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Sample records for leu-phe knockdown resistance

  1. Association of the N-formyl-Met-Leu-Phe receptor in human neutrophils with a GTP-binding protein sensitive to pertussis toxin.

    PubMed Central

    Lad, P M; Olson, C V; Smiley, P A

    1985-01-01

    Pertussis toxin inhibits the N-formyl-Met-Leu-Phe (fMet-Leu-Phe) mediated human neutrophil functions of enzyme release, superoxide generation, aggregation, and chemotaxis. As pertussis toxin modifies the GTP binding receptor-regulatory protein "Ni," the association of the fMet-Leu-Phe receptor with such a protein was further examined in purified neutrophil plasma membranes. Both fMet-Leu-Phe-mediated guanine nucleotide exchange and nucleotide-mediated regulation of the fMet-Leu-Phe receptor are inhibited by pertussis toxin. In addition, membrane pretreatment with pertussis toxin abolishes the fMet-Leu-Phe-mediated inhibition of adenylate cyclase. Actions of pertussis toxin are due to the ADP-ribosylation of a single subunit at 41 kDa in the neutrophil plasma membrane, which comigrates on NaDodSO4 gels with the Ni GTP-binding protein in the platelet plasma membrane. Our results suggest that (i) the fMet-Leu-Phe receptor is associated with a Ni GTP regulatory protein, and (ii) a fMet-Leu-Phe-Ni complex is important in the control of several neutrophil functions, probably involving multiple transduction systems, including adenylate cyclase. Images PMID:2983319

  2. Chemotactic peptide analogues. Synthesis and chemotactic activity of N-formyl-Met-Leu-Phe analogues containing (S)-phenylalaninol derivatives.

    PubMed

    Zecchini, G P; Paradisi, M P; Torrini, I; Spisani, S

    1995-09-01

    The synthesis and the biological activity towards human neutrophils of some N-formyl-Met-Leu-Phe-OMe analogues containing (S)-phenylalaninol (Pheol) or its derivatives in place of the native phenylalanine are reported. While the analogue containing Pheol (4) was found to be devoid of significant biological activity, its esters 3 and 5, although inactive as chemoattractants, are able to strongly stimulate superoxide production and are active with a lower efficacy in the lysozyme release. PMID:7487425

  3. Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) regulates f Met-Leu-Phe receptors on human neutrophils.

    PubMed Central

    Atkinson, Y H; Lopez, A F; Marasco, W A; Lucas, C M; Wong, G G; Burns, G F; Vadas, M A

    1988-01-01

    The regulation of mature human neutrophil function by recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was studied. Preincubation of neutrophils with this CSF did not stimulate superoxide anion directly but enhanced the subsequent release of superoxide anion in response to stimulation with the bacterial product formylmethionylleucyl-phenylalanine (f Met-Leu-Phe). Enhanced superoxide anion production was evident by 5 min and reached a plateau at 30 min. In contrast, neutrophils preincubated with rH GM-CSF exhibited reduced chemotaxis under agarose in response to a gradient of f Met-Leu-Phe. The inhibition of neutrophil migration was dependent on the dose of rH GM-CSF and exhibited a time-course similar to the effect on superoxide production. Binding studies of f Met-Leu-[3H]Phe to purified human neutrophils revealed heterogeneous binding to unstimulated cells. Two affinity components were identified. The high-affinity component consisted of approximately 2000 sites/cell and had an average Kd of 4 +/- 2 nM (n = 6). The low-affinity component consisted of approximately 40,000 sites/cell and had an average Kd of 220 +/- 130 nM (n = 6). rH GM-CSF caused conversion to a linear Scatchard plot showing no significant change in total binding sites but a single Kd of 30 +/- 10 nM. These data indicate that rH GM-CSF may influence neutrophil responses to f Met-Leu-Phe by regulating the affinity of f Met-Leu-Phe receptors. PMID:2842255

  4. Platelets abrogate leukotriene B(4) generation by human blood neutrophils stimulated with monosodium urate monohydrate or f-Met-Leu-Phe in vitro.

    PubMed

    Chabannes, Bernard; Poubelle, Patrice E; Molière, Patrick; De Médicis, Rinaldo; Lussier, André; Lagarde, Michel

    2003-04-01

    Neutrophils are physiologically associated with platelets in whole blood. Inflammatory reactions can be modulated by the presence of platelets. To investigate the influence of platelets on neutrophil activity, we studied the 5-lipoxygenase (5-LOX) metabolic pathway in normal human blood neutrophils stimulated with f-Met-Leu-Phe (fMLP) or monosodium urate monohydrate (MSUM) in the presence of autologous platelets. Platelets inhibited by more than 90% the synthesis of leukotriene B(4) and 5-HETE in neutrophils activated with fMLP or MSUM. The addition of exogenous arachidonic acid did not reverse the inhibitory effect of platelets on 5-LOX-generated metabolites in fMLP- or MSUM-activated neutrophils. Preincubation of neutrophils with adenosine deaminase reversed the inhibitory effect of platelets in fMLP-treated neutrophils, indicating that adenosine was responsible for the platelet inhibition of leukotriene B(4) and 5-HETE formation. In contrast, adenosine deaminase had no influence on the inhibitory effects of platelets in MSUM-stimulated cells. These results suggest that platelets can inhibit the synthesis of 5-LOX products (a). by acting mainly downstream to phospholipase A(2) in cells stimulated by fMLP or MSUM, (b). through adenosine when neutrophils are activated with fMLP, and (c). by an adenosine-independent mechanism in MSUM-activated neutrophils by an as-yet-unidentified mediator.

  5. Relationship between knockdown resistance, metabolic detoxification and organismal resistance to pyrethroids in Anopheles sinensis.

    PubMed

    Zhong, Daibin; Chang, Xuelian; Zhou, Guofa; He, Zhengbo; Fu, Fengyang; Yan, Zhentian; Zhu, Guoding; Xu, Tielong; Bonizzoni, Mariangela; Wang, Mei-Hui; Cui, Liwang; Zheng, Bin; Chen, Bin; Yan, Guiyun

    2013-01-01

    Anopheles sinensis is the most important vector of malaria in Southeast Asia, including China. Currently, the most effective measure to prevent malaria transmission relies on vector control through the use of insecticides, primarily pyrethroids. Extensive use of insecticides poses strong selection pressure on mosquito populations for resistance. Resistance to insecticides can arise due to mutations in the insecticide target site (target site resistance), which in the case of pyrethroids is the para-type sodium channel gene, and/or the catabolism of the insecticide by detoxification enzymes before it reaches its target (metabolic detoxification resistance). In this study, we examined deltamethrin resistance in An. sinensis from China and investigated the relative importance of target site versus metabolic detoxification mechanisms in resistance. A high frequency (>85%) of nonsynonymous mutations in the para gene was found in populations from central China, but not in populations from southern China. Metabolic detoxification as measured by the activity of monooxygenases and glutathione S-transferases (GSTs) was detected in populations from both central and southern China. Monooxygenase activity levels were significantly higher in the resistant than the susceptible mosquitoes, independently of their geographic origin. Stepwise multiple regression analyses in mosquito populations from central China found that both knockdown resistance (kdr) mutations and monooxygenase activity were significantly associated with deltamethrin resistance, with monooxygenase activity playing a stronger role. These results demonstrate the importance of metabolic detoxification in pyrethroid resistance in An. sinensis, and suggest that different mechanisms of resistance could evolve in geographically different populations.

  6. Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils. Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase.

    PubMed Central

    Matsumoto, T; Tao, W; Sha'afi, R I

    1988-01-01

    The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their

  7. Role of HSF activation for resistance to heat, cold and high-temperature knock-down.

    PubMed

    Nielsen, Morten Muhlig; Overgaard, Johannes; Sørensen, Jesper Givskov; Holmstrup, Martin; Justesen, Just; Loeschcke, Volker

    2005-12-01

    Regulation of heat shock proteins (Hsps) by the heat shock factor (HSF) and the importance of these proteins for resistance to heat stress is well documented. Less characterized is the importance of Hsps for cold stress resistance although Hsp70 is known to be induced following long-term cold exposure in Drosophila melanogaster. In this study, a temperature-sensitive HSF mutant line was used to investigate the role of HSF activation following heat hardening, rapid cold hardening (RCH) and long-term cold acclimation (LTCA) on heat and cold resistance, and this was correlated with Hsp70 expression. In addition, the effect of HSF activation on high-temperature knock-down resistance was evaluated. We found a significantly decreased HSF activation in the mutant line as compared to a corresponding control line following heat hardening, and this was correlated with decreased heat resistance of the mutant line. However, we did not find this difference in HSF activity to be important for resistance to cold stress or high-temperature knock-down. The findings indicate that induction of stress genes regulated by HSF, such as Hsps, although occurring following LTCA, are not of major importance for cold stress resistance and neither for RCH nor high-temperature knock-down resistance in D. melanogaster. PMID:16169555

  8. Response of Two Heat Shock Genes to Selection for Knockdown Heat Resistance in Drosophila Melanogaster

    PubMed Central

    McColl, G.; Hoffmann, A. A.; McKechnie, S. W.

    1996-01-01

    To identify genes involved in stress resistance and heat hardening, replicate lines of Drosophila melanogaster were selected for increased resistance to knockdown by a 39° heat stress. Two selective regimes were used, one with and one without prior hardening. Mean knockdown times were increased from ~5 min to >20 min after 18 generations. Initial realized heritabilities were as high as 10% for lines selected without hardening, and crosses between lines indicated simple additive gene effects for the selected phenotypes. To survey allelic variation and correlated selection responses in two candidate stress genes, hsr-omega and hsp68, we applied denaturing gradient gel electrophoresis to amplified DNA sequences from small regions of these genes. After eight generations of selection, allele frequencies at both loci showed correlated responses for selection following hardening, but not without hardening. The hardening process itself was associated with a hsp68 frequency change in the opposite direction to that associated with selection that followed hardening. These stress loci are closely linked on chromosome III, and the hardening selection established a disequilibrium, suggesting an epistatic effect on resistance. The data indicate that molecular variation in both hsr-omega and hsp68 contribute to natural heritable variation for hardened heat resistance. PMID:8844150

  9. Knockdown Resistance (kdr) Mutations in Indian Anopheles stephensi (Diptera: Culicidae) Populations.

    PubMed

    Dykes, Cherry L; Das, Manoj K; Eapen, Alex; Batra, Chandra P; Ghosh, Susanta K; Vijayan, V A; Mishra, Shobhna; Singh, Om P

    2016-03-01

    Knockdown resistance (kdr) in insects resulting from mutation(s) in the voltage-gated sodium channel (VGSC) gene is one of the mechanisms of resistance against DDT and the pyrethroid group of insecticides. Earlier, we reported the presence of two classic kdr mutations, i.e., L1014F and L1014S in Anopheles stephensi Liston, a major Indian malaria vector affecting mainly urban areas. This report presents the distribution of these alleles in different An. stephensi populations. Seven populations of An. stephensi from six states of India were screened for the presence of two alternative kdr mutations L1014F and L1014S using allele-specific polymerase chain reaction assays. We recorded the presence of both kdr mutations in northern Indian populations (Alwar and Gurgaon), with the preponderance of L1014S, whereas only L1014F was present in Raipur (central India) and Chennai (southern India). None of the kdr mutations were found in Ranchi in eastern India and in Mangaluru and Mysuru in southern India. This study provides evidence for a focal pattern of distribution of kdr alleles in India. PMID:26747858

  10. Knockdown Resistance Allele Frequencies in North American Head Louse (Anoplura: Pediculidae) Populations

    PubMed Central

    Yoon, Kyong Sup; Previte, Domenic J.; Hodgdon, Hilliary E.; Poole, Bryan C.; Kwon, Deok Ho; El-Ghar, Gamal E. Abo; Lee, Si Hyeock; Clark, J. Marshall

    2014-01-01

    The study examines the extent and frequency of a knockdown-type resistance allele (kdr type) in North American populations of human head lice. Lice were collected from 32 locations in Canada and the United States. DNA was extracted from individual lice and used to determine their zygosity using the serial invasive signal amplification technique to detect the kdr-type T917I (TI) mutation, which is most responsible for nerve insensitivity that results in the kdr phenotype and permethrin resistance. Previously sampled sites were resampled to determine if the frequency of the TI mutation was changing. The TI frequency was also reevaluated using a quantitative sequencing method on pooled DNA samples from selected sites to validate this population genotyping method. Genotyping substantiated that TI occurs at high levels in North American lice (88.4%). Overall, the TI frequency in U.S. lice was 84.4% from 1999 to 2009, increased to 99.6% from 2007 to 2009, and was 97.1% in Canadian lice in 2008. Genotyping results using the serial invasive signal amplification reaction (99.54%) and quantitative sequencing (99.45%) techniques were highly correlated. Thus, the frequencies of TI in North American head louse populations were found to be uniformly high, which may be due to the high selection pressure from the intensive and widespread use of the pyrethrins- or pyrethroid-based pediculicides over many years, and is likely a main cause of increased pediculosis and failure of pyrethrins- or permethrin-based products in Canada and the United States. Alternative approaches to treatment of head lice infestations are critically needed. PMID:24724296

  11. Knockdown of c-MET induced apoptosis in ABCB1-overexpressed multidrug-resistance cancer cell lines.

    PubMed

    Hung, T-H; Li, Y-H; Tseng, C-P; Lan, Y-W; Hsu, S-C; Chen, Y-H; Huang, T-T; Lai, H-C; Chen, C-M; Choo, K-B; Chong, K-Y

    2015-05-01

    Inappropriate c-MET signaling in cancer can enhance tumor cell proliferation, survival, motility, and invasion. Inhibition of c-MET signaling induces apoptosis in a variety of cancers. It has also been recognized as a novel anticancer therapy approach. Furthermore, reports have also indicated that constitutive expression of P-glycoprotein (ABCB1) is involved in the HGF/c-MET-related pathway of multidrug resistance ABCB1-positive human hepatocellular carcinoma cell lines. We previously reported that elevated expression levels of PKCδ and AP-1 downstream genes, and HGF receptor (c-MET) and ABCB1, in the drug-resistant MES-SA/Dx5 cells. Moreover, leukemia cell lines overexpressing ABCB1 have also been shown to be more resistant to the tyrosine kinase inhibitor imatinib mesylate. These findings suggest that chemoresistant cancer cells may also develop a similar mechanism against chemotherapy agents. To circumvent clinical complications arising from drug resistance during cancer therapy, the present study was designed to investigate apoptosis induction in ABCB1-overexpressed cancer cells using c-MET-targeted RNA interference technology in vitro and in vivo. The results showed that cell viability decreased and apoptosis rate increased in c-MET shRNA-transfected HGF/c-MET pathway-positive MES-SA/Dx5 and MCF-7/ADR2 cell lines in a dose-dependent manner. In vivo reduction of tumor volume in mice harboring c-MET shRNA-knockdown MES-SA/Dx5 cells was clearly demonstrated. Our study demonstrated that downregulation of c-MET by shRNA-induced apoptosis in a multidrug resistance cell line.

  12. Expansion of the Knockdown Resistance Frequency Map for Human Head Lice (Phthiraptera: Pediculidae) in the United States Using Quantitative Sequencing

    PubMed Central

    Gellatly, Kyle J.; Krim, Sarah; Palenchar, Daniel J.; Shepherd, Katie; Yoon, Kyong Sup; Rhodes, Christopher J.; Lee, Si Hyeock; Marshall Clark, J.

    2016-01-01

    Pediculosis is a prevalent parasitic infestation of humans, which is increasing due, in part, to the selection of lice resistant to either the pyrethrins or pyrethroid insecticides by the knockdown resistance (kdr) mechanism. To determine the extent and magnitude of the kdr-type mutations responsible for this resistance, lice were collected from 138 collection sites in 48 U.S. states from 22 July 2013 to 11 May 2015 and analyzed by quantitative sequencing. Previously published data were used for comparisons of the changes in the frequency of the kdr-type mutations over time. Mean percent resistance allele frequency (mean % RAF) values across the three mutation loci were determined from each collection site. The overall mean % RAF (±SD) for all analyzed lice was 98.3 ± 10%. 132/138 sites (95.6%) had a mean % RAF of 100%, five sites (3.7%) had intermediate values, and only a single site had no mutations (0.0%). Forty-two states (88%) had a mean % RAF of 100%. The frequencies of kdr-type mutations did not differ regardless of the human population size that the lice were collected from, indicating a uniformly high level of resistant alleles. The loss of efficacy of the Nix formulation (Prestige Brand, Tarrytown, NY) from 1998 to 2013 was correlated to the increase in kdr-type mutations. These data provide a plausible reason for the decrease in the effectiveness of permethrin in the Nix formulation, which is the parallel increase of kdr-type mutations in lice over time. PMID:27032417

  13. The cytochrome P450 CYP6P4 is responsible for the high pyrethroid resistance in knockdown resistance-free Anopheles arabiensis

    PubMed Central

    Ibrahim, Sulaiman S.; Riveron, Jacob M.; Stott, Robert; Irving, Helen; Wondji, Charles S.

    2016-01-01

    Pyrethroid insecticides are the front line vector control tools used in bed nets to reduce malaria transmission and its burden. However, resistance in major vectors such as Anopheles arabiensis is posing a serious challenge to the success of malaria control. Herein, we elucidated the molecular and biochemical basis of pyrethroid resistance in a knockdown resistance-free Anopheles arabiensis population from Chad, Central Africa. Using heterologous expression of P450s in Escherichia coli coupled with metabolism assays we established that the over-expressed P450 CYP6P4, located in the major pyrethroid resistance (rp1) quantitative trait locus (QTL), is responsible for resistance to Type I and Type II pyrethroid insecticides, with the exception of deltamethrin, in correlation with field resistance profile. However, CYP6P4 exhibited no metabolic activity towards non-pyrethroid insecticides, including DDT, bendiocarb, propoxur and malathion. Combining fluorescent probes inhibition assays with molecular docking simulation, we established that CYP6P4 can bind deltamethrin but cannot metabolise it. This is possibly due to steric hindrance because of the large vdW radius of bromine atoms of the dihalovinyl group of deltamethrin which docks into the heme catalytic centre. The establishment of CYP6P4 as a partial pyrethroid resistance gene explained the observed field resistance to permethrin, and its inability to metabolise deltamethrin probably explained the high mortality from deltamethrin exposure in the field populations of this Sudano-Sahelian An. arabiensis. These findings describe the heterogeneity in resistance towards insecticides, even from the same class, highlighting the need to thoroughly understand the molecular basis of resistance before implementing resistance management/control tools. PMID:26548743

  14. The cytochrome P450 CYP6P4 is responsible for the high pyrethroid resistance in knockdown resistance-free Anopheles arabiensis.

    PubMed

    Ibrahim, Sulaiman S; Riveron, Jacob M; Stott, Robert; Irving, Helen; Wondji, Charles S

    2016-01-01

    Pyrethroid insecticides are the front line vector control tools used in bed nets to reduce malaria transmission and its burden. However, resistance in major vectors such as Anopheles arabiensis is posing a serious challenge to the success of malaria control. Herein, we elucidated the molecular and biochemical basis of pyrethroid resistance in a knockdown resistance-free Anopheles arabiensis population from Chad, Central Africa. Using heterologous expression of P450s in Escherichia coli coupled with metabolism assays we established that the over-expressed P450 CYP6P4, located in the major pyrethroid resistance (rp1) quantitative trait locus (QTL), is responsible for resistance to Type I and Type II pyrethroid insecticides, with the exception of deltamethrin, in correlation with field resistance profile. However, CYP6P4 exhibited no metabolic activity towards non-pyrethroid insecticides, including DDT, bendiocarb, propoxur and malathion. Combining fluorescent probes inhibition assays with molecular docking simulation, we established that CYP6P4 can bind deltamethrin but cannot metabolise it. This is possibly due to steric hindrance because of the large vdW radius of bromine atoms of the dihalovinyl group of deltamethrin which docks into the heme catalytic centre. The establishment of CYP6P4 as a partial pyrethroid resistance gene explained the observed field resistance to permethrin, and its inability to metabolise deltamethrin probably explained the high mortality from deltamethrin exposure in the field populations of this Sudano-Sahelian An. arabiensis. These findings describe the heterogeneity in resistance towards insecticides, even from the same class, highlighting the need to thoroughly understand the molecular basis of resistance before implementing resistance management/control tools.

  15. NAMPT knockdown attenuates atherosclerosis and promotes reverse cholesterol transport in ApoE KO mice with high-fat-induced insulin resistance.

    PubMed

    Li, Shengbing; Wang, Cong; Li, Ke; Li, Ling; Tian, Mingyuan; Xie, Jing; Yang, Mengliu; Jia, Yanjun; He, Junying; Gao, Lin; Boden, Guenther; Liu, Hua; Yang, Gangyi

    2016-01-01

    NAMPT has been suggested association with atherosclerosis and insulin resistance. However, the impact of NAMPT on atherosclerosis remained unknown. Therefore, the objective of this study was to use a NAMPT loss-of-function approach to investigate the effect of NAMPT on atherosclerosis in hypercholesterolemic mice. We demonstrated that a specific NAMPT knockdown increased plasma HDL-C levels, reduced the plaque area of the total aorta en face and the cross-sectional aortic sinus, decreased macrophage number and apoptosis, and promoted RCT in HFD-fed ApoE KO mice. These changes were accompanied by increased PPARα, LXRα, ABCA1 and ABCG1 expressions in the liver. NAMPT knockdown also facilitated cholesterol efflux in RAW264.7 cells. We further investigated the effect of NAMPT knockdown on the PPARα-LXRα pathway of cholesterol metabolism with MK886 (a selective inhibitor of PPARα) in RAW264.7 macrophages. MK886 abolished the ability of NAMPT knockdown to decrease intracellular cholesterol levels to enhance the rate of (3)H-cholesterol efflux and to increase ABCA1/G1 and LXRα expressions in RAW264.7 macrophages. Our observations demonstrate that NAMPT knockdown exerted antiatherogenic effects by promoting cholesterol efflux and macrophage RCT through the PPARα- LXRα- ABCA1/G1pathway in vitro and in vivo. PMID:27229177

  16. NAMPT knockdown attenuates atherosclerosis and promotes reverse cholesterol transport in ApoE KO mice with high-fat-induced insulin resistance

    PubMed Central

    Li, Shengbing; Wang, Cong; Li, Ke; Li, Ling; Tian, Mingyuan; Xie, Jing; Yang, Mengliu; Jia, Yanjun; He, Junying; Gao, Lin; Boden, Guenther; Liu, Hua; Yang, Gangyi

    2016-01-01

    NAMPT has been suggested association with atherosclerosis and insulin resistance. However, the impact of NAMPT on atherosclerosis remained unknown. Therefore, the objective of this study was to use a NAMPT loss-of-function approach to investigate the effect of NAMPT on atherosclerosis in hypercholesterolemic mice. We demonstrated that a specific NAMPT knockdown increased plasma HDL-C levels, reduced the plaque area of the total aorta en face and the cross-sectional aortic sinus, decreased macrophage number and apoptosis, and promoted RCT in HFD-fed ApoE KO mice. These changes were accompanied by increased PPARα, LXRα, ABCA1 and ABCG1 expressions in the liver. NAMPT knockdown also facilitated cholesterol efflux in RAW264.7 cells. We further investigated the effect of NAMPT knockdown on the PPARα-LXRα pathway of cholesterol metabolism with MK886 (a selective inhibitor of PPARα) in RAW264.7 macrophages. MK886 abolished the ability of NAMPT knockdown to decrease intracellular cholesterol levels to enhance the rate of 3H-cholesterol efflux and to increase ABCA1/G1 and LXRα expressions in RAW264.7 macrophages. Our observations demonstrate that NAMPT knockdown exerted antiatherogenic effects by promoting cholesterol efflux and macrophage RCT through the PPARα- LXRα- ABCA1/G1pathway in vitro and in vivo. PMID:27229177

  17. Detection of a knockdown resistance mutation associated with permethrin resistance in the body louse Pediculus humanus corporis by use of melting curve analysis genotyping.

    PubMed

    Drali, Rezak; Benkouiten, Samir; Badiaga, Sékéné; Bitam, Idir; Rolain, Jean Marc; Brouqui, Philippe

    2012-07-01

    Louse-borne diseases are prevalent in the homeless, and body louse eradication has thus far been unsuccessful in this population. We aim to develop a rapid and robust genotyping method usable in large field-based clinical studies to monitor permethrin resistance in the human body louse Pediculus humanus corporis. We assessed a melting curve analysis genotyping method based on real-time PCR using hybridization probes to detect the M815I-T917I-L920F knockdown resistance (kdr) mutation in the paraorthologous voltage-sensitive sodium channel (VSSC) α subunit gene, which is associated with permethrin resistance. The 908-bp DNA fragment of the VSSC gene, encoding the α subunit of the sodium channel and encompassing the three mutation sites, was PCR sequenced from 65 lice collected from a homeless population. We noted a high prevalence of the 3 indicated mutations in the body lice collected from homeless people (100% for the M815I and L920F mutations and 56.73% for the T917I mutation). These results were confirmed by melting curve analysis genotyping, which had a calculated sensitivity of 100% for the M815I and T917I mutations and of 98% for the L920F mutation. The specificity was 100% for M815I and L920F and 96% for T917I. Melting curve analysis genotyping is a fast, sensitive, and specific tool that is fully compatible with the analysis of a large number of samples in epidemiological surveys, allowing the simultaneous genotyping of 96 samples in just over an hour (75 min). Thus, it is perfectly suited for the epidemiological monitoring of permethrin resistance in human body lice in large-scale clinical studies. PMID:22573588

  18. Knockdown of linc-POU3F3 suppresses the proliferation, apoptosis, and migration resistance of colorectal cancer

    PubMed Central

    Shan, Ti-Dong; Xu, Ji-Hao; Yu, Tao; Li, Jie-Yao; Zhao, Lin-Na; Ouyang, Hui; Luo, Su; Lu, Xi-Ji; Huang, Can-Ze; Lan, Qiu-Shen; Zhong, Wa; Chen, Qi-Kui

    2016-01-01

    Long intergenic noncoding RNAs (lincRNAs) play important roles in regulating the biological functions and underlying molecular mechanisms of colorectal cancer (CRC). Here, we investigated the association of linc-POU3F3 and prognosis in CRC. We demonstrated that linc-POU3F3 was overexpressed in CRC tissues and positively correlated with tumor grade and N stage. Inhibition of linc-POU3F3 resulted in inhibition of cell proliferation and G1 cell cycle arrest, which was mediated by cyclin D1, CDK4, p18, Rb, and phosphorylated Rb. Inhibition of linc-POU3F3 induced apoptosis, and suppressed migration and invasion in LOVO and SW480 cell lines. This inhibition also increased the expressions of epithelial markers and decreased the expressions of mesenchymal markers, thus inhibiting the cancer epithelial-mesenchymal transition. The decreased migration and invasion following linc-POU3F3 knockdown were mediated by an increased BMP signal. Furthermore, autophagy was enhanced by linc-POU3F3 knockdown, suggesting the involvement of autophagy in the induced apoptosis. Collectively, linc-POU3F3 might be crucial in pro-proliferation, anti-apoptosis, and metastasis in LOVO and SW480 cells by regulating the cell cycle, intrinsic apoptosis, BMP signaling and autophagy. Thus, linc-POU3F3 is a potential therapeutic target and novel molecular biomarker for CRC. PMID:26510906

  19. Knockdown of HOXA10 reverses the multidrug resistance of human chronic mylogenous leukemia K562/ADM cells by downregulating P-gp and MRP-1.

    PubMed

    Yi, Ying-Jie; Jia, Xiu-Hong; Wang, Jian-Yong; Li, You-Jie; Wang, Hong; Xie, Shu-Yang

    2016-05-01

    Multidrug resistance (MDR) of leukemia cells is a major obstacle in chemotherapeutic treatment. The high expression and constitutive activation of P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP-1) have been reported to play a vital role in enhancing cell resistance to anticancer drugs in many tumors. The present study aimed to investigate the reversal of MDR by silencing homeobox A10 (HOXA10) in adriamycin (ADR)-resistant human chronic myelogenous leukemia (CML) K562/ADM cells by modulating the expression of P-gp and MRP-1. K562/ADM cells were stably transfected with HOXA10-targeted short hairpin RNA (shRNA). The results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis showed that the mRNA and protein expression of HOXA10 was markedly suppressed following transfection with a shRNA-containing vector. The sensitivity of the K562/ADM cells to ADR was enhanced by the silencing of HOXA10, due to the increased intracellular accumulation of ADR. The accumulation of ADR induced by the silencing of HOXA10 may be due to the downregulation of P-gp and MRP-1. Western blot analysis revealed that downregulating HOXA10 inhibited the protein expression of P-gp and MRP-1. Taken together, these results suggest that knockdown of HOXA10 combats resistance and that HOXA10 is a potential target for resistant human CML. PMID:27035504

  20. Molecular evidence for historical presence of knock-down resistance in Anopheles albimanus, a key malaria vector in Latin America

    PubMed Central

    2013-01-01

    Background Anopheles albimanus is a key malaria vector in the northern neotropics. Current vector control measures in the region are based on mass distributions of long-lasting insecticidal nets (LLINs) and focal indoor residual spraying (IRS) with pyrethroids. Resistance to pyrethroid insecticides can be mediated by increased esterase and/or multi-function oxidase activity and/or mutations in the voltage-gated sodium channel gene. The aim of this work was to characterize the homologous kdr region of the voltage-gated sodium channel gene in An. albimanus and to conduct a preliminary retrospective analysis of field samples collected in the 1990’s, coinciding with a time of intense pyrethroid application related to agricultural and public health insect control in the region. Methods Degenerate primers were designed to amplify the homologous kdr region in a pyrethroid-susceptible laboratory strain (Sanarate) of An. albimanus. Subsequently, a more specific primer pair was used to amplify and sequence the region that contains the 1014 codon associated with pyrethroid resistance in other Anopheles spp. (L1014F, L1014S or L1014C). Results Direct sequencing of the PCR products confirmed the presence of the susceptible kdr allele in the Sanarate strain (L1014) and the presence of homozygous-resistant kdr alleles in field-collected individuals from Mexico (L1014F), Nicaragua (L1014C) and Costa Rica (L1014C). Conclusions For the first time, the kdr region in An. albimanus is described. Furthermore, molecular evidence suggests the presence of kdr-type resistance in field-collected An. albimanus in Mesoamerica in the 1990s. Further research is needed to conclusively determine an association between the genotypes and resistant phenotypes, and to what extent they may compromise current vector control efforts. PMID:24330978

  1. Small interfering RNA-mediated selective knockdown of Na(V)1.8 tetrodotoxin-resistant sodium channel reverses mechanical allodynia in neuropathic rats.

    PubMed

    Dong, X-W; Goregoaker, S; Engler, H; Zhou, X; Mark, L; Crona, J; Terry, R; Hunter, J; Priestley, T

    2007-05-11

    The biophysical properties of a tetrodotoxin resistant (TTXr) sodium channel, Na(V)1.8, and its restricted expression to the peripheral sensory neurons suggest that blocking this channel might have therapeutic potential in various pain states and may offer improved tolerability compared with existing sodium channel blockers. However, the role of Na(V)1.8 in nociception cannot be tested using a traditional pharmacological approach with small molecules because currently available sodium channel blockers do not distinguish between sodium channel subtypes. We sought to determine whether small interfering RNAs (siRNAs) might be capable of achieving the desired selectivity. Using Northern blot analysis and membrane potential measurement, several siRNAs were identified that were capable of a highly-selective attenuation of Na(V)1.8 message as well as functional expression in clonal ND7/23 cells which were stably transfected with the rat Na(V)1.8 gene. Functional knockdown of the channel was confirmed using whole-cell voltage-clamp electrophysiology. One of the siRNA probes showing a robust knockdown of Na(V)1.8 current was evaluated for in vivo efficacy in reversing an established tactile allodynia in the rat chronic constriction nerve-injury (CCI) model. The siRNA, which was delivered to lumbar dorsal root ganglia (DRG) via an indwelling epidural cannula, caused a significant reduction of Na(V)1.8 mRNA expression in lumbar 4 and 5 (L4-L5) DRG neurons and consequently reversed mechanical allodynia in CCI rats. We conclude that silencing of Na(V)1.8 channel using a siRNA approach is capable of producing pain relief in the CCI model and further support a role for Na(V)1.8 in pathological sensory dysfunction. PMID:17367951

  2. RNAi-Mediated CCR5 Knockdown Provides HIV-1 Resistance to Memory T Cells in Humanized BLT Mice.

    PubMed

    Shimizu, Saki; Ringpis, Gene-Errol; Marsden, Matthew D; Cortado, Ruth V; Wilhalme, Holly M; Elashoff, David; Zack, Jerome A; Chen, Irvin S Y; An, Dong Sung

    2015-01-01

    Transplantation of hematopoietic stem/progenitor cells (HSPC) modified with a lentiviral vector bearing a potent nontoxic short hairpin RNA (sh1005) directed to the HIV coreceptor CCR5 is capable of continuously producing CCR5 downregulated CD4+ T lymphocytes. Here, we characterized HIV-1 resistance of the sh1005-modified CD4+ T lymphocytes in vivo in humanized bone marrow/liver/thymus (hu BLT) mice. The sh1005-modified CD4+ T lymphocytes were positively selected in CCR5-tropic HIV-1-challenged mice. The sh1005-modified memory CD4+ T lymphocytes (the primary target of CCR5-tropic HIV-1) expressing sh1005 were maintained in lymphoid tissues in CCR5-tropic HIV-1-challenged mice. Frequencies of HIV-1 p24 expressing cells were significantly reduced in the sh1005-modified splenocytes by ex vivo cell stimulation confirming that CCR5 downregulated sh1005 modified cells are protected from viral infection. These results demonstrate that stable CCR5 downregulation through genetic modification of human HSPC by lentivirally delivered sh1005 is highly effective in providing HIV-1 resistance. Our results provide in vivo evidence in a relevant small animal model that sh1005 is a potent early-step anti-HIV reagent that has potential as a novel anti-HIV-1 HSPC gene therapeutic reagent for human applications. PMID:25689223

  3. QTL for the thermotolerance effect of heat hardening, knockdown resistance to heat and chill-coma recovery in an intercontinental set of recombinant inbred lines of Drosophila melanogaster.

    PubMed

    Norry, Fabian M; Scannapieco, Alejandra C; Sambucetti, Pablo; Bertoli, Carlos I; Loeschcke, Volker

    2008-10-01

    The thermotolerance effect of heat hardening (also called short-term acclimation), knockdown resistance to high temperature (KRHT) with and without heat hardening and chill-coma recovery (CCR) are important phenotypes of thermal adaptation in insects and other organisms. Drosophila melanogaster from Denmark and Australia were previously selected for low and high KRHT, respectively. These flies were crossed to construct recombinant inbred lines (RIL). KRHT was higher in heat-hardened than in nonhardened RIL. We quantify the heat-hardening effect (HHE) as the ratio in KRHT between heat-hardened and nonhardened RIL. Composite interval mapping revealed a more complex genetic architecture for KRHT without heat-hardening than for KRHT in heat-hardened insects. Five quantitative trait loci (QTL) were found for KRHT, but only two of them were significant after heat hardening. KRHT and CCR showed trade-off associations for QTL both in the middle of chromosome 2 and the right arm of chromosome 3, which should be the result of either pleiotropy or linkage. The major QTL on chromosome 2 explained 18% and 27-33% of the phenotypic variance in CCR and KRHT in nonhardened flies, respectively, but its KRHT effects decreased by heat hardening. We discuss candidate loci for each QTL. One HHE-QTL was found in the region of small heat-shock protein genes. However, HHE-QTL explained only a small fraction of the phenotypic variance. Most heat-resistance QTL did not colocalize with CCR-QTL. Large-effect QTL for CCR and KRHT without hardening (basal thermotolerance) were consistent across continents, with apparent transgressive segregation for CCR. HHE (inducible thermotolerance) was not regulated by large-effect QTL.

  4. Two Novel Bioassays to Assess the Effects of Pyrethroid-Treated Netting on Knockdown-Susceptible Versus Resistant Strains of Aedes aegypti.

    PubMed

    Denham, Steven; Eisen, Lars; Beaty, Meaghan; Beaty, Barry J; Black, William C; Saavedra-Rodriguez, Karla

    2015-03-01

    We describe 2 new mosquito bioassays for use with insecticide-treated netting or other textiles. The 1st is a cylinder bioassay in which a mosquito is forced to contact treated material regardless of where it lands within the bioassay construct. The 2nd is a repellency/irritancy and biting-inhibition bioassay (RIBB) in which human arms and breath are used as attractants. Mosquitoes have the choice to pass through holes cut in untreated or treated netting to move from a center release chamber into side chambers to reach arms and potentially bite. Trials were conducted with pyrethroid-susceptible (New Orleans), moderately resistant (Hunucmá), and highly resistant (Vergel) strains of Aedes aegypti. Tests with netting treated with different pyrethroids demonstrated the utility of the cylinder bioassay to quantify knockdown and mortality following exposure to treated netting, and of the RIBB to quantify spatial repellency/contact irritancy of the treated netting and biting inhibition after females land on and then pass through holes in the treated netting. Both tested brands of pyrethroid-treated mosquitocidal netting (DuraNet® and NetProtect®) were effective against New Orleans but ineffective against Vergel strains. Mortality in the cylinder bioassay was 100% for New Orleans for all tested brands of treated netting, but only 10-14% for Vergel. Rates of passage through treated netting to reach a human arm in the RIBB were 10-15% for New Orleans versus 24-37% for Vergel. The reduction in biting after passage through treated netting, compared with untreated netting in the same trial replicates, was 12-39% for New Orleans versus ≤9% for Vergel. PMID:25843176

  5. Two Novel Bioassays to Assess the Effects of Pyrethroid-Treated Netting on Knockdown-Susceptible Versus Resistant Strains of Aedes aegypti.

    PubMed

    Denham, Steven; Eisen, Lars; Beaty, Meaghan; Beaty, Barry J; Black, William C; Saavedra-Rodriguez, Karla

    2015-03-01

    We describe 2 new mosquito bioassays for use with insecticide-treated netting or other textiles. The 1st is a cylinder bioassay in which a mosquito is forced to contact treated material regardless of where it lands within the bioassay construct. The 2nd is a repellency/irritancy and biting-inhibition bioassay (RIBB) in which human arms and breath are used as attractants. Mosquitoes have the choice to pass through holes cut in untreated or treated netting to move from a center release chamber into side chambers to reach arms and potentially bite. Trials were conducted with pyrethroid-susceptible (New Orleans), moderately resistant (Hunucmá), and highly resistant (Vergel) strains of Aedes aegypti. Tests with netting treated with different pyrethroids demonstrated the utility of the cylinder bioassay to quantify knockdown and mortality following exposure to treated netting, and of the RIBB to quantify spatial repellency/contact irritancy of the treated netting and biting inhibition after females land on and then pass through holes in the treated netting. Both tested brands of pyrethroid-treated mosquitocidal netting (DuraNet® and NetProtect®) were effective against New Orleans but ineffective against Vergel strains. Mortality in the cylinder bioassay was 100% for New Orleans for all tested brands of treated netting, but only 10-14% for Vergel. Rates of passage through treated netting to reach a human arm in the RIBB were 10-15% for New Orleans versus 24-37% for Vergel. The reduction in biting after passage through treated netting, compared with untreated netting in the same trial replicates, was 12-39% for New Orleans versus ≤9% for Vergel.

  6. Co-occurrence and distribution of East (L1014S) and West (L1014F) African knock-down resistance in Anopheles gambiae sensu lato population of Tanzania

    PubMed Central

    Kabula, Bilali; Kisinza, William; Tungu, Patrick; Ndege, Chacha; Batengana, Benard; Kollo, Douglas; Malima, Robert; Kafuko, Jessica; Mohamed, Mahdi; Magesa, Stephen

    2014-01-01

    Objective Insecticide resistance molecular markers can provide sensitive indicators of resistance development in Anopheles vector populations. Assaying these makers is of paramount importance in the resistance monitoring programme. We investigated the presence and distribution of knock-down resistance (kdr) mutations in Anopheles gambiae s.l. in Tanzania. Methods Indoor-resting Anopheles mosquitoes were collected from 10 sites and tested for insecticide resistance using the standard WHO protocol. Polymerase chain reaction-based molecular diagnostics were used to genotype mosquitoes and detect kdr mutations. Results The An. gambiae tested were resistance to lambdacyhalothrin in Muheza, Arumeru and Muleba. Out of 350 An. gambiae s.l. genotyped, 35% were An. gambiae s.s. and 65% An. arabiensis. L1014S and L1014F mutations were detected in both An. gambiae s.s. and An. arabiensis. L1014S point mutation was found at the allelic frequency of 4–33%, while L1014F was at the allelic frequency 6–41%. The L1014S mutation was much associated with An. gambiae s.s. (χ2 = 23.41; P < 0.0001) and L1014F associated with An. arabiensis (χ2 = 11.21; P = 0.0008). The occurrence of the L1014S allele was significantly associated with lambdacyhalothrin resistance mosquitoes (Fisher exact P < 0.001). Conclusion The observed co-occurrence of L1014S and L1014F mutations coupled with reports of insecticide resistance in the country suggest that pyrethroid resistance is becoming a widespread phenomenon among our malaria vector populations. The presence of L1014F mutation in this East African mosquito population indicates the spreading of this gene across Africa. The potential operational implications of these findings on malaria control need further exploration. Objectif Les marqueurs moléculaires de la résistance aux insecticides peuvent fournir des indicateurs sensibles du développement de la résistance dans les populations de vecteurs Anopheles. Le test de ces

  7. Knockdown of NADPH-cytochrome P450 reductase results in reduced resistance to buprofezin in the small brown planthopper, Laodelphax striatellus (fallén).

    PubMed

    Zhang, Yueliang; Wang, Yaming; Wang, Lihua; Yao, Jing; Guo, Huifang; Fang, Jichao

    2016-02-01

    NADPH-cytochrome P450 reductase (CPR) plays an important role in cytochrome P450 function, and CPR knockdown in several insects leads to increased susceptibility to insecticides. However, a putative CPR gene has not yet been fully characterized in the small brown planthopper Laodelphax striatellus, a notorious agricultural pest in rice that causes serious damage by transmitting rice stripe and rice black-streaked dwarf viruses. The objective of this study was to clone the cDNA and to knock down the expression of the gene that encodes L. striatellus CPR (LsCPR) to further determine whether P450s are involved in the resistance of L. striatellus to buprofezin. First, the full-length cDNA of LsCPR was cloned and found to contain an open reading frame (ORF) encoding a polypeptide of 679 amino acids with a calculated molecular mass and isoelectric point of 76.92kDa and 5.37, respectively. The deduced amino acid sequence shares high identity with the CPRs of other insects (98%, 97%, 75% and 68% for Sogatella furcifera, Nilaparvata lugens, Cimex lectularius and Anopheles gambiae, respectively) and possesses the characteristic features of classical CPRs, such as an N-terminal membrane anchor and conserved domains for flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding. Phylogenetic analysis revealed that LsCPR is located in a branch along with the CPRs of other hemipteran insects. LsCPR mRNA was detectable in all examined body parts and developmental stages of L. striatellus, as determined by real-time quantitative PCR (qPCR), and transcripts were most abundant in the adult abdomen and in first-instar nymphs and adults. Ingestion of 200μg/mL of LsCPR double-stranded RNA (dsLsCPR) by the planthopper for 5days significantly reduced the transcription level of LsCPR. Moreover, silencing of LsCPR caused increased susceptibility to buprofezin in a buprofezin-resistant (YN-BPF) strain but not in a

  8. Knockdown of NADPH-cytochrome P450 reductase results in reduced resistance to buprofezin in the small brown planthopper, Laodelphax striatellus (fallén).

    PubMed

    Zhang, Yueliang; Wang, Yaming; Wang, Lihua; Yao, Jing; Guo, Huifang; Fang, Jichao

    2016-02-01

    NADPH-cytochrome P450 reductase (CPR) plays an important role in cytochrome P450 function, and CPR knockdown in several insects leads to increased susceptibility to insecticides. However, a putative CPR gene has not yet been fully characterized in the small brown planthopper Laodelphax striatellus, a notorious agricultural pest in rice that causes serious damage by transmitting rice stripe and rice black-streaked dwarf viruses. The objective of this study was to clone the cDNA and to knock down the expression of the gene that encodes L. striatellus CPR (LsCPR) to further determine whether P450s are involved in the resistance of L. striatellus to buprofezin. First, the full-length cDNA of LsCPR was cloned and found to contain an open reading frame (ORF) encoding a polypeptide of 679 amino acids with a calculated molecular mass and isoelectric point of 76.92kDa and 5.37, respectively. The deduced amino acid sequence shares high identity with the CPRs of other insects (98%, 97%, 75% and 68% for Sogatella furcifera, Nilaparvata lugens, Cimex lectularius and Anopheles gambiae, respectively) and possesses the characteristic features of classical CPRs, such as an N-terminal membrane anchor and conserved domains for flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding. Phylogenetic analysis revealed that LsCPR is located in a branch along with the CPRs of other hemipteran insects. LsCPR mRNA was detectable in all examined body parts and developmental stages of L. striatellus, as determined by real-time quantitative PCR (qPCR), and transcripts were most abundant in the adult abdomen and in first-instar nymphs and adults. Ingestion of 200μg/mL of LsCPR double-stranded RNA (dsLsCPR) by the planthopper for 5days significantly reduced the transcription level of LsCPR. Moreover, silencing of LsCPR caused increased susceptibility to buprofezin in a buprofezin-resistant (YN-BPF) strain but not in a

  9. Susceptibility of Adult Cat Fleas (Siphonaptera: Pulicidae) to Insecticides and Status of Insecticide Resistance Mutations at the Rdl and Knockdown Resistance Loci.

    PubMed

    Rust, Michael K; Vetter, Richard; Denholm, Ian; Blagburn, Byron; Williamson, Martin S; Kopp, Steven; Coleman, Glen; Hostetler, Joe; Davis, Wendell; Mencke, Norbert; Rees, Robert; Foit, Sabrina; Böhm, Claudia; Tetzner, Kathrin

    2015-08-01

    The susceptibility of 12 field-collected isolates and 4 laboratory strains of cat fleas, Ctenocephalides felis was determined by topical application of some of the insecticides used as on-animal therapies to control them. In the tested field-collected flea isolates the LD50 values for fipronil and imidacloprid ranged from 0.09 to 0.35 ng/flea and 0.02 to 0.19 ng/flea, respectively, and were consistent with baseline figures published previously. The extent of variation in response to four pyrethroid insecticides differed between compounds with the LD50 values for deltamethrin ranging from 2.3 to 28.2 ng/flea, etofenprox ranging from 26.7 to 86.7 ng/flea, permethrin ranging from 17.5 to 85.6 ng/flea, and d-phenothrin ranging from 14.5 to 130 ng/flea. A comparison with earlier data for permethrin and deltamethrin implied a level of pyrethroid resistance in all isolates and strains. LD50 values for tetrachlorvinphos ranged from 20.0 to 420.0 ng/flea. The rdl mutation (conferring target-site resistance to cyclodiene insecticides) was present in most field-collected and laboratory strains, but had no discernible effect on responses to fipronil, which acts on the same receptor protein as cyclodienes. The kdr and skdr mutations conferring target-site resistance to pyrethroids but segregated in opposition to one another, precluding the formation of genotypes homozygous for both mutations.

  10. Shrimp with knockdown of LvSOCS2, a negative feedback loop regulator of JAK/STAT pathway in Litopenaeus vannamei, exhibit enhanced resistance against WSSV.

    PubMed

    Wang, Sheng; Song, Xuan; Zhang, Zijian; Li, Haoyang; L, Kai; Yin, Bin; He, Jianguo; Li, Chaozheng

    2016-12-01

    JAK/STAT pathway is one of cytokine signaling pathways and mediates diversity immune responses to protect host from viral infection. In this study, LvSOCS2, a member of suppressor of cytokine signaling (SOCS) families, has been cloned and identified from Litopenaeus vannamei. The full length of LvSOCS2 is 1601 bp, including an 1194 bp open reading frame (ORF) coding for a putative protein of 397 amino acids with a calculated molecular weight of ∼42.3 kDa. LvSOCS2 expression was most abundant in gills and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly (I: C) and white spot syndrome virus (WSSV). There are several STAT binding motifs presented in the proximal promoter region of LvSOCS2 and its expression was induced by LvJAK or LvSTAT protein in a dose dependent manner, suggesting LvSOCS2 could be the transcriptional target gene of JAK/STAT pathway. Moreover, the transcription of DmVir-1, a read out of the activation of JAK/STAT pathway in Drosophila, was promoted by LvJAK but inhibited by LvSOCS2, indicating that LvSOCS2 could be a negative regulator in this pathway and thus can form a negative feedback loop. Our previous study indicated that shrimp JAK/STAT pathway played a positive role against WSSV. In this study, RNAi-mediated knockdown of LvSOCS2 shrimps showed lower susceptibility to WSSV infection and caused lessened virus loads, which further demonstrated that the JAK/STAT pathway could function as an anti-viral immunity in shrimp. PMID:27497874

  11. Shrimp with knockdown of LvSOCS2, a negative feedback loop regulator of JAK/STAT pathway in Litopenaeus vannamei, exhibit enhanced resistance against WSSV.

    PubMed

    Wang, Sheng; Song, Xuan; Zhang, Zijian; Li, Haoyang; L, Kai; Yin, Bin; He, Jianguo; Li, Chaozheng

    2016-12-01

    JAK/STAT pathway is one of cytokine signaling pathways and mediates diversity immune responses to protect host from viral infection. In this study, LvSOCS2, a member of suppressor of cytokine signaling (SOCS) families, has been cloned and identified from Litopenaeus vannamei. The full length of LvSOCS2 is 1601 bp, including an 1194 bp open reading frame (ORF) coding for a putative protein of 397 amino acids with a calculated molecular weight of ∼42.3 kDa. LvSOCS2 expression was most abundant in gills and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly (I: C) and white spot syndrome virus (WSSV). There are several STAT binding motifs presented in the proximal promoter region of LvSOCS2 and its expression was induced by LvJAK or LvSTAT protein in a dose dependent manner, suggesting LvSOCS2 could be the transcriptional target gene of JAK/STAT pathway. Moreover, the transcription of DmVir-1, a read out of the activation of JAK/STAT pathway in Drosophila, was promoted by LvJAK but inhibited by LvSOCS2, indicating that LvSOCS2 could be a negative regulator in this pathway and thus can form a negative feedback loop. Our previous study indicated that shrimp JAK/STAT pathway played a positive role against WSSV. In this study, RNAi-mediated knockdown of LvSOCS2 shrimps showed lower susceptibility to WSSV infection and caused lessened virus loads, which further demonstrated that the JAK/STAT pathway could function as an anti-viral immunity in shrimp.

  12. Knockdown and lethal effects of eight commercial nonconventional and two pyrethroid insecticides against moderately permethrin-resistant adult bed bugs, Cimex lectularius (L.) (Hemiptera: Cimicidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The common bed bug, Cimex lectularius (L.) (Hemiptera: Cimicidae) is undergoing a rapid resurgence in the United States during the last decade which has created a notable pest management challenge largely because the pest has developed resistance against DDT, organophosphates, carbamates, and pyreth...

  13. Knock-Down of Both eIF4E1 and eIF4E2 Genes Confers Broad-Spectrum Resistance against Potyviruses in Tomato

    PubMed Central

    Mazier, Marianne; Flamain, Fabrice; Nicolaï, Maryse; Sarnette, Verane; Caranta, Carole

    2011-01-01

    Background The eukaryotic translation initiation factor eIF4E plays a key role in plant-potyvirus interactions. eIF4E belongs to a small multigenic family and three genes, eIF4E1, eIF4E2 and eIF(iso)4E, have been identified in tomato. It has been demonstrated that eIF4E-mediated natural recessive resistances against potyviruses result from non-synonymous mutations in an eIF4E protein, which impair its direct interaction with the potyviral protein VPg. In tomato, the role of eIF4E proteins in potyvirus resistance is still unclear because natural or induced mutations in eIF4E1 confer only a narrow resistance spectrum against potyviruses. This contrasts with the broad spectrum resistance identified in the natural diversity of tomato. These results suggest that more than one eIF4E protein form is involved in the observed broad spectrum resistance. Methodology/Principal Findings To gain insight into the respective contribution of each eIF4E protein in tomato-potyvirus interactions, two tomato lines silenced for both eIF4E1 and eIF4E2 (RNAi-4E) and two lines silenced for eIF(iso)4E (RNAi-iso4E) were obtained and characterized. RNAi-4E lines are slightly impaired in their growth and fertility, whereas no obvious growth defects were observed in RNAi-iso4E lines. The F1 hybrid between RNAi-4E and RNAi-iso4E lines presented a pronounced semi-dwarf phenotype. Interestingly, the RNAi-4E lines silenced for both eIF4E1 and eIF4E2 showed broad spectrum resistance to potyviruses while the RNAi-iso4E lines were fully susceptible to potyviruses. Yeast two-hybrid interaction assays between the three eIF4E proteins and a set of viral VPgs identified two types of VPgs: those that interacted only with eIF4E1 and those that interacted with either eIF4E1 or with eIF4E2. Conclusion/Significance These experiments provide evidence for the involvement of both eIF4E1 and eIF4E2 in broad spectrum resistance of tomato against potyviruses and suggest a role for eIF4E2 in tomato

  14. Knockdown of RNA Interference Pathway Genes in Western Corn Rootworms (Diabrotica virgifera virgifera Le Conte) Demonstrates a Possible Mechanism of Resistance to Lethal dsRNA.

    PubMed

    Vélez, Ana María; Khajuria, Chitvan; Wang, Haichuan; Narva, Kenneth E; Siegfried, Blair D

    2016-01-01

    RNA interference (RNAi) is being developed as a potential tool for insect pest management. Increased understanding of the RNAi pathway in target insect pests will provide information to use this technology effectively and to inform decisions related to resistant management strategies for RNAi based traits. Dicer 2 (Dcr2), an endonuclease responsible for formation of small interfering RNA's and Argonaute 2 (Ago2), an essential catalytic component of the RNA-induced silencing complex (RISC) have both been associated with the RNAi pathway in a number of different insect species including the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). We identified both genes from a transcriptome library generated from different tissues and developmental stages of the western corn rootworm, an important target pest for transgenic plants expressing dsRNA targeting essential genes. The expression of these genes was suppressed by more than 90% after injecting gene specific dsRNA into adult rootworms. The injected beetles were then fed vATPase A dsRNA which has previously been demonstrated to cause mortality in western corn rootworm adults. The suppression of both RNAi pathway genes resulted in reduced mortality after subsequent exposure to lethal concentrations of vATPase A dsRNA as well as increased vATPase A expression relative to control treatments. Injections with dsRNA for a non-lethal target sequence (Laccase 2) did not affect mortality or expression caused by vATPase A dsRNA indicating that the results observed with Argo and Dicer dsRNA were not caused by simple competition among different dsRNA's. These results confirm that both genes play an important role in the RNAi pathway for western corn rootworms and indicate that selection pressures that potentially affect the expression of these genes may provide a basis for future studies to understand potential mechanisms of resistance. PMID:27310918

  15. Knockdown of RNA Interference Pathway Genes in Western Corn Rootworms (Diabrotica virgifera virgifera Le Conte) Demonstrates a Possible Mechanism of Resistance to Lethal dsRNA

    PubMed Central

    Vélez, Ana María; Khajuria, Chitvan; Wang, Haichuan; Narva, Kenneth E.; Siegfried, Blair D.

    2016-01-01

    RNA interference (RNAi) is being developed as a potential tool for insect pest management. Increased understanding of the RNAi pathway in target insect pests will provide information to use this technology effectively and to inform decisions related to resistant management strategies for RNAi based traits. Dicer 2 (Dcr2), an endonuclease responsible for formation of small interfering RNA’s and Argonaute 2 (Ago2), an essential catalytic component of the RNA-induced silencing complex (RISC) have both been associated with the RNAi pathway in a number of different insect species including the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). We identified both genes from a transcriptome library generated from different tissues and developmental stages of the western corn rootworm, an important target pest for transgenic plants expressing dsRNA targeting essential genes. The expression of these genes was suppressed by more than 90% after injecting gene specific dsRNA into adult rootworms. The injected beetles were then fed vATPase A dsRNA which has previously been demonstrated to cause mortality in western corn rootworm adults. The suppression of both RNAi pathway genes resulted in reduced mortality after subsequent exposure to lethal concentrations of vATPase A dsRNA as well as increased vATPase A expression relative to control treatments. Injections with dsRNA for a non-lethal target sequence (Laccase 2) did not affect mortality or expression caused by vATPase A dsRNA indicating that the results observed with Argo and Dicer dsRNA were not caused by simple competition among different dsRNA’s. These results confirm that both genes play an important role in the RNAi pathway for western corn rootworms and indicate that selection pressures that potentially affect the expression of these genes may provide a basis for future studies to understand potential mechanisms of resistance. PMID:27310918

  16. Temporal frequency of knockdown resistance mutations, F1534C and V1016G, in Aedes aegypti in Chiang Mai city, Thailand and the impact of the mutations on the efficiency of thermal fogging spray with pyrethroids.

    PubMed

    Plernsub, Suriya; Saingamsook, Jassada; Yanola, Jintana; Lumjuan, Nongkran; Tippawangkosol, Pongsri; Walton, Catherine; Somboon, Pradya

    2016-10-01

    In Thailand, control of dengue outbreaks is currently attained by the use of space sprays, particularly thermal fogging using pyrethroids, with the aim of killing infected Aedes mosquito vectors in epidemic areas. However, the principal dengue vector, Aedes aegypti, is resistant to pyrethroids conferred mainly by mutations in the voltage-gated sodium channel gene, F1534C and V1016G, termed knockdown resistance (kdr). The objectives of this study were to determine the temporal frequencies of F1534C and V1016G in Ae. aegypti populations in relation to pyrethroid resistance in Chiang Mai city, and to evaluate the impact of the mutations on the efficacy of thermal fogging with the pyrethroid deltamethrin. Larvae and pupae were collected from several areas around Chiang Mai city during 2011-2015 and reared to adulthood for bioassays for deltamethrin susceptibility. These revealed no trend of increasing deltamethrin resistance during the study period (mortality 58.0-69.5%, average 62.8%). This corresponded to no overall change in the frequencies of the C1534 allele (0.55-0.66, average 0.62) and G1016 allele (0.34-0.45, average 0.38), determined using allele specific amplification. Only three genotypes of kdr mutations were detected: C1534 homozygous (VV/CC); G1016/C1534 double heterozygous (VG/FC); and G1016 homozygous (GG/FF) indicating that the F1534C and V1016G mutations occurred on separate haplotypic backgrounds and a lack of recombination between them to date. The F1 progeny females were used to evaluate the efficacy of thermal fogging spray with Damthrin-SP(®) (deltamethrin+S-bioallethrin+piperonyl butoxide) using a caged mosquito bioassay. The thermal fogging spray killed 100% and 61.3% of caged mosquito bioassay placed indoors and outdoors, respectively. The outdoor spray had greater killing effect on C1534 homozygous and had partially effect on double heterozygous mosquitoes, but did not kill any G1016 homozygous mutants living outdoors. As this selection

  17. PCTAIRE1-knockdown sensitizes cancer cells to TNF family cytokines.

    PubMed

    Yanagi, Teruki; Shi, Ranxin; Aza-Blanc, Pedro; Reed, John C; Matsuzawa, Shu-ichi

    2015-01-01

    While PCTAIRE1/PCTK1/Cdk16 is overexpressed in malignant cells and is crucial in tumorigenesis, its function in apoptosis remains unclear. Here we investigated the role of PCTAIRE1 in apoptosis, especially in the extrinsic cell death pathway. Gene-knockdown of PCTAIRE1 sensitized prostate cancer PPC1 and Du145 cells, and breast cancer MDA-MB-468 cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, PCTAIRE1-knockdown did not sensitize non-malignant cells, including diploid fibroblasts IMR-90 and the immortalized prostate epithelial cell line 267B1. PCTAIRE1-knockdown did not up-regulate death receptor expression on the cell surface or affect caspase-8, FADD and FLIP expression levels. PCTAIRE1-knockdown did promote caspase-8 cleavage and RIPK1 degradation, while RIPK1 mRNA knockdown sensitized PPC1 cells to TNF-family cytokines. Furthermore, the kinase inhibitor SNS-032, which inhibits PCTAIRE1 kinase activity, sensitized PPC1 cells to TRAIL-induced apoptosis. Together these results suggest that PCTAIRE1 contributes to the resistance of cancer cell lines to apoptosis induced by TNF-family cytokines, which implies that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines for cytodestruction of cancer cells. PMID:25790448

  18. Knockdown of Slit2 promotes growth and motility in gastric cancer cells via activation of AKT/β-catenin.

    PubMed

    Shi, Rongliang; Yang, Zhen; Liu, Weiyan; Liu, Bingya; Xu, Ziping; Zhang, Ziping

    2014-02-01

    We previously showed that Slit2 was highly expressed in gastric cancer tissues that exhibit less advanced clinicopathological features, suggesting a tumor suppressor role for Slit2. In the present study, we investigated the effects of Slit2 knockdown on gastric cancer cells. Slit2-specific shRNAs were used to generate Slit2-knockdown SGC-7901 gastric cancer cells. Cell proliferation assay, Annexin V/PI double staining and cell cycle analysis were used to investigate the role of Slit2 knockdown in cell growth. Wound-healing and in vitro migration/invasion assays were performed. Subcutaneous tumor formation and peritoneal spreading in nude mice were employed to examine the in vivo effects of Slit2 knockdown. Cell signaling changes induced by Slit2 knockdown were analyzed by immunoblotting. Slit2 knockdown increased gastric cancer cell growth in monolayer and soft agar/Matrigel 3D culture. Slit2 knockdown inhibited apoptosis but did not alter cell cycle progression. Slit2-knockdown cells formed larger tumors and produced more peritoneal metastatic nodules in nude mice. Slit2 knockdown increased AKT phosphorylation, activated anti-apoptotic signaling, suppressed GSK3β activity and induced β-catenin activation. Blocking the effects of PI3K/AKT using pharmacological inhibitors abolished the ability of Slit2 knockdown to induce apoptosis resistance and cell migration/invasion. These results indicate that Slit2 knockdown promotes gastric cancer growth and metastasis through activation of the AKT/β‑catenin-mediated signaling pathway.

  19. Glucocorticoid receptor knockdown decreases the antioxidant protection of B16 melanoma cells: an endocrine system-related mechanism that compromises metastatic cell resistance to vascular endothelium-induced tumor cytotoxicity.

    PubMed

    Obrador, Elena; Valles, Soraya L; Benlloch, María; Sirerol, J Antoni; Pellicer, José A; Alcácer, Javier; Coronado, Javier Alcácer-F; Estrela, José M

    2014-01-01

    We previously reported an interorgan system in which stress-related hormones (corticosterone and noradrenaline), interleukin-6, and glutathione (GSH) coordinately regulate metastatic growth of highly aggressive B16-F10 melanoma cells. Corticosterone, at levels measured in tumor-bearing mice, also induces apoptotic cell death in metastatic cells with low GSH content. In the present study we explored the potential role of glucocorticoids in the regulation of metastatic cell death/survival during the early stages of organ invasion. Glucocorticoid receptor (GCR) knockdown decreased the expression and activity of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting step in GSH synthesis, in metastatic cells in vivo independent of the tumor location (liver, lung, or subcutaneous). The decrease in γ-GCS activity was associated with lower intracellular GSH levels. Nrf2- and p53-dependent down-regulation of γ-GCS was associated with a decrease in the activities of superoxide dismutase 1 and 2, catalase, glutathione peroxidase, and glutathione reductase, but not of the O2--generating NADPH oxidase. The GCR knockdown-induced decrease in antioxidant protection caused a drastic decrease in the survival of metastatic cells during their interaction with endothelial cells, both in vitro and in vivo; only 10% of cancer cells attached to the endothelium survived compared to 90% survival observed in the controls. This very low rate of metastatic cell survival was partially increased (up to 52%) in vivo by inoculating B16-F10 cells preloaded with GSH ester, which enters the cell and delivers free GSH. Taken together, our results indicate that glucocorticoid signaling influences the survival of metastatic cells during their interaction with the vascular endothelium.

  20. Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia

    PubMed Central

    Rochmijati Wuliandari, Juli; Lee, Siu Fai; White, Vanessa Linley; Tantowijoyo, Warsito; Hoffmann, Ary Anthony; Endersby-Harshman, Nancy Margaret

    2015-01-01

    Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations. PMID:26463408

  1. Knockdown of Lmo7 inhibits chick myogenesis.

    PubMed

    Possidonio, Ana C B; Soares, Carolina P; Fontenele, Marcio; Morris, Eduardo R; Mouly, Vincent; Costa, Manoel L; Mermelstein, Claudia

    2016-02-01

    The multifunctional protein Lmo7 has been implicated in some aspects of myogenesis in mammals. Here we studied the distribution and expression of Lmo7 and the effects of Lmo7 knockdown in primary cultures of chick skeletal muscle cells. Lmo7 was localized within the nuclei of myoblasts and at the perinuclear region of myotubes. Knockdown of Lmo7 using siRNA specific to chick reduces the number and width of myotubes and the number of MyoD positive-myoblasts. Both Wnt3a enriched medium and Bio, activators of the Wnt/beta-catenin pathway, could rescue the effects of the Lmo7 knockdown suggesting a crosstalk between the Wnt/beta-catenin and Lmo7-mediated signaling pathways. Our data shows a role of Lmo7 during the initial events of chick skeletal myogenesis, particularly in myoblast survival.

  2. Genotype effect on lifespan following vitellogenin knockdown.

    PubMed

    Ihle, Kate E; Fondrk, M Kim; Page, Robert E; Amdam, Gro V

    2015-01-01

    Honey bee workers display remarkable flexibility in the aging process. This plasticity is closely tied to behavioral maturation. Workers who initiate foraging behavior at earlier ages have shorter lifespans, and much of the variation in total lifespan can be explained by differences in pre-foraging lifespan. Vitellogenin (Vg), a yolk precursor protein, influences worker lifespan both as a regulator of behavioral maturation and through anti-oxidant and immune functions. Experimental reduction of Vg mRNA, and thus Vg protein levels, in wild-type bees results in precocious foraging behavior, decreased lifespan, and increased susceptibility to oxidative damage. We sought to separate the effects of Vg on lifespan due to behavioral maturation from those due to immune and antioxidant function using two selected strains of honey bees that differ in their phenotypic responsiveness to Vg gene knockdown. Surprisingly, we found that lifespans lengthen in the strain described as behaviorally and hormonally insensitive to Vg reduction. We then performed targeted gene expression analyses on genes hypothesized to mediate aging and lifespan: the insulin-like peptides (Ilp1 and 2) and manganese superoxide dismutase (mnSOD). The two honey bee Ilps are the most upstream components in the insulin-signaling pathway, which influences lifespan in Drosophila melanogaster and other organisms, while manganese superoxide dismutase encodes an enzyme with antioxidant functions in animals. We found expression differences in the llps in fat body related to behavior (llp1 and 2) and genetic background (Ilp2), but did not find strain by treatment effects. Expression of mnSOD was also affected by behavior and genetic background. Additionally, we observed a differential response to Vg knockdown in fat body expression of mnSOD, suggesting that antioxidant pathways may partially explain the strain-specific lifespan responses to Vg knockdown.

  3. Cu(II)-catalyzed reactions in ternary [Cu(AA)(AA - H)]+ complexes (AA = Gly, Ala, Val, Leu, Ile, t-Leu, Phe).

    PubMed

    Wang, Ping; Ohanessian, Gilles; Wesdemiotis, Chrys

    2009-01-01

    The unimolecular chemistry of [Cu(II)AA(AA - H)](+) complexes, composed of an intact and a deprotonated amino acid (AA) ligand, have been probed in the gas phase by tandem and multistage mass spectrometry in an electrospray ionization quadrupole ion trap mass spectrometer. The amino acids examined include Gly, Ala, Val, Leu, Ile, t-Leu and Phe. Upon collisionally-activated dissociation (CAD), the [Cu(II)AA(AA - H)](+) complexes undergo decarboxylation with simultaneous reduction of Cu(II) to Cu(I); during this process, a radical site is created at the alpha-carbon of the decarboxylated ligand (H(2)N(1) - (*)C(alpha)H - C(beta)H(2) - R; R = side chain substituent). The radical site is able to move along the backbone of the decarboxylated amino acid to form two new radicals (HN(1)(*) - C(alpha)H(2) - C(beta)H(2) - R and H(2)N(1) - C(alpha)H(2) - (*)C(beta)H - R). From the complexes of Gly and t-Leu, only C(alpha) and N(1) radicals can be formed. The whole radical ligand can be lost to form [Cu(I)AA](+) from these three isomeric radicals. Alternatively, further radical induced dissociations can take place along the backbone of the decarboxylated amino acid ligand to yield [Cu(II)AA(AA - 2H - CO(2))](+), [Cu(I)AA((*)NH(2))](+), [Cu(I)AA(HN = C(alpha)H(2))](+), or [Cu(I)AA(H(2)N - C(alpha)H = C(beta)H - R'](+) (R' = partial side chain substituent). The sodiated copper complexes, [Cu(II)(AA - H + Na)(AA - H)](+), show the same fragmentation patterns as their non-sodiated counterparts; sodium ion is retained on the intact amino acid ligand and is not involved in the CAD pathways. The amino groups of both AA units, the carbonyl group of the intact amino acid, and the deprotonated hydroxyl oxygen coordinate Cu(II) in square-planar fashion. Ab initio calculations indicate that the metal ion facilitates hydrogen atom shuttling between the N(1), C(alpha) and C(beta) atoms of the decarboxylated amino acid ligand. The dissociations of the decarboxylated radical ions unveil important insight about the so far largely unknown intrinsic chemistry of alpha-amino acid and peptide radicals, which are implicated as intermediates in numerous pathogenic biological processes. PMID:19423917

  4. Knockdown of FIBRILLIN4 Gene Expression in Apple Decreases Plastoglobule Plastoquinone Content

    PubMed Central

    Singh, Dharmendra K.; Laremore, Tatiana N.; Smith, Philip B.; Maximova, Siela N.; McNellis, Timothy W.

    2012-01-01

    Fibrillin4 (FBN4) is a protein component of plastoglobules, which are antioxidant-rich sub-compartments attached to the chloroplast thylakoid membranes. FBN4 is required for normal plant biotic and abiotic stress resistance, including bacterial pathogens, herbicide, high light intensity, and ozone; FBN4 is also required for the accumulation of osmiophilic material inside plastoglobules. In this study, the contribution of FBN4 to plastoglobule lipid composition was examined using cultivated apple trees in which FBN4 gene expression was knocked down using RNA interference. Chloroplasts and plastoglobules were isolated from leaves of wild-type and fbn4 knock-down trees. Total lipids were extracted from chloroplasts and plastoglobules separately, and analyzed using liquid chromatography-mass spectrometry (LC–MS). Three lipids were consistently present at lower levels in the plastoglobules from fbn4 knock-down apple leaves compared to the wild-type as determined by LC-MS multiple ion monitoring. One of these species had a molecular mass and fragmentation pattern that identified it as plastoquinone, a known major component of plastoglobules. The plastoquinone level in fbn4 knock-down plastoglobules was less than 10% of that in wild-type plastoglobules. In contrast, plastoquinone was present at similar levels in the lipid extracts of whole chloroplasts from leaves of wild-type and fbn4 knock-down trees. These results suggest that the partitioning of plastoquinone between the plastoglobules and the rest of the chloroplast is disrupted in fbn4 knock-down leaves. These results indicate that FBN4 is required for high-level accumulation of plastoquinone and some other lipids in the plastoglobule. The dramatic decrease in plastoquinone content in fbn4 knock-down plastoglobules is consistent with the decreased plastoglobule osmiophilicity previously described for fbn4 knock-down plastoglobules. Failure to accumulate the antioxidant plastoquinone in the fbn4 knock-down

  5. Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine

    PubMed Central

    Pessina, Stefano; Lenzi, Luisa; Perazzolli, Michele; Campa, Manuela; Dalla Costa, Lorenza; Urso, Simona; Valè, Giampiero; Salamini, Francesco; Velasco, Riccardo; Malnoy, Mickael

    2016-01-01

    Erysiphe necator is the causal agent of powdery mildew (PM), one of the most destructive diseases of grapevine. PM is controlled by sulfur-based and synthetic fungicides, which every year are dispersed into the environment. This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production. PM resistance can be achieved in other crops by knocking out susceptibility S-genes, such as those residing at genetic loci known as MLO (Mildew Locus O). All MLO S-genes of dicots belong to the phylogenetic clade V, including grapevine genes VvMLO7, 11 and 13, which are upregulated during PM infection, and VvMLO6, which is not upregulated. Before adopting a gene-editing approach to knockout candidate S-genes, the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary. This paper reports the knockdown through RNA interference of VvMLO6, 7, 11 and 13. The knockdown of VvMLO6, 11 and 13 did not decrease PM severity, whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%. The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance, whereas a role for VvMLO11 does not seem likely. Cell wall appositions (papillae) were present in both resistant and susceptible lines in response to PM attack. Thirteen genes involved in defense were less upregulated in infected mlo plants, highlighting the early mlo-dependent disruption of PM invasion. PMID:27390621

  6. Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine.

    PubMed

    Pessina, Stefano; Lenzi, Luisa; Perazzolli, Michele; Campa, Manuela; Dalla Costa, Lorenza; Urso, Simona; Valè, Giampiero; Salamini, Francesco; Velasco, Riccardo; Malnoy, Mickael

    2016-01-01

    Erysiphe necator is the causal agent of powdery mildew (PM), one of the most destructive diseases of grapevine. PM is controlled by sulfur-based and synthetic fungicides, which every year are dispersed into the environment. This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production. PM resistance can be achieved in other crops by knocking out susceptibility S-genes, such as those residing at genetic loci known as MLO (Mildew Locus O). All MLO S-genes of dicots belong to the phylogenetic clade V, including grapevine genes VvMLO7, 11 and 13, which are upregulated during PM infection, and VvMLO6, which is not upregulated. Before adopting a gene-editing approach to knockout candidate S-genes, the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary. This paper reports the knockdown through RNA interference of VvMLO6, 7, 11 and 13. The knockdown of VvMLO6, 11 and 13 did not decrease PM severity, whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%. The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance, whereas a role for VvMLO11 does not seem likely. Cell wall appositions (papillae) were present in both resistant and susceptible lines in response to PM attack. Thirteen genes involved in defense were less upregulated in infected mlo plants, highlighting the early mlo-dependent disruption of PM invasion. PMID:27390621

  7. CERKL Knockdown Causes Retinal Degeneration in Zebrafish

    PubMed Central

    Riera, Marina; Burguera, Demian; Garcia-Fernàndez, Jordi; Gonzàlez-Duarte, Roser

    2013-01-01

    The human CERKL gene is responsible for common and severe forms of retinal dystrophies. Despite intense in vitro studies at the molecular and cellular level and in vivo analyses of the retina of murine knockout models, CERKL function remains unknown. In this study, we aimed to approach the developmental and functional features of cerkl in Danio rerio within an Evo-Devo framework. We show that gene expression increases from early developmental stages until the formation of the retina in the optic cup. Unlike the high mRNA-CERKL isoform multiplicity shown in mammals, the moderate transcriptional complexity in fish facilitates phenotypic studies derived from gene silencing. Moreover, of relevance to pathogenicity, teleost CERKL shares the two main human protein isoforms. Morpholino injection has been used to generate a cerkl knockdown zebrafish model. The morphant phenotype results in abnormal eye development with lamination defects, failure to develop photoreceptor outer segments, increased apoptosis of retinal cells and small eyes. Our data support that zebrafish Cerkl does not interfere with proliferation and neural differentiation during early developmental stages but is relevant for survival and protection of the retinal tissue. Overall, we propose that this zebrafish model is a powerful tool to unveil CERKL contribution to human retinal degeneration. PMID:23671706

  8. Fascin-1 knock-down of human glioma cells reduces their microvilli/filopodia while improving their susceptibility to lymphocyte-mediated cytotoxicity

    PubMed Central

    Hoa, Neil T; Ge, Lisheng; Erickson, Kate L; Kruse, Carol A; Cornforth, Andrew N; Kuznetsov, Yurii; McPherson, Alex; Martini, Filippo; Jadus, Martin R

    2015-01-01

    Cancer cells derived from Glioblastoma multiforme possess membranous protrusions allowing these cells to infiltrate surrounding tissue, while resisting lymphocyte cytotoxicity. Microvilli and filopodia are supported by actin filaments cross-linked by fascin. Fascin-1 was genetically silenced within human U251 glioma cells; these knock-down glioma cells lost their microvilli/filopodia. The doubling time of these fascin-1 knock-down cells was doubled that of shRNA control U251 cells. Fascin-1 knock-down cells lost their transmigratory ability responding to interleukin-6 or insulin-like growth factor-1. Fascin-1 silenced U251 cells were more easily killed by cytolytic lymphocytes. Fascin-1 knock-down provides unique opportunities to augment glioma immunotherapy by simultaneously targeting several key glioma functions: like cell transmigration, cell division and resisting immune responses. PMID:25901196

  9. Enhanced toxic cloud knockdown spray system for decontamination applications

    SciTech Connect

    Betty, Rita G.; Tucker, Mark D.; Brockmann, John E.; Lucero, Daniel A.; Levin, Bruce L.; Leonard, Jonathan

    2011-09-06

    Methods and systems for knockdown and neutralization of toxic clouds of aerosolized chemical or biological warfare (CBW) agents and toxic industrial chemicals using a non-toxic, non-corrosive aqueous decontamination formulation.

  10. Knockdown of retinoblastoma protein may sensitize glioma cells to cisplatin through inhibition of autophagy.

    PubMed

    Liu, Xiangyu; Sun, Kangjian; Wang, Handong; Dai, Yuyuan

    2016-05-01

    Glioblastoma multiforme (GBM) is one of the deadliest forms of cancer due to its limited sensitivity to chemotherapy and radiotherapy. Cisplatin (CCDP) is a widely used chemotherapeutic agent for tumors, but the agent often results in the development of chemo-resistance. In several cancers, cisplatin resistance is associated with autophagy induction. Here, we found that in glioma cells cisplatin treatment induced autophagy. Our data indicates that the autophagy induction plays a critical role in cisplatin resistance of glioma cells, knockdown of RB inhibited autophagy induced by cisplatin, and inhibition of autophagy improved cisplatin-induced apoptosis. It suggests that a combination of autophagy inhibitors with cisplatin may improve the therapeutic efficiency of cisplatin towards GBM with acquired resistance. PMID:27048711

  11. Knockdown of Drosophila hemoglobin suggests a role in O2 homeostasis.

    PubMed

    Gleixner, Eva; Ripp, Fabian; Gorr, Thomas A; Schuh, Reinhard; Wolf, Christian; Burmester, Thorsten; Hankeln, Thomas

    2016-05-01

    Almost all insects are equipped with a tracheal system, which appears to be sufficient for O2 supply even in phases of high metabolic activity. Therefore, with the exception of a few species dwelling in hypoxic habitats, specialized respiratory proteins had been considered unnecessary in insects. The recent discovery and apparently universal presence of intracellular hemoglobins in insects has remained functionally unexplained. The fruitfly Drosophila melanogaster harbors three different globin genes (referred to as glob1-3). Glob1 is the most highly expressed globin and essentially occurs in the tracheal system and the fat body. To better understand the functions of insect globins, the levels of glob1 were modulated in Drosophila larvae and adults by RNAi-mediated knockdown and transgenic over-expression. No effects on the development were observed in flies with manipulated glob1 levels. However, the knockdown of glob1 led to a significantly reduced survival rate of adult flies under hypoxia (5% and 1.5% O2). Surprisingly, the glob1 knockdown flies also displayed increased resistance towards the reactive oxygen species-forming agent paraquat, which may be explained by a restricted availability of O2 resulting in decreased formation of harmful O2(-). In summary, our results suggest an important functional role of glob1 in O2 homeostasis, possibly by enhancing O2 supply. PMID:27001071

  12. Effects of simultaneous knockdown of HER2 and PTK6 on malignancy and tumor progression in human breast cancer cells.

    PubMed

    Ludyga, Natalie; Anastasov, Natasa; Rosemann, Michael; Seiler, Jana; Lohmann, Nadine; Braselmann, Herbert; Mengele, Karin; Schmitt, Manfred; Höfler, Heinz; Aubele, Michaela

    2013-04-01

    Breast cancer is the most common malignancy in women of the Western world. One prominent feature of breast cancer is the co- and overexpression of HER2 and protein tyrosine kinase 6 (PTK6). According to the current clinical cancer therapy guidelines, HER2-overexpressing tumors are routinely treated with trastuzumab, a humanized monoclonal antibody targeting HER2. Approximately, 30% of HER2-overexpressing breast tumors at least initially respond to the anti-HER2 therapy, but a subgroup of these tumors develops resistance shortly after the administration of trastuzumab. A PTK6-targeted therapy does not yet exist. Here, we show for the first time that the simultaneous knockdown in vitro, compared with the single knockdown of HER2 and PTK6, in particular in the trastuzumab-resistant JIMT-1 cells, leads to a significantly decreased phosphorylation of crucial signaling proteins: mitogen-activated protein kinase 1/3 (MAPK 1/3, ERK 1/2) and p38 MAPK, and (phosphatase and tensin homologue deleted on chromosome ten) PTEN that are involved in tumorigenesis. In addition, dual knockdown strongly reduced the migration and invasion of the JIMT-1 cells. Moreover, the downregulation of HER2 and PTK6 led to an induction of p27, and the dual knockdown significantly diminished cell proliferation in JIMT-1 and T47D cells. In vivo experiments showed significantly reduced levels of tumor growth following HER2 or PTK6 knockdown. Our results indicate a novel strategy also for the treatment of trastuzumab resistance in tumors. Thus, the inhibition of these two signaling proteins may lead to a more effective control of breast cancer.

  13. The knock-down of the expression of MdMLO19 reduces susceptibility to powdery mildew (Podosphaera leucotricha) in apple (Malus domestica).

    PubMed

    Pessina, Stefano; Angeli, Dario; Martens, Stefan; Visser, Richard G F; Bai, Yuling; Salamini, Francesco; Velasco, Riccardo; Schouten, Henk J; Malnoy, Mickael

    2016-10-01

    Varieties resistant to powdery mildew (PM; caused by Podosphaera leucotricha) are a major component of sustainable apple production. Resistance can be achieved by knocking-out susceptibility S-genes to be singled out among members of the MLO (Mildew Locus O) gene family. Candidates are MLO S-genes of phylogenetic clade V up-regulated upon PM inoculation, such as MdMLO11 and 19 (clade V) and MdMLO18 (clade VII). We report the knock-down through RNA interference of MdMLO11 and 19, as well as the complementation of resistance with MdMLO18 in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 reduced PM disease severity by 75%, whereas the knock-down of MdMLO11, alone or in combination with MdMLO19, did not result in any reduction or additional reduction of susceptibility compared with MdMLO19 alone. The test in A. thaliana excluded a role for MdMLO18 in PM susceptibility. Cell wall appositions (papillae) were present in both PM-resistant and PM-susceptible plants, but were larger in resistant lines. No obvious negative phenotype was observed in plants with mlo genes knocked down. Apparently, MdMLO19 plays the pivotal role in apple PM susceptibility and its knock-down induces a very significant level of resistance. PMID:26997489

  14. The knock-down of the expression of MdMLO19 reduces susceptibility to powdery mildew (Podosphaera leucotricha) in apple (Malus domestica).

    PubMed

    Pessina, Stefano; Angeli, Dario; Martens, Stefan; Visser, Richard G F; Bai, Yuling; Salamini, Francesco; Velasco, Riccardo; Schouten, Henk J; Malnoy, Mickael

    2016-10-01

    Varieties resistant to powdery mildew (PM; caused by Podosphaera leucotricha) are a major component of sustainable apple production. Resistance can be achieved by knocking-out susceptibility S-genes to be singled out among members of the MLO (Mildew Locus O) gene family. Candidates are MLO S-genes of phylogenetic clade V up-regulated upon PM inoculation, such as MdMLO11 and 19 (clade V) and MdMLO18 (clade VII). We report the knock-down through RNA interference of MdMLO11 and 19, as well as the complementation of resistance with MdMLO18 in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 reduced PM disease severity by 75%, whereas the knock-down of MdMLO11, alone or in combination with MdMLO19, did not result in any reduction or additional reduction of susceptibility compared with MdMLO19 alone. The test in A. thaliana excluded a role for MdMLO18 in PM susceptibility. Cell wall appositions (papillae) were present in both PM-resistant and PM-susceptible plants, but were larger in resistant lines. No obvious negative phenotype was observed in plants with mlo genes knocked down. Apparently, MdMLO19 plays the pivotal role in apple PM susceptibility and its knock-down induces a very significant level of resistance.

  15. Catalytic in vivo protein knockdown by small-molecule PROTACs

    PubMed Central

    Bondeson, Daniel P; Mares, Alina; Smith, Ian E D; Ko, Eunhwa; Campos, Sebastien; Miah, Afjal H; Mulholland, Katie E; Routly, Natasha; Buckley, Dennis L; Gustafson, Jeffrey L; Zinn, Nico; Grandi, Paola; Shimamura, Satoko; Bergamini, Giovanna; Faelth-Savitski, Maria; Bantscheff, Marcus; Cox, Carly; Gordon, Deborah A; Willard, Ryan R; Flanagan, John J; Casillas, Linda N; Votta, Bartholomew J; den Besten, Willem; Famm, Kristoffer; Kruidenier, Laurens; Carter, Paul S; Harling, John D; Churcher, Ian; Crews, Craig M

    2015-01-01

    The current predominant theapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target’s ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR. PMID:26075522

  16. Comparative phosphoproteomics of zebrafish Fyn/Yes morpholino knockdown embryos.

    PubMed

    Lemeer, Simone; Jopling, Chris; Gouw, Joost; Mohammed, Shabaz; Heck, Albert J R; Slijper, Monique; den Hertog, Jeroen

    2008-11-01

    The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

  17. The Effect of Msh2 Knockdown on Toxicity Induced by tert-Butyl-hydroperoxide, Potassium Bromate, and Hydrogen Peroxide in Base Excision Repair Proficient and Deficient Cells

    PubMed Central

    Cooley, N.; Elder, R. H.; Povey, A. C.

    2013-01-01

    The DNA mismatch repair (MMR) and base excision repair (BER) systems are important determinants of cellular toxicity following exposure to agents that cause oxidative DNA damage. To examine the interactions between these different repair systems, we examined whether toxicity, induced by t-BOOH and KBrO3, differs in BER proficient (Mpg+/+, Nth1+/+) and deficient (Mpg−/−, Nth1−/−) mouse embryonic fibroblasts (MEFs) following Msh2 knockdown of between 79 and 88% using an shRNA expression vector. Msh2 knockdown in Nth1+/+ cells had no effect on t-BOOH and KBrO3 induced toxicity as assessed by an MTT assay; knockdown in Nth1−/− cells resulted in increased resistance to t-BOOH and KBrO3, a result consistent with Nth1 removing oxidised pyrimidines. Msh2 knockdown in Mpg+/+ cells had no effect on t-BOOH toxicity but increased resistance to KBrO3; in Mpg−/− cells, Msh2 knockdown increased cellular sensitivity to KBrO3 but increased resistance to t-BOOH, suggesting a role for Mpg in removing DNA damage induced by these agents. MSH2 dependent and independent pathways then determine cellular toxicity induced by oxidising agents. A complex interaction between MMR and BER repair systems, that is, exposure dependent, also exists to determine cellular toxicity. PMID:23984319

  18. PTEN knockdown alters dendritic spine/protrusion morphology, not density

    PubMed Central

    Haws, Michael E.; Jaramillo, Thomas C.; Espinosa-Becerra, Felipe; Widman, Allie; Stuber, Garret D.; Sparta, Dennis R.; Tye, Kay M.; Russo, Scott J.; Parada, Luis F.; Kaplitt, Michael; Bonci, Antonello; Powell, Craig M.

    2014-01-01

    Mutations in phosphatase and tensin homolog deleted on chromosome ten (PTEN) are implicated in neuropsychiatric disorders including autism. Previous studies report that PTEN knockdown in neurons in vivo leads to increased spine density and synaptic activity. To better characterize synaptic changes in neurons lacking PTEN, we examined the effects of shRNA knockdown of PTEN in basolateral amygdala neurons on synaptic spine density and morphology using fluorescent dye confocal imaging. Contrary to previous studies in dentate gyrus, we find that knockdown of PTEN in basolateral amygdala leads to a significant decrease in total spine density in distal dendrites. Curiously, this decreased spine density is associated with increased miniature excitatory post-synaptic current frequency and amplitude, suggesting an increase in number and function of mature spines. These seemingly contradictory findings were reconciled by spine morphology analysis demonstrating increased mushroom spine density and size with correspondingly decreased thin protrusion density at more distal segments. The same analysis of PTEN conditional deletion in dentate gyrus demonstrated that loss of PTEN does not significantly alter total density of dendritic protrusions in the dentate gyrus, but does decrease thin protrusion density and increases density of more mature mushroom spines. These findings suggest that, contrary to previous reports, PTEN knockdown may not induce de novo spinogenesis, but instead may increase synaptic activity by inducing morphological and functional maturation of spines. Furthermore, behavioral analysis of basolateral amygdala PTEN knockdown suggests that these changes limited only to the basolateral amygdala complex may not be sufficient to induce increased anxiety-related behaviors. PMID:24264880

  19. AP endonuclease knockdown enhances methyl methanesulfonate hypersensitivity of DNA polymerase β knockout mouse embryonic fibroblasts.

    PubMed

    Yamamoto, Ryohei; Umetsu, Makio; Yamamoto, Mizuki; Matsuyama, Satoshi; Takenaka, Shigeo; Ide, Hiroshi; Kubo, Kihei

    2015-05-01

    Apurinic/apyrimidinic (AP) endonuclease (Apex) is required for base excision repair (BER), which is the major mechanism of repair for small DNA lesions such as alkylated bases. Apex incises the DNA strand at an AP site to leave 3'-OH and 5'-deoxyribose phosphate (5'-dRp) termini. DNA polymerase β (PolB) plays a dominant role in single nucleotide (Sn-) BER by incorporating a nucleotide and removing 5'-dRp. Methyl methanesulfonate (MMS)-induced damage is repaired by Sn-BER, and thus mouse embryonic fibroblasts (MEFs) deficient in PolB show significantly increased sensitivity to MMS. However, the survival curve for PolB-knockout MEFs (PolBKOs) has a shoulder, and increased sensitivity is only apparent at relatively high MMS concentrations. In this study, we prepared Apex-knockdown/PolB-knockout MEFs (AKDBKOs) to examine whether BER is related to the apparent resistance of PolBKOs at low MMS concentrations. The viability of PolBKOs immediately after MMS treatment was significantly lower than that of wild-type MEFs, but there was essentially no effect of Apex-knockdown on cell viability in the presence or absence of PolB. In contrast, relative counts of MEFs after repair were decreased by Apex knockdown. Parental PolBKOs showed especially high sensitivity at >1.5 mM MMS, suggesting that PolBKOs have another repair mechanism in addition to PolB-dependent Sn-BER, and that the back-up mechanism is unable to repair damage induced by high MMS concentrations. Interestingly, AKDBKOs were hypersensitive to MMS in a relative cell growth assay, suggesting that MMS-induced damage in PolB-knockout MEFs is repaired by Apex-dependent repair mechanisms, presumably including long-patch BER.

  20. Ribosomal Protein Gene Knockdown Causes Developmental Defects in Zebrafish

    PubMed Central

    Uechi, Tamayo; Nakajima, Yukari; Nakao, Akihiro; Torihara, Hidetsugu; Chakraborty, Anirban; Inoue, Kunio; Kenmochi, Naoya

    2006-01-01

    The ribosomal proteins (RPs) form the majority of cellular proteins and are mandatory for cellular growth. RP genes have been linked, either directly or indirectly, to various diseases in humans. Mutations in RP genes are also associated with tissue-specific phenotypes, suggesting a possible role in organ development during early embryogenesis. However, it is not yet known how mutations in a particular RP gene result in specific cellular changes, or how RP genes might contribute to human diseases. The development of animal models with defects in RP genes will be essential for studying these questions. In this study, we knocked down 21 RP genes in zebrafish by using morpholino antisense oligos to inhibit their translation. Of these 21, knockdown of 19 RPs resulted in the development of morphants with obvious deformities. Although mutations in RP genes, like other housekeeping genes, would be expected to result in nonspecific developmental defects with widespread phenotypes, we found that knockdown of some RP genes resulted in phenotypes specific to each gene, with varying degrees of abnormality in the brain, body trunk, eyes, and ears at about 25 hours post fertilization. We focused further on the organogenesis of the brain. Each knocked-down gene that affected the morphogenesis of the brain produced a different pattern of abnormality. Among the 7 RP genes whose knockdown produced severe brain phenotypes, 3 human orthologs are located within chromosomal regions that have been linked to brain-associated diseases, suggesting a possible involvement of RP genes in brain or neurological diseases. The RP gene knockdown system developed in this study could be a powerful tool for studying the roles of ribosomes in human diseases. PMID:17183665

  1. Fatty acids increase neuronal hypertrophy of Pten knockdown neurons

    PubMed Central

    Fricano, Catherine J.; DeSpenza, Tyrone; Frazel, Paul W.; Li, Meijie; O'Malley, A. James; Westbrook, Gary L.; Luikart, Bryan W.

    2014-01-01

    Phosphatase and tensin homolog (Pten) catalyzes the reverse reaction of PI3K by dephosphorylating PIP3 to PIP2. This negatively regulates downstream Akt/mTOR/S6 signaling resulting in decreased cellular growth and proliferation. Co-injection of a lentivirus knocking Pten down with a control lentivirus allows us to compare the effects of Pten knockdown between individual neurons within the same animal. We find that knockdown of Pten results in neuronal hypertrophy by 21 days post-injection. This neuronal hypertrophy is correlated with increased p-S6 and p-mTOR in individual neurons. We used this system to test whether an environmental factor that has been implicated in cellular hypertrophy could influence the severity of the Pten knockdown-induced hypertrophy. Implantation of mini-osmotic pumps delivering fatty acids results in increased neuronal hypertrophy and p-S6/p-mTOR staining. These hypertrophic effects were reversed in response to rapamycin treatment. However, we did not observe a similar increase in hypertrophy in response to dietary manipulations of fatty acids. Thus, we conclude that by driving growth signaling with fatty acids and knocking down a critical regulator of growth, Pten, we are able to observe an additive morphological phenotype of increased soma size mediated by the mTOR pathway. PMID:24795563

  2. MRP4 knockdown enhances migration, suppresses apoptosis, and produces aggregated morphology in human retinal vascular endothelial cells

    SciTech Connect

    Tagami, Mizuki; Kusuhara, Sentaro; Imai, Hisanori; Uemura, Akiyoshi; Honda, Shigeru; Tsukahara, Yasutomo; Negi, Akira

    2010-10-01

    Research highlights: {yields} Exogenous VEGF decreases MRP4 expression in a dose-dependent manner. {yields} MRP4 knockdown leads to enhanced cell migration. {yields} MRP4 knockdown suppresses caspase-3-mediated cell apoptosis. {yields} MRP4 knockdown produces cell assembly and cell aggregation. -- Abstract: The multidrug resistance protein (MRP) MRP4/ABCC4 is an ATP-binding cassette transporter that actively effluxes endogenous and xenobiotic substrates out of cells. In the rodent retina, Mrp4 mRNA and protein are exclusively expressed in vascular endothelial cells, but the angiogenic properties of Mrp4 are poorly understood so far. This study aims to explore the angiogenic properties of MRP4 in human retinal microvascular endothelial cells (HRECs) utilizing the RNA interference (RNAi) technique. MRP4 expression was decreased at the mRNA and protein levels after stimulation with exogenous vascular endothelial growth factor in a dose-dependent manner. RNAi-mediated MRP4 knockdown in HRECs do not affect cell proliferation but enhances cell migration. Moreover, cell apoptosis induced by serum starvation was less prominent in MRP4 siRNA-treated HRECs as compared to control siRNA-treated HRECs. In a Matrigel-based tube-formation assay, although MRP4 knockdown did not lead to a significant change in the total tube length, MRP4 siRNA-treated HRECs assembled and aggregated into a massive tube-like structure, which was not observed in control siRNA-treated HRECs. These results suggest that MRP4 is uniquely involved in retinal angiogenesis.

  3. In Vivo GFP Knockdown by Cationic Nanogel-siRNA Polyplexes

    PubMed Central

    Shrivats, Arun R.; Mishina, Yuji; Averick, Saadyah; Matyjaszewski, Krzysztof; Hollinger, Jeffrey O.

    2016-01-01

    RNA interference (RNAi) is a powerful tool to treat diseases and elucidate target gene function. Prior to clinical implementation, however, challenges including the safe, efficient and targeted delivery of siRNA must be addressed. Here, we report cationic nanogel nanostructured polymers (NSPs) prepared by atom transfer radical polymerization (ATRP) for in vitro and in vivo siRNA delivery in mammalian models. Outcomes from siRNA protection studies suggested that nanogel NSPs reduce enzymatic degradation of siRNA within polyplexes. Further, the methylation of siRNA may enhance nuclease resistance without compromising gene knockdown potency. NSP-mediated RNAi treatments against Gapdh significantly reduced GAPDH enzyme activity in mammalian cell culture models supplemented with 10% serum. Moreover, nanogel NSP-mediated siRNA delivery significantly inhibited in vivo GFP expression in a mouse model. GFP knockdown was siRNA sequence-dependent and facilitated by nanogel NSP carriers. Continued testing of NSP/siRNA compositions in disease models may produce important new therapeutic options for patient care. PMID:27280121

  4. siRNA Knock-Down of RANK Signaling to Control Osteoclast-Mediated Bone Resorption

    PubMed Central

    Wang, Yuwei; Grainger, David W.

    2010-01-01

    Purpose To demonstrate the ability of small interfering (si)RNA targeting the cell receptor, RANK, to control osteoclast function in cultures of both primary and secondary osteoclasts and their precursor cells. Methods siRNA targeting RANK was transfected into both RAW264.7 and primary bone marrow cell cultures. RANK knock-down by siRNA and functional inhibition were assessed in both mature osteoclast and their precursor cell cultures. RANK mRNA message and protein expression after the transfections were analyzed by PCR and Western blot, respectively. Off-target effects were assessed. The inhibition of osteoclast formation was evaluated using tartrate-resistant acid phosphatase (TRAP) assay, and subsequent bone resorption was determined by resorption pit assay. Results Both osteoclasts and osteoclast precursors can be targeted by siRNA in serum-containing media. Delivery of siRNA targeting RANK to both RAW 264.7 and primary bone marrow cell cultures produces short term repression of RANK expression without off-targeting effects, and significantly inhibits both osteoclast formation and bone resorption. Moreover, data support successful RANK knock-down by siRNA specifically in mature osteoclast cultures. Conclusions RANK is demonstrated to be an attractive target for siRNA control of osteoclast activity, with utility for development of new therapeutics for low bone mass pathologies or osteoporosis. PMID:20333451

  5. Manipulating the in vivo immune response by targeted gene knockdown

    PubMed Central

    Lieberman, Judy

    2015-01-01

    Aptamers, nucleic acids selected for high affinity binding to proteins, can be used to activate or antagonize immune mediators or receptors in a location and cell-type specific manner and to enhance antigen presentation. They can also be linked to other molecules (other aptamers, siRNAs or miRNAs, proteins, toxins) to produce multifunctional compounds for targeted immune modulation in vivo. Aptamer-siRNA chimeras (AsiCs) that induce efficient cell-specific knockdown in immune cells in vitro and in vivo can be used as an immunological research tool or potentially as an immunomodulating therapeutic. PMID:26149459

  6. Hepatic ATGL knockdown uncouples glucose intolerance from liver TAG accumulation.

    PubMed

    Ong, Kuok Teong; Mashek, Mara T; Bu, So Young; Mashek, Douglas G

    2013-01-01

    Adipose triglyceride lipase (ATGL) is the predominant triacylglycerol (TAG) hydrolase in mammals; however, the tissue-specific effects of ATGL outside of adipose tissue have not been well characterized. Hence, we tested the contribution of hepatic ATGL on mediating glucose tolerance and insulin action. Glucose or insulin tolerance tests and insulin signaling were performed in C57BL/6 mice administered control (nongene specific shRNA) or Atgl shRNA adenoviruses. Glucose and lipid metabolism assays were conducted in primary hepatocytes isolated from mice transduced with control or Atgl shRNA adenoviruses. Knocking down hepatic ATGL completely abrogated the increase in serum insulin following either 1 or 12 wk of feeding a high-fat (HF) diet despite higher hepatic TAG content. Glucose tolerance tests demonstrated that ATGL knockdown normalized glucose tolerance in HF-diet-fed mice. The observed improvements in glucose tolerance were present despite unaltered hepatic insulin signaling and increased liver TAG. Mice with suppressed hepatic ATGL had reduced hepatic glucose production in vivo, and hepatocytes isolated from Atgl shRNA-treated mice displayed a 26% decrease in glucose production and a 38% increase in glucose oxidation compared to control cells. Taken together, these data suggest that hepatic ATGL knockdown enhances glucose tolerance by increasing hepatic glucose utilization and uncouples impairments in insulin action from hepatic TAG accumulation.

  7. A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

    PubMed Central

    Huang, Liwei; Xiao, An; Wecker, Andrea; McBride, Daniel A.; Choi, Soo Young; Zhou, Weibin; Lipschutz, Joshua H.

    2015-01-01

    Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to “off-target” effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney. PMID:25489842

  8. A possible zebrafish model of polycystic kidney disease: knockdown of wnt5a causes cysts in zebrafish kidneys.

    PubMed

    Huang, Liwei; Xiao, An; Wecker, Andrea; McBride, Daniel A; Choi, Soo Young; Zhou, Weibin; Lipschutz, Joshua H

    2014-12-02

    Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to "off-target" effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

  9. ENHANCING ADULT NERVE REGENERATION THROUGH THE KNOCKDOWN OF RETINOBLASTOMA PROTEIN

    PubMed Central

    Christie, Kimberly J.; Krishnan, Anand; Martinez, Jose A.; Purdy, Kaylynn; Singh, Bhagat; Eaton, Shane; Zochodne, Douglas

    2016-01-01

    Tumour suppressor pathways may offer novel targets capable of altering the plasticity of post-mitotic adult neurons. Here we describe a role for retinoblastoma (Rb) protein, widely expressed in adult sensory neurons and their axons, during regeneration. In adult sensory neurons, Rb siRNA knockdown or Rb1 deletion in vitro enhances neurite outgrowth and branching. Plasticity is achieved in part through upregulation of neuronal PPARγ; its antagonism inhibits Rb siRNA plasticity whereas a PPARγ agonist increases growth. In an in vivo regenerative paradigm following complete peripheral nerve trunk transection, direct delivery of Rb siRNA prompts increased outgrowth of axons from proximal stumps and entrains Schwann cells to accompany them for greater distances. Similarly Rb siRNA delivery following a nerve crush improves behavioural indices of motor and sensory recovery in mice. The overall findings indicate that inhibition of tumour suppressor molecules has a role to play in promoting adult neuron regeneration. PMID:24752312

  10. Knockdown of neuropeptide Y in the dorsomedial hypothalamus reverses high-fat diet-induced obesity and impaired glucose tolerance in rats.

    PubMed

    Kim, Yonwook J; Bi, Sheng

    2016-01-15

    Neuropeptide Y (NPY) in the dorsomedial hypothalamus (DMH) plays an important role in the regulation of energy balance. While DMH NPY overexpression causes hyperphagia and obesity in rats, knockdown of NPY in the DMH via adeno-associated virus (AAV)-mediated RNAi (AAVshNPY) ameliorates these alterations. Whether this knockdown has a therapeutic effect on obesity and glycemic disorder has yet to be determined. The present study sought to test this potential using a rat model of high-fat diet (HFD)-induced obesity and insulin resistance, mimicking human obesity with impaired glucose homeostasis. Rats had ad libitum access to rodent regular chow (RC) or HFD. Six weeks later, an oral glucose tolerance test (OGTT) was performed for verifying HFD-induced glucose intolerance. After verification, obese rats received bilateral DMH injections of AAVshNPY or the control vector AAVshCTL, and OGTT and insulin tolerance test (ITT) were performed at 16 and 18 wk after viral injection (23 and 25 wk on HFD), respectively. Rats were killed at 26 wk on HFD. We found that AAVshCTL rats on HFD remained hyperphagic, obese, glucose intolerant, and insulin resistant relative to lean control RC-fed rats receiving DMH injection of AAVshCTL, whereas these alterations were reversed in NPY knockdown rats fed a HFD. NPY knockdown rats exhibited normal food intake, body weight, glucose tolerance, and insulin sensitivity, as seen in lean control rats. Together, these results demonstrate a therapeutic action of DMH NPY knockdown against obesity and impaired glucose homeostasis in rats, providing a potential target for the treatment of obesity and diabetes.

  11. Focal Scn1a knockdown induces cognitive impairment without seizures.

    PubMed

    Bender, Alex C; Natola, Heather; Ndong, Christian; Holmes, Gregory L; Scott, Rod C; Lenck-Santini, Pierre-Pascal

    2013-06-01

    Cognitive impairment is a common comorbidity in pediatric epilepsy that can severely affect quality of life. In many cases, antiepileptic treatments fail to improve cognition. Therefore, a fundamental question is whether underlying brain abnormalities may contribute to cognitive impairment through mechanisms independent of seizures. Here, we examined the possible effects on cognition of Nav1.1 down-regulation, a sodium channel principally involved in Dravet syndrome but also implicated in other cognitive disorders, including autism and Alzheimer's disease. Using an siRNA approach to knockdown Nav1.1 selectively in the basal forebrain region, we were able to target a learning and memory network while avoiding the generation of spontaneous seizures. We show that reduction of Nav1.1 expression in the medial septum and diagonal band of Broca leads to a dysregulation of hippocampal oscillations in association with a spatial memory deficit. We propose that the underlying etiology responsible for Dravet syndrome may directly contribute to cognitive impairment in a manner that is independent from seizures.

  12. RNAi knockdown of parafusin inhibits the secretory pathway.

    PubMed

    Liu, Li; Wyroba, Elzbieta; Satir, Birgit H

    2011-10-01

    Several glycolytic enzymes and their isoforms have been found to be important in cell signaling unrelated to glycolysis. The involvement of parafusin (PFUS), a member of the phosphoglucomutase (PGM) superfamily with no phosphoglucomutase activity, in Ca(2+)-dependent exocytosis has been controversial. This protein was first described in Paramecium tetraurelia, but is widely found. Earlier work showed that parafusin is a secretory vesicle scaffold component with unusual post-translational modifications (cyclic phosphorylation and phosphoglucosylation) coupled to stages in the exocytic process. Using RNAi, we demonstrate that parafusin synthesis can be reversibly blocked, with minor or no effect on other PGM isoforms. PFUS knockdown produces an inhibition of dense core secretory vesicle (DCSV) synthesis leading to an exo(-) phenotype. Although cell growth is unaffected, vesicle content is not packaged properly and no new DCSVs are formed. We conclude that PFUS and its orthologs are necessary for proper scaffold maturation. Because of this association, parafusin is an important signaling component for regulatory control of the secretory pathway.

  13. Nanolayered siRNA dressing for sustained localized knockdown.

    PubMed

    Castleberry, Steven; Wang, Mary; Hammond, Paula T

    2013-06-25

    The success of RNA interference (RNAi) in medicine relies on the development of technology capable of successfully delivering it to tissues of interest. Significant research has focused on the difficult task of systemic delivery of RNAi; however its local delivery could be a more easily realized approach. Localized delivery is of particular interest for many medical applications, including the treatment of localized diseases, the modulation of cellular response to implants or tissue engineering constructs, and the management of wound healing and regenerative medicine. In this work we present an ultrathin electrostatically assembled coating for localized and sustained delivery of short interfering RNA (siRNA). This film was applied to a commercially available woven nylon dressing commonly used for surgical applications and was demonstrated to sustain significant knockdown of protein expression in multiple cell types for more than one week in vitro. Significantly, this coating can be easily applied to a medically relevant device and requires no externally delivered transfection agents for effective delivery of siRNA. These results present promising opportunities for the localized administration of RNAi. PMID:23672676

  14. Specific in vivo knockdown of protein function by intrabodies

    PubMed Central

    Marschall, Andrea LJ; Dübel, Stefan; Böldicke, Thomas

    2015-01-01

    Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e., their antigens). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, miRNA and small molecule inhibitors, by-passing various limitations and disadvantages of these methods. The advantages of intrabodies include very high specificity for the target, the possibility to knock down several protein isoforms by one intrabody and targeting of specific splice variants or even post-translational modifications. Different types of intrabodies must be designed to target proteins at different locations, typically either in the cytoplasm, in the nucleus or in the endoplasmic reticulum (ER). Most straightforward is the use of intrabodies retained in the ER (ER intrabodies) to knock down the function of proteins passing the ER, which disturbs the function of members of the membrane or plasma proteomes. More effort is needed to functionally knock down cytoplasmic or nuclear proteins because in this case antibodies need to provide an inhibitory effect and must be able to fold in the reducing milieu of the cytoplasm. In this review, we present a broad overview of intrabody technology, as well as applications both of ER and cytoplasmic intrabodies, which have yielded valuable insights in the biology of many targets relevant for drug development, including α-synuclein, TAU, BCR-ABL, ErbB-2, EGFR, HIV gp120, CCR5, IL-2, IL-6, β-amyloid protein and p75NTR. Strategies for the generation of intrabodies and various designs of their applications are also reviewed. PMID:26252565

  15. Knockdown of phosphoethanolamine transmethylation enzymes decreases viability of Haemonchus contortus.

    PubMed

    Witola, William H; Cooks-Fagbodun, Sheritta; Ordonez, Adriana Reyes; Matthews, Kwame; Abugri, Daniel A; McHugh, Mark

    2016-06-15

    The phosphobase methylation pathway, in which phosphoethanolamine N-methyltransferases (PMTs) successively catalyze the methylation of phosphoethanolamine to phosphocholine, is essential in the free-living nematode Caenorhabditis elegans. Two PMT-encoding genes (HcPMT1 and HcPMT2) cloned from Haemonchus contortus have been shown, by in vitro assays, to possess enzymatic characteristics similar to those of C. elegans PMTs, but their physiological significance in H. contortus is yet to be elucidated. Therefore, in this study, we endeavored to determine the importance of HcPMT1 and HcPMT2 in the survival of H. contortus by adapting the use of phosphorodiamidate morpholino oligomers (PPMO) antisense approach to block the translation of HcPMT1 and HcPMT2 in the worms. We found that PPMOs targeting HcPMT1 and HcPMT2 down-regulated the expression of HcPMT1 and HcPMT2 proteins in adult H. contortus. Analysis of the effect of HcPMT1 and HcPMT2 knockdown showed that it significantly decreased worm motility and viability, thus validating HcPMT1 and HcPMT2 as essential enzymes for survival of H. contortus. Studies of gene function in H. contortus have been constrained by limited forward and reverse genetic technologies for use in H. contortus. Thus, our success in adaptation of use of PPMO antisense approach in H. contortus provides an important reverse genetic technological advance for studying this parasitic nematode of veterinary significance. PMID:27198768

  16. Phosphoribosylpyrophosphate Synthetase 1 Knockdown Suppresses Tumor Formation of Glioma CD133+ Cells Through Upregulating Cell Apoptosis.

    PubMed

    Li, Chen; Yan, Zhongjie; Cao, Xuhua; Zhang, Xiaowei; Yang, Liang

    2016-10-01

    Relapse is the main cause of mortality in patients with glioblastoma multiforme (GBM). Treatment options involve surgical resection followed by a combination of radiotheraphy and chemotherapy with temozolomide. Several genes and genetic pathways have been identified to contribute to therapeutic resistance, giving rise to recurrence of the malignancy. In the last decades, glioma stem cells (GSCs) with the capacity of self-renewal have been demonstrated to maintain tumor propagation and treatment resistance. Here, we isolated CD133-positive (CD133+) and CD133-negative (CD133-) cells from glioblastoma U98G and U87MG cell lines. The role of phosphoribosylpyrophosphate synthetase 1 (PRPS1), which catalyzes the first step of the synthesis of nucleotide, in proliferation and apoptosis was investigated. We found that PRPS1 had a remarkable effect on cell proliferation and sphere formation in both CD133+ and CD133- cells. Compared to CD133- cells, CD133+ cells exhibited more significant results in cell apoptosis assay. CD133+ T98G and U87MG cells were used in xenograft mouse model of tumor formation. Interestingly, the mice implanted with PRPS1 knockdown T98G or U87MG stem cells exhibited prolonged survival time and reduced tumor volume. By immunostaining caspase-3 in tumor tissues of these mice, we demonstrated that the apoptotic activities in tumor cells were positively correlated to the survival time but negatively correlated to PRPS1 expression. Our results indicate that PRPS1 plays an important role in proliferation and apoptosis in GSCs and provide new clues for potential PRPS1-targeted therapy in GBM treatment. PMID:27343059

  17. Vitellogenin knockdown strongly affects cotton boll weevil egg viability but not the number of eggs laid by females.

    PubMed

    Coelho, Roberta R; de Souza Júnior, José Dijair Antonino; Firmino, Alexandre A P; de Macedo, Leonardo L P; Fonseca, Fernando C A; Terra, Walter R; Engler, Gilbert; de Almeida Engler, Janice; da Silva, Maria Cristina M; Grossi-de-Sa, Maria Fatima

    2016-09-01

    Vitellogenin (Vg), a yolk protein precursor, is the primary egg nutrient source involved in insect reproduction and embryo development. The Cotton Boll weevil (CBW) Anthonomus grandis Boheman, the most important cotton pest in Americas, accumulates large amounts of Vg during reproduction. However, the precise role of this protein during embryo development in this insect remains unknown. Herein, we investigated the effects of vitellogenin (AgraVg) knockdown on the egg-laying and egg viability in A. grandis females, and also characterized morphologically the unviable eggs. AgraVg transcripts were found during all developmental stages of A. grandis, with highest abundance in females. Silencing of AgraVg culminated in a significant reduction in transcript amount, around 90%. Despite this transcriptional reduction, egg-laying was not affected in dsRNA-treated females but almost 100% of the eggs lost their viability. Eggs from dsRNA-treated females showed aberrant embryos phenotype suggesting interference at different stages of embryonic development. Unlike for other insects, the AgraVg knockdown did not affect the egg-laying ability of A. grandis, but hampered A. grandis reproduction by perturbing embryo development. We concluded that the Vg protein is essential for A. grandis reproduction and a good candidate to bio-engineer the resistance against this devastating cotton pest. PMID:27419079

  18. Vitellogenin knockdown strongly affects cotton boll weevil egg viability but not the number of eggs laid by females.

    PubMed

    Coelho, Roberta R; de Souza Júnior, José Dijair Antonino; Firmino, Alexandre A P; de Macedo, Leonardo L P; Fonseca, Fernando C A; Terra, Walter R; Engler, Gilbert; de Almeida Engler, Janice; da Silva, Maria Cristina M; Grossi-de-Sa, Maria Fatima

    2016-09-01

    Vitellogenin (Vg), a yolk protein precursor, is the primary egg nutrient source involved in insect reproduction and embryo development. The Cotton Boll weevil (CBW) Anthonomus grandis Boheman, the most important cotton pest in Americas, accumulates large amounts of Vg during reproduction. However, the precise role of this protein during embryo development in this insect remains unknown. Herein, we investigated the effects of vitellogenin (AgraVg) knockdown on the egg-laying and egg viability in A. grandis females, and also characterized morphologically the unviable eggs. AgraVg transcripts were found during all developmental stages of A. grandis, with highest abundance in females. Silencing of AgraVg culminated in a significant reduction in transcript amount, around 90%. Despite this transcriptional reduction, egg-laying was not affected in dsRNA-treated females but almost 100% of the eggs lost their viability. Eggs from dsRNA-treated females showed aberrant embryos phenotype suggesting interference at different stages of embryonic development. Unlike for other insects, the AgraVg knockdown did not affect the egg-laying ability of A. grandis, but hampered A. grandis reproduction by perturbing embryo development. We concluded that the Vg protein is essential for A. grandis reproduction and a good candidate to bio-engineer the resistance against this devastating cotton pest.

  19. Knockdown of dual specificity phosphatase 4 enhances the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin

    SciTech Connect

    Liu, Yu; Du, Feiya; Chen, Wei; Yao, Minya; Lv, Kezhen; Fu, Peifen

    2013-12-10

    Background: Breast cancer is the major cause of cancer-related deaths in females world-wide. Doxorubicin-based therapy has limited efficacy in breast cancer due to drug resistance, which has been shown to be associated with the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms linking the EMT and drug resistance in breast cancer cells remain unclear. Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is associated with cellular proliferation and differentiation; however, its role in breast cancer progression is controversial. Methods: We used cell viability assays, Western blotting and immunofluorescent staining, combined with siRNA interference, to evaluate chemoresistance and the EMT in MCF-7 and adriamycin-resistant MCF-7/ADR breast cancer cells, and investigate the underlying mechanisms. Results: Knockdown of DUSP4 significantly increased the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin, and MCF-7/ADR cells which expressed high levels of DUSP4 had a mesenchymal phenotype. Furthermore, knockdown of DUSP4 reversed the EMT in MCF-7/ADR cells, as demonstrated by upregulation of epithelial biomarkers and downregulation of mesenchymal biomarkers, and also increased the chemosensitivity of MCF-7/ADR cells to doxorubicin. Conclusions: DUSP4 might represent a potential drug target for inhibiting drug resistance and regulating the process of the EMT during the treatment of breast cancer. - Highlights: • We used different technologies to prove our conclusion. • DUSP4 knockdown increased doxorubicin chemosensitivity in breast cancer cells. • DUSP4 is a potential target for combating drug resistance in breast cancer. • DUSP4 is a potential target for regulating the EMT in breast cancer.

  20. Knockdown of actin and caspase gene expression by RNA interference in the symbiotic anemone Aiptasia pallida.

    PubMed

    Dunn, Simon R; Phillips, Wendy S; Green, Douglas R; Weis, Virginia M

    2007-06-01

    Since the discovery of the ancient eukaryotic process of RNA-mediated gene silencing, the reverse-genetics technique RNA interference (RNAi) has increasingly been used to examine gene function in vertebrate and invertebrate systems. In this study, we report on the use of RNAi, adapted from studies on animal model systems, to manipulate gene expression in a symbiotic marine cnidarian. We describe gene knockdown of actin and of acasp--a cysteine protease, or caspase--in the symbiotic sea anemone Aiptasia pallida. Knockdown was assessed qualitatively with in situ hybridizations for both genes. Quantitative PCR and caspase activity assays were used as a quantitative measure of knockdown for acasp. PMID:17565114

  1. Knock-down of Kaiso induces proliferation and blocks granulocytic differentiation in blast crisis of chronic myeloid leukemia

    PubMed Central

    2012-01-01

    Background Kaiso protein has been identified as a new member of the POZ-ZF subfamily of transcription factors that are involved in development and cancer. There is consistent evidence of the role of Kaiso and its involvement in human tumorigenesis but there is no evidence about its role in hematopoietic differentiation or establishment of chronic myeloid leukemia (CML). We used, normal K562 cell line, established from a CML patient in blast crisis, and imatinib-resistant K562 cell line, to investigate the specific distribution of Kaiso and their contribution to the cell differentiation status of the blast crisis of CML (CML-BP). Results We found cytoplasmic expression of Kaiso, in K562 cells and patients, confirmed by immunofluorescence, immunohistochemistry and western blot of cytoplasmic protein fraction. Kaiso was weakly expressed in the imatinib-resistant K562 cell line confirmed by immunofluorescence and western blot. The cytoplasmic expression of Kaiso was not modified when the K562 cells were treated for 16 h with imatinib 0.1 and 1 μM. In our study, small interfering RNA (siRNA) was introduced to down regulate the expression of Kaiso and p120ctn in K562 cell line. Kaiso and p120ctn were down regulated individually (siRNA-Kaiso or siRNA-p120ctn) or in combination using a simultaneous co-transfection (siRNA-Kaiso/p120ctn). We next investigated whether knockdown either Kaiso or p120ctn alone or in combination affects the cell differentiation status in K562 cells. After down regulation we analyzed the expression of hematopoietic cell differentiation and proliferation genes: SCF, PU-1, c-MyB, C/EBPα, Gata-2 and maturation markers of hematopoietic cells expressed in the plasma membrane: CD15, CD11b, CD33, CD117. The levels of SCF and c-MyB were increased by 1000% and 65% respectively and PU-1, Gata-2 and C/EBPα were decreased by 66%, 50% and 80% respectively, when Kaiso levels were down regulated by siRNA. The results were similar when both Kaiso and p120

  2. RNAi-mediated gene knockdown and in vivo diuresis assay in adult female Aedes aegypti mosquitoes.

    PubMed

    Drake, Lisa L; Price, David P; Aguirre, Sarah E; Hansen, Immo A

    2012-07-14

    This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis. The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

  3. Agitation during lipoplex formation improves the gene knockdown effect of siRNA.

    PubMed

    Barichello, Jose Mario; Kizuki, Shinji; Tagami, Tatsuaki; Asai, Tomohiro; Ishida, Tatsuhiro; Kikuchi, Hiroshi; Oku, Naoto; Kiwada, Hiroshi

    2011-05-30

    The successful delivery of therapeutic siRNA to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful RNAi therapy. In the present study, we focused on the siRNA-lipoplex preparation procedure and its effect on the gene-knockdown efficiency of siRNA in vitro. Agitation (vortex-mixing) during siRNA-lipoplex (vor-LTsiR) preparation and its effect on the gene-knockdown efficiency of stably expressed cell GFP was investigated, and their efficiency was compared with that of spontaneously formed lipoplex (spo-LTsiR). A dramatic difference in size between lipoplexes was observed at the N/P ratio of 7.62 (siRNA dose of 30 nM), even though both lipoplexes were positively charged. With the siRNA dose of 30 nM, vor-LTsiR accomplished a 50% gene-knockdown, while spo-LTsiR managed a similar knockdown effect at the 120 nM level, suggesting that the preparation procedure remarkably affects the gene-knockdown efficacy of siRNA. The uptake of vor-LTsiR was mainly via clathrin-mediated endocytosis, whereas that of spo-LTsiR was via membrane fusion. In addition, by inhibiting clathrin-mediated endocytosis, the gene-knockdown efficiency was significantly lowered. The size of the lipoplex, promoted by the preparation procedure, is likely to define the entry pathway, resulting in an increased amount of siRNA internalized in cells and an enhanced gene-knockdown efficacy. The results of the present study definitively show that a proper siRNA-lipoplex preparation procedure makes a significant contribution to the efficiency of cellular uptake, and thereby, to the gene-knockdown efficiency of siRNA. PMID:21392562

  4. Lentivirus-mediated Knockdown of HDAC1 Uncovers Its Role in Esophageal Cancer Metastasis and Chemosensitivity

    PubMed Central

    Song, Min; He, Gang; Wang, Yan; Pang, Xueli; Zhang, Bo

    2016-01-01

    Histone deacetylationase 1 (HDAC1) is ubiquitously expressed in various cell lines and tissues and play an important role of regulation gene expression. Overexpression of HDAC1 has been observed in various types of cancers, which indicated that it might be a target for cancer therapy. To test HDAC1 inhibition for cancer treatment, the gene expression of HDAC1 was knockdown mediated by a lentivirus system. Our data showed the gene expression of HDAC1 could be efficiently knockdown by RNAi mediated by lentivirus in esophageal carcinoma EC109 cells. Knockdown of HDAC1 led to significant decrease of cell growth and altered cell cycle distribution. The result of transwell assay showed that the numbers of cells travelled through the micropore membrane was significantly decreased as HDAC1 expression was knockdown. Moreover, HDAC1 knockdown inhibited the migration of EC109 cells as determining by scratch test. Additionally, enhancement of cisplatin-stimulated apoptosis was detected by HDAC1 knockdown. Our data suggested inhibition of HDAC1 expression by lentivirus mediated shRNA might be further applied for esophageal cancer chemotherapy. PMID:27698906

  5. Lentivirus-mediated Knockdown of HDAC1 Uncovers Its Role in Esophageal Cancer Metastasis and Chemosensitivity

    PubMed Central

    Song, Min; He, Gang; Wang, Yan; Pang, Xueli; Zhang, Bo

    2016-01-01

    Histone deacetylationase 1 (HDAC1) is ubiquitously expressed in various cell lines and tissues and play an important role of regulation gene expression. Overexpression of HDAC1 has been observed in various types of cancers, which indicated that it might be a target for cancer therapy. To test HDAC1 inhibition for cancer treatment, the gene expression of HDAC1 was knockdown mediated by a lentivirus system. Our data showed the gene expression of HDAC1 could be efficiently knockdown by RNAi mediated by lentivirus in esophageal carcinoma EC109 cells. Knockdown of HDAC1 led to significant decrease of cell growth and altered cell cycle distribution. The result of transwell assay showed that the numbers of cells travelled through the micropore membrane was significantly decreased as HDAC1 expression was knockdown. Moreover, HDAC1 knockdown inhibited the migration of EC109 cells as determining by scratch test. Additionally, enhancement of cisplatin-stimulated apoptosis was detected by HDAC1 knockdown. Our data suggested inhibition of HDAC1 expression by lentivirus mediated shRNA might be further applied for esophageal cancer chemotherapy.

  6. Knockdown of TWIST1 enhances arsenic trioxide- and ionizing radiation-induced cell death in lung cancer cells by promoting mitochondrial dysfunction

    SciTech Connect

    Seo, Sung-Keum; Kim, Jae-Hee; Choi, Ha-Na; Choe, Tae-Boo; Hong, Seok-Il; Yi, Jae-Youn; Hwang, Sang-Gu; Lee, Hyun-Gyu; Lee, Yun-Han; Park, In-Chul

    2014-07-11

    Highlights: • Knockdown of TWIST1 enhanced ATO- and IR-induced cell death in NSCLCs. • Intracellular ROS levels were increased in cells treated with TWIST1 siRNA. • TWIST1 siRNA induced MMP loss and mitochondrial fragmentation. • TWIST1 siRNA upregulated the fission-related proteins FIS1 and DRP1. - Abstract: TWIST1 is implicated in the process of epithelial mesenchymal transition, metastasis, stemness, and drug resistance in cancer cells, and therefore is a potential target for cancer therapy. In the present study, we found that knockdown of TWIST1 by small interfering RNA (siRNA) enhanced arsenic trioxide (ATO)- and ionizing radiation (IR)-induced cell death in non-small-cell lung cancer cells. Interestingly, intracellular reactive oxygen species levels were increased in cells treated with TWIST1 siRNA and further increased by co-treatment with ATO or IR. Pretreatment of lung cancer cells with the antioxidant N-acetyl-cysteine markedly suppressed the cell death induced by combined treatment with TWIST1 siRNA and ATO or IR. Moreover, treatment of cells with TWIST1 siRNA induced mitochondrial membrane depolarization and significantly increased mitochondrial fragmentation (fission) and upregulated the fission-related proteins FIS1 and DRP1. Collectively, our results demonstrate that siRNA-mediated TWIST1 knockdown induces mitochondrial dysfunction and enhances IR- and ATO-induced cell death in lung cancer cells.

  7. FRZB knockdown upregulates β-catenin activity and enhances cell aggressiveness in gastric cancer.

    PubMed

    Qin, Shuai; Zhang, Zhuo; Li, Jianfang; Zang, Lu

    2014-05-01

    Studies have shown that FRZB correlates with gastric tumorigenicity and may play role in regulating the Wnt/β‑catenin signaling pathway. In the present study, we investigated the correlation between FRZB and the Wnt/β‑catenin signaling pathway using gastric cancer tissues and an FRZB‑knockdown gastric cancer cell line model. The protein levels of FRZB and β‑catenin were examined using immunohistochemical staining. FRZB-specific shRNAs were used to generate FRZB‑knockdown MKN45 gastric cancer cells. Cell proliferation assay, suspending culture and Annexin V/PI double staining analysis were used to investigate the role of FRZB knockdown in cell growth. In vitro migration/invasion assays were performed. The expression of Wnt/β‑catenin downstream targets was analyzed by RT-PCR. FRZB mRNA levels showed negative correlation with β‑catenin levels in paired non-tumor and tumor tissues. FRZB protein levels were negatively correlated with β‑catenin levels analyzed by IHC staining. Furthermore, high FRZB protein levels were correlated with membrane localization of β‑catenin. FRZB knockdown increased gastric cancer cell growth in monolayer and soft agar culture; it increased cell aggregates in suspending culture and rendered less apoptosis which indicated increased anti-anoikis growth. FRZB knockdown increased cell migration and invasion and increased the expression of Wnt/β‑catenin downstream targets such as MMP7 and cyclin D1. Our studies revealed that FRZB levels were correlated with β‑catenin subcellular localization. Knockdown of FRZB in gastric cancer cells increased cell growth and migration/invasion which was also accompanied by activation of Wnt/β‑catenin downstream targets. FRZB knockdown may upregulate the Wnt/β‑catenin pathway and promote aggressiveness in gastric cancer. PMID:24676361

  8. ZEB1 knockdown mediated using polypeptide cationic micelles inhibits metastasis and effects sensitization to a chemotherapeutic drug for cancer therapy

    NASA Astrophysics Data System (ADS)

    Fang, Shengtao; Wu, Lei; Li, Mingxing; Yi, Huqiang; Gao, Guanhui; Sheng, Zonghai; Gong, Ping; Ma, Yifan; Cai, Lintao

    2014-08-01

    Metastasis and drug resistance are the main causes for the failure in clinical cancer therapy. Emerging evidence suggests an intricate role of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) in metastasis and drug resistance. The EMT-activator ZEB1 is crucial in malignant tumor progression by linking EMT-activation and stemness-maintenance. Here, we used multifunctional polypeptide micelle nanoparticles (NP) as nanocarriers for the delivery of ZEB1 siRNA and doxorubicin (DOX). The nanocarriers could effectively deliver siRNA to the cytoplasm and knockdown the target gene in H460 cells and H460 xenograft tumors, leading to reduced EMT and repressed CSC properties in vitro and in vivo. The complex micelle nanoparticles with ZEB1 siRNA (siRNA-NP) significantly reduced metastasis in the lung. When DOX and siRNA were co-delivered by the nanocarriers (siRNA-DOX-NP), a synergistic therapeutic effect was observed, resulting in dramatic inhibition of tumor growth in a H460 xenograft model. These results demonstrated that the siRNA-NP or siRNA-DOX-NP complex targeting ZEB1 could be developed into a new therapeutic approach for non-small cell lung cancer (NSCLC) treatment.Metastasis and drug resistance are the main causes for the failure in clinical cancer therapy. Emerging evidence suggests an intricate role of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) in metastasis and drug resistance. The EMT-activator ZEB1 is crucial in malignant tumor progression by linking EMT-activation and stemness-maintenance. Here, we used multifunctional polypeptide micelle nanoparticles (NP) as nanocarriers for the delivery of ZEB1 siRNA and doxorubicin (DOX). The nanocarriers could effectively deliver siRNA to the cytoplasm and knockdown the target gene in H460 cells and H460 xenograft tumors, leading to reduced EMT and repressed CSC properties in vitro and in vivo. The complex micelle nanoparticles with ZEB1 siRNA (siRNA-NP) significantly reduced

  9. Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules

    PubMed Central

    Lopes, Mark William; Sapio, Matthew R.; Leal, Rodrigo B.; Fricker, Lloyd D.

    2016-01-01

    Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency. PMID:27050163

  10. Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

    SciTech Connect

    Huang, Sujun; Wu, Binwen; Li, Dongfeng; Zhou, Weihong; Deng, Gang; Zhang, Kaijun; Li, Youjia

    2014-02-14

    Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2{sup ∗}, -193b and -193a, and inversely inhibit miR-31 and -9{sup ∗}. Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC.

  11. Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules.

    PubMed

    Lopes, Mark William; Sapio, Matthew R; Leal, Rodrigo B; Fricker, Lloyd D

    2016-01-01

    Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency. PMID:27050163

  12. Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis

    PubMed Central

    Miyoshi, N; Wittner, B S; Shioda, K; Hitora, T; Ito, T; Ramaswamy, S; Isselbacher, K J; Sgroi, D C; Shioda, T

    2015-01-01

    Although RNA interference (RNAi) knockdown screening of cancer cell cultures is an effective approach to predict drug targets or therapeutic/prognostic biomarkers, interactions among identified targets often remain obscure. Here, we introduce the nodes-and-connections RNAi knockdown screening that generates a map of target interactions through systematic iterations of in silico prediction of targets and their experimental validation. An initial RNAi knockdown screening of MCF-7 human breast cancer cells targeting 6560 proteins identified four signaling molecules required for their fulvestrant-induced apoptosis. Signaling molecules physically or functionally interacting with these four primary node targets were computationally predicted and experimentally validated, resulting in identification of four second-generation nodes. Three rounds of further iterations of the prediction–validation cycle generated third, fourth and fifth generation of nodes, completing a 19-node interaction map that contained three predicted nodes but without experimental validation because of technical limitations. The interaction map involved all three members of the death-associated protein kinases (DAPKs) as well as their upstream and downstream signaling molecules (calmodulins and myosin light chain kinases), suggesting that DAPKs play critical roles in the cytocidal action of fulvestrant. The in silico Kaplan–Meier analysis of previously reported human breast cancer cohorts demonstrated significant prognostic predictive power for five of the experimentally validated nodes and for three of the prediction-only nodes. Immunohistochemical studies on the expression of 10 nodal proteins in human breast cancer tissues not only supported their prognostic prediction power but also provided statistically significant evidence of their synchronized expression, implying functional interactions among these nodal proteins. Thus, the Nodes-and-Connections approach to RNAi knockdown screening yields

  13. Knockdown of p53 suppresses Nanog expression in embryonic stem cells

    SciTech Connect

    Abdelalim, Essam Mohamed; Tooyama, Ikuo

    2014-01-10

    Highlights: •We investigate the role of p53 in ESCs in the absence of DNA damage. •p53 knockdown suppresses ESC proliferation. •p53 knockdown downregulates Nanog expression. •p53 is essential for mouse ESC self-renewal. -- Abstract: Mouse embryonic stem cells (ESCs) express high levels of cytoplasmic p53. Exposure of mouse ESCs to DNA damage leads to activation of p53, inducing Nanog suppression. In contrast to earlier studies, we recently reported that chemical inhibition of p53 suppresses ESC proliferation. Here, we confirm that p53 signaling is involved in the maintenance of mouse ESC self-renewal. RNA interference-mediated knockdown of p53 induced downregulation of p21 and defects in ESC proliferation. Furthermore, p53 knockdown resulted in a significant downregulation in Nanog expression at 24 and 48 h post-transfection. p53 knockdown also caused a reduction in Oct4 expression at 48 h post-transfection. Conversely, exposure of ESCs to DNA damage caused a higher reduction of Nanog expression in control siRNA-treated cells than in p53 siRNA-treated cells. These data show that in the absence of DNA damage, p53 is required for the maintenance of mouse ESC self-renewal by regulating Nanog expression.

  14. Mitochondria-Targeted Antioxidant Prevents Cardiac Dysfunction Induced by Tafazzin Gene Knockdown in Cardiac Myocytes

    PubMed Central

    He, Quan; Harris, Nicole; Ren, Jun; Han, Xianlin

    2014-01-01

    Tafazzin, a mitochondrial acyltransferase, plays an important role in cardiolipin side chain remodeling. Previous studies have shown that dysfunction of tafazzin reduces cardiolipin content, impairs mitochondrial function, and causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS) have been implicated in the development of cardiomyopathy and are also the obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown increases ROS production from mitochondria, and a mitochondria-targeted antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac dysfunction. We employed cardiac myocytes transduced with an adenovirus containing tafazzin shRNA as a model to investigate the effects of the mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady state levels of cardiolipin and increased mitochondrial ROS. Treatment of cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced mitochondrial ROS production and cellular ATP decline. Mito-Tempo also significantly abrogated tafazzin knockdown induced cardiac hypertrophy, contractile dysfunction, and cell death. We conclude that mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth syndrome and other dilated cardiomyopathies resulting from mitochondrial oxidative stress. PMID:25247053

  15. Selenoprotein X Gene Knockdown Aggravated H2O2-Induced Apoptosis in Liver LO2 Cells.

    PubMed

    Tang, Jiayong; Cao, Lei; Li, Qiang; Wang, Longqiong; Jia, Gang; Liu, Guangmang; Chen, Xiaoling; Cai, Jingyi; Shang, Haiying; Zhao, Hua

    2016-09-01

    To determine the roles of selenoprotein X gene (Selx) in protecting liver cells against oxidative damage, the influences of Selx knockdown on H2O2-induced apoptosis in human normal hepatocyte (LO2) cells were studied. pSilencer 3.1 was used to develop knockdown vector targeting the 3'-UTR of human Selx. The Selx knockdown and control cells were further exposed to H2O2, and cell viability, cell apoptosis rate, and the expression levels of mRNA and protein of apoptosis-related genes were detected. The results showed that vector targeting the 3'-UTR of Selx successfully silenced mRNA or protein expression of SelX in LO2 cells. Selx knockdown resulted in decreased cell viability, increased percentage of early apoptotic cells, decreased Bcl2A1 and Bcl-2 expression, and increased phosphorylation of P38 in LO2 cells. When Selx knockdown LO2 cells were exposed to H2O2, characteristics of H2O2-induced cell dysfunctions were further exacerbated. Taken together, our findings suggested that SelX played important roles in protecting LO2 cells against oxidative damage and reducing H2O2-induced apoptosis in liver cells.

  16. Size-dependent knockdown potential of siRNA-loaded cationic nanohydrogel particles.

    PubMed

    Nuhn, Lutz; Tomcin, Stephanie; Miyata, Kanjiro; Mailänder, Volker; Landfester, Katharina; Kataoka, Kazunori; Zentel, Rudolf

    2014-11-10

    To overcome the poor pharmacokinetic conditions of short double-stranded RNA molecules in RNA interference therapies, cationic nanohydrogel particles can be considered as alternative safe and stable carriers for oligonucleotide delivery. For understanding key parameters during this process, two different types of well-defined cationic nanohydrogel particles were synthesized, which provided nearly identical physicochemical properties with regards to their material composition and resulting siRNA loading characteristics. Yet, according to the manufacturing process using amphiphilic reactive ester block copolymers of pentafluorophenyl methacrylate (PFPMA) and tri(ethylene glycol)methyl ether methacrylate (MEO3MA) with similar compositions but different molecular weights, the resulting nanohydrogel particles differed in size after cross-linking with spermine (average diameter 40 vs 100 nm). This affected their knockdown potential significantly. Only the 40 nm sized cationic nanogel particles were able to generate moderate gene knockdown levels, which lasted, however, up to 3 days. Interestingly, primary cell uptake and colocalization studies with lysosomal compartments revealed that only these small sized nanogels were able to avoid acidic compartments of endolysosomal uptake pathways, which may contribute to their knockdown ability exclusively. To that respect, this size-dependent intracellular distribution behavior may be considered as an essential key parameter for tuning the knockdown potential of siRNA nanohydrogel particles, which may further contribute to the development of advanced siRNA carrier systems with improved knockdown potential.

  17. Knockdown of cullin 4A inhibits growth and increases chemosensitivity in lung cancer cells.

    PubMed

    Hung, Ming-Szu; Chen, I-Chuan; You, Liang; Jablons, David M; Li, Ya-Chin; Mao, Jian-Hua; Xu, Zhidong; Lung, Jr-Hau; Yang, Cheng-Ta; Liu, Shih-Tung

    2016-07-01

    Cullin 4A (Cul4A) has been observed to be overexpressed in various cancers. In this study, the role of Cul4A in the growth and chemosensitivity in lung cancer cells were studied. We showed that Cul4A is overexpressed in lung cancer cells and tissues. Knockdown of the Cul4A expression by shRNA in lung cancer cells resulted in decreased cellular proliferation and growth in lung cancer cells. Increased sensitivity to gemcitabine, a chemotherapy drug, was also noted in those Cul4A knockdown lung cancer cells. Moreover, increased expression of p21, transforming growth factor (TGF)-β inducible early gene-1 (TIEG1) and TGF beta-induced (TGFBI) was observed in lung cancer cells after Cul4A knockdown, which may be partially related to increased chemosensitivity to gemcitabine. G0/G1 cell cycle arrest was also noted after Cul4A knockdown. Notably, decreased tumour growth and increased chemosensitivity to gemcitabine were also noted after Cul4A knockdown in lung cancer xenograft nude mice models. In summary, our study showed that targeting Cul4A with RNAi or other techniques may provide a possible insight to the development of lung cancer therapy in the future.

  18. Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

    SciTech Connect

    Li, Jie; Yang, Xi-fei; Ren, Xiao-hu; Meng, Xiao-jing; Huang, Hai-yan; Zhao, Qiong-hui; Yuan, Jian-hui; Hong, Wen-xu; Xia, Bo; Huang, Xin-feng; Zhou, Li; Liu, Jian-jun; Zou, Fei

    2014-10-10

    Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we explore the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.

  19. Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes

    PubMed Central

    Nakagaki, Brenda Naemi; Mendonça-Neto, Rondon Pessoa; Canavaci, Adriana Monte Cassiano; Souza Melo, Normanda; Martinelli, Patrícia Massara; Fernandes, Ana Paula; daRocha, Wanderson Duarte; Teixeira, Santuza M. R.

    2015-01-01

    Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole. PMID:26641088

  20. MRP1 knockdown down-regulates the deposition of collagen and leads to a reduced hypertrophic scar fibrosis.

    PubMed

    Li, Yan; Yang, Longlong; Zheng, Zhao; Shi, Jihong; Wu, Xue; Guan, Hao; Jia, Yanhui; Tao, Ke; Wang, Hongtao; Han, Shichao; Gao, Jianxin; Zhao, Bin; Su, Linlin; Hu, Dahai

    2015-10-01

    Multidrug resistance-associated protein 1 (MRP1) belongs to ATP-binding cassette transporters family. The overexpression of MRP1 is predominantly related with the failure of chemo-radiotherapy in various tumors. However, its possible role in hypertrophic scar (HS) is hardly investigated. Here we showed that the mRNA level and protein expression of MRP1 were higher in HS and HS derived fibroblasts (HSFs) than that in normal skin (NS) and NS derived fibroblasts (NSFs). Immunohistochemistry and immunofluorescence showed that the percentage of positive cells was higher in HS and HSFs. Meanwhile, the co-localization of MRP1 and α-SMA was stronger in HS. MRP1 knockdown in HSFs provoked a significant reduction in the protein expressions of collagen 3 and α-SMA in vitro. Moreover, MRP1 siRNA transfection could decrease the deposition of collagen in cultured tissues ex vivo and inhibit the scar formation in rabbit ear scar model in vivo. H&E staining and Masson trichrome staining revealed thinner and more orderly arranged collagen fiber in the MRP1 siRNA transfection group. The appearance of scar was improved as well. All these results indicate that MRP1 plays an important role in the formation of HS, MRP1 knockdown could be a potential method to reduce the accumulation of collagen and to improve the abnormal deposition of extracellular matrix in HS, which indicates that down-regulation of MRP1 has the potential therapeutic effect in the treatment and prophylaxis of HS.

  1. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    PubMed

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC. PMID:23924858

  2. Small Interfering RNA-Mediated Connexin Gene Knockdown in Vascular Endothelial and Smooth Muscle Cells.

    PubMed

    Good, Miranda E; Begandt, Daniela; DeLalio, Leon J; Johnstone, Scott R; Isakson, Brant E

    2016-01-01

    Global knockout of vascular connexins can result in premature/neonatal death, severe developmental complications, or compensatory up-regulation of different connexin isoforms. Thus, specific connexin gene knockdown using RNAi-mediated technologies is a technique that allows investigators to efficiently monitor silencing effects of single or multiple connexin gene products. The present chapter describes the transient knockdown of connexins in vitro and ex vivo for cells of the blood vessel wall. In detail, different transfection methods for primary endothelial cells and ex vivo thoracodorsal arteries are described. Essential controls for validating transfection efficiency as well as targeted gene knockdown are explained. These protocols provide researchers with the ability to modify connexin gene expression levels in a multitude of experimental setups. PMID:27207287

  3. Knockdown of Pokemon protein expression inhibits hepatocellular carcinoma cell proliferation by suppression of AKT activity.

    PubMed

    Zhu, Xiaosan; Dai, Yichen; Chen, Zhangxin; Xie, Junpei; Zeng, Wei; Lin, Yuanyuan

    2013-01-01

    Overexpression of Pokemon, which is an erythroid myeloid ontogenic factor protein, occurs in different cancers, including hepatocellular carcinoma (HCC). Pokemon is also reported to have an oncogenic activity in various human cancers. This study investigated the effect of Pokemon knockdown on the regulation of HCC growth. POK shRNA suppressed the expression of Pokemon protein in HepG2 cells compared to the negative control vector-transfected HCC cells. Pokemon knockdown also reduced HCC cell viability and enhanced cisplatin-induced apoptosis in HCC cells. AKT activation and the expression of various cell cycle-related genes were inhibited following Pokemon knockdown. These data demonstrate that Pokemon may play a role in HCC progression, suggesting that inhibition of Pokemon expression using Pokemon shRNA should be further evaluated as a novel target for the control of HCC.

  4. FTO knockdown in rat ventromedial hypothalamus does not affect energy balance

    PubMed Central

    van Gestel, Margriet A.; Sanders, Loek E.; de Jong, Johannes W.; Luijendijk, Mieneke C. M.; Adan, Roger A. H.

    2014-01-01

    Abstract Single nucleotide polymorphisms (SNPs) clustered in the first intron of the fat mass and obesity‐associated (FTO) gene has been associated with obesity. FTO expression is ubiquitous, with particularly high levels in the hypothalamic area of the brain. To investigate the region‐specific role of FTO, AAV technology was applied to knockdown FTO in the ventromedial hypothalamus (VMH). No effect of FTO knockdown was observed on bodyweight or parameters of energy balance. Animals were exposed twice to an overnight fast, followed by a high‐fat high‐sucrose (HFHS) diet for 1 week. FTO knockdown did not result in a different response to the diets. A region‐specific role for FTO in the VMH in the regulation of energy balance could not be found. PMID:25501432

  5. Sleeping Beauty-mediated knockdown of sheep myostatin by RNA interference.

    PubMed

    Hu, Shengwei; Ni, Wei; Sai, Wujiafu; Zhang, Hui; Cao, Xudong; Qiao, Jun; Sheng, Jinliang; Guo, Fei; Chen, Chuangfu

    2011-10-01

    Myostatin is a negative regulator of skeletal muscle growth. Myostatin dysfunction therefore offers a strategy for promoting animal muscle growth in livestock production. Knockdown of myostatin was achieved by combining RNA interference and the Sleeping Beauty (SB) transposon system in sheep cells. Four targeting sites of sheep myostatin were designed and measured for myostatin silencing in sheep fetal fibroblasts by real-time PCR. The sh3 construct induced significant decrease of myostatin gene expression by 90% (P<0.05). Myostatin silencing induced by SB-mediated sh3 was further tested in stably transfected cells. SB transposition increased the integration frequency of genes into sheep genomes and mediated a more efficient myostatin knockdown than random integration of sh3. We suggest that SB-mediated shRNA provides a novel potential tool for gene knockdown in the donor cells of animal cloning. PMID:21698446

  6. Withaferin A inhibits in vivo growth of breast cancer cells accelerated by Notch2 knockdown.

    PubMed

    Kim, Su-Hyeong; Hahm, Eun-Ryeong; Arlotti, Julie A; Samanta, Suman K; Moura, Michelle B; Thorne, Stephen H; Shuai, Yongli; Anderson, Carolyn J; White, Alexander G; Lokshin, Anna; Lee, Joomin; Singh, Shivendra V

    2016-05-01

    The present study offers novel insights into the molecular circuitry of accelerated in vivo tumor growth by Notch2 knockdown in triple-negative breast cancer (TNBC) cells. Therapeutic vulnerability of Notch2-altered growth to a small molecule (withaferin A, WA) is also demonstrated. MDA-MB-231 and SUM159 cells were used for the xenograft studies. A variety of technologies were deployed to elucidate the mechanisms underlying tumor growth augmentation by Notch2 knockdown and its reversal by WA, including Fluorescence Molecular Tomography for measurement of tumor angiogenesis in live mice, Seahorse Flux analyzer for ex vivo measurement of tumor metabolism, proteomics, and Luminex-based cytokine profiling. Stable knockdown of Notch2 resulted in accelerated in vivo tumor growth in both cells reflected by tumor volume and/or latency. For example, the wet tumor weight from mice bearing Notch2 knockdown MDA-MB-231 cells was about 7.1-fold higher compared with control (P < 0.0001). Accelerated tumor growth by Notch2 knockdown was highly sensitive to inhibition by a promising steroidal lactone (WA) derived from a medicinal plant. Molecular underpinnings for tumor growth intensification by Notch2 knockdown included compensatory increase in Notch1 activation, increased cellular proliferation and/or angiogenesis, and increased plasma or tumor levels of growth stimulatory cytokines. WA administration reversed many of these effects providing explanation for its remarkable anti-cancer efficacy. Notch2 functions as a tumor growth suppressor in TNBC and WA offers a novel therapeutic strategy for restoring this function. PMID:27097807

  7. Knockdown of FAK inhibits the invasion and metastasis of Tca‑8113 cells in vitro.

    PubMed

    Xiao, Wenbo; Jiang, Mingxin; Li, Hongdan; Li, Chunshan; Su, Rongjian; Huang, Keqiang

    2013-08-01

    Tongue cancer originating on the surface of the tongue is most commonly squamous cell carcinoma, which has a higher invasive ability and a lower survival rate compared with other forms of tongue cancer. Notably, tongue squamous cell carcinomas metastasize into lymph nodes at early stages. Focal adhesion kinase (FAK) is an important protein tyrosine kinase involved in invasion and metastasis of cancer cells. In the present study, the role of FAK in the invasion and metastasis of tongue cancer was evaluated and the underlying mechanisms involved in this process were explored. FAK knockdown was performed using shRNA in the tongue cancer cell line, Tca‑8113, and the invasion and metastasis potentials were analyzed using wound healing and transwell assays, respectively. Cytoskeletal arrangement was detected by fluorescence using TRITC‑conjugated phalloidin staining. The activity of matrix metalloproteinase (MMP)‑2 and ‑9 was examined by gelatin zymography. Paxillin distribution was observed by immunofluorescence. The levels of E‑cadherin, N‑cadherin, MMP‑2 and ‑9, and c‑Jun N‑terminal kinase (JNK) was detected by western blot analysis. Wound healing and transwell assays demonstrated that FAK knockdown inhibited the invasion and metastasis of Tca‑8113 cells. Further analysis revealed that FAK knockdown caused the rearrangement of the cytoskeleton and decreased the activity of MMP‑2 and ‑9. Immunofluorescence analysis revealed that downregulation of FAK induced the relocalization of paxillin. Paxillin accumulated as dots and patches at the cell membrane in control cells. By contrast, in FAK knockdown cells, paxillin was distributed homogeneously in the cytoplasm. Western blot analysis revealed that FAK knockdown inhibited epithelial-mesenchymal transition (EMT) and decreased levels of MMP‑2 and ‑9, and p‑JNK. Knockdown of FAK inhibits the invasion and metastasis of Tca‑8113 by decreasing MMP‑2 and ‑9 activities and led to the

  8. Prolonged gene knockdown in the tsetse fly Glossina by feeding double stranded RNA.

    PubMed

    Walshe, D P; Lehane, S M; Lehane, M J; Haines, L R

    2009-02-01

    Reverse genetic studies based on RNA interference (RNAi) have revolutionized analysis of gene function in most insects. However the necessity of injecting double stranded RNA (dsRNA) inevitably compromises many investigations particularly those on immunity. Additionally, injection of tsetse flies often causes significant mortality. We demonstrate, at transcript and protein level, that delivering dsRNA in the bloodmeal to Glossina morsitans morsitans is as effective as injection in knockdown of the immunoresponsive midgut-expressed gene TsetseEP. However, feeding dsRNA fails to knockdown the fat body expressed transferrin gene, 2A192, previously shown to be silenced by dsRNA injection. Mortality rates of the dsRNA fed flies were significantly reduced compared to injected flies 14 days after treatment (Fed: 10.1%+/- 1.8%; injected: 37.9% +/- 3.6% (Mean +/- SEM)). This is the first demonstration in Diptera of gene knockdown by feeding and the first example of knockdown in a blood-sucking insect by including dsRNA in the bloodmeal. PMID:19016913

  9. Sustained conditional knockdown reveals intracellular bone sialoprotein as essential for breast cancer skeletal metastasis.

    PubMed

    Kovacheva, Marineta; Zepp, Michael; Berger, Stefan M; Berger, Martin R

    2014-07-30

    Increased bone sialoprotein (BSP) serum levels are related to breast cancer skeletal metastasis, but their relevance is unknown. We elucidated novel intracellular BSP functions by a conditional knockdown of BSP. Conditional MDA-MB-231 subclones were equipped with a novel gene expression cassette containing a tet-reg-ulated miRNA providing knockdown of BSP production. These clones were used to assess the effect of BSP on morphology, proliferation, migration, colony formation and gene expression in vitro, and on soft tissue and osteolytic le-sions in a xenograft model by three imaging methods. BSP knockdown caused significant anti-proliferative, anti-migratory and anti-clonogenic effects in vitro (p<0.001). In vivo, significant de-creases of soft tissue and osteolytic lesions (p<0.03) were recorded after 3 weeks of miRNA treatment, leading to complete remission within 6 weeks. Microarray data revealed that 0.3% of genes were modulated in response to BSP knockdown. Upregulated genes included the endoplasmic reticulum stress genes ATF3 and DDIT3, the tumor suppressor gene EGR1, ID2 (related to breast epithelial differentiation), c-FOS and SERPINB2, whereas the metastasis associated genes CD44 and IL11 were downregulated. Also, activation of apoptotic pathways was demonstrated. These results implicate that intracellular BSP is essential for breast cancer skeletal metastasis and a target for treating these lesions.

  10. TET1 knockdown inhibits the odontogenic differentiation potential of human dental pulp cells

    PubMed Central

    Rao, Li-Jia; Yi, Bai-Cheng; Li, Qi-Meng; Xu, Qiong

    2016-01-01

    Human dental pulp cells (hDPCs) possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten–eleven translocation 1 (TET1) is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TET1-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration. PMID:27357322

  11. Strength knock-down assessment of porosity in composites: modelling, characterising and specimen manufacture

    NASA Astrophysics Data System (ADS)

    McMillan, Alison J.; Archer, Edward; McIlhagger, Alistair; Lelong, Guillaume

    2012-08-01

    Porosity and inclusion of foreign material is known to reduce the strength of materials, and this paper addresses the particular problem of strength knock-down assessment due to porosity in composite materials. Porosity is often measured in terms of percentage of voids per unit volume of a component, because this can be related directly to ultra-sound absorption. Nevertheless, this is a poor indicator of actual strength knock-down, as it provides little information about void size, shape, orientation and whether they are evenly distributed or are clustered. Characterisation of void clustering enables a link between a cluster characteristic and the strength knock-down. Laboratory based testing achieves controlled porosity in specimens by introducing pin-holes into the RTM in-flow pipework, which entrains voids into the body of the preform within mould tooling. Specimens are manufactured to create resin regions bounded by a fibre reinforced picture frame, to allow for easy load application. Strength knock-downs from test are related to the theoretical expectations.

  12. The functional genetic link of NLGN4X knockdown and neurodevelopment in neural stem cells

    PubMed Central

    Shi, Lingling; Chang, Xiao; Zhang, Peilin; Coba, Marcelo P.; Lu, Wange; Wang, Kai

    2013-01-01

    Genetic mutations in NLGN4X (neuroligin 4), including point mutations and copy number variants (CNVs), have been associated with susceptibility to autism spectrum disorders (ASDs). However, it is unclear how mutations in NLGN4X result in neurodevelopmental defects. Here, we used neural stem cells (NSCs) as in vitro models to explore the impacts of NLGN4X knockdown on neurodevelopment. Using two shRNAmir-based vectors targeting NLGN4X and one control shRNAmir vector, we modulated NLGN4X expression and differentiated these NSCs into mature neurons. We monitored the neurodevelopmental process at Weeks 0, 0.5, 1, 2, 4 and 6, based on morphological analysis and whole-genome gene expression profiling. At the cellular level, in NSCs with NLGN4X knockdown, we observed increasingly delayed neuronal development and compromised neurite formation, starting from Week 2 through Week 6 post differentiation. At the molecular level, we identified multiple pathways, such as neurogenesis, neuron differentiation and muscle development, which are increasingly disturbed in cells with NLGN4X knockdown. Notably, several postsynaptic genes, including DLG4, NLGN1 and NLGN3, also have decreased expression. Based on in vitro models, NLGN4X knockdown directly impacts neurodevelopmental process during the formation of neurons and their connections. Our functional genomics study highlights the utility of NSCs models in understanding the functional roles of CNVs in affecting neurodevelopment and conferring susceptibility to neurodevelopmental diseases. PMID:23710042

  13. TET1 knockdown inhibits the odontogenic differentiation potential of human dental pulp cells.

    PubMed

    Rao, Li-Jia; Yi, Bai-Cheng; Li, Qi-Meng; Xu, Qiong

    2016-01-01

    Human dental pulp cells (hDPCs) possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten-eleven translocation 1 (TET1) is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TET1-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration. PMID:27357322

  14. Anti-tumor effect of estrogen-related receptor alpha knockdown on uterine endometrial cancer

    PubMed Central

    Matsushima, Hiroshi; Mori, Taisuke; Ito, Fumitake; Yamamoto, Takuro; Akiyama, Makoto; Kokabu, Tetsuya; Yoriki, Kaori; Umemura, Shiori; Akashi, Kyoko; Kitawaki, Jo

    2016-01-01

    Estrogen-related receptor (ERR)α presents structural similarities with estrogen receptor (ER)α. However, it is an orphan receptor not binding to naturally occurring estrogens. This study was designed to investigate the role of ERRα in endometrial cancer progression. Immunohistochemistry analysis on 50 specimens from patients with endometrial cancer showed that ERRα was expressed in all examined tissues and the elevated expression levels of ERRα were associated with advanced clinical stages and serous histological type (p < 0.01 for each). ERRα knockdown with siRNA suppressed angiogenesis via VEGF and cell proliferation in vitro (p < 0.01). Cell cycle and apoptosis assays using flow cytometry and western blot revealed that ERRα knockdown induced cell cycle arrest during the mitotic phase followed by apoptosis initiated by caspase-3. Additionally, ERRα knockdown sensitized cells to paclitaxel. A significant reduction of tumor growth and angiogenesis was also observed in ERRα knockdown xenografts (p < 0.01). These findings indicate that ERRα may serve as a novel molecular target for the treatment of endometrial cancer. PMID:27153547

  15. Knockdown of asparagine synthetase by RNAi suppresses cell growth in human melanoma cells and epidermoid carcinoma cells.

    PubMed

    Li, Hui; Zhou, Fusheng; Du, Wenhui; Dou, Jinfa; Xu, Yu; Gao, Wanwan; Chen, Gang; Zuo, Xianbo; Sun, Liangdan; Zhang, Xuejun; Yang, Sen

    2016-05-01

    Melanoma, the most aggressive form of skin cancer, causes more than 40,000 deaths each year worldwide. And epidermoid carcinoma is another major form of skin cancer, which could be studied together with melanoma in several aspects. Asparagine synthetase (ASNS) gene encodes an enzyme that catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine, and its expression is associated with the chemotherapy resistance and prognosis in several human cancers. The present study aims to explore the potential role of ASNS in melanoma cells A375 and human epidermoid carcinoma cell line A431. We applied a lentivirus-mediated RNA interference (RNAi) system to study its function in cell growth of both cells. The results revealed that inhibition of ASNS expression by RNAi significantly suppressed the growth of melanoma cells and epidermoid carcinoma cells, and induced a G0/G1 cell cycle arrest in melanoma cells. Knockdown of ASNS in A375 cells remarkably downregulated the expression levels of CDK4, CDK6, and Cyclin D1, and upregulated the expression of p21. Therefore, our study provides evidence that ASNS may represent a potential therapeutic target for the treatment of melanoma.

  16. Knockdown of asparagine synthetase by RNAi suppresses cell growth in human melanoma cells and epidermoid carcinoma cells.

    PubMed

    Li, Hui; Zhou, Fusheng; Du, Wenhui; Dou, Jinfa; Xu, Yu; Gao, Wanwan; Chen, Gang; Zuo, Xianbo; Sun, Liangdan; Zhang, Xuejun; Yang, Sen

    2016-05-01

    Melanoma, the most aggressive form of skin cancer, causes more than 40,000 deaths each year worldwide. And epidermoid carcinoma is another major form of skin cancer, which could be studied together with melanoma in several aspects. Asparagine synthetase (ASNS) gene encodes an enzyme that catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine, and its expression is associated with the chemotherapy resistance and prognosis in several human cancers. The present study aims to explore the potential role of ASNS in melanoma cells A375 and human epidermoid carcinoma cell line A431. We applied a lentivirus-mediated RNA interference (RNAi) system to study its function in cell growth of both cells. The results revealed that inhibition of ASNS expression by RNAi significantly suppressed the growth of melanoma cells and epidermoid carcinoma cells, and induced a G0/G1 cell cycle arrest in melanoma cells. Knockdown of ASNS in A375 cells remarkably downregulated the expression levels of CDK4, CDK6, and Cyclin D1, and upregulated the expression of p21. Therefore, our study provides evidence that ASNS may represent a potential therapeutic target for the treatment of melanoma. PMID:25858017

  17. Cathepsin L knockdown enhances curcumin-mediated inhibition of growth, migration, and invasion of glioma cells.

    PubMed

    Fei, Yao; Xiong, Yajie; Zhao, Yifan; Wang, Wenjuan; Han, Meilin; Wang, Long; Tan, Caihong; Liang, Zhongqin

    2016-09-01

    Curcumin can be used to prevent and treat cancer. However, its exact underlying molecular mechanisms remain poorly understood. Cathepsin L, a lysosomal cysteine protease, is overexpressed in several cancer types. This study aimed to determine the role of cathepsin L in curcumin-mediated inhibition of growth, migration, and invasion of glioma cells. Results revealed that the activity of cathepsin L was enhanced in curcumin-treated glioma cells. Cathepsin L knockdown induced by RNA interference significantly promoted curcumin-induced cytotoxicity, apoptosis, and cell cycle arrest. The knockdown also inhibited the migration and invasion of glioma cells. Our results suggested that the inhibition of cathepsin L can enhance the sensitivity of glioma cells to curcumin. Therefore, cathepsin L may be a new target to enhance the efficacy of curcumin against cancers. PMID:27373979

  18. Lentivirus-Mediated knockdown of tectonic family member 1 inhibits medulloblastoma cell proliferation

    PubMed Central

    Jing, Junjie; Wang, Chengfeng; Liang, Qinchuan; Zhao, Yang; Zhao, Qingshuang; Wang, Shousen; Ma, Jie

    2015-01-01

    Tectonic family member 1 (TCTN1) encodes a member of the tectonic family which are evolutionarily conserved secreted and transmembrane proteins, involving in a diverse variety of developmental processes. It has been demonstrated that tectonics expressed in regions that participate in Hedgehog (Hh) signaling during mouse embryonic development and was imperative for Hh-mediated patterning of the ventral neural tube. However, the expression and regulation of tectonics in human tumor is still not clear. In this study, shRNA-expressing lentivirus was constructed to knockdown TCTN1 in medulloblastoma cell line Daoy. The results showed that knockdown of TCTN1 inhibited cell proliferation and colony formation in Daoy cell line, also caused cell cycle arrest at the G2/M boundary. Taken all together, our data suggest that TCTN1 might play an important role in the progression of medulloblastoma. PMID:26550235

  19. Effects of Shell-Buckling Knockdown Factors in Large Cylindrical Shells

    NASA Technical Reports Server (NTRS)

    Hrinda, Glenn A.

    2012-01-01

    Shell-buckling knockdown factors (SBKF) have been used in large cylindrical shell structures to account for uncertainty in buckling loads. As the diameter of the cylinder increases, achieving the manufacturing tolerances becomes increasingly more difficult. Knockdown factors account for manufacturing imperfections in the shell geometry by decreasing the allowable buckling load of the cylinder. In this paper, large-diameter (33 ft) cylinders are investigated by using various SBKF's. An investigation that is based on finite-element analysis (FEA) is used to develop design sensitivity relationships. Different manufacturing imperfections are modeled into a perfect cylinder to investigate the effects of these imperfections on buckling. The analysis results may be applicable to large- diameter rockets, cylindrical tower structures, bulk storage tanks, and silos.

  20. RNAi-mediated double gene knockdown and gustatory perception measurement in honey bees (Apis mellifera).

    PubMed

    Wang, Ying; Baker, Nicholas; Amdam, Gro V

    2013-07-25

    This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception. RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species. The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

  1. Knockdown of pre-mRNA cleavage factor Im 25 kDa promotes neurite outgrowth

    SciTech Connect

    Fukumitsu, Hidefumi; Soumiya, Hitomi; Furukawa, Shoei

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer CFIm25 knockdown promoted NGF-induced neurite out growth from PC12 cells. Black-Right-Pointing-Pointer Depletion of CFIm25 did not influence the morphology of proliferating PC12 cells. Black-Right-Pointing-Pointer CFIm regulated NGF-induced neurite outgrowth via coordinating RhoA activity. Black-Right-Pointing-Pointer CFIm25 knockdown increase the number of primary dendrites of hippocampal neurons. -- Abstract: Mammalian precursor mRNA (pre-mRNA) cleavage factor I (CFIm) plays important roles in the selection of poly(A) sites in a 3 Prime -untranslated region (3 Prime -UTR), producing mRNAs with variable 3 Prime ends. Because 3 Prime -UTRs often contain cis elements that impact stability or localization of mRNA or translation, alternative polyadenylation diversifies utilization of primary transcripts in mammalian cells. However, the physiological role of CFIm remains unclear. CFIm acts as a heterodimer comprising a 25 kDa subunit (CFIm25) and one of the three large subunits-CFIm59, CFIm68, or CFIm72. CFIm25 binds directly to RNA and introduces and anchors the larger subunit. To examine the physiological roles of CFIm, we knocked down the CFIm25 gene in neuronal cells using RNA interference. Knockdown of CFIm25 increased the number of primary dendrites of developing hippocampal neurons and promoted nerve growth factor (NGF)-induced neurite extension from rat pheochromocytoma PC12 cells without affecting the morphology of proliferating PC12 cells. On the other hand, CFIm25 knockdown did not influence constitutively active or dominantly negative RhoA suppression or promotion of NGF-induced neurite extension from PC12 cells, respectively. Taken together, our results indicate that endogenous CFIm may promote neuritogenesis in developing neurons by coordinating events upstream of NGF-induced RhoA inactivation.

  2. Stable knockdown of Kif5b in MDCK cells leads to epithelial–mesenchymal transition

    SciTech Connect

    Cui, Ju; Jin, Guoxiang; Yu, Bin; Wang, Zai; Lin, Raozhou; Huang, Jian-Dong

    2015-07-17

    Polarization of epithelial cells requires vectorial sorting and transport of polarity proteins to apical or basolateral domains. Kif5b is the mouse homologue of the human ubiquitous Kinesin Heavy Chain (uKHC). To investigate the function of Kif5b in epithelial cells, we examined the phenotypes of Kif5b-deficient MDCK cells. Stable knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate, profound changes in cell morphology, loss of epithelial cell marker, and gain of mesenchymal marker, as well as increased cell migration, invasion, and tumorigenesis abilities. E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells, and their expression levels were decreased in Kif5b-deficient MDCK cells. Overexpression of E-cadherin and NMMIIA in Kif5b depleted MDCK cells could decrease mesenchymal marker expression and cell migration ability. These results indicate that stable knockdown of Kif5b in MDCK cells can lead to epithelial–mesenchymal transition, which is mediated by defective E-cadherin and NMMIIA expression. - Highlights: • Knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate. • Kif5b deficient MDCK cells underwent epithelial–mesenchymal transition. • E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells. • Decreased E-cadherin and NMMIIA levels mediate EMT in Kif5b deficient MDCK cells. • Overexpression of E-cadherin and NMMIIA reverse the effects of Kif5b knockdown.

  3. Knockdown of FoxP2 alters spine density in Area X of the zebra finch.

    PubMed

    Schulz, S B; Haesler, S; Scharff, C; Rochefort, C

    2010-10-01

    Mutations in the gene encoding the transcription factor FoxP2 impair human speech and language. We have previously shown that deficits in vocal learning occur in zebra finches after reduction of FoxP2 in Area X, a striatal nucleus involved in song acquisition. We recently showed that FoxP2 is expressed in newly generated spiny neurons (SN) in adult Area X as well as in the ventricular zone (VZ) from which the SN originates. Moreover, their recruitment to Area X increases transiently during the song learning phase. The present report therefore investigated whether FoxP2 is involved in the structural plasticity of Area X. We assessed the proliferation, differentiation and morphology of SN after lentivirally mediated knockdown of FoxP2 in Area X or in the VZ during the song learning phase. Proliferation rate was not significantly affected by knockdown of FoxP2 in the VZ. In addition, FoxP2 reduction both in the VZ and in Area X did not affect the number of new neurons in Area X. However, at the fine-structural level, SN in Area X bore fewer spines after FoxP2 knockdown. This effect was even more pronounced when neurons received the knockdown before differentiation, i.e. as neuroblasts in the VZ. These results suggest that FoxP2 might directly or indirectly regulate spine dynamics in Area X and thereby influence song plasticity. Together, these data present the first evidence for a role of FoxP2 in the structural plasticity of dendritic spines and complement the emerging evidence of physiological synaptic plasticity in FoxP2 mouse models.

  4. CHIP Knockdown Reduced Heat Shock Response and Protein Quality Control Capacity in Lens Epithelial Cells.

    PubMed

    Zhang, W; Liu, Z; Bao, X; Qin, Y; Taylor, A; Shang, F; Wu, M

    2015-01-01

    Protein quality control (PQC) systems, including molecular chaperones and ubiquitin-proteasome pathway (UPP), plays an important role in maintaining intracellular protein homeostasis. Carboxyl terminus of Hsc70- interacting protein (CHIP) links the chaperone and UPPs, thus contributing to the repair or removal of damaged proteins. Over-expression of CHIP had previously been used to protect cells from environmental stress. In order to gain a more physiologic mechanism of the advantage conferred by CHIP, we induced a CHIP knockdown and monitored the ability of cells to cope with environmental stress. To knockdown CHIP, the human lens epithelial cell line HLE B3 was transfected with lentiviral particles that encode a CHIP short hairpin RNA (shRNA) or negative control lentiviral particles. Stable CHIP-knock down cells (KD) and negative control cells (NC) were selected with puromycin. After exposure to heat shock stress, there was no change observed in the expression of Hsp90. In contrast, Hsp70 levels increased significantly in NC cells but less so in KD cells. Hsp27 levels also increased after heat shock, but only in NC cells. Protein ubiquitination was reduced when CHIP was knocked down. CHIP knockdown reduced the ability to clear aggregation proteins. When same levels of aggregation-prone RFP-mutant crystallin fusion protein, RFP/V76D-γD, was expressed, there was ~9- fold more aggregates in KD cells as compared to that observed in NC cells. Furthermore, KD cells were more sensitive to toxicity of amino acid analog canavanine as compared to NC cells. Together, these data indicate that CHIP is required for PQC and that CHIP knockdown diminished cellular PQC capacity in lens cells.

  5. LRRK2 knockdown in zebrafish causes developmental defects, neuronal loss, and synuclein aggregation.

    PubMed

    Prabhudesai, Shubhangi; Bensabeur, Fatima Zahra; Abdullah, Rashed; Basak, Indranil; Baez, Solange; Alves, Guido; Holtzman, Nathalia G; Larsen, Jan Petter; Møller, Simon Geir

    2016-08-01

    Although mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinson's disease, their function is largely unknown. LRRK2 is pleiotropic in nature, shown to be involved in neurodegeneration and in more peripheral processes, including kidney functions, in rats and mice. Recent studies in zebrafish have shown conflicting evidence that removal of the LRRK2 WD40 domain may or may not affect dopaminergic neurons and/or locomotion. This study shows that ∼50% LRRK2 knockdown in zebrafish causes not only neuronal loss but also developmental perturbations such as axis curvature defects, ocular abnormalities, and edema in the eyes, lens, and otic vesicles. We further show that LRRK2 knockdown results in significant neuronal loss, including a reduction of dopaminergic neurons. Immunofluorescence demonstrates that endogenous LRRK2 is expressed in the lens, brain, heart, spinal cord, and kidney (pronephros), which mirror the LRRK2 morphant phenotypes observed. LRRK2 knockdown results further in the concomitant upregulation of β-synuclein, PARK13, and SOD1 and causes β-synuclein aggregation in the diencephalon, midbrain, hindbrain, and postoptic commissure. LRRK2 knockdown causes mislocalization of the Na(+) /K(+) ATPase protein in the pronephric ducts, suggesting that the edema might be linked to renal malfunction and that LRRK2 might be associated with pronephric duct epithelial cell differentiation. Combined, our study shows that LRRK2 has multifaceted roles in zebrafish and that zebrafish represent a complementary model to further our understanding of this central protein. © 2016 Wiley Periodicals, Inc. PMID:27265751

  6. CHIP Knockdown Reduced Heat Shock Response and Protein Quality Control Capacity in Lens Epithelial Cells.

    PubMed

    Zhang, W; Liu, Z; Bao, X; Qin, Y; Taylor, A; Shang, F; Wu, M

    2015-01-01

    Protein quality control (PQC) systems, including molecular chaperones and ubiquitin-proteasome pathway (UPP), plays an important role in maintaining intracellular protein homeostasis. Carboxyl terminus of Hsc70- interacting protein (CHIP) links the chaperone and UPPs, thus contributing to the repair or removal of damaged proteins. Over-expression of CHIP had previously been used to protect cells from environmental stress. In order to gain a more physiologic mechanism of the advantage conferred by CHIP, we induced a CHIP knockdown and monitored the ability of cells to cope with environmental stress. To knockdown CHIP, the human lens epithelial cell line HLE B3 was transfected with lentiviral particles that encode a CHIP short hairpin RNA (shRNA) or negative control lentiviral particles. Stable CHIP-knock down cells (KD) and negative control cells (NC) were selected with puromycin. After exposure to heat shock stress, there was no change observed in the expression of Hsp90. In contrast, Hsp70 levels increased significantly in NC cells but less so in KD cells. Hsp27 levels also increased after heat shock, but only in NC cells. Protein ubiquitination was reduced when CHIP was knocked down. CHIP knockdown reduced the ability to clear aggregation proteins. When same levels of aggregation-prone RFP-mutant crystallin fusion protein, RFP/V76D-γD, was expressed, there was ~9- fold more aggregates in KD cells as compared to that observed in NC cells. Furthermore, KD cells were more sensitive to toxicity of amino acid analog canavanine as compared to NC cells. Together, these data indicate that CHIP is required for PQC and that CHIP knockdown diminished cellular PQC capacity in lens cells. PMID:26321754

  7. The exocyst protein Sec10 interacts with Polycystin-2 and knockdown causes PKD-phenotypes.

    PubMed

    Fogelgren, Ben; Lin, Shin-Yi; Zuo, Xiaofeng; Jaffe, Kimberly M; Park, Kwon Moo; Reichert, Ryan J; Bell, P Darwin; Burdine, Rebecca D; Lipschutz, Joshua H

    2011-04-01

    Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown-including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation-suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes.

  8. Let-7a gene knockdown protects against cerebral ischemia/reperfusion injury

    PubMed Central

    Wang, Zhong-kun; Liu, Fang-fang; Wang, Yu; Jiang, Xin-mei; Yu, Xue-fan

    2016-01-01

    The microRNA (miRNA) let-7 was one of the first miRNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1 (MKP1) is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury. PMID:27073379

  9. shRNA-mediated knockdown of Bmi-1 inhibit lung adenocarcinoma cell migration and metastasis.

    PubMed

    Meng, Xiuxiang; Wang, Yifang; Zheng, Xiangyu; Liu, Chunqing; Su, Benli; Nie, Huiling; Zhao, Baoxia; Zhao, Xinyu; Yang, Hong

    2012-07-01

    Bmi-1 has been implicated in cancer cell growth and metastasis in a variety of tumor types. In this study, we sought to evaluate the expression of Bmi-1 in lung adenocarcinoma samples, and to determine if a correlation exists between Bmi-1 expression and clinical features of lung cancer, such as metastasis. Our results showed that Bmi-1 expression is increased in lung cancer tissues compared to adjacent non-cancerous tissues, and is associated with clinical features of lung cancer, including clinical stage and distant metastasis. We were then interested in determining if shRNA-mediated knockdown of Bmi-1 would inhibit metastasis of lung adenocarcinoma cells. To this end, we chose the most efficient shRNA duplexes targeting Bmi-1, and constructed two stably transfected lung adenocarcinoma cell lines (A549 and SPCA1). The shRNA-mediated knockdown of Bmi-1 significantly reduced migration in vitro, and metastasis in vivo, of A549 and SPCA1 cells. More importantly, knockdown of Bmi-1 also upregulated PTEN expression, and downregulated p-Akt and VEGF expression. These data support the hypothesis that Bmi-1 regulates key pathways involved in the metastasis of lung adenocarcinoma cells.

  10. PDIA3 Knockdown Exacerbates Free Fatty Acid-Induced Hepatocyte Steatosis and Apoptosis

    PubMed Central

    Yu, Chao-hui; Xu, Cheng-fu; Xu, Lei; Li, You-ming; Chen, Wei-xing

    2015-01-01

    Nonalcoholic fatty liver disease (NAFLD) has emerged as one of the most common chronic liver disease over the past decades. Endoplasmic reticulum stress (ERS) plays a pivotal role during the development of NAFLD. This study aims to analyze the potential role of protein disulfide isomerase A3 precursor (PDIA3), one of the ER chaperones, in free fatty acid-induced cell model of NAFLD. Human liver L02 cell line was treated with sodium palmitate for 24 hours, which developed severe intracellular lipid accumulation. The increased protein level of PDIA3 was detected via immunoblotting analysis in the fat loaded cell models of NAFLD. siRNA-mediated knockdown of PDIA3 in L02 cells not only increased the cellular lipid accumulation, but also exacerbated hepatocytes apoptosis induced by sodium palmitate. Further investigation revealed that knockdown of PDIA3 up-regulated protein expression of fatty acid synthase (FAS), a key enzyme involved in fatty acid synthesis. PDIA3 knockdown also up-regulated key molecules of ERS pathway, including glucose-regulated protein 78 (GRP78), phospho-PKR-like ER kinase (p-PERK), and C/EBP homologous protein (CHOP). Our results suggested that ER chaperone PDIA3 plays a pivotal role in FFA-induced hepatocyte steatosis and apoptosis. PMID:26214517

  11. Gene expression profiling of selenophosphate synthetase 2 knockdown in Drosophila melanogaster.

    PubMed

    Li, Gaopeng; Liu, Liying; Li, Ping; Chen, Luonan; Song, Haiyun; Zhang, Yan

    2016-03-01

    Selenium (Se) is an important trace element for many organisms and is incorporated into selenoproteins as selenocysteine (Sec). In eukaryotes, selenophosphate synthetase SPS2 is essential for Sec biosynthesis. In recent years, genetic disruptions of both Sec biosynthesis genes and selenoprotein genes have been investigated in different animal models, which provide important clues for understanding the Se metabolism and function in these organisms. However, a systematic study on the knockdown of SPS2 has not been performed in vivo. Herein, we conducted microarray experiments to study the transcriptome of fruit flies with knockdown of SPS2 in larval and adult stages. Several hundred differentially expressed genes were identified in each stage. In spite that the expression levels of other Sec biosynthesis genes and selenoprotein genes were not significantly changed, it is possible that selenoprotein translation might be reduced without impacting the mRNA level. Functional enrichment and network-based analyses revealed that although different sets of differentially expressed genes were obtained in each stage, they were both significantly enriched in the carbohydrate metabolism and redox processes. Furthermore, protein-protein interaction (PPI)-based network clustering analysis implied that several hub genes detected in the top modules, such as Nimrod C1 and regucalcin, could be considered as key regulators that are responsible for the complex responses caused by SPS2 knockdown. Overall, our data provide new insights into the relationship between Se utilization and several fundamental cellular processes as well as diseases. PMID:26824785

  12. Sod2 knock-down in the musculature has whole organism consequences in Drosophila

    PubMed Central

    Martin, Ian; Jones, Melanie A.; Rhodenizer, Devin; Zheng, Jie; Warrick, John M.; Seroude, Laurent; Grotewiel, Mike

    2009-01-01

    Oxidative damage to cell macromolecules by reactive oxygen species is associated with numerous diseases and aging. In Drosophila, RNAi-mediated silencing of the mitochondrial antioxidant manganese superoxide dismutase (SOD2) throughout the body dramatically reduces life span, accelerates senescence of locomotor function, and enhances sensitivity to applied oxidative stress. Here, we show that Sod2 knock-down in the musculature alone is sufficient to cause the shortened life span and accelerated locomotor declines observed with knock-down of Sod2 throughout the body, indicating that Sod2 deficiency in muscle is central to these phenotypes. Knock-down of Sod2 in the muscle also increased caspase activity (a marker for apoptosis) and caused a mitochondrial pathology characterized by swollen mitochondria, decreased mitochondrial content and reduced ATP levels. These findings indicate that Sod2 plays a crucial role in the musculature in Drosophila and that the consequences of Sod2 loss in this tissue extend to the viability of the organism as a whole. PMID:19545620

  13. AAV-mediated in vivo knockdown of luciferase using combinatorial RNAi and U1i.

    PubMed

    Koornneef, A; van Logtenstein, R; Timmermans, E; Pisas, L; Blits, B; Abad, X; Fortes, P; Petry, H; Konstantinova, P; Ritsema, T

    2011-09-01

    RNA interference (RNAi) has been successfully employed for specific inhibition of gene expression; however, safety and delivery of RNAi remain critical issues. We investigated the combinatorial use of RNAi and U1 interference (U1i). U1i is a gene-silencing technique that acts on the pre-mRNA by preventing polyadenylation. RNAi and U1i have distinct mechanisms of action in different cellular compartments and their combined effect allows usage of minimal doses, thereby avoiding toxicity while retaining high target inhibition. As a proof of concept, we investigated knockdown of the firefly luciferase reporter gene by combinatorial use of RNAi and U1i, and evaluated their inhibitory potential both in vitro and in vivo. Co-transfection of RNAi and U1i constructs showed additive reduction of luciferase expression up to 95% in vitro. We attained similar knockdown when RNAi and U1i constructs were hydrodynamically transfected into murine liver, demonstrating for the first time successful in vivo application of U1i. Moreover, we demonstrated long-term gene silencing by AAV-mediated transduction of murine muscle with RNAi/U1i constructs targeting firefly luciferase. In conclusion, these results provide a proof of principle for the combinatorial use of RNAi and U1i to enhance target gene knockdown in vivo.

  14. Effects of ezrin knockdown on the structure of gastric glandular epithelia.

    PubMed

    Yoshida, Saori; Yamamoto, Hiroto; Tetsui, Takahito; Kobayakawa, Yuka; Hatano, Ryo; Mukaisho, Ken-ichi; Hattori, Takanori; Sugihara, Hiroyuki; Asano, Shinji

    2016-01-01

    Ezrin, an adaptor protein that cross-links plasma membrane-associated proteins with the actin cytoskeleton, is concentrated on apical surfaces of epithelial cells, especially in microvilli of the small intestine and stomach. In the stomach, ezrin is predominantly expressed on the apical canalicular membrane of parietal cells. Transgenic ezrin knockdown mice in which the expression level of ezrin was reduced to <7% compared with the wild-type suffered from achlorhydria because of impairment of membrane fusion between tubulovesicles and apical membranes. We observed, for the first time, hypergastrinemia and foveolar hyperplasia in the gastric fundic region of the knockdown mice. Dilation of fundic glands was observed, the percentage of parietal and chief cells was reduced, and that of mucous-secreting cells was increased. The parietal cells of knockdown mice contained dilated tubulovesicles and abnormal mitochondria, and subsets of these cells contained abnormal vacuoles and multilamellar structures. Therefore, lack of ezrin not only causes achlorhydria and hypergastrinemia but also changes the structure of gastric glands, with severe perturbation of the secretory membranes of parietal cells. PMID:26329936

  15. Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression

    SciTech Connect

    Chang, C.-J.; Hsu, C.-C.; Yung, M.-C.; Chen, K.-Y.; Tzao Ching; Wu, W.-F.; Chou, H.-Y.; Lee, Y.-Y.; Lu, K.-H.; Chiou, S.-H.; Ma, H.-I

    2009-03-06

    CD133-expressing glioma cells play a critical role in tumor recovery after treatment and are resistant to radiotherapy. Herein, we demonstrated that glioblastoma-derived CD133-positive cells (GBM-CD133{sup +}) are capable of self-renewal and express high levels of embryonic stem cell genes and SirT1 compared to GBM-CD133{sup -} cells. To evaluate the role of SirT1 in GBM-CD133{sup +}, we used a lentiviral vector expressing shRNA to knock-down SirT1 expression (sh-SirT1) in GBM-CD133{sup +}. Silencing of SirT1 significantly enhanced the sensitivity of GBM-CD133{sup +} to radiation and increased the level of radiation-mediated apoptosis. Importantly, knock-down of SirT1 increased the effectiveness of radiotherapy in the inhibition of tumor growth in nude mice transplanted with GBM-CD133{sup +}. Kaplan-Meier survival analysis indicated that the mean survival rate of GBM-CD133{sup +} mice treated with radiotherapy was significantly improved by Sh-SirT1 as well. In sum, these results suggest that SirT1 is a potential target for increasing the sensitivity of GBM and glioblastoma-associated cancer stem cells to radiotherapy.

  16. In vivo identification of Bacillus thuringiensis Cry4Ba toxin receptors by RNA interference knockdown of glycosylphosphatidylinositol-linked aminopeptidase N transcripts in Aedes aegypti larvae.

    PubMed

    Saengwiman, Suchada; Aroonkesorn, Aratee; Dedvisitsakul, Plaipol; Sakdee, Somsri; Leetachewa, Somphob; Angsuthanasombat, Chanan; Pootanakit, Kusol

    2011-04-22

    Bacillus thuringiensis Cry4Ba toxin selectively kills Aedes aegypti mosquito larvae as it is in part due to the presence of specific membrane-bound protein receptors. In this study, using data mining approach, we initially identified three potential glycosylphosphatidylinositol-linked aminopeptidase N (GPI-APN) isoforms, APN2778, APN2783 and APN5808, which are believed to act as Cry4Ba toxin receptors. These three isoforms that are functionally expressed in the larval midgut can be sequence-specific knocked down (ranging from ∼80 % to 95 %) by soaking the Aedes aegypti larvae in buffer of long double-stranded GPI-APN RNAs (∼300-680 bp). Finally, to see the physiological effect of APN knockdowns, the larvae were fed with Escherichia coli expressing Cry4Ba toxin. The results revealed that all the three identified GPI-APN isoforms may possibly function as a Cry4Ba receptor, particularly for APN2783 as those larvae with this transcript knockdown showed a dramatic increase in resistance to Cry4Ba toxicity. PMID:21439264

  17. Knockdown of Inner Arm Protein IC138 in Trypanosoma brucei Causes Defective Motility and Flagellar Detachment

    PubMed Central

    Wilson, Corinne S.; Chang, Alex J.; Greene, Rebecca; Machado, Sulynn; Parsons, Matthew W.; Takats, Taylor A.; Zambetti, Luke J.; Springer, Amy L.

    2015-01-01

    Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei. PMID:26555902

  18. β-catenin knockdown inhibits the proliferation of human glioma cells in vitro and in vivo

    PubMed Central

    WANG, ZHONG; CHEN, QIANXUE

    2016-01-01

    β-catenin is a crucial oncogene that is capable of regulating cancer progression. The aim of the present study was to clarify whether β-catenin was associated with the proliferation and progress of glioma. In order to knockdown the expression of β-catenin in human U251 glioma cells, three pairs of small interfering (si)RNA were designed and synthesized and the most effective siRNA was selected and used for silencing the endogenous β-catenin, which was detected by western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Proliferation was subsequently detected using a methylthiazolyl-tetrazolium bromide assay and the results demonstrated that knockdown of β-catenin significantly inhibited the proliferation of U251 cells in a time- and dose-dependent manner (P<0.01). Cell apoptosis rate was analyzed using flow cytometry and Annexin V-fluorescein isothiocyanate/propidium iodide staining demonstrated that β-catenin siRNA significantly increased the apoptosis of U251 cells (P<0.01). Furthermore, the results of an in vitro scratch assay demonstrated that β-catenin silencing suppressed the proliferation of U251 cells, as compared with the control group (P<0.01). In vivo, β-catenin expression levels in U251 cells were significantly inhibited (P<0.01) following β-catenin short hairpin (sh)RNA lentiviral-vector transfection, as detected by western blot analysis and RT-qPCR. Tumorigenicity experiments demonstrated that β-catenin inhibition significantly increased the survival rate of nude mice. The results of the present study demonstrated that knockdown of β-catenin expression significantly inhibited the progression of human glioma cancer cells, in vitro and in vivo; thus suggesting that β-catenin silencing may be a novel therapy for the treatment of human glioma. PMID:26998037

  19. Knockdown of Inner Arm Protein IC138 in Trypanosoma brucei Causes Defective Motility and Flagellar Detachment.

    PubMed

    Wilson, Corinne S; Chang, Alex J; Greene, Rebecca; Machado, Sulynn; Parsons, Matthew W; Takats, Taylor A; Zambetti, Luke J; Springer, Amy L

    2015-01-01

    Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei. PMID:26555902

  20. Lifespan and reproduction in brain-specific miR-29-knockdown mouse.

    PubMed

    Takeda, Toru; Tanabe, Hiroyuki

    2016-03-18

    The microRNA miR-29 is widely distributed and highly expressed in adult mouse brain during the mouse's lifetime. We recently created conditional mutant mice whose miR-29 was brain-specifically knocked down through overexpression of an antisense RNA transgene against miR-29. To explore a role for brain miR-29 in maximizing organismal fitness, we assessed somatic growth, reproduction, and lifespan in the miR-29-knockdown (KD) mice and their wild-type (WT) littermates. The KD mice were developmentally indistinguishable from WT mice with respect to gross morphology and physical activity. Fertility testing revealed that KD males were subfertile, whereas KD females were hyperfertile, only in terms of reproductive success, when compared to their gender-matched WT correspondents. Another phenotypic difference between KD and WT animals appeared in their lifespan data; KD males displayed an overall increasing tendency in post-reproductive survival relative to WT males. In contrast, KD females were prone to shorter lifespans than WT females. These results clarify that brain-targeted miR-29 knockdown affects both lifespan and reproduction in a gender-dependent manner, and moreover that the reciprocal responsiveness to the miR-29 knockdown between these two phenotypes in both genders closely follow life-course models based on the classical trade-off prediction wherein elaborate early-life energetic investment in reproduction entails accelerated late-life declines in survival, and vice versa. Thus, this study identified miR-29 as the first mammalian miRNA that is directly implicated in the lifetime trade-off between the two major fitness components, lifespan and reproduction.

  1. AHR2 morpholino knockdown reduces the toxicity of total particulate matter to zebrafish embryos.

    PubMed

    Massarsky, Andrey; Bone, Audrey J; Dong, Wu; Hinton, David E; Prasad, G L; Di Giulio, Richard T

    2016-10-15

    The zebrafish embryo has been proposed as a 'bridge model' to study the effects of cigarette smoke on early development. Previous studies showed that exposure to total particulate matter (TPM) led to adverse effects in developing zebrafish, and suggested that the antioxidant and aryl hydrocarbon receptor (AHR) pathways play important roles. This study investigated the roles of these two pathways in mediating TPM toxicity. The study consisted of four experiments. In experiment I, zebrafish embryos were exposed from 6h post fertilization (hpf) until 96hpf to TPM0.5 and TPM1.0 (corresponding to 0.5 and 1.0μg/mL equi-nicotine units) in the presence or absence of an antioxidant (N-acetyl cysteine/NAC) or a pro-oxidant (buthionine sulfoximine/BSO). In experiment II, TPM exposures were performed in embryos that were microinjected with nuclear factor erythroid 2-related factor 2 (Nrf2), AHR2, cytochrome P450 1A (CYP1A), or CYP1B1 morpholinos, and deformities were assessed. In experiment III, embryos were exposed to TPM, and embryos/larvae were collected at 24, 48, 72, and 96hpf to assess several genes associated with the antioxidant and AHR pathways. Lastly, experiment IV assessed the activity and protein levels of CYP1A and CYP1B1 after exposure to TPM. We demonstrate that the incidence of TPM-induced deformities was generally not affected by NAC/BSO treatments or Nrf2 knockdown. In contrast, AHR2 knockdown reduced, while CYP1A or CYP1B1 knockdowns elevated the incidence of some deformities. Moreover, as shown by gene expression the AHR pathway, but not the antioxidant pathway, was induced in response to TPM exposure, providing further evidence for its importance in mediating TPM toxicity.

  2. AHR2 morpholino knockdown reduces the toxicity of total particulate matter to zebrafish embryos.

    PubMed

    Massarsky, Andrey; Bone, Audrey J; Dong, Wu; Hinton, David E; Prasad, G L; Di Giulio, Richard T

    2016-10-15

    The zebrafish embryo has been proposed as a 'bridge model' to study the effects of cigarette smoke on early development. Previous studies showed that exposure to total particulate matter (TPM) led to adverse effects in developing zebrafish, and suggested that the antioxidant and aryl hydrocarbon receptor (AHR) pathways play important roles. This study investigated the roles of these two pathways in mediating TPM toxicity. The study consisted of four experiments. In experiment I, zebrafish embryos were exposed from 6h post fertilization (hpf) until 96hpf to TPM0.5 and TPM1.0 (corresponding to 0.5 and 1.0μg/mL equi-nicotine units) in the presence or absence of an antioxidant (N-acetyl cysteine/NAC) or a pro-oxidant (buthionine sulfoximine/BSO). In experiment II, TPM exposures were performed in embryos that were microinjected with nuclear factor erythroid 2-related factor 2 (Nrf2), AHR2, cytochrome P450 1A (CYP1A), or CYP1B1 morpholinos, and deformities were assessed. In experiment III, embryos were exposed to TPM, and embryos/larvae were collected at 24, 48, 72, and 96hpf to assess several genes associated with the antioxidant and AHR pathways. Lastly, experiment IV assessed the activity and protein levels of CYP1A and CYP1B1 after exposure to TPM. We demonstrate that the incidence of TPM-induced deformities was generally not affected by NAC/BSO treatments or Nrf2 knockdown. In contrast, AHR2 knockdown reduced, while CYP1A or CYP1B1 knockdowns elevated the incidence of some deformities. Moreover, as shown by gene expression the AHR pathway, but not the antioxidant pathway, was induced in response to TPM exposure, providing further evidence for its importance in mediating TPM toxicity. PMID:27576004

  3. [Knockdown of Puma protects cord blood CD34(+) cells against γ- irradiation].

    PubMed

    Zhao, Lei; Zhang, Hong-Yan; Pang, Ya-Kun; Gu, Hai-Hui; Xu, Jing; Yuan, Wei-Ping; Cheng, Tao

    2014-04-01

    Puma (P53 upregulated modulator of apoptosis) is a BCL-2 homology 3 (BH3)-only BCL-1 family member and a critical mediator of P53-dependent and -independent apoptosis. Puma plays an essential role in the apoptosis of hematopoietic stem cells exposed to irradiation without an increased risk of malignancies. This study was purposed to develop an effective lentiviral vector to target Puma in human hematopoietic cells and to investigate the effect of Puma gene knockdown on the biological function of human cord blood CD34(+) cells. SF-LV-shPuma-EGFP and control vectors were constructed, and packaged with the pSPAX2/pMD2.G packaging plasmids via 293T cells to produce pseudo-type lentiviruses. SF-LV-shPuma-EGFP or control lentiviruses were harvested within 72 hours after transfection and then were used to transduce human cord blood CD34(+) cells. GFP(+) transduced cells were sorted by flow cytometry (FCM) for subsequent studies. Semi-quantitative real time RT PCR, Western blot, FCM with Annexin V-PE/7-AAD double staining, Ki67 staining, colony forming cell assay (CFC), CCK-8 assay and BrdU incorporation were performed to determine the expression of Puma and its effect on the cord blood CD34(+) cells. The results showed that Puma was significantly knocked down in cord blood CD34(+) cells and the low expression of Puma conferred a radio-protective effect on the cord blood CD34(+) cells. This effect was achieved through reduced apoptosis and sustained quiescence after irradiation due to Puma knockdown. It is concluded that knockdown of puma gene in CD34(+) hematopoietic stem cells of human cord blood possesses the radioprotective effect, maintains the cells in silence targeting Puma in human hematopoietic cells may have a similar effect with that on mouse hematopoietic cells as previously shown, and our lentiviral targeting system for Puma provides a valuable tool for future translational studies with human cells.

  4. Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium

    PubMed Central

    Cass, Cynthia L.; Peraldi, Antoine; Dowd, Patrick F.; Mottiar, Yaseen; Santoro, Nicholas; Karlen, Steven D.; Bukhman, Yury V.; Foster, Cliff E.; Thrower, Nick; Bruno, Laura C.; Moskvin, Oleg V.; Johnson, Eric T.; Willhoit, Megan E.; Phutane, Megha; Ralph, John; Mansfield, Shawn D.; Nicholson, Paul; Sedbrook, John C.

    2015-01-01

    The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops. PMID:26093023

  5. Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium.

    PubMed

    Cass, Cynthia L; Peraldi, Antoine; Dowd, Patrick F; Mottiar, Yaseen; Santoro, Nicholas; Karlen, Steven D; Bukhman, Yury V; Foster, Cliff E; Thrower, Nick; Bruno, Laura C; Moskvin, Oleg V; Johnson, Eric T; Willhoit, Megan E; Phutane, Megha; Ralph, John; Mansfield, Shawn D; Nicholson, Paul; Sedbrook, John C

    2015-07-01

    The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops. PMID:26093023

  6. Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction.

    PubMed

    Bagci, Enise; Heijlen, Marjolein; Vergauwen, Lucia; Hagenaars, An; Houbrechts, Anne M; Esguerra, Camila V; Blust, Ronny; Darras, Veerle M; Knapen, Dries

    2015-01-01

    Thyroid hormone (TH) balance is essential for vertebrate development. Deiodinase type 1 (D1) and type 2 (D2) increase and deiodinase type 3 (D3) decreases local intracellular levels of T3, the most important active TH. The role of deiodinase-mediated TH effects in early vertebrate development is only partially understood. Therefore, we investigated the role of deiodinases during early development of zebrafish until 96 hours post fertilization at the level of the transcriptome (microarray), biochemistry, morphology and physiology using morpholino (MO) knockdown. Knockdown of D1+D2 (D1D2MO) and knockdown of D3 (D3MO) both resulted in transcriptional regulation of energy metabolism and (muscle) development in abdomen and tail, together with reduced growth, impaired swim bladder inflation, reduced protein content and reduced motility. The reduced growth and impaired swim bladder inflation in D1D2MO could be due to lower levels of T3 which is known to drive growth and development. The pronounced upregulation of a large number of transcripts coding for key proteins in ATP-producing pathways in D1D2MO could reflect a compensatory response to a decreased metabolic rate, also typically linked to hypothyroidism. Compared to D1D2MO, the effects were more pronounced or more frequent in D3MO, in which hyperthyroidism is expected. More specifically, increased heart rate, delayed hatching and increased carbohydrate content were observed only in D3MO. An increase of the metabolic rate, a decrease of the metabolic efficiency and a stimulation of gluconeogenesis using amino acids as substrates may have been involved in the observed reduced protein content, growth and motility in D3MO larvae. Furthermore, expression of transcripts involved in purine metabolism coupled to vision was decreased in both knockdown conditions, suggesting that both may impair vision. This study provides new insights, not only into the role of deiodinases, but also into the importance of a correct TH balance

  7. Deiodinase Knockdown during Early Zebrafish Development Affects Growth, Development, Energy Metabolism, Motility and Phototransduction

    PubMed Central

    Bagci, Enise; Heijlen, Marjolein; Vergauwen, Lucia; Hagenaars, An; Houbrechts, Anne M.; Esguerra, Camila V.; Blust, Ronny; Darras, Veerle M.; Knapen, Dries

    2015-01-01

    Thyroid hormone (TH) balance is essential for vertebrate development. Deiodinase type 1 (D1) and type 2 (D2) increase and deiodinase type 3 (D3) decreases local intracellular levels of T3, the most important active TH. The role of deiodinase-mediated TH effects in early vertebrate development is only partially understood. Therefore, we investigated the role of deiodinases during early development of zebrafish until 96 hours post fertilization at the level of the transcriptome (microarray), biochemistry, morphology and physiology using morpholino (MO) knockdown. Knockdown of D1+D2 (D1D2MO) and knockdown of D3 (D3MO) both resulted in transcriptional regulation of energy metabolism and (muscle) development in abdomen and tail, together with reduced growth, impaired swim bladder inflation, reduced protein content and reduced motility. The reduced growth and impaired swim bladder inflation in D1D2MO could be due to lower levels of T3 which is known to drive growth and development. The pronounced upregulation of a large number of transcripts coding for key proteins in ATP-producing pathways in D1D2MO could reflect a compensatory response to a decreased metabolic rate, also typically linked to hypothyroidism. Compared to D1D2MO, the effects were more pronounced or more frequent in D3MO, in which hyperthyroidism is expected. More specifically, increased heart rate, delayed hatching and increased carbohydrate content were observed only in D3MO. An increase of the metabolic rate, a decrease of the metabolic efficiency and a stimulation of gluconeogenesis using amino acids as substrates may have been involved in the observed reduced protein content, growth and motility in D3MO larvae. Furthermore, expression of transcripts involved in purine metabolism coupled to vision was decreased in both knockdown conditions, suggesting that both may impair vision. This study provides new insights, not only into the role of deiodinases, but also into the importance of a correct TH balance

  8. The effects of Kiaa0319 knockdown on cortical and subcortical anatomy in male rats.

    PubMed

    Szalkowski, Caitlin E; Fiondella, Christopher F; Truong, Dongnhu T; Rosen, Glenn D; LoTurco, Joseph J; Fitch, Roslyn H

    2013-04-01

    Developmental dyslexia is a disorder characterized by a specific deficit in reading despite adequate overall intelligence and educational resources. The neurological substrate underlying these significant behavioral impairments is not known. Studies of post mortem brain tissue from male and female dyslexic individuals revealed focal disruptions of neuronal migration concentrated in the left hemisphere, along with aberrant symmetry of the right and left the planum temporale, and changes in cell size distribution within the medial geniculate nucleus of the thalamus (Galaburda et al., 1985; Humphreys et al., 1990). More recent neuroimaging studies have identified several changes in the brains of dyslexic individuals, including regional changes in gray matter, changes in white matter, and changes in patterns of functional activation. In a further effort to elucidate the etiology of dyslexia, epidemiological and genetic studies have identified several candidate dyslexia susceptibility genes. Some recent work has investigated associations between some of these genetic variants and structural changes in the brain. Variants of one candidate dyslexia susceptibility gene, KIAA0319, have been linked to morphological changes in the cerebellum and functional activational changes in the superior temporal sulcus (Jamadar et al., 2011; Pinel et al., 2012). Animal models have been used to create a knockdown of Kiaa0319 (the rodent homolog of the human gene) via in utero RNA interference in order to study the gene's effects on brain development and behavior. Studies using this animal model have demonstrated that knocking down the gene leads to focal disruptions of neuronal migration in the form of ectopias and heterotopias, similar to those observed in the brains of human dyslexics. However, further changes to the structure of the brain have not been studied following this genetic disruption. The current study sought to determine the effects of embryonic Kiaa0319 knockdown on volume

  9. NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells

    NASA Technical Reports Server (NTRS)

    Zhang, Ying; Lim, Chang U K.; Williams, Eli S.; Zhou, Junqing; Zhang, Qinming; Fox, Michael H.; Bailey, Susan M.; Liber, Howard L.

    2005-01-01

    Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

  10. Ron Knockdown and Ron Monoclonal Antibody IMC-RON8 Sensitize Pancreatic Cancer to Histone Deacetylase Inhibitors (HDACi)

    PubMed Central

    Zou, Yi; Howell, Gillian M.; Humphrey, Lisa E.; Wang, Jing; Brattain, Michael G.

    2013-01-01

    Recepteur d’origine nantais (Ron) is overexpressed in a panel of pancreatic cancer cells and tissue samples from pancreatic cancer patients. Ron can be activated by its ligand macrophage stimulating protein (MSP), thereby activating oncogenic signaling pathways. Crosstalk between Ron and EGFR, c-Met, or IGF-1R may provide a mechanism underlying drug resistance. Thus, targeting Ron may represent a novel therapeutic strategy. IMC-RON8 is the first Ron monoclonal antibody (mAb) entering clinical trial for targeting Ron overexpression. Our studies show IMC-RON8 downmodulated Ron expression in pancreatic cancer cells and significantly blocked MSP-stimulated Ron activation, downstream Akt and ERK phosphorylation, and survivin mRNA expression. IMC-RON8 hindered MSP-induced cell migration and reduced cell transformation. Histone deacetylase inhibitors (HDACi) are reported to target expression of various genes through modification of nucleosome histones and non-histone proteins. Our work shows HDACi TSA and Panobinostat (PS) decreased Ron mRNA and protein expression in pancreatic cancer cells. PS also reduced downstream signaling of pAkt, survivin, and XIAP, as well as enhanced cell apoptosis. Interestingly, PS reduced colony formation in Ron knockdown cells to a greater extent than Ron scramble control cells in colony formation and soft agarose assays. IMC-RON8 could also sensitize pancreatic cancer cells to PS, as reflected by reduced colony numbers and size in combination treatment with IMC-RON8 and PS compared to single treatment alone. The co-treatment further reduced Ron expression and pAkt, and increased PARP cleavage compared to either treatment alone. This study suggests the potential for a novel combination approach which may ultimately be of value in treatment of pancreatic cancer. PMID:23922886

  11. Pharmacological Characterization of an Antisense Knockdown Zebrafish Model of Dravet Syndrome: Inhibition of Epileptic Seizures by the Serotonin Agonist Fenfluramine

    PubMed Central

    Copmans, Daniëlle; Langlois, Mélanie; Crawford, Alexander D.; Ceulemans, Berten; Lagae, Lieven; de Witte, Peter A. M.; Esguerra, Camila V.

    2015-01-01

    Dravet syndrome (DS) is one of the most pharmacoresistant and devastating forms of childhood epilepsy syndromes. Distinct de novo mutations in the SCN1A gene are responsible for over 80% of DS cases. While DS is largely resistant to treatment with existing anti-epileptic drugs, promising results have been obtained in clinical trials with human patients treated with the serotonin agonist fenfluramine as an add-on therapeutic. We developed a zebrafish model of DS using morpholino antisense oligomers (MOs) targeting scn1Lab, the zebrafish ortholog of SCN1A. Zebrafish larvae with an antisense knockdown of scn1Lab (scn1Lab morphants) were characterized by automated behavioral tracking and high-resolution video imaging, in addition to measuring brain activity through local field potential recordings. Our findings reveal that scn1Lab morphants display hyperactivity, convulsive seizure-like behavior, loss of posture, repetitive jerking and a myoclonic seizure-like pattern. The occurrence of spontaneous seizures was confirmed by local field potential recordings of the forebrain, measuring epileptiform discharges. Furthermore, we show that these larvae are remarkably sensitive to hyperthermia, similar to what has been described for mouse models of DS, as well as for human DS patients. Pharmacological evaluation revealed that sodium valproate and fenfluramine significantly reduce epileptiform discharges in scn1Lab morphants. Our findings for this zebrafish model of DS are in accordance with clinical data for human DS patients. To our knowledge, this is the first study demonstrating effective seizure inhibition of fenfluramine in an animal model of Dravet syndrome. Moreover, these results provide a basis for identifying novel analogs with improved activity and significantly milder or no side effects. PMID:25965391

  12. Ron knockdown and Ron monoclonal antibody IMC-RON8 sensitize pancreatic cancer to histone deacetylase inhibitors (HDACi).

    PubMed

    Zou, Yi; Howell, Gillian M; Humphrey, Lisa E; Wang, Jing; Brattain, Michael G

    2013-01-01

    Recepteur d'origine nantais (Ron) is overexpressed in a panel of pancreatic cancer cells and tissue samples from pancreatic cancer patients. Ron can be activated by its ligand macrophage stimulating protein (MSP), thereby activating oncogenic signaling pathways. Crosstalk between Ron and EGFR, c-Met, or IGF-1R may provide a mechanism underlying drug resistance. Thus, targeting Ron may represent a novel therapeutic strategy. IMC-RON8 is the first Ron monoclonal antibody (mAb) entering clinical trial for targeting Ron overexpression. Our studies show IMC-RON8 downmodulated Ron expression in pancreatic cancer cells and significantly blocked MSP-stimulated Ron activation, downstream Akt and ERK phosphorylation, and survivin mRNA expression. IMC-RON8 hindered MSP-induced cell migration and reduced cell transformation. Histone deacetylase inhibitors (HDACi) are reported to target expression of various genes through modification of nucleosome histones and non-histone proteins. Our work shows HDACi TSA and Panobinostat (PS) decreased Ron mRNA and protein expression in pancreatic cancer cells. PS also reduced downstream signaling of pAkt, survivin, and XIAP, as well as enhanced cell apoptosis. Interestingly, PS reduced colony formation in Ron knockdown cells to a greater extent than Ron scramble control cells in colony formation and soft agarose assays. IMC-RON8 could also sensitize pancreatic cancer cells to PS, as reflected by reduced colony numbers and size in combination treatment with IMC-RON8 and PS compared to single treatment alone. The co-treatment further reduced Ron expression and pAkt, and increased PARP cleavage compared to either treatment alone. This study suggests the potential for a novel combination approach which may ultimately be of value in treatment of pancreatic cancer.

  13. Knock-down of heat-shock protein 90 and isocitrate lyase gene expression reduced root-knot nematode reproduction.

    PubMed

    Lourenço-Tessutti, Isabela Tristan; Souza Junior, José Dijair Antonino; Martins-de-Sa, Diogo; Viana, Antônio Américo Barbosa; Carneiro, Regina Maria Dechechi Gomes; Togawa, Roberto Coiti; de Almeida-Engler, Janice; Batista, João Aguiar Nogueira; Silva, Maria Cristina Mattar; Fragoso, Rodrigo Rocha; Grossi-de-Sa, Maria Fatima

    2015-05-01

    Crop losses caused by nematode infections are estimated to be valued at USD 157 billion per year. Meloidogyne incognita, a root-knot nematode (RKN), is considered to be one of the most important plant pathogens due to its worldwide distribution and the austere damage it can cause to a large variety of agronomically important crops. RNA interference (RNAi), a gene silencing process, has proven to be a valuable biotechnology alternative method for RKN control. In this study, the RNAi approach was applied, using fragments of M. incognita genes that encode for two essential molecules, heat-shock protein 90 (HSP90) and isocitrate lyase (ICL). Plant-mediated RNAi of these genes led to a significant level of resistance against M. incognita in the transgenic Nicotiana tabacum plants. Bioassays of plants expressing HSP90 dsRNA demonstrated a delay in gall formation and up to 46% reduction in eggs compared with wild-type plants. A reduction in the level of HSP90 transcripts was observed in recovered eggs from plants expressing dsRNA, indicating that gene silencing persisted and was passed along to first progeny. The ICL knock-down had no clear effect on gall formation but resulted in up to 77% reduction in egg oviposition compared with wild-type plants. Our data suggest that both genes may be involved in RKN development and reproduction. Thus, in this paper, we describe essential candidate genes that could be applied to generate genetically modified crops, using the RNAi strategy to control RKN parasitism.

  14. REST/NRSF Knockdown Alters Survival, Lineage Differentiation and Signaling in Human Embryonic Stem Cells.

    PubMed

    Thakore-Shah, Kaushali; Koleilat, Tasneem; Jan, Majib; John, Alan; Pyle, April D

    2015-01-01

    REST (RE1 silencing transcription factor), also known as NRSF (neuron-restrictive silencer factor), is a well-known transcriptional repressor of neural genes in non-neural tissues and stem cells. Dysregulation of REST activity is thought to play a role in diverse diseases including epilepsy, cancer, Down's syndrome and Huntington's disease. The role of REST/NRSF in control of human embryonic stem cell (hESC) fate has never been examined. To evaluate the role of REST in hESCs we developed an inducible REST knockdown system and examined both growth and differentiation over short and long term culture. Interestingly, we have found that altering REST levels in multiple hESC lines does not result in loss of self-renewal but instead leads to increased survival. During differentiation, REST knockdown resulted in increased MAPK/ERK and WNT signaling and increased expression of mesendoderm differentiation markers. Therefore we have uncovered a new role for REST in regulation of growth and early differentiation decisions in human embryonic stem cells. PMID:26690059

  15. The Exocyst Protein Sec10 Interacts with Polycystin-2 and Knockdown Causes PKD-Phenotypes

    PubMed Central

    Zuo, Xiaofeng; Jaffe, Kimberly M.; Park, Kwon Moo; Reichert, Ryan J.; Bell, P. Darwin; Burdine, Rebecca D.; Lipschutz, Joshua H.

    2011-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown—including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation—suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes. PMID:21490950

  16. Knockdown of Pentraxin 3 suppresses tumorigenicity and metastasis of human cervical cancer cells

    PubMed Central

    Ying, Tsung-Ho; Lee, Chien-Hsing; Chiou, Hui-Ling; Yang, Shun-Fa; Lin, Chu-Liang; Hung, Chia-Hung; Tsai, Jen-Pi; Hsieh, Yi-Hsien

    2016-01-01

    Pentraxin 3 (PTX3) as an inflammatory molecule has been shown to be involved in immune response, inflammation, and cancer. However, the effects of PTX3 on the biological features of cervical cancer cells in vitro and in vivo have not been delineated. Immunohistochemical staining showed that increased PTX3 expression was significantly associated with tumor grade (P < 0.011) and differentiation (P < 0.019). Knocking down PTX3 with lentivirus-mediated small hairpin RNA (shRNA) in cervical cancer cell lines resulted in inhibited cell viability, diminished colony-forming ability, and induced cell cycle arrest at the G2/M phase of the cell cycle, along with downregulated expression of cyclin B1, cdc2, and cdc25c, and upregulated expression of p-cdc2, p-cdc25c, p21, and p27. Furthermore, knockdown of PTX3 significantly decreased the potential of migration and invasion of cervical cancer cells by inhibiting matrix metalloproteidase-2 (MMP-2), MMP-9, and urokinase plasminogen activator (uPA). Moreover, in vivo functional studies showed PTX3-knockdown in mice suppressed tumorigenicity and lung metastatic potential. Conversely, overexpression of PTX3 enhanced proliferation and invasion both in vitro and in vivo. Our results demonstrated that PTX3 contributes to tumorigenesis and metastasis of human cervical cancer cells. Further studies are warranted to demonstrate PTX3 as a novel therapeutic biomarker for human cervical cancer. PMID:27377307

  17. Deiodinase knockdown affects zebrafish eye development at the level of gene expression, morphology and function.

    PubMed

    Houbrechts, Anne M; Vergauwen, Lucia; Bagci, Enise; Van Houcke, Jolien; Heijlen, Marjolein; Kulemeka, Bernard; Hyde, David R; Knapen, Dries; Darras, Veerle M

    2016-03-15

    Retinal development in vertebrates relies extensively on thyroid hormones. Their local availability is tightly controlled by several regulators, including deiodinases (Ds). Here we used morpholino technology to explore the roles of Ds during eye development in zebrafish. Transcriptome analysis at 3 days post fertilization (dpf) revealed a pronounced effect of knockdown of both T4-activating Ds (D1D2MO) or knockdown of T3-inactivating D3 (D3bMO) on phototransduction and retinoid recycling. This was accompanied by morphological defects (studied from 1 to 7 dpf) including reduced eye size, disturbed retinal lamination and strong reduction in rods and all four cone types. Defects were more prominent and persistent in D3-deficient fish. Finally, D3-deficient zebrafish larvae had disrupted visual function at 4 dpf and were less sensitive to a light stimulus at 5 dpf. These data demonstrate the importance of TH-activating and -inactivating Ds for correct zebrafish eye development, and point to D3b as a central player. PMID:26802877

  18. Gene expression profiling of NB4 cells following knockdown of nucleostemin using DNA microarrays

    PubMed Central

    SUN, XIAOLI; JIA, YU; WEI, YUANYU; LIU, SHUAI; YUE, BAOHONG

    2016-01-01

    Nucleostemin (NS) is mainly expressed in stem and tumor cells, and is necessary for the maintenance of their self-renewal and proliferation. Originally, NS was thought to exert its effects through inhibiting p53, while recent studies have revealed that NS is also able to function independently of p53. The present study performed a gene expression profiling analysis of p53-mutant NB4 leukeima cells following knockdown of NS in order to elucidate the p53-independent NS pathway. NS expression was silenced using lentivirus-mediated RNA interference technology, and gene expression profiling of NB4 cells was performed by DNA microarray analysis. A total of 1,953 genes were identified to be differentially expressed (fold change ≥2 or ≤0.5) following knockdown of NS expression. Furthermore, reverse-transcription quantitative polymerase chain reaction analysis was used to detect the expression of certain candidate genes, and the results were in agreement with the micaroarray data. Pathway analysis indicated that aberrant genes were enhanced in endoplasmic, c-Jun N-terminal kinase and mineral absorption pathways. The present study shed light on the mechanisms of the p54-independent NS pathway in NB4 cells and provided a foundation for the discovery of promising targets for the treatment of p53-mutant leukemia. PMID:27374947

  19. Knockdown of TRAF4 expression suppresses osteosarcoma cell growth in vitro and in vivo.

    PubMed

    Yao, Weitao; Wang, Xin; Cai, Qiqing; Gao, Songtao; Wang, Jiaqiang; Zhang, Peng

    2014-12-01

    Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is an adapter molecule that is overexpressed in certain cancers. TRAF4 is overexpressed in osteosarcoma tissues and osteosarcoma cells. Using the technique of RNA interference, the expression of TRAF4 in the human osteosarcoma Saos-2 cell line was shown to be downregulated. The proliferation, cell cycle arrest and apoptosis ability of Saos‑2 cells were examined, as was tumor development in a xenograft mouse model. The results showed that the TRAF4 knockdown exerts inhibitory effects on the proliferation ability of Saos-2 cells and tumor development in a xenograft mouse model. Simultaneously, it was found that TRAF4 knockdown led to cell cycle arrest in the G1 phase and promoted Saos-2 cell apoptosis. Following TNF-α treatment, the expression of nuclear factor κB was significantly reduced in the TRAF4‑small interfering RNA group. These results indicate that TRAF4 regulated osteosarcoma cell growth in vitro and in vivo, and offers a candidate molecular target for osteosarcoma prevention and therapy. PMID:25270078

  20. Effects of Buckling Knockdown Factor, Internal Pressure and Material on the Design of Stiffened Cylinders

    NASA Technical Reports Server (NTRS)

    Lovejoy, Andrew E.; Hilburger, Mark W.; Chunchu, Prasad B.

    2010-01-01

    A design study was conducted to investigate the effect shell buckling knockdown factor (SBKF), internal pressure and aluminum alloy material selection on the structural weight of stiffened cylindrical shells. Two structural optimization codes were used for the design study to determine the optimum minimum-weight design for a series of design cases, and included an in-house developed genetic algorithm (GA) code and PANDA2. Each design case specified a unique set of geometry, material, knockdown factor combinations and loads. The resulting designs were examined and compared to determine the effects of SBKF, internal pressure and material selection on the acreage design weight and controlling failure mode. This design study shows that use of less conservative SBKF values, including internal pressure, and proper selection of material alloy can result in significant weight savings for stiffened cylinders. In particular, buckling-critical cylinders with integrally machined stiffener construction can benefit from the use of thicker plate material that enables taller stiffeners, even when the stiffness, strength and density properties of these materials appear to be inferior.

  1. Raman spectroscopic study of keratin 8 knockdown oral squamous cell carcinoma derived cells

    NASA Astrophysics Data System (ADS)

    Singh, S. P.; Alam, Hunain; Dmello, Crismita; Vaidya, Milind M.; Krishna, C. Murali

    2012-03-01

    Keratins are one of most widely used markers for oral cancers. Keratin 8 and 18 are expressed in simple epithelia and perform both mechanical and regulatory functions. Their expression are not seen in normal oral tissues but are often expressed in oral squamous cell carcinoma. Aberrant expression of keratins 8 and 18 is most common change in human oral cancer. Optical-spectroscopic methods are sensitive to biochemical changes and being projected as novel diagnostic tools for cancer diagnosis. Aim of this study was to evaluate potentials of Raman spectroscopy in detecting minor changes associated with differential level of keratin expression in tongue-cancer-derived AW13516 cells. Knockdown clones for K8 were generated and synchronized by growing under serum-free conditions. Cell pellets of three independent experiments in duplicate were used for recording Raman spectra with fiberoptic-probe coupled HE-785 Raman-instrument. A total of 123 and 96 spectra from knockdown clones and vector controls respectively in 1200-1800 cm-1 region were successfully utilized for classification using LDA. Two separate clusters with classification-efficiency of ~95% were obtained. Leave-one-out cross-validation yielded ~63% efficiency. Findings of the study demonstrate the potentials of Raman spectroscopy in detecting even subtle changes such as variations in keratin expression levels. Future studies towards identifying Raman signals from keratin in oral cells can help in precise cancer diagnosis.

  2. Inducible Knockdown of Plasmodium Gene Expression Using the glmS Ribozyme

    PubMed Central

    Prommana, Parichat; Uthaipibull, Chairat; Wongsombat, Chayaphat; Kamchonwongpaisan, Sumalee; Yuthavong, Yongyuth; Knuepfer, Ellen; Holder, Anthony A.; Shaw, Philip J.

    2013-01-01

    Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3′ untranslated region are efficiently knocked down in transgenic P. falciparum parasites in response to glucosamine inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme may thus be a tool for study of essential genes in P. falciparum and other parasite species amenable to transfection. PMID:24023691

  3. Paraquat exposure and Sod2 knockdown have dissimilar impacts on the Drosophila melanogaster carbonylated protein proteome

    PubMed Central

    Narayanasamy, Suresh K.; Simpson, David C.; Martin, Ian; Grotewiel, Mike; Gronert, Scott

    2014-01-01

    Exposure to Paraquat and RNA interference knockdown of Mn or mitochondrial superoxide dismutase (Sod2) are known to result in significant lifespan reduction, locomotor dysfunction, and mitochondrial degeneration in Drosophila melanogaster. Both perturbations increase the flux of the progenitor ROS, superoxide, but the molecular underpinnings of the resulting phenotypes are poorly understood. Improved understanding of such processes could lead to advances in the treatment of numerous age-related disorders. Superoxide toxicity can act through protein carbonylation. Analysis of carbonylated proteins is attractive since carbonyl groups are not present in the twenty canonical amino acids and are amenable to labeling and enrichment strategies. Here, carbonylated proteins were labeled with biotin hydrazide and enriched on streptavidin beads. On-bead digestion was used to release carbonylated protein peptides, with relative abundance ratios versus controls obtained using the iTRAQ MS-based proteomics approach. Western blotting and biotin quantitation assay approaches were also investigated. By both western blotting and proteomics, Paraquat exposure, but not Sod2 knockdown, resulted in increased carbonylated protein relative abundance. For Paraquat exposure versus control, the median carbonylated protein relative abundance ratio (1.53) determined using MS-based proteomics was in good agreement with that obtained using a commercial biotin quantitation kit (1.36). PMID:25091824

  4. CXCL5 knockdown expression inhibits human bladder cancer T24 cells proliferation and migration

    SciTech Connect

    Zheng, Jiajia; Zhu, Xi; Zhang, Jie

    2014-03-28

    Highlights: • We first demonstrated CXCL5 is highly expressed in human bladder tumor tissues and cells. • CXCL5 knockdown inhibits proliferation, migration and promotes apoptosis in T24 cells. • CXCL5 knockdown inhibits Snail, PI3K-AKT and ERK1/2 signaling pathways in T24 cells. • CXCL5 is critical for bladder tumor growth and progression. - Abstract: CXCL5 (epithelial neutrophil activating peptide-78) which acts as a potent chemoattractant and activator of neutrophil function was reported to play a multifaceted role in tumorigenesis. To investigate the role of CXCL5 in bladder cancer progression, we examined the CXCL5 expression in bladder cancer tissues by real-time PCR and Western blot, additionally, we used shRNA-mediated silencing to generate stable CXCL5 silenced bladder cancer T24 cells and defined its biological functions. Our results demonstrated that mRNA and protein of CXCL5 is increased in human bladder tumor tissues and cell lines, down-regulation of CXCL5 in T24 cells resulted in significantly decreased cell proliferation, migration and increased cell apoptosis in vitro through Snail, PI3K-AKT and ERK1/2 signaling pathways. These data suggest that CXCL5 is critical for bladder tumor growth and progression, it may represent a potential application in cancer diagnosis and therapy.

  5. Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells.

    PubMed

    Ikon, Nikita; Su, Betty; Hsu, Fong-Fu; Forte, Trudy M; Ryan, Robert O

    2015-08-21

    The concentration and composition of cardiolipin (CL) in mitochondria are altered in age-related heart disease, Barth Syndrome, and other rare genetic disorders, resulting in mitochondrial dysfunction. To explore whether exogenous CL can be delivered to cells, CL was combined with apolipoprotein A-I to generate water-soluble, nanoscale complexes termed nanodisks (ND). Mass spectrometry of HL60 myeloid progenitor cell extracts revealed a 30-fold increase in cellular CL content following incubation with CL-ND. When CL-ND containing a fluorescent CL analogue was employed, confocal microscopy revealed CL localization to mitochondria. The ability of CL-ND to elicit a physiological response was examined in an HL60 cell culture model of Barth Syndrome neutropenia. siRNA knockdown of the phospholipid transacylase, tafazzin (TAZ), induced apoptosis in these cells. When TAZ knockdown cells were incubated with CL-ND, the apoptotic response was attenuated. Thus, CL-ND represent a potential intervention strategy for replenishment of CL in Barth Syndrome, age-related heart disease, and other disorders characterized by depletion of this key mitochondrial phospholipid.

  6. Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

    SciTech Connect

    Byrne, Ann; McLaren, Rajashree P.; Mason, Paul; Chai, Lilly; Dufault, Michael R.; Huang, Yinyin; Liang, Beirong; Gans, Joseph D.; Zhang, Mindy; Carter, Kara; Gladysheva, Tatiana B.; Teicher, Beverly A.; Biemann, Hans-Peter N.; Booker, Michael; Goldberg, Mark A.; Klinger, Katherine W.; Lillie, James; Madden, Stephen L.; Jiang, Yide

    2010-01-15

    The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21{sup /Cip} and p27{sup /Kip1}. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

  7. Knockdown of linc-UFC1 suppresses proliferation and induces apoptosis of colorectal cancer

    PubMed Central

    Yu, T; Shan, T-D; Li, J-Y; Huang, C-Z; Wang, S-Y; Ouyang, H; Lu, X-J; Xu, J-H; Zhong, W; Chen, Q-K

    2016-01-01

    Long intergenic noncoding RNAs (lincRNAs) have important roles in biological functions, molecular mechanisms and prognostic values in colorectal cancer (CRC). In this context, the roles of linc-UFC1 remain to be elucidated. In this study, linc-UFC1 was overexpressed in CRC patient tissues and positively correlated with tumor grade, N stage and M stage. Inhibition of linc-UFC1 resulted in cell proliferation inhibition and G1 cell cycle arrest, which was mediated by cyclin D1, CDK4, Rb and phosphorylated Rb. In addition, inhibition of linc-UFC1 induced cell apoptosis through the intrinsic apoptosis signaling pathway, as evidenced by the activation of caspase-9 and caspase-3. An investigation of the signaling pathway revealed that the effects on proliferation and apoptosis following linc-UFC1 knockdown were mediated by suppression of β-catenin and activation of phosphorylated P38. Furthermore, the P38 inhibitor SB203580 could attenuate the apoptotic effect achieved by linc-UFC1 knockdown, confirming the involvement of P38 signaling in the induced apoptosis. Taken together, linc-UFC1 might have a critical role in pro-proliferation and anti-apoptosis in CRC by regulating the cell cycle, intrinsic apoptosis, and β-catenin and P38 signaling. Thus, linc-UFC1 could be a potential therapeutic target and novel molecular biomarker for CRC. PMID:27195675

  8. Knockdown of Pentraxin 3 suppresses tumorigenicity and metastasis of human cervical cancer cells.

    PubMed

    Ying, Tsung-Ho; Lee, Chien-Hsing; Chiou, Hui-Ling; Yang, Shun-Fa; Lin, Chu-Liang; Hung, Chia-Hung; Tsai, Jen-Pi; Hsieh, Yi-Hsien

    2016-01-01

    Pentraxin 3 (PTX3) as an inflammatory molecule has been shown to be involved in immune response, inflammation, and cancer. However, the effects of PTX3 on the biological features of cervical cancer cells in vitro and in vivo have not been delineated. Immunohistochemical staining showed that increased PTX3 expression was significantly associated with tumor grade (P < 0.011) and differentiation (P < 0.019). Knocking down PTX3 with lentivirus-mediated small hairpin RNA (shRNA) in cervical cancer cell lines resulted in inhibited cell viability, diminished colony-forming ability, and induced cell cycle arrest at the G2/M phase of the cell cycle, along with downregulated expression of cyclin B1, cdc2, and cdc25c, and upregulated expression of p-cdc2, p-cdc25c, p21, and p27. Furthermore, knockdown of PTX3 significantly decreased the potential of migration and invasion of cervical cancer cells by inhibiting matrix metalloproteidase-2 (MMP-2), MMP-9, and urokinase plasminogen activator (uPA). Moreover, in vivo functional studies showed PTX3-knockdown in mice suppressed tumorigenicity and lung metastatic potential. Conversely, overexpression of PTX3 enhanced proliferation and invasion both in vitro and in vivo. Our results demonstrated that PTX3 contributes to tumorigenesis and metastasis of human cervical cancer cells. Further studies are warranted to demonstrate PTX3 as a novel therapeutic biomarker for human cervical cancer.

  9. Zebrafish ambra1a and ambra1b knockdown impairs skeletal muscle development.

    PubMed

    Skobo, Tatjana; Benato, Francesca; Grumati, Paolo; Meneghetti, Giacomo; Cianfanelli, Valentina; Castagnaro, Silvia; Chrisam, Martina; Di Bartolomeo, Sabrina; Bonaldo, Paolo; Cecconi, Francesco; Dalla Valle, Luisa

    2014-01-01

    The essential role of autophagy in muscle homeostasis has been clearly demonstrated by phenotype analysis of mice with muscle-specific inactivation of genes encoding autophagy-related proteins. Ambra1 is a key component of the Beclin 1 complex and, in zebrafish, it is encoded by two paralogous genes, ambra1a and ambra1b, both required for normal embryogenesis and larval development. In this study we focused on the function of Ambra1, a positive regulator of the autophagic process, during skeletal muscle development by means of morpholino (MO)-mediated knockdown and compared the phenotype of zebrafish Ambra1-depleted embryos with that of Ambra1gt/gt mouse embryos. Morphological analysis of zebrafish morphant embryos revealed that silencing of ambra1 impairs locomotor activity and muscle development, as well as myoD1 expression. Skeletal muscles in ATG-morphant embryos displayed severe histopathological changes and contained only small areas of organized myofibrils that were widely dispersed throughout the cell. Double knockdown of ambra1a and ambra1b resulted in a more severe phenotype whereas defects were much less evident in splice-morphants. The morphants phenotypes were effectively rescued by co-injection with human AMBRA1 mRNA. Together, these results indicate that ambra1a and ambra1b are required for the correct development and morphogenesis of skeletal muscle. PMID:24922546

  10. REST/NRSF Knockdown Alters Survival, Lineage Differentiation and Signaling in Human Embryonic Stem Cells

    PubMed Central

    Thakore-Shah, Kaushali; Koleilat, Tasneem; Jan, Majib; John, Alan; Pyle, April D.

    2015-01-01

    REST (RE1 silencing transcription factor), also known as NRSF (neuron-restrictive silencer factor), is a well-known transcriptional repressor of neural genes in non-neural tissues and stem cells. Dysregulation of REST activity is thought to play a role in diverse diseases including epilepsy, cancer, Down’s syndrome and Huntington’s disease. The role of REST/NRSF in control of human embryonic stem cell (hESC) fate has never been examined. To evaluate the role of REST in hESCs we developed an inducible REST knockdown system and examined both growth and differentiation over short and long term culture. Interestingly, we have found that altering REST levels in multiple hESC lines does not result in loss of self-renewal but instead leads to increased survival. During differentiation, REST knockdown resulted in increased MAPK/ERK and WNT signaling and increased expression of mesendoderm differentiation markers. Therefore we have uncovered a new role for REST in regulation of growth and early differentiation decisions in human embryonic stem cells. PMID:26690059

  11. Acute sterol o-acyltransferase 2 (SOAT2) knockdown rapidly mobilizes hepatic cholesterol for fecal excretion.

    PubMed

    Marshall, Stephanie M; Gromovsky, Anthony D; Kelley, Kathryn L; Davis, Matthew A; Wilson, Martha D; Lee, Richard G; Crooke, Rosanne M; Graham, Mark J; Rudel, Lawrence L; Brown, J Mark; Temel, Ryan E

    2014-01-01

    The primary risk factor for atherosclerotic cardiovascular disease is LDL cholesterol, which can be reduced by increasing cholesterol excretion from the body. Fecal cholesterol excretion can be driven by a hepatobiliary as well as a non-biliary pathway known as transintestinal cholesterol efflux (TICE). We previously showed that chronic knockdown of the hepatic cholesterol esterifying enzyme sterol O-acyltransferase 2 (SOAT2) increased fecal cholesterol loss via TICE. To elucidate the initial events that stimulate TICE, C57Bl/6 mice were fed a high cholesterol diet to induce hepatic cholesterol accumulation and were then treated for 1 or 2 weeks with an antisense oligonucleotide targeting SOAT2. Within 2 weeks of hepatic SOAT2 knockdown (SOAT2HKD), the concentration of cholesteryl ester in the liver was reduced by 70% without a reciprocal increase in hepatic free cholesterol. The rapid mobilization of hepatic cholesterol stores resulted in a ∼ 2-fold increase in fecal neutral sterol loss but no change in biliary cholesterol concentration. Acute SOAT2HKD increased plasma cholesterol carried primarily in lipoproteins enriched in apoB and apoE. Collectively, our data suggest that acutely reducing SOAT2 causes hepatic cholesterol to be swiftly mobilized and packaged onto nascent lipoproteins that feed cholesterol into the TICE pathway for fecal excretion.

  12. Acute Sterol O-Acyltransferase 2 (SOAT2) Knockdown Rapidly Mobilizes Hepatic Cholesterol for Fecal Excretion

    PubMed Central

    Marshall, Stephanie M.; Gromovsky, Anthony D.; Kelley, Kathryn L.; Davis, Matthew A.; Wilson, Martha D.; Lee, Richard G.; Crooke, Rosanne M.; Graham, Mark J.; Rudel, Lawrence L.

    2014-01-01

    The primary risk factor for atherosclerotic cardiovascular disease is LDL cholesterol, which can be reduced by increasing cholesterol excretion from the body. Fecal cholesterol excretion can be driven by a hepatobiliary as well as a non-biliary pathway known as transintestinal cholesterol efflux (TICE). We previously showed that chronic knockdown of the hepatic cholesterol esterifying enzyme sterol O-acyltransferase 2 (SOAT2) increased fecal cholesterol loss via TICE. To elucidate the initial events that stimulate TICE, C57Bl/6 mice were fed a high cholesterol diet to induce hepatic cholesterol accumulation and were then treated for 1 or 2 weeks with an antisense oligonucleotide targeting SOAT2. Within 2 weeks of hepatic SOAT2 knockdown (SOAT2HKD), the concentration of cholesteryl ester in the liver was reduced by 70% without a reciprocal increase in hepatic free cholesterol. The rapid mobilization of hepatic cholesterol stores resulted in a ∼2-fold increase in fecal neutral sterol loss but no change in biliary cholesterol concentration. Acute SOAT2HKD increased plasma cholesterol carried primarily in lipoproteins enriched in apoB and apoE. Collectively, our data suggest that acutely reducing SOAT2 causes hepatic cholesterol to be swiftly mobilized and packaged onto nascent lipoproteins that feed cholesterol into the TICE pathway for fecal excretion. PMID:24901470

  13. Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells

    PubMed Central

    Ikon, Nikita; Su, Betty; Hsu, Fong-Fu; Forteand, Trudy M.; Ryan, Robert O.

    2015-01-01

    The concentration and composition of cardiolipin (CL) in mitochondria are altered in age-related heart disease, Barth Syndrome, and other rare genetic disorders, resulting in mitochondrial dysfunction. To explore whether exogenous CL can be delivered to cells, CL was combined with apolipoprotein A-I to generate water-soluble, nanoscale complexes termed nanodisks (ND). Mass spectrometry HL60 myeloid progenitor cell extracts revealed a 30-fold increase in cellular CL content following incubation with CL-ND. When CL-ND containing a fluorescent CL analogue was employed, confocal microscopy revealed CL localization to mitochondria. The ability of CL-ND to elicit a physiological response was examined in an HL60 cell culture model of Barth Syndrome neutropenia. siRNA knockdown of the phospholipid transacylase, tafazzin (TAZ), induced apoptosis in these cells. When TAZ knockdown cells were incubated with CL-ND, the apoptotic response was attenuated. Thus, CL-ND represent a potential intervention strategy for replenishment of CL in Barth Syndrome, age-related heart disease, and other disorders characterized by depletion of this key mitochondrial phospholipid. PMID:26164234

  14. Aquaporin 4 knockdown exacerbates streptozotocin-induced diabetic retinopathy through aggravating inflammatory response.

    PubMed

    Cui, Bei; Sun, Jin-Hua; Xiang, Fen-Fen; Liu, Lin; Li, Wen-Jie

    2012-05-01

    Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Diabetes is known to alter the amount of retinal expression of the water-selective channels aquaporin 4 (AQP4). However, the function and impact of AQP4 in diabetic retinopathy is not well understood. In the present work, diabetes was induced by intraperitoneal injection of streptozotocin in Sprague-Dawley rats. Two weeks later, AQP4 shRNA (r) lentiviral particles or negative lentiviral particles were delivered by intravitreal injection to the eyes. Gene delivery was confirmed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blotting analysis. Eight weeks later, BRB breakdown was measured using Evans blue dye. Images of retinal sections were obtained and the thicknesses of the retinas were determined. Retinal leukostasis measurement was performed using acridine orange leukocyte fluorography. The mRNA levels of IL-1β, IL-6, intercellular adhesion molecule 1 (ICAM-1), glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) were determined using qRT-PCR method. AQP4 shRNA (r) lentiviral particles or negative lentiviral particles were transfected into rMC-1 cells to investigate its effect on inflammation induced by high glucose. Incubation with IL-1β or IL-6 was performed to test their effect on AQP4 expression in rMC-1 cells. In the current work, it was found that AQP4 expression was enhanced in the retina of diabetic rats. AQP4 knockdown led to exacerbation of retinopathy including enhancing retinal vascular permeability, retinal thickness, pro-inflammatory factors expression, and VEGF and GFAP expression in retinas of diabetic rats. AQP4 knockdown enhanced the expression of pro-inflammatory cytokines induced by high glucose in rMC-1 cells. In addition, AQP4 knockdown enhanced the release of IL-6 and VEGF from rMC-1 cells into the medium. Moreover, it was found that incubation with IL-1β or IL-6 suppressed AQP4

  15. shRNA-Induced Gene Knockdown In Vivo to Investigate Neutrophil Function.

    PubMed

    Basit, Abdul; Tang, Wenwen; Wu, Dianqing

    2016-01-01

    To silence genes in neutrophils efficiently, we exploited the RNA interference and developed an shRNA-based gene knockdown technique. This method involves transfection of mouse bone marrow-derived hematopoietic stem cells with retroviral vector carrying shRNA directed at a specific gene. Transfected stem cells are then transplanted into irradiated wild-type mice. After engraftment of stem cells, the transplanted mice have two sets of circulating neutrophils. One set has a gene of interest knocked down while the other set has full complement of expressed genes. This efficient technique provides a unique way to directly compare the response of neutrophils with a knocked-down gene to that of neutrophils with the full complement of expressed genes in the same environment. PMID:27271902

  16. Identification of Genetic Suppressors of the Sin3A Knockdown Wing Phenotype

    PubMed Central

    Fox, Stephanie; Gammouh, Sarah; Pile, Lori A.

    2012-01-01

    The role of the Sin3A transcriptional corepressor in regulating the cell cycle is established in various metazoans. Little is known, however, about the signaling pathways that trigger or are triggered by Sin3A function. To discover genes that work in similar or opposing pathways to Sin3A during development, we have performed an unbiased screen of deficiencies of the Drosophila third chromosome. Additionally, we have performed a targeted loss of function screen to identify cell cycle genes that genetically interact with Sin3A. We have identified genes that encode proteins involved in regulation of gene expression, signaling pathways and cell cycle that can suppress the curved wing phenotype caused by the knockdown of Sin3A. These data indicate that Sin3A function is quite diverse and impacts a wide variety of cellular processes. PMID:23166712

  17. Early protective role of MST1 knockdown in response to experimental diabetic nephropathy

    PubMed Central

    Wu, Weihua; Zhang, Maoping; Ou, Santao; Liu, Xing; Xue, Ling; Liu, Jian; Wu, Yuke; Li, Ying; Liu, Qi

    2016-01-01

    Diabetic nephropathy (DN) is a progressive kidney disease caused by the damage of capillaries in kidney’s glomeruli. Mammalian Sterile 20-like kinase 1 (MST1) has been reported to play an important role in many disease, such as diabetes, cardiac disease and ect. However, the potential role of MST1 pathway in DN has not been fully evaluated. In this study, we hypothesized that MST1 could be involved in DN, and MST1 knockdown would attenuate the DN injury in experimental diabetic nephropathy induced by streptozotocin (STZ). The sieving method was used to generate primary cultures of rat podocytes, and cultured according to the previous reports. The clinical data were analyzed for vein specimens from ESRD. Real-time quantitative PCR was used to examine the mRNA levels. Immuno-fluorescence assay was used for primary podocyte in vitro. Lectrophoretic mobility shift assay was used for DNA binding activity of NF-κB. HE staining for histological examination and western blot assay for protein expression were employed. The average GBM thickness (GBMT) was measured By using the electron microscopy. In vitro, MST1 level increased significantly in primary rat podocyte cultured in hyperglycemia condition. In vivo experiment, diabetes induced by a single STZ injection (50 mg/kg) in SD rats. Knockdown of MST1 expression by lentiviral mediated gene transfer partly reduced the proteinuria and the level of FASL, and improved the pathological changes of the diabetic kidney. In conclusion, the MST1 could be involved in DN pathogenesis and may serve as the target for development of new therapies for DN. PMID:27186267

  18. RNAi knockdown of fatty acid elongase1 alters fatty acid composition in Brassica napus.

    PubMed

    Shi, Jianghua; Lang, Chunxiu; Wu, Xuelong; Liu, Renhu; Zheng, Tao; Zhang, Dongqing; Chen, Jinqing; Wu, Guanting

    2015-10-23

    The quality and end-use of oil from oilseed crops is determined by its fatty acid composition. In particular, the relative proportions of erucic and oleic acids are key selection traits for breeders. The goal of our research is to genetically improve the nutritional quality of Brassica napus cultivar CY2, the oil of which is high in erucic acid (about 40%) and low in oleic acid (about 20%). Here, we report the use of a seed-specific napin A promoter to drive the knockdown of BnFAE1 in transgenic CY2. Southern blotting results confirmed the presence of the transgene. RT-PCR analysis showed that the levels of BnFAE1 were greatly decreased in BnFAE1-Ri lines compared with the CY2 cultivar. Knockdown of BnFAE1 sharply decreased the levels of erucic acid (less than 3%), largely increased the contents of oleic acid (more than 60%) and slightly increased the polyunsaturated chain fatty acids. Compared with high erucic acid parents, expression of BnFAE1 was dramatically decreased in developing F1 seeds derived from reciprocally crossed BnFAE1-Ri lines and high erucic acid cultivars. In addition, F1 seeds derived from reciprocal crosses between BnFAE1-Ri lines and high erucic acid cultivars showed significantly increased oleic acid (more than 52%) and sharply decreased erucic acid (less than 4%), demonstrating that the RNAi construct of BnFAE1 can effectively interfere with the target gene in F1 seeds. Taken together, our results demonstrate that BnFAE1 is a reliable target for genetic improvement of rapeseed in seed oil quality promotion.

  19. Knockdown of the Cell Cycle Inhibitor p21 Enhances Cartilage Formation by Induced Pluripotent Stem Cells

    PubMed Central

    Diekman, Brian O.; Thakore, Pratiksha I.; O'Connor, Shannon K.; Willard, Vincent P.; Brunger, Jonathan M.; Christoforou, Nicolas; Leong, Kam W.

    2015-01-01

    The limited regenerative capacity of articular cartilage contributes to progressive joint dysfunction associated with cartilage injury or osteoarthritis. Cartilage tissue engineering seeks to provide a biological substitute for repairing damaged or diseased cartilage, but requires a cell source with the capacity for extensive expansion without loss of chondrogenic potential. In this study, we hypothesized that decreased expression of the cell cycle inhibitor p21 would enhance the proliferative and chondrogenic potential of differentiated induced pluripotent stem cells (iPSCs). Murine iPSCs were directed to differentiate toward the chondrogenic lineage with an established protocol and then engineered to express a short hairpin RNA (shRNA) to reduce the expression of p21. Cells expressing the p21 shRNA demonstrated higher proliferative potential during monolayer expansion and increased synthesis of glycosaminoglycans (GAGs) in pellet cultures. Furthermore, these cells could be expanded ∼150-fold over three additional passages without a reduction in the subsequent production of GAGs, while control cells showed reduced potential for GAG synthesis with three additional passages. In pellets from extensively passaged cells, knockdown of p21 attenuated the sharp decrease in cell number that occurred in control cells, and immunohistochemical analysis showed that p21 knockdown limited the production of type I and type X collagen while maintaining synthesis of cartilage-specific type II collagen. These findings suggest that manipulating the cell cycle can augment the monolayer expansion and preserve the chondrogenic capacity of differentiated iPSCs, providing a strategy for enhancing iPSC-based cartilage tissue engineering. PMID:25517798

  20. Knockdown of the cell cycle inhibitor p21 enhances cartilage formation by induced pluripotent stem cells.

    PubMed

    Diekman, Brian O; Thakore, Pratiksha I; O'Connor, Shannon K; Willard, Vincent P; Brunger, Jonathan M; Christoforou, Nicolas; Leong, Kam W; Gersbach, Charles A; Guilak, Farshid

    2015-04-01

    The limited regenerative capacity of articular cartilage contributes to progressive joint dysfunction associated with cartilage injury or osteoarthritis. Cartilage tissue engineering seeks to provide a biological substitute for repairing damaged or diseased cartilage, but requires a cell source with the capacity for extensive expansion without loss of chondrogenic potential. In this study, we hypothesized that decreased expression of the cell cycle inhibitor p21 would enhance the proliferative and chondrogenic potential of differentiated induced pluripotent stem cells (iPSCs). Murine iPSCs were directed to differentiate toward the chondrogenic lineage with an established protocol and then engineered to express a short hairpin RNA (shRNA) to reduce the expression of p21. Cells expressing the p21 shRNA demonstrated higher proliferative potential during monolayer expansion and increased synthesis of glycosaminoglycans (GAGs) in pellet cultures. Furthermore, these cells could be expanded ∼150-fold over three additional passages without a reduction in the subsequent production of GAGs, while control cells showed reduced potential for GAG synthesis with three additional passages. In pellets from extensively passaged cells, knockdown of p21 attenuated the sharp decrease in cell number that occurred in control cells, and immunohistochemical analysis showed that p21 knockdown limited the production of type I and type X collagen while maintaining synthesis of cartilage-specific type II collagen. These findings suggest that manipulating the cell cycle can augment the monolayer expansion and preserve the chondrogenic capacity of differentiated iPSCs, providing a strategy for enhancing iPSC-based cartilage tissue engineering.

  1. Dynamics of mitochondrial Ca2+ uptake in MICU1-knockdown cells.

    PubMed

    de la Fuente, Sergio; Matesanz-Isabel, Jessica; Fonteriz, Rosalba I; Montero, Mayte; Alvarez, Javier

    2014-02-15

    MICU1 (Ca2+ uptake protein 1, mitochondrial) is an important regulator of the MCU (Ca2+ uniporter protein, mitochondrial) that has been shown recently to act as a gatekeeper of the MCU at low [Ca2+]c (cytosolic [Ca2+]). In the present study we have investigated in detail the dynamics of MCU activity after shRNA-knockdown of MICU1 and we have found several new interesting properties. In MICU1-knockdown cells, the rate of mitochondrial Ca2+ uptake was largely increased at a low [Ca2+]c (<2 μM), but it was decreased at a high [Ca2+]c (>4 μM). In the 2-4 μM range a mixed behaviour was observed, where mitochondrial Ca2+ uptake started earlier in the MICU1-silenced cells, but at a lower rate than in the controls. The sensitivity of Ca2+ uptake to Ruthenium Red and Ru360 was similar at both high and low [Ca2+]c, indicating that the same Ca2+ pathway was operating in both cases. The increased Ca2+-uptake rate observed at a [Ca2+]c below 2 μM was transient and became inhibited during Ca2+ entry. Development of this inhibition was slow, requiring 5 min for completion, and was hardly reversible. Therefore MICU1 acts both as a MCU gatekeeper at low [Ca2+]c and as a cofactor necessary to reach the maximum Ca2+-uptake rate at high [Ca2+]c. Moreover, in the absence of MICU1, the MCU becomes sensitive to a slow-developing inhibition that requires prolonged increases in [Ca2+]c in the low micromolar range.

  2. [Knockdown of PRDX6 in microglia reduces neuron viability after OGD/R injury].

    PubMed

    Tan, Li; Zhao, Yong; Jiang, Beibei; Yang, Bo; Zhang, Hui

    2016-08-01

    Objective To observe the effects of peroxiredoxin 6 (PRDX6) knockdown in the microglia on neuron viability after oxygen-glucose deprivation and reoxygenation (OGD/R). Methods Microglia was treated with lentivirus PRDX6-siRNA and Ca(2+)-independent phospholipase A2 (iPLA2) inhibitor, 1-hexadecyl-3-(trifluoroethgl)-sn-glycerol-2 phosphomethanol (MJ33). Twenty-four hours later, it was co-cultured with primary neuron to establish the microglia-neuron co-culture OGD/R model. According to the different treatment of microglia, the cells were divided into normal group, OGD/R group, negative control-siRNA treated OGD/R group, PRDX6-siRNA treated OGD/R group and PRDX6-siRNA combined with MJ33 treated OGD/R group. Western blot analysis and real-time quantitative PCR were respectively performed to detect PRDX6 protein and mRNA levels after knockdown of PRDX6 in microglia. The iPLA2 activity was measured by ELISA. MTS and lactate dehydrogenase (LDH) assay were used to measure neuron viability and cell damage. The oxidative stress level of neuron was determined by measuring superoxide dismutase (SOD) and malonaldehyde (MDA) content. Results In PRDX6-siRNA group, neuron viability was inhibited and oxidative stress damage was aggravated compared with OGD/R group. In PRDX6-siRNA combined with MJ33 group, cell viability was promoted and oxidative stress damage was alleviated compared with PRDX6-siRNA group. Conclusion PRDX6 in microglia protects neuron against OGD/R-induced injury, and iPLA2 activity has an effect on PRDX6. PMID:27412928

  3. Morpholino oligonucleotide knockdown of the extracellular calcium-sensing receptor impairs early skeletal development in zebrafish.

    PubMed

    Herberger, Amanda L; Loretz, Christopher A

    2013-11-01

    The complex vertebrate skeleton depends on regulated cell activities to lay down protein matrix and mineral components of bone. As a distinctive vertebrate characteristic, bone is a storage site for physiologically-important calcium ion. The extracellular calcium-sensing receptor (CaSR) is linked to homeostatic regulation of calcium through its expression in endocrine glands that secrete calcium homeostatic hormones, in Ca(2+)- and ion-transporting epithelia, and in skeleton. Since CaSR is restricted in its presence to the chordate-vertebrate evolutionary lineage, we propose there to be important functional ties between CaSRs and vertebrate skeleton in the context of that group's characteristic form of calcium-mineralized skeleton. Since little is known about CaSR in the skeletal biology of non-mammalian vertebrates, reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization and immunohistochemistry were applied to adult and embryonic zebrafish to reveal CaSR transcript and protein expression in several tissues, including, among these, chondrocytes and developing bone and notochord as components in skeletal development. Morpholino oligonucleotide (MO) knockdown technique was used to probe CaSR role(s) in the zebrafish model system. By RT-PCR assessment, injection of a splice-inhibiting CaSR MO reduced normally-spliced Casr gene transcript expression measured at 2days postfertilization (dpf). Corresponding to the knockdown of normally-spliced mRNA by the CaSR MO, we observed a morphant phenotype characterized by stunted growth and disorganization of the notochord and axial skeleton by 1dpf. We conclude that, like its critically important role in normal bone development in mammals, CaSR is essential in skeletogenesis in fishes.

  4. Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors

    PubMed Central

    Reddy, B. K. Kishore; Landge, Sudhir; Ravishankar, Sudha; Patil, Vikas; Shinde, Vikas; Tantry, Subramanyam; Kale, Manoj; Raichurkar, Anandkumar; Menasinakai, Sreenivasaiah; Mudugal, Naina Vinay; Ambady, Anisha; Ghosh, Anirban; Tunduguru, Ragadeepthi; Kaur, Parvinder; Singh, Ragini; Kumar, Naveen; Bharath, Sowmya; Sundaram, Aishwarya; Bhