID:21559518

miR-300 regulates cellular radiosensitivity through targeting p53 and apaf1 in human lung cancer cells.

PubMed

He, Jinpeng; Feng, Xiu; Hua, Junrui; Wei, Li; Lu, Zhiwei; Wei, Wenjun; Cai, Hui; Wang, Bing; Shi, Wengui; Ding, Nan; Li, He; Zhang, Yanan; Wang, Jufang

2017-10-18

microRNAs (miRNAs) play a crucial role in mediation of the cellular sensitivity to ionizing radiation (IR). Previous studies revealed that miR-300 was involved in the cellular response to IR or chemotherapy drug. However, whether miR-300 could regulate the DNA damage responses induced by extrinsic genotoxic stress in human lung cancer and the underlying mechanism remain unknown. In this study, the expression of miR-300 was examined in lung cancer cells treated with IR, and the effects of miR-300 on DNA damage repair, cell cycle arrest, apoptosis and senescence induced by IR were investigated. It was found that IR induced upregulation of endogenous miR-300, and ectopic expression of miR-300 by transfected with miR-300 mimics not only greatly enhanced the cellular DNA damage repair ability but also substantially abrogated the G2 cell cycle arrest and apoptosis induced by IR. Bioinformatic analysis predicted that p53 and apaf1 were potential targets of miR-300, and the luciferase reporter assay showed that miR-300 significantly suppressed the luciferase activity through binding to the 3'-UTR of p53 or apaf1 mRNA. In addition, overexpression of miR-300 significantly reduced p53/apaf1 and/or IR-induced p53/apaf1 protein expression levels. Flow cytomertry analysis and colony formation assay showed that miR-300 desensitized lung cancer cells to IR by suppressing p53-dependent G2 cell cycle arrest, apoptosis and senescence. These data demonstrate that miR-300 regulates the cellular sensitivity to IR through targeting p53 and apaf1 in lung cancer cells.

Delivery of kinesin spindle protein targeting siRNA in solid lipid nanoparticles to cellular models of tumor vasculature

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

Ying, Bo; Campbell, Robert B., E-mail: robert.campbell@mcphs.edu

2014-04-04

Highlights: • siRNA-lipid nanoparticles are solid particles not lipid bilayers with aqueous core. • High, but not low, PEG content can prevent nanoparticle encapsulation of siRNA. • PEG reduces cellular toxicity of cationic nanoparticles in vitro. • PEG reduces zeta potential while improving gene silencing of siRNA nanoparticles. • Kinesin spindle protein can be an effective target for tumor vascular targeting. - Abstract: The ideal siRNA delivery system should selectively deliver the construct to the target cell, avoid enzymatic degradation, and evade uptake by phagocytes. In the present study, we evaluated the importance of polyethylene glycol (PEG) on lipid-based carriermore » systems for encapsulating, and delivering, siRNA to tumor vessels using cellular models. Lipid nanoparticles containing different percentage of PEG were evaluated based on their physical chemical properties, density compared to water, siRNA encapsulation, toxicity, targeting efficiency and gene silencing in vitro. siRNA can be efficiently loaded into lipid nanoparticles (LNPs) when DOTAP is included in the formulation mixture. However, the total amount encapsulated decreased with increase in PEG content. In the presence of siRNA, the final formulations contained a mixed population of particles based on density. The major population which contains the majority of siRNA exhibited a density of 4% glucose, and the minor fraction associated with a decreased amount of siRNA had a density less than PBS. The inclusion of 10 mol% PEG resulted in a greater amount of siRNA associated with the minor fraction. Finally, when kinesin spindle protein (KSP) siRNA was encapsulated in lipid nanoparticles containing a modest amount of PEG, the proliferation of endothelial cells was inhibited due to the efficient knock down of KSP mRNA. The presence of siRNA resulted in the formation of solid lipid nanoparticles when prepared using the thin film and hydration method. LNPs with a relatively modest

Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

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

Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.

2016-02-12

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative tomore » other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.« less

Using the CPTAC Assay Portal to Identify and Implement Highly Characterized Targeted Proteomics Assays.

PubMed

Whiteaker, Jeffrey R; Halusa, Goran N; Hoofnagle, Andrew N; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John A; Kennedy, Jacob; Mani, D R; Zimmerman, Lisa J; Meyer, Matthew R; Mesri, Mehdi; Boja, Emily; Carr, Steven A; Chan, Daniel W; Chen, Xian; Chen, Jing; Davies, Sherri R; Ellis, Matthew J C; Fenyö, David; Hiltke, Tara; Ketchum, Karen A; Kinsinger, Chris; Kuhn, Eric; Liebler, Daniel C; Liu, Tao; Loss, Michael; MacCoss, Michael J; Qian, Wei-Jun; Rivers, Robert; Rodland, Karin D; Ruggles, Kelly V; Scott, Mitchell G; Smith, Richard D; Thomas, Stefani; Townsend, R Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G

2016-01-01

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and posttranslational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

mTOR Regulates Cellular Iron Homeostasis through Tristetraprolin

PubMed Central

Bayeva, Marina; Khechaduri, Arineh; Puig, Sergi; Chang, Hsiang-Chun; Patial, Sonika; Blackshear, Perry J.; Ardehali, Hossein

2013-01-01

SUMMARY Iron is an essential cofactor with unique redox properties. Iron regulatory proteins 1 and 2 (IRP1/2) have been established as important regulators of cellular iron homeostasis, but little is known about the role of other pathways in this process. Here we report that the mammalian target of rapamycin (mTOR) regulates iron homeostasis by modulating transferrin receptor 1 (TfR1) stability and altering cellular iron flux. Mechanistic studies identify tristetraprolin (TTP), a protein involved in anti-inflammatory response, as the downstream target of mTOR that binds to and enhances degradation of TfR1 mRNA. We also show that TTP is strongly induced by iron chelation, promotes downregulation of iron-requiring genes in both mammalian and yeast cells, and modulates survival in low-iron states. Taken together, our data uncover a link between metabolic, inflammatory, and iron regulatory pathways, and point towards the existence of a yeast-like TTP-mediated iron conservation program in mammals. PMID:23102618

Quantum dot tailored to single wall carbon nanotubes: a multifunctional hybrid nanoconstruct for cellular imaging and targeted photothermal therapy.

PubMed

Nair, Lakshmi V; Nagaoka, Yutaka; Maekawa, Toru; Sakthikumar, D; Jayasree, Ramapurath S

2014-07-23

Hybrid nanomaterial based on quantum dots and SWCNTs is used for cellular imaging and photothermal therapy. Furthermore, the ligand conjugated hybrid system (FaQd@CNT) enables selective targeting in cancer cells. The imaging capability of quantum dots and the therapeutic potential of SWCNT are available in a single system with cancer targeting property. Heat generated by the system is found to be high enough to destroy cancer cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human T-cell leukemia virus-I tax oncoprotein functionally targets a subnuclear complex involved in cellular DNA damage-response.

PubMed

Haoudi, Abdelali; Daniels, Rodney C; Wong, Eric; Kupfer, Gary; Semmes, O John

2003-09-26

The virally encoded oncoprotein Tax has been implicated in HTLV-1-mediated cellular transformation. The exact mechanism by which this protein contributes to the oncogenic process is not known. However, it has been hypothesized that Tax induces genomic instability via repression of cellular DNA repair. We examined the effect of de novo Tax expression upon the cell cycle, because appropriate activation of cell cycle checkpoints is essential to a robust damage-repair response. Upon induction of tax expression, Jurkat T-cells displayed a pronounced accumulation in G2/M that was reversible by caffeine. We examined the G2-specific checkpoint signaling response in these cells and found activation of the ATM/chk2-mediated pathway, whereas the ATR/chk1-mediated response was unaffected. Immunoprecipitation with anti-chk2 antibody results in co-precipitation of Tax demonstrating a direct interaction of Tax with a chk2-containing complex. We also show that Tax targets a discrete nuclear site and co-localizes with chk2 and not chk1. This nuclear site, previously identified as Tax Speckled Structures (TSS), also contains the early damage response factor 53BP1. The recruitment of 53BP1 to TSS is dependent upon ATM signaling and requires expression of Tax. Specifically, Tax expression induces redistribution of diffuse nuclear 53BP1 to the TSS foci. Taken together these data suggest that the TSS describe a unique nuclear site involved in DNA damage recognition, repair response, and cell cycle checkpoint activation. We suggest that association of Tax with this multifunctional subnuclear site results in disruption of a subset of the site-specific activities and contributes to cellular genomic instability.

Target identification by image analysis.

PubMed

Fetz, V; Prochnow, H; Brönstrup, M; Sasse, F

2016-05-04

Covering: 1997 to the end of 2015Each biologically active compound induces phenotypic changes in target cells that are characteristic for its mode of action. These phenotypic alterations can be directly observed under the microscope or made visible by labelling structural elements or selected proteins of the cells with dyes. A comparison of the cellular phenotype induced by a compound of interest with the phenotypes of reference compounds with known cellular targets allows predicting its mode of action. While this approach has been successfully applied to the characterization of natural products based on a visual inspection of images, recent studies used automated microscopy and analysis software to increase speed and to reduce subjective interpretation. In this review, we give a general outline of the workflow for manual and automated image analysis, and we highlight natural products whose bacterial and eucaryotic targets could be identified through such approaches.

Recurrent Targeted Genes of Hepatitis B Virus in the Liver Cancer Genomes Identified by a Next-Generation Sequencing–Based Approach

PubMed Central

Ding, Dong; Lou, Xiaoyan; Hua, Dasong; Yu, Wei; Li, Lisha; Wang, Jun; Gao, Feng; Zhao, Na; Ren, Guoping; Li, Lanjuan; Lin, Biaoyang

2012-01-01

Integration of the viral DNA into host chromosomes was found in most of the hepatitis B virus (HBV)–related hepatocellular carcinomas (HCCs). Here we devised a massive anchored parallel sequencing (MAPS) method using next-generation sequencing to isolate and sequence HBV integrants. Applying MAPS to 40 pairs of HBV–related HCC tissues (cancer and adjacent tissues), we identified 296 HBV integration events corresponding to 286 unique integration sites (UISs) with precise HBV–Human DNA junctions. HBV integration favored chromosome 17 and preferentially integrated into human transcript units. HBV targeted genes were enriched in GO terms: cAMP metabolic processes, T cell differentiation and activation, TGF beta receptor pathway, ncRNA catabolic process, and dsRNA fragmentation and cellular response to dsRNA. The HBV targeted genes include 7 genes (PTPRJ, CNTN6, IL12B, MYOM1, FNDC3B, LRFN2, FN1) containing IPR003961 (Fibronectin, type III domain), 7 genes (NRG3, MASP2, NELL1, LRP1B, ADAM21, NRXN1, FN1) containing IPR013032 (EGF-like region, conserved site), and three genes (PDE7A, PDE4B, PDE11A) containing IPR002073 (3′, 5′-cyclic-nucleotide phosphodiesterase). Enriched pathways include hsa04512 (ECM-receptor interaction), hsa04510 (Focal adhesion), and hsa04012 (ErbB signaling pathway). Fewer integration events were found in cancers compared to cancer-adjacent tissues, suggesting a clonal expansion model in HCC development. Finally, we identified 8 genes that were recurrent target genes by HBV integration including fibronectin 1 (FN1) and telomerase reverse transcriptase (TERT1), two known recurrent target genes, and additional novel target genes such as SMAD family member 5 (SMAD5), phosphatase and actin regulator 4 (PHACTR4), and RNA binding protein fox-1 homolog (C. elegans) 1 (RBFOX1). Integrating analysis with recently published whole-genome sequencing analysis, we identified 14 additional recurrent HBV target genes, greatly expanding the HBV recurrent

Plant-Derived Polyphenols in Human Health: Biological Activity, Metabolites and Putative Molecular Targets.

PubMed

Olivares-Vicente, Marilo; Barrajon-Catalan, Enrique; Herranz-Lopez, Maria; Segura-Carretero, Antonio; Joven, Jorge; Encinar, Jose Antonio; Micol, Vicente

2018-01-01

Hibiscus sabdariffa, Lippia citriodora, Rosmarinus officinalis and Olea europaea, are rich in bioactive compounds that represent most of the phenolic compounds' families and have exhibited potential benefits in human health. These plants have been used in folk medicine for their potential therapeutic properties in human chronic diseases. Recent evidence leads to postulate that polyphenols may account for such effects. Nevertheless, the compounds or metabolites that are responsible for reaching the molecular targets are unknown. data based on studies directly using complex extracts on cellular models, without considering metabolic aspects, have limited applicability. In contrast, studies exploring the absorption process, metabolites in the blood circulation and tissues have become essential to identify the intracellular final effectors that are responsible for extracts bioactivity. Once the cellular metabolites are identified using high-resolution mass spectrometry, docking techniques suppose a unique tool for virtually screening a large number of compounds on selected targets in order to elucidate their potential mechanisms. we provide an updated overview of the in vitro and in vivo studies on the toxicity, absorption, permeability, pharmacokinetics and cellular metabolism of bioactive compounds derived from the abovementioned plants to identify the potential compounds that are responsible for the observed health effects. we propose the use of targeted metabolomics followed by in silico studies to virtually screen identified metabolites on selected protein targets, in combination with the use of the candidate metabolites in cellular models, as the methods of choice for elucidating the molecular mechanisms of these compounds. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Double activity imaging reveals distinct cellular targets of haloperidol, clozapine and dopamine D(3) receptor selective RGH-1756.

PubMed

Kovács, K J; Csejtei, M; Laszlovszky, I

2001-03-01

Acute administration of typical (haloperidol) and atypical (clozapine) antipsychotics results in distinct and overlapping regions of immediate-early gene expression in the rat brain. RGH-1756 is a recently developed atypical antipsychotic with high affinity to dopamine D(3) receptors that results in a unique pattern of c-Fos induction. A single injection of either antipsychotic results in c-fos mRNA expression that peaks around 30 min after drug administration, while the maximum of c-Fos protein induction is seen 2 h after challenge. The transient and distinct temporal inducibility of c-fos mRNA and c-Fos protein was exploited to reveal and compare cellular targets of different antipsychotic drugs by concomitant localization of c-fos mRNA and c-Fos immunoreactivity in brain sections of rats that were timely challenged with two different antipsychotics. Double activity imaging revealed that haloperidol, clozapine and RGH-1756 share cellular targets in the nucleus accumbens, where 40% of all labeled neurons displayed both c-fos mRNA and c-Fos protein. Haloperidol activates cells in the caudate putamen, while clozapine-responsive, single labeled neurons were dominant in the prefrontal cortex and major island of Calleja. RGH-1756 targets haloperidol-sensitive cells in the caudate putamen, but cells that are activated by clozapine and RGH-1756 in the major island of Calleja are different.

Affinity resins as new tools for identifying target proteins of ascorbic acid.

PubMed

Iwaoka, Yuji; Nishino, Kohei; Ishikawa, Takahiro; Ito, Hideyuki; Sawa, Yoshihiro; Tai, Akihiro

2018-02-12

l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

Functional kinomics identifies candidate therapeutic targets in head and neck cancer

PubMed Central

Moser, Russell; Xu, Chang; Kao, Michael; Annis, James; Lerma, Luisa Angelica; Schaupp, Christopher M.; Gurley, Kay E.; Jang, In Sock; Biktasova, Asel; Yarbrough, Wendell G.; Margolin, Adam A.; Grandori, Carla; Kemp, Christopher J.; Méndez, Eduardo

2014-01-01

Purpose To identify novel therapeutic drug targets for p53 mutant head and neck squamous cell carcinoma (HNSCC). Experimental Design RNAi kinome viability screens were performed on HNSCC cells including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19Arf. Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was utilized to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets utilizing multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition utilizing a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Results Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2/M cell cycle checkpoint, SFK, PI3K and FAK pathways. RNAi mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53 mutant HNSCC xenograft model. Conclusions WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. PMID:25125259

Functional kinomics identifies candidate therapeutic targets in head and neck cancer.

PubMed

Moser, Russell; Xu, Chang; Kao, Michael; Annis, James; Lerma, Luisa Angelica; Schaupp, Christopher M; Gurley, Kay E; Jang, In Sock; Biktasova, Asel; Yarbrough, Wendell G; Margolin, Adam A; Grandori, Carla; Kemp, Christopher J; Méndez, Eduardo

2014-08-15

To identify novel therapeutic drug targets for p53-mutant head and neck squamous cell carcinoma (HNSCC). RNAi kinome viability screens were performed on HNSCC cells, including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19(Arf). Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was used to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets using multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition using a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2-M cell-cycle checkpoint, SFK, PI3K, and FAK pathways. RNAi-mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53-mutant HNSCC xenograft model. WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. ©2014 American Association for Cancer Research.

Initial targets and cellular responses to PDT

NASA Astrophysics Data System (ADS)

Rodriguez, Myriam E.; Azizuddin, Kashif; Chiu, Song-mao; Delos Santos, Grace; Joseph, Sheeba; Xue, Liang-yan; Oleinick, Nancy L.

2007-02-01

Pc 4, a photosensitizer first synthesized at Case Western Reserve University and now in clinical trial at University Hospitals of Cleveland, has been shown to bind preferentially and with high affinity to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components are photodamaged. In most cancer cells, apoptosis is triggered by the initial photodamage; however, in cells deficient in one of the critical intermediates of apoptosis, this process does not occur, although the cells remain as sensitive to the lethal effects of Pc 4-PDT as the apoptosis-competent cells, when cell death is determined by colony formation. Here we report that an alternative death process, autophagy, is induced in all cells tested and becomes the dominant pathway for elimination of lethally damaged cells when apoptosis is compromised. The anti-apoptotic protein Bcl-2, when overexpressed, protects only apoptosis-competent cells against loss of clonogenicity, while the autophagy inhibitor 3-methyladenine provides a markedly greater protection to apoptosis-deficient cells. The results suggest that the primary determinant of cell death is not the final pathway for elimination of the cells but the initial photodamage to critical membrane targets. In attempts to identify those targets, we have studied the role of different membrane phospholipids in the localization of Pc 4. Cardiolipin (CL) is a phospholipid found exclusively in the mitochondrial inner membrane and at the contact sites between the inner and outer membranes. Previous fluorescence resonance energy transfer studies revealed colocalization of Pc 4 and CL, which points to CL as a possible binding site and target for Pc 4. Unilamellar liposomes with different lipid compositions were used as membrane models to test the affinity of Pc 4. As revealed by the binding constants, Pc 4 does not display preferential binding to CL in these systems. Moreover, binding affinities appear to be

Fine tuning cellular recognition: The function of the leucine rich repeat (LRR) trans-membrane protein, LRT, in muscle targeting to tendon cells.

PubMed

Gilsohn, Eli; Volk, Talila

2010-01-01

The formation of complex tissues during embryonic development is often accompanied by directed cellular migration towards a target tissue. Specific mutual recognition between the migrating cell and its target tissue leads to the arrest of the cell migratory behavior and subsequent contact formation between the two interacting cell types. Recent studies implicated a novel family of surface proteins containing a trans-membrane domain and single leucine-rich repeat (LRR) domain in inter-cellular recognition and the arrest of cell migration. Here, we describe the involvement of a novel LRR surface protein, LRT, in targeting migrating muscles towards their corresponding tendon cells in the Drosophila embryo. LRT is specifically expressed by the target tendon cells and is essential for arresting the migratory behavior of the muscle cells. Additional studies in Drosophila S2 cultured cells suggest that LRT forms a protein complex with the Roundabout (Robo) receptor, essential for guiding muscles towards their tendon partners. Genetic analysis supports a model in which LRT performs its activity non-autonomously through its interaction with the Robo receptors expressed on the muscle surfaces. These results suggest a novel mechanism of intercellular recognition through interactions between LRR family members and Robo receptors.

Biased and unbiased strategies to identify biologically active small molecules.

PubMed

Abet, Valentina; Mariani, Angelica; Truscott, Fiona R; Britton, Sébastien; Rodriguez, Raphaël

2014-08-15

Small molecules are central players in chemical biology studies. They promote the perturbation of cellular processes underlying diseases and enable the identification of biological targets that can be validated for therapeutic intervention. Small molecules have been shown to accurately tune a single function of pluripotent proteins in a reversible manner with exceptional temporal resolution. The identification of molecular probes and drugs remains a worthy challenge that can be addressed by the use of biased and unbiased strategies. Hypothesis-driven methodologies employs a known biological target to synthesize complementary hits while discovery-driven strategies offer the additional means of identifying previously unanticipated biological targets. This review article provides a general overview of recent synthetic frameworks that gave rise to an impressive arsenal of biologically active small molecules with unprecedented cellular mechanisms. Copyright © 2014. Published by Elsevier Ltd.

Identification of KasA as the cellular target of an anti-tubercular scaffold

PubMed Central

Abrahams, Katherine A.; Chung, Chun-wa; Ghidelli-Disse, Sonja; Rullas, Joaquín; Rebollo-López, María José; Gurcha, Sudagar S.; Cox, Jonathan A. G.; Mendoza, Alfonso; Jiménez-Navarro, Elena; Martínez-Martínez, María Santos; Neu, Margarete; Shillings, Anthony; Homes, Paul; Argyrou, Argyrides; Casanueva, Ruth; Loman, Nicholas J.; Moynihan, Patrick J.; Lelièvre, Joël; Selenski, Carolyn; Axtman, Matthew; Kremer, Laurent; Bantscheff, Marcus; Angulo-Barturen, Iñigo; Izquierdo, Mónica Cacho; Cammack, Nicholas C.; Drewes, Gerard; Ballell, Lluis; Barros, David; Besra, Gurdyal S.; Bates, Robert H.

2016-01-01

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis. PMID:27581223

A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies.

PubMed

Vij, Rajesh; Lin, Zhonghua; Chiang, Nancy; Vernes, Jean-Michel; Storek, Kelly M; Park, Summer; Chan, Joyce; Meng, Y Gloria; Comps-Agrar, Laetitia; Luan, Peng; Lee, Sophia; Schneider, Kellen; Bevers, Jack; Zilberleyb, Inna; Tam, Christine; Koth, Christopher M; Xu, Min; Gill, Avinash; Auerbach, Marcy R; Smith, Peter A; Rutherford, Steven T; Nakamura, Gerald; Seshasayee, Dhaya; Payandeh, Jian; Koerber, James T

2018-05-08

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP β-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a "targeted boost-and-sort" strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of β-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.

Using Click Chemistry to Identify Potential Drug Targets in Plasmodium

DTIC Science & Technology

2015-04-01

step of the Plasmodium mammalian cycle . Inhibiting this step can block malaria at an early step. However, few anti-malarials target liver infection...points in the life cycle of malaria parasites. PLoS Biol 12: e1001806. 2. Falae A, Combe A, Amaladoss A, Carvalho T, Menard R, et al. (2010) Role of...AWARD NUMBER: W81XWH-13-1-0429 TITLE: Using "Click Chemistry" to Identify Potential Drug Targets in Plasmodium PRINCIPAL INVESTIGATOR: Dr. Purnima

Identifying protein kinase target preferences using mass spectrometry

PubMed Central

Douglass, Jacqueline; Gunaratne, Ruwan; Bradford, Davis; Saeed, Fahad; Hoffert, Jason D.; Steinbach, Peter J.; Pisitkun, Trairak

2012-01-01

A general question in molecular physiology is how to identify candidate protein kinases corresponding to a known or hypothetical phosphorylation site in a protein of interest. It is generally recognized that the amino acid sequence surrounding the phosphorylation site provides information that is relevant to identification of the cognate protein kinase. Here, we present a mass spectrometry-based method for profiling the target specificity of a given protein kinase as well as a computational tool for the calculation and visualization of the target preferences. The mass spectrometry-based method identifies sites phosphorylated in response to in vitro incubation of protein mixtures with active recombinant protein kinases followed by standard phosphoproteomic methodologies. The computational tool, called “PhosphoLogo,” uses an information-theoretic algorithm to calculate position-specific amino acid preferences and anti-preferences from the mass-spectrometry data (http://helixweb.nih.gov/PhosphoLogo/). The method was tested using protein kinase A (catalytic subunit α), revealing the well-known preference for basic amino acids in positions −2 and −3 relative to the phosphorylated amino acid. It also provides evidence for a preference for amino acids with a branched aliphatic side chain in position +1, a finding compatible with known crystal structures of protein kinase A. The method was also employed to profile target preferences and anti-preferences for 15 additional protein kinases with potential roles in regulation of epithelial transport: CK2, p38, AKT1, SGK1, PKCδ, CaMK2δ, DAPK1, MAPKAPK2, PKD3, PIM1, OSR1, STK39/SPAK, GSK3β, Wnk1, and Wnk4. PMID:22723110

Mitochondria targeting by environmental stressors: Implications for redox cellular signaling.

PubMed

Blajszczak, Chuck; Bonini, Marcelo G

2017-11-01

Mitochondria are cellular powerhouses as well as metabolic and signaling hubs regulating diverse cellular functions, from basic physiology to phenotypic fate determination. It is widely accepted that reactive oxygen species (ROS) generated in mitochondria participate in the regulation of cellular signaling, and that some mitochondria chronically operate at a high ROS baseline. However, it is not completely understood how mitochondria adapt to persistently high ROS states and to environmental stressors that disturb the redox balance. Here we will review some of the current concepts regarding how mitochondria resist oxidative damage, how they are replaced when excessive oxidative damage compromises function, and the effect of environmental toxicants (i.e. heavy metals) on the regulation of mitochondrial ROS (mtROS) production and subsequent impact. Copyright © 2017 Elsevier B.V. All rights reserved.

Integrative biology approach identifies cytokine targeting strategies for psoriasis.

PubMed

Perera, Gayathri K; Ainali, Chrysanthi; Semenova, Ekaterina; Hundhausen, Christian; Barinaga, Guillermo; Kassen, Deepika; Williams, Andrew E; Mirza, Muddassar M; Balazs, Mercedesz; Wang, Xiaoting; Rodriguez, Robert Sanchez; Alendar, Andrej; Barker, Jonathan; Tsoka, Sophia; Ouyang, Wenjun; Nestle, Frank O

2014-02-12

Cytokines are critical checkpoints of inflammation. The treatment of human autoimmune disease has been revolutionized by targeting inflammatory cytokines as key drivers of disease pathogenesis. Despite this, there exist numerous pitfalls when translating preclinical data into the clinic. We developed an integrative biology approach combining human disease transcriptome data sets with clinically relevant in vivo models in an attempt to bridge this translational gap. We chose interleukin-22 (IL-22) as a model cytokine because of its potentially important proinflammatory role in epithelial tissues. Injection of IL-22 into normal human skin grafts produced marked inflammatory skin changes resembling human psoriasis. Injection of anti-IL-22 monoclonal antibody in a human xenotransplant model of psoriasis, developed specifically to test potential therapeutic candidates, efficiently blocked skin inflammation. Bioinformatic analysis integrating both the IL-22 and anti-IL-22 cytokine transcriptomes and mapping them onto a psoriasis disease gene coexpression network identified key cytokine-dependent hub genes. Using knockout mice and small-molecule blockade, we show that one of these hub genes, the so far unexplored serine/threonine kinase PIM1, is a critical checkpoint for human skin inflammation and potential future therapeutic target in psoriasis. Using in silico integration of human data sets and biological models, we were able to identify a new target in the treatment of psoriasis.

Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches

PubMed Central

Ekland, Eric H.; Schneider, Jessica; Fidock, David A.

2011-01-01

Malarial parasites have evolved resistance to all previously used therapies, and recent evidence suggests emerging resistance to the first-line artemisinins. To identify antimalarials with novel mechanisms of action, we have developed a high-throughput screen targeting the apicoplast organelle of Plasmodium falciparum. Antibiotics known to interfere with this organelle, such as azithromycin, exhibit an unusual phenotype whereby the progeny of drug-treated parasites die. Our screen exploits this phenomenon by assaying for “delayed death” compounds that exhibit a higher potency after two cycles of intraerythrocytic development compared to one. We report a primary assay employing parasites with an integrated copy of a firefly luciferase reporter gene and a secondary flow cytometry-based assay using a nucleic acid stain paired with a mitochondrial vital dye. Screening of the U.S. National Institutes of Health Clinical Collection identified known and novel antimalarials including kitasamycin. This inexpensive macrolide, used for agricultural applications, exhibited an in vitro IC50 in the 50 nM range, comparable to the 30 nM activity of our control drug, azithromycin. Imaging and pharmacologic studies confirmed kitasamycin action against the apicoplast, and in vivo activity was observed in a murine malaria model. These assays provide the foundation for high-throughput campaigns to identify novel chemotypes for combination therapies to treat multidrug-resistant malaria.—Ekland, E. H., Schneider, J., Fidock, D. A. Identifying apicoplast-targeting antimalarials using high-throughput compatible approaches. PMID:21746861

Regulation of cellular senescence by the essential caveolar component PTRF/Cavin-1

PubMed Central

Bai, Lin; Deng, Xiaoli; Li, Juanjuan; Wang, Miao; Li, Qian; An, Wei; A, Deli; Cong, Yu-Sheng

2011-01-01

Polymerase I and transcript release factor (PTRF, also known as Cavin-1) is an essential component in the biogenesis and function of caveolae. Here, we show that PTRF expression is increased in senescent human fibroblasts. Importantly, overexpression of PTRF induced features characteristic of cellular senescence, whereas reduced PTRF expression extended the cellular replicative lifespan. Interestingly, we found that PTRF localized primarily to the nuclei of young and quiescent WI-38 human fibroblasts, but translocated to the cytosol and plasma membrane during cellular senescence. Furthermore, electron microscopic analysis demonstrated an increased number of caveolar structures in senescent and PTRF-transfected WI-38 cells. Our data suggest that the role of PTRF in cellular senescence is dependent on its targeting to caveolae and its interaction with caveolin-1, which appeared to be regulated by the phosphorylation of PTRF. Taken together, our findings identify PTRF as a novel regulator of cellular senescence that acts through the p53/p21 and caveolar pathways. PMID:21445100

A pharmacologically-based array to identify targets of cyclosporine A-induced toxicity in cultured renal proximal tubule cells

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

Sarró, Eduard, E-mail: eduard.sarro@vhir.org; Renal Physiopathology, CIBBIM-Nanomedicine, Vall d'Hebron Research Institute; Jacobs-Cachá, Conxita, E-mail: conxita.jacobs@vhir.org

2012-01-15

Mechanisms of cyclosporine A (CsA)-induced nephrotoxicity were generally thought to be hemodynamic in origin; however, there is now accumulating evidence of a direct tubular effect. Although genomic and proteomic experiments by our group and others provided overall information on genes and proteins up- or down-regulated by CsA in proximal tubule cells (PTC), a comprehensive view of events occurring after CsA exposure remains to be described. For this purpose, we applied a pharmacologic approach based on the use of known activities of a large panel of potentially protective compounds and evaluated their efficacy in preventing CsA toxicity in cultured mouse PTC.more » Our results show that compounds that blocked protein synthesis and apoptosis, together with the CK2 inhibitor DMAT and the PI3K inhibitor apigenin, were the most efficient in preventing CsA toxicity. We also identified GSK3, MMPs and PKC pathways as potential targets to prevent CsA damage. Additionally, heparinase-I and MAPK inhibitors afforded partial but significant protection. Interestingly, antioxidants and calcium metabolism-related compounds were unable to ameliorate CsA-induced cytotoxicity. Subsequent experiments allowed us to clarify the hierarchical relationship of targeted pathways after CsA treatment, with ER stress identified as an early effector of CsA toxicity, which leads to ROS generation, phenotypical changes and cell death. In summary, this work presents a novel experimental approach to characterizing cellular responses to cytotoxics while pointing to new targets to prevent CsA-induced toxicity in proximal tubule cells. Highlights: ► We used a novel pharmacological approach to elucidate cyclosporine (CsA) toxicity. ► The ability of a broad range of compounds to prevent CsA toxicity was evaluated. ► CsA toxicity was monitored using LDH release assay and PARP cleavage. ► Protein synthesis, PI3K, GSK3, MMP, PKC and caspase inhibitors prevented CsA toxicity. ► We also

Metal complexes of curcumin for cellular imaging, targeting, and photoinduced anticancer activity.

PubMed

Banerjee, Samya; Chakravarty, Akhil R

2015-07-21

Curcumin is a polyphenolic species. As an active ingredient of turmeric, it is well-known for its traditional medicinal properties. The therapeutic values include antioxidant, anti-inflammatory, antiseptic, and anticancer activity with the last being primarily due to inhibition of the transcription factor NF-κB besides affecting several biological pathways to arrest tumor growth and its progression. Curcumin with all these positive qualities has only remained a potential candidate for cancer treatment over the years without seeing any proper usage because of its hydrolytic instability involving the diketo moiety in a cellular medium and its poor bioavailability. The situation has changed considerably in recent years with the observation that curcumin in monoanionic form could be stabilized on binding to a metal ion. The reports from our group and other groups have shown that curcumin in the metal-bound form retains its therapeutic potential. This has opened up new avenues to develop curcumin-based metal complexes as anticancer agents. Zinc(II) complexes of curcumin are shown to be stable in a cellular medium. They display moderate cytotoxicity against prostate cancer and neuroblastoma cell lines. A similar stabilization and cytotoxic effect is reported for (arene)ruthenium(II) complexes of curcumin against a variety of cell lines. The half-sandwich 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane (RAPTA)-type ruthenium(II) complexes of curcumin are shown to be promising cytotoxic agents with low micromolar concentrations for a series of cancer cell lines. In a different approach, cobalt(III) complexes of curcumin are used for its cellular delivery in hypoxic tumor cells using intracellular agents that reduce the metal and release curcumin as a cytotoxin. Utilizing the photophysical and photochemical properties of the curcumin dye, we have designed and synthesized photoactive curcumin metal complexes that are used for cellular imaging by fluorescence microscopy and

Use of an activated beta-catenin to identify Wnt pathway target genes in caenorhabditis elegans, including a subset of collagen genes expressed in late larval development.

PubMed

Jackson, Belinda M; Abete-Luzi, Patricia; Krause, Michael W; Eisenmann, David M

2014-04-16

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

The cellular prion protein identifies bipotential cardiomyogenic progenitors.

PubMed

Hidaka, Kyoko; Shirai, Manabu; Lee, Jong-Kook; Wakayama, Takanari; Kodama, Itsuo; Schneider, Michael D; Morisaki, Takayuki

2010-01-08

The paucity of specific surface markers for cardiomyocytes and their progenitors has impeded the development of embryonic or pluripotent stem cell-based transplantation therapy. Identification of relevant surface markers may also enhance our understanding of the mechanisms underlying differentiation. Here, we show that cellular prion protein (PrP) serves as an effective surface marker for isolating nascent cardiomyocytes as well as cardiomyogenic progenitors. Embryonic stem (or embryo-derived) cells were analyzed using flow cytometry to detect surface expression of PrP and intracellular myosin heavy chain (Myhc) proteins. Sorted cells were then analyzed for their differentiation potential. PrP+ cells from beating embryoid bodies (EBs) frequently included nascent Myhc+ cardiomyocytes. Cultured PrP+ cells further differentiated, giving rise to cardiac troponin I+ definitive cardiomyocytes with either an atrial or a ventricular identity. These cells were electrophysiologically functional and able to survive in vivo after transplantation. Combining PrP with a second marker, platelet-derived growth factor receptor (PDGFR)alpha, enabled us to identify an earlier cardiomyogenic population from prebeating EBs, the PrP+PDGFRalpha+ (PRa) cells. The Myhc- PRa cells expressed cardiac transcription factors, such as Nkx2.5, T-box transcription factor 5, and Isl1 (islet LIM homeobox 1), although they were not completely committed. In mouse embryos, PRa cells in cardiac crescent at the 1 to 2 somite stage were Myhc+, whereas they were Myhc- at headfold stages. PRa cells clonally expanded in methlycellulose cultures. Furthermore, single Myhc- PRa cell-derived colonies contained both cardiac and smooth muscle cells. Thus, PrP demarcates a population of bipotential cardiomyogenic progenitor cells that can differentiate into cardiac or smooth muscle cells.

N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection.

PubMed

Jagdeo, Julienne M; Dufour, Antoine; Klein, Theo; Solis, Nestor; Kleifeld, Oded; Kizhakkedathu, Jayachandran; Luo, Honglin; Overall, Christopher M; Jan, Eric

2018-04-15

Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed t erminal a mine i sotopic l abeling of s ubstrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3C pro s) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3C pro in vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3C pro -targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3C pro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3C pro substrates in vivo , we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner. IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection

N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection

PubMed Central

Jagdeo, Julienne M.; Dufour, Antoine; Klein, Theo; Solis, Nestor; Kleifeld, Oded; Kizhakkedathu, Jayachandran; Luo, Honglin; Overall, Christopher M.

2018-01-01

ABSTRACT Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed terminal amine isotopic labeling of substrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3Cpros) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3Cpro in vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3Cpro-targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3Cpro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3Cpro substrates in vivo, we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner. IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection

Macrophages with cellular backpacks for targeted drug delivery to the brain.

PubMed

Klyachko, Natalia L; Polak, Roberta; Haney, Matthew J; Zhao, Yuling; Gomes Neto, Reginaldo J; Hill, Michael C; Kabanov, Alexander V; Cohen, Robert E; Rubner, Michael F; Batrakova, Elena V

2017-09-01

Most potent therapeutics are unable to cross the blood-brain barrier following systemic administration, which necessitates the development of unconventional, clinically applicable drug delivery systems. With the given challenges, biologically active vehicles are crucial to accomplishing this task. We now report a new method for drug delivery that utilizes living cells as vehicles for drug carriage across the blood brain barrier. Cellular backpacks, 7-10 μm diameter polymer patches of a few hundred nanometers in thickness, are a potentially interesting approach, because they can act as drug depots that travel with the cell-carrier, without being phagocytized. Backpacks loaded with a potent antioxidant, catalase, were attached to autologous macrophages and systemically administered into mice with brain inflammation. Using inflammatory response cells enabled targeted drug transport to the inflamed brain. Furthermore, catalase-loaded backpacks demonstrated potent therapeutic effects deactivating free radicals released by activated microglia in vitro. This approach for drug carriage and release can accelerate the development of new drug formulations for all the neurodegenerative disorders. Copyright © 2017. Published by Elsevier Ltd.

Anti-EGFR Targeted Monoclonal Antibody Isotype Influences Antitumor Cellular Immunity in Head and Neck Cancer Patients.

PubMed

Trivedi, Sumita; Srivastava, Raghvendra M; Concha-Benavente, Fernando; Ferrone, Soldano; Garcia-Bates, Tatiana M; Li, Jing; Ferris, Robert L

2016-11-01

EGF receptor (EGFR) is highly overexpressed on several cancers and two targeted anti-EGFR antibodies which differ by isotype are FDA-approved for clinical use. Cetuximab (IgG1 isotype) inhibits downstream signaling of EGFR and activates antitumor, cellular immune mechanisms. As panitumumab (IgG2 isotype) may inhibit downstream EGFR signaling similar to cetuximab, it might also induce adaptive immunity. We measured in vitro activation of cellular components of the innate and adaptive immune systems. We also studied the in vivo activation of components of the adaptive immune system in patient specimens from two recent clinical trials using cetuximab or panitumumab. Both monoclonal antibodies (mAb) primarily activate natural killer (NK) cells, although cetuximab is significantly more potent than panitumumab. Cetuximab-activated neutrophils mediate antibody-dependent cellular cytotoxicity (ADCC) against head and neck squamous cell carcinomas (HNSCC) tumor cells, and interestingly, this effect was FcγRIIa- and FcγRIIIa genotype-dependent. Panitumumab may activate monocytes through CD32 (FcγRIIa); however, monocytes activated by either mAb are not able to mediate ADCC. Cetuximab enhanced dendritic cell (DC) maturation to a greater extent than panitumumab, which was associated with improved tumor antigen cross-presentation by cetuximab compared with panitumumab. This correlated with increased EGFR-specific cytotoxic CD8 + T cells in patients treated with cetuximab compared with those treated with panitumumab. Although panitumumab effectively inhibits EGFR signaling to a similar extent as cetuximab, it is less effective at triggering antitumor, cellular immune mechanisms which may be crucial for effective therapy of HNSCC. Clin Cancer Res; 22(21); 5229-37. ©2016 AACR. ©2016 American Association for Cancer Research.

Patterns of HIV-1 Protein Interaction Identify Perturbed Host-Cellular Subsystems

PubMed Central

MacPherson, Jamie I.; Dickerson, Jonathan E.; Pinney, John W.; Robertson, David L.

2010-01-01

Human immunodeficiency virus type 1 (HIV-1) exploits a diverse array of host cell functions in order to replicate. This is mediated through a network of virus-host interactions. A variety of recent studies have catalogued this information. In particular the HIV-1, Human Protein Interaction Database (HHPID) has provided a unique depth of protein interaction detail. However, as a map of HIV-1 infection, the HHPID is problematic, as it contains curation error and redundancy; in addition, it is based on a heterogeneous set of experimental methods. Based on identifying shared patterns of HIV-host interaction, we have developed a novel methodology to delimit the core set of host-cellular functions and their associated perturbation from the HHPID. Initially, using biclustering, we identify 279 significant sets of host proteins that undergo the same types of interaction. The functional cohesiveness of these protein sets was validated using a human protein-protein interaction network, gene ontology annotation and sequence similarity. Next, using a distance measure, we group host protein sets and identify 37 distinct higher-level subsystems. We further demonstrate the biological significance of these subsystems by cross-referencing with global siRNA screens that have been used to detect host factors necessary for HIV-1 replication, and investigate the seemingly small intersect between these data sets. Our results highlight significant host-cell subsystems that are perturbed during the course of HIV-1 infection. Moreover, we characterise the patterns of interaction that contribute to these perturbations. Thus, our work disentangles the complex set of HIV-1-host protein interactions in the HHPID, reconciles these with siRNA screens and provides an accessible and interpretable map of infection. PMID:20686668

Molecular imaging with targeted perfluorocarbon nanoparticles: Quantification of the concentration dependence of contrast enhancement for binding to sparse cellular epitopes

PubMed Central

Marsh, Jon N.; Partlow, Kathryn C.; Abendschein, Dana R.; Scott, Michael J.; Lanza, Gregory M.; Wickline, Samuel A.

2007-01-01

Targeted, liquid perfluorocarbon nanoparticles are effective agents for acoustic contrast enhancement of abundant cellular epitopes (e.g. fibrin in thrombi) and for lower prevalence binding sites, such as integrins associated with tumor neovasculature. In this study we sought to delineate the quantitative relationship between the extent of contrast enhancement of targeted surfaces and the density (and concentration) of bound perfluorocarbon (PFC) nanoparticles. Two dramatically different substrates were utilized for targeting. In one set of experiments, the surfaces of smooth, flat, avidin-coated agar disks were exposed to biotinylated nanoparticles to yield a thin layer of targeted contrast. For the second set of measurements, we targeted PFC nanoparticles applied in thicker layers to cultured smooth muscle cells expressing the transmembrane glycoprotein “tissue factor” at the cell surface. An acoustic microscope was used to characterize reflectivity for all samples as a function of bound PFC (determined via gas chromatography). We utilized a formulation of low-scattering nanoparticles having oil-based cores to compete against high-scattering PFC nanoparticles for binding, to elucidate the dependence of contrast enhancement on PFC concentration. The relationship between reflectivity enhancement and bound PFC content varied in a curvilinear fashion, and exhibited an apparent asymptote (approximately 16 dB and 9 dB enhancement for agar and cell samples, respectively) at the maximum concentrations (~150 μg and ~1000 μg PFOB for agar and cell samples, respectively). Samples targeted with only oil-based nanoparticles exhibited mean backscatter values that were nearly identical to untreated samples (<1 dB difference), confirming the oil particles’ low-scattering behavior. The results of this study indicate that substantial contrast enhancement with liquid perfluorocarbon nanoparticles can be realized even in cases of partial surface coverage (as might be

Design of ligand-targeted nanoparticles for enhanced cancer targeting

NASA Astrophysics Data System (ADS)

Stefanick, Jared F.

Ligand-targeted nanoparticles are increasingly used as drug delivery vehicles for cancer therapy, yet have not consistently produced successful clinical outcomes. Although these inconsistencies may arise from differences in disease models and target receptors, nanoparticle design parameters can significantly influence therapeutic efficacy. By employing a multifaceted synthetic strategy to prepare peptide-targeted nanoparticles with high purity, reproducibility, and precisely controlled stoichiometry of functionalities, this work evaluates the roles of polyethylene glycol (PEG) coating, ethylene glycol (EG) peptide-linker length, peptide hydrophilicity, peptide density, and nanoparticle size on tumor targeting in a systematic manner. These parameters were analyzed in multiple disease models by targeting human epidermal growth factor receptor 2 (HER2) in breast cancer and very late antigen-4 (VLA-4) in multiple myeloma to demonstrate the widespread applicability of this approach. By increasing the hydrophilicity of the targeting peptide sequence and simultaneously optimizing the EG peptide-linker length, the in vitro cellular uptake of targeted liposomes was significantly enhanced. Specifically, including a short oligolysine chain adjacent to the targeting peptide sequence effectively increased cellular uptake ~80-fold using an EG6 peptide-linker compared to ~10-fold using an EG45 linker. In vivo, targeted liposomes prepared in a traditional manner lacking the oligolysine chain demonstrated similar biodistribution and tumor uptake to non-targeted liposomes. However, by including the oligolysine chain, targeted liposomes using an EG45 linker significantly improved tumor uptake ~8-fold over non-targeted liposomes, while the use of an EG6 linker decreased tumor accumulation and uptake, owing to differences in cellular uptake kinetics, clearance mechanisms, and binding site barrier effects. To further improve tumor targeting and enhance the selectivity of targeted

Genomes2Drugs: Identifies Target Proteins and Lead Drugs from Proteome Data

PubMed Central

Toomey, David; Hoppe, Heinrich C.; Brennan, Marian P.; Nolan, Kevin B.; Chubb, Anthony J.

2009-01-01

Background Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. Methodology/Principal Findings To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i) homologous to previously crystallized proteins or (ii) targets of known drugs, but are (iii) not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. Conclusions/Significance Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under ‘change-of-application’ patents. PMID:19593435

Wide screening of phage-displayed libraries identifies immune targets in planta.

PubMed

Rioja, Cristina; Van Wees, Saskia C; Charlton, Keith A; Pieterse, Corné M J; Lorenzo, Oscar; García-Sánchez, Susana

2013-01-01

Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 2 × 10(7) different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well

TimeXNet Web: Identifying cellular response networks from diverse omics time-course data.

PubMed

Tan, Phit Ling; López, Yosvany; Nakai, Kenta; Patil, Ashwini

2018-05-14

Condition-specific time-course omics profiles are frequently used to study cellular response to stimuli and identify associated signaling pathways. However, few online tools allow users to analyze multiple types of high-throughput time-course data. TimeXNet Web is a web server that extracts a time-dependent gene/protein response network from time-course transcriptomic, proteomic or phospho-proteomic data, and an input interaction network. It classifies the given genes/proteins into time-dependent groups based on the time of their highest activity and identifies the most probable paths connecting genes/proteins in consecutive groups. The response sub-network is enriched in activated genes/proteins and contains novel regulators that do not show any observable change in the input data. Users can view the resultant response network and analyze it for functional enrichment. TimeXNet Web supports the analysis of high-throughput data from multiple species by providing high quality, weighted protein-protein interaction networks for 12 model organisms. http://txnet.hgc.jp/. ashwini@hgc.jp. Supplementary data are available at Bioinformatics online.

Intravenous phage display identifies peptide sequences that target the burn-injured intestine.

PubMed

Costantini, Todd W; Eliceiri, Brian P; Putnam, James G; Bansal, Vishal; Baird, Andrew; Coimbra, Raul

2012-11-01

The injured intestine is responsible for significant morbidity and mortality after severe trauma and burn; however, targeting the intestine with therapeutics aimed at decreasing injury has proven difficult. We hypothesized that we could use intravenous phage display technology to identify peptide sequences that target the injured intestinal mucosa in a murine model, and then confirm the cross-reactivity of this peptide sequence with ex vivo human gut. Four hours following 30% TBSA burn we performed an in vivo, intravenous systemic administration of phage library containing 10(12) phage in balb/c mice to biopan for gut-targeting peptides. In vivo assessment of the candidate peptide sequences identified after 4 rounds of internalization was performed by injecting 1×10(12) copies of each selected phage clone into sham or burned animals. Internalization into the gut was assessed using quantitative polymerase chain reaction. We then incubated this gut-targeting peptide sequence with human intestine and visualized fluorescence using confocal microscopy. We identified 3 gut-targeting peptide sequences which caused collapse of the phage library (4-1: SGHQLLLNKMP, 4-5: ILANDLTAPGPR, 4-11: SFKPSGLPAQSL). Sequence 4-5 was internalized into the intestinal mucosa of burned animals 9.3-fold higher than sham animals injected with the same sequence (2.9×10(5)vs. 3.1×10(4) particles per mg tissue). Sequences 4-1 and 4-11 were both internalized into the gut, but did not demonstrate specificity for the injured mucosa. Phage sequence 4-11 demonstrated cross-reactivity with human intestine. In the future, this gut-targeting peptide sequence could serve as a platform for the delivery of biotherapeutics. Copyright © 2012 Elsevier Inc. All rights reserved.

Rapid detection of biothreat agents based on cellular machinery.

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

Lane, Todd W.; Gantt, Richard W.

This research addresses rapid and sensitive identification of biological agents in a complex background. We attempted to devise a method by which the specificity of the cellular transcriptional machinery could be used to detect and identify bacterial bio-terror agents in a background of other organisms. Bacterial cells contain RNA polymerases and transcription factors that transcribe genes into mRNA for translation into proteins. RNA polymerases in conjunction with transcription factors recognize regulatory elements (promoters) upstream of the gene. These promoters are, in many cases, recognized by the polymerase and transcription factor combinations of one species only. We have engineered a plasmid,more » for Escherichia coli, containing the virA promoter from the target species Shigella flexneri. This promoter was fused to a reporter gene Green Fluorescent Protein (GFP). In theory the indicator strain (carrying the plasmid) is mixed with the target strain and the two are lysed. The cellular machinery from both cells mixes and the GFP is produced. This report details the results of testing this system.« less

Protein expression profiling identifies molecular targets of quercetin as a major dietary flavonoid in human colon cancer cells.

PubMed

Wenzel, Uwe; Herzog, Angelika; Kuntz, Sabine; Daniel, Hannelore

2004-07-01

A high dietary intake of plant foods is thought to contribute to the prevention of colorectal cancers in humans and flavonoids as part of such a diet are considered to contribute to those protective effects. Quercetin is a major dietary flavonoid consumed with a diet rich in onions, tea, and apples. We used HT-29 human colon cancer cells and investigated the effects of quercetin on proliferation, apoptosis, and differentiation as processes shown to be disregulated during cancer development. To identify the cellular targets of quercetin action, two-dimensional gel electrophoresis was performed and proteins altered in expression level after quercetin exposure of cells were identified by mass spectrometry of peptide fragments generated by tryptic digestion. Quercetin inhibited the proliferation of HT-29 cells with an IC(50)-value of 81.2 +/- 6.6 microM. Cell differentiation based on surface expression of alkaline phosphatase was enhanced 4-fold and the activity of the pro-apoptotic effector caspase-3 increased 3-fold. Those effects were associated with the regulation of heat-shock proteins and annexins shown to both play a crucial role in the process of apoptosis. Cytoskeletal caspase substrates were found as regulated as well and various proteins involved in intermediary metabolism and in gene regulation showed altered steady-state expression levels upon quercetin treatment of cells. In conclusion, quercetin alters the levels of a variety of proteins involved in growth, differentiation, and apoptosis of colon cancer cells. Their identification as molecular targets of quercetin may explain the anti-cancer activities of this flavonoid.

Combined expressional analysis, bioinformatics and targeted proteomics identify new potential therapeutic targets in glioblastoma stem cells.

PubMed

Stangeland, Biljana; Mughal, Awais A; Grieg, Zanina; Sandberg, Cecilie Jonsgar; Joel, Mrinal; Nygård, Ståle; Meling, Torstein; Murrell, Wayne; Vik Mo, Einar O; Langmoen, Iver A

2015-09-22

Glioblastoma (GBM) is both the most common and the most lethal primary brain tumor. It is thought that GBM stem cells (GSCs) are critically important in resistance to therapy. Therefore, there is a strong rationale to target these cells in order to develop new molecular therapies.To identify molecular targets in GSCs, we compared gene expression in GSCs to that in neural stem cells (NSCs) from the adult human brain, using microarrays. Bioinformatic filtering identified 20 genes (PBK/TOPK, CENPA, KIF15, DEPDC1, CDC6, DLG7/DLGAP5/HURP, KIF18A, EZH2, HMMR/RHAMM/CD168, NOL4, MPP6, MDM1, RAPGEF4, RHBDD1, FNDC3B, FILIP1L, MCC, ATXN7L4/ATXN7L1, P2RY5/LPAR6 and FAM118A) that were consistently expressed in GSC cultures and consistently not expressed in NSC cultures. The expression of these genes was confirmed in clinical samples (TCGA and REMBRANDT). The first nine genes were highly co-expressed in all GBM subtypes and were part of the same protein-protein interaction network. Furthermore, their combined up-regulation correlated negatively with patient survival in the mesenchymal GBM subtype. Using targeted proteomics and the COGNOSCENTE database we linked these genes to GBM signalling pathways.Nine genes: PBK, CENPA, KIF15, DEPDC1, CDC6, DLG7, KIF18A, EZH2 and HMMR should be further explored as targets for treatment of GBM.

Stabilization of exosome-targeting peptides via engineered glycosylation.

PubMed

Hung, Michelle E; Leonard, Joshua N

2015-03-27

Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion*

PubMed Central

Hung, Chien-Wen; Klein, Tobias; Cassidy, Liam; Linke, Dennis; Lange, Sabrina; Anders, Uwe; Bureik, Matthias; Heinzle, Elmar; Schneider, Konstantin; Tholey, Andreas

2016-01-01

Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe. Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016. PMID:27477394

Murine Hyperglycemic Vasculopathy and Cardiomyopathy: Whole-Genome Gene Expression Analysis Predicts Cellular Targets and Regulatory Networks Influenced by Mannose Binding Lectin

PubMed Central

Zou, Chenhui; La Bonte, Laura R.; Pavlov, Vasile I.; Stahl, Gregory L.

2012-01-01

Hyperglycemia, in the absence of type 1 or 2 diabetes, is an independent risk factor for cardiovascular disease. We have previously demonstrated a central role for mannose binding lectin (MBL)-mediated cardiac dysfunction in acute hyperglycemic mice. In this study, we applied whole-genome microarray data analysis to investigate MBL’s role in systematic gene expression changes. The data predict possible intracellular events taking place in multiple cellular compartments such as enhanced insulin signaling pathway sensitivity, promoted mitochondrial respiratory function, improved cellular energy expenditure and protein quality control, improved cytoskeleton structure, and facilitated intracellular trafficking, all of which may contribute to the organismal health of MBL null mice against acute hyperglycemia. Our data show a tight association between gene expression profile and tissue function which might be a very useful tool in predicting cellular targets and regulatory networks connected with in vivo observations, providing clues for further mechanistic studies. PMID:22375142

Cellular effects of the microtubule-targeting agent peloruside A in hypoxia-conditioned colorectal carcinoma cells.

PubMed

Řehulka, Jiří; Annadurai, Narendran; Frydrych, Ivo; Znojek, Pawel; Džubák, Petr; Northcote, Peter; Miller, John H; Hajdúch, Marián; Das, Viswanath

2017-07-01

Hypoxia is a prominent feature of solid tumors, dramatically remodeling microtubule structures and cellular pathways and contributing to paclitaxel resistance. Peloruside A (PLA), a microtubule-targeting agent, has shown promising anti-tumor effects in preclinical studies. Although it has a similar mode of action to paclitaxel, it binds to a distinct site on β-tubulin that differs from the classical taxane site. In this study, we examined the unexplored effects of PLA in hypoxia-conditioned colorectal HCT116 cancer cells. Cytotoxicity of PLA was determined by cell proliferation assay. The effects of a pre-exposure to hypoxia on PLA-induced cell cycle alterations and apoptosis were examined by flow cytometry, time-lapse imaging, and western blot analysis of selected markers. The hypoxia effect on stabilization of microtubules by PLA was monitored by an intracellular tubulin polymerization assay. Our findings show that the cytotoxicity of PLA is not altered in hypoxia-conditioned cells compared to paclitaxel and vincristine. Furthermore, hypoxia does not alter PLA-induced microtubule stabilization nor the multinucleation of cells. PLA causes cyclin B1 and G2/M accumulation followed by apoptosis. The cellular and molecular effects of PLA have been determined in normoxic conditions, but there are no reports of PLA effects in hypoxic cells. Our findings reveal that hypoxia preconditioning does not alter the sensitivity of HCT116 to PLA. These data report on the cellular and molecular effects of PLA in hypoxia-conditioned cells for the first time, and will encourage further exploration of PLA as a promising anti-tumor agent. Copyright © 2017 Elsevier B.V. All rights reserved.

Converging cellular themes for the hereditary spastic paraplegias.

PubMed

Blackstone, Craig

2018-05-10

Hereditary spastic paraplegias (HSPs) are neurologic disorders characterized by prominent lower-extremity spasticity, resulting from a length-dependent axonopathy of corticospinal upper motor neurons. They are among the most genetically-diverse neurologic disorders, with >80 distinct genetic loci and over 60 identified genes. Studies investigating the molecular pathogenesis underlying HSPs have emphasized the importance of converging cellular pathogenic themes in the most common forms of HSP, providing compelling targets for therapy. Most notably, these include organelle shaping and biogenesis as well as membrane and cargo trafficking. Published by Elsevier Ltd.

Interaction of cellular proteins with BCL-xL targeted to cytoplasmic inclusion bodies in adenovirus infected cells.

PubMed

Subramanian, T; Vijayalingam, S; Kuppuswamy, M; Chinnadurai, G

2015-09-01

Adenovirus-mediated apoptosis was suppressed when cellular anti-apoptosis proteins (BCL-2 and BCL-xL) were substituted for the viral E1B-19K. For unbiased proteomic analysis of proteins targeted by BCL-xL in adenovirus-infected cells and to visualize the interactions with target proteins, BCL-xL was targeted to cytosolic inclusion bodies utilizing the orthoreovirus µNS protein sequences. The chimeric protein was localized in non-canonical cytosolic factory-like sites and promoted survival of virus-infected cells. The BCL-xL-associated proteins were isolated from the cytosolic inclusion bodies in adenovirus-infected cells and analyzed by LC-MS. These proteins included BAX, BAK, BID, BIK and BIM as well as mitochondrial proteins such as prohibitin 2, ATP synthase and DNA-PKcs. Our studies suggested that in addition to the interaction with various pro-apoptotic proteins, the association with certain mitochondrial proteins such as DNA-PKcs and prohibitins might augment the survival function of BCL-xL in virus infected cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Global functional analyses of cellular responses to pore-forming toxins.

PubMed

Kao, Cheng-Yuan; Los, Ferdinand C O; Huffman, Danielle L; Wachi, Shinichiro; Kloft, Nicole; Husmann, Matthias; Karabrahimi, Valbona; Schwartz, Jean-Louis; Bellier, Audrey; Ha, Christine; Sagong, Youn; Fan, Hui; Ghosh, Partho; Hsieh, Mindy; Hsu, Chih-Shen; Chen, Li; Aroian, Raffi V

2011-03-01

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

Identification of TRIM27 as a novel degradation target of herpes simplex virus 1 ICP0.

PubMed

Conwell, Sara E; White, Anne E; Harper, J Wade; Knipe, David M

2015-01-01

The herpes simplex virus 1 (HSV-1) immediate early protein ICP0 performs many functions during infection, including transactivation of viral gene expression, suppression of innate immune responses, and modification and eviction of histones from viral chromatin. Although these functions of ICP0 have been characterized, the detailed mechanisms underlying ICP0's complex role during infection warrant further investigation. We thus undertook an unbiased proteomic approach to identifying viral and cellular proteins that interact with ICP0 in the infected cell. Cellular candidates resulting from our analysis included the ubiquitin-specific protease USP7, the transcriptional repressor TRIM27, DNA repair proteins NBN and MRE11A, regulators of apoptosis, including BIRC6, and the proteasome. We also identified two HSV-1 early proteins involved in nucleotide metabolism, UL39 and UL50, as novel candidate interactors of ICP0. Because TRIM27 was the most statistically significant cellular candidate, we investigated the relationship between TRIM27 and ICP0. We observed rapid, ICP0-dependent loss of TRIM27 during HSV-1 infection. TRIM27 protein levels were restored by disrupting the RING domain of ICP0 or by inhibiting the proteasome, arguing that TRIM27 is a novel degradation target of ICP0. A mutant ICP0 lacking E3 ligase activity interacted with endogenous TRIM27 during infection as demonstrated by reciprocal coimmunoprecipitation and supported by immunofluorescence data. Surprisingly, ICP0-null mutant virus yields decreased upon TRIM27 depletion, arguing that TRIM27 has a positive effect on infection despite being targeted for degradation. These results illustrate a complex interaction between TRIM27 and viral infection with potential positive or negative effects of TRIM27 on HSV under different infection conditions. During productive infection, a virus must simultaneously redirect multiple cellular pathways to replicate itself while evading detection by the host's defenses. To

Identifying the cellular mechanisms of symbiont-induced epithelial morphogenesis in the squid-vibrio association

PubMed Central

Koropatnick, Tanya; Goodson, Michael S.; Heath-Heckman, Elizabeth A. C.; McFall-Ngai, Margaret

2014-01-01

The symbiotic association between the Hawaiian bobtail squid Euprymna scolopes and the luminous marine bacterium Vibrio fischeri provides a unique opportunity to study epithelial morphogenesis. Shortly after hatching, the squid host harvests bacteria from the seawater using currents created by two elaborate fields of ciliated epithelia on the surface of the juvenile light organ. After light organ colonization, the symbiont population signals the gradual loss of the ciliated epithelia through apoptosis of the cells, which culminates in the complete regression of these tissues. Whereas aspects of this process have been studied at the morphological, biochemical and molecular levels, no in-depth analysis of the cellular events has been reported. Here we describe the cellular structure of the epithelial field and present evidence that the symbiosis-induced regression occurs in two steps. Using confocal microscopic analyses, we observed an initial epithelial remodeling, which serves to disable the function of the harvesting apparatus, followed by a protracted regression involving actin rearrangements and epithelial cell extrusion. We identified a metal-dependent gelatinolytic activity in the symbiont-induced morphogenic epithelial fields, suggesting the involvement of Zn-dependent matrix metalloproteinase(s) (MMP) in light organ morphogenesis. These data show that the bacterial symbionts not only induce apoptosis of the field, but also change the form, function and biochemistry of the cells as part of the morphogenic program. PMID:24648207

Identifying the cellular mechanisms of symbiont-induced epithelial morphogenesis in the squid-Vibrio association.

PubMed

Koropatnick, Tanya; Goodson, Michael S; Heath-Heckman, Elizabeth A C; McFall-Ngai, Margaret

2014-02-01

The symbiotic association between the Hawaiian bobtail squid Euprymna scolopes and the luminous marine bacterium Vibrio fischeri provides a unique opportunity to study epithelial morphogenesis. Shortly after hatching, the squid host harvests bacteria from the seawater using currents created by two elaborate fields of ciliated epithelia on the surface of the juvenile light organ. After light organ colonization, the symbiont population signals the gradual loss of the ciliated epithelia through apoptosis of the cells, which culminates in the complete regression of these tissues. Whereas aspects of this process have been studied at the morphological, biochemical, and molecular levels, no in-depth analysis of the cellular events has been reported. Here we describe the cellular structure of the epithelial field and present evidence that the symbiosis-induced regression occurs in two steps. Using confocal microscopic analyses, we observed an initial epithelial remodeling, which serves to disable the function of the harvesting apparatus, followed by a protracted regression involving actin rearrangements and epithelial cell extrusion. We identified a metal-dependent gelatinolytic activity in the symbiont-induced morphogenic epithelial fields, suggesting the involvement of Zn-dependent matrix metalloproteinase(s) (MMP) in light organ morphogenesis. These data show that the bacterial symbionts not only induce apoptosis of the field, but also change the form, function, and biochemistry of the cells as part of the morphogenic program.

CPTAC Collaborates with Molecular & Cellular Proteomics to Address Reproducibility in Targeted Assay Development | Office of Cancer Clinical Proteomics Research

Cancer.gov

The journal Molecular & Cellular Proteomics (MCP), in collaboration with the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI), part of the National Institutes of Health, announce new guidelines and requirements for papers describing the development and application of targeted mass spectrometry measurements of peptides, modified peptides and proteins (Mol Cell Proteomics 2017; PMID: 28183812).  NCI’s participation is part of NIH’s overall effort to address the r

Antibiotic efficacy is linked to bacterial cellular respiration

PubMed Central

Lobritz, Michael A.; Belenky, Peter; Porter, Caroline B. M.; Gutierrez, Arnaud; Yang, Jason H.; Schwarz, Eric G.; Dwyer, Daniel J.; Khalil, Ahmad S.; Collins, James J.

2015-01-01

Bacteriostatic and bactericidal antibiotic treatments result in two fundamentally different phenotypic outcomes—the inhibition of bacterial growth or, alternatively, cell death. Most antibiotics inhibit processes that are major consumers of cellular energy output, suggesting that antibiotic treatment may have important downstream consequences on bacterial metabolism. We hypothesized that the specific metabolic effects of bacteriostatic and bactericidal antibiotics contribute to their overall efficacy. We leveraged the opposing phenotypes of bacteriostatic and bactericidal drugs in combination to investigate their activity. Growth inhibition from bacteriostatic antibiotics was associated with suppressed cellular respiration whereas cell death from most bactericidal antibiotics was associated with accelerated respiration. In combination, suppression of cellular respiration by the bacteriostatic antibiotic was the dominant effect, blocking bactericidal killing. Global metabolic profiling of bacteriostatic antibiotic treatment revealed that accumulation of metabolites involved in specific drug target activity was linked to the buildup of energy metabolites that feed the electron transport chain. Inhibition of cellular respiration by knockout of the cytochrome oxidases was sufficient to attenuate bactericidal lethality whereas acceleration of basal respiration by genetically uncoupling ATP synthesis from electron transport resulted in potentiation of the killing effect of bactericidal antibiotics. This work identifies a link between antibiotic-induced cellular respiration and bactericidal lethality and demonstrates that bactericidal activity can be arrested by attenuated respiration and potentiated by accelerated respiration. Our data collectively show that antibiotics perturb the metabolic state of bacteria and that the metabolic state of bacteria impacts antibiotic efficacy. PMID:26100898

Target-similarity search using Plasmodium falciparum proteome identifies approved drugs with anti-malarial activity and their possible targets

PubMed Central

Akala, Hoseah M.; Macharia, Rosaline W.; Juma, Dennis W.; Cheruiyot, Agnes C.; Andagalu, Ben; Brown, Mathew L.; El-Shemy, Hany A.; Nyanjom, Steven G.

2017-01-01

Malaria causes about half a million deaths annually, with Plasmodium falciparum being responsible for 90% of all the cases. Recent reports on artemisinin resistance in Southeast Asia warrant urgent discovery of novel drugs for the treatment of malaria. However, most bioactive compounds fail to progress to treatments due to safety concerns. Drug repositioning offers an alternative strategy where drugs that have already been approved as safe for other diseases could be used to treat malaria. This study screened approved drugs for antimalarial activity using an in silico chemogenomics approach prior to in vitro verification. All the P. falciparum proteins sequences available in NCBI RefSeq were mined and used to perform a similarity search against DrugBank, TTD and STITCH databases to identify similar putative drug targets. Druggability indices of the potential P. falciparum drug targets were obtained from TDR targets database. Functional amino acid residues of the drug targets were determined using ConSurf server which was used to fine tune the similarity search. This study predicted 133 approved drugs that could target 34 P. falciparum proteins. A literature search done at PubMed and Google Scholar showed 105 out of the 133 drugs to have been previously tested against malaria, with most showing activity. For further validation, drug susceptibility assays using SYBR Green I method were done on a representative group of 10 predicted drugs, eight of which did show activity against P. falciparum 3D7 clone. Seven had IC50 values ranging from 1 μM to 50 μM. This study also suggests drug-target association and hence possible mechanisms of action of drugs that did show antiplasmodial activity. The study results validate the use of proteome-wide target similarity approach in identifying approved drugs with activity against P. falciparum and could be adapted for other pathogens. PMID:29088219

Parasitoid wasp venom SERCA regulates Drosophila calcium levels and inhibits cellular immunity.

PubMed

Mortimer, Nathan T; Goecks, Jeremy; Kacsoh, Balint Z; Mobley, James A; Bowersock, Gregory J; Taylor, James; Schlenke, Todd A

2013-06-04

Because parasite virulence factors target host immune responses, identification and functional characterization of these factors can provide insight into poorly understood host immune mechanisms. The fruit fly Drosophila melanogaster is a model system for understanding humoral innate immunity, but Drosophila cellular innate immune responses remain incompletely characterized. Fruit flies are regularly infected by parasitoid wasps in nature and, following infection, flies mount a cellular immune response culminating in the cellular encapsulation of the wasp egg. The mechanistic basis of this response is largely unknown, but wasps use a mixture of virulence proteins derived from the venom gland to suppress cellular encapsulation. To gain insight into the mechanisms underlying wasp virulence and fly cellular immunity, we used a joint transcriptomic/proteomic approach to identify venom genes from Ganaspis sp.1 (G1), a previously uncharacterized Drosophila parasitoid species, and found that G1 venom contains a highly abundant sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. Accordingly, we found that fly immune cells termed plasmatocytes normally undergo a cytoplasmic calcium burst following infection, and that this calcium burst is required for activation of the cellular immune response. We further found that the plasmatocyte calcium burst is suppressed by G1 venom in a SERCA-dependent manner, leading to the failure of plasmatocytes to become activated and migrate toward G1 eggs. Finally, by genetically manipulating plasmatocyte calcium levels, we were able to alter fly immune success against G1 and other parasitoid species. Our characterization of parasitoid wasp venom proteins led us to identify plasmatocyte cytoplasmic calcium bursts as an important aspect of fly cellular immunity.

Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

PubMed Central

Liu, Bing; Gao, Yankun; Ruan, Hai-Bin; Chen, Yue

2016-01-01

Proline hydroxylation is a critical cellular mechanism regulating oxygen-response pathways in tumor initiation and progression. Yet, its substrate diversity and functions remain largely unknown. Here, we report a system-wide analysis to characterize proline hydroxylation substrates in cancer cells using an immunoaffinity-purification assisted proteomics strategy. We identified 562 sites from 272 proteins in HeLa cells. Bioinformatic analysis revealed that proline hydroxylation substrates are significantly enriched with mRNA processing and stress-response cellular pathways with canonical and diverse flanking sequence motifs. Structural analysis indicates a significant enrichment of proline hydroxylation participating in the secondary structure of substrate proteins. Our study identified and validated Brd4, a key transcription factor, as a novel proline hydroxylation substrate. Functional analysis showed that the inhibition of proline hydroxylation pathway significantly reduced the proline hydroxylation abundance on Brd4 and affected Brd4-mediated transcriptional activity as well as cell proliferation in AML leukemia cells. Taken together, our study identified a broad regulatory role of proline hydroxylation in cellular oxygen-sensing pathways and revealed potentially new targets that dynamically respond to hypoxia microenvironment in tumor cells. PMID:27764789

Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins

PubMed Central

Harden, Mallory E.; Prasad, Nripesh; Griffiths, Anthony

2017-01-01

ABSTRACT The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation. PMID:28049151

HIV-1 Tat targets Tip60 to impair the apoptotic cell response to genotoxic stresses

PubMed Central

Col, Edwige; Caron, Cécile; Chable-Bessia, Christine; Legube, Gaelle; Gazzeri, Sylvie; Komatsu, Yasuhiko; Yoshida, Minoru; Benkirane, Monsef; Trouche, Didier; Khochbin, Saadi

2005-01-01

HIV-1 transactivator Tat uses cellular acetylation signalling by targeting several cellular histone acetyltransferases (HAT) to optimize its various functions. Although Tip60 was the first HAT identified to interact with Tat, the biological significance of this interaction has remained obscure. We had previously shown that Tat represses Tip60 HAT activity. Here, a new mechanism of Tip60 neutralization by Tat is described, where Tip60 is identified as a substrate for the newly reported p300/CBP-associated E4-type ubiquitin-ligase activity, and Tat uses this mechanism to induce the polyubiquitination and degradation of Tip60. Tip60 targeting by Tat results in a dramatic impairment of the Tip60-dependent apoptotic cell response to DNA damage. These data reveal yet unknown strategies developed by HIV-1 to increase cell resistance to genotoxic stresses and show a role of Tat as a modulator of cellular protein ubiquitination. PMID:16001085

Cellular Immune Responses against Simian T-Lymphotropic Virus Type 1 Target Tax in Infected Baboons

PubMed Central

Castro, Iris; Giret, Teresa M.; Magnani, Diogo M.; Maxwell, Helen S.; Umland, Oliver; Perry, Jessica K.; Pecotte, Jerilyn K.; Brasky, Kathleen M.; Barber, Glen N.; Desrosiers, Ronald C.

2016-01-01

ABSTRACT There are currently 5 million to 10 million human T-lymphotropic virus type 1 (HTLV-1)-infected people, and many of them will develop severe complications resulting from this infection. A vaccine is urgently needed in areas where HTLV-1 is endemic. Many vaccines are best tested in nonhuman primate animal models. As a first step in designing an effective HTLV-1 vaccine, we defined the CD8+ and CD4+ T cell response against simian T-lymphotropic virus type 1 (STLV-1), a virus closely related to HTLV-1, in olive baboons (Papio anubis). Consistent with persistent antigenic exposure, we observed that STLV-1-specific CD8+ T cells displayed an effector memory phenotype and usually expressed CD107a, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α). To assess the viral targets of the cellular immune response in STLV-1-infected animals, we used intracellular cytokine staining to detect responses against overlapping peptides covering the entire STLV-1 proteome. Our results show that, similarly to humans, the baboon CD8+ T cell response narrowly targeted the Tax protein. Our findings suggest that the STLV-1-infected baboon model may recapitulate some of the important aspects of the human response against HTLV-1 and could be an important tool for the development of immune-based therapy and prophylaxis. IMPORTANCE HTLV-1 infection can lead to many different and often fatal conditions. A vaccine deployed in areas of high prevalence might reduce the incidence of HTLV-1-induced disease. Unfortunately, there are very few animal models of HTLV-1 infection useful for testing vaccine approaches. Here we describe cellular immune responses in baboons against a closely related virus, STLV-1. We show for the first time that the immune response against STLV-1 in naturally infected baboons is largely directed against the Tax protein. Similar findings in humans and the sequence similarity between the human and baboon viruses suggest that the STLV-1-infected baboon

Identifying Drug-Target Interactions with Decision Templates.

PubMed

Yan, Xiao-Ying; Zhang, Shao-Wu

2018-01-01

During the development process of new drugs, identification of the drug-target interactions wins primary concerns. However, the chemical or biological experiments bear the limitation in coverage as well as the huge cost of both time and money. Based on drug similarity and target similarity, chemogenomic methods can be able to predict potential drug-target interactions (DTIs) on a large scale and have no luxurious need about target structures or ligand entries. In order to reflect the cases that the drugs having variant structures interact with common targets and the targets having dissimilar sequences interact with same drugs. In addition, though several other similarity metrics have been developed to predict DTIs, the combination of multiple similarity metrics (especially heterogeneous similarities) is too naïve to sufficiently explore the multiple similarities. In this paper, based on Gene Ontology and pathway annotation, we introduce two novel target similarity metrics to address above issues. More importantly, we propose a more effective strategy via decision template to integrate multiple classifiers designed with multiple similarity metrics. In the scenarios that predict existing targets for new drugs and predict approved drugs for new protein targets, the results on the DTI benchmark datasets show that our target similarity metrics are able to enhance the predictive accuracies in two scenarios. And the elaborate fusion strategy of multiple classifiers has better predictive power than the naïve combination of multiple similarity metrics. Compared with other two state-of-the-art approaches on the four popular benchmark datasets of binary drug-target interactions, our method achieves the best results in terms of AUC and AUPR for predicting available targets for new drugs (S2), and predicting approved drugs for new protein targets (S3).These results demonstrate that our method can effectively predict the drug-target interactions. The software package can

Landscape phages and their fusion proteins targeted to breast cancer cells

PubMed Central

Fagbohun, Olusegun A.; Bedi, Deepa; Grabchenko, Natalia I.; Deinnocentes, Patricia A.; Bird, Richard C.; Petrenko, Valery A.

2012-01-01

Breast cancer is a leading cause of death among women in the USA. The efficacy of existing anticancer therapeutics can be improved by targeting them through conjugation with ligands binding to cellular receptors. Recently, we developed a novel drug targeting strategy based on the use of pre-selected cancer-specific ‘fusion pVIII proteins’ (fpVIII), as targeting ligands. To study the efficiency of this approach in animal models, we developed a panel of breast cancer cell-binding phages as a source of targeted fpVIIIs. Two landscape phage peptide libraries (8-mer f8/8 and 9-mer f8/9) were screened to isolate 132 phage variants that recognize breast carcinoma cells MCF-7 and ZR-75-1 and internalize into the cells. When tested for their interaction with the breast cancer cells in comparison with liver cancer cells HepG2, human mammary cells MCF-10A cells and serum, 16 of the phage probes selectively interacted with the breast cancer cells whereas 32 bound both breast and liver cancer cells. The most prominent cancer-specific phage DMPGTVLP, demonstrating sub-nanomolar Kd in interaction with target cells, was used for affinity chromatography of cellular membrane molecules to reveal its potential binding receptor. The isolated protein was identified by direct sequencing as cellular surface nucleolin. This conclusion was confirmed by inhibition of the phage–cell interaction with nucleolin antibodies. Other prominent phage binders VPTDTDYS, VEEGGYIAA, and DWRGDSMDS demonstrate consensus motifs common to previously identified cancer-specific peptides. Isolated phage proteins exhibit inherent binding specificity towards cancer cells, demonstrating the functional activity of the selected fused peptides. The selected phages, their peptide inserts and intact fusion proteins can serve as promising ligands for the development of targeted nanomedicines and their study in model mice with xenograft of human cells MCF-7 and ZR-75-1. PMID:22490956

Soybean extracts increase cell surface ZIP4 abundance and cellular zinc levels: a potential novel strategy to enhance zinc absorption by ZIP4 targeting.

PubMed

Hashimoto, Ayako; Ohkura, Katsuma; Takahashi, Masakazu; Kizu, Kumiko; Narita, Hiroshi; Enomoto, Shuichi; Miyamae, Yusaku; Masuda, Seiji; Nagao, Masaya; Irie, Kazuhiro; Ohigashi, Hajime; Andrews, Glen K; Kambe, Taiho

2015-12-01

Dietary zinc deficiency puts human health at risk, so we explored strategies for enhancing zinc absorption. In the small intestine, the zinc transporter ZIP4 functions as an essential component of zinc absorption. Overexpression of ZIP4 protein increases zinc uptake and thereby cellular zinc levels, suggesting that food components with the ability to increase ZIP4 could potentially enhance zinc absorption via the intestine. In the present study, we used mouse Hepa cells, which regulate mouse Zip4 (mZip4) in a manner indistinguishable from that in intestinal enterocytes, to screen for suitable food components that can increase the abundance of ZIP4. Using this ZIP4-targeting strategy, two such soybean extracts were identified that were specifically able to decrease mZip4 endocytosis in response to zinc. These soybean extracts also effectively increased the abundance of apically localized mZip4 in transfected polarized Caco2 and Madin-Darby canine kidney cells and, moreover, two apically localized mZip4 acrodermatitis enteropathica mutants. Soybean components were purified from one extract and soyasaponin Bb was identified as an active component that increased both mZip4 protein abundance and zinc levels in Hepa cells. Finally, we confirmed that soyasaponin Bb is capable of enhancing cell surface endogenous human ZIP4 in human cells. Our results suggest that ZIP4 targeting may represent a new strategy to improve zinc absorption in humans. © 2015 Authors; published by Portland Press Limited.

Identifying relationships between unrelated pharmaceutical target proteins on the basis of shared active compounds.

PubMed

Miljković, Filip; Kunimoto, Ryo; Bajorath, Jürgen

2017-08-01

Computational exploration of small-molecule-based relationships between target proteins from different families. Target annotations of drugs and other bioactive compounds were systematically analyzed on the basis of high-confidence activity data. A total of 286 novel chemical links were established between distantly related or unrelated target proteins. These relationships involved a total of 1859 bioactive compounds including 147 drugs and 141 targets. Computational analysis of large amounts of compounds and activity data has revealed unexpected relationships between diverse target proteins on the basis of compounds they share. These relationships are relevant for drug discovery efforts. Target pairs that we have identified and associated compound information are made freely available.

Biochemical and Cellular Assessment of Acetabular Chondral Flaps Identified During Hip Arthroscopy.

PubMed

Hariri, Sanaz; Truntzer, Jeremy; Smith, Robert Lane; Safran, Marc R

2015-06-01

To analyze chondral flaps debrided during hip arthroscopy to determine their biochemical and cellular composition. Thirty-one full-thickness acetabular chondral flaps were collected during hip arthroscopy. Biochemical analysis was undertaken in 21 flaps from 20 patients, and cellular viability was determined in 10 flaps from 10 patients. Biochemical analysis included concentrations of (1) DNA (an indicator of chondrocyte content), (2) hydroxyproline (an indicator of collagen content), and (3) glycosaminoglycan (an indicator of chondrocyte biosynthesis). Higher values for these parameters indicated more healthy tissue. The flaps were examined to determine the percentage of viable chondrocytes. The percentage of acetabular chondral flap specimens that had concentrations within 1 SD of the mean values reported in previous normal cartilage studies was 38% for DNA, 0% for glycosaminoglycan, and 43% for hydroxyproline. The average cellular viability of our acetabular chondral flap specimens was 39% (SD, 14%). Only 2 of the 10 specimens had more than half the cells still viable. There was no correlation between (1) the gross examination of the joint or knowledge of the patient's demographic characteristics and symptoms and (2) biochemical properties and cell viability of the flap, with one exception: a degenerative appearance of the surrounding cartilage correlated with a higher hydroxyproline concentration. Although full-thickness acetabular chondral flaps can appear normal grossly, the biochemical properties and percentage of live chondrocytes in full-thickness chondral flaps encountered in hip arthroscopy show that this tissue is not normal. There has been recent interest in repairing chondral flaps encountered during hip arthroscopy. These data suggest that acetabular chondral flaps are not biochemically and cellularly normal. Although these flaps may still be valuable mechanically and/or as a scaffold in some conductive or inductive capacity, further study is

Computational analysis of ribonomics datasets identifies long non-coding RNA targets of γ-herpesviral miRNAs.

PubMed

Sethuraman, Sunantha; Thomas, Merin; Gay, Lauren A; Renne, Rolf

2018-05-29

Ribonomics experiments involving crosslinking and immuno-precipitation (CLIP) of Ago proteins have expanded the understanding of the miRNA targetome of several organisms. These techniques, collectively referred to as CLIP-seq, have been applied to identifying the mRNA targets of miRNAs expressed by Kaposi's Sarcoma-associated herpes virus (KSHV) and Epstein-Barr virus (EBV). However, these studies focused on identifying only those RNA targets of KSHV and EBV miRNAs that are known to encode proteins. Recent studies have demonstrated that long non-coding RNAs (lncRNAs) are also targeted by miRNAs. In this study, we performed a systematic re-analysis of published datasets from KSHV- and EBV-driven cancers. We used CLIP-seq data from lymphoma cells or EBV-transformed B cells, and a crosslinking, ligation and sequencing of hybrids dataset from KSHV-infected endothelial cells, to identify novel lncRNA targets of viral miRNAs. Here, we catalog the lncRNA targetome of KSHV and EBV miRNAs, and provide a detailed in silico analysis of lncRNA-miRNA binding interactions. Viral miRNAs target several hundred lncRNAs, including a subset previously shown to be aberrantly expressed in human malignancies. In addition, we identified thousands of lncRNAs to be putative targets of human miRNAs, suggesting that miRNA-lncRNA interactions broadly contribute to the regulation of gene expression.

Identifying mRNA sequence elements for target recognition by human Argonaute proteins

PubMed Central

Li, Jingjing; Kim, TaeHyung; Nutiu, Razvan; Ray, Debashish; Hughes, Timothy R.; Zhang, Zhaolei

2014-01-01

It is commonly known that mammalian microRNAs (miRNAs) guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments that coimmunoprecipitate the Argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs that lack seed complementarity with miRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide miRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (∼10 nucleotides in length) that alone can accurately predict AGO–mRNA associations, independent of the presence of miRNA binding sites. Within this region, we further identified an enriched motif that was replicable on independent AGO-immunoprecipitation data sets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences and validated the AGO motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid miRNAs in recognizing their targets with high specificity. PMID:24663241

Factor Analysis of Therapist-Identified Treatment Targets in Community-Based Children's Mental Health.

PubMed

Love, Allison R; Okado, Izumi; Orimoto, Trina E; Mueller, Charles W

2018-01-01

The present study used exploratory and confirmatory factor analyses to identify underlying latent factors affecting variation in community therapists' endorsement of treatment targets. As part of a statewide practice management program, therapist completed monthly reports of treatment targets (up to 10 per month) for a sample of youth (n = 790) receiving intensive in-home therapy. Nearly 75 % of youth were diagnosed with multiple co-occurring disorders. Five factors emerged: Disinhibition, Societal Rules Evasion, Social Engagement Deficits, Emotional Distress, and Management of Biodevelopmental Outcomes. Using logistic regression, primary diagnosis predicted therapist selection of Disinhibition and Emotional Distress targets. Client age predicted endorsement of Societal Rules Evasion targets. Practice-to-research implications are discussed.

Cellular genetic therapy.

PubMed

Del Vecchio, F; Filareto, A; Spitalieri, P; Sangiuolo, F; Novelli, G

2005-01-01

Cellular genetic therapy is the ultimate frontier for those pathologies that are consequent to a specific nonfunctional cellular type. A viable cure for there kinds of diseases is the replacement of sick cells with healthy ones, which can be obtained from the same patient or a different donor. In fact, structures can be corrected and strengthened with the introduction of undifferentiated cells within specific target tissues, where they will specialize into the desired cellular types. Furthermore, consequent to the recent results obtained with the transdifferentiation experiments, a process that allows the in vitro differentiation of embryonic and adult stem cells, it has also became clear that many advantages may be obtained from the use of stem cells to produce drugs, vaccines, and therapeutic molecules. Since stem cells can sustain lineage potentials, the capacity for differentiation, and better tolerance for the introduction of exogenous genes, they are also considered as feasible therapeutic vehicles for gene therapy. In fact, it is strongly believed that the combination of cellular genetic and gene therapy approaches will definitely allow the development of new therapeutic strategies as well as the production of totipotent cell lines to be used as experimental models for the cure of genetic disorders.

Targeting dendritic cells through gold nanoparticles: A review on the cellular uptake and subsequent immunological properties.

PubMed

Ahmad, Suhana; Zamry, Anes Ateqah; Tan, Hern-Tze Tina; Wong, Kah Keng; Lim, JitKang; Mohamud, Rohimah

2017-11-01

Gold nanoparticles (NPs) have been proposed as a highly potential tool in immunotherapies due to its advantageous properties including customizable size and shapes, surface functionality and biocompatibility. Dendritic cells (DCs), the sentinels of immune response, have been of interest to be manipulated by using gold NPs for targeted delivery of immunotherapeutic agent. Researches done especially in human DCs showed a variation of gold NPs effects on cellular uptake and internalization, DC maturation and subsequent T cells priming as well as cytotoxicity. In this review, we describe the synthesis and physiochemical properties of gold NPs as well as the importance of gold NPs in immunotherapies through their actions on human DCs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

DOE PAGES

Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric; ...

2015-03-30

Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

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

Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric

Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

Targeting Virus-host Interactions of HIV Replication.

PubMed

Weydert, Caroline; De Rijck, Jan; Christ, Frauke; Debyser, Zeger

2016-01-01

Cellular proteins that are hijacked by HIV in order to complete its replication cycle, form attractive new targets for antiretroviral therapy. In particular, the protein-protein interactions between these cellular proteins (cofactors) and viral proteins are of great interest to develop new therapies. Research efforts have led to the validation of different cofactors and some successes in therapeutic applications. Maraviroc, the first cofactor inhibitor approved for human medicinal use, provided a proof of concept. Furthermore, compounds developed as Integrase-LEDGF/p75 interaction inhibitors (LEDGINs) have advanced to early clinical trials. Other compounds targeting cofactors and cofactor-viral protein interactions are currently under development. Likewise, interactions between cellular restriction factors and their counteracting HIV protein might serve as interesting targets in order to impair HIV replication. In this respect, compounds targeting the Vif-APOBEC3G interaction have been described. In this review, we focus on compounds targeting the Integrase- LEDGF/p75 interaction, the Tat-P-TEFb interaction and the Vif-APOBEC3G interaction. Additionally we give an overview of currently discovered compounds presumably targeting cellular cofactor-HIV protein interactions.

Adaptive Cellular Stress Pathways as Therapeutic Targets of Dietary Phytochemicals: Focus on the Nervous System

PubMed Central

Jo, Dong-Gyu; Park, Daeui; Chung, Hae Young

2014-01-01

During the past 5 decades, it has been widely promulgated that the chemicals in plants that are good for health act as direct scavengers of free radicals. Here we review evidence that favors a different hypothesis for the health benefits of plant consumption, namely, that some phytochemicals exert disease-preventive and therapeutic actions by engaging one or more adaptive cellular response pathways in cells. The evolutionary basis for the latter mechanism is grounded in the fact that plants produce natural antifeedant/noxious chemicals that discourage insects and other organisms from eating them. However, in the amounts typically consumed by humans, the phytochemicals activate one or more conserved adaptive cellular stress response pathways and thereby enhance the ability of cells to resist injury and disease. Examplesof such pathways include those involving the transcription factors nuclear factor erythroid 2-related factor 2, nuclear factor-κB, hypoxia-inducible factor 1α, peroxisome proliferator-activated receptor γ, and forkhead box subgroup O, as well as the production and action of trophic factors and hormones. Translational research to develop interventions that target these pathways may lead to new classes of therapeutic agents that act by stimulating adaptive stress response pathways to bolster endogenous defenses against tissue injury and disease. Because neurons are particularly sensitive to potentially noxious phytochemicals, we focus on the nervous system but also include findings from other cell types in which actions of phytochemicals on specific signal transduction pathways have been more thoroughly studied. PMID:24958636

Identifying Belief-Based Targets for the Promotion of Leisure-Time Walking

ERIC Educational Resources Information Center

Rhodes, Ryan E.; Blanchard, Chris M.; Courneya, Kerry S.; Plotnikoff, Ronald C.

2009-01-01

Walking is the most common type of physical activity (PA) and the likely target of efforts to increase PA. No studies, however, have identified the belief-level correlates for walking using the theory of planned behavior. This study elicits salient beliefs about walking and evaluates beliefs that may be most important for walking-promotion…

Transcriptome Analysis in Prenatal IGF1-Deficient Mice Identifies Molecular Pathways and Target Genes Involved in Distal Lung Differentiation

PubMed Central

Hernández-Porras, Isabel; López, Icíar Paula; De Las Rivas, Javier; Pichel, José García

2013-01-01

Background Insulin-like Growth Factor 1 (IGF1) is a multifunctional regulator of somatic growth and development throughout evolution. IGF1 signaling through IGF type 1 receptor (IGF1R) controls cell proliferation, survival and differentiation in multiple cell types. IGF1 deficiency in mice disrupts lung morphogenesis, causing altered prenatal pulmonary alveologenesis. Nevertheless, little is known about the cellular and molecular basis of IGF1 activity during lung development. Methods/Principal Findings Prenatal Igf1−/− mutant mice with a C57Bl/6J genetic background displayed severe disproportional lung hypoplasia, leading to lethal neonatal respiratory distress. Immuno-histological analysis of their lungs showed a thickened mesenchyme, alterations in extracellular matrix deposition, thinner smooth muscles and dilated blood vessels, which indicated immature and delayed distal pulmonary organogenesis. Transcriptomic analysis of Igf1−/− E18.5 lungs using RNA microarrays identified deregulated genes related to vascularization, morphogenesis and cellular growth, and to MAP-kinase, Wnt and cell-adhesion pathways. Up-regulation of immunity-related genes was verified by an increase in inflammatory markers. Increased expression of Nfib and reduced expression of Klf2, Egr1 and Ctgf regulatory proteins as well as activation of ERK2 MAP-kinase were corroborated by Western blot. Among IGF-system genes only IGFBP2 revealed a reduction in mRNA expression in mutant lungs. Immuno-staining patterns for IGF1R and IGF2, similar in both genotypes, correlated to alterations found in specific cell compartments of Igf1−/− lungs. IGF1 addition to Igf1−/− embryonic lungs cultured ex vivo increased airway septa remodeling and distal epithelium maturation, processes accompanied by up-regulation of Nfib and Klf2 transcription factors and Cyr61 matricellular protein. Conclusions/Significance We demonstrated the functional tissue specific implication of IGF1 on fetal lung

Growth hormone is a cellular senescence target in pituitary and nonpituitary cells

PubMed Central

Chesnokova, Vera; Zhou, Cuiqi; Ben-Shlomo, Anat; Zonis, Svetlana; Tani, Yuji; Ren, Song-Guang; Melmed, Shlomo

2013-01-01

Premature proliferative arrest in benign or early-stage tumors induced by oncoproteins, chromosomal instability, or DNA damage is associated with p53/p21 activation, culminating in either senescence or apoptosis, depending on cell context. Growth hormone (GH) elicits direct peripheral metabolic actions as well as growth effects mediated by insulin-like growth factor 1 (IGF1). Locally produced peripheral tissue GH, in contrast to circulating pituitary-derived endocrine GH, has been proposed to be both proapoptotic and prooncogenic. Pituitary adenomas expressing and secreting GH are invariably benign and exhibit DNA damage and a senescent phenotype. We therefore tested effects of nutlin-induced p53-mediated senescence in rat and human pituitary cells. We show that DNA damage senescence induced by nutlin triggers the p53/p21 senescent pathway, with subsequent marked induction of intracellular pituitary GH in vitro. In contrast, GH is not induced in cells devoid of p53. Furthermore we show that p53 binds specific GH promoter motifs and enhances GH transcription and secretion in senescent pituitary adenoma cells and also in nonpituitary (human breast and colon) cells. In vivo, treatment with nutlin results in up-regulation of both p53 and GH in the pituitary gland, as well as increased GH expression in nonpituitary tissues (lung and liver). Intracrine GH acts in pituitary cells as an apoptosis switch for p53-mediated senescence, likely protecting the pituitary adenoma from progression to malignancy. Unlike in the pituitary, in nonpituitary cells GH exerts antiapoptotic properties. Thus, the results show that GH is a direct p53 transcriptional target and fulfills criteria as a p53 target gene. Induced GH is a readily measurable cell marker for p53-mediated cellular senescence. PMID:23940366

Navigating novel mechanisms of cellular plasticity with the NAD+ precursor and nutrient nicotinamide.

PubMed

Li, Faqi; Chong, Zhao Zhong; Maiese, Kenneth

2004-09-01

Interest in neuroprotectants for the central nervous system continues to garner significant attention. Nicotinamide, the amide form of niacin (vitamin B3), is the precursor for the coenzyme beta-nicotinamide adenine dinucleotide (NAD+) and is considered to be necessary for cellular function and metabolism. However, recent work has focused on the development of nicotinamide as a novel agent that is critical for modulating cellular plasticity, longevity, and inflammatory microglial function. The ability of nicotinamide to preserve both neuronal and vascular cell populations in the brain during injury is intriguing, but further knowledge of the specific cellular mechanisms that determine protection by this agent is required. The capacity of nicotinamide to govern not only intrinsic cellular integrity, but also extrinsic cellular inflammation rests with the modulation of a host of cellular targets that involve protein kinase B, glycogen synthase kinase-3 beta (GSK-3 beta), Forkhead transcription factors, mitochondrial dysfunction, poly(ADP-ribose) polymerase, cysteine proteases, and microglial activation. Intimately tied to the cytoprotection of nicotinamide is the modulation of an early and late phase of apoptotic injury that is triggered by the loss of membrane asymmetry. Identifying robust cytoprotective agents as nicotinamide in conjunction with the elucidation of the cellular mechanisms responsible for cell survival will continue to solidify the development of therapeutic strategies against neurodegenerative diseases

Targeting distinct myeloid cell populations in vivo using polymers, liposomes and microbubbles.

PubMed

Ergen, Can; Heymann, Felix; Al Rawashdeh, Wa'el; Gremse, Felix; Bartneck, Matthias; Panzer, Ulf; Pola, Robert; Pechar, Michal; Storm, Gert; Mohr, Nicole; Barz, Matthias; Zentel, Rudolf; Kiessling, Fabian; Trautwein, Christian; Lammers, Twan; Tacke, Frank

2017-01-01

Identifying intended or accidental cellular targets for drug delivery systems is highly relevant for evaluating therapeutic and toxic effects. However, limited knowledge exists on the distribution of nano- and micrometer-sized carrier systems at the cellular level in different organs. We hypothesized that clinically relevant carrier materials, differing in composition and size, are able to target distinct myeloid cell subsets that control inflammatory processes, such as macrophages, neutrophils, monocytes and dendritic cells. Therefore, we analyzed the biodistribution and in vivo cellular uptake of intravenously injected poly(N-(2-hydroxypropyl) methacrylamide) polymers, PEGylated liposomes and poly(butyl cyanoacrylate) microbubbles in mice, using whole-body imaging (computed tomography - fluorescence-mediated tomography), intra-organ imaging (intravital multi-photon microscopy) and cellular analysis (flow cytometry of blood, liver, spleen, lung and kidney). While the three carrier materials shared accumulation in tissue macrophages in liver and spleen, they notably differed in uptake by other myeloid subsets. Kupffer cells and splenic red pulp macrophages rapidly take up microbubbles. Liposomes efficiently reach dendritic cells in liver, lung and kidney. Polymers exhibit the longest circulation half-life and target endothelial cells in the liver, neutrophils and alveolar macrophages. The identification of such previously unrecognized target cell populations might open up new avenues for more efficient drug delivery. Copyright © 2016 Elsevier Ltd. All rights reserved.

Growth cone travel in space and time: the cellular ensemble of cytoskeleton, adhesion, and membrane.

PubMed

Vitriol, Eric A; Zheng, James Q

2012-03-22

Growth cones, found at the tip of axonal projections, are the sensory and motile organelles of developing neurons that enable axon pathfinding and target recognition for precise wiring of the neural circuitry. To date, many families of conserved guidance molecules and their corresponding receptors have been identified that work in space and time to ensure billions of axons to reach their targets. Research in the past two decades has also gained significant insight into the ways in which growth cones translate extracellular signals into directional migration. This review aims to examine new progress toward understanding the cellular mechanisms underlying directional motility of the growth cone and to discuss questions that remain to be addressed. Specifically, we will focus on the cellular ensemble of cytoskeleton, adhesion, and membrane and examine how the intricate interplay between these processes orchestrates the directed movement of growth cones. Copyright © 2012 Elsevier Inc. All rights reserved.

MEDICI: Mining Essentiality Data to Identify Critical Interactions for Cancer Drug Target Discovery and Development | Office of Cancer Genomics

Cancer.gov

Protein-protein interactions (PPIs) mediate the transmission and regulation of oncogenic signals that are essential to cellular proliferation and survival, and thus represent potential targets for anti-cancer therapeutic discovery. Despite their significance, there is no method to experimentally disrupt and interrogate the essentiality of individual endogenous PPIs. The ability to computationally predict or infer PPI essentiality would help prioritize PPIs for drug discovery and help advance understanding of cancer biology.

Targeting cellular and molecular drivers of head and neck squamous cell carcinoma: current options and emerging perspectives

PubMed Central

Ausoni, Simonetta; Boscolo-Rizzo, Paolo; Singh, Bhuvanesh; da Mosto, Maria Cristina; Spinato, Giacomo; Tirelli, Giancarlo; Spinato, Roberto; Azzarello, Giuseppe

2017-01-01

Despite improvements in functional outcomes attributable to advances in radiotherapy, chemotherapy, surgical techniques, and imaging techniques, survival in head and neck squamous cell carcinoma (HNSCC) patients has improved only marginally during the last couple of decades, and optimal therapy has yet to be devised. Genomic complexity and intratumoral genetic heterogeneity may contribute to treatment resistance and the propensity for locoregional recurrence. Countering this, demands a significant effort from both basic and clinical scientists in the search for more-effective targeted therapies. Recent genomewide studies have provided valuable insights into the genetic basis of HNSCC, uncovering potential new therapeutic opportunities. In addition, several studies have elucidated how inflammatory, immune, and stromal cells contribute to the particular properties of these neoplasms. In the present review, we introduce recent findings on genomic aberrations resulting from whole-genome sequencing of HNSCC, we discuss how the particular microenvironment affects the pathogenesis of this disease, and we describe clinical trials exploring new perspectives on the use of combined genetic and cellular targeted therapies. PMID:27194534

In silico molecular comparisons of C. elegans and mammalian pharmacology identify distinct targets that regulate feeding.

PubMed

Lemieux, George A; Keiser, Michael J; Sassano, Maria F; Laggner, Christian; Mayer, Fahima; Bainton, Roland J; Werb, Zena; Roth, Bryan L; Shoichet, Brian K; Ashrafi, Kaveh

2013-11-01

Phenotypic screens can identify molecules that are at once penetrant and active on the integrated circuitry of a whole cell or organism. These advantages are offset by the need to identify the targets underlying the phenotypes. Additionally, logistical considerations limit screening for certain physiological and behavioral phenotypes to organisms such as zebrafish and C. elegans. This further raises the challenge of elucidating whether compound-target relationships found in model organisms are preserved in humans. To address these challenges we searched for compounds that affect feeding behavior in C. elegans and sought to identify their molecular mechanisms of action. Here, we applied predictive chemoinformatics to small molecules previously identified in a C. elegans phenotypic screen likely to be enriched for feeding regulatory compounds. Based on the predictions, 16 of these compounds were tested in vitro against 20 mammalian targets. Of these, nine were active, with affinities ranging from 9 nM to 10 µM. Four of these nine compounds were found to alter feeding. We then verified the in vitro findings in vivo through genetic knockdowns, the use of previously characterized compounds with high affinity for the four targets, and chemical genetic epistasis, which is the effect of combined chemical and genetic perturbations on a phenotype relative to that of each perturbation in isolation. Our findings reveal four previously unrecognized pathways that regulate feeding in C. elegans with strong parallels in mammals. Together, our study addresses three inherent challenges in phenotypic screening: the identification of the molecular targets from a phenotypic screen, the confirmation of the in vivo relevance of these targets, and the evolutionary conservation and relevance of these targets to their human orthologs.

Viperin targets flavivirus virulence by inducing assembly of non-infectious capsid particles.

PubMed

Vonderstein, Kirstin; Nilsson, Emma; Hubel, Philipp; Nygård Skalman, Lars; Upadhyay, Arunkumar; Pasto, Jenny; Pichlmair, Andreas; Lundmark, Richard; Överby, Anna K

2017-10-18

Efficient antiviral immunity requires interference with virus replication at multiple layers targeting diverse steps in the viral life cycle. Here we describe a novel flavivirus inhibition mechanism that results in interferon-mediated obstruction of tick-borne encephalitis virus particle assembly, and involves release of malfunctional membrane associated capsid (C) particles. This mechanism is controlled by the activity of the interferon-induced protein viperin, a broad spectrum antiviral interferon stimulated gene. Through analysis of the viperin-interactome, we identified the Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1), as the cellular protein targeted by viperin. Viperin-induced antiviral activity as well as C-particle release was stimulated by GBF1 inhibition and knock down, and reduced by elevated levels of GBF1. Our results suggest that viperin targets flavivirus virulence by inducing the secretion of unproductive non-infectious virus particles, by a GBF1-dependent mechanism. This yet undescribed antiviral mechanism allows potential therapeutic intervention. Importance The interferon response can target viral infection on almost every level, however, very little is known about interference of flavivirus assembly. Here we show that interferon, through the action of viperin, can disturb assembly of tick-borne encephalitis virus. The viperin protein is highly induced after viral infection and exhibit broad-spectrum antiviral activity. However, the mechanism of action is still elusive and appear to vary between the different viruses, indicating that cellular targets utilized by several viruses might be involved. In this study we show that viperin induce capsid particle release by interacting and inhibiting the function of the cellular protein Golgi Brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1). GBF1 is a key protein in the cellular secretory pathway and essential in the life cycle of many viruses, also targeted by

Novel target for high-risk neuroblastoma identified in pre-clinical research | Center for Cancer Research

Cancer.gov

Pre-clinical research by investigators at the Center for Cancer Research and their colleagues have identified a number of novel epigenetic targets for high-risk neuroblastoma and validated a promising new targeted inhibitor in pre-clinical models.  Read more...

GRIL-Seq, a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

PubMed Central

Han, Kook; Tjaden, Brian; Lory, Stephen

2017-01-01

The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base-pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique (referred to as GRIL-Seq) is based on preferential ligation of sRNAs to ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimeras. In addition to the RNA chaperone Hfq, the GRIL-Seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrate that direct regulatory targets of this sRNA can be readily identified. Therefore, GRIL-Seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but can also result in uncovering novel roles for sRNAs and their targets in complex regulatory networks. PMID:28005055

Models for discovery of targeted therapy in genetic epileptic encephalopathies.

PubMed

Maljevic, Snezana; Reid, Christopher A; Petrou, Steven

2017-10-01

Epileptic encephalopathies are severe disorders emerging in the first days to years of life that commonly include refractory seizures, various types of movement disorders, and different levels of developmental delay. In recent years, many de novo occurring variants have been identified in individuals with these devastating disorders. To unravel disease mechanisms, the functional impact of detected variants associated with epileptic encephalopathies is investigated in a range of cellular and animal models. This review addresses efforts to advance and use such models to identify specific molecular and cellular targets for the development of novel therapies. We focus on ion channels as the best-studied group of epilepsy genes. Given the clinical and genetic heterogeneity of epileptic encephalopathy disorders, experimental models that can reflect this complexity are critical for the development of disease mechanisms-based targeted therapy. The convergence of technological advances in gene sequencing, stem cell biology, genome editing, and high throughput functional screening together with massive unmet clinical needs provides unprecedented opportunities and imperatives for precision medicine in epileptic encephalopathies. © 2017 International Society for Neurochemistry.

Using Bioinformatic Approaches to Identify Pathways Targeted by Human Leukemogens

PubMed Central

Thomas, Reuben; Phuong, Jimmy; McHale, Cliona M.; Zhang, Luoping

2012-01-01

We have applied bioinformatic approaches to identify pathways common to chemical leukemogens and to determine whether leukemogens could be distinguished from non-leukemogenic carcinogens. From all known and probable carcinogens classified by IARC and NTP, we identified 35 carcinogens that were associated with leukemia risk in human studies and 16 non-leukemogenic carcinogens. Using data on gene/protein targets available in the Comparative Toxicogenomics Database (CTD) for 29 of the leukemogens and 11 of the non-leukemogenic carcinogens, we analyzed for enrichment of all 250 human biochemical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The top pathways targeted by the leukemogens included metabolism of xenobiotics by cytochrome P450, glutathione metabolism, neurotrophin signaling pathway, apoptosis, MAPK signaling, Toll-like receptor signaling and various cancer pathways. The 29 leukemogens formed 18 distinct clusters comprising 1 to 3 chemicals that did not correlate with known mechanism of action or with structural similarity as determined by 2D Tanimoto coefficients in the PubChem database. Unsupervised clustering and one-class support vector machines, based on the pathway data, were unable to distinguish the 29 leukemogens from 11 non-leukemogenic known and probable IARC carcinogens. However, using two-class random forests to estimate leukemogen and non-leukemogen patterns, we estimated a 76% chance of distinguishing a random leukemogen/non-leukemogen pair from each other. PMID:22851955

Combining functional genomics and chemical biology to identify targets of bioactive compounds.

PubMed

Ho, Cheuk Hei; Piotrowski, Jeff; Dixon, Scott J; Baryshnikova, Anastasia; Costanzo, Michael; Boone, Charles

2011-02-01

Genome sequencing projects have revealed thousands of suspected genes, challenging researchers to develop efficient large-scale functional analysis methodologies. Determining the function of a gene product generally requires a means to alter its function. Genetically tractable model organisms have been widely exploited for the isolation and characterization of activating and inactivating mutations in genes encoding proteins of interest. Chemical genetics represents a complementary approach involving the use of small molecules capable of either inactivating or activating their targets. Saccharomyces cerevisiae has been an important test bed for the development and application of chemical genomic assays aimed at identifying targets and modes of action of known and uncharacterized compounds. Here we review yeast chemical genomic assays strategies for drug target identification. Copyright © 2010 Elsevier Ltd. All rights reserved.

Targeted Cellular Drug Delivery using Tailored Dendritic Nanostructures

NASA Astrophysics Data System (ADS)

Kannan, Rangaramanujam; Kolhe, Parag; Kannan, Sujatha; Lieh-Lai, Mary

2002-03-01

Dendrimers and hyperbranched polymers possess highly branched architectures, with a large number of controllable, tailorble, ‘peripheral’ functionalities. Since the surface chemistry of these materials can be modified with relative ease, these materials have tremendous potential in targeted drug and gene delivery. The large number of end groups can also be tailored to create special affinity to targeted cells, and can also encapsulate drugs and deliver them in a controlled manner. We are developing tailor-modified dendritic systems for drug delivery. Synthesis, in-vitro drug loading, in-vitro drug delivery, and the targeting efficiency to the cell are being studied systematically using a wide variety of experimental tools. Polyamidoamine and Polyol dendrimers, with different generations and end-groups are studied, with drugs such as Ibuprofen and Methotrexate. Our results indicate that a large number of drug molecules can be encapsulated/attached to the dendrimers, depending on the end groups. The drug-encapsulated dendrimer is able to enter the cells rapidly and deliver the drug. Targeting strategies being explored

Genomic Target Database (GTD): A database of potential targets in human pathogenic bacteria

PubMed Central

Barh, Debmalya; Kumar, Anil; Misra, Amarendra Narayana

2009-01-01

A Genomic Target Database (GTD) has been developed having putative genomic drug targets for human bacterial pathogens. The selected pathogens are either drug resistant or vaccines are yet to be developed against them. The drug targets have been identified using subtractive genomics approaches and these are subsequently classified into Drug targets in pathogen specific unique metabolic pathways,Drug targets in host-pathogen common metabolic pathways, andMembrane localized drug targets. HTML code is used to link each target to its various properties and other available public resources. Essential resources and tools for subtractive genomic analysis, sub-cellular localization, vaccine and drug designing are also mentioned. To the best of authors knowledge, no such database (DB) is presently available that has listed metabolic pathways and membrane specific genomic drug targets based on subtractive genomics. Listed targets in GTD are readily available resource in developing drug and vaccine against the respective pathogen, its subtypes, and other family members. Currently GTD contains 58 drug targets for four pathogens. Shortly, drug targets for six more pathogens will be listed. Availability GTD is available at IIOAB website http://www.iioab.webs.com/GTD.htm. It can also be accessed at http://www.iioabdgd.webs.com.GTD is free for academic research and non-commercial use only. Commercial use is strictly prohibited without prior permission from IIOAB. PMID:20011153

Cellular and Molecular Targets of Menthol Actions

PubMed Central

Oz, Murat; El Nebrisi, Eslam G.; Yang, Keun-Hang S.; Howarth, Frank C.; Al Kury, Lina T.

2017-01-01

Menthol belongs to monoterpene class of a structurally diverse group of phytochemicals found in plant-derived essential oils. Menthol is widely used in pharmaceuticals, confectionary, oral hygiene products, pesticides, cosmetics, and as a flavoring agent. In addition, menthol is known to have antioxidant, anti-inflammatory, and analgesic effects. Recently, there has been renewed awareness in comprehending the biological and pharmacological effects of menthol. TRP channels have been demonstrated to mediate the cooling actions of menthol. There has been new evidence demonstrating that menthol can significantly influence the functional characteristics of a number of different kinds of ligand and voltage-gated ion channels, indicating that at least some of the biological and pharmacological effects of menthol can be mediated by alterations in cellular excitability. In this article, we examine the results of earlier studies on the actions of menthol with voltage and ligand-gated ion channels. PMID:28769802

Distinguishing between biochemical and cellular function: Are there peptide signatures for cellular function of proteins?

PubMed

Jain, Shruti; Bhattacharyya, Kausik; Bakshi, Rachit; Narang, Ankita; Brahmachari, Vani

2017-04-01

The genome annotation and identification of gene function depends on conserved biochemical activity. However, in the cell, proteins with the same biochemical function can participate in different cellular pathways and cannot complement one another. Similarly, two proteins of very different biochemical functions are put in the same class of cellular function; for example, the classification of a gene as an oncogene or a tumour suppressor gene is not related to its biochemical function, but is related to its cellular function. We have taken an approach to identify peptide signatures for cellular function in proteins with known biochemical function. ATPases as a test case, we classified ATPases (2360 proteins) and kinases (517 proteins) from the human genome into different cellular function categories such as transcriptional, replicative, and chromatin remodelling proteins. Using publicly available tool, MEME, we identify peptide signatures shared among the members of a given category but not between cellular functional categories; for example, no motif sharing is seen between chromatin remodelling and transporter ATPases, similarly between receptor Serine/Threonine Kinase and Receptor Tyrosine Kinase. There are motifs shared within each category with significant E value and high occurrence. This concept of signature for cellular function was applied to developmental regulators, the polycomb and trithorax proteins which led to the prediction of the role of INO80, a chromatin remodelling protein, in development. This has been experimentally validated earlier for its role in homeotic gene regulation and its interaction with regulatory complexes like the Polycomb and Trithorax complex. Proteins 2017; 85:682-693. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

RAGE is a key cellular target for Aβ-induced perturbation in Alzheimer's disease

PubMed Central

Yan, Shirley ShiDu; Chen, Doris; Yan, Shiqian; Guo, Lan; Chen, John Xi

2013-01-01

RAGE, a receptor for advanced glycation endproducts, is an immunoglobulin-like cell surface receptor that is often described as a pattern recognition receptor due to the structural heterogeneity of its ligand. RAGE is an important cellular cofactor for amyloid β-peptide (Aβ)-mediated cellular perturbation relevant to the pathogenesis of Alzheimer's disease (AD). The interaction of RAGE with Aβ in neurons, microglia, and vascular cells accelerates and amplifies deleterious effects on neuronal and synaptic function. RAGE-dependent signaling contributes to Aβ-mediated amyloid pathology and cognitive dysfunction observed in the AD mouse model. Blockade of RAGE significantly attenuates neuronal and synaptic injury. In this review, we summarize the role of RAGE in the pathogenesis of AD, specifically in Aβ-induced cellular perturbation. PMID:22202057

Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer

PubMed Central

Azorsa, David O; Gonzales, Irma M; Basu, Gargi D; Choudhary, Ashish; Arora, Shilpi; Bisanz, Kristen M; Kiefer, Jeffrey A; Henderson, Meredith C; Trent, Jeffrey M; Von Hoff, Daniel D; Mousses, Spyro

2009-01-01

Background Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement. Methods In order to improve gemcitabine response in pancreatic cancer cells, we utilized a synthetic lethal RNAi screen targeting 572 known kinases to identify genes that when silenced would sensitize pancreatic cancer cells to gemcitabine. Results Results from the RNAi screens identified several genes that, when silenced, potentiated the growth inhibitory effects of gemcitabine in pancreatic cancer cells. The greatest potentiation was shown by siRNA targeting checkpoint kinase 1 (CHK1). Validation of the screening results was performed in MIA PaCa-2 and BxPC3 pancreatic cancer cells by examining the dose response of gemcitabine treatment in the presence of either CHK1 or CHK2 siRNA. These results showed a three to ten-fold decrease in the EC50 for CHK1 siRNA-treated cells versus control siRNA-treated cells while treatment with CHK2 siRNA resulted in no change compared to controls. CHK1 was further targeted with specific small molecule inhibitors SB 218078 and PD 407824 in combination with gemcitabine. Results showed that treatment of MIA PaCa-2 cells with either of the CHK1 inhibitors SB 218078 or PD 407824 led to sensitization of the pancreatic cancer cells to gemcitabine. Conclusion These findings demonstrate the effectiveness of synthetic lethal RNAi screening as a tool for identifying sensitizing targets to chemotherapeutic agents. These results also indicate that CHK1 could serve as a putative therapeutic target for sensitizing pancreatic cancer cells to gemcitabine. PMID

GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation.

PubMed

Han, Kook; Tjaden, Brian; Lory, Stephen

2016-12-22

The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique, referred to as global small non-coding RNA target identification by ligation and sequencing (GRIL-seq), is based on preferential ligation of sRNAs to the ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimaeras. In addition to the RNA chaperone Hfq, the GRIL-seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrated that direct regulatory targets of this sRNA can readily be identified. Therefore, GRIL-seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but also for uncovering novel roles for sRNAs and their targets in complex regulatory networks.

Omen: identifying potential spear-phishing targets before the email is sent.

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

Wendt, Jeremy Daniel.

2013-07-01

We present the results of a two year project focused on a common social engineering attack method called "spear phishing". In a spear phishing attack, the user receives an email with information specifically focused on the user. This email contains either a malware-laced attachment or a link to download the malware that has been disguised as a useful program. Spear phishing attacks have been one of the most effective avenues for attackers to gain initial entry into a target network. This project focused on a proactive approach to spear phishing. To create an effective, user-specific spear phishing email, the attackermore » must research the intended recipient. We believe that much of the information used by the attacker is provided by the target organization's own external website. Thus when researching potential targets, the attacker leaves signs of his research in the webserver's logs. We created tools and visualizations to improve cybersecurity analysts' abilities to quickly understand a visitor's visit patterns and interests. Given these suspicious visitors and log-parsing tools, analysts can more quickly identify truly suspicious visitors, search for potential spear-phishing targeted users, and improve security around those users before the spear phishing email is sent.« less

Cellular sources and targets of IFN-gamma-mediated protection against viral demyelination and neurological deficits.

PubMed

Murray, Paul D; McGavern, Dorian B; Pease, Larry R; Rodriguez, Moses

2002-03-01

IFN-gamma is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-gamma, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler's virus model of multiple sclerosis. During viral infections, IFN-gamma is produced by natural killer (NK) cells, CD4(+) and CD8(+) T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-gamma-mediated protection. To determine the lymphocyte subsets that produce IFN-gamma to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-gamma-production. We demonstrate that IFN-gamma production by both CD4(+) and CD8(+) T cell subsets is critical for resistance to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4(+) T cells make a greater contribution to IFN-gamma-mediated protection. To determine the cellular targets of IFN-gamma-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-gamma receptor. We demonstrate that IFN-gamma receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination.

Cellular sources and targets of IFN-γ-mediated protection against viral demyelination and neurological deficits

PubMed Central

Murray, Paul D.; McGavern, Dorian B.; Pease, Larry R.; Rodriguez, Moses

2017-01-01

IFN-γ is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-γ, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler’s virus model of multiple sclerosis. During viral infections, IFN-γ is produced by natural killer (NK) cells, CD4+ and CD8+ T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-γ-mediated protection. To determine the lymphocyte subsets that produce IFN-γ to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-γ-production. We demonstrate that IFN-γ production by both CD4+ and CD8+ T cell subsets is critical for resistance to Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4+ T cells make a greater contribution to IFN-γ-mediated protection. To determine the cellular targets of IFN-γ-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-γ receptor. We demonstrate that IFN-γ receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination. PMID:11857334

Large-Scale Chemical Similarity Networks for Target Profiling of Compounds Identified in Cell-Based Chemical Screens

PubMed Central

Lo, Yu-Chen; Senese, Silvia; Li, Chien-Ming; Hu, Qiyang; Huang, Yong; Damoiseaux, Robert; Torres, Jorge Z.

2015-01-01

Target identification is one of the most critical steps following cell-based phenotypic chemical screens aimed at identifying compounds with potential uses in cell biology and for developing novel disease therapies. Current in silico target identification methods, including chemical similarity database searches, are limited to single or sequential ligand analysis that have limited capabilities for accurate deconvolution of a large number of compounds with diverse chemical structures. Here, we present CSNAP (Chemical Similarity Network Analysis Pulldown), a new computational target identification method that utilizes chemical similarity networks for large-scale chemotype (consensus chemical pattern) recognition and drug target profiling. Our benchmark study showed that CSNAP can achieve an overall higher accuracy (>80%) of target prediction with respect to representative chemotypes in large (>200) compound sets, in comparison to the SEA approach (60–70%). Additionally, CSNAP is capable of integrating with biological knowledge-based databases (Uniprot, GO) and high-throughput biology platforms (proteomic, genetic, etc) for system-wise drug target validation. To demonstrate the utility of the CSNAP approach, we combined CSNAP's target prediction with experimental ligand evaluation to identify the major mitotic targets of hit compounds from a cell-based chemical screen and we highlight novel compounds targeting microtubules, an important cancer therapeutic target. The CSNAP method is freely available and can be accessed from the CSNAP web server (http://services.mbi.ucla.edu/CSNAP/). PMID:25826798

Cellular Angiofibroma of the Nasopharynx.

PubMed

Erdur, Zülküf Burak; Yener, Haydar Murat; Yilmaz, Mehmet; Karaaltin, Ayşegül Batioğlu; Inan, Hakki Caner; Alaskarov, Elvin; Gozen, Emine Deniz

2017-11-01

Angiofibroma is a common tumor of the nasopharynx region but cellular type is extremely rare in head and neck. A 13-year-old boy presented with frequent epistaxis and nasal obstruction persisting for 6 months. According to the clinical symptoms and imaging studies juvenile angiofibroma was suspected. Following angiographic embolization total excision of the lesion by midfacial degloving approach was performed. Histological examination revealed that the tumor consisted of staghorn blood vessels and irregular fibrous stroma. Stellate fibroblasts with small pyknotic to large vesicular nuclei were seen in a highly cellular stroma. These findings identified cellular angiofibroma mimicking juvenile angiofibroma. This article is about a very rare patient of cellular angiofibroma of nasopharynx.

A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18*

PubMed Central

Yang, Zhaoshou; Hou, Yongheng; Hao, Taofang; Rho, Hee-Sool; Wan, Jun; Luan, Yizhao; Gao, Xin; Yao, Jianping; Pan, Aihua; Xie, Zhi; Qian, Jiang; Liao, Wanqin; Zhu, Heng; Zhou, Xingwang

2017-01-01

Toxoplasma kinase ROP18 is a key molecule responsible for the virulence of Toxoplasma gondii; however, the mechanisms by which ROP18 exerts parasite virulence via interaction with host proteins remain limited to a small number of identified substrates. To identify a broader array of ROP18 substrates, we successfully purified bioactive mature ROP18 and used it to probe a human proteome array. Sixty eight new putative host targets were identified. Functional annotation analysis suggested that these proteins have a variety of functions, including metabolic process, kinase activity and phosphorylation, cell growth, apoptosis and cell death, and immunity, indicating a pleiotropic role of ROP18 kinase. Among these proteins, four candidates, p53, p38, UBE2N, and Smad1, were further validated. We demonstrated that ROP18 targets p53, p38, UBE2N, and Smad1 for degradation. Importantly, we demonstrated that ROP18 phosphorylates Smad1 Ser-187 to trigger its proteasome-dependent degradation. Further functional characterization of the substrates of ROP18 may enhance understanding of the pathogenesis of Toxoplasma infection and provide new therapeutic targets. Similar strategies could be used to identify novel host targets for other microbial kinases functioning at the pathogen-host interface. PMID:28087594

Targeted Approach to Identify Genetic Loci Associated with ...

EPA Pesticide Factsheets

Extreme tolerance to highly toxic dioxin-like contaminants (DLCs) has evolved independently and contemporaneously in (at least) four populations of Atlantic killifish (Fundulus heteroclitus). Surprisingly, the magnitude and phenotype of DLC tolerance is similar among these killifish populations that have adapted to varied, but highly contaminated urban/industrialized estuaries of the US Atlantic coast. We hypothesized that comparisons among tolerant populations and in contrast to their sensitive neighboring killifish might reveal genetic loci associated with DLC tolerance. Since the aryl hydrocarbon receptor (AHR) pathway partly or fully mediates DLC toxicity in vertebrates, we identified single nucleotide polymorphisms (SNPs) from 43 genes associated with the AHR to serve as targeted markers. Wild fish from the four highly tolerant killifish populations and four nearby sensitive populations were genotyped using 59 SNP markers. Consistent with other killifish population genetic analyses, our results revealed strong genetic differentiation among populations, consistent with isolation by distance models. Pairwise comparisons of nearby tolerant and sensitive populations revealed differentiation among these loci: AHR 1 and 2, cathepsin Z, the cytochrome P450s (CYP) 1A and 3A30, and the NADH ubiquinone oxidoreductase MLRQ subunit. By grouping tolerant versus sensitive populations, we also identified cytochrome P450 1A and the AHR2 loci as under selection, lend

Targeted proteomics identifies liquid-biopsy signatures for extracapsular prostate cancer

PubMed Central

Kim, Yunee; Jeon, Jouhyun; Mejia, Salvador; Yao, Cindy Q; Ignatchenko, Vladimir; Nyalwidhe, Julius O; Gramolini, Anthony O; Lance, Raymond S; Troyer, Dean A; Drake, Richard R; Boutros, Paul C; Semmes, O. John; Kislinger, Thomas

2016-01-01

Biomarkers are rapidly gaining importance in personalized medicine. Although numerous molecular signatures have been developed over the past decade, there is a lack of overlap and many biomarkers fail to validate in independent patient cohorts and hence are not useful for clinical application. For these reasons, identification of novel and robust biomarkers remains a formidable challenge. We combine targeted proteomics with computational biology to discover robust proteomic signatures for prostate cancer. Quantitative proteomics conducted in expressed prostatic secretions from men with extraprostatic and organ-confined prostate cancers identified 133 differentially expressed proteins. Using synthetic peptides, we evaluate them by targeted proteomics in a 74-patient cohort of expressed prostatic secretions in urine. We quantify a panel of 34 candidates in an independent 207-patient cohort. We apply machine-learning approaches to develop clinical predictive models for prostate cancer diagnosis and prognosis. Our results demonstrate that computationally guided proteomics can discover highly accurate non-invasive biomarkers. PMID:27350604

CD9 monoclonal antibody-conjugated PEGylated liposomes for targeted delivery of rapamycin in the treatment of cellular senescence

NASA Astrophysics Data System (ADS)

Thuy Nguyen, Hanh; Thapa, Raj Kumar; Shin, Beom Soo; Jeong, Jee-Heon; Kim, Jae-Ryong; Yong, Chul Soon; Kim, Jong Oh

2017-03-01

Premature cellular senescence refers to the state of irreversible cell cycle arrest due to DNA damage or other stresses. In this study, CD9 monoclonal antibody (CD9mAb) was successfully conjugated to the surface of PEGylated liposomes for targeted delivery of rapamycin (LR-CD9mAb) to overcome senescence of CD9 receptor-overexpressing cells. LR-CD9mAb has a small particle size (143.3 ± 2.4 nm), narrow size distribution (polydispersity index: 0.220 ± 0.036), and negative zeta potential (-14.6 ± 1.2 mV). The uptake of CD9-targeted liposomes by premature senescent human dermal fibroblasts (HDFs) was higher than that by young HDFs, as displayed by confocal microscopic images. The senescence might not be reversed by treatment with rapamycin; however, the drug promoted cell proliferation and reduced the number of cells that expressed the senescence-associated-β-galactosidase (SA-β-gal). These effects were further confirmed by cell viability, cell cycle, and Western blotting analyses. Moreover, CD9-targeted liposomes showed better anti-senescence activity, in comparison with free rapamycin or the conventional liposomal formulation, suggesting the potential application of this system in further in vivo studies.

Identifying antimalarial compounds targeting dihydrofolate reductase-thymidylate synthase (DHFR-TS) by chemogenomic profiling.

PubMed

Aroonsri, Aiyada; Akinola, Olugbenga; Posayapisit, Navaporn; Songsungthong, Warangkhana; Uthaipibull, Chairat; Kamchonwongpaisan, Sumalee; Gbotosho, Grace O; Yuthavong, Yongyuth; Shaw, Philip J

2016-07-01

The mode of action of many antimalarial drugs is unknown. Chemogenomic profiling is a powerful method to address this issue. This experimental approach entails disruption of gene function and phenotypic screening for changes in sensitivity to bioactive compounds. Here, we describe the application of reverse genetics for chemogenomic profiling in Plasmodium. Plasmodium falciparum parasites harbouring a transgenic insertion of the glmS ribozyme downstream of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene were used for chemogenomic profiling of antimalarial compounds to identify those which target DHFR-TS. DHFR-TS expression can be attenuated by exposing parasites to glucosamine. Parasites with attenuated DHFR-TS expression were significantly more sensitive to antifolate drugs known to target DHFR-TS. In contrast, no change in sensitivity to other antimalarial drugs with different modes of action was observed. Chemogenomic profiling was performed using the Medicines for Malaria Venture (Switzerland) Malaria Box compound library, and two compounds were identified as novel DHFR-TS inhibitors. We also tested the glmS ribozyme in Plasmodium berghei, a rodent malaria parasite. The expression of reporter genes with downstream glmS ribozyme could be attenuated in transgenic parasites comparable with that obtained in P. falciparum. The chemogenomic profiling method was applied in a P. berghei line expressing a pyrimethamine-resistant Toxoplasma gondii DHFR-TS reporter gene under glmS ribozyme control. Parasites with attenuated expression of this gene were significantly sensitised to antifolates targeting DHFR-TS, but not other drugs with different modes of action. In conclusion, these data show that the glmS ribozyme reverse genetic tool can be applied for identifying primary targets of antimalarial compounds in human and rodent malaria parasites. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Enhanced cellular uptake of LHRH-conjugated PEG-coated magnetite nanoparticles for specific targeting of triple negative breast cancer cells.

PubMed

Hu, J; Obayemi, J D; Malatesta, K; Košmrlj, A; Soboyejo, W O

2018-07-01

Targeted therapy is an emerging technique in cancer detection and treatment. This paper presents the results of a combined experimental and theoretical study of the specific targeting and entry of luteinizing hormone releasing hormone (LHRH)-conjugated PEG-coated magnetite nanoparticles into triple negative breast cancer (TNBC) cells and normal breast cells. The conjugated nanoparticles structures, cellular uptake of PEG-coated magnetite nanoparticles (MNPs) and LHRH-conjugated PEG-coated magnetite nanoparticles (LHRH-MNPs) into breast cancer cells and normal breast cells were investigated using a combination of transmission electron microscope, optical and confocal fluorescence microscopy techniques. The results show that the presence of LHRH enhances the uptake of LHRH-MNPs into TNBC cells. Nanoparticle entry into breast cancer cells is also studied using a combination of thermodynamics and kinetics models. The trends in the predicted nanoparticle entry times (into TNBC cells) and the size ranges of the engulfed nanoparticles (within the TNBC cells) are shown to be consistent with experimental observations. The implications of the results are then discussed for the specific targeting of TNBCs with LHRH-conjugated PEG-coated magnetite nanoparticles for the early detection and treatment of TNBC. Copyright © 2018. Published by Elsevier B.V.

Synthesis, characterization and target protein binding of drug-conjugated quantum dots in vitro and in living cells

NASA Astrophysics Data System (ADS)

Choi, Youngseon; Kim, Minjung; Cho, Yoojin; Yun, Eunsuk; Song, Rita

2013-02-01

Elucidation of unknown target proteins of a drug is of great importance in understanding cell biology and drug discovery. There have been extensive studies to discover and identify target proteins in the cell. Visualization of targets using drug-conjugated probes has been an important approach to gathering mechanistic information of drug action at the cellular level. As quantum dot (QD) nanocrystals have attracted much attention as a fluorescent probe in the bioimaging area, we prepared drug-conjugated QD to explore the potential of target discovery. As a model drug, we selected a well-known anticancer drug, methotrexate (MTX), which has been known to target dihydrofolate reductase (DHFR) with high affinity binding (Kd = 0.54 nM). MTX molecules were covalently attached to amino-PEG-polymer-coated QDs. Specific interactions of MTX-conjugated QDs with DHFR were identified using agarose gel electrophoresis and fluorescence microscopy. Cellular uptake of the MTX-conjugated QDs in living CHO cells was investigated with regard to their localization and distribution pattern. MTX-QD was found to be internalized into the cells via caveolae-medicated endocytosis without significant sequestration in endosomes. A colocalization experiment of the MTX-QD conjugate with antiDHFR-TAT-QD also confirmed that MTX-QD binds to the target DHFR. This study showed the potential of the drug-QD conjugate to identify or visualize drug-target interactions in the cell, which is currently of great importance in the area of drug discovery and chemical biology.

Aptamers as tools for target prioritization and lead identification.

PubMed

Burgstaller, Petra; Girod, Anne; Blind, Michael

2002-12-15

The increasing number of potential drug target candidates has driven the development of novel technologies designed to identify functionally important targets and enhance the subsequent lead discovery process. Highly specific synthetic nucleic acid ligands--also known as aptamers--offer a new exciting route in the drug discovery process by linking target validation directly with HTS. Recently, aptamers have proven to be valuable tools for modulating the function of endogenous cellular proteins in their natural environment. A set of technologies has been developed to use these sophisticated ligands for the validation of potential drug targets in disease models. Moreover, aptamers that are specific antagonists of protein function can act as substitute interaction partners in HTS assays to facilitate the identification of small-molecule lead compounds.

Cellular Homeostasis and Aging.

PubMed

Hartl, F Ulrich

2016-06-02

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

RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing's sarcoma.

PubMed

Arora, Shilpi; Gonzales, Irma M; Hagelstrom, R Tanner; Beaudry, Christian; Choudhary, Ashish; Sima, Chao; Tibes, Raoul; Mousses, Spyro; Azorsa, David O

2010-08-18

Ewing's sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewing's sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewing's sarcoma cells. Four Ewing's sarcoma cell lines TC-32, TC-71, SK-ES-1 and RD-ES were tested in high throughput-RNAi screens using a siRNA library targeting 572 kinases. Knockdown of 25 siRNAs reduced the growth of all four Ewing's sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewing's sarcoma cells as compared to normal fibroblasts. Secondary validation and preliminary mechanistic studies highlighted the kinases STK10 and TNK2 as having important roles in growth and survival of Ewing's sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis. In summary, RNAi-based phenotypic profiling proved to be a powerful gene target discovery strategy, leading to successful identification and validation of STK10 and TNK2 as two novel potential therapeutic targets for Ewing's sarcoma.

Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach.

PubMed

Triboulet, Sarah; Aude-Garcia, Catherine; Armand, Lucie; Gerdil, Adèle; Diemer, Hélène; Proamer, Fabienne; Collin-Faure, Véronique; Habert, Aurélie; Strub, Jean-Marc; Hanau, Daniel; Herlin, Nathalie; Carrière, Marie; Van Dorsselaer, Alain; Rabilloud, Thierry

2014-06-07

Two different zinc oxide nanoparticles, as well as zinc ions, are used to study the cellular responses of the RAW 264 macrophage cell line. A proteomic screen is used to provide a wide view of the molecular effects of zinc, and the most prominent results are cross-validated by targeted studies. Furthermore, the alteration of important macrophage functions (e.g. phagocytosis) by zinc is also investigated. The intracellular dissolution/uptake of zinc is also studied to further characterize zinc toxicity. Zinc oxide nanoparticles dissolve readily in the cells, leading to high intracellular zinc concentrations, mostly as protein-bound zinc. The proteomic screen reveals a rather weak response in the oxidative stress response pathway, but a strong response both in the central metabolism and in the proteasomal protein degradation pathway. Targeted experiments confirm that carbohydrate catabolism and proteasome are critical determinants of sensitivity to zinc, which also induces DNA damage. Conversely, glutathione levels and phagocytosis appear unaffected at moderately toxic zinc concentrations.

Novel theranostic zinc phthalocyanine-phospholipid complex self-assembled nanoparticles for imaging-guided targeted photodynamic treatment with controllable ROS production and shape-assisted enhanced cellular uptake.

PubMed

Ma, Jinyuan; Li, Yang; Liu, Guihua; Li, Ai; Chen, Yilin; Zhou, Xinyi; Chen, Dengyue; Hou, Zhenqing; Zhu, Xuan

2018-02-01

The novel drug delivery system based on self-assembly of zinc phthalocyanine-soybean phosphatidylcholine (ZnPc-SPC) complex was developed by a co-solvent method followed by a nanoprecipitaion technique. DSPE-PEG-methotrexate (DSPE-PEG-MTX) was introduced on the surface of ZnPc-SPC self-assembled nanoparticles (ZS) to endow them with folate receptor-targeting property. NMR, XRD, FTIR, and UV-vis-NIR analysis demonstrated the weak molecular interaction between ZnPc and SPC. The ZS functionalized with DSPE-PEG-MTX (ZSPM) was successfully constructed with an average particle size of ∼170nm, a narrow size distribution, and could remain physiologically stable for at least 7days. In vitro cellular uptake and cytotoxicity studies demonstrated that ZSPM exhibited stronger cellular uptake efficacy and photodynamic cytotoxicity against HeLa and MCF-7 cells than ZS functionalized with DSPE-mPEG (ZSP) and free ZnPc. More importantly, ZSPM showed the enhanced accumulation effect at the tumor region compared with ZSP by the active-plus-passive targeting via enhanced permeability and retention (EPR) effect and folate receptor-mediated endocytosis. Furthermore, in vivo antitumor effect and histological analysis demonstrated the superior tumor growth inhibition effect of ZSPM. In addition, the needle-shape ZSP (ZSPN) exhibited better in vitro cellular uptake and in vivo tumor accumulation compared with ZSP due to the shape-assisted effect. Moreover, the interesting off-on switch effect of reactive oxygen species (ROS) production of ZnPc-SPC complex-based nanoparticles was discovered to achieve photodynamic treatment in a controllable way. These findings suggested that the ZnPc-SPC complex-based self-assembled nanoparticles could serve as a promising and effective formulation to achieve tumor-targeting fluorescence imaging and enhanced photodynamic treatment. Copyright © 2017. Published by Elsevier B.V.

A screen to identify drug resistant variants to target-directed anti-cancer agents

PubMed Central

Azam, Mohammad; Raz, Tal; Nardi, Valentina; Opitz, Sarah L.

2003-01-01

The discovery of oncogenes and signal transduction pathways important for mitogenesis has triggered the development of target-specific small molecule anti-cancer compounds. As exemplified by imatinib (Gleevec), a specific inhibitor of the Chronic Myeloid Leukemia (CML)-associated Bcr-Abl kinase, these agents promise impressive activity in clinical trials, with low levels of clinical toxicity. However, such therapy is susceptible to the emergence of drug resistance due to amino acid substitutions in the target protein. Defining the spectrum of such mutations is important for patient monitoring and the design of next-generation inhibitors. Using imatinib and BCR/ABL as a paradigm for a drug-target pair, we recently reported a retroviral vector-based screening strategy to identify the spectrum of resistance-conferring mutations. Here we provide a detailed methodology for the screen, which can be generally applied to any drug-target pair. PMID:14615817

Cellular Therapies Clinical Research Roadmap: lessons learned on how to move a cellular therapy into a clinical trial.

PubMed

Ouseph, Stacy; Tappitake, Darah; Armant, Myriam; Wesselschmidt, Robin; Derecho, Ivy; Draxler, Rebecca; Wood, Deborah; Centanni, John M

2015-04-01

A clinical research roadmap has been developed as a resource for researchers to identify critical areas and potential pitfalls when transitioning a cellular therapy product from the research laboratory, by means of an Investigational New Drug (IND) application, into early-phase clinical trials. The roadmap describes four key areas: basic and preclinical research, resource development, translational research and Good Manufacturing Practice (GMP) and IND assembly and submission. Basic and preclinical research identifies a new therapeutic concept and demonstrates its potential value with the use of a model of the relevant disease. During resource development, the appropriate specialists and the required expertise to bring this product into the clinic are identified (eg, researchers, regulatory specialists, GMP manufacturing staff, clinicians and clinical trials staff, etc). Additionally, the funds required to achieve this goal (or a plan to procure them) are identified. In the next phase, the plan to translate the research product into a clinical-grade therapeutic is developed. Finally regulatory approval to start the trial must be obtained. In the United States, this is done by filing an IND application with the Food and Drug Administration. The National Heart, Lung and Blood Institute-funded Production Assistance for Cellular Therapies program has facilitated the transition of a variety of cellular therapy products from the laboratory into Phase1/2 trials. The five Production Assistance for Cellular Therapies facilities have assisted investigators by performing translational studies and GMP manufacturing to ensure that cellular products met release specifications and were manufactured safely, reproducibly and at the appropriate scale. The roadmap resulting from this experience is the focus of this article. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Unbiased Combinatorial Genomic Approaches to Identify Alternative Therapeutic Targets within the TSC Signaling Network

DTIC Science & Technology

2015-09-01

assessed the specificity of mutation in Drosophila S2R+ cells. We generated a quantitative mutation reporter vector in which an sgRNA target sequence ...phosphatases (563 genes) in the Drosophila genome (Figure 4). 65 samples that displayed synthetic lethality (15 genes) or synthetic increases in viability...targeting all kinases and phosphatases (563 genes) in the Drosophila genome . . Identified three hits (mRNA-Cap, Pitslre and CycT) that scored as

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target.

PubMed

Manguso, Robert T; Pope, Hans W; Zimmer, Margaret D; Brown, Flavian D; Yates, Kathleen B; Miller, Brian C; Collins, Natalie B; Bi, Kevin; LaFleur, Martin W; Juneja, Vikram R; Weiss, Sarah A; Lo, Jennifer; Fisher, David E; Miao, Diana; Van Allen, Eliezer; Root, David E; Sharpe, Arlene H; Doench, John G; Haining, W Nicholas

2017-07-27

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

Sub-cellular mRNA localization modulates the regulation of gene expression by small RNAs in bacteria

NASA Astrophysics Data System (ADS)

Teimouri, Hamid; Korkmazhan, Elgin; Stavans, Joel; Levine, Erel

2017-10-01

Small non-coding RNAs can exert significant regulatory activity on gene expression in bacteria. In recent years, substantial progress has been made in understanding bacterial gene expression by sRNAs. However, recent findings that demonstrate that families of mRNAs show non-trivial sub-cellular distributions raise the question of how localization may affect the regulatory activity of sRNAs. Here we address this question within a simple mathematical model. We show that the non-uniform spatial distributions of mRNA can alter the threshold-linear response that characterizes sRNAs that act stoichiometrically, and modulate the hierarchy among targets co-regulated by the same sRNA. We also identify conditions where the sub-cellular organization of cofactors in the sRNA pathway can induce spatial heterogeneity on sRNA targets. Our results suggest that under certain conditions, interpretation and modeling of natural and synthetic gene regulatory circuits need to take into account the spatial organization of the transcripts of participating genes.

Regulation of cellular growth by the Drosophila target of rapamycin dTOR

PubMed Central

Zhang, Hongbing; Stallock, James P.; Ng, Joyce C.; Reinhard, Christoph; Neufeld, Thomas P.

2000-01-01

The TOR protein kinases (TOR1 and TOR2 in yeast; mTOR/FRAP/RAFT1 in mammals) promote cellular proliferation in response to nutrients and growth factors, but their role in development is poorly understood. Here, we show that the Drosophila TOR homolog dTOR is required cell autonomously for normal growth and proliferation during larval development, and for increases in cellular growth caused by activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. As in mammalian cells, the kinase activity of dTOR is required for growth factor-dependent phosphorylation of p70 S6 kinase (p70S6K) in vitro, and we demonstrate that overexpression of p70S6K in vivo can rescue dTOR mutant animals to viability. Loss of dTOR also results in cellular phenotypes characteristic of amino acid deprivation, including reduced nucleolar size, lipid vesicle aggregation in the larval fat body, and a cell type-specific pattern of cell cycle arrest that can be bypassed by overexpression of the S-phase regulator cyclin E. Our results suggest that dTOR regulates growth during animal development by coupling growth factor signaling to nutrient availability. PMID:11069888

Complementary Approaches to Existing Target Based Drug Discovery for Identifying Novel Drug Targets.

PubMed

Vasaikar, Suhas; Bhatia, Pooja; Bhatia, Partap G; Chu Yaiw, Koon

2016-11-21

In the past decade, it was observed that the relationship between the emerging New Molecular Entities and the quantum of R&D investment has not been favorable. There might be numerous reasons but few studies stress the introduction of target based drug discovery approach as one of the factors. Although a number of drugs have been developed with an emphasis on a single protein target, yet identification of valid target is complex. The approach focuses on an in vitro single target, which overlooks the complexity of cell and makes process of validation drug targets uncertain. Thus, it is imperative to search for alternatives rather than looking at success stories of target-based drug discovery. It would be beneficial if the drugs were developed to target multiple components. New approaches like reverse engineering and translational research need to take into account both system and target-based approach. This review evaluates the strengths and limitations of known drug discovery approaches and proposes alternative approaches for increasing efficiency against treatment.

Researchers identify potential therapeutic targets for a rare childhood cancer | Center for Cancer Research

Cancer.gov

CCR researchers have identified the mechanism behind a rare but extremely aggressive childhood cancer called alveolar rhabdomyosarcoma (ARMS) and have pinpointed a potential drug target for its treatment. Learn more...

Acceleration of recovery in acute renal failure: from cellular mechanisms of tubular repair to innovative targeted therapies.

PubMed

Abbate, M; Remuzzi, G

1996-05-01

Kidney repair from injury is a major focus of interest for research, both clinical and basic, in the field of acute renal failure. This is so because very little progress has been made during the past several years to improve mortality in hospitalized patients with acute renal failure despite the unique potential of the kidney for complete structural and functional recovery. Novel therapeutic options have recently emerged from the knowledge of molecular mechanisms of tissue injury after ischemia, including pathways of endothelial-leukocyte interaction and epithelial cell aggregation mediated by integrin molecules. These strategies are promising because they may target early mechanisms of leukocyte infiltration and tubular obstruction. However, it seems clear that additional interventions should address the reparative program that potentially leads to the full restoration of kidney structure and function. Thus, acceleration of repair from acute renal failure is achieved experimentally by growth factors which besides different renal actions seem to have in common the ability to stimulate proliferation of surviving tubular epithelial cells. We direct attention to cellular processes which characterize, and possibly have role in, renal repair from acute tubular injury as potential targets of therapy. In addition to proliferation, they include epithelial differentiation and apoptosis. Further investigation in the biology of repair should set the stage for rational design of targeted therapies which may accelerate the pace of recovery and hopefully decrease mortality in such a dramatic and potentially reversible setting.

The influenza virus NS1 protein as a therapeutic target.

PubMed

Engel, Daniel A

2013-09-01

Nonstructural protein 1 (NS1) of influenza A virus plays a central role in virus replication and blockade of the host innate immune response, and is therefore being considered as a potential therapeutic target. The primary function of NS1 is to dampen the host interferon (IFN) response through several distinct molecular mechanisms that are triggered by interactions with dsRNA or specific cellular proteins. Sequestration of dsRNA by NS1 results in inhibition of the 2'-5' oligoadenylate synthetase/RNase L antiviral pathway, and also inhibition of dsRNA-dependent signaling required for new IFN production. Binding of NS1 to the E3 ubiquitin ligase TRIM25 prevents activation of RIG-I signaling and subsequent IFN induction. Cellular RNA processing is also targeted by NS1, through recognition of cleavage and polyadenylation specificity factor 30 (CPSF30), leading to inhibition of IFN-β mRNA processing as well as that of other cellular mRNAs. In addition NS1 binds to and inhibits cellular protein kinase R (PKR), thus blocking an important arm of the IFN system. Many additional proteins have been reported to interact with NS1, either directly or indirectly, which may serve its anti-IFN and additional functions, including the regulation of viral and host gene expression, signaling pathways and viral pathogenesis. Many of these interactions are potential targets for small-molecule intervention. Structural, biochemical and functional studies have resulted in hypotheses for drug discovery approaches that are beginning to bear experimental fruit, such as targeting the dsRNA-NS1 interaction, which could lead to restoration of innate immune function and inhibition of virus replication. This review describes biochemical, cell-based and nucleic acid-based approaches to identifying NS1 antagonists. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

The influenza virus NS1 protein as a therapeutic target

PubMed Central

Engel, Daniel A.

2015-01-01

Nonstructural protein 1 (NS1) of influenza A virus plays a central role in virus replication and blockade of the host innate immune response, and is therefore being considered as a potential therapeutic target. The primary function of NS1 is to dampen the host interferon (IFN) response through several distinct molecular mechanisms that are triggered by interactions with dsRNA or specific cellular proteins. Sequestration of dsRNA by NS1 results in inhibition of the 2’-5’ oligoadenylate synthetase/RNase L antiviral pathway, and also inhibition of dsRNA-dependent signaling required for new IFN production. Binding of NS1 to the E3 ubiquitin ligase TRIM25 prevents activation of RIG-I signaling and subsequent IFN induction. Cellular RNA processing is also targeted by NS1, through recognition of cleavage and polyadenylation specificity factor 30 (CPSF30), leading to inhibition of IFN- mRNA processing as well as that of other cellular mRNAs. In addition NS1 binds to and inhibits cellular protein kinase R (PKR), thus blocking an important arm of the IFN system. Many additional proteins have been reported to interact with NS1, either directly or indirectly, which may serve its anti-IFN and additional functions, including the regulation of viral and host gene expression, signaling pathways and viral pathogenesis. Many of these interactions are potential targets for small-molecule intervention. Structural, biochemical and functional studies have resulted in hypotheses for drug discovery approaches that are beginning to bear experimental fruit, such as targeting the dsRNA-NS1 interaction, which could lead to restoration of innate immune function and inhibition of virus replication. This review describes biochemical, cell-based and nucleic acid-based approaches to identifying NS1 antagonists. PMID:23796981

Identification and Herc5-mediated ISGylation of novel target proteins.

PubMed

Takeuchi, Tomoharu; Inoue, Satoshi; Yokosawa, Hideyoshi

2006-09-22

ISG15, a protein containing two ubiquitin-like domains, is an interferon-stimulated gene product that functions in antiviral response and is conjugated to various cellular proteins (ISGylation) upon interferon stimulation. ISGylation occurs via a pathway similar to the pathway for ubiquitination that requires the sequential action of E1/E2/E3: the E1 (UBE1L), E2 (UbcH8), and E3 (Efp/Herc5) enzymes for ISGylation have been hitherto identified. In this study, we identified six novel candidate target proteins for ISGylation by a proteomic approach. Four candidate target proteins were demonstrated to be ISGylated in UBE1L- and UbcH8-dependent manners, and ISGylation of the respective target proteins was stimulated by Herc5. In addition, Herc5 was capable of binding with the respective target proteins. Thus, these results suggest that Herc5 functions as a general E3 ligase for protein ISGylation.

Targeting tumor highly-expressed LAT1 transporter with amino acid-modified nanoparticles: Toward a novel active targeting strategy in breast cancer therapy.

PubMed

Li, Lin; Di, Xingsheng; Wu, Mingrui; Sun, Zhisu; Zhong, Lu; Wang, Yongjun; Fu, Qiang; Kan, Qiming; Sun, Jin; He, Zhonggui

2017-04-01

Designing active targeting nanocarriers with increased cellular accumulation of chemotherapeutic agents is a promising strategy in cancer therapy. Herein, we report a novel active targeting strategy based on the large amino acid transporter 1 (LAT1) overexpressed in a variety of cancers. Glutamate was conjugated to polyoxyethylene stearate as a targeting ligand to achieve LAT1-targeting PLGA nanoparticles. The targeting efficiency of nanoparticles was investigated in HeLa and MCF-7 cells. Significant increase in cellular uptake and cytotoxicity was observed in LAT1-targeting nanoparticles compared to the unmodified ones. More interestingly, the internalized LAT1 together with targeting nanoparticles could recycle back to the cell membrane within 3 h, guaranteeing sufficient transporters on cell membrane for continuous cellular uptake. The LAT1 targeting nanoparticles exhibited better tumor accumulation and antitumor effects. These results suggested that the overexpressed LAT1 on cancer cells holds a great potential to be a high-efficiency target for the rational design of active-targeting nanosystems. Copyright © 2016 Elsevier Inc. All rights reserved.

Targeting ubiquitination for cancer therapies.

PubMed

Morrow, John Kenneth; Lin, Hui-Kuan; Sun, Shao-Cong; Zhang, Shuxing

2015-01-01

Ubiquitination, the structured degradation and turnover of cellular proteins, is regulated by the ubiquitin-proteasome system (UPS). Most proteins that are critical for cellular regulations and functions are targets of the process. Ubiquitination is comprised of a sequence of three enzymatic steps, and aberrations in the pathway can lead to tumor development and progression as observed in many cancer types. Recent evidence indicates that targeting the UPS is effective for certain cancer treatment, but many more potential targets might have been previously overlooked. In this review, we will discuss the current state of small molecules that target various elements of ubiquitination. Special attention will be given to novel inhibitors of E3 ubiquitin ligases, especially those in the SCF family.

The mTOR inhibitor sirolimus suppresses renal, hepatic, and cardiac tissue cellular respiration.

PubMed

Albawardi, Alia; Almarzooqi, Saeeda; Saraswathiamma, Dhanya; Abdul-Kader, Hidaya Mohammed; Souid, Abdul-Kader; Alfazari, Ali S

2015-01-01

The purpose of this in vitro study was to develop a useful biomarker (e.g., cellular respiration, or mitochondrial O2 consumption) for measuring activities of mTOR inhibitors. It measured the effects of commonly used immunosuppressants (sirolimus-rapamycin, tacrolimus, and cyclosporine) on cellular respiration in target tissues (kidney, liver, and heart) from C57BL/6 mice. The mammalian target of rapamycin (mTOR), a serine/ threonine kinase that supports nutrient-dependent cell growth and survival, is known to control energy conversion processes within the mitochondria. Consistently, inhibitors of mTOR (e.g., rapamycin, also known as sirolimus or Rapamune®) have been shown to impair mitochondrial function. Inhibitors of the calcium-dependent serine/threonine phosphatase calcineurin (e.g., tacrolimus and cyclosporine), on the other hand, strictly prevent lymphokine production leading to a reduced T-cell function. Sirolimus (10 μM) inhibited renal (22%, P=0.002), hepatic (39%, P<0.001), and cardiac (42%, P=0.005) cellular respiration. Tacrolimus and cyclosporine had no or minimum effects on cellular respiration in these tissues. Thus, these results clearly demonstrate that impaired cellular respiration (bioenergetics) is a sensitive biomarker of the immunosuppressants that target mTOR.

What determines organ size differences between species? A meta-analysis of the cellular basis.

PubMed

Gázquez, Ayelén; Beemster, Gerrit T S

2017-07-01

Little is known about how the characteristic differences in organ size between species are regulated. At the cellular level, the size of an organ is strictly regulated by cell division and expansion during its development. We performed a meta-analysis of the growth parameters of roots, and Graminae and eudicotyledonous leaves, to address the question of how quantitative variation in these two processes contributes to size differences across a range of species. We extracted or derived cellular parameters from published kinematic growth analyses. These data were subjected to linear regression analyses to identify the parameters that determine differences in organ growth. Our results demonstrate that, across all species and organs, similar conclusions can be made: cell number rather than cell size determines the final size of plant organs; cell number is determined by meristem size rather than the rate at which cells divide; cells that are small when leaving the meristem compensate by expanding for longer; mature cell size is primarily determined by the duration of cell expansion. These results identify the regulation of the transition from cell division to expansion as the key cellular mechanism targeted by the evolution of organ size. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Cellular immunotherapy for malignant gliomas.

PubMed

Lin, Yi; Okada, Hideho

2016-10-01

Cancer immunotherapy has made much progress in recent years. Clinical trials evaluating a variety of immunotherapeutic approaches are underway in patients with malignant gliomas. Thanks to recent advancements in cell engineering technologies, infusion of ex vivo prepared immune cells have emerged as promising strategies of cancer immunotherapy. Herein, the authors review recent and current studies using cellular immunotherapies for malignant gliomas. Specifically, they cover the following areas: a) cellular vaccine approaches using tumor cell-based or dendritic cell (DC)-based vaccines, and b) adoptive cell transfer (ACT) approaches, including lymphokine-activated killer (LAK) cells, γδ T cells, tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor (CAR)-T cells and T-cell receptor (TCR) transduced T cells. While some of the recent studies have shown promising results, the ultimate success of cellular immunotherapy in brain tumor patients would require improvements in the following areas: 1) feasibility in producing cellular therapeutics; 2) identification and characterization of targetable antigens given the paucity and heterogeneity of tumor specific antigens; 3) the development of strategies to promote effector T-cell trafficking; 4) overcoming local and systemic immune suppression, and 5) proper interpretation of imaging data for brain tumor patients receiving immunotherapy.

Inhibition of NFkappaB reduces cellular viability in GH3 pituitary adenoma cells.

PubMed

Vender, John R; Laird, Melissa D; Dhandapani, Krishnan M

2008-05-01

Adenomas of the pituitary gland are among the most common types of tumors of the adult brain. Although adenomas are histologically benign, they may be associated with significant morbidity and mortality, mostly because of their invasive growth pattern and hormone hypersecretion. Current medical therapies are suppressive, acting at a receptor level. Thus, there is a need to identify novel cellular and molecular targets for pituitary tumors. We investigated the possible role of the NFkappaB transcription factor in pituitary tumor cell growth. The effect of NFkappaB pathway inhibition on cellular viability was studied in the GH3 pituitary adenoma cell line, a well-characterized rat cell line that secretes growth hormone and prolactin. Cells were treated with mechanistically diverse pharmacological NFkappaB pathway inhibitors or with molecular inhibitors that were overexpressed in tumor cells before the assessment of cellular viability. NFkappaB activity was also assessed in GH3 cells using deoxyribonucleic acid binding assays. GH3 cells exhibited constitutive NFkappaB activity, which contributed to increased cellular proliferation. Treatment with wedelolactone, an IkappaB kinase inhibitor, or overexpression of an IkappaB super-repressor reduced cell viability, further implicating NFkappaB in pituitary tumor cell growth. Pharmacological or molecular inhibition of Akt similarly reduced GH3 viability and NFkappaB binding, suggesting that constitutive activation of NFkappaB may be, at least in part, mediated by Akt. Directed targeting of the Akt and NFkappaB signaling pathways may be a useful adjunct in the clinical management of pituitary tumors. Further elucidation of this pathway may yield novel information regarding the behavior of pituitary tumors in humans.

A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system

PubMed Central

2014-01-01

Background Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. Results We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu’s positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson’s disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. Conclusions A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations. PMID:24708151

Predicting selective drug targets in cancer through metabolic networks

PubMed Central

Folger, Ori; Jerby, Livnat; Frezza, Christian; Gottlieb, Eyal; Ruppin, Eytan; Shlomi, Tomer

2011-01-01

The interest in studying metabolic alterations in cancer and their potential role as novel targets for therapy has been rejuvenated in recent years. Here, we report the development of the first genome-scale network model of cancer metabolism, validated by correctly identifying genes essential for cellular proliferation in cancer cell lines. The model predicts 52 cytostatic drug targets, of which 40% are targeted by known, approved or experimental anticancer drugs, and the rest are new. It further predicts combinations of synthetic lethal drug targets, whose synergy is validated using available drug efficacy and gene expression measurements across the NCI-60 cancer cell line collection. Finally, potential selective treatments for specific cancers that depend on cancer type-specific downregulation of gene expression and somatic mutations are compiled. PMID:21694718

Vascular Targeting of Nanocarriers: Perplexing Aspects of the Seemingly Straightforward Paradigm

PubMed Central

2015-01-01

Targeted nanomedicine holds promise to find clinical use in many medical areas. Endothelial cells that line the luminal surface of blood vessels represent a key target for treatment of inflammation, ischemia, thrombosis, stroke, and other neurological, cardiovascular, pulmonary, and oncological conditions. In other cases, the endothelium is a barrier for tissue penetration or a victim of adverse effects. Several endothelial surface markers including peptidases (e.g., ACE, APP, and APN) and adhesion molecules (e.g., ICAM-1 and PECAM) have been identified as key targets. Binding of nanocarriers to these molecules enables drug targeting and subsequent penetration into or across the endothelium, offering therapeutic effects that are unattainable by their nontargeted counterparts. We analyze diverse aspects of endothelial nanomedicine including (i) circulation and targeting of carriers with diverse geometries, (ii) multivalent interactions of carrier with endothelium, (iii) anchoring to multiple determinants, (iv) accessibility of binding sites and cellular response to their engagement, (v) role of cell phenotype and microenvironment in targeting, (vi) optimization of targeting by lowering carrier avidity, (vii) endocytosis of multivalent carriers via molecules not implicated in internalization of their ligands, and (viii) modulation of cellular uptake and trafficking by selection of specific epitopes on the target determinant, carrier geometry, and hydrodynamic factors. Refinement of these aspects and improving our understanding of vascular biology and pathology is likely to enable the clinical translation of vascular endothelial targeting of nanocarriers. PMID:24787360

Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets

PubMed Central

Arango, Daniel; Morohashi, Kengo; Yilmaz, Alper; Kuramochi, Kouji; Parihar, Arti; Brahimaj, Bledi; Grotewold, Erich; Doseff, Andrea I.

2013-01-01

Flavonoids constitute the largest class of dietary phytochemicals, adding essential health value to our diet, and are emerging as key nutraceuticals. Cellular targets for dietary phytochemicals remain largely unknown, posing significant challenges for the regulation of dietary supplements and the understanding of how nutraceuticals provide health value. Here, we describe the identification of human cellular targets of apigenin, a flavonoid abundantly present in fruits and vegetables, using an innovative high-throughput approach that combines phage display with second generation sequencing. The 160 identified high-confidence candidate apigenin targets are significantly enriched in three main functional categories: GTPase activation, membrane transport, and mRNA metabolism/alternative splicing. This last category includes the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), a factor involved in splicing regulation, mRNA stability, and mRNA transport. Apigenin binds to the C-terminal glycine-rich domain of hnRNPA2, preventing hnRNPA2 from forming homodimers, and therefore, it perturbs the alternative splicing of several human hnRNPA2 targets. Our results provide a framework to understand how dietary phytochemicals exert their actions by binding to many functionally diverse cellular targets. In turn, some of them may modulate the activity of a large number of downstream genes, which is exemplified here by the effects of apigenin on the alternative splicing activity of hnRNPA2. Hence, in contrast to small-molecule pharmaceuticals designed for defined target specificity, dietary phytochemicals affect a large number of cellular targets with varied affinities that, combined, result in their recognized health benefits. PMID:23697369

Myeloid-derived suppressor cells: Cellular missiles to target tumors.

PubMed

Chandra, Dinesh; Gravekamp, Claudia

2013-11-01

While conventional anticancer therapies, including surgical resection, radiotherapy, and/or chemotherapy, are relatively efficient at eliminating primary tumors, these treatment modalities are largely ineffective against metastases. At least in part, this reflects the rather inefficient delivery of conventional anticancer agents to metastatic lesions. We have recently demonstrated that myeloid-derived suppressor cells (MDSCs) can be used as cellular missiles to selectively deliver a radioisotope-coupled attenuated variant of Listeria monocytogenes to both primary and metastatic neoplastic lesions in mice with pancreatic cancer. This novel immunotherapeutic intervention robustly inhibited tumor growth while promoting a dramatic decrease in the number of metastases.

Identification of Cellular Proteins Required for Replication of Human Immunodeficiency Virus Type 1

PubMed Central

Dziuba, Natallia; Ferguson, Monique R.; O'Brien, William A.; Sanchez, Anthony; Prussia, Andrew J.; McDonald, Natalie J.; Friedrich, Brian M.; Li, Guangyu; Shaw, Michael W.; Sheng, Jinsong; Hodge, Thomas W.; Rubin, Donald H.

2012-01-01

Abstract Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent β-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation. PMID:22404213

Identification of cellular proteins required for replication of human immunodeficiency virus type 1.

PubMed

Dziuba, Natallia; Ferguson, Monique R; O'Brien, William A; Sanchez, Anthony; Prussia, Andrew J; McDonald, Natalie J; Friedrich, Brian M; Li, Guangyu; Shaw, Michael W; Sheng, Jinsong; Hodge, Thomas W; Rubin, Donald H; Murray, James L

2012-10-01

Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent β-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation.

New concept: cellular senescence in pathophysiology of cholangiocarcinoma.

PubMed

Sasaki, Motoko; Nakanuma, Yasuni

2016-01-01

Cholangiocarcinoma, a malignant tumor arising in the hepatobiliary system, presents with poor prognosis because of difficulty in its early detection/diagnosis. Recent progress revealed that cellular senescence may be involved in the pathophysiology of cholangiocarcinoma. Cellular senescence is defined as permanent growth arrest caused by several cellular injuries, such as oncogenic mutations and oxidative stress. "Oncogene-induced" and/or stress-induced senescence may occur in the process of multi-step cholangiocarcinogenesis, and overexpression of a polycomb group protein EZH2 may play a role in the escape from, and/or bypassing of, senescence. Furthermore, senescent cells may play important roles in tumor development and progression via the production of senescence-associated secretory phenotypes. Cellular senescence may be a new target for the prevention, early diagnosis, and therapy of cholangiocarcinoma in the near future.

Celastrol Analogs as Inducers of the Heat Shock Response. Design and Synthesis of Affinity Probes for the Identification of Protein Targets

PubMed Central

Klaić, Lada; Morimoto, Richard I.; Silverman, Richard B.

2012-01-01

The natural product celastrol (1) possesses numerous beneficial therapeutic properties and affects numerous cellular pathways. The mechanism of action and cellular target(s) of celastrol, however, remain unresolved. While a number of studies have proposed that the activity of celastrol is mediated through reaction with cysteine residues, these observations have been based on studies with specific proteins or by in vitro analysis of a small fraction of the proteome. In this study, we have investigated the spatial and structural requirements of celastrol for the design of suitable affinity probes to identify cellular binding partners of celastrol. Although celastrol has several potential sites for modification, some of these were not synthetically amenable or yielded unstable analogs. Conversion of the carboxylic acid functionality to amides and long-chain analogs, however, yielded bioactive compounds that induced the heat shock response (HSR) and antioxidant response and inhibited Hsp90 activity. This led to the synthesis of biotinylated celastrols (23 and 24) that were used as affinity reagents in extracts of human Panc-1 cells to identify Annexin II, eEF1A, and β-tubulin as potential targets of celastrol. PMID:22380712

Molecular Chaperone Hsp90 Is a Therapeutic Target for Noroviruses

PubMed Central

Urena, Luis; Gonzalez-Hernandez, Mariam B.; Choi, Jayoung; de Rougemont, Alexis; Rocha-Pereira, Joana; Neyts, Johan; Hwang, Seungmin; Wobus, Christiane E.

2015-01-01

ABSTRACT Human noroviruses (HuNoV) are a significant cause of acute gastroenteritis in the developed world, and yet our understanding of the molecular pathways involved in norovirus replication and pathogenesis has been limited by the inability to efficiently culture these viruses in the laboratory. Using the murine norovirus (MNV) model, we have recently identified a network of host factors that interact with the 5′ and 3′ extremities of the norovirus RNA genome. In addition to a number of well-known cellular RNA binding proteins, the molecular chaperone Hsp90 was identified as a component of the ribonucleoprotein complex. Here, we show that the inhibition of Hsp90 activity negatively impacts norovirus replication in cell culture. Small-molecule-mediated inhibition of Hsp90 activity using 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) revealed that Hsp90 plays a pleiotropic role in the norovirus life cycle but that the stability of the viral capsid protein is integrally linked to Hsp90 activity. Furthermore, we demonstrate that both the MNV-1 and the HuNoV capsid proteins require Hsp90 activity for their stability and that targeting Hsp90 in vivo can significantly reduce virus replication. In summary, we demonstrate that targeting cellular proteostasis can inhibit norovirus replication, identifying a potential novel therapeutic target for the treatment of norovirus infections. IMPORTANCE HuNoV are a major cause of acute gastroenteritis around the world. RNA viruses, including noroviruses, rely heavily on host cell proteins and pathways for all aspects of their life cycle. Here, we identify one such protein, the molecular chaperone Hsp90, as an important factor required during the norovirus life cycle. We demonstrate that both murine and human noroviruses require the activity of Hsp90 for the stability of their capsid proteins. Furthermore, we demonstrate that targeting Hsp90 activity in vivo using small molecule inhibitors also reduces

Identification of cellular targets involved in cardiac failure caused by PKI in oncology: an approach combining pharmacovigilance and pharmacodynamics.

PubMed

Patras de Campaigno, Emilie; Bondon-Guitton, Emmanuelle; Laurent, Guy; Montastruc, Francois; Montastruc, Jean-Louis; Lapeyre-Mestre, Maryse; Despas, Fabien

2017-07-01

The aims of the present study were to evaluate the risk of cardiac failure (CF) associated with 15 anticancer protein kinase inhibitors (PKIs) through a case/noncase analysis and to identify which PK(s) and pathways are involved in PKI-induced CF. In order to evaluate the risk of CF, adjusted reporting odds ratios (aRORs) were calculated for the 15 anticancer PKIs in the World Health Organization safety report database (VigiBase®). We realised a literature review to identify 21 protein kinases (PKs) that were possibly involved in CF caused by PKIs. Pearson correlation coefficients (r) between aRORs and affinity data of the 15 PKIs for the 21 PKs were calculated to identify the cellular target most likely to be involved in PKI-induced CF. A total of 141 601 individual case safety reports (ICSRs) were extracted from VigiBase® for the following PKIs: afatinib, axitinib, bosutinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ruxolitinib, sorafenib, sunitinib and vandetanib. Among them, 2594 ICSRs concerned CF. The disproportionality analysis revealed that, for dasatinib, imatinib, bosutinib, sunitinib and nilotinib, disproportionality for CF was significantly higher than for other PKIs, with aRORs of 2.52 [95% CI 2.26, 2.82], 1.79 (95% CI 1.57, 2.03), 1.73 (95% CI 1.18, 2.54), 1.67 (95% CI 1.51, 1.84) and 1.38 (95% CI 1.18, 1.61), respectively. Significant values for correlation coefficients between the product of dissociation constant (pKd) and aROR were observed for two non-receptor protein kinases: ABL1 (non-phosphorylated and phosphorylated forms) and ABL2 protein kinases, with values of r = 0.83 (P = 0.0001), r = 0.75 (P = 0.0014) and r = 0.78 (P = 0.0006), respectively. We observed a higher disproportionality for CF with dasatinib, imatinib, bosutinib, sunitinib and nilotinib than with other PKIs. In addition, the study highlighted the role of ABL tyrosine kinases in CF caused by anticancer PKIs. © 2017 The British

A proteomic approach to identifying new drug targets (potentiating topoisomerase II poisons).

PubMed

Jenkins, J R

2008-10-01

Topoisomerase II poisons are an established part of best clinical practice for the treatment of a number of solid tumours and haematological malignancies. However, toxicity and resistance to chemotherapeutic drugs often complicate the treatment. Furthermore, topoisomerase II poisons can also induce sister chromatid exchange, chromosomal recombination and chromosome aberrations and are associated with a significant risk of secondary leukaemia. It would therefore be of great clinical benefit if the efficacy of topoisomerase II inhibitors could be enhanced without the increased toxic side effects. It is proposed that clinical agents targeting topoisomerase II can be enhanced by inhibiting proteins that modulate topoisomerase II. The aim is to identify proteins, that by the nature of their interaction with topoisomerase II, represent putative drug targets.

An integrated chemical biology approach identifies specific vulnerability of Ewing's sarcoma to combined inhibition of Aurora kinases A and B.

PubMed

Winter, Georg E; Rix, Uwe; Lissat, Andrej; Stukalov, Alexey; Müllner, Markus K; Bennett, Keiryn L; Colinge, Jacques; Nijman, Sebastian M; Kubicek, Stefan; Kovar, Heinrich; Kontny, Udo; Superti-Furga, Giulio

2011-10-01

Ewing's sarcoma is a pediatric cancer of the bone that is characterized by the expression of the chimeric transcription factor EWS-FLI1 that confers a highly malignant phenotype and results from the chromosomal translocation t(11;22)(q24;q12). Poor overall survival and pronounced long-term side effects associated with traditional chemotherapy necessitate the development of novel, targeted, therapeutic strategies. We therefore conducted a focused viability screen with 200 small molecule kinase inhibitors in 2 different Ewing's sarcoma cell lines. This resulted in the identification of several potential molecular intervention points. Most notably, tozasertib (VX-680, MK-0457) displayed unique nanomolar efficacy, which extended to other cell lines, but was specific for Ewing's sarcoma. Furthermore, tozasertib showed strong synergies with the chemotherapeutic drugs etoposide and doxorubicin, the current standard agents for Ewing's sarcoma. To identify the relevant targets underlying the specific vulnerability toward tozasertib, we determined its cellular target profile by chemical proteomics. We identified 20 known and unknown serine/threonine and tyrosine protein kinase targets. Additional target deconvolution and functional validation by RNAi showed simultaneous inhibition of Aurora kinases A and B to be responsible for the observed tozasertib sensitivity, thereby revealing a new mechanism for targeting Ewing's sarcoma. We further corroborated our cellular observations with xenograft mouse models. In summary, the multilayered chemical biology approach presented here identified a specific vulnerability of Ewing's sarcoma to concomitant inhibition of Aurora kinases A and B by tozasertib and danusertib, which has the potential to become a new therapeutic option.

Genome and transcriptome sequencing identifies breeding targets in the orphan crop tef (Eragrostis tef).

PubMed

Cannarozzi, Gina; Plaza-Wüthrich, Sonia; Esfeld, Korinna; Larti, Stéphanie; Wilson, Yi Song; Girma, Dejene; de Castro, Edouard; Chanyalew, Solomon; Blösch, Regula; Farinelli, Laurent; Lyons, Eric; Schneider, Michel; Falquet, Laurent; Kuhlemeier, Cris; Assefa, Kebebew; Tadele, Zerihun

2014-07-09

Tef (Eragrostis tef), an indigenous cereal critical to food security in the Horn of Africa, is rich in minerals and protein, resistant to many biotic and abiotic stresses and safe for diabetics as well as sufferers of immune reactions to wheat gluten. We present the genome of tef, the first species in the grass subfamily Chloridoideae and the first allotetraploid assembled de novo. We sequenced the tef genome for marker-assisted breeding, to shed light on the molecular mechanisms conferring tef's desirable nutritional and agronomic properties, and to make its genome publicly available as a community resource. The draft genome contains 672 Mbp representing 87% of the genome size estimated from flow cytometry. We also sequenced two transcriptomes, one from a normalized RNA library and another from unnormalized RNASeq data. The normalized RNA library revealed around 38000 transcripts that were then annotated by the SwissProt group. The CoGe comparative genomics platform was used to compare the tef genome to other genomes, notably sorghum. Scaffolds comprising approximately half of the genome size were ordered by syntenic alignment to sorghum producing tef pseudo-chromosomes, which were sorted into A and B genomes as well as compared to the genetic map of tef. The draft genome was used to identify novel SSR markers, investigate target genes for abiotic stress resistance studies, and understand the evolution of the prolamin family of proteins that are responsible for the immune response to gluten. It is highly plausible that breeding targets previously identified in other cereal crops will also be valuable breeding targets in tef. The draft genome and transcriptome will be of great use for identifying these targets for genetic improvement of this orphan crop that is vital for feeding 50 million people in the Horn of Africa.

Positive Newborn Screen for Methylmalonic Aciduria Identifies the First Mutation in TCblR/CD320, the Gene for Cellular Uptake of Transcobalamin-bound Vitamin B12

PubMed Central

Quadros, Edward V.; Lai, Shao-Chiang; Nakayama, Yasumi; Sequeira, Jeffrey M.; Hannibal, Luciana; Wang, Sihe; Jacobsen, Donald W.; Fedosov, Sergey; Wright, Erica; Gallagher, Renata C.; Anastasio, Natascia; Watkins, David; Rosenblatt, David S.

2010-01-01

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low-density lipoprotein receptor type A-like domain. Inserting the codon by site-directed mutagenesis fully restored TCblR function. PMID:20524213

Drug target identification using network analysis: Taking active components in Sini decoction as an example

NASA Astrophysics Data System (ADS)

Chen, Si; Jiang, Hailong; Cao, Yan; Wang, Yun; Hu, Ziheng; Zhu, Zhenyu; Chai, Yifeng

2016-04-01

Identifying the molecular targets for the beneficial effects of active small-molecule compounds simultaneously is an important and currently unmet challenge. In this study, we firstly proposed network analysis by integrating data from network pharmacology and metabolomics to identify targets of active components in sini decoction (SND) simultaneously against heart failure. To begin with, 48 potential active components in SND against heart failure were predicted by serum pharmacochemistry, text mining and similarity match. Then, we employed network pharmacology including text mining and molecular docking to identify the potential targets of these components. The key enriched processes, pathways and related diseases of these target proteins were analyzed by STRING database. At last, network analysis was conducted to identify most possible targets of components in SND. Among the 25 targets predicted by network analysis, tumor necrosis factor α (TNF-α) was firstly experimentally validated in molecular and cellular level. Results indicated that hypaconitine, mesaconitine, higenamine and quercetin in SND can directly bind to TNF-α, reduce the TNF-α-mediated cytotoxicity on L929 cells and exert anti-myocardial cell apoptosis effects. We envisage that network analysis will also be useful in target identification of a bioactive compound.

Drug target identification using network analysis: Taking active components in Sini decoction as an example

PubMed Central

Chen, Si; Jiang, Hailong; Cao, Yan; Wang, Yun; Hu, Ziheng; Zhu, Zhenyu; Chai, Yifeng

2016-01-01

Identifying the molecular targets for the beneficial effects of active small-molecule compounds simultaneously is an important and currently unmet challenge. In this study, we firstly proposed network analysis by integrating data from network pharmacology and metabolomics to identify targets of active components in sini decoction (SND) simultaneously against heart failure. To begin with, 48 potential active components in SND against heart failure were predicted by serum pharmacochemistry, text mining and similarity match. Then, we employed network pharmacology including text mining and molecular docking to identify the potential targets of these components. The key enriched processes, pathways and related diseases of these target proteins were analyzed by STRING database. At last, network analysis was conducted to identify most possible targets of components in SND. Among the 25 targets predicted by network analysis, tumor necrosis factor α (TNF-α) was firstly experimentally validated in molecular and cellular level. Results indicated that hypaconitine, mesaconitine, higenamine and quercetin in SND can directly bind to TNF-α, reduce the TNF-α-mediated cytotoxicity on L929 cells and exert anti-myocardial cell apoptosis effects. We envisage that network analysis will also be useful in target identification of a bioactive compound. PMID:27095146

Autoimmune therapies targeting costimulation and emerging trends in multivalent therapeutics.

PubMed

Chittasupho, Chuda; Siahaan, Teruna J; Vines, Charlotte M; Berkland, Cory

2011-07-01

Proteins participating in immunological signaling have emerged as important targets for controlling the immune response. A multitude of receptor-ligand pairs that regulate signaling pathways of the immune response have been identified. In the complex milieu of immune signaling, therapeutic agents targeting mediators of cellular signaling often either activate an inflammatory immune response or induce tolerance. This review is primarily focused on therapeutics that inhibit the inflammatory immune response by targeting membrane-bound proteins regulating costimulation or mediating immune-cell adhesion. Many of these signals participate in larger, organized structures such as the immunological synapse. Receptor clustering and arrangement into organized structures is also reviewed and emerging trends implicating a potential role for multivalent therapeutics is posited.

Cellular, synaptic and biochemical features of resilient cognition in Alzheimer’s disease

PubMed Central

Arnold, Steven. E.; Louneva, Natalia; Cao, Kajia; Wang, Li-San; Han, Li-Ying; Wolk, David A.; Negash, Selamawit; Leurgans, Sue E.; Schneider, Julie A.; Buchman, Aron S.; Wilson, Robert S.; Bennett, David A.

2012-01-01

While neuritic plaques and neurofibrillary tangles in older adults are correlated with cognitive impairment and severity of dementia, it has long been recognized that the relationship is imperfect as some people exhibit normal cognition despite high levels of AD pathology. We compared the cellular, synaptic and biochemical composition of midfrontal cortices in female subjects from the Religious Orders Study who were stratified into three subgroups: 1) pathological AD with normal cognition (“AD-Resilient”), 2) pathological AD with AD-typical dementia (“AD-Dementia)” and 3) pathologically normal with normal cognition (“Normal Comparison”). The AD-Resilient group exhibited preserved densities of synaptophysin-labeled presynaptic terminals and synaptopodin-labeled dendritic spines compared to the AD-Dementia group, and increased densities of GFAP astrocytes compared to both the AD-Dementia and Normal Comparison group. Further, in a discovery antibody microarray protein analysis we identified a number of candidate protein abnormalities that were associated with diagnostic group. These data characterize cellular and synaptic features and identify novel biochemical targets that may be associated with resilient cognitive brain aging in the setting of pathological AD. PMID:22554416

Targeting the Human Papillomavirus E6 and E7 Oncogenes through Expression of the Bovine Papillomavirus Type 1 E2 Protein Stimulates Cellular Motility▿†

PubMed Central

Morrison, Monique A.; Morreale, Richard J.; Akunuru, Shailaja; Kofron, Matthew; Zheng, Yi; Wells, Susanne I.

2011-01-01

Expression of the high-risk human papillomavirus (HPV) E6 and E7 oncogenes is essential for the initiation and maintenance of cervical cancer. The repression of both was previously shown to result in activation of their respective tumor suppressor targets, p53 and pRb, and subsequent senescence induction in cervical cancer cells. Consequently, viral oncogene suppression is a promising approach for the treatment of HPV-positive tumors. One well-established method of E6/E7 repression involves the reexpression of the viral E2 protein which is usually deleted in HPV-positive cancer cells. Here, we show that, surprisingly, bovine papillomavirus type 1 (BPV1) E2 but not RNA interference-mediated E6/E7 repression in HPV-positive cervical cancer cells stimulates cellular motility and invasion. Migration correlated with the dynamic formation of cellular protrusions and was dependent upon cell-to-cell contact. While E2-expressing migratory cells were senescent, migration was not a general feature of cellular senescence or cell cycle arrest and was specifically observed in HPV-positive cervical cancer cells. Interestingly, E2-expressing cells not only were themselves motile but also conferred increased motility to admixed HeLa cervical cancer cells. Together, our data suggest that repression of the viral oncogenes by E2 stimulates the motility of E6/E7-targeted cells as well as adjacent nontargeted cancer cells, thus raising the possibility that E2 expression may unfavorably increase the local invasiveness of HPV-positive tumors. PMID:21835799

Aspartate β-hydroxylase modulates cellular senescence via glycogen synthase kinase 3β in hepatocellular carcinoma

PubMed Central

Iwagami, Yoshifumi; Huang, Chiung-Kuei; Olsen, Mark J.; Thomas, John-Michael; Jang, Grace; Kim, Miran; Lin, Qiushi; Carlson, Rolf I.; Wagner, Carl E.; Dong, Xiaoqun; Wands, Jack R.

2015-01-01

Background & Aims Aspartate β-hydroxylase (ASPH) is an enzyme overexpressed in human hepatocellular carcinoma (HCC) tumors and participates in the malignant transformation process. We determined if ASPH was a therapeutic target by exerting effects on cellular senescence to retard HCC progression. Methods ASPH knockdown or knockout was achieved by shRNAs or CRISPR/Cas9 system, respectively, whereas enzymatic inhibition was rendered by a potent 2nd generation small molecule inhibitor (SMI) of ASPH. Alterations of cell proliferation, colony formation and cellular senescence were evaluated in human HCC cell lines. The potential mechanisms for activating cellular senescence were explored using murine subcutaneous and orthotopic xenograft models. Results Inhibition of ASPH expression and enzymatic activity significantly reduced cell proliferation and colony formation, but induced tumor cell senescence. Following inhibition of ASPH activity, phosphorylation of GSK3β and p16 expression were increased to promote senescence whereas cyclin D1 and PCNA were decreased to reduce cell proliferation. The mechanisms involved demonstrate that ASPH binds to GSK3β and inhibits its subsequent interactions with AKT and p38 upstream kinases as shown by co-immunoprecipitation. In vivo experiments demonstrated that the SMI treatment of HCC bearing mice resulted in significant dose-dependent reduced tumor growth, induced phosphorylation of GSK3β, enhanced p16 expression in tumor cells and promoted cellular senescence. Conclusions We have identified a new mechanism that promotes HCC growth and progression by modulating senescence of tumor cells. These findings suggest that ASPH enzymatic activity is a novel therapeutic target for HCC. PMID:26683595

PACCMIT/PACCMIT-CDS: identifying microRNA targets in 3' UTRs and coding sequences.

PubMed

Šulc, Miroslav; Marín, Ray M; Robins, Harlan S; Vaníček, Jiří

2015-07-01

The purpose of the proposed web server, publicly available at http://paccmit.epfl.ch, is to provide a user-friendly interface to two algorithms for predicting messenger RNA (mRNA) molecules regulated by microRNAs: (i) PACCMIT (Prediction of ACcessible and/or Conserved MIcroRNA Targets), which identifies primarily mRNA transcripts targeted in their 3' untranslated regions (3' UTRs), and (ii) PACCMIT-CDS, designed to find mRNAs targeted within their coding sequences (CDSs). While PACCMIT belongs among the accurate algorithms for predicting conserved microRNA targets in the 3' UTRs, the main contribution of the web server is 2-fold: PACCMIT provides an accurate tool for predicting targets also of weakly conserved or non-conserved microRNAs, whereas PACCMIT-CDS addresses the lack of similar portals adapted specifically for targets in CDS. The web server asks the user for microRNAs and mRNAs to be analyzed, accesses the precomputed P-values for all microRNA-mRNA pairs from a database for all mRNAs and microRNAs in a given species, ranks the predicted microRNA-mRNA pairs, evaluates their significance according to the false discovery rate and finally displays the predictions in a tabular form. The results are also available for download in several standard formats. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Potential Mechanisms of Action of Dietary Phytochemicals for Cancer Prevention by Targeting Cellular Signaling Transduction Pathways.

PubMed

Chen, Hongyu; Liu, Rui Hai

2018-04-04

Cancer is a severe health problem that significantly undermines life span and quality. Dietary approach helps provide preventive, nontoxic, and economical strategies against cancer. Increased intake of fruits, vegetables, and whole grains are linked to reduced risk of cancer and other chronic diseases. The anticancer activities of plant-based foods are related to the actions of phytochemicals. One potential mechanism of action of anticancer phytochemicals is that they regulate cellular signal transduction pathways and hence affects cancer cell behaviors such as proliferation, apoptosis, and invasion. Recent publications have reported phytochemicals to have anticancer activities through targeting a wide variety of cell signaling pathways at different levels, such as transcriptional or post-transcriptional regulation, protein activation and intercellular messaging. In this review, we discuss major groups of phytochemicals and their regulation on cell signaling transduction against carcinogenesis via key participators, such as Nrf2, CYP450, MAPK, Akt, JAK/STAT, Wnt/β-catenin, p53, NF-κB, and cancer-related miRNAs.

Cellular Restriction Factors of Feline Immunodeficiency Virus

PubMed Central

Zielonka, Jörg; Münk, Carsten

2011-01-01

Lentiviruses are known for their narrow cell- and species-tropisms, which are determined by cellular proteins whose absence or presence either support viral replication (dependency factors, cofactors) or inhibit viral replication (restriction factors). Similar to Human immunodeficiency virus type 1 (HIV-1), the cat lentivirus Feline immunodeficiency virus (FIV) is sensitive to recently discovered cellular restriction factors from non-host species that are able to stop viruses from replicating. Of particular importance are the cellular proteins APOBEC3, TRIM5α and tetherin/BST-2. In general, lentiviruses counteract or escape their species’ own variant of the restriction factor, but are targeted by the orthologous proteins of distantly related species. Most of the knowledge regarding lentiviral restriction factors has been obtained in the HIV-1 system; however, much less is known about their effects on other lentiviruses. We describe here the molecular mechanisms that explain how FIV maintains its replication in feline cells, but is largely prevented from cross-species infections by cellular restriction factors. PMID:22069525

Silent genetic alterations identified by targeted next-generation sequencing in pheochromocytoma/paraganglioma: A clinicopathological correlations.

PubMed

Pillai, Suja; Gopalan, Vinod; Lo, Chung Y; Liew, Victor; Smith, Robert A; Lam, Alfred King Y

2017-02-01

The goal of this pilot study was to develop a customized, cost-effective amplicon panel (Ampliseq) for target sequencing in a cohort of patients with sporadic phaeochromocytoma/paraganglioma. Phaeochromocytoma/paragangliomas from 25 patients were analysed by targeted next-generation sequencing approach using an Ion Torrent PGM instrument. Primers for 15 target genes (NF1, RET, VHL, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, MEN1, KIF1Bβ, EPAS1, CDKN2 & PHD2) were designed using ion ampliseq designer. Ion Reporter software and Ingenuity® Variant Analysis™ software (www.ingenuity.com/variants) from Ingenuity Systems were used to analysis these results. Overall, 713 variants were identified. The variants identified from the Ion Reporter ranged from 64 to 161 per patient. Single nucleotide variants (SNV) were the most common. Further annotation with the help of Ingenuity variant analysis revealed 29 of these 713variants were deletions. Of these, six variants were non-pathogenic and four were likely to be pathogenic. The remaining 19 variants were of uncertain significance. The most frequently altered gene in the cohort was KIF1B followed by NF1. Novel KIF1B pathogenic variant c.3375+1G>A was identified. The mutation was noted in a patient with clinically confirmed neurofibromatosis. Chromosome 1 showed the presence of maximum number of variants. Use of targeted next-generation sequencing is a sensitive method for the detecting genetic changes in patients with phaeochromocytoma/paraganglioma. The precise detection of these genetic changes helps in understanding the pathogenesis of these tumours. Copyright © 2016 Elsevier Inc. All rights reserved.

Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer

DTIC Science & Technology

2015-09-01

AWARD NUMBER: W81XWH-14-1-0177 TITLE: Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer PRINCIPAL INVESTIGATOR: Katerina Politi...CONTRACT NUMBER Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer 5b. GRANT NUMBER W81XWH-14-1-0177 5c. PROGRAM ELEMENT NUMBER 6...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Phenotypic changes have been observed in EGFR mutant lung cancers that become resistant to targeted

Sleep- and circadian rhythm-associated pathways as therapeutic targets in bipolar disorder.

PubMed

Bellivier, Frank; Geoffroy, Pierre-Alexis; Etain, Bruno; Scott, Jan

2015-06-01

Disruptions in sleep and circadian rhythms are observed in individuals with bipolar disorders (BD), both during acute mood episodes and remission. Such abnormalities may relate to dysfunction of the molecular circadian clock and could offer a target for new drugs. This review focuses on clinical, actigraphic, biochemical and genetic biomarkers of BDs, as well as animal and cellular models, and highlights that sleep and circadian rhythm disturbances are closely linked to the susceptibility to BDs and vulnerability to mood relapses. As lithium is likely to act as a synchronizer and stabilizer of circadian rhythms, we will review pharmacogenetic studies testing circadian gene polymorphisms and prophylactic response to lithium. Interventions such as sleep deprivation, light therapy and psychological therapies may also target sleep and circadian disruptions in BDs efficiently for treatment and prevention of bipolar depression. We suggest that future research should clarify the associations between sleep and circadian rhythm disturbances and alterations of the molecular clock in order to identify critical targets within the circadian pathway. The investigation of such targets using human cellular models or animal models combined with 'omics' approaches are crucial steps for new drug development.

Cellular immunotherapy for malignant gliomas

PubMed Central

Lin, Yi

2016-01-01

Introduction Cancer immunotherapy has made much progress in recent years. Clinical trials evaluating a variety of immunotherapeutic approaches are underway in patients with malignant gliomas. Thanks to recent advancements in cell engineering technologies, infusion of ex vivo prepared immune cells have emerged as promising strategies of cancer immunotherapy. Areas covered Herein, the authors review recent and current studies using cellular immunotherapies for malignant gliomas. Specifically, they cover the following areas: a) cellular vaccine approaches using tumor cell-based or dendritic cell (DC)-based vaccines, and b) adoptive cell transfer (ACT) approaches, including lymphokine-activated killer (LAK) cells, γδ T cells, tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor (CAR)-T cells and T-cell receptor (TCR) transduced T cells. Expert opinion While some of the recent studies have shown promising results, the ultimate success of cellular immunotherapy in brain tumor patients would require improvements in the following areas: 1) feasibility in producing cellular therapeutics; 2) identification and characterization of targetable antigens given the paucity and heterogeneity of tumor specific antigens; 3) the development of strategies to promote effector T-cell trafficking; 4) overcoming local and systemic immune suppression, and 5) proper interpretation of imaging data for brain tumor patients receiving immunotherapy. PMID:27434205

Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements

PubMed Central

Mumbach, Maxwell R; Satpathy, Ansuman T; Boyle, Evan A; Dai, Chao; Gowen, Benjamin G; Cho, Seung Woo; Nguyen, Michelle L; Rubin, Adam J; Granja, Jeffrey M; Kazane, Katelynn R; Wei, Yuning; Nguyen, Trieu; Greenside, Peyton G; Corces, M Ryan; Tycko, Josh; Simeonov, Dimitre R; Suliman, Nabeela; Li, Rui; Xu, Jin; Flynn, Ryan A; Kundaje, Anshul; Khavari, Paul A; Marson, Alexander; Corn, Jacob E; Quertermous, Thomas; Greenleaf, William J; Chang, Howard Y

2018-01-01

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer–promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases. PMID:28945252

Vitiligo blood transcriptomics provides new insights into disease mechanisms and identifies potential novel therapeutic targets.

PubMed

Dey-Rao, Rama; Sinha, Animesh A

2017-01-28

Significant gaps remain regarding the pathomechanisms underlying the autoimmune response in vitiligo (VL), where the loss of self-tolerance leads to the targeted killing of melanocytes. Specifically, there is incomplete information regarding alterations in the systemic environment that are relevant to the disease state. We undertook a genome-wide profiling approach to examine gene expression in the peripheral blood of VL patients and healthy controls in the context of our previously published VL-skin gene expression profile. We used several in silico bioinformatics-based analyses to provide new insights into disease mechanisms and suggest novel targets for future therapy. Unsupervised clustering methods of the VL-blood dataset demonstrate a "disease-state"-specific set of co-expressed genes. Ontology enrichment analysis of 99 differentially expressed genes (DEGs) uncovers a down-regulated immune/inflammatory response, B-Cell antigen receptor (BCR) pathways, apoptosis and catabolic processes in VL-blood. There is evidence for both type I and II interferon (IFN) playing a role in VL pathogenesis. We used interactome analysis to identify several key blood associated transcriptional factors (TFs) from within (STAT1, STAT6 and NF-kB), as well as "hidden" (CREB1, MYC, IRF4, IRF1, and TP53) from the dataset that potentially affect disease pathogenesis. The TFs overlap with our reported lesional-skin transcriptional circuitry, underscoring their potential importance to the disease. We also identify a shared VL-blood and -skin transcriptional "hot spot" that maps to chromosome 6, and includes three VL-blood dysregulated genes (PSMB8, PSMB9 and TAP1) described as potential VL-associated genetic susceptibility loci. Finally, we provide bioinformatics-based support for prioritizing dysregulated genes in VL-blood or skin as potential therapeutic targets. We examined the VL-blood transcriptome in context with our (previously published) VL-skin transcriptional profile to address

Comparative genome analysis identifies novel nucleic acid diagnostic targets for use in the specific detection of Haemophilus influenzae.

PubMed

Coughlan, Helena; Reddington, Kate; Tuite, Nina; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Clancy, Eoin; Barry, Thomas

2015-10-01

Haemophilus influenzae is recognised as an important human pathogen associated with invasive infections, including bloodstream infection and meningitis. Currently used molecular-based diagnostic assays lack specificity in correctly detecting and identifying H. influenzae. As such, there is a need to develop novel diagnostic assays for the specific identification of H. influenzae. Whole genome comparative analysis was performed to identify putative diagnostic targets, which are unique in nucleotide sequence to H. influenzae. From this analysis, we identified 2H. influenzae putative diagnostic targets, phoB and pstA, for use in real-time PCR diagnostic assays. Real-time PCR diagnostic assays using these targets were designed and optimised to specifically detect and identify all 55H. influenzae strains tested. These novel rapid assays can be applied to the specific detection and identification of H. influenzae for use in epidemiological studies and could also enable improved monitoring of invasive disease caused by these bacteria. Copyright © 2015 Elsevier Inc. All rights reserved.

Integrative screening approach identifies regulators of polyploidization and targets for acute megakaryocytic leukemia

PubMed Central

Wen, Qiang; Goldenson, Benjamin; Silver, Serena J.; Schenone, Monica; Dancik, Vladimir; Huang, Zan; Wang, Ling-Zhi; Lewis, Timothy; An, W. Frank; Li, Xiaoyu; Bray, Mark-Anthony; Thiollier, Clarisse; Diebold, Lauren; Gilles, Laure; Vokes, Martha S.; Moore, Christopher B.; Bliss-Moreau, Meghan; VerPlank, Lynn; Tolliday, Nicola J.; Mishra, Rama; Vemula, Sasidhar; Shi, Jianjian; Wei, Lei; Kapur, Reuben; Lopez, Cécile K.; Gerby, Bastien; Ballerini, Paola; Pflumio, Francoise; Gilliland, D. Gary; Goldberg, Liat; Birger, Yehudit; Izraeli, Shai; Gamis, Alan S.; Smith, Franklin O.; Woods, William G.; Taub, Jeffrey; Scherer, Christina A.; Bradner, James; Goh, Boon-Cher; Mercher, Thomas; Carpenter, Anne E.; Gould, Robert J.; Clemons, Paul A.; Carr, Steven A.; Root, David E.; Schreiber, Stuart L.; Stern, Andrew M.; Crispino, John D.

2012-01-01

Summary The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. We found that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. A broadly applicable, highly integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora A kinase (AURKA), which has not been studied extensively in megakaryocytes. Moreover, we discovered that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in AMKL blasts and displayed potent anti-AMKL activity in vivo. This research provides the rationale to support clinical trials of MLN8237 and other inducers of polyploidization in AMKL. Finally, we have identified five networks of kinases that regulate the switch to polyploidy. PMID:22863010

The Near-Earth Object Human Space Flight Accessible Targets Study (NHATS) List of Near-Earth Asteroids: Identifying Potential Targets for Future Exploration

NASA Technical Reports Server (NTRS)

Abell, Paul A.; Barbee, B. W.; Mink, R. G.; Alberding, C. M.; Adamo, D. R.; Mazanek, D. D.; Johnson, L. N.; Yeomans, D. K.; Chodas, P. W.; Chamberlin, A. B.;

Drug Target Discovery Methods In Targeting Neurotropic Parasitic Amoebae.

PubMed

Baig, Abdul Mannan; Waliani, Nuzair; Karim, Saiqa

2018-02-21

Neurotropic parasitic amoebal infections have imposed an enormous challenge to chemotherapy in patients who fall victims to the infections caused by them. Conventional antibiotics that are given to treat these infections have a low patient compliance because of the serious adverse effects that are associated with their use. Additionally, the growing incidence of the development of drug resistance by the neurotropic parasites like Naegleria fowleri, Balamuthia mandrillaris, and Acanthamoeba spp has made the drug therapy more challenging. Recent studies have reported some cellular targets in the neurotropic parasitic Acanthamoeba that are used as receptors by human neurotransmitters like acetylcholine. This Viewpoint attempts to highlight the novel methodologies that use drug assays and structural modeling to uncover cellular targets of diverse groups of drugs and the safety issues of the drugs proposed for their use in brain infections caused by the neurotropic parasitic amoebae.

Cellular Factors Shape 3D Genome Landscape

Cancer.gov

Researchers, using novel large-scale imaging technology, have mapped the spatial location of individual genes in the nucleus of human cells and identified 50 cellular factors required for the proper 3D positioning of genes. These spatial locations play important roles in gene expression, DNA repair, genome stability, and other cellular activities.

Targeted PEG-based bioconjugates enhance the cellular uptake and transport of a HIV-1 TAT nonapeptide.

PubMed

Ramanathan, S; Qiu, B; Pooyan, S; Zhang, G; Stein, S; Leibowitz, M J; Sinko, P J

2001-12-13

We previously described the enhanced cell uptake and transport of R.I-K(biotin)-Tat9, a large ( approximately 1500 Da) peptidic inhibitor of HIV-1 Tat protein, via SMVT, the intestinal biotin transporter. The aim of the present study was to investigate the feasibility of targeting biotinylated PEG-based conjugates to SMVT in order to enhance cell uptake and transport of Tat9. The 29 kDa peptide-loaded bioconjugate (PEG:(R.I-Cys-K(biotin)-Tat9)8) used in these studies contained eight copies of R.I-K(biotin)-Tat9 appended to PEG by means of a cysteine linkage. The absorptive transport of biotin-PEG-3400 (0.6-100 microM) and the bioconjugate (0.1-30 microM) was studied using Caco-2 cell monolayers. Inhibition of biotin-PEG-3400 by positive controls (biotin, biocytin, and desthiobiotin) was also determined. Uptake of these two compounds was also determined in CHO cells transfected with human SMVT (CHO/hSMVT) and control cells (CHO/pSPORT) over the concentration ranges of 0.05-12.5 microM and 0.003-30 microM, respectively. Nonbiotinylated forms of these two compounds, PEG-3350 and PEG:(R.I-Cys-K-Tat9)8, were used in the control studies. Biotin-PEG-3400 transport was found to be concentration-dependent and saturable in Caco-2 cells (K(m)=6.61 microM) and CHO/hSMVT cells (K(m)=1.26 microM). Transport/uptake was significantly inhibited by positive control substrates of SMVT. PEG:(R.I-Cys-K(biotin)Tat9)8 also showed saturable transport kinetics in Caco-2 cells (K(m)=6.13 microM) and CHO/hSMVT cells (K(m)=8.19 microM). Maximal uptake in molar equivalents of R.I-Cys-K(biotin)Tat9 was 5.7 times greater using the conjugate versus the biotinylated peptide alone. Transport of the nonbiotinylated forms was significantly lower (P<0.001) in all cases. The present results demonstrate that biotin-PEG-3400 and PEG:(R.I-Cys-K(biotin)Tat9)8 interact with human SMVT to enhance the cellular uptake and transport of these larger molecules and that targeted bioconjugates may have potential

In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line.

PubMed

Wang, Jigang; Zhang, Jianbin; Zhang, Chong-Jing; Wong, Yin Kwan; Lim, Teck Kwang; Hua, Zi-Chun; Liu, Bin; Tannenbaum, Steven R; Shen, Han-Ming; Lin, Qingsong

2016-02-26

To date, the exact targets and mechanism of action of curcumin, a natural product with anti-inflammatory and anti-cancer properties, remain elusive. Here we synthesized a cell permeable curcumin probe (Cur-P) with an alkyne moiety, which can be tagged with biotin for affinity enrichment, or with a fluorescent dye for visualization of the direct-binding protein targets of curcumin in situ. iTRAQ(TM) quantitative proteomics approach was applied to distinguish the specific binding targets from the non-specific ones. In total, 197 proteins were confidently identified as curcumin binding targets from HCT116 colon cancer cell line. Gene Ontology analysis showed that the targets are broadly distributed and enriched in the nucleus, mitochondria and plasma membrane, and they are involved in various biological functions including metabolic process, regulation, response to stimulus and cellular process. Ingenuity Pathway Analysis(TM) (IPA) suggested that curcumin may exert its anticancer effects over multiple critical biological pathways including the EIF2, eIF4/p70S6K, mTOR signaling and mitochondrial dysfunction pathways. Functional validations confirmed that curcumin downregulates cellular protein synthesis, and induces autophagy, lysosomal activation and increased ROS production, thus leading to cell death.

In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line

PubMed Central

Wang, Jigang; Zhang, Jianbin; Zhang, Chong-Jing; Wong, Yin Kwan; Lim, Teck Kwang; Hua, Zi-Chun; Liu, Bin; Tannenbaum, Steven R.; Shen, Han-Ming; Lin, Qingsong

2016-01-01

To date, the exact targets and mechanism of action of curcumin, a natural product with anti-inflammatory and anti-cancer properties, remain elusive. Here we synthesized a cell permeable curcumin probe (Cur-P) with an alkyne moiety, which can be tagged with biotin for affinity enrichment, or with a fluorescent dye for visualization of the direct-binding protein targets of curcumin in situ. iTRAQTM quantitative proteomics approach was applied to distinguish the specific binding targets from the non-specific ones. In total, 197 proteins were confidently identified as curcumin binding targets from HCT116 colon cancer cell line. Gene Ontology analysis showed that the targets are broadly distributed and enriched in the nucleus, mitochondria and plasma membrane, and they are involved in various biological functions including metabolic process, regulation, response to stimulus and cellular process. Ingenuity Pathway AnalysisTM (IPA) suggested that curcumin may exert its anticancer effects over multiple critical biological pathways including the EIF2, eIF4/p70S6K, mTOR signaling and mitochondrial dysfunction pathways. Functional validations confirmed that curcumin downregulates cellular protein synthesis, and induces autophagy, lysosomal activation and increased ROS production, thus leading to cell death. PMID:26915414

Identifying biomarkers for asthma diagnosis using targeted metabolomics approaches.

PubMed

Checkley, William; Deza, Maria P; Klawitter, Jost; Romero, Karina M; Klawitter, Jelena; Pollard, Suzanne L; Wise, Robert A; Christians, Uwe; Hansel, Nadia N

2016-12-01

The diagnosis of asthma in children is challenging and relies on a combination of clinical factors and biomarkers including methacholine challenge, lung function, bronchodilator responsiveness, and presence of airway inflammation. No single test is diagnostic. We sought to identify a pattern of inflammatory biomarkers that was unique to asthma using a targeted metabolomics approach combined with data science methods. We conducted a nested case-control study of 100 children living in a peri-urban community in Lima, Peru. We defined cases as children with current asthma, and controls as children with no prior history of asthma and normal lung function. We further categorized enrollment following a factorial design to enroll equal numbers of children as either overweight or not. We obtained a fasting venous blood sample to characterize a comprehensive panel of targeted markers using a metabolomics approach based on high performance liquid chromatography-mass spectrometry. A statistical comparison of targeted metabolites between children with asthma (n = 50) and healthy controls (n = 49) revealed distinct patterns in relative concentrations of several metabolites: children with asthma had approximately 40-50% lower relative concentrations of ascorbic acid, 2-isopropylmalic acid, shikimate-3-phosphate, and 6-phospho-d-gluconate when compared to children without asthma, and 70% lower relative concentrations of reduced glutathione (all p < 0.001 after Bonferroni correction). Moreover, a combination of 2-isopropylmalic acid and betaine strongly discriminated between children with asthma (2-isopropylmalic acid ≤ 13 077 normalized counts/second) and controls (2-isopropylmalic acid > 13 077 normalized counts/second and betaine ≤ 16 47 121 normalized counts/second). By using a metabolomics approach applied to serum, we were able to discriminate between children with and without asthma by revealing different metabolic patterns. These results suggest that

Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling.

PubMed

Cottet, Sandra; Corthésy-Theulaz, Irène; Spertini, François; Corthésy, Blaise

2002-09-13

Molecular dissection of the mechanisms underlying Helicobacter pylori infection suffers from the lack of in vitro systems mimicking in vivo observations. A system was developed whereby human epithelial cells (Caco-2) grown as polarized monolayers and bacteria can communicate with each other under culture conditions optimal for each partner. Caco-2 cells grown on filter supports were inserted in a vertical position into diffusion chambers equilibrated with air and 5% CO(2) at their basolateral surface (aerophilic conditions) and 5% CO(2), 5% O(2), 90% N(2) (microaerophilic conditions) in the apical compartment. Remarkably, the epithelial polarized layer was stable under these asymmetric culture conditions for at least 24 h, and the presence of Caco-2 cells was necessary to maintain H. pylori growth. In contrast to previous studies conducted with non-polarized Caco-2 cells and other cell lines kept under aerophilic conditions, we found H. pylori-dependent stimulation of cytokine secretion (MCP-1 (monocyte chemoattractant protein-1), GRO-alpha (growth-regulated oncogene-alpha), RANTES (regulated on activation normal T cell expressed and secreted)). This correlated with nuclear translocation of NF-kappaB p50 and p65 subunits. Tyrosine phosphorylation of nine cellular proteins was induced or enhanced; we identified p120(RasGAP), p190(RhoGAP), p62dok (downstream of tyrosine kinases), and cortactin as H. pylori-inducible targets. Moreover, reduction of H. pylori urease expression was observed in adherent bacteria as compared with bacteria in suspension. In addition to mimicking several observations seen in the inflamed gastric mucosa, the novel in vitro system was allowed to underscore complex cellular events not seen in classical in vitro analyses of microaerophilic bacteria-epithelial cell cross-talk.

Systematic MicroRNA Analysis Identifies ATP6V0C as an Essential Host Factor for Human Cytomegalovirus Replication

PubMed Central

Pavelin, Jon; Reynolds, Natalie; Chiweshe, Stephen; Wu, Guanming; Tiribassi, Rebecca; Grey, Finn

2013-01-01

Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions. PMID:24385903

Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases.

PubMed

Connolly, Niamh M C; Theurey, Pierre; Adam-Vizi, Vera; Bazan, Nicolas G; Bernardi, Paolo; Bolaños, Juan P; Culmsee, Carsten; Dawson, Valina L; Deshmukh, Mohanish; Duchen, Michael R; Düssmann, Heiko; Fiskum, Gary; Galindo, Maria F; Hardingham, Giles E; Hardwick, J Marie; Jekabsons, Mika B; Jonas, Elizabeth A; Jordán, Joaquin; Lipton, Stuart A; Manfredi, Giovanni; Mattson, Mark P; McLaughlin, BethAnn; Methner, Axel; Murphy, Anne N; Murphy, Michael P; Nicholls, David G; Polster, Brian M; Pozzan, Tullio; Rizzuto, Rosario; Satrústegui, Jorgina; Slack, Ruth S; Swanson, Raymond A; Swerdlow, Russell H; Will, Yvonne; Ying, Zheng; Joselin, Alvin; Gioran, Anna; Moreira Pinho, Catarina; Watters, Orla; Salvucci, Manuela; Llorente-Folch, Irene; Park, David S; Bano, Daniele; Ankarcrona, Maria; Pizzo, Paola; Prehn, Jochen H M

2018-03-01

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.

Cellular Therapies Clinical Research Roadmap: Lessons learned on how to move a cellular therapy into a clinical trial

PubMed Central

Ouseph, Stacy; Tappitake, Darah; Armant, Myriam; Wesselschmidt, Robin; Derecho, Ivy; Draxler, Rebecca; Wood, Deborah; Centanni, John M.

2014-01-01

A clinical research roadmap has been developed as a resource for researchers to identify critical areas and potential pitfalls when transitioning a cellular therapy product from the research laboratory, via and Investigational New Drug (IND) application, into early phase clinical trials. The roadmap describes four key areas; basic and preclinical research, resource development, translational research and good manufacturing practice (GMP), and IND assembly and submission. Basic and preclinical research identifies a new therapeutic concept and demonstrates its potential value using a model of the relevant disease. During resource development the appropriate specialists and the required expertise to bring this product into the clinic are identified (e.g., researchers, regulatory specialists, GMP manufacturing staff, clinicians, and clinical trials staff, etc.). Additionally, the funds required to achieve this goal (or a plan to procure them) are identified. In the next phase the plan to translate the research product into a clinical grade therapeutic is developed. Finally regulatory approval to start the trial must be obtained. In the United States this is done by filing an IND application with the Food and Drug Administration. The NHLBI-funded Production Assistance for Cellular Therapies (PACT) program has facilitated the transition of a variety of cellular therapy products from the laboratory into Phase1/2 trials. The five PACT facilities have assisted investigators by performing translational studies and GMP manufacturing to ensure that cellular products met release specifications and were manufactured safely, reproducibly, and at the appropriate scale. The roadmap resulting from this experience is the focus of this article. PMID:25484311

Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells

PubMed Central

Lake, Michael P.; Bouchard, Louis-S.

2017-01-01

Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging. PMID:28636640

Identifying mechanism-of-action targets for drugs and probes

PubMed Central

Gregori-Puigjané, Elisabet; Setola, Vincent; Hert, Jérôme; Crews, Brenda A.; Irwin, John J.; Lounkine, Eugen; Marnett, Lawrence; Roth, Bryan L.; Shoichet, Brian K.

2012-01-01

Notwithstanding their key roles in therapy and as biological probes, 7% of approved drugs are purported to have no known primary target, and up to 18% lack a well-defined mechanism of action. Using a chemoinformatics approach, we sought to “de-orphanize” drugs that lack primary targets. Surprisingly, targets could be easily predicted for many: Whereas these targets were not known to us nor to the common databases, most could be confirmed by literature search, leaving only 13 Food and Drug Administration—approved drugs with unknown targets; the number of drugs without molecular targets likely is far fewer than reported. The number of worldwide drugs without reasonable molecular targets similarly dropped, from 352 (25%) to 44 (4%). Nevertheless, there remained at least seven drugs for which reasonable mechanism-of-action targets were unknown but could be predicted, including the antitussives clemastine, cloperastine, and nepinalone; the antiemetic benzquinamide; the muscle relaxant cyclobenzaprine; the analgesic nefopam; and the immunomodulator lobenzarit. For each, predicted targets were confirmed experimentally, with affinities within their physiological concentration ranges. Turning this question on its head, we next asked which drugs were specific enough to act as chemical probes. Over 100 drugs met the standard criteria for probes, and 40 did so by more stringent criteria. A chemical information approach to drug-target association can guide therapeutic development and reveal applications to probe biology, a focus of much current interest. PMID:22711801

Dynamic behavior of cellular materials and cellular structures: Experiments and modeling

NASA Astrophysics Data System (ADS)

Gao, Ziyang

Cellular solids, including cellular materials and cellular structures (CMS), have attracted people's great interests because of their low densities and novel physical, mechanical, thermal, electrical and acoustic properties. They offer potential for lightweight structures, energy absorption, thermal management, etc. Therefore, the studies of cellular solids have become one of the hottest research fields nowadays. From energy absorption point of view, any plastically deformed structures can be divided into two types (called type I and type II), and the basic cells of the CMS may take the configurations of these two types of structures. Accordingly, separated discussions are presented in this thesis. First, a modified 1-D model is proposed and numerically solved for a typical type II structure. Good agreement is achieved with the previous experimental data, hence is used to simulate the dynamic behavior of a type II chain. Resulted from different load speeds, interesting collapse modes are observed, and the parameters which govern the cell's post-collapse behavior are identified through a comprehensive non-dimensional analysis on general cellular chains. Secondly, the MHS specimens are chosen as an example of type I foam materials because of their good uniformity of the cell geometry. An extensive experimental study was carried out, where more attention was paid to their responses to dynamic loadings. Great enhancement of the stress-strain curve was observed in dynamic cases, and the energy absorption capacity is found to be several times higher than that of the commercial metal foams. Based on the experimental study, finite elemental simulations and theoretical modeling are also conducted, achieving good agreements and demonstrating the validities of those models. It is believed that the experimental, numerical and analytical results obtained in the present study will certainly deepen the understanding of the unsolved fundamental issues on the mechanical behavior of

Investigating the cellular fate of a DNA-targeted platinum-based anticancer agent by orthogonal double-click chemistry

PubMed Central

Qiao, Xin; Ding, Song; Liu, Fang; Kucera, Gregory L.

2014-01-01

Confocal fluorescence microscopy was used to study a platinum-based anticancer agent in intact NCI-H460 lung cancer cells. Orthogonal copper-catalyzed azide–alkyne cycloaddition (click) reactions were used to simultaneously determine the cell-cycle-specific localization of the azide-functionalized platinum–acridine agent 1 and monitor its effects on nucleic acid metabolism. Copper-catalyzed postlabeling showed advantages over copper-free click chemistry using a dibenzocyclooctyne (DIBO)-modified reporter dye, which produced high background levels in microscopic images and failed to efficiently label platinum adducts in chromatin. Compound 1 was successfully labeled with the fluorophore DIBO to yield 1* (characterized by in-line high-performance liquid chromatography/electrospray mass spectrometry). 1 and 1* show a high degree of colocalization in the confocal images, but the ability of 1* to target the (compacted) chromatin was markedly reduced, most likely owing to the steric bulk introduced by the DIBO tag. Nuclear platinum levels correlated inversely with the ability of the cells to synthesize DNA and cause cell cycle arrest, as confirmed by bivariate flow cytometry analysis. In addition, a decrease in the level of cellular transcription, shrinkage of the nucleolar regions, and redistribution of RNA into the cytosol were observed. Postlabeling in conjunction with colocalization experiments is a useful tool for studying the cell killing mechanism of this type of DNA-targeted agent. PMID:24407462

Genome-wide identification of translationally inhibited and degraded miR-155 targets using RNA-interacting protein-IP

PubMed Central

Meier, Jan; Hovestadt, Volker; Zapatka, Marc; Pscherer, Armin; Lichter, Peter; Seiffert, Martina

2013-01-01

MicroRNAs (miRNAs) are single-stranded, small, non-coding RNAs, which fine-tune protein expression by degrading and/or translationally inhibiting mRNAs. Manipulation of miRNA expression in animal models frequently results in severe phenotypes indicating their relevance in controlling cellular functions, most likely by interacting with multiple targets. To better understand the effect of miRNA activities, genome-wide analysis of their targets are required. MicroRNA profiling as well as transcriptome analysis upon enforced miRNA expression were frequently used to investigate their relevance. However, these approaches often fail to identify relevant miRNAs targets. Therefore, we tested the precision of RNA-interacting protein immunoprecipitation (RIP) using AGO2-specific antibodies, a core component of the “RNA-induced silencing complex” (RISC), followed by RNA sequencing (Seq) in a defined cellular system, the HEK293T cells with stable, ectopic expression of miR-155. Thereby, we identified 100 AGO2-associated mRNAs in miR-155-expressing cells, of which 67 were in silico predicted miR-155 target genes. An integrated analysis of the corresponding expression profiles indicated that these targets were either regulated by mRNA decay or by translational repression. Of the identified miR-155 targets, 17 were related to cell cycle control, suggesting their involvement in the observed increase in cell proliferation of HEK293T cells upon miR-155 expression. Additional, secondary changes within the gene expression profile were detected and might contribute to this phenotype as well. Interestingly, by analyzing RIP-Seq data of HEK-293T cells and two B-cell lines we identified a recurrent disproportional enrichment of several miRNAs, including miR-155 and miRNAs of the miR-17-92 cluster, in the AGO2-associated precipitates, suggesting discrepancies in miRNA expression and activity. PMID:23673373

A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs

PubMed Central

Karlas, Alexander; Berre, Stefano; Couderc, Thérèse; Varjak, Margus; Braun, Peter; Meyer, Michael; Gangneux, Nicolas; Karo-Astover, Liis; Weege, Friderike; Raftery, Martin; Schönrich, Günther; Klemm, Uwe; Wurzlbauer, Anne; Bracher, Franz; Merits, Andres; Meyer, Thomas F.; Lecuit, Marc

2016-01-01

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents. PMID:27177310

A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs.

PubMed

Karlas, Alexander; Berre, Stefano; Couderc, Thérèse; Varjak, Margus; Braun, Peter; Meyer, Michael; Gangneux, Nicolas; Karo-Astover, Liis; Weege, Friderike; Raftery, Martin; Schönrich, Günther; Klemm, Uwe; Wurzlbauer, Anne; Bracher, Franz; Merits, Andres; Meyer, Thomas F; Lecuit, Marc

2016-05-12

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents.

Sparse feature selection methods identify unexpected global cellular response to strontium-containing materials

PubMed Central

Autefage, Hélène; Littmann, Elena; Hedegaard, Martin A. B.; Von Erlach, Thomas; O’Donnell, Matthew; Burden, Frank R.; Winkler, David A.; Stevens, Molly M.

2015-01-01

Despite the increasing sophistication of biomaterials design and functional characterization studies, little is known regarding cells’ global response to biomaterials. Here, we combined nontargeted holistic biological and physical science techniques to evaluate how simple strontium ion incorporation within the well-described biomaterial 45S5 bioactive glass (BG) influences the global response of human mesenchymal stem cells. Our objective analyses of whole gene-expression profiles, confirmed by standard molecular biology techniques, revealed that strontium-substituted BG up-regulated the isoprenoid pathway, suggesting an influence on both sterol metabolite synthesis and protein prenylation processes. This up-regulation was accompanied by increases in cellular and membrane cholesterol and lipid raft contents as determined by Raman spectroscopy mapping and total internal reflection fluorescence microscopy analyses and by an increase in cellular content of phosphorylated myosin II light chain. Our unexpected findings of this strong metabolic pathway regulation as a response to biomaterial composition highlight the benefits of discovery-driven nonreductionist approaches to gain a deeper understanding of global cell–material interactions and suggest alternative research routes for evaluating biomaterials to improve their design. PMID:25831522

Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR

PubMed Central

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V.; Tai, TC; Saleh, Mazen; Parrillo, Joseph E.; Kumar, Anand

2012-01-01

Severe sepsis and the ensuing septic shock are serious life threatening conditions. These diseases are triggered by the host's over exuberant systemic response to the infecting pathogen. Several surveillance mechanisms have evolved to discriminate self from foreign RNA and accordingly trigger effective cellular responses to target the pathogenic threats. The RNA-dependent protein kinase (PKR) is a key component of the cytoplasmic RNA sensors involved in the recognition of viral double-stranded RNA (dsRNA). Here, we identify bacterial RNA as a distinct pathogenic pattern recognized by PKR. Our results indicate that natural RNA derived from bacteria directly binds to and activates PKR. We further show that bacterial RNA induces human cardiac myocyte apoptosis and identify the requirement for PKR in mediating this response. In addition to bacterial immunity, the results presented here may also have implications in cardiac pathophysiology. PMID:22833816

Cellular zinc fluxes and the regulation of apoptosis/gene-directed cell death.

PubMed

Truong-Tran, A Q; Ho, L H; Chai, F; Zalewski, P D

2000-05-01

The maintenance of discrete subcellular pools of zinc (Zn) is critical for the functional and structural integrity of cells. Among the important biological processes influenced by Zn is apoptosis, a process that is important in cellular homeostasis (an important cellular homeostatic process). It has also been identified as a major mechanism contributing to cell death in response to toxins and in disease, offering hope that novel therapies that target apoptotic pathways may be developed. Because Zn levels in the body can be increased in a relatively nontoxic manner, it may be possible to prevent or ameliorate degenerative disorders that are associated with high rates of apoptotic cell death. This review begins with brief introductions that address, first, the cellular biology of Zn, especially the critical labile Zn pools, and, second, the phenomenon of apoptosis. We then review the evidence relating Zn to apoptosis and address three major hypotheses: (1) that a specific pool or pools of intracellular labile Zn regulates apoptosis; (2) that systemic changes in Zn levels in the body, due to dietary factors, altered physiological states or disease, can influence cell susceptibility to apoptosis, and (3) that this altered susceptibility to apoptosis contributes to pathophysiological changes in the body. Other key issues are the identity of the molecular targets of Zn in the apoptotic cascade, the types of cells and tissues most susceptible to Zn-regulated apoptosis, the role of Zn as a coordinate regulator of mitosis and apoptosis and the apparent release of tightly bound intracellular pools of Zn during the later stages of apoptosis. This review concludes with a section highlighting areas of priority for future studies.

Amiodarone affects Ebola virus binding and entry into target cells.

PubMed

Salata, Cristiano; Munegato, Denis; Martelli, Francesco; Parolin, Cristina; Calistri, Arianna; Baritussio, Aldo; Palù, Giorgio

2018-03-02

Ebola Virus Disease is one of the most lethal transmissible infections characterized by a high fatality rate. Several research studies have aimed to identify effective antiviral agents. Amiodarone, a drug used for the treatment of arrhythmias, has been shown to inhibit filovirus infection in vitro by acting at the early step of the viral replication cycle. Here we demonstrate that amiodarone reduces virus binding to target cells and slows down the progression of the viral particles along the endocytic pathway. Overall our data support the notion that amiodarone interferes with Ebola virus infection by affecting cellular pathways/targets involved in the viral entry process.

The Emerging Role of Skeletal Muscle Metabolism as a Biological Target and Cellular Regulator of Cancer-Induced Muscle Wasting

PubMed Central

Carson, James A.; Hardee, Justin P.; VanderVeen, Brandon N.

2015-01-01

While skeletal muscle mass is an established primary outcome related to understanding cancer cachexia mechanisms, considerable gaps exist in our understanding of muscle biochemical and functional properties that have recognized roles in systemic health. Skeletal muscle quality is a classification beyond mass, and is aligned with muscle’s metabolic capacity and substrate utilization flexibility. This supplies an additional role for the mitochondria in cancer-induced muscle wasting. While the historical assessment of mitochondria content and function during cancer-induced muscle loss was closely aligned with energy flux and wasting susceptibility, this understanding has expanded to link mitochondria dysfunction to cellular processes regulating myofiber wasting. The primary objective of this article is to highlight muscle mitochondria and oxidative metabolism as a biological target of cancer cachexia and also as a cellular regulator of cancer-induced muscle wasting. Initially, we examine the role of muscle metabolic phenotype and mitochondria content in cancer-induced wasting susceptibility. We then assess the evidence for cancer-induced regulation of skeletal muscle mitochondrial biogenesis, dynamics, mitophagy, and oxidative stress. In addition, we discuss environments associated with cancer cachexia that can impact the regulation of skeletal muscle oxidative metabolism. The article also examines the role of cytokine-mediated regulation of mitochondria function regulation, followed by the potential role of cancer-induced hypogonadism. Lastly, a role for decreased muscle use in cancer-induced mitochondrial dysfunction is reviewed. PMID:26593326

Cellular death, reactive oxygen species (ROS) and diabetic complications.

PubMed

Volpe, Caroline Maria Oliveira; Villar-Delfino, Pedro Henrique; Dos Anjos, Paula Martins Ferreira; Nogueira-Machado, José Augusto

2018-01-25

Chronic or intermittent hyperglycemia is associated with the development of diabetic complications. Several signaling pathways can be altered by having hyperglycemia in different tissues, producing oxidative stress, the formation of advanced glycation end products (AGEs), as well as the secretion of the pro-inflammatory cytokines and cellular death (pathological autophagy and/or apoptosis). However, the signaling pathways that are directly triggered by hyperglycemia appear to have a pivotal role in diabetic complications due to the production of reactive oxygen species (ROS), oxidative stress, and cellular death. The present review will discuss the role of cellular death in diabetic complications, and it will suggest the cause and the consequences between the hyperglycemia-induced signaling pathways and cell death. The signaling pathways discussed in this review are to be described step-by-step, together with their respective inhibitors. They involve diacylglycerol, the activation of protein kinase C (PKC) and NADPH-oxidase system, and the consequent production of ROS. This was initially entitled the "dangerous metabolic route in diabetes". The historical usages and the recent advancement of new drugs in controlling possible therapeutical targets have been highlighted, in order to evaluate the evolution of knowledge in this sensitive area. It has recently been shown that the metabolic responses to stimuli (i.e., hyperglycemia) involve an integrated network of signaling pathways, in order to define the exact responses. Certain new drugs have been experimentally tested-or suggested and proposed-for their ability to modulate the possible biochemical therapeutical targets for the downregulation of retinopathy, nephropathy, neuropathy, heart disease, angiogenesis, oxidative stress, and cellular death. The aim of this study was to critically and didactically evaluate the exact steps of these signaling pathways and hence mark the indicated sites for the actions of such

Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters

PubMed Central

Baseler, Laura; Scott, Dana P.; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz

2016-01-01

Background Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Methodology/Principal Findings Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Conclusions/Significance Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central

Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters.

PubMed

Baseler, Laura; Scott, Dana P; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz; de Wit, Emmie

2016-11-01

Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central nervous system pathology.

Cellular and molecular actions of binary toxins possessing ADP-ribosyltransferase activity.

PubMed

Considine, R V; Simpson, L L

1991-01-01

Clostridial organisms produce a number of binary toxins. Thus far, three complete toxins (botulinum, perfringens and spiroforme) and one incomplete toxin (difficile) have been identified. In the case of complete toxins, there is a heavy chain component (Mr approximately 100,000) that binds to target cells and helps create a docking site for the light chain component (Mr approximately 50,000). The latter is an enzyme that possesses mono(ADP-ribosyl)transferase activity. The toxins appear to proceed through a three step sequence to exert their effects, including a binding step, an internalization step and an intracellular poisoning step. The substrate for the toxins is G-actin. By virtue of ADP-ribosylating monomeric actin, the toxins prevent polymerization as well as promoting depolymerization. The most characteristic cellular effect of the toxins is alteration of the cytoskeleton, which leads directly to changes in cellular morphology and indirectly to changes in cell function (e.g. release of chemical mediators). Binary toxins capable of modifying actin are likely to be useful tools in the study of cell biology.

Time scale of diffusion in molecular and cellular biology

NASA Astrophysics Data System (ADS)

Holcman, D.; Schuss, Z.

2014-05-01

Diffusion is the driver of critical biological processes in cellular and molecular biology. The diverse temporal scales of cellular function are determined by vastly diverse spatial scales in most biophysical processes. The latter are due, among others, to small binding sites inside or on the cell membrane or to narrow passages between large cellular compartments. The great disparity in scales is at the root of the difficulty in quantifying cell function from molecular dynamics and from simulations. The coarse-grained time scale of cellular function is determined from molecular diffusion by the mean first passage time of molecular Brownian motion to a small targets or through narrow passages. The narrow escape theory (NET) concerns this issue. The NET is ubiquitous in molecular and cellular biology and is manifested, among others, in chemical reactions, in the calculation of the effective diffusion coefficient of receptors diffusing on a neuronal cell membrane strewn with obstacles, in the quantification of the early steps of viral trafficking, in the regulation of diffusion between the mother and daughter cells during cell division, and many other cases. Brownian trajectories can represent the motion of a molecule, a protein, an ion in solution, a receptor in a cell or on its membrane, and many other biochemical processes. The small target can represent a binding site or an ionic channel, a hidden active site embedded in a complex protein structure, a receptor for a neurotransmitter on the membrane of a neuron, and so on. The mean time to attach to a receptor or activator determines diffusion fluxes that are key regulators of cell function. This review describes physical models of various subcellular microdomains, in which the NET coarse-grains the molecular scale to a higher cellular-level, thus clarifying the role of cell geometry in determining subcellular function.

PACCMIT/PACCMIT-CDS: identifying microRNA targets in 3′ UTRs and coding sequences

PubMed Central

Šulc, Miroslav; Marín, Ray M.; Robins, Harlan S.; Vaníček, Jiří

2015-01-01

The purpose of the proposed web server, publicly available at http://paccmit.epfl.ch, is to provide a user-friendly interface to two algorithms for predicting messenger RNA (mRNA) molecules regulated by microRNAs: (i) PACCMIT (Prediction of ACcessible and/or Conserved MIcroRNA Targets), which identifies primarily mRNA transcripts targeted in their 3′ untranslated regions (3′ UTRs), and (ii) PACCMIT-CDS, designed to find mRNAs targeted within their coding sequences (CDSs). While PACCMIT belongs among the accurate algorithms for predicting conserved microRNA targets in the 3′ UTRs, the main contribution of the web server is 2-fold: PACCMIT provides an accurate tool for predicting targets also of weakly conserved or non-conserved microRNAs, whereas PACCMIT-CDS addresses the lack of similar portals adapted specifically for targets in CDS. The web server asks the user for microRNAs and mRNAs to be analyzed, accesses the precomputed P-values for all microRNA–mRNA pairs from a database for all mRNAs and microRNAs in a given species, ranks the predicted microRNA–mRNA pairs, evaluates their significance according to the false discovery rate and finally displays the predictions in a tabular form. The results are also available for download in several standard formats. PMID:25948580

Molecular and cellular targets affected by green tea extracts in vascular cells

USDA-ARS?s Scientific Manuscript database

Consumption of green or black tea has been associated with a lower risk for the development of cardiovascular diseases, but despite many studies, a firm connection has not been delineated. Several molecular and cellular mechanisms may play a role in the preventive activity of tea. As reviewed here, ...

Targeting chemotherapy-resistant leukemia by combining DNT cellular therapy with conventional chemotherapy.

PubMed

Chen, Branson; Lee, Jong Bok; Kang, Hyeonjeong; Minden, Mark D; Zhang, Li

2018-04-24

While conventional chemotherapy is effective at eliminating the bulk of leukemic cells, chemotherapy resistance in acute myeloid leukemia (AML) is a prevalent problem that hinders conventional therapies and contributes to disease relapse, and ultimately patient death. We have recently shown that allogeneic double negative T cells (DNTs) are able to target the majority of primary AML blasts in vitro and in patient-derived xenograft models. However, some primary AML blast samples are resistant to DNT cell therapy. Given the differences in the modes of action of DNTs and chemotherapy, we hypothesize that DNT therapy can be used in combination with conventional chemotherapy to further improve their anti-leukemic effects and to target chemotherapy-resistant disease. Drug titration assays and flow-based cytotoxicity assays using ex vivo expanded allogeneic DNTs were performed on multiple AML cell lines to identify therapy-resistance. Primary AML samples were also tested to validate our in vitro findings. Further, a xenograft model was employed to demonstrate the feasibility of combining conventional chemotherapy and adoptive DNT therapy to target therapy-resistant AML. Lastly, blocking assays with neutralizing antibodies were employed to determine the mechanism by which chemotherapy increases the susceptibility of AML to DNT-mediated cytotoxicity. Here, we demonstrate that KG1a, a stem-like AML cell line that is resistant to DNTs and chemotherapy, and chemotherapy-resistant primary AML samples both became more susceptible to DNT-mediated cytotoxicity in vitro following pre-treatment with daunorubicin. Moreover, chemotherapy treatment followed by adoptive DNT cell therapy significantly decreased bone marrow engraftment of KG1a in a xenograft model. Mechanistically, daunorubicin increased the expression of NKG2D and DNAM-1 ligands on KG1a; blocking of these pathways attenuated DNT-mediated cytotoxicity. Our results demonstrate the feasibility and benefit of using DNTs as

Bioinformatics approaches to predict target genes from transcription factor binding data.

PubMed

Essebier, Alexandra; Lamprecht, Marnie; Piper, Michael; Bodén, Mikael

2017-12-01

Transcription factors regulate gene expression and play an essential role in development by maintaining proliferative states, driving cellular differentiation and determining cell fate. Transcription factors are capable of regulating multiple genes over potentially long distances making target gene identification challenging. Currently available experimental approaches to detect distal interactions have multiple weaknesses that have motivated the development of computational approaches. Although an improvement over experimental approaches, existing computational approaches are still limited in their application, with different weaknesses depending on the approach. Here, we review computational approaches with a focus on data dependency, cell type specificity and usability. With the aim of identifying transcription factor target genes, we apply available approaches to typical transcription factor experimental datasets. We show that approaches are not always capable of annotating all transcription factor binding sites; binding sites should be treated disparately; and a combination of approaches can increase the biological relevance of the set of genes identified as targets. Copyright © 2017 Elsevier Inc. All rights reserved.

Constitutional Flavonoids Derived from Epimedium Dose-Dependently Reduce Incidence of Steroid-Associated Osteonecrosis Not via Direct Action by Themselves on Potential Cellular Targets

PubMed Central

Xie, Xin-Hui; He, Yi-Xin; Yao, Xin-Sheng; Li, Zi-Rong; Lee, Kwong-Man; He, Wei; Leung, Kwok-Sui; Qin, Ling

2009-01-01

Intravascular-thrombosis and extravascular-lipid-deposit are the two key pathogenic events considered to interrupt intraosseous blood supply during development of steroid-associated osteonecrosis (ON). However, there are no clinically employed agents capable of simultaneously targeting these two key pathogenic events. The present experimental study demonstrated that constitutional flavonoid glycosides derived from herb Epimedium (EF, composed of seven flavonoid compounds with common stem nuclear) exerted dose-dependent effect on inhibition of both thrombosis and lipid-deposition and accordingly reducing incidence of steroid-associated ON in rabbits, which was not via direct action by themselves rather by their common metabolite on potential cellular targets involved in the two pathogenic pathways. The underlying mechanism could be explained by counteracting endothelium injury and excessive adipogenesis. These findings encourage designing clinical trials to investigate potential of EF in prevention of steroid-associated ON. PMID:19641620

Cellular Contraction and Polarization Drive Collective Cellular Motion.

PubMed

Notbohm, Jacob; Banerjee, Shiladitya; Utuje, Kazage J C; Gweon, Bomi; Jang, Hwanseok; Park, Yongdoo; Shin, Jennifer; Butler, James P; Fredberg, Jeffrey J; Marchetti, M Cristina

2016-06-21

Coordinated motions of close-packed multicellular systems typically generate cooperative packs, swirls, and clusters. These cooperative motions are driven by active cellular forces, but the physical nature of these forces and how they generate collective cellular motion remain poorly understood. Here, we study forces and motions in a confined epithelial monolayer and make two experimental observations: 1) the direction of local cellular motion deviates systematically from the direction of the local traction exerted by each cell upon its substrate; and 2) oscillating waves of cellular motion arise spontaneously. Based on these observations, we propose a theory that connects forces and motions using two internal state variables, one of which generates an effective cellular polarization, and the other, through contractile forces, an effective cellular inertia. In agreement with theoretical predictions, drugs that inhibit contractility reduce both the cellular effective elastic modulus and the frequency of oscillations. Together, theory and experiment provide evidence suggesting that collective cellular motion is driven by at least two internal variables that serve to sustain waves and to polarize local cellular traction in a direction that deviates systematically from local cellular velocity. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

More Haste, Less Speed: Could Public–Private Partnerships Advance Cellular Immunotherapies?

PubMed Central

Bubela, Tania; Bonter, Katherine; Lachance, Silvy; Delisle, Jean-Sébastien; Gold, E. Richard

2017-01-01

Cellular immunotherapies promise to transform cancer care. However, they must overcome serious challenges, including: (1) the need to identify and characterize novel cancer antigens to expand the range of therapeutic targets; (2) the need to develop strategies to minimize serious adverse events, such as cytokine release syndrome and treatment-related toxicities; and (3) the need to develop efficient production/manufacturing processes to reduce costs. Here, we discuss whether these challenges might better be addressed through forms of public–private research collaborations, including public–private partnerships (PPPs), or whether these challenges are best addressed by way of standard market transactions. We reviewed 14 public–private relationships and 25 underlying agreements for the clinical development of cancer cellular immunotherapies in the US. Most were based on bilateral research agreements and pure market transactions in the form of service contracts and technology licenses, which is representative of the commercialization focus of the field. We make the strategic case that multiparty PPPs may better advance cancer antigen discovery and characterization and improved cell processing/manufacturing and related activities. In the rush toward the competitive end of the translational continuum for cancer cellular immunotherapy and the attendant focus on commercialization, many gaps have appeared in our understanding of cellular biology, immunology, and bioengineering. We conclude that the model of bilateral agreements between leading research institutions and the private sector may be inadequate to efficiently harness the interdisciplinary skills and knowledge of the public and private sectors to bring these promising therapies to the clinic for the benefit of cancer patients. PMID:28861415

More Haste, Less Speed: Could Public-Private Partnerships Advance Cellular Immunotherapies?

PubMed

Bubela, Tania; Bonter, Katherine; Lachance, Silvy; Delisle, Jean-Sébastien; Gold, E Richard

2017-01-01

Cellular immunotherapies promise to transform cancer care. However, they must overcome serious challenges, including: (1) the need to identify and characterize novel cancer antigens to expand the range of therapeutic targets; (2) the need to develop strategies to minimize serious adverse events, such as cytokine release syndrome and treatment-related toxicities; and (3) the need to develop efficient production/manufacturing processes to reduce costs. Here, we discuss whether these challenges might better be addressed through forms of public-private research collaborations, including public-private partnerships (PPPs), or whether these challenges are best addressed by way of standard market transactions. We reviewed 14 public-private relationships and 25 underlying agreements for the clinical development of cancer cellular immunotherapies in the US. Most were based on bilateral research agreements and pure market transactions in the form of service contracts and technology licenses, which is representative of the commercialization focus of the field. We make the strategic case that multiparty PPPs may better advance cancer antigen discovery and characterization and improved cell processing/manufacturing and related activities. In the rush toward the competitive end of the translational continuum for cancer cellular immunotherapy and the attendant focus on commercialization, many gaps have appeared in our understanding of cellular biology, immunology, and bioengineering. We conclude that the model of bilateral agreements between leading research institutions and the private sector may be inadequate to efficiently harness the interdisciplinary skills and knowledge of the public and private sectors to bring these promising therapies to the clinic for the benefit of cancer patients.

Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes

PubMed Central

2004-01-01

14-3-3 proteins exert an extraordinarily widespread influence on cellular processes in all eukaryotes. They operate by binding to specific phosphorylated sites on diverse target proteins, thereby forcing conformational changes or influencing interactions between their targets and other molecules. In these ways, 14-3-3s ‘finish the job’ when phosphorylation alone lacks the power to drive changes in the activities of intracellular proteins. By interacting dynamically with phosphorylated proteins, 14-3-3s often trigger events that promote cell survival – in situations from preventing metabolic imbalances caused by sudden darkness in leaves to mammalian cell-survival responses to growth factors. Recent work linking specific 14-3-3 isoforms to genetic disorders and cancers, and the cellular effects of 14-3-3 agonists and antagonists, indicate that the cellular complement of 14-3-3 proteins may integrate the specificity and strength of signalling through to different cellular responses. PMID:15167810

Nano/microvehicles for efficient delivery and (bio)sensing at the cellular level

PubMed Central

Esteban-Fernández de Ávila, B.; Yáñez-Sedeño, P.

2017-01-01

A perspective review of recent strategies involving the use of nano/microvehicles to address the key challenges associated with delivery and (bio)sensing at the cellular level is presented. The main types and characteristics of the different nano/microvehicles used for these cellular applications are discussed, including fabrication pathways, propulsion (catalytic, magnetic, acoustic or biological) and navigation strategies, and relevant parameters affecting their propulsion performance and sensing and delivery capabilities. Thereafter, selected applications are critically discussed. An emphasis is made on enhancing the extra- and intra-cellular biosensing capabilities, fast cell internalization, rapid inter- or intra-cellular movement, efficient payload delivery and targeted on-demand controlled release in order to greatly improve the monitoring and modulation of cellular processes. A critical discussion of selected breakthrough applications illustrates how these smart multifunctional nano/microdevices operate as nano/microcarriers and sensors at the intra- and extra-cellular levels. These advances allow both the real-time biosensing of relevant targets and processes even at a single cell level, and the delivery of different cargoes (drugs, functional proteins, oligonucleotides and cells) for therapeutics, gene silencing/transfection and assisted fertilization, while overcoming challenges faced by current affinity biosensors and delivery vehicles. Key challenges for the future and the envisioned opportunities and future perspectives of this remarkably exciting field are discussed. PMID:29147499

Predicting essential genes for identifying potential drug targets in Aspergillus fumigatus.

PubMed

Lu, Yao; Deng, Jingyuan; Rhodes, Judith C; Lu, Hui; Lu, Long Jason

2014-06-01

Aspergillus fumigatus (Af) is a ubiquitous and opportunistic pathogen capable of causing acute, invasive pulmonary disease in susceptible hosts. Despite current therapeutic options, mortality associated with invasive Af infections remains unacceptably high, increasing 357% since 1980. Therefore, there is an urgent need for the development of novel therapeutic strategies, including more efficacious drugs acting on new targets. Thus, as noted in a recent review, "the identification of essential genes in fungi represents a crucial step in the development of new antifungal drugs". Expanding the target space by rapidly identifying new essential genes has thus been described as "the most important task of genomics-based target validation". In previous research, we were the first to show that essential gene annotation can be reliably transferred between distantly related four Prokaryotic species. In this study, we extend our machine learning approach to the much more complex Eukaryotic fungal species. A compendium of essential genes is predicted in Af by transferring known essential gene annotations from another filamentous fungus Neurospora crassa. This approach predicts essential genes by integrating diverse types of intrinsic and context-dependent genomic features encoded in microbial genomes. The predicted essential datasets contained 1674 genes. We validated our results by comparing our predictions with known essential genes in Af, comparing our predictions with those predicted by homology mapping, and conducting conditional expressed alleles. We applied several layers of filters and selected a set of potential drug targets from the predicted essential genes. Finally, we have conducted wet lab knockout experiments to verify our predictions, which further validates the accuracy and wide applicability of the machine learning approach. The approach presented here significantly extended our ability to predict essential genes beyond orthologs and made it possible to

Identifying Minefields and Verifying Clearance: Adapting Statistical Methods for UXO Target Detection

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

Gilbert, Richard O.; O'Brien, Robert F.; Wilson, John E.

2003-09-01

It may not be feasible to completely survey large tracts of land suspected of containing minefields. It is desirable to develop a characterization protocol that will confidently identify minefields within these large land tracts if they exist. Naturally, surveying areas of greatest concern and most likely locations would be necessary but will not provide the needed confidence that an unknown minefield had not eluded detection. Once minefields are detected, methods are needed to bound the area that will require detailed mine detection surveys. The US Department of Defense Strategic Environmental Research and Development Program (SERDP) is sponsoring the development ofmore » statistical survey methods and tools for detecting potential UXO targets. These methods may be directly applicable to demining efforts. Statistical methods are employed to determine the optimal geophysical survey transect spacing to have confidence of detecting target areas of a critical size, shape, and anomaly density. Other methods under development determine the proportion of a land area that must be surveyed to confidently conclude that there are no UXO present. Adaptive sampling schemes are also being developed as an approach for bounding the target areas. These methods and tools will be presented and the status of relevant research in this area will be discussed.« less

Creating the Chemistry in Cellular Respiration Concept Inventory (CCRCI)

NASA Astrophysics Data System (ADS)

Forshee, Jay Lance, II

Students at our institution report cellular respiration to be the most difficult concept they encounter in undergraduate biology, but why students find this difficult is unknown. Students may find cellular respiration difficult because there is a large amount of steps, or because there are persistent, long-lasting misconceptions and misunderstandings surrounding their knowledge of chemistry, which affect their performance on cellular respiration assessments. Most studies of cellular respiration focus on student macro understanding of the process related to breathing, and matter and energy. To date, no studies identify which chemistry concepts are most relevant to students' development of an understanding of the process of cellular respiration or have developed an assessment to measure student understanding of them. Following the Delphi method, the researchers conducted expert interviews with faculty members from four-year, masters-, and PhD-granting institutions who teach undergraduate general biology, and are experts in their respective fields of biology. From these interviews, researchers identified twelve chemistry concepts important to understanding cellular respiration and using surveys, these twelve concepts were refined into five (electron transfer, energy transfer, thermodynamics (law/conservation), chemical reactions, and gradients). The researchers then interviewed undergraduate introductory biology students at a large Midwestern university to identify their knowledge and misconceptions of the chemistry concepts that the faculty had identified previously as important. The CCRCI was developed using the five important chemistry concepts underlying cellular respiration. The final version of the CCRCI was administered to n=160 introductory biology students during the spring 2017 semester. Reliability of the CCRCI was evaluated using Cronbach's alpha (=.7) and split-half reliability (=.769), and validity of the instrument was assessed through content validity

Genetically targeted 3D visualisation of Drosophila neurons under Electron Microscopy and X-Ray Microscopy using miniSOG

PubMed Central

Ng, Julian; Browning, Alyssa; Lechner, Lorenz; Terada, Masako; Howard, Gillian; Jefferis, Gregory S. X. E.

2016-01-01

Large dimension, high-resolution imaging is important for neural circuit visualisation as neurons have both long- and short-range patterns: from axons and dendrites to the numerous synapses at terminal endings. Electron Microscopy (EM) is the favoured approach for synaptic resolution imaging but how such structures can be segmented from high-density images within large volume datasets remains challenging. Fluorescent probes are widely used to localise synapses, identify cell-types and in tracing studies. The equivalent EM approach would benefit visualising such labelled structures from within sub-cellular, cellular, tissue and neuroanatomical contexts. Here we developed genetically-encoded, electron-dense markers using miniSOG. We demonstrate their ability in 1) labelling cellular sub-compartments of genetically-targeted neurons, 2) generating contrast under different EM modalities, and 3) segmenting labelled structures from EM volumes using computer-assisted strategies. We also tested non-destructive X-ray imaging on whole Drosophila brains to evaluate contrast staining. This enabled us to target specific regions for EM volume acquisition. PMID:27958322

Inforna 2.0: A Platform for the Sequence-Based Design of Small Molecules Targeting Structured RNAs.

PubMed

Disney, Matthew D; Winkelsas, Audrey M; Velagapudi, Sai Pradeep; Southern, Mark; Fallahi, Mohammad; Childs-Disney, Jessica L

2016-06-17

The development of small molecules that target RNA is challenging yet, if successful, could advance the development of chemical probes to study RNA function or precision therapeutics to treat RNA-mediated disease. Previously, we described Inforna, an approach that can mine motifs (secondary structures) within target RNAs, which is deduced from the RNA sequence, and compare them to a database of known RNA motif-small molecule binding partners. Output generated by Inforna includes the motif found in both the database and the desired RNA target, lead small molecules for that target, and other related meta-data. Lead small molecules can then be tested for binding and affecting cellular (dys)function. Herein, we describe Inforna 2.0, which incorporates all known RNA motif-small molecule binding partners reported in the scientific literature, a chemical similarity searching feature, and an improved user interface and is freely available via an online web server. By incorporation of interactions identified by other laboratories, the database has been doubled, containing 1936 RNA motif-small molecule interactions, including 244 unique small molecules and 1331 motifs. Interestingly, chemotype analysis of the compounds that bind RNA in the database reveals features in small molecule chemotypes that are privileged for binding. Further, this updated database expanded the number of cellular RNAs to which lead compounds can be identified.

Identifying Novel Regulators of Vacuolar Trafficking by Combining Fluorescence Imaging-Based Forward Genetic Screening and In Vitro Pollen Germination.

PubMed

Feng, Qiang-Nan; Zhang, Yan

2017-01-01

Subcellular targeting of vacuolar proteins depends on cellular machinery regulating vesicular trafficking. Plant-specific vacuolar trafficking routes have been reported. However, regulators mediating these processes are obscure. By combining a fluorescence imaging-based forward genetic approach and in vitro pollen germination system, we show an efficient protocol of identifying regulators of plant-specific vacuolar trafficking routes.

Systems pharmacology identifies drug targets for Stargardt disease–associated retinal degeneration

PubMed Central

Chen, Yu; Palczewska, Grazyna; Mustafi, Debarshi; Golczak, Marcin; Dong, Zhiqian; Sawada, Osamu; Maeda, Tadao; Maeda, Akiko; Palczewski, Krzysztof

2013-01-01

A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases. PMID:24231350

Recent developments in emerging therapeutic targets of osteoarthritis.

PubMed

Sun, Margaret Man-Ger; Beier, Frank; Pest, Michael A

2017-01-01

Despite the tremendous individual suffering and socioeconomic burden caused by osteoarthritis, there are currently no effective disease-modifying treatment options. This is in part because of our incomplete understanding of osteoarthritis disease mechanism. This review summarizes recent developments in therapeutic targets identified from surgical animal models of osteoarthritis that provide novel insight into osteoarthritis pathology and possess potential for progression into preclinical studies. Several candidate pathways and processes that have been identified include chondrocyte autophagy, growth factor signaling, inflammation, and nociceptive signaling. Major strategies that possess therapeutic potential at the cellular level include inhibiting autophagy suppression and decreasing reactive oxygen species (ROS) production. Cartilage anabolism and prevention of cartilage degradation has been shown to result from growth factor signaling modulation, such as TGF-β, TGF-α, and FGF; however, the results are context-dependent and require further investigation. Pain assessment studies in rodent surgical models have demonstrated potential in employing anti-NGF strategies for minimizing osteoarthritis-associated pain. Studies of potential therapeutic targets in osteoarthritis using animal surgical models are helping to elucidate osteoarthritis pathology and propel therapeutics development. Further studies should continue to elucidate pathological mechanisms and therapeutic targets in various joint tissues to improve overall joint health.

Sphingolipid and Ceramide Homeostasis: Potential Therapeutic Targets

PubMed Central

Young, Simon A.; Mina, John G.; Denny, Paul W.; Smith, Terry K.

2012-01-01

Sphingolipids are ubiquitous in eukaryotic cells where they have been attributed a plethora of functions from the formation of structural domains to polarized cellular trafficking and signal transduction. Recent research has identified and characterised many of the key enzymes involved in sphingolipid metabolism and this has led to a heightened interest in the possibility of targeting these processes for therapies against cancers, Alzheimer's disease, and numerous important human pathogens. In this paper we outline the major pathways in eukaryotic sphingolipid metabolism and discuss these in relation to disease and therapy for both chronic and infectious conditions. PMID:22400113

Identify mutation in amyotrophic lateral sclerosis cases using HaloPlex target enrichment system.

PubMed

Liu, Zhi-Jun; Li, Hong-Fu; Tan, Guo-He; Tao, Qing-Qing; Ni, Wang; Cheng, Xue-Wen; Xiong, Zhi-Qi; Wu, Zhi-Ying

2014-12-01

To date, at least 18 causative genes have been identified in amyotrophic lateral sclerosis (ALS). Because of the clinical and genetic heterogeneity, molecular diagnosis for ALS faces great challenges. HaloPlex target enrichment system is a new targeted sequencing approach, which can detect already known mutations or candidate genes. We performed this approach to screen 18 causative genes of ALS, including SOD1, SETX, FUS, ANG, TARDBP, ALS2, FIG4, VAPB, OPTN, DAO, VCP, UBQLN2, SPG11, SIGMAR1, DCTN1, SQSTM1, PFN1, and CHMP2B in 8 ALS probands. Using this approach, we got an average of 9.5 synonymous or missense mutations per sample. After validation by Sanger sequencing, we identified 3 documented SOD1 mutations (p.F21C, p.G148D, and p.C147R) and 1 novel DCTN1 p.G59R mutation in 4 probands. The novel DCTN1 mutation appeared to segregate with the disease in the pedigree and was absent in 200 control subjects. The high throughput and efficiency of this approach indicated that it could be applied to diagnose ALS and other inherited diseases with multiple causative genes in clinical practice. Copyright © 2014 Elsevier Inc. All rights reserved.

CRISPR-mediated HDAC2 disruption identifies two distinct classes of target genes in human cells.

PubMed

Somanath, Priyanka; Herndon Klein, Rachel; Knoepfler, Paul S

2017-01-01

The transcriptional functions of the class I histone deacetylases (HDACs) HDAC1 and HDAC2 are mainly viewed as both repressive and redundant based on murine knockout studies, but they may have additional independent roles and their physiological functions in human cells are not as clearly defined. To address the individual epigenomic functions of HDAC2, here we utilized CRISPR-Cas9 to disrupt HDAC2 in human cells. We find that while HDAC2 null cells exhibited signs of cross-regulation between HDAC1 and HDAC2, specific epigenomic phenotypes were still apparent using RNA-seq and ChIP assays. We identified specific targets of HDAC2 repression, and defined a novel class of genes that are actively expressed in a partially HDAC2-dependent manner. While HDAC2 was required for the recruitment of HDAC1 to repressed HDAC2-gene targets, HDAC2 was dispensable for HDAC1 binding to HDAC2-activated targets, supporting the notion of distinct classes of targets. Both active and repressed classes of gene targets demonstrated enhanced histone acetylation and methylation in HDAC2-null cells. Binding of the HDAC1/2-associated SIN3A corepressor was altered at most HDAC2-targets, but without a clear pattern. Overall, our study defines two classes of HDAC2 targets in human cells, with a dependence of HDAC1 on HDAC2 at one class of targets, and distinguishes unique functions for HDAC2.

Differential cellular recognition pattern to M. tuberculosis targets defined by IFN-γ and IL-17 production in blood from TB + patients from Honduras as compared to health care workers: TB and immune responses in patients from Honduras.

PubMed

Alvarez-Corrales, Nancy; Ahmed, Raija K; Rodriguez, Carol A; Balaji, Kithiganahalli N; Rivera, Rebeca; Sompallae, Ramakrishna; Vudattu, Nalini K; Hoffner, Sven E; Zumla, Alimuddin; Pineda-Garcia, Lelany; Maeurer, Markus

2013-03-06

A better understanding of the quality of cellular immune responses directed against molecularly defined targets will guide the development of TB diagnostics and identification of molecularly defined, clinically relevant M.tb vaccine candidates. Recombinant proteins (n = 8) and peptide pools (n = 14) from M. tuberculosis (M.tb) targets were used to compare cellular immune responses defined by IFN-γ and IL-17 production using a Whole Blood Assay (WBA) in a cohort of 148 individuals, i.e. patients with TB + (n = 38), TB- individuals with other pulmonary diseases (n = 81) and individuals exposed to TB without evidence of clinical TB (health care workers, n = 29). M.tb antigens Rv2958c (glycosyltransferase), Rv2962c (mycolyltransferase), Rv1886c (Ag85B), Rv3804c (Ag85A), and the PPE family member Rv3347c were frequently recognized, defined by IFN-γ production, in blood from healthy individuals exposed to M.tb (health care workers). A different recognition pattern was found for IL-17 production in blood from M.tb exposed individuals responding to TB10.4 (Rv0288), Ag85B (Rv1886c) and the PPE family members Rv0978c and Rv1917c. The pattern of immune target recognition is different in regard to IFN-γ and IL-17 production to defined molecular M.tb targets in PBMCs from individuals frequently exposed to M.tb. The data represent the first mapping of cellular immune responses against M.tb targets in TB patients from Honduras.

A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

PubMed

Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

2016-11-09

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication. Published by Elsevier Inc.

Characteristics of excessive cellular phone use in Korean adolescents.

PubMed

Ha, Jee Hyun; Chin, Bumsu; Park, Doo-Heum; Ryu, Seung-Ho; Yu, Jaehak

2008-12-01

Abstract The objective of this study was to evaluate the possible psychological problems related to excessive cellular phone use in adolescents. Results from 595 participants showed that the potentially excessive user group had a tendency to identify themselves with their cellular phones and to have difficulties in controlling usage. They expressed more depressive symptoms, higher interpersonal anxiety, and lower self-esteem. A positive correlation was also observed between excessive cellular phone use and Internet addiction.

Targeting LKB1 in cancer – exposing and exploiting vulnerabilities

PubMed Central

Momcilovic, M; Shackelford, D B

2015-01-01

The LKB1 tumour suppressor is a serine/threonine kinase that functions as master regulator of cell growth, metabolism, survival and polarity. LKB1 is frequently mutated in human cancers and research spanning the last two decades have begun decoding the cellular pathways deregulated following LKB1 inactivation. This work has led to the identification of vulnerabilities present in LKB1-deficient tumour cells. Pre-clinical studies have now identified therapeutic strategies targeting this subset of tumours that promise to benefit this large patient population harbouring LKB1 mutations. Here, we review the current efforts that are underway to translate pre-clinical discovery of therapeutic strategies targeting LKB1 mutant cancers into clinical practice. PMID:26196184

CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

PubMed Central

Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

2016-01-01

Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

DIGE Proteome Analysis Reveals Suitability of Ischemic Cardiac In Vitro Model for Studying Cellular Response to Acute Ischemia and Regeneration

PubMed Central

Haas, Sina; Jahnke, Heinz-Georg; Moerbt, Nora; von Bergen, Martin; Aharinejad, Seyedhossein; Andrukhova, Olena; Robitzki, Andrea A.

2012-01-01

Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy. With the establishment of our in vitro disease model for ischemia injury target identification via proteomic research becomes independent from rare human material and will create new possibilities in cardiac research. PMID:22384053

Terminal addition in a cellular world.

PubMed

Torday, J S; Miller, William B

2018-07-01

Recent advances in our understanding of evolutionary development permit a reframed appraisal of Terminal Addition as a continuous historical process of cellular-environmental complementarity. Within this frame of reference, evolutionary terminal additions can be identified as environmental induction of episodic adjustments to cell-cell signaling patterns that yield the cellular-molecular pathways that lead to differing developmental forms. Phenotypes derive, thereby, through cellular mutualistic/competitive niche constructions in reciprocating responsiveness to environmental stresses and epigenetic impacts. In such terms, Terminal Addition flows according to a logic of cellular needs confronting environmental challenges over space-time. A reconciliation of evolutionary development and Terminal Addition can be achieved through a combined focus on cell-cell signaling, molecular phylogenies and a broader understanding of epigenetic phenomena among eukaryotic organisms. When understood in this manner, Terminal Addition has an important role in evolutionary development, and chronic disease might be considered as a form of 'reverse evolution' of the self-same processes. Copyright © 2017. Published by Elsevier Ltd.

Turbulence Measurement in the Atmospheric Boundary Layer Using Cellular Telephone Signals

DTIC Science & Technology

2012-03-01

TURBULENCE MEASUREMENT IN THE ATMOSPHERIC BOUNDARY LAYER USING CELLULAR TELEPHONE SIGNALS THESIS Lee R. Burchett, Civilian AFIT/APPLPHY/ENP/12 - M01...85 xiv TURBULENCE MEASUREMENT IN THE ATMOSPHERIC BOUNDARY LAYER USING CELLULAR TELEPHONE SIGNALS I. Introduction What follows is an...efficient use of these systems. For example, the effective range of a laser weapon is limited by the strength of turbulence on the path to the target

Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization.

PubMed

Ozawa, Akihiko; Brunori, Gloria; Mercatelli, Daniela; Wu, Jinhua; Cippitelli, Andrea; Zou, Bende; Xie, Xinmin Simon; Williams, Melissa; Zaveri, Nurulain T; Low, Sarah; Scherrer, Grégory; Kieffer, Brigitte L; Toll, Lawrence

2015-08-19

The nociceptin/orphanin FQ (NOP) receptor, the fourth member of the opioid receptor family, is involved in many processes common to the opioid receptors including pain and drug abuse. To better characterize receptor location and trafficking, knock-in mice were created by inserting the gene encoding enhanced green fluorescent protein (eGFP) into the NOP receptor gene (Oprl1) and producing mice expressing a functional NOP-eGFP C-terminal fusion in place of the native NOP receptor. The NOP-eGFP receptor was present in brain of homozygous knock-in animals in concentrations somewhat higher than in wild-type mice and was functional when tested for stimulation of [(35)S]GTPγS binding in vitro and in patch-clamp electrophysiology in dorsal root ganglia (DRG) neurons and hippocampal slices. Inhibition of morphine analgesia was equivalent when tested in knock-in and wild-type mice. Imaging revealed detailed neuroanatomy in brain, spinal cord, and DRG and was generally consistent with in vitro autoradiographic imaging of receptor location. Multicolor immunohistochemistry identified cells coexpressing various spinal cord and DRG cellular markers, as well as coexpression with μ-opioid receptors in DRG and brain regions. Both in tissue slices and primary cultures, the NOP-eGFP receptors appear throughout the cell body and in processes. These knock-in mice have NOP receptors that function both in vitro and in vivo and appear to be an exceptional tool to study receptor neuroanatomy and correlate with NOP receptor function. The NOP receptor, the fourth member of the opioid receptor family, is involved in pain, drug abuse, and a number of other CNS processes. The regional and cellular distribution has been difficult to determine due to lack of validated antibodies for immunohistochemical analysis. To provide a new tool for the investigation of receptor localization, we have produced knock-in mice with a fluorescent-tagged NOP receptor in place of the native NOP receptor. These

ROCK inhibition as a therapy for spinal muscular atrophy: understanding the repercussions on multiple cellular targets

PubMed Central

Coque, Emmanuelle; Raoul, Cédric; Bowerman, Mélissa

2014-01-01

Spinal muscular atrophy (SMA) is the most common genetic disease causing infant death, due to an extended loss of motoneurons. This neuromuscular disorder results from deletions and/or mutations within the Survival Motor Neuron 1 (SMN1) gene, leading to a pathological decreased expression of functional full-length SMN protein. Emerging studies suggest that the small GTPase RhoA and its major downstream effector Rho kinase (ROCK), which both play an instrumental role in cytoskeleton organization, contribute to the pathology of motoneuron diseases. Indeed, an enhanced activation of RhoA and ROCK has been reported in the spinal cord of an SMA mouse model. Moreover, the treatment of SMA mice with ROCK inhibitors leads to an increased lifespan as well as improved skeletal muscle and neuromuscular junction pathology, without preventing motoneuron degeneration. Although motoneurons are the primary target in SMA, an increasing number of reports show that other cell types inside and outside the central nervous system contribute to SMA pathogenesis. As administration of ROCK inhibitors to SMA mice was systemic, the improvement in survival and phenotype could therefore be attributed to specific effects on motoneurons and/or on other non-neuronal cell types. In the present review, we will present the various roles of the RhoA/ROCK pathway in several SMA cellular targets including neurons, myoblasts, glial cells, cardiomyocytes and pancreatic cells as well as discuss how ROCK inhibition may ameliorate their health and function. It is most likely a concerted influence of ROCK modulation on all these cell types that ultimately lead to the observed benefits of pharmacological ROCK inhibition in SMA mice. PMID:25221469

Identifying On-Orbit Test Targets for Space Fence Operational Testing

NASA Astrophysics Data System (ADS)

Pechkis, D.; Pacheco, N.; Botting, T.

2014-09-01

Space Fence will be an integrated system of two ground-based, S-band (2 to 4 GHz) phased-array radars located in Kwajalein and perhaps Western Australia [1]. Space Fence will cooperate with other Space Surveillance Network sensors to provide space object tracking and radar characterization data to support U.S. Strategic Command space object catalog maintenance and other space situational awareness needs. We present a rigorous statistical test design intended to test Space Fence to the letter of the program requirements as well as to characterize the system performance across the entire operational envelope. The design uses altitude, size, and inclination as independent factors in statistical tests of dependent variables (e.g., observation accuracy) linked to requirements. The analysis derives the type and number of necessary test targets. Comparing the resulting sample sizes with the number of currently known targets, we identify those areas where modelling and simulation methods are needed. Assuming hypothetical Kwajalein radar coverage and a conservative number of radar passes per object per day, we conclude that tests involving real-world space objects should take no more than 25 days to evaluate all operational requirements; almost 60 percent of the requirements can be tested in a single day and nearly 90 percent can be tested in one week or less. Reference: [1] L. Haines and P. Phu, Space Fence PDR Concept Development Phase, 2011 AMOS Conference Technical Papers.

High-throughput screening identifies microRNAs that target Nox2 and improve function after acute myocardial infarction.

PubMed

Yang, Junyu; Brown, Milton E; Zhang, Hanshuo; Martinez, Mario; Zhao, Zhihua; Bhutani, Srishti; Yin, Shenyi; Trac, David; Xi, Jianzhong Jeff; Davis, Michael E

2017-05-01

Myocardial infarction (MI) is the most common cause of heart failure. Excessive production of ROS plays a key role in the pathogenesis of cardiac remodeling after MI. NADPH with NADPH oxidase (Nox)2 as the catalytic subunit is a major source of superoxide production, and expression is significantly increased in the infarcted myocardium, especially by infiltrating macrophages. While microRNAs (miRNAs) are potent regulators of gene expression and play an important role in heart disease, there still lacks efficient ways to identify miRNAs that target important pathological genes for treating MI. Thus, the overall objective was to establish a miRNA screening and delivery system for improving heart function after MI using Nox2 as a critical target. With the use of the miRNA-target screening system composed of a self-assembled cell microarray (SAMcell), three miRNAs, miR-106b, miR-148b, and miR-204, were identified that could regulate Nox2 expression and its downstream products in both human and mouse macrophages. Each of these miRNAs were encapsulated into polyketal (PK3) nanoparticles that could effectively deliver miRNAs into macrophages. Both in vitro and in vivo studies in mice confirmed that PK3-miRNAs particles could inhibit Nox2 expression and activity and significantly improve infarct size and acute cardiac function after MI. In conclusion, our results show that miR-106b, miR-148b, and miR-204 were able to improve heart function after myocardial infarction in mice by targeting Nox2 and possibly altering inflammatory cytokine production. This screening system and delivery method could have broader implications for miRNA-mediated therapeutics for cardiovascular and other diseases. NEW & NOTEWORTHY NADPH oxidase (Nox)2 is a promising target for treating cardiovascular disease, but there are no specific inhibitors. Finding endogenous signals that can target Nox2 and other inflammatory molecules is of great interest. In this study, we used high-throughput screening

Identifying target processes for microbial electrosynthesis by elementary mode analysis.

PubMed

Kracke, Frauke; Krömer, Jens O

2014-12-30

Microbial electrosynthesis and electro fermentation are techniques that aim to optimize microbial production of chemicals and fuels by regulating the cellular redox balance via interaction with electrodes. While the concept is known for decades major knowledge gaps remain, which make it hard to evaluate its biotechnological potential. Here we present an in silico approach to identify beneficial production processes for electro fermentation by elementary mode analysis. Since the fundamentals of electron transport between electrodes and microbes have not been fully uncovered yet, we propose different options and discuss their impact on biomass and product yields. For the first time 20 different valuable products were screened for their potential to show increased yields during anaerobic electrically enhanced fermentation. Surprisingly we found that an increase in product formation by electrical enhancement is not necessarily dependent on the degree of reduction of the product but rather the metabolic pathway it is derived from. We present a variety of beneficial processes with product yield increases of maximal 36% in reductive and 84% in oxidative fermentations and final theoretical product yields up to 100%. This includes compounds that are already produced at industrial scale such as succinic acid, lysine and diaminopentane as well as potential novel bio-commodities such as isoprene, para-hydroxybenzoic acid and para-aminobenzoic acid. Furthermore, it is shown that the way of electron transport has major impact on achievable biomass and product yields. The coupling of electron transport to energy conservation could be identified as crucial for most processes. This study introduces a powerful tool to determine beneficial substrate and product combinations for electro-fermentation. It also highlights that the maximal yield achievable by bio electrochemical techniques depends strongly on the actual electron transport mechanisms. Therefore it is of great importance to

Functional recognition imaging using artificial neural networks: applications to rapid cellular identification via broadband electromechanical response

NASA Astrophysics Data System (ADS)

Nikiforov, M. P.; Reukov, V. V.; Thompson, G. L.; Vertegel, A. A.; Guo, S.; Kalinin, S. V.; Jesse, S.

2009-10-01

Functional recognition imaging in scanning probe microscopy (SPM) using artificial neural network identification is demonstrated. This approach utilizes statistical analysis of complex SPM responses at a single spatial location to identify the target behavior, which is reminiscent of associative thinking in the human brain, obviating the need for analytical models. We demonstrate, as an example of recognition imaging, rapid identification of cellular organisms using the difference in electromechanical activity over a broad frequency range. Single-pixel identification of model Micrococcus lysodeikticus and Pseudomonas fluorescens bacteria is achieved, demonstrating the viability of the method.

Targetable vulnerabilities in T- and NK-cell lymphomas identified through preclinical models.

PubMed

Ng, Samuel Y; Yoshida, Noriaki; Christie, Amanda L; Ghandi, Mahmoud; Dharia, Neekesh V; Dempster, Joshua; Murakami, Mark; Shigemori, Kay; Morrow, Sara N; Van Scoyk, Alexandria; Cordero, Nicolas A; Stevenson, Kristen E; Puligandla, Maneka; Haas, Brian; Lo, Christopher; Meyers, Robin; Gao, Galen; Cherniack, Andrew; Louissaint, Abner; Nardi, Valentina; Thorner, Aaron R; Long, Henry; Qiu, Xintao; Morgan, Elizabeth A; Dorfman, David M; Fiore, Danilo; Jang, Julie; Epstein, Alan L; Dogan, Ahmet; Zhang, Yanming; Horwitz, Steven M; Jacobsen, Eric D; Santiago, Solimar; Ren, Jian-Guo; Guerlavais, Vincent; Annis, D Allen; Aivado, Manuel; Saleh, Mansoor N; Mehta, Amitkumar; Tsherniak, Aviad; Root, David; Vazquez, Francisca; Hahn, William C; Inghirami, Giorgio; Aster, Jon C; Weinstock, David M; Koch, Raphael

2018-05-22

T- and NK-cell lymphomas (TCL) are a heterogenous group of lymphoid malignancies with poor prognosis. In contrast to B-cell and myeloid malignancies, there are few preclinical models of TCLs, which has hampered the development of effective therapeutics. Here we establish and characterize preclinical models of TCL. We identify multiple vulnerabilities that are targetable with currently available agents (e.g., inhibitors of JAK2 or IKZF1) and demonstrate proof-of-principle for biomarker-driven therapies using patient-derived xenografts (PDXs). We show that MDM2 and MDMX are targetable vulnerabilities within TP53-wild-type TCLs. ALRN-6924, a stapled peptide that blocks interactions between p53 and both MDM2 and MDMX has potent in vitro activity and superior in vivo activity across 8 different PDX models compared to the standard-of-care agent romidepsin. ALRN-6924 induced a complete remission in a patient with TP53-wild-type angioimmunoblastic T-cell lymphoma, demonstrating the potential for rapid translation of discoveries from subtype-specific preclinical models.

SUMO proteases as potential targets for cancer therapy.

PubMed

Bialik, Piotr; Woźniak, Katarzyna

2017-12-08

Sumoylation is one of the post-translational modifications of proteins, responsible for the regulation of many cellular processes, such as DNA replication and repair, transcription, signal transduction and nuclear transport. During sumoylation, SUMO proteins are covalently attached to the ε-amino group of lysine in target proteins via an enzymatic cascade that requires the sequential action of E1, E2 and E3 enzymes. An important aspect of sumoylation is its reversibility, which involves SUMO-specific proteases called SENPs. SENPs (sentrin/SUMO-specific proteases) catalyze the deconjugation of SUMO proteins using their isopeptidase activity. These enzymes participate through hydrolase activity in the reaction of SUMO protein maturation, which involves the removal of a short fragment on the C-terminus of SUMO inactive form and exposure two glycine residues. SENPs are important for maintaining the balance between sumoylated and desumoylated proteins required for normal cellular physiology. Six SENP isoforms (SENP1, SENP2, SENP3, SENP5, SENP6 and SENP7) have been identified in mammals. These SENPs can be divided into three subfamilies based on their sequence homology, substrate specificity and subcellular localization. Results of studies indicate the role of SUMO proteases in the development of human diseases including cancer, suggesting that these proteins may be attractive targets for new drugs.

Hydrogen Peroxide Probes Directed to Different Cellular Compartments

PubMed Central

Malinouski, Mikalai; Zhou, You; Belousov, Vsevolod V.; Hatfield, Dolph L.; Gladyshev, Vadim N.

2011-01-01

Background Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells. Principal Findings Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events. Conclusions We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells. PMID:21283738

Acoustic Parametric Array for Identifying Standoff Targets

NASA Astrophysics Data System (ADS)

Hinders, M. K.; Rudd, K. E.

2010-02-01

An integrated simulation method for investigating nonlinear sound beams and 3D acoustic scattering from any combination of complicated objects is presented. A standard finite-difference simulation method is used to model pulsed nonlinear sound propagation from a source to a scattering target via the KZK equation. Then, a parallel 3D acoustic simulation method based on the finite integration technique is used to model the acoustic wave interaction with the target. Any combination of objects and material layers can be placed into the 3D simulation space to study the resulting interaction. Several example simulations are presented to demonstrate the simulation method and 3D visualization techniques. The combined simulation method is validated by comparing experimental and simulation data and a demonstration of how this combined simulation method assisted in the development of a nonlinear acoustic concealed weapons detector is also presented.

Cellular delivery of PEGylated PLGA nanoparticles.

PubMed

Pamujula, Sarala; Hazari, Sidhartha; Bolden, Gevoni; Graves, Richard A; Chinta, Dakshinamurthy Devanga; Dash, Srikanta; Kishore, Vimal; Mandal, Tarun K

2012-01-01

The objective of this study was to investigate the efficiency of uptake of PEGylated polylactide-co-gycolide (PLGA) nanoparticles by breast cancer cells. Nanoparticles of PLGA containing various amounts of polyethylene glycol (PEG, 5%-15%) were prepared using a double emulsion solvent evaporation method. The nanoparticles were loaded with coumarin-6 (C6) as a fluorescence marker. The particles were characterized for surface morphology, particle size, zeta potential, and for cellular uptake by 4T1 murine breast cancer cells. Irrespective of the amount of PEG, all formulations yielded smooth spherical particles. However, a comparison of the particle size of various formulations showed bimodal distribution of particles. Each formulation was later passed through a 1.2 µm filter to obtain target size particles (114-335 nm) with zeta potentials ranging from -2.8 mV to -26.2 mV. While PLGA-PEG di-block (15% PEG) formulation showed significantly higher 4T1 cellular uptake than all other formulations, there was no statistical difference in cellular uptake among PLGA, PLGA-PEG-PLGA tri-block (10% PEG), PLGA-PEG di-block (5% PEG) and PLGA-PEG di-block (10% PEG) nanoparticles. These preliminary findings indicated that the nanoparticle formulation prepared with 15% PEGylated PLGA showed maximum cellular uptake due to it having the smallest particle size and lowest zeta potential. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

Studying a Drug-like, RNA-Focused Small Molecule Library Identifies Compounds That Inhibit RNA Toxicity in Myotonic Dystrophy.

PubMed

Rzuczek, Suzanne G; Southern, Mark R; Disney, Matthew D

2015-12-18

There are many RNA targets in the transcriptome to which small molecule chemical probes and lead therapeutics are desired. However, identifying compounds that bind and modulate RNA function in cellulo is difficult. Although rational design approaches have been developed, they are still in their infancies and leave many RNAs "undruggable". In an effort to develop a small molecule library that is biased for binding RNA, we computationally identified "drug-like" compounds from screening collections that have favorable properties for binding RNA and for suitability as lead drugs. As proof-of-concept, this collection was screened for binding to and modulating the cellular dysfunction of the expanded repeating RNA (r(CUG)(exp)) that causes myotonic dystrophy type 1. Hit compounds bind the target in cellulo, as determined by the target identification approach Competitive Chemical Cross-Linking and Isolation by Pull-down (C-ChemCLIP), and selectively improve several disease-associated defects. The best compounds identified from our 320-member library are more potent in cellulo than compounds identified by high-throughput screening (HTS) campaigns against this RNA. Furthermore, the compound collection has a higher hit rate (9% compared to 0.01-3%), and the bioactive compounds identified are not charged; thus, RNA can be "drugged" with compounds that have favorable pharmacological properties. Finally, this RNA-focused small molecule library may serve as a useful starting point to identify lead "drug-like" chemical probes that affect the biological (dys)function of other RNA targets by direct target engagement.

Cotransporting Ion is a Trigger for Cellular Endocytosis of Transporter-Targeting Nanoparticles: A Case Study of High-Efficiency SLC22A5 (OCTN2)-Mediated Carnitine-Conjugated Nanoparticles for Oral Delivery of Therapeutic Drugs.

PubMed

Kou, Longfa; Yao, Qing; Sun, Mengchi; Wu, Chunnuan; Wang, Jia; Luo, Qiuhua; Wang, Gang; Du, Yuqian; Fu, Qiang; Wang, Jian; He, Zhonggui; Ganapathy, Vadivel; Sun, Jin

2017-09-01

OCTN2 (SLC22A5) is a Na + -coupled absorption transporter for l-carnitine in small intestine. This study tests the potential of this transporter for oral delivery of therapeutic drugs encapsulated in l-carnitine-conjugated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (LC-PLGA NPs) and discloses the molecular mechanism for cellular endocytosis of transporter-targeting nanoparticles. Conjugation of l-carnitine to a surface of PLGA-NPs enhances the cellular uptake and intestinal absorption of encapsulated drug. In both cases, the uptake process is dependent on cotransporting ion Na + . Computational OCTN2 docking analysis shows that the presence of Na + is important for the formation of the energetically stable intermediate complex of transporter-Na + -LC-PLGA NPs, which is also the first step in cellular endocytosis of nanoparticles. The transporter-mediated intestinal absorption of LC-PLGA NPs occurs via endocytosis/transcytosis rather than via the traditional transmembrane transport. The portal blood versus the lymphatic route is evaluated by the plasma appearance of the drug in the control and lymph duct-ligated rats. Absorption via the lymphatic system is the predominant route in the oral delivery of the NPs. In summary, LC-PLGA NPs can effectively target OCTN2 on the enterocytes for enhancing oral delivery of drugs and the critical role of cotransporting ions should be noticed in designing transporter-targeting nanoparticles. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting bacterial central metabolism for drug development.

PubMed

Murima, Paul; McKinney, John D; Pethe, Kevin

2014-11-20

Current antibiotics, derived mainly from natural sources, inhibit a narrow spectrum of cellular processes, namely DNA replication, protein synthesis, and cell wall biosynthesis. With the worldwide explosion of drug resistance, there is renewed interest in the investigation of alternate essential cellular processes, including bacterial central metabolic pathways, as a drug target space for the next generation of antibiotics. However, the validation of targets in central metabolism is more complex, as essentiality of such targets can be conditional and/or contextual. Bearing in mind our enhanced understanding of prokaryotic central metabolism, a key question arises: can central metabolism be bacteria's Achilles' heel and a therapeutic target for the development of new classes of antibiotics? In this review, we draw lessons from oncology and attempt to address some of the open questions related to feasibility of targeting bacterial central metabolism as a strategy for developing new antibacterial drugs. Copyright © 2014 Elsevier Ltd. All rights reserved.

An enhanced genome-scale metabolic reconstruction of Streptomyces clavuligerus identifies novel strain improvement strategies.

PubMed

Toro, León; Pinilla, Laura; Avignone-Rossa, Claudio; Ríos-Estepa, Rigoberto

2018-05-01

In this work, we expanded and updated a genome-scale metabolic model of Streptomyces clavuligerus. The model includes 1021 genes and 1494 biochemical reactions; genome-reaction information was curated and new features related to clavam metabolism and to the biomass synthesis equation were incorporated. The model was validated using experimental data from the literature and simulations were performed to predict cellular growth and clavulanic acid biosynthesis. Flux balance analysis (FBA) showed that limiting concentrations of phosphate and an excess of ammonia accumulation are unfavorable for growth and clavulanic acid biosynthesis. The evaluation of different objective functions for FBA showed that maximization of ATP yields the best predictions for cellular behavior in continuous cultures, while the maximization of growth rate provides better predictions for batch cultures. Through gene essentiality analysis, 130 essential genes were found using a limited in silico media, while 100 essential genes were identified in amino acid-supplemented media. Finally, a strain design was carried out to identify candidate genes to be overexpressed or knocked out so as to maximize antibiotic biosynthesis. Interestingly, potential metabolic engineering targets, identified in this study, have not been tested experimentally.

Method of forming a continuous polymeric skin on a cellular foam material

DOEpatents

Duchane, David V.; Barthell, Barry L.

1985-01-01

Hydrophobic cellular material is coated with a thin hydrophilic polymer skin which stretches tightly over the outer surface of the foam but which does not fill the cells of the foam, thus resulting in a polymer-coated foam structure having a smoothness which was not possible in the prior art. In particular, when the hydrophobic cellular material is a specially chosen hydrophobic polymer foam and is formed into arbitrarily chosen shapes prior to the coating with hydrophilic polymer, inertial confinement fusion (ICF) targets of arbitrary shapes can be produced by subsequently coating the shapes with metal or with any other suitable material. New articles of manufacture are produced, including improved ICF targets, improved integrated circuits, and improved solar reflectors and solar collectors. In the coating method, the cell size of the hydrophobic cellular material, the viscosity of the polymer solution used to coat, and the surface tensin of the polymer solution used to coat are all very important to the coating.

Plate-impact loading of cellular structures formed by selective laser melting

NASA Astrophysics Data System (ADS)

Winter, R. E.; Cotton, M.; Harris, E. J.; Maw, J. R.; Chapman, D. J.; Eakins, D. E.; McShane, G.

2014-03-01

Porous materials are of great interest because of improved energy absorption over their solid counterparts. Their properties, however, have been difficult to optimize. Additive manufacturing has emerged as a potential technique to closely define the structure and properties of porous components, i.e. density, strut width and pore size; however, the behaviour of these materials at very high impact energies remains largely unexplored. We describe an initial study of the dynamic compression response of lattice materials fabricated through additive manufacturing. Lattices consisting of an array of intersecting stainless steel rods were fabricated into discs using selective laser melting. The resulting discs were impacted against solid stainless steel targets at velocities ranging from 300 to 700 m s-1 using a gas gun. Continuum CTH simulations were performed to identify key features in the measured wave profiles, while 3D simulations, in which the individual cells were modelled, revealed details of microscale deformation during collapse of the lattice structure. The validated computer models have been used to provide an understanding of the deformation processes in the cellular samples. The study supports the optimization of cellular structures for application as energy absorbers.

Many si/shRNAs can kill cancer cells by targeting multiple survival genes through an off-target mechanism

PubMed Central

van Dongen, Stijn; Haluck-Kangas, Ashley; Sarshad, Aishe A; Bartom, Elizabeth T; Kim, Kwang-Youn A; Scholtens, Denise M; Hafner, Markus; Zhao, Jonathan C; Murmann, Andrea E

2017-01-01

Over 80% of multiple-tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3’UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer-deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that specific toxic RNAi-active sequences present in the genome can kill cancer cells. PMID:29063830

Use of eQTL Analysis for the Discovery of Target Genes Identified by GWAS

DTIC Science & Technology

2013-04-01

the biologic pathways affected by these inherited factors, and ultimately to identify targets for disease prediction, risk stratification and...quality using an Agilent chip technology. Cases having a RIN number of 7.0 or greater were considered good quality. Once completed, the optimum set of...AD_________________ Award Number: W81XWH-11-1-0261 TITLE: Use of eQTL Analysis for the Discovery of

Targeting inflammation in pancreatic cancer: Clinical translation

PubMed Central

Steele, Colin William; Kaur Gill, Nina Angharad; Jamieson, Nigel Balfour; Carter, Christopher Ross

2016-01-01

Preclinical modelling studies are beginning to aid development of therapies targeted against key regulators of pancreatic cancer progression. Pancreatic cancer is an aggressive, stromally-rich tumor, from which few people survive. Within the tumor microenvironment cellular and extracellular components exist, shielding tumor cells from immune cell clearance, and chemotherapy, enhancing progression of the disease. The cellular component of this microenvironment consists mainly of stellate cells and inflammatory cells. New findings suggest that manipulation of the cellular component of the tumor microenvironment is possible to promote immune cell killing of tumor cells. Here we explore possible immunogenic therapeutic strategies. Additionally extracellular stromal elements play a key role in protecting tumor cells from chemotherapies targeted at the pancreas. We describe the experimental findings and the pitfalls associated with translation of stromally targeted therapies to clinical trial. Finally, we discuss the key inflammatory signal transducers activated subsequent to driver mutations in oncogenic Kras in pancreatic cancer. We present the preclinical findings that have led to successful early trials of STAT3 inhibitors in pancreatic adenocarcinoma. PMID:27096033

Nipah virus matrix protein: expert hacker of cellular machines.

PubMed

Watkinson, Ruth E; Lee, Benhur

2016-08-01

Nipah virus (NiV, Henipavirus) is a highly lethal emergent zoonotic paramyxovirus responsible for repeated human outbreaks of encephalitis in South East Asia. There are no approved vaccines or treatments, thus improved understanding of NiV biology is imperative. NiV matrix protein recruits a plethora of cellular machinery to scaffold and coordinate virion budding. Intriguingly, matrix also hijacks cellular trafficking and ubiquitination pathways to facilitate transient nuclear localization. While the biological significance of matrix nuclear localization for an otherwise cytoplasmic virus remains enigmatic, the molecular details have begun to be characterized, and are conserved among matrix proteins from divergent paramyxoviruses. Matrix protein appropriation of cellular machinery will be discussed in terms of its early nuclear targeting and later role in virion assembly. © 2016 Federation of European Biochemical Societies.

TP53, PIK3CA, FBXW7 and KRAS Mutations in Esophageal Cancer Identified by Targeted Sequencing.

PubMed

Zheng, Huili; Wang, Yan; Tang, Chuanning; Jones, Lindsey; Ye, Hua; Zhang, Guangchun; Cao, Weihai; Li, Jingwen; Liu, Lifeng; Liu, Zhencong; Zhang, Chao; Lou, Feng; Liu, Zhiyuan; Li, Yangyang; Shi, Zhenfen; Zhang, Jingbo; Zhang, Dandan; Sun, Hong; Dong, Haichao; Dong, Zhishou; Guo, Baishuai; Yan, H E; Lu, Qingyu; Huang, Xue; Chen, Si-Yi

2016-01-01

Esophageal cancer (EC) is a common malignancy with significant morbidity and mortality. As individual cancers exhibit unique mutation patterns, identifying and characterizing gene mutations in EC that may serve as biomarkers might help predict patient outcome and guide treatment. Traditionally, personalized cancer DNA sequencing was impractical and expensive. Recent technological advancements have made targeted DNA sequencing more cost- and time-effective with reliable results. This technology may be useful for clinicians to direct patient treatment. The Ion PGM and AmpliSeq Cancer Panel was used to identify mutations at 737 hotspot loci of 45 cancer-related genes in 64 EC samples from Chinese patients. Frequent mutations were found in TP53 and less frequent mutations in PIK3CA, FBXW7 and KRAS. These results demonstrate that targeted sequencing can reliably identify mutations in individual tumors that make this technology a possibility for clinical use. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Non-coding RNAs—Novel targets in neurotoxicity

PubMed Central

Tal, Tamara L.; Tanguay, Robert L.

2012-01-01

Over the past ten years non-coding RNAs (ncRNAs) have emerged as pivotal players in fundamental physiological and cellular processes and have been increasingly implicated in cancer, immune disorders, and cardiovascular, neurodegenerative, and metabolic diseases. MicroRNAs (miRNAs) represent a class of ncRNA molecules that function as negative regulators of post-transcriptional gene expression. miRNAs are predicted to regulate 60% of all human protein-coding genes and as such, play key roles in cellular and developmental processes, human health, and disease. Relative to counterparts that lack bindings sites for miRNAs, genes encoding proteins that are post-transcriptionally regulated by miRNAs are twice as likely to be sensitive to environmental chemical exposure. Not surprisingly, miRNAs have been recognized as targets or effectors of nervous system, developmental, hepatic, and carcinogenic toxicants, and have been identified as putative regulators of phase I xenobiotic-metabolizing enzymes. In this review, we give an overview of the types of ncRNAs and highlight their roles in neurodevelopment, neurological disease, activity-dependent signaling, and drug metabolism. We then delve into specific examples that illustrate their importance as mediators, effectors, or adaptive agents of neurotoxicants or neuroactive pharmaceutical compounds. Finally, we identify a number of outstanding questions regarding ncRNAs and neurotoxicity. PMID:22394481

A Behavioral Intervention to Reduce Child Exposure to Indoor Air Pollution: Identifying Possible Target Behaviors

ERIC Educational Resources Information Center

Barnes, Brendon R.; Mathee, Angela; Shafritz, Lonna B.; Krieger, Laurie; Zimicki, Susan

2004-01-01

Indoor air pollution has been causally linked to acute lower respiratory infections in children younger than 5. The aim of this study was to identify target behaviors for a behavioral intervention to reduce child exposure to indoor air pollution by attempting to answer two research questions: Which behaviors are protective of child respiratory…

Pericyte-targeting drug delivery and tissue engineering.

PubMed

Kang, Eunah; Shin, Jong Wook

2016-01-01

Pericytes are contractile mural cells that wrap around the endothelial cells of capillaries and venules. Depending on the triggers by cellular signals, pericytes have specific functionality in tumor microenvironments, properties of potent stem cells, and plasticity in cellular pathology. These features of pericytes can be activated for the promotion or reduction of angiogenesis. Frontier studies have exploited pericyte-targeting drug delivery, using pericyte-specific peptides, small molecules, and DNA in tumor therapy. Moreover, the communication between pericytes and endothelial cells has been applied to the induction of vessel neoformation in tissue engineering. Pericytes may prove to be a novel target for tumor therapy and tissue engineering. The present paper specifically reviews pericyte-specific drug delivery and tissue engineering, allowing insight into the emerging research targeting pericytes.

Algorithm for cellular reprogramming.

PubMed

Ronquist, Scott; Patterson, Geoff; Muir, Lindsey A; Lindsly, Stephen; Chen, Haiming; Brown, Markus; Wicha, Max S; Bloch, Anthony; Brockett, Roger; Rajapakse, Indika

2017-11-07

The day we understand the time evolution of subcellular events at a level of detail comparable to physical systems governed by Newton's laws of motion seems far away. Even so, quantitative approaches to cellular dynamics add to our understanding of cell biology. With data-guided frameworks we can develop better predictions about, and methods for, control over specific biological processes and system-wide cell behavior. Here we describe an approach for optimizing the use of transcription factors (TFs) in cellular reprogramming, based on a device commonly used in optimal control. We construct an approximate model for the natural evolution of a cell-cycle-synchronized population of human fibroblasts, based on data obtained by sampling the expression of 22,083 genes at several time points during the cell cycle. To arrive at a model of moderate complexity, we cluster gene expression based on division of the genome into topologically associating domains (TADs) and then model the dynamics of TAD expression levels. Based on this dynamical model and additional data, such as known TF binding sites and activity, we develop a methodology for identifying the top TF candidates for a specific cellular reprogramming task. Our data-guided methodology identifies a number of TFs previously validated for reprogramming and/or natural differentiation and predicts some potentially useful combinations of TFs. Our findings highlight the immense potential of dynamical models, mathematics, and data-guided methodologies for improving strategies for control over biological processes. Copyright © 2017 the Author(s). Published by PNAS.

Kidney disease models: tools to identify mechanisms and potential therapeutic targets

PubMed Central

Bao, Yin-Wu; Yuan, Yuan; Chen, Jiang-Hua; Lin, Wei-Qiang

2018-01-01

Acute kidney injury (AKI) and chronic kidney disease (CKD) are worldwide public health problems affecting millions of people and have rapidly increased in prevalence in recent years. Due to the multiple causes of renal failure, many animal models have been developed to advance our understanding of human nephropathy. Among these experimental models, rodents have been extensively used to enable mechanistic understanding of kidney disease induction and progression, as well as to identify potential targets for therapy. In this review, we discuss AKI models induced by surgical operation and drugs or toxins, as well as a variety of CKD models (mainly genetically modified mouse models). Results from recent and ongoing clinical trials and conceptual advances derived from animal models are also explored. PMID:29515089

Plasma Membrane Proteomics of Human Breast Cancer Cell Lines Identifies Potential Targets for Breast Cancer Diagnosis and Treatment

PubMed Central

Ziegler, Yvonne S.; Moresco, James J.; Tu, Patricia G.; Yates, John R.; Nardulli, Ann M.

2014-01-01

The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease. PMID:25029196

Targeting cancer stem-like cells in glioblastoma and colorectal cancer through metabolic pathways.

PubMed

Kahlert, U D; Mooney, S M; Natsumeda, M; Steiger, H-J; Maciaczyk, J

2017-01-01

Cancer stem-like cells (CSCs) are thought to be the main cause of tumor occurrence, progression and therapeutic resistance. Strong research efforts in the last decade have led to the development of several tailored approaches to target CSCs with some very promising clinical trials underway; however, until now no anti-CSC therapy has been approved for clinical use. Given the recent improvement in our understanding of how onco-proteins can manipulate cellular metabolic networks to promote tumorigenesis, cancer metabolism research may well lead to innovative strategies to identify novel regulators and downstream mediators of CSC maintenance. Interfering with distinct stages of CSC-associated metabolics may elucidate novel, more efficient strategies to target this highly malignant cell population. Here recent discoveries regarding the metabolic properties attributed to CSCs in glioblastoma (GBM) and malignant colorectal cancer (CRC) were summarized. The association between stem cell markers, the response to hypoxia and other environmental stresses including therapeutic insults as well as developmentally conserved signaling pathways with alterations in cellular bioenergetic networks were also discussed. The recent developments in metabolic imaging to identify CSCs were also summarized. This summary should comprehensively update basic and clinical scientists on the metabolic traits of CSCs in GBM and malignant CRC. © 2016 UICC.

Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification.

PubMed

Deans, Richard M; Morgens, David W; Ökesli, Ayşe; Pillay, Sirika; Horlbeck, Max A; Kampmann, Martin; Gilbert, Luke A; Li, Amy; Mateo, Roberto; Smith, Mark; Glenn, Jeffrey S; Carette, Jan E; Khosla, Chaitan; Bassik, Michael C

2016-05-01

Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and limitations of each screening method for identifying drug targets, and demonstrate the utility of parallel knockdown and knockout screens for comprehensive probing of drug activity.

The HIV Nef protein modulates cellular and exosomal miRNA profiles in human monocytic cells.

PubMed

Aqil, Madeeha; Naqvi, Afsar Raza; Mallik, Saurav; Bandyopadhyay, Sanghamitra; Maulik, Ujjwal; Jameel, Shahid

2014-01-01

The HIV Nef protein is a multifunctional virulence factor that perturbs intracellular membranes and signalling and is secreted into exosomes. While Nef-containing exosomes have a distinct proteomic profile, no comprehensive analysis of their miRNA cargo has been carried out. Since Nef functions as a viral suppressor of RNA interference and disturbs the distribution of RNA-induced silencing complex proteins between cells and exosomes, we hypothesized that it might also affect the export of miRNAs into exosomes. Exosomes were purified from human monocytic U937 cells that stably expressed HIV-1 Nef. The RNA from cells and exosomes was profiled for 667 miRNAs using a Taqman Low Density Array. Selected miRNAs and their mRNA targets were validated by quantitative RT-PCR. Bioinformatics analyses were used to identify targets and predict pathways. Nef expression affected a significant fraction of miRNAs in U937 cells. Our analysis showed 47 miRNAs to be selectively secreted into Nef exosomes and 2 miRNAs to be selectively retained in Nef-expressing cells. The exosomal miRNAs were predicted to target several cellular genes in inflammatory cytokine and other pathways important for HIV pathogenesis, and an overwhelming majority had targets within the HIV genome. This is the first study to report miRnome analysis of HIV Nef expressing monocytes and exosomes. Our results demonstrate that Nef causes large-scale dysregulation of cellular miRNAs, including their secretion through exosomes. We suggest this to be a novel viral strategy to affect pathogenesis and to limit the effects of RNA interference on viral replication and persistence.

Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy.

PubMed

Huang, Xiao-Yan; Zhuang, Hong; Wu, Ji-Hong; Li, Jian-Kang; Hu, Fang-Yuan; Zheng, Yu; Tellier, Laurent Christian Asker M; Zhang, Sheng-Hai; Gao, Feng-Juan; Zhang, Jian-Guo; Xu, Ge-Zhi

2017-01-01

Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [ LRP5 , c.4053 DelC (p.Ile1351IlefsX88); TSPAN12 , EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. We identified two novel heterozygous deletion mutations [ LRP5 , c.4053 DelC (p.Ile1351IlefsX88); TSPAN12 , EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype-phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling.

Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy

PubMed Central

Huang, Xiao-Yan; Zhuang, Hong; Wu, Ji-Hong; Li, Jian-Kang; Hu, Fang-Yuan; Zheng, Yu; Tellier, Laurent Christian Asker M.; Zhang, Sheng-Hai; Gao, Feng-Juan; Zhang, Jian-Guo

2017-01-01

Purpose Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. Methods To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). Results Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. Conclusions We identified two novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype–phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling. PMID:28867931

microRNA expression profiling in fetal single ventricle malformation identified by deep sequencing.

PubMed

Yu, Zhang-Bin; Han, Shu-Ping; Bai, Yun-Fei; Zhu, Chun; Pan, Ya; Guo, Xi-Rong

2012-01-01

microRNAs (miRNAs) have emerged as key regulators in many biological processes, particularly cardiac growth and development, although the specific miRNA expression profile associated with this process remains to be elucidated. This study aimed to characterize the cellular microRNA profile involved in the development of congenital heart malformation, through the investigation of single ventricle (SV) defects. Comprehensive miRNA profiling in human fetal SV cardiac tissue was performed by deep sequencing. Differential expression of 48 miRNAs was revealed by sequencing by oligonucleotide ligation and detection (SOLiD) analysis. Of these, 38 were down-regulated and 10 were up-regulated in differentiated SV cardiac tissue, compared to control cardiac tissue. This was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Predicted target genes of the 48 differentially expressed miRNAs were analyzed by gene ontology and categorized according to cellular process, regulation of biological process and metabolic process. Pathway-Express analysis identified the WNT and mTOR signaling pathways as the most significant processes putatively affected by the differential expression of these miRNAs. The candidate genes involved in cardiac development were identified as potential targets for these differentially expressed microRNAs and the collaborative network of microRNAs and cardiac development related-mRNAs was constructed. These data provide the basis for future investigation of the mechanism of the occurrence and development of fetal SV malformations.

Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulation.

PubMed

Chan, Kwok Keung; Wong, Corinne Kung Yen; Lui, Vincent Chi Hang; Tam, Paul Kwong Hang; Sham, Mai Har

2003-10-15

SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis

A Mouse Polyomavirus-encoded microRNA Targets the Cellular Apoptosis Pathway through Smad2 Inhibition

PubMed Central

Sung, Chang Kyoo; Yim, Hyungshin; Andrews, Erik; Benjamin, Thomas L.

2014-01-01

Some viruses and most eukaryotic cells have microRNAs that regulate the expression of many genes. Although many viral miRNAs have been identified, only a few have been included in in vivo functional studies. Here we show that a Py-encoded miRNA downregulates the expression of the pro-apoptotic factor Smad2, resulting in the suppression of the apoptosis pathway. To study the Py miRNA in an in vivo context, a miRNA-deficient mutant virus was created on the background of the LID virus strain which establishes a rapid and lethal infection in newborn mice. Apoptosis analysis on kidney tissues indicates that the pro-apoptotic pathway is targeted in the infected host as well. Suppression of apoptosis through targeting of Smad2 by the Py miRNA is expected to synergize with anti-apoptotic effects previously attributed to the polyoma tumor antigens in support of virus replication in the natural host. PMID:25146733

Impact of Upfront Cellular Enrichment by Laser Capture Microdissection on Protein and Phosphoprotein Drug Target Signaling Activation Measurements in Human Lung Cancer: Implications for Personalized Medicine

PubMed Central

Elisa, Baldelli; B., Haura Eric; Lucio, Crinò; Douglas, Cress W.; Vienna, Ludovini; B., Schabath Matthew; A., Liotta Lance; F., Petricoin Emanuel; Mariaelena, Pierobon

2015-01-01

Purpose The aim of this study was to evaluate whether upfront cellular enrichment via laser capture microdissection is necessary for accurately quantifying predictive biomarkers in non-small cell lung cancer tumors. Experimental design Fifteen snap frozen surgical biopsies were analyzed. Whole tissue lysate and matched highly enriched tumor epithelium via laser capture microdissection (LCM) were obtained for each patient. The expression and activation/phosphorylation levels of 26 proteins were measured by reverse phase protein microarray. Differences in signaling architecture of dissected and undissected matched pairs were visualized using unsupervised clustering analysis, bar graphs, and scatter plots. Results Overall patient matched LCM and undissected material displayed very distinct and differing signaling architectures with 93% of the matched pairs clustering separately. These differences were seen regardless of the amount of starting tumor epithelial content present in the specimen. Conclusions and clinical relevance These results indicate that LCM driven upfront cellular enrichment is necessary to accurately determine the expression/activation levels of predictive protein signaling markers although results should be evaluated in larger clinical settings. Upfront cellular enrichment of the target cell appears to be an important part of the workflow needed for the accurate quantification of predictive protein signaling biomarkers. Larger independent studies are warranted. PMID:25676683

Advances in identification and validation of protein targets of natural products without chemical modification.

PubMed

Chang, J; Kim, Y; Kwon, H J

2016-05-04

Covering: up to February 2016Identification of the target proteins of natural products is pivotal to understanding the mechanisms of action to develop natural products for use as molecular probes and potential therapeutic drugs. Affinity chromatography of immobilized natural products has been conventionally used to identify target proteins, and has yielded good results. However, this method has limitations, in that labeling or tagging for immobilization and affinity purification often result in reduced or altered activity of the natural product. New strategies have recently been developed and applied to identify the target proteins of natural products and synthetic small molecules without chemical modification of the natural product. These direct and indirect methods for target identification of label-free natural products include drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP), and bioinformatics-based analysis of connectivity. This review focuses on and reports case studies of the latest advances in target protein identification methods for label-free natural products. The integration of newly developed technologies will provide new insights and highlight the value of natural products for use as biological probes and new drug candidates.

Literature-based discovery of diabetes- and ROS-related targets

PubMed Central

2010-01-01

Background Reactive oxygen species (ROS) are known mediators of cellular damage in multiple diseases including diabetic complications. Despite its importance, no comprehensive database is currently available for the genes associated with ROS. Methods We present ROS- and diabetes-related targets (genes/proteins) collected from the biomedical literature through a text mining technology. A web-based literature mining tool, SciMiner, was applied to 1,154 biomedical papers indexed with diabetes and ROS by PubMed to identify relevant targets. Over-represented targets in the ROS-diabetes literature were obtained through comparisons against randomly selected literature. The expression levels of nine genes, selected from the top ranked ROS-diabetes set, were measured in the dorsal root ganglia (DRG) of diabetic and non-diabetic DBA/2J mice in order to evaluate the biological relevance of literature-derived targets in the pathogenesis of diabetic neuropathy. Results SciMiner identified 1,026 ROS- and diabetes-related targets from the 1,154 biomedical papers (http://jdrf.neurology.med.umich.edu/ROSDiabetes/). Fifty-three targets were significantly over-represented in the ROS-diabetes literature compared to randomly selected literature. These over-represented targets included well-known members of the oxidative stress response including catalase, the NADPH oxidase family, and the superoxide dismutase family of proteins. Eight of the nine selected genes exhibited significant differential expression between diabetic and non-diabetic mice. For six genes, the direction of expression change in diabetes paralleled enhanced oxidative stress in the DRG. Conclusions Literature mining compiled ROS-diabetes related targets from the biomedical literature and led us to evaluate the biological relevance of selected targets in the pathogenesis of diabetic neuropathy. PMID:20979611

Regression-Based Identification of Behavior-Encoding Neurons During Large-Scale Optical Imaging of Neural Activity at Cellular Resolution

PubMed Central

Miri, Andrew; Daie, Kayvon; Burdine, Rebecca D.; Aksay, Emre

2011-01-01

The advent of methods for optical imaging of large-scale neural activity at cellular resolution in behaving animals presents the problem of identifying behavior-encoding cells within the resulting image time series. Rapid and precise identification of cells with particular neural encoding would facilitate targeted activity measurements and perturbations useful in characterizing the operating principles of neural circuits. Here we report a regression-based approach to semiautomatically identify neurons that is based on the correlation of fluorescence time series with quantitative measurements of behavior. The approach is illustrated with a novel preparation allowing synchronous eye tracking and two-photon laser scanning fluorescence imaging of calcium changes in populations of hindbrain neurons during spontaneous eye movement in the larval zebrafish. Putative velocity-to-position oculomotor integrator neurons were identified that showed a broad spatial distribution and diversity of encoding. Optical identification of integrator neurons was confirmed with targeted loose-patch electrical recording and laser ablation. The general regression-based approach we demonstrate should be widely applicable to calcium imaging time series in behaving animals. PMID:21084686

Cellular mRNA decay protein AUF1 negatively regulates enterovirus and human rhinovirus infections.

PubMed

Cathcart, Andrea L; Rozovics, Janet M; Semler, Bert L

2013-10-01

To successfully complete their replication cycles, picornaviruses modify several host proteins to alter the cellular environment to favor virus production. One such target of viral proteinase cleavage is AU-rich binding factor 1 (AUF1), a cellular protein that binds to AU-rich elements, or AREs, in the 3' noncoding regions (NCRs) of mRNAs to affect the stability of the RNA. Previous studies found that, during poliovirus or human rhinovirus infection, AUF1 is cleaved by the viral proteinase 3CD and that AUF1 can interact with the long 5' NCR of these viruses in vitro. Here, we expand on these initial findings to demonstrate that all four isoforms of AUF1 bind directly to stem-loop IV of the poliovirus 5' NCR, an interaction that is inhibited through proteolytic cleavage of AUF1 by the viral proteinase 3CD. Endogenous AUF1 was observed to relocalize to the cytoplasm of infected cells in a viral protein 2A-driven manner and to partially colocalize with the viral protein 3CD. We identify a negative role for AUF1 in poliovirus infection, as AUF1 inhibited viral translation and, ultimately, overall viral titers. Our findings also demonstrate that AUF1 functions as an antiviral factor during infection by coxsackievirus or human rhinovirus, suggesting a common mechanism that targets these related picornaviruses.

Cellular mRNA Decay Protein AUF1 Negatively Regulates Enterovirus and Human Rhinovirus Infections

PubMed Central

Cathcart, Andrea L.; Rozovics, Janet M.

2013-01-01

To successfully complete their replication cycles, picornaviruses modify several host proteins to alter the cellular environment to favor virus production. One such target of viral proteinase cleavage is AU-rich binding factor 1 (AUF1), a cellular protein that binds to AU-rich elements, or AREs, in the 3′ noncoding regions (NCRs) of mRNAs to affect the stability of the RNA. Previous studies found that, during poliovirus or human rhinovirus infection, AUF1 is cleaved by the viral proteinase 3CD and that AUF1 can interact with the long 5′ NCR of these viruses in vitro. Here, we expand on these initial findings to demonstrate that all four isoforms of AUF1 bind directly to stem-loop IV of the poliovirus 5′ NCR, an interaction that is inhibited through proteolytic cleavage of AUF1 by the viral proteinase 3CD. Endogenous AUF1 was observed to relocalize to the cytoplasm of infected cells in a viral protein 2A-driven manner and to partially colocalize with the viral protein 3CD. We identify a negative role for AUF1 in poliovirus infection, as AUF1 inhibited viral translation and, ultimately, overall viral titers. Our findings also demonstrate that AUF1 functions as an antiviral factor during infection by coxsackievirus or human rhinovirus, suggesting a common mechanism that targets these related picornaviruses. PMID:23903828

A new method of identifying target groups for pronatalist policy applied to Australia.

PubMed

Chen, Mengni; Lloyd, Chris J; Yip, Paul S F

2018-01-01

A country's total fertility rate (TFR) depends on many factors. Attributing changes in TFR to changes of policy is difficult, as they could easily be correlated with changes in the unmeasured drivers of TFR. A case in point is Australia where both pronatalist effort and TFR increased in lock step from 2001 to 2008 and then decreased. The global financial crisis or other unobserved confounders might explain both the reducing TFR and pronatalist incentives after 2008. Therefore, it is difficult to estimate causal effects of policy using econometric techniques. The aim of this study is to instead look at the structure of the population to identify which subgroups most influence TFR. Specifically, we build a stochastic model relating TFR to the fertility rates of various subgroups and calculate elasticity of TFR with respect to each rate. For each subgroup, the ratio of its elasticity to its group size is used to evaluate the subgroup's potential cost effectiveness as a pronatalist target. In addition, we measure the historical stability of group fertility rates, which measures propensity to change. Groups with a high effectiveness ratio and also high propensity to change are natural policy targets. We applied this new method to Australian data on fertility rates broken down by parity, age and marital status. The results show that targeting parity 3+ is more cost-effective than lower parities. This study contributes to the literature on pronatalist policies by investigating the targeting of policies, and generates important implications for formulating cost-effective policies.

A new method of identifying target groups for pronatalist policy applied to Australia

PubMed Central

Chen, Mengni; Lloyd, Chris J.

2018-01-01

A country’s total fertility rate (TFR) depends on many factors. Attributing changes in TFR to changes of policy is difficult, as they could easily be correlated with changes in the unmeasured drivers of TFR. A case in point is Australia where both pronatalist effort and TFR increased in lock step from 2001 to 2008 and then decreased. The global financial crisis or other unobserved confounders might explain both the reducing TFR and pronatalist incentives after 2008. Therefore, it is difficult to estimate causal effects of policy using econometric techniques. The aim of this study is to instead look at the structure of the population to identify which subgroups most influence TFR. Specifically, we build a stochastic model relating TFR to the fertility rates of various subgroups and calculate elasticity of TFR with respect to each rate. For each subgroup, the ratio of its elasticity to its group size is used to evaluate the subgroup’s potential cost effectiveness as a pronatalist target. In addition, we measure the historical stability of group fertility rates, which measures propensity to change. Groups with a high effectiveness ratio and also high propensity to change are natural policy targets. We applied this new method to Australian data on fertility rates broken down by parity, age and marital status. The results show that targeting parity 3+ is more cost-effective than lower parities. This study contributes to the literature on pronatalist policies by investigating the targeting of policies, and generates important implications for formulating cost-effective policies. PMID:29425220

The imperative for controlled mechanical stresses in unraveling cellular mechanisms of mechanotransduction

PubMed Central

Anderson, Eric J; Falls, Thomas D; Sorkin, Adam M; Tate, Melissa L Knothe

2006-01-01

Background In vitro mechanotransduction studies are designed to elucidate cell behavior in response to a well-defined mechanical signal that is imparted to cultured cells, e.g. through fluid flow. Typically, flow rates are calculated based on a parallel plate flow assumption, to achieve a targeted cellular shear stress. This study evaluates the performance of specific flow/perfusion chambers in imparting the targeted stress at the cellular level. Methods To evaluate how well actual flow chambers meet their target stresses (set for 1 and 10 dyn/cm2 for this study) at a cellular level, computational models were developed to calculate flow velocity components and imparted shear stresses for a given pressure gradient. Computational predictions were validated with micro-particle image velocimetry (μPIV) experiments. Results Based on these computational and experimental studies, as few as 66% of cells seeded along the midplane of commonly implemented flow/perfusion chambers are subjected to stresses within ±10% of the target stress. In addition, flow velocities and shear stresses imparted through fluid drag vary as a function of location within each chamber. Hence, not only a limited number of cells are exposed to target stress levels within each chamber, but also neighboring cells may experience different flow regimes. Finally, flow regimes are highly dependent on flow chamber geometry, resulting in significant variation in magnitudes and spatial distributions of stress between chambers. Conclusion The results of this study challenge the basic premise of in vitro mechanotransduction studies, i.e. that a controlled flow regime is applied to impart a defined mechanical stimulus to cells. These results also underscore the fact that data from studies in which different chambers are utilized can not be compared, even if the target stress regimes are comparable. PMID:16672051

The emerging role of skeletal muscle oxidative metabolism as a biological target and cellular regulator of cancer-induced muscle wasting.

PubMed

Carson, James A; Hardee, Justin P; VanderVeen, Brandon N

2016-06-01

While skeletal muscle mass is an established primary outcome related to understanding cancer cachexia mechanisms, considerable gaps exist in our understanding of muscle biochemical and functional properties that have recognized roles in systemic health. Skeletal muscle quality is a classification beyond mass, and is aligned with muscle's metabolic capacity and substrate utilization flexibility. This supplies an additional role for the mitochondria in cancer-induced muscle wasting. While the historical assessment of mitochondria content and function during cancer-induced muscle loss was closely aligned with energy flux and wasting susceptibility, this understanding has expanded to link mitochondria dysfunction to cellular processes regulating myofiber wasting. The primary objective of this article is to highlight muscle mitochondria and oxidative metabolism as a biological target of cancer cachexia and also as a cellular regulator of cancer-induced muscle wasting. Initially, we examine the role of muscle metabolic phenotype and mitochondria content in cancer-induced wasting susceptibility. We then assess the evidence for cancer-induced regulation of skeletal muscle mitochondrial biogenesis, dynamics, mitophagy, and oxidative stress. In addition, we discuss environments associated with cancer cachexia that can impact the regulation of skeletal muscle oxidative metabolism. The article also examines the role of cytokine-mediated regulation of mitochondria function, followed by the potential role of cancer-induced hypogonadism. Lastly, a role for decreased muscle use in cancer-induced mitochondrial dysfunction is reviewed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cellular Automata

NASA Astrophysics Data System (ADS)

Gutowitz, Howard

1991-08-01

Cellular automata, dynamic systems in which space and time are discrete, are yielding interesting applications in both the physical and natural sciences. The thirty four contributions in this book cover many aspects of contemporary studies on cellular automata and include reviews, research reports, and guides to recent literature and available software. Chapters cover mathematical analysis, the structure of the space of cellular automata, learning rules with specified properties: cellular automata in biology, physics, chemistry, and computation theory; and generalizations of cellular automata in neural nets, Boolean nets, and coupled map lattices. Current work on cellular automata may be viewed as revolving around two central and closely related problems: the forward problem and the inverse problem. The forward problem concerns the description of properties of given cellular automata. Properties considered include reversibility, invariants, criticality, fractal dimension, and computational power. The role of cellular automata in computation theory is seen as a particularly exciting venue for exploring parallel computers as theoretical and practical tools in mathematical physics. The inverse problem, an area of study gaining prominence particularly in the natural sciences, involves designing rules that possess specified properties or perform specified task. A long-term goal is to develop a set of techniques that can find a rule or set of rules that can reproduce quantitative observations of a physical system. Studies of the inverse problem take up the organization and structure of the set of automata, in particular the parameterization of the space of cellular automata. Optimization and learning techniques, like the genetic algorithm and adaptive stochastic cellular automata are applied to find cellular automaton rules that model such physical phenomena as crystal growth or perform such adaptive-learning tasks as balancing an inverted pole. Howard Gutowitz is

Cellular automata simulation of topological effects on the dynamics of feed-forward motifs

PubMed Central

Apte, Advait A; Cain, John W; Bonchev, Danail G; Fong, Stephen S

2008-01-01

Background Feed-forward motifs are important functional modules in biological and other complex networks. The functionality of feed-forward motifs and other network motifs is largely dictated by the connectivity of the individual network components. While studies on the dynamics of motifs and networks are usually devoted to the temporal or spatial description of processes, this study focuses on the relationship between the specific architecture and the overall rate of the processes of the feed-forward family of motifs, including double and triple feed-forward loops. The search for the most efficient network architecture could be of particular interest for regulatory or signaling pathways in biology, as well as in computational and communication systems. Results Feed-forward motif dynamics were studied using cellular automata and compared with differential equation modeling. The number of cellular automata iterations needed for a 100% conversion of a substrate into a target product was used as an inverse measure of the transformation rate. Several basic topological patterns were identified that order the specific feed-forward constructions according to the rate of dynamics they enable. At the same number of network nodes and constant other parameters, the bi-parallel and tri-parallel motifs provide higher network efficacy than single feed-forward motifs. Additionally, a topological property of isodynamicity was identified for feed-forward motifs where different network architectures resulted in the same overall rate of the target production. Conclusion It was shown for classes of structural motifs with feed-forward architecture that network topology affects the overall rate of a process in a quantitatively predictable manner. These fundamental results can be used as a basis for simulating larger networks as combinations of smaller network modules with implications on studying synthetic gene circuits, small regulatory systems, and eventually dynamic whole-cell models

The Near-Earth Object Human Space Flight Accessible Targets Study (NHATS) List of Near-Earth Asteroids: Identifying Potential Targets for Future Exploration

NASA Astrophysics Data System (ADS)

Abell, Paul; Barbee, B. W.; Mink, R. G.; Adamo, D. R.; Alberding, C. M.; Mazanek, D. D.; Johnson, L. N.; Yeomans, D. K.; Chodas, P. W.; Chamberlin, A. B.; Benner, L. A. M.; Drake, B. G.; Friedensen, V. P.

2012-10-01

Introduction: Much attention has recently been focused on human exploration of near-Earth asteroids (NEAs). Detailed planning for deep space exploration and identification of potential NEA targets for human space flight requires selecting objects from the growing list of known NEAs. NASA therefore initiated the Near-Earth Object Human Space Flight Accessible Target Study (NHATS), which uses dynamical trajectory performance constraints to identify potentially accessible NEAs. Accessibility Criteria: Future NASA human space flight capability is being defined while the Orion Multi-Purpose Crew Vehicle and Space Launch System are under development. Velocity change and mission duration are two of the most critical factors in any human spaceflight endeavor, so the most accessible NEAs tend to be those with orbits similar to Earth’s. To be classified as NHATS-compliant, a NEA must offer at least one round-trip trajectory solution satisfying purposely inclusive constraints, including total mission change in velocity ≤ 12 km/s, mission duration ≤ 450 days (with at least 8 days at the NEA), Earth departure between Jan 1, 2015 and Dec 31, 2040, Earth departure C3 ≤ 60 km2/s2, and Earth return atmospheric entry speed ≤ 12 km/s. Monitoring and Updates: The NHATS list of potentially accessible targets is continuously updated as NEAs are discovered and orbit solutions for known NEAs are improved. The current list of accessible NEAs identified as potentially viable for future human exploration under the NHATS criteria is available to the international community via a website maintained by NASA’s NEO Program Office (http://neo.jpl.nasa.gov/nhats/). This website also lists predicted optical and radar observing opportunities for each NHATS-compliant NEA to facilitate acquisition of follow-up observations. Conclusions: This list of NEAs will be useful for analyzing robotic mission opportunities, identifying optimal round trip human space flight trajectories, and

Quantum cellular automata

NASA Astrophysics Data System (ADS)

Porod, Wolfgang; Lent, Craig S.; Bernstein, Gary H.

1994-06-01

The Notre Dame group has developed a new paradigm for ultra-dense and ultra-fast information processing in nanoelectronic systems. These Quantum Cellular Automata (QCA's) are the first concrete proposal for a technology based on arrays of coupled quantum dots. The basic building block of these cellular arrays is the Notre Dame Logic Cell, as it has been called in the literature. The phenomenon of Coulomb exclusion, which is a synergistic interplay of quantum confinement and Coulomb interaction, leads to a bistable behavior of each cell which makes possible their use in large-scale cellular arrays. The physical interaction between neighboring cells has been exploited to implement logic functions. New functionality may be achieved in this fashion, and the Notre Dame group invented a versatile majority logic gate. In a series of papers, the feasibility of QCA wires, wire crossing, inverters, and Boolean logic gates was demonstrated. A major finding is that all logic functions may be integrated in a hierarchial fashion which allows the design of complicated QCA structures. The most complicated system which was simulated to date is a one-bit full adder consisting of some 200 cells. In addition to exploring these new concepts, efforts are under way to physically realize such structures both in semiconductor and metal systems. Extensive modeling work of semiconductor quantum dot structures has helped identify optimum design parameters for QCA experimental implementations.

Boron Stress Responsive MicroRNAs and Their Targets in Barley

PubMed Central

Ozhuner, Esma; Eldem, Vahap; Ipek, Arif; Okay, Sezer; Sakcali, Serdal; Zhang, Baohong; Boke, Hatice; Unver, Turgay

2013-01-01

Boron stress is an environmental factor affecting plant development and production. Recently, microRNAs (miRNAs) have been found to be involved in several plant processes such as growth regulation and stress responses. In this study, miRNAs associated with boron stress were identified and characterized in barley. miRNA profiles were also comparatively analyzed between root and leave samples. A total of 31 known and 3 new miRNAs were identified in barley; 25 of them were found to respond to boron treatment. Several miRNAs were expressed in a tissue specific manner; for example, miR156d, miR171a, miR397, and miR444a were only detected in leaves. Additionally, a total of 934 barley transcripts were found to be specifically targeted and degraded by miRNAs. In silico analysis of miRNA target genes demonstrated that many miRNA targets are conserved transcription factors such as Squamosa promoter-binding protein, Auxin response factor (ARF), and the MYB transcription factor family. A majority of these targets were responsible for plant growth and response to environmental changes. We also propose that some of the miRNAs in barley such as miRNA408 might play critical roles against boron exposure. In conclusion, barley may use several pathways and cellular processes targeted by miRNAs to cope with boron stress. PMID:23555702

Haloacetic Acid Water Disinfection Byproducts Affect Pyruvate Dehydrogenase Activity and Disrupt Cellular Metabolism.

PubMed

Dad, Azra; Jeong, Clara H; Wagner, Elizabeth D; Plewa, Michael J

2018-02-06

The disinfection of drinking water has been a major public health achievement. However, haloacetic acids (HAAs), generated as byproducts of water disinfection, are cytotoxic, genotoxic, mutagenic, carcinogenic, and teratogenic. Previous studies of monoHAA-induced genotoxicity and cell stress demonstrated that the toxicity was due to inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to disruption of cellular metabolism and energy homeostasis. DiHAAs and triHAAs are also produced during water disinfection, and whether they share mechanisms of action with monoHAAs is unknown. In this study, we evaluated the effects of mono-, di-, and tri-HAAs on cellular GAPDH enzyme kinetics, cellular ATP levels, and pyruvate dehydrogenase complex (PDC) activity. Here, treatments conducted in Chinese hamster ovary (CHO) cells revealed differences among mono-, di-, and triHAAs in their molecular targets. The monoHAAs, iodoacetic acid and bromoacetic acid, were the strongest inhibitors of GAPDH and greatly reduced cellular ATP levels. Chloroacetic acid, diHAAs, and triHAAs were weaker inhibitors of GAPDH and some increased the levels of cellular ATP. HAAs also affected PDC activity, with most HAAs activating PDC. The primary finding of this work is that mono- versus multi-HAAs address different molecular targets, and the results are generally consistent with a model in which monoHAAs activate the PDC through GAPDH inhibition-mediated disruption in cellular metabolites, including altering ATP-to-ADP and NADH-to-NAD ratios. The monoHAA-mediated reduction in cellular metabolites results in accelerated PDC activity by way of metabolite-ratio-dependent PDC regulation. DiHAAs and triHAAs are weaker inhibitors of GAPDH, but many also increase cellular ATP levels, and we suggest that they increase PDC activity by inhibiting pyruvate dehydrogenase kinase.

Mequindox induced cellular DNA damage via generation of reactive oxygen species.

PubMed

Liu, Jing; Ouyang, Man; Jiang, Jun; Mu, Peiqiang; Wu, Jun; Yang, Qi; Zhang, Caihui; Xu, Weiying; Wang, Lijuan; Huen, Michael S Y; Deng, Yiqun

2012-01-24

Mequindox, a quinoxaline-N-dioxide derivative that possesses antibacterial properties, has been widely used as a feed additive in the stockbreeding industry in China. While recent pharmacological studies have uncovered potential hazardous effects of mequindox, exactly how mequindox induces pathological changes and the cellular responses associated with its consumption remain largely unexplored. In this study, we investigated the cellular responses associated with mequindox treatment. We report here that mequindox inhibits cell proliferation by arresting cells at the G2/M phase of the cell cycle. Interestingly, this mequindox-associated deleterious effect on cell proliferation was observed in human, pig as well as chicken cells, suggesting that mequindox acts on evolutionarily conserved target(s). To further understand the mequindox-host interaction and the mechanism underlying mequindox-induced cell cycle arrest, we measured the cellular content of DNA damage, which is known to perturb cell proliferation and compromise cell survival. Accordingly, using γ-H2AX as a surrogate marker for DNA damage, we found that mequindox treatment induced cellular DNA damage, which paralleled the chemical-induced elevation of reactive oxygen species (ROS) levels. Importantly, expression of the antioxidant enzyme catalase partially alleviated these mequindox-associated effects. Taken together, our results suggest that mequindox cytotoxicity is attributable, in part, to its role as a potent inducer of DNA damage via ROS. © 2011 Elsevier B.V. All rights reserved.

Potential field cellular automata model for pedestrian flow

NASA Astrophysics Data System (ADS)

Zhang, Peng; Jian, Xiao-Xia; Wong, S. C.; Choi, Keechoo

2012-02-01

This paper proposes a cellular automata model of pedestrian flow that defines a cost potential field, which takes into account the costs of travel time and discomfort, for a pedestrian to move to an empty neighboring cell. The formulation is based on a reconstruction of the density distribution and the underlying physics, including the rule for resolving conflicts, which is comparable to that in the floor field cellular automaton model. However, we assume that each pedestrian is familiar with the surroundings, thereby minimizing his or her instantaneous cost. This, in turn, helps reduce the randomness in selecting a target cell, which improves the existing cellular automata modelings, together with the computational efficiency. In the presence of two pedestrian groups, which are distinguished by their destinations, the cost distribution for each group is magnified due to the strong interaction between the two groups. As a typical phenomenon, the formation of lanes in the counter flow is reproduced.

Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways.

PubMed

Khor, S C; Mohd Yusof, Y A; Wan Ngah, W Z; Makpol, S

Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs). Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein. Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells. Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.

Using Cellular Proteins to Reveal Mechanisms of HIV Infection | Center for Cancer Research

Cancer.gov

A vital step in HIV infection is the insertion of viral DNA into the genome of the host cell. In order for the insertion to occur, viral nucleic acid must be transported through the membrane that separates the main cellular compartment (the cytoplasm) from the nucleus, where the host DNA is located. Scientists are actively studying the mechanism used to transport viral DNA into the nucleus in the hopes of targeting this step with future anti-HIV treatments. Up to this point, researchers have identified some of the viral components that play a role in nuclear transport, but they have not determined how viral interactions with other molecules in the cell contribute to the process.

Personalized Therapy: Tumor Antigen Discovery for Adoptive Cellular Therapy.

PubMed

Yee, Cassian; Lizee, Gregory A

Adoptive cell therapy using endogenous T cells involves the ex vivo isolation and expansion of antigen-specific T cells from the peripheral blood and is uniquely suited for validating and translating antigen discovery. Endogenous T-cell therapy does not require accessible tumor as a source of infiltrating T cells and is free of regulatory and logistical constraints associated with engineering T cells. Candidate epitope peptides identified through antigen discovery may be rapidly implemented as targets in clinical trials of endogenous T-cell therapy and even incorporated as an "ad hoc" approach to personalized treatment when autologous tumor is available. Several first-in-human studies using a uniform population of antigen-specific T cells defined by phenotype and specificity have provided a means to confirm candidate antigens as potential tumor rejection antigens and to evaluate the reasons for success or failure using as a "transferrable cellular biomarker" the adoptively transferred T cells.

Integrated genomic analysis identifies the mitotic checkpoint kinase WEE1 as a novel therapeutic target in medulloblastoma

PubMed Central

2014-01-01

Background Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients do poorly with significant morbidity. Methods To identify new molecular targets, we performed an integrated genomic analysis using structural and functional methods. Gene expression profiling in 16 medulloblastoma patient samples and subsequent gene set enrichment analysis indicated that cell cycle-related kinases were associated with disease development. In addition a kinome-wide small interfering RNA (siRNA) screen was performed to identify kinases that, when inhibited, could prevent cell proliferation. The two genome-scale analyses were combined to identify key vulnerabilities in medulloblastoma. The inhibition of one of the identified targets was further investigated using RNAi and a small molecule inhibitor. Results Combining the two analyses revealed that mitosis-related kinases were critical determinants of medulloblastoma cell proliferation. RNA interference (RNAi)-mediated knockdown of WEE1 kinase and other mitotic kinases was sufficient to reduce medulloblastoma cell proliferation. These data prompted us to examine the effects of inhibiting WEE1 by RNAi and by a small molecule inhibitor of WEE1, MK-1775, in medulloblastoma cell lines. MK-1775 inhibited the growth of medulloblastoma cell lines, induced apoptosis and increased DNA damage at nanomolar concentrations. Further, MK-1775 was synergistic with cisplatin in reducing medulloblastoma cell proliferation and resulted in an associated increase in cell death. In vivo MK-1775 suppressed medulloblastoma tumor growth as a single agent. Conclusions Taken together, these findings highlight mitotic kinases and, in particular, WEE1 as a rational therapeutic target for medulloblastoma. PMID:24661910

Insights into the Sigma-1 receptor chaperone’s cellular functions: a microarray report

PubMed Central

Tsai, Shang-Yi; Rothman, Richard Kyle; Su, Tsung-Ping

2013-01-01

We previously demonstrated that Sig-1Rs are critical regulators in neuronal morphogenesis and development via the regulation of oxidative stress and mitochondrial functions. In the present study, we sought to identify pathways and genes that are affected by Sig-1R. Gene expression profiles were examined in rat hippocampal neurons that had been cultured for18 days in vitro (DIV). The cells were transduced with AAV siRNA targeting Sig-1R on DIV 10 for 7 days, followed by gene expression analysis using a rat genome cDNA array. The gene array results indicated that Sig-1R knockdown hampered cellular functions including steroid biogenesis, protein ubiquitination, actin cytoskeleton network, and Nrf-2 mediated oxidative stress. Many of the cellular components important for actin polymerization and synapse plasticity, including F-actin capping protein and neurofilaments, were significantly changed in AAV-siSig-1R neurons. Further, cytochrome c was reduced in AAV-Sig-1R neurons whereas free-radical generating enzymes including cytochrome p450 and cytochrome b-245 were increased. The microarray results also suggest that Sig-1Rs may regulate genes that are involved in the pathogenesis of many CNS diseases including Alzheimer’s disease and Parkinson’s disease. These data further confirmed that Sig-1Rs play critical roles in the CNS and thus these findings may aid in future development of therapeutic treatments targeting neurodegenerative disorders. PMID:21905129

Multiplexed Thiol Reactivity Profiling for Target Discovery of Electrophilic Natural Products.

PubMed

Tian, Caiping; Sun, Rui; Liu, Keke; Fu, Ling; Liu, Xiaoyu; Zhou, Wanqi; Yang, Yong; Yang, Jing

2017-11-16

Electrophilic groups, such as Michael acceptors, expoxides, are common motifs in natural products (NPs). Electrophilic NPs can act through covalent modification of cysteinyl thiols on functional proteins, and exhibit potent cytotoxicity and anti-inflammatory/cancer activities. Here we describe a new chemoproteomic strategy, termed multiplexed thiol reactivity profiling (MTRP), and its use in target discovery of electrophilic NPs. We demonstrate the utility of MTRP by identifying cellular targets of gambogic acid, an electrophilic NP that is currently under evaluation in clinical trials as anticancer agent. Moreover, MTRP enables simultaneous comparison of seven structurally diversified α,β-unsaturated γ-lactones, which provides insights into the relative proteomic reactivity and target preference of diverse structural scaffolds coupled to a common electrophilic motif and reveals various potential druggable targets with liganded cysteines. We anticipate that this new method for thiol reactivity profiling in a multiplexed manner will find broad application in redox biology and drug discovery. Copyright © 2017 Elsevier Ltd. All rights reserved.

Discovery of host-targeted covalent inhibitors of dengue virus

PubMed Central

de Wispelaere, Mélissanne; Carocci, Margot; Liang, Yanke; Liu, Qingsong; Sun, Eileen; Vetter, Michael L.; Wang, Jinhua; Gray, Nathanael S.; Yang, Priscilla L.

2017-01-01

We report here on an approach targeting the host reactive cysteinome to identify inhibitors of host factors required for the infectious cycle of Flaviviruses and other viruses. We used two parallel cellular phenotypic screens to identify a series of covalent inhibitors, exemplified by QL-XII-47, that are active against dengue virus. We show that the compounds effectively block viral protein expression and that this inhibition is associated with repression of downstream processes of the infectious cycle, and thus significantly contributes to the potent antiviral activity of these compounds. We demonstrate that QL-XII-47’s antiviral activity requires selective, covalent modification of a host target by showing that the compound's antiviral activity is recapitulated when cells are preincubated with QL-XII-47 and then washed prior to viral infection and by showing that QL-XII-47R, a non-reactive analog, lacks antiviral activity at concentrations more than 20-fold higher than QL-XII-47's IC90. QL-XII-47’s inhibition of Zika virus, West Nile virus, hepatitis C virus, and poliovirus further suggests that it acts via a target mediating inhibition of these other medically relevant viruses. These results demonstrate the utility of screens targeting the host reactive cysteinome for rapid identification of compounds with potent antiviral activity. PMID:28034743

Targeted gene insertion for molecular medicine.

PubMed

Voigt, Katrin; Izsvák, Zsuzsanna; Ivics, Zoltán

2008-11-01

Genomic insertion of a functional gene together with suitable transcriptional regulatory elements is often required for long-term therapeutical benefit in gene therapy for several genetic diseases. A variety of integrating vectors for gene delivery exist. Some of them exhibit random genomic integration, whereas others have integration preferences based on attributes of the targeted site, such as primary DNA sequence and physical structure of the DNA, or through tethering to certain DNA sequences by host-encoded cellular factors. Uncontrolled genomic insertion bears the risk of the transgene being silenced due to chromosomal position effects, and can lead to genotoxic effects due to mutagenesis of cellular genes. None of the vector systems currently used in either preclinical experiments or clinical trials displays sufficient preferences for target DNA sequences that would ensure appropriate and reliable expression of the transgene and simultaneously prevent hazardous side effects. We review in this paper the advantages and disadvantages of both viral and non-viral gene delivery technologies, discuss mechanisms of target site selection of integrating genetic elements (viruses and transposons), and suggest distinct molecular strategies for targeted gene delivery.

A screen of cell-surface molecules identifies leucine-rich repeat proteins as key mediators of synaptic target selection in the Drosophila neuromuscular system

PubMed Central

Kurusu, Mitsuhiko; Cording, Amy; Taniguchi, Misako; Menon, Kaushiki; Suzuki, Emiko; Zinn, Kai

2008-01-01

Summary In Drosophila embryos and larvae, a small number of identified motor neurons innervate body wall muscles in a highly stereotyped pattern. Although genetic screens have identified many proteins that are required for axon guidance and synaptogenesis in this system, little is known about the mechanisms by which muscle fibers are defined as targets for specific motor axons. To identify potential target labels, we screened 410 genes encoding cell-surface and secreted proteins, searching for those whose overexpression on all muscle fibers causes motor axons to make targeting errors. Thirty such genes were identified, and a number of these were members of a large gene family encoding proteins whose extracellular domains contain leucine-rich repeat (LRR) sequences, which are protein interaction modules. By manipulating gene expression in muscle 12, we showed that four LRR proteins participate in the selection of this muscle as the appropriate synaptic target for the RP5 motor neuron. PMID:18817735

Cellular Aspects of Prion Replication In Vitro

PubMed Central

Grassmann, Andrea; Wolf, Hanna; Hofmann, Julia; Graham, James; Vorberg, Ina

2013-01-01

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders in mammals that are caused by unconventional agents predominantly composed of aggregated misfolded prion protein (PrP). Prions self-propagate by recruitment of host-encoded PrP into highly ordered β-sheet rich aggregates. Prion strains differ in their clinical, pathological and biochemical characteristics and are likely to be the consequence of distinct abnormal prion protein conformers that stably replicate their alternate states in the host cell. Understanding prion cell biology is fundamental for identifying potential drug targets for disease intervention. The development of permissive cell culture models has greatly enhanced our knowledge on entry, propagation and dissemination of TSE agents. However, despite extensive research, the precise mechanism of prion infection and potential strain effects remain enigmatic. This review summarizes our current knowledge of the cell biology and propagation of prions derived from cell culture experiments. We discuss recent findings on the trafficking of cellular and pathologic PrP, the potential sites of abnormal prion protein synthesis and potential co-factors involved in prion entry and propagation. PMID:23340381

Identifying mechanisms for facilitating knowledge to action strategies targeting the built environment.

PubMed

Fazli, Ghazal S; Creatore, Maria I; Matheson, Flora I; Guilcher, Sara; Kaufman-Shriqui, Vered; Manson, Heather; Johns, Ashley; Booth, Gillian L

2017-01-03

In recent years, obesity-related diseases have been on the rise globally resulting in major challenges for health systems and society as a whole. Emerging research in population health suggests that interventions targeting the built environment may help reduce the burden of obesity and type 2 diabetes. However, translation of the evidence on the built environment into effective policy and planning changes requires engagement and collaboration between multiple sectors and government agencies for designing neighborhoods that are more conducive to healthy and active living. In this study, we identified knowledge gaps and other barriers to evidence-based decision-making and policy development related to the built environment; as well as the infrastructure, processes, and mechanisms needed to drive policy changes in this area. We conducted a qualitative thematic analysis of data collected through consultations with a broad group of stakeholders (N = 42) from Southern Ontario, Canada, within various sectors (public health, urban planning, and transportation) and levels of government (federal, provincial, and municipalities). Relevant themes were classified based on the specific phase of the knowledge-to-action cycle (research, translation, and implementation) in which they were most closely aligned. We identified 5 themes including: 1) the need for policy-informed and actionable research (e.g. health economic analyses and policy evaluations); 2) impactful messaging that targets all relevant sectors to create the political will necessary to drive policy change; 3) common measures and tools to increase capacity for monitoring and surveillance of built environment changes; (4) intersectoral collaboration and alignment within and between levels of government to enable collective actions and provide mechanisms for sharing of resources and expertise, (5) aligning public and private sector priorities to generate public demand and support for community action; and, (6

Functional precision medicine identifies novel druggable targets and therapeutic options in head and neck cancer. | Office of Cancer Genomics

Cancer.gov

Purpose: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with high mortality and a lack of targeted therapies. To identify and prioritize druggable targets, we performed genome analysis together with genome-scale siRNA and oncology drug profiling using low passage tumor cells derived from a patient with a treatmentresistant HPV-negative HNSCC.

Cellular target of weak magnetic fields: ionic conduction along actin filaments of microvilli.

PubMed

Gartzke, Joachim; Lange, Klaus

2002-11-01

The interaction of weak electromagnetic fields (EMF) with living cells is a most important but still unresolved biophysical problem. For this interaction, thermal and other types of noise appear to cause severe restrictions in the action of weak signals on relevant components of the cell. A recently presented general concept of regulation of ion and substrate pathways through microvilli provides a possible theoretical basis for the comprehension of physiological effects of even extremely low magnetic fields. The actin-based core of microfilaments in microvilli is proposed to represent a cellular interaction site for magnetic fields. Both the central role of F-actin in Ca2+ signaling and its polyelectrolyte nature eliciting specific ion conduction properties render the microvillar actin filament bundle an ideal interaction site for magnetic and electric fields. Ion channels at the tip of microvilli are connected with the cytoplasm by a bundle of microfilaments forming a diffusion barrier system. Because of its polyelectrolyte nature, the microfilament core of microvilli allows Ca2+ entry into the cytoplasm via nonlinear cable-like cation conduction through arrays of condensed ion clouds. The interaction of ion clouds with periodically applied EMFs and field-induced cation pumping through the cascade of potential barriers on the F-actin polyelectrolyte follows well-known physical principles of ion-magnetic field (MF) interaction and signal discrimination as described by the stochastic resonance and Brownian motor hypotheses. The proposed interaction mechanism is in accord with our present knowledge about Ca2+ signaling as the biological main target of MFs and the postulated extreme sensitivity for coherent excitation by very low field energies within specific amplitude and frequency windows. Microvillar F-actin bundles shielded by a lipid membrane appear to function like electronic integration devices for signal-to-noise enhancement; the influence of coherent signals

Novel Modeling of Combinatorial miRNA Targeting Identifies SNP with Potential Role in Bone Density

PubMed Central

Coronnello, Claudia; Hartmaier, Ryan; Arora, Arshi; Huleihel, Luai; Pandit, Kusum V.; Bais, Abha S.; Butterworth, Michael; Kaminski, Naftali; Stormo, Gary D.; Oesterreich, Steffi; Benos, Panayiotis V.

2012-01-01

MicroRNAs (miRNAs) are post-transcriptional regulators that bind to their target mRNAs through base complementarity. Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions. Until recently, very few algorithms considered the dynamic nature of the interactions, including the effect of less specific interactions, the miRNA expression level, and the effect of combinatorial miRNA binding. Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications, including efficient miRNA-related SNP evaluation. We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders. Modeling combinatorial miRNA targeting is a natural extension of this model. Two other algorithms show improved prediction efficiency when combinatorial binding models were considered. ComiR (Combinatorial miRNA targeting), a novel algorithm we developed, incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning. Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination. ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding. As proof of principle, ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction, which we confirmed in vitro. We also found rs17737058 to be significantly associated with decreased bone mineral density (BMD) in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women. With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential tool for mi

Covalent small-molecule-RNA complex formation enables cellular profiling of small-molecule-RNA interactions.

PubMed

Guan, Lirui; Disney, Matthew D

2013-09-16

Won't let you go! A strategy is described to design small molecules that react with their cellular RNA targets. This approach not only improves the activity of compounds targeting RNA in cell culture by a factor of about 2500 but also enables cell-wide profiling of its RNA targets. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cellular Senescence, Neurological Function, and Redox State.

PubMed

Maciel-Barón, Luis Ángel; Moreno-Blas, Daniel; Morales-Rosales, Sandra Lizbeth; González-Puertos, Viridiana Yazmín; López-Díazguerrero, Norma Edith; Torres, Claudio; Castro-Obregón, Susana; Königsberg, Mina

2018-06-20

Cellular senescence, characterized by permanent cell cycle arrest, has been extensively studied in mitotic cells such as fibroblasts. However, senescent cells have also been observed in the brain. Even though it is recognized that cellular energetic metabolism and redox homeostasis are perturbed in the aged brain and neurodegenerative diseases (NDDs), it is still unknown which alterations in the overall physiology can stimulate cellular senescence induction and their relationship with the former events. Recent Advances: Recent findings have shown that during prolonged inflammatory and pathologic events, the blood-brain barrier could be compromised and immune cells might enter the brain; this fact along with the brain's high oxygen dependence might result in oxidative damage to macromolecules and therefore senescence induction. Thus, cellular senescence in different brain cell types is revised here. Most information related to cellular senescence in the brain has been obtained from research in glial cells since it has been assumed that the senescent phenotype is a feature exclusive to mitotic cells. Nevertheless, neurons with senescence hallmarks have been observed in old mouse brains. Therefore, although this is a controversial topic in the field, here we summarize and integrate the observations from several studies and propose that neurons indeed senesce. It is still unknown which alterations in the overall metabolism can stimulate senescence induction in the aged brain, what are the mechanisms and signaling pathways, and what is their relationship to NDD development. The understanding of these processes will expose new targets to intervene age-associated pathologies.-Antioxid. Redox Signal. 28, 1704-1723.

COMPARATIVE DIVERSITY OF FECAL BACTERIA IN AGRICULTURALLY SIGNIFICANT ANIMALS TO IDENTIFY ALTERNATIVE TARGETS FOR MICROBIAL SOURCE TRACKING

EPA Science Inventory

Animals of agricultural significance contribute a large percentage of fecal pollution to waterways via runoff contamination. The premise of microbial source tracking is to utilize fecal bacteria to identify target populations which are directly correlated to specific animal feces...

A generalized target theory and its applications.

PubMed

Zhao, Lei; Mi, Dong; Hu, Bei; Sun, Yeqing

2015-09-28

Different radiobiological models have been proposed to estimate the cell-killing effects, which are very important in radiotherapy and radiation risk assessment. However, most applied models have their own scopes of application. In this work, by generalizing the relationship between "hit" and "survival" in traditional target theory with Yager negation operator in Fuzzy mathematics, we propose a generalized target model of radiation-induced cell inactivation that takes into account both cellular repair effects and indirect effects of radiation. The simulation results of the model and the rethinking of "the number of targets in a cell" and "the number of hits per target" suggest that it is only necessary to investigate the generalized single-hit single-target (GSHST) in the present theoretical frame. Analysis shows that the GSHST model can be reduced to the linear quadratic model and multitarget model in the low-dose and high-dose regions, respectively. The fitting results show that the GSHST model agrees well with the usual experimental observations. In addition, the present model can be used to effectively predict cellular repair capacity, radiosensitivity, target size, especially the biologically effective dose for the treatment planning in clinical applications.

RORα, a Potential Tumor Suppressor and Therapeutic Target of Breast Cancer

PubMed Central

Du, Jun; Xu, Ren

2012-01-01

The function of the nuclear receptor (NR) in breast cancer progression has been investigated for decades. The majority of the nuclear receptors have well characterized natural ligands, but a few of them are orphan receptors for which no ligand has been identified. RORα, one member of the retinoid orphan nuclear receptor (ROR) subfamily of orphan receptors, regulates various cellular and pathological activities. RORα is commonly down-regulated and/or hypoactivated in breast cancer compared to normal mammary tissue. Expression of RORα suppresses malignant phenotypes in breast cancer cells, in vitro and in vivo. Activity of RORα can be categorized into the canonical and non-canonical nuclear receptor pathways, which in turn regulate various breast cancer cellular function, including cell proliferation, apoptosis and invasion. This information suggests that RORα is a potent tumor suppressor and a potential therapeutic target for breast cancer. PMID:23443091

Arylacetamide deacetylase: a novel host factor with important roles in the lipolysis of cellular triacylglycerol stores, VLDL assembly and HCV production.

PubMed

Nourbakhsh, Mahra; Douglas, Donna N; Pu, Christopher Hao; Lewis, Jamie T; Kawahara, Toshiyasu; Lisboa, Luiz F; Wei, Enhui; Asthana, Sonal; Quiroga, Ariel D; Law, Lok Man John; Chen, Chao; Addison, William R; Nelson, Randy; Houghton, Michael; Lehner, Richard; Kneteman, Norman M

2013-08-01

Very low density lipoproteins (VLDLs) are triacylglycerol (TG)-rich lipoproteins produced by the human liver. VLDLs derive the majority of their TG cargo from the lipolysis of TG stored in hepatocellular lipid droplets (LDs). Important roles for LDs and the VLDL secretory pathway in the cell culture production of infectious hepatitis C virus (HCV) have been established. We hypothesized that TG lipolysis and VLDL production are impaired during HCV infection so that these cellular processes can be diverted towards HCV production. We used an HCV permissive cell culture system (JFH-1/HuH7.5 cells) to examine the relationship between TG lipolysis, VLDL assembly, and the HCV lifecycle using standard biochemical approaches. Lipolysis of cellular TG and VLDL production were impaired in HCV infected cells during the early peak of viral infection. This was partially explained by an apparent deficiency of a putative TG lipase, arylacetamide deacetylase (AADAC). The re-introduction of AADAC to infected cells restored cellular TG lipolysis, indicating a role for HCV-mediated downregulation of AADAC in this process. Defective lipolysis of cellular TG stores and VLDL production were also observed in HuH7.5 cells stably expressing a short hairpin RNA targeting AADAC expression, proving AADAC deficiency contributes to these defective pathways. Finally, impaired production of HCV was observed with AADAC knockdown cells, demonstrating a role for AADAC in the HCV lifecycle. This insight into the biology of HCV infection and possibly pathogenesis identifies AADAC as a novel and translationally relevant therapeutic target. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

iTAR: a web server for identifying target genes of transcription factors using ChIP-seq or ChIP-chip data.

PubMed

Yang, Chia-Chun; Andrews, Erik H; Chen, Min-Hsuan; Wang, Wan-Yu; Chen, Jeremy J W; Gerstein, Mark; Liu, Chun-Chi; Cheng, Chao

2016-08-12

Chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) or microarray hybridization (ChIP-chip) has been widely used to determine the genomic occupation of transcription factors (TFs). We have previously developed a probabilistic method, called TIP (Target Identification from Profiles), to identify TF target genes using ChIP-seq/ChIP-chip data. To achieve high specificity, TIP applies a conservative method to estimate significance of target genes, with the trade-off being a relatively low sensitivity of target gene identification compared to other methods. Additionally, TIP's output does not render binding-peak locations or intensity, information highly useful for visualization and general experimental biological use, while the variability of ChIP-seq/ChIP-chip file formats has made input into TIP more difficult than desired. To improve upon these facets, here we present are fined TIP with key extensions. First, it implements a Gaussian mixture model for p-value estimation, increasing target gene identification sensitivity and more accurately capturing the shape of TF binding profile distributions. Second, it enables the incorporation of TF binding-peak data by identifying their locations in significant target gene promoter regions and quantifies their strengths. Finally, for full ease of implementation we have incorporated it into a web server ( http://syslab3.nchu.edu.tw/iTAR/ ) that enables flexibility of input file format, can be used across multiple species and genome assembly versions, and is freely available for public use. The web server additionally performs GO enrichment analysis for the identified target genes to reveal the potential function of the corresponding TF. The iTAR web server provides a user-friendly interface and supports target gene identification in seven species, ranging from yeast to human. To facilitate investigating the quality of ChIP-seq/ChIP-chip data, the web server generates the chart of the

Peptide-conjugated micelles as a targeting nanocarrier for gene delivery

NASA Astrophysics Data System (ADS)

Lin, Wen Jen; Chien, Wei Hsuan

2015-09-01

The aim of this study was to develop peptide-conjugated micelles possessing epidermal growth factor receptor (EGFR) targeting ability for gene delivery. A sequence-modified dodecylpeptide, GE11(2R), with enhancing EGF receptor binding affinity, was applied in this study as a targeting ligand. The active targeting micelles were composed of poly( d,l-lactide- co-glycolide)-poly(ethylene glycol) (PLGA-PEG) copolymer conjugated with GE11(2R)-peptide. The particle sizes of peptide-free and peptide-conjugated micelles were 277.0 ± 5.1 and 308.7 ± 14.5 nm, respectively. The peptide-conjugated micelles demonstrated the cellular uptake significantly higher than peptide-free micelles in EGFR high-expressed MDA-MB-231 and MDA-MB-468 cells due to GE11(2R)-peptide specificity. Furthermore, the peptide-conjugated micelles were able to encapsulate plasmid DNA and expressed cellular transfection higher than peptide-free micelles in EGFR high-expressed cells. The EGFR-targeting delivery micelles enhanced DNA internalized into cells and achieved higher cellular transfection in EGFR high-expressed cells.

Meta-Analysis Identifies NF-κB as a Therapeutic Target in Renal Cancer

PubMed Central

Peri, Suraj; Devarajan, Karthik; Yang, Dong-Hua; Knudson, Alfred G.; Balachandran, Siddharth

2013-01-01

Objective To determine the expression patterns of NF-κB regulators and target genes in clear cell renal cell carcinoma (ccRCC), their correlation with von Hippel Lindau (VHL) mutational status, and their association with survival outcomes. Methods Meta-analyses were carried out on published ccRCC gene expression datasets by RankProd, a non-parametric statistical method. DEGs with a False Discovery Rate of < 0.05 by this method were considered significant, and intersected with a curated list of NF-κB regulators and targets to determine the nature and extent of NF-κB deregulation in ccRCC. Results A highly-disproportionate fraction (~40%; p < 0.001) of NF-κB regulators and target genes were found to be up-regulated in ccRCC, indicative of elevated NF-κB activity in this cancer. A subset of these genes, comprising a key NF-κB regulator (IKBKB) and established mediators of the NF-κB cell-survival and pro-inflammatory responses (MMP9, PSMB9, and SOD2), correlated with higher relative risk, poorer prognosis, and reduced overall patient survival. Surprisingly, levels of several interferon regulatory factors (IRFs) and interferon target genes were also elevated in ccRCC, indicating that an ‘interferon signature’ may represent a novel feature of this disease. Loss of VHL gene expression correlated strongly with the appearance of NF-κB- and interferon gene signatures in both familial and sporadic cases of ccRCC. As NF-κB controls expression of key interferon signaling nodes, our results suggest a causal link between VHL loss, elevated NF-κB activity, and the appearance of an interferon signature during ccRCC tumorigenesis. Conclusions These findings identify NF-κB and interferon signatures as clinical features of ccRCC, provide strong rationale for the incorporation of NF-κB inhibitors and/or and the exploitation of interferon signaling in the treatment of ccRCC, and supply new NF-κB targets for potential therapeutic intervention in this currently

Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer*

PubMed Central

Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S.; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

2015-01-01

Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. PMID:26330541

Matrix Rigidity Regulates Cancer Cell Growth by Modulating Cellular Metabolism and Protein Synthesis

PubMed Central

Tilghman, Robert W.; Blais, Edik M.; Cowan, Catharine R.; Sherman, Nicholas E.; Grigera, Pablo R.; Jeffery, Erin D.; Fox, Jay W.; Blackman, Brett R.; Tschumperlin, Daniel J.; Papin, Jason A.; Parsons, J. Thomas

2012-01-01

Background Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy. Methodology/Principal Findings This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates. Compared to cells growing on more rigid/stiff substrates (>10,000 Pa), cells on soft substrates (150–300 Pa) exhibited a longer cell cycle, due predominantly to an extension of the G1 phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis. Using stable isotope labeling of amino acids in culture (SILAC) and mass spectrometry, we measured the rates of protein synthesis of over 1200 cellular proteins under growth conditions on soft and rigid/stiff substrates. We identified cellular proteins whose syntheses were either preferentially inhibited or preserved on soft matrices. The former category included proteins that regulate cytoskeletal structures (e.g., tubulins) and glycolysis (e.g., phosphofructokinase-1), whereas the latter category included proteins that regulate key metabolic pathways required for survival, e.g., nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway. Conclusions/Significance The cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of dormant cancer cells, e.g., slow growth rate and reduced metabolism. We suggest that the use of relatively soft gels as cell culture substrates would allow molecular pathways to be studied under

Systemic analysis of genome-wide expression profiles identified potential therapeutic targets of demethylation drugs for glioblastoma.

PubMed

Ning, Tongbo; Cui, Hao; Sun, Feng; Zou, Jidian

2017-09-05

Glioblastoma represents one of the most aggressive malignant brain tumors with high morbidity and motility. Demethylation drugs have been developed for its treatment with little efficacy has been observed. The purpose of this study was to screen therapeutic targets of demethylation drugs or bioactive molecules for glioblastoma through systemic bioinformatics analysis. We firstly downloaded genome-wide expression profiles from the Gene Expression Omnibus (GEO) and conducted the primary analysis through R software, mainly including preprocessing of raw microarray data, transformation between probe ID and gene symbol and identification of differential expression genes (DEGs). Secondly, functional enrichment analysis was conducted via the Database for Annotation, Visualization and Integrated Discovery (DAVID) to explore biological processes involved in the development of glioblastoma. Thirdly, we constructed protein-protein interaction (PPI) network of interested genes and conducted cross analysis for multi datasets to obtain potential therapeutic targets for glioblastoma. Finally, we further confirmed the therapeutic targets through real-time RT-PCR. As a result, biological processes that related to cancer development, amino metabolism, immune response and etc. were found to be significantly enriched in genes that differential expression in glioblastoma and regulated by 5'aza-dC. Besides, network and cross analysis identified ACAT2, UFC1 and CYB5R1 as novel therapeutic targets of demethylation drugs which also confirmed by real time RT-PCR. In conclusions, our study identified several biological processes and genes that involved in the development of glioblastoma and regulated by 5'aza-dC, which would be helpful for the treatment of glioblastoma. Copyright © 2017 Elsevier B.V. All rights reserved.

Phosphotyrosine profiling identifies ephrin receptor A2 as a potential therapeutic target in esophageal squamous‐cell carcinoma

PubMed Central

Syed, Nazia; Barbhuiya, Mustafa A.; Pinto, Sneha M.; Nirujogi, Raja Sekhar; Renuse, Santosh; Datta, Keshava K.; Khan, Aafaque Ahmad; Srikumar, Kotteazeth; Prasad, T. S. Keshava; Kumar, M. Vijaya; Kumar, Rekha Vijay; Chatterjee, Aditi; Pandey, Akhilesh

2015-01-01

Esophageal squamous‐cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early‐stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non‐neoplastic Het‐1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry‐based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA‐based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation. PMID:25366905

Classification accuracy of claims-based methods for identifying providers failing to meet performance targets.

PubMed

Hubbard, Rebecca A; Benjamin-Johnson, Rhondee; Onega, Tracy; Smith-Bindman, Rebecca; Zhu, Weiwei; Fenton, Joshua J

2015-01-15

Quality assessment is critical for healthcare reform, but data sources are lacking for measurement of many important healthcare outcomes. With over 49 million people covered by Medicare as of 2010, Medicare claims data offer a potentially valuable source that could be used in targeted health care quality improvement efforts. However, little is known about the operating characteristics of provider profiling methods using claims-based outcome measures that may estimate provider performance with error. Motivated by the example of screening mammography performance, we compared approaches to identifying providers failing to meet guideline targets using Medicare claims data. We used data from the Breast Cancer Surveillance Consortium and linked Medicare claims to compare claims-based and clinical estimates of cancer detection rate. We then demonstrated the performance of claim-based estimates across a broad range of operating characteristics using simulation studies. We found that identification of poor performing providers was extremely sensitive to algorithm specificity, with no approach identifying more than 65% of poor performing providers when claims-based measures had specificity of 0.995 or less. We conclude that claims have the potential to contribute important information on healthcare outcomes to quality improvement efforts. However, to achieve this potential, development of highly accurate claims-based outcome measures should remain a priority. Copyright © 2014 John Wiley & Sons, Ltd.

Identifying novel members of the Wntless interactome through genetic and candidate gene approaches.

PubMed

Petko, Jessica; Tranchina, Trevor; Patel, Goral; Levenson, Robert; Justice-Bitner, Stephanie

2018-04-01

Wnt signaling is an important pathway that regulates several aspects of embryogenesis, stem cell maintenance, and neural connectivity. We have recently determined that opioids decrease Wnt secretion, presumably by inhibiting the recycling of the Wnt trafficking protein Wntless (Wls). This effect appears to be mediated by protein-protein interaction between Wls and the mu-opioid receptor (MOR), the primary cellular target of opioid drugs. The goal of this study was to identify novel protein interactors of Wls that are expressed in the brain and may also play a role in reward or addiction. Using genetic and candidate gene approaches, we show that among a variety of protein, Wls interacts with the dopamine transporter (target of cocaine), cannabinoid receptors (target of THC), Adenosine A2A receptor (target of caffeine), and SGIP1 (endocytic regulator of cannabinoid receptors). Our study shows that aside from opioid receptors, Wntless interacts with additional proteins involved in reward and/or addiction. Future studies will determine whether Wntless and WNT signaling play a more universal role in these processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets.

PubMed

Brooks, Lauren E; Ul-Hasan, Sabah; Chan, Benjamin K; Sistrom, Mark J

2018-01-01

Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection-representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments-specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been developed to date

Quantifying the Evolutionary Conservation of Genes Encoding Multidrug Efflux Pumps in the ESKAPE Pathogens To Identify Antimicrobial Drug Targets

PubMed Central

Ul-Hasan, Sabah; Chan, Benjamin K.; Sistrom, Mark J.

2018-01-01

ABSTRACT Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selection—representing targets which can facilitate broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that divergent selection may underlie some of the problems encountered in the development of effective treatments—specifically in relation to the NorA system in S. aureus. This study provides a comprehensive evolutionary context to efflux in the ESKAPE pathogens, which will provide critical context to the evaluation of efflux systems as antibiotic targets. IMPORTANCE Increasing rates of antibiotic-resistant bacterial infection are one of the most pressing contemporary global health concerns. The ESKAPE pathogen group represents the leading cause of these infections, and upregulation of efflux pump expression is a significant mechanism of resistance in these pathogens. This has resulted in substantial interest in the development of efflux pump inhibitors to combat antibiotic-resistant infections; however, no widespread treatments have been

Mitochondria as Sub-cellular Targets of Space Radiation

NASA Astrophysics Data System (ADS)

Hei, Tom; Zhang, Bo; Davidson, Mercy

High linear energy transfer (LET) radiation including alpha particles and heavy ions is the major type of radiation find in space and is considered a potential health risk for astronauts. Even though the chance that these high LET particles traversing through the cytoplasm of cells is higher than that through the nuclei, the contribution of targeted cytoplasmic irradiation, to the induction of genomic instability and other chromosomal damages induced by high LET radiation is not known. Mitochondria are the sole energy center of a cell and normal mitochondria are highly dynamic organelles that move along microtubules or microfilaments and continuously fuse and divide in healthy cells. A balance between mitochondrial fusion and fission is essential to maintain normal mitochondrial function. Targeted cytoplasmic irradiation by high LET alpha particles induced DNA oxidative damage and double strand breaks in wild type rho+ human small airway epithelial (SAE) cells. Furthermore, there was a significant increase in autophagy and micronuclei, which is an indication of genomic instability, together with the activation of nuclear factor kappa-B (NF-kappaB) and mitochondrial inducible nitric oxide synthase (iNOS) signaling pathways in rho+ SAE cells. In contrast, SAE cells with depleted mitochondrial DNA (rho0) and, therefore, no oxidative metabolic functions, exhibited a significantly lower response to these same endpoints examined after cytoplasmic irradiation with high LET alpha particles. The results indicate that normal mitochondrial function is essential in mediating radiation induced genotoxic damages in mammalian cells. Furthermore, the findings may shed some light in the design of countermeasures for space radiation protection.

The nucleus is the target for radiation-induced chromosomal instability

NASA Technical Reports Server (NTRS)

Kaplan, M. I.; Morgan, W. F.

1998-01-01

We have previously described chromosomal instability in cells of a human-hamster hybrid cell line after exposure to X rays. Chromosomal instability in these cells is characterized by the appearance of novel chromosomal rearrangements multiple generations after exposure to ionizing radiation. To identify the cellular target(s) for radiation-induced chromosomal instability, cells were treated with 125I-labeled compounds and frozen. Radioactive decays from 125I cause damage to the cell primarily at the site of their decay, and freezing the cells allows damage to accumulate in the absence of other cellular processes. We found that the decay of 125I-iododeoxyuridine, which is incorporated into the DNA, caused chromosomal instability. While cell killing and first-division chromosomal rearrangements increased with increasing numbers of 125I decays, the frequency of chromosomal instability was independent of dose. Chromosomal instability could also be induced from incorporation of 125I-iododeoxyuridine without freezing the cells for accumulation of decays. This indicates that DNA double-strand breaks in frozen cells resulting from 125I decays failed to lead to instability. Incorporation of an 125I-labeled protein (125I-succinyl-concanavalin A), which was internalized into the cell and/or bound to the plasma membrane, neither caused chromosomal instability nor potentiated chromosomal instability induced by 125I-iododeoxyuridine. These results show that the target for radiation-induced chromosomal instability in these cells is the nucleus.

Researchers Use a Kinome Screen to Identify New Therapeutic Targets | Office of Cancer Genomics

Cancer.gov

The tumor suppressor p53 is mutated in over 50% of head and neck squamous cell carcinomas (HNSCC), yet there are currently no available therapies to target it. CTD2 researchers at the Fred Hutchison Cancer Research Center hypothesized that HNSCC cancer cells with p53 mutations are dependent on particular kinases for survival. In a study published in Clinical Cancer Research, they sought to identify these kinases using RNAi against known kinase genes in mouse and human cell lines.

Drugs in the brain--cellular imaging with receptor microscopic autoradiography.

PubMed

Stumpf, Walter E

2012-03-01

For cell and tissue localization of drugs, receptor microscopic autoradiography is reviewed, including its development history, multiple testing, extensive applications and significant discoveries. This sensitive high-resolution imaging method is based on the use of radiolabeled compounds (esp. tagged with (3)H or (125)I), preservation through freezing of in vivo localization of tissue constituents, cutting thin frozen sections, and close contact with the recording nuclear emulsion. After extensive testing of the utility of this method, the distribution of radiolabeled compounds has been identified and characterized for estradiol, progestagens, adrenal steroids, thyroid hormone, ecdysteroids, vitamin D, retinoic acid, metabolic indicators glucose and 2-deoxyglucose, as well as extracellular space indicators. Target cells and associated tissues have been characterized with special stains, fluorescing compounds, or combined autoradiography-immunocytochemistry with antibodies to dopamine-beta-hydroxylase, GABA, enkephalin, specific receptor proteins, or other cellular products. Blood-brain barrier and brain entries via capillary endothelium, ependyma, or circumventricular recess organs have been visualized for (3)H-dexamethasone, (210)Pb lead, and (3)H-1,25(OH)(2) vitamin D(3). With this histopharmacologic approach, cellular details and tissue integrative overviews can be assessed in the same preparation. As a result, information has been gained that would have been difficult or impossible otherwise. Maps of brain drug distribution have been developed and relevant target circuits have been recognized. Examples include the stria terminalis that links septal-amygdaloid-thalamic-hypothalamic structures and telencephalic limbic system components which extend as the periventricular autonomic-neuroendocrine ABC (Allocortex-Brainstem-Circuitry) system into the mid- and hindbrain. Discoveries with radiolabeled substances challenged existing paradigms, engendering new concepts

PDT-based combinations in overcoming chemoresistance from stromal and heterotypic cellular communication (Conference Presentation)

NASA Astrophysics Data System (ADS)

Rizvi, Imran; Bulin, Anne-Laure; Anbil, Sriram R.; Briars, Emma A.; Vecchio, Daniela; Celli, Jonathan P.; Broekgaarden, Mans; Hasan, Tayyaba

2017-02-01

Targeting the molecular and cellular cues that influence treatment resistance in tumors is critical to effectively treating unresponsive populations of stubborn disease. The informed design of mechanism-based combinations is emerging as increasingly important to targeting resistance and improving the efficacy of conventional treatments, while minimizing toxicity. Photodynamic therapy (PDT) has been shown to synergize with conventional agents and to overcome the evasion pathways that cause resistance. Increasing evidence shows that PDT-based combinations cooperate mechanistically with, and improve the therapeutic index of, traditional chemotherapies. These and other findings emphasize the importance of including PDT as part of comprehensive treatment plans for cancer, particularly in complex disease sites. Identifying effective combinations requires a multi-faceted approach that includes the development of bioengineered cancer models and corresponding image analysis tools. The molecular and phenotypic basis of verteporfin-mediated PDT-based enhancement of chemotherapeutic efficacy and predictability in complex 3D models for ovarian cancer will be presented.

Discovery of cellular substrates for protein kinase A using a peptide array screening protocol.

PubMed

Smith, F Donelson; Samelson, Bret K; Scott, John D

2011-08-15

Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser(119) inside cells and that this mode of regulation may control its ability to affect cell growth. © The Authors Journal compilation © 2011 Biochemical Society

Global analysis of glycoproteins identifies markers of endotoxin tolerant monocytes and GPR84 as a modulator of TNFα expression.

PubMed

Müller, Mario M; Lehmann, Roland; Klassert, Tilman E; Reifenstein, Stella; Conrad, Theresia; Moore, Christoph; Kuhn, Anna; Behnert, Andrea; Guthke, Reinhard; Driesch, Dominik; Slevogt, Hortense

2017-04-12

Exposure of human monocytes to lipopolysaccharide (LPS) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance. In this study, we investigated the LPS-induced global glycoprotein expression changes of tolerant human monocytes and THP-1 cells to identify markers and glycoprotein targets capable to modulate the immunosuppressive state. Using hydrazide chemistry and LC-MS/MS analysis, we analyzed glycoprotein expression changes during a 48 h LPS time course. The cellular snapshots at different time points identified 1491 glycoproteins expressed by monocytes and THP-1 cells. Label-free quantitative analysis revealed transient or long-lasting LPS-induced expression changes of secreted or membrane-anchored glycoproteins derived from intracellular membrane coated organelles or from the plasma membrane. Monocytes and THP-1 cells demonstrated marked differences in glycoproteins differentially expressed in the tolerant state. Among the shared differentially expressed glycoproteins G protein-coupled receptor 84 (GPR84) was identified as being capable of modulating pro-inflammatory TNFα mRNA expression in the tolerant cell state when activated with its ligand Decanoic acid.

The use of functional chemical-protein associations to identify multi-pathway renoprotectants.

PubMed

Xu, Jia; Meng, Kexin; Zhang, Rui; Yang, He; Liao, Chang; Zhu, Wenliang; Jiao, Jundong

2014-01-01

Typically, most nephropathies can be categorized as complex human diseases in which the cumulative effect of multiple minor genes, combined with environmental and lifestyle factors, determines the disease phenotype. Thus, multi-target drugs would be more likely to facilitate comprehensive renoprotection than single-target agents. In this study, functional chemical-protein association analysis was performed to retrieve multi-target drugs of high pathway wideness from the STITCH 3.1 database. Pathway wideness of a drug evaluated the efficiency of regulation of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in quantity. We identified nine experimentally validated renoprotectants that exerted remarkable impact on KEGG pathways by targeting a limited number of proteins. We selected curcumin as an illustrative compound to display the advantage of multi-pathway drugs on renoprotection. We compared curcumin with hemin, an agonist of heme oxygenase-1 (HO-1), which significantly affects only one KEGG pathway, porphyrin and chlorophyll metabolism (adjusted p = 1.5×10-5). At the same concentration (10 µM), both curcumin and hemin equivalently mitigated oxidative stress in H2O2-treated glomerular mesangial cells. The benefit of using hemin was derived from its agonistic effect on HO-1, providing relief from oxidative stress. Selective inhibition of HO-1 completely blocked the action of hemin but not that of curcumin, suggesting simultaneous multi-pathway intervention by curcumin. Curcumin also increased cellular autophagy levels, enhancing its protective effect; however, hemin had no effects. Based on the fact that the dysregulation of multiple pathways is implicated in the etiology of complex diseases, we proposed a feasible method for identifying multi-pathway drugs from compounds with validated targets. Our efforts will help identify multi-pathway agents capable of providing comprehensive protection against renal injuries.

Functional DNA-Containing Nanomaterials: Cellular Applications in Biosensing, Imaging, and Targeted Therapy

PubMed Central

2015-01-01

Conspectus DNA performs a vital function as a carrier of genetic code, but in the field of nanotechnology, DNA molecules can catalyze chemical reactions in the cell, that is, DNAzymes, or bind with target-specific ligands, that is, aptamers. These functional DNAs with different modifications have been developed for sensing, imaging, and therapeutic systems. Thus, functional DNAs hold great promise for future applications in nanotechnology and bioanalysis. However, these functional DNAs face challenges, especially in the field of biomedicine. For example, functional DNAs typically require the use of cationic transfection reagents to realize cellular uptake. Such reagents enter the cells, increasing the difficulty of performing bioassays in vivo and potentially damaging the cell’s nucleus. To address this obstacle, nanomaterials, such as metallic, carbon, silica, or magnetic materials, have been utilized as DNA carriers or assistants. In this Account, we describe selected examples of functional DNA-containing nanomaterials and their applications from our recent research and those of others. As models, we have chosen to highlight DNA/nanomaterial complexes consisting of gold nanoparticles, graphene oxides, and aptamer–micelles, and we illustrate the potential of such complexes in biosensing, imaging, and medical diagnostics. Under proper conditions, multiple ligand–receptor interactions, decreased steric hindrance, and increased surface roughness can be achieved from a high density of DNA that is bound to the surface of nanomaterials, resulting in a higher affinity for complementary DNA and other targets. In addition, this high density of DNA causes a high local salt concentration and negative charge density, which can prevent DNA degradation. For example, DNAzymes assembled on gold nanoparticles can effectively catalyze chemical reactions even in living cells. And it has been confirmed that DNA–nanomaterial complexes can enter cells more easily than free

Functional DNA-containing nanomaterials: cellular applications in biosensing, imaging, and targeted therapy.

PubMed

Liang, Hao; Zhang, Xiao-Bing; Lv, Yifan; Gong, Liang; Wang, Ruowen; Zhu, Xiaoyan; Yang, Ronghua; Tan, Weihong

2014-06-17

CONSPECTUS: DNA performs a vital function as a carrier of genetic code, but in the field of nanotechnology, DNA molecules can catalyze chemical reactions in the cell, that is, DNAzymes, or bind with target-specific ligands, that is, aptamers. These functional DNAs with different modifications have been developed for sensing, imaging, and therapeutic systems. Thus, functional DNAs hold great promise for future applications in nanotechnology and bioanalysis. However, these functional DNAs face challenges, especially in the field of biomedicine. For example, functional DNAs typically require the use of cationic transfection reagents to realize cellular uptake. Such reagents enter the cells, increasing the difficulty of performing bioassays in vivo and potentially damaging the cell's nucleus. To address this obstacle, nanomaterials, such as metallic, carbon, silica, or magnetic materials, have been utilized as DNA carriers or assistants. In this Account, we describe selected examples of functional DNA-containing nanomaterials and their applications from our recent research and those of others. As models, we have chosen to highlight DNA/nanomaterial complexes consisting of gold nanoparticles, graphene oxides, and aptamer-micelles, and we illustrate the potential of such complexes in biosensing, imaging, and medical diagnostics. Under proper conditions, multiple ligand-receptor interactions, decreased steric hindrance, and increased surface roughness can be achieved from a high density of DNA that is bound to the surface of nanomaterials, resulting in a higher affinity for complementary DNA and other targets. In addition, this high density of DNA causes a high local salt concentration and negative charge density, which can prevent DNA degradation. For example, DNAzymes assembled on gold nanoparticles can effectively catalyze chemical reactions even in living cells. And it has been confirmed that DNA-nanomaterial complexes can enter cells more easily than free single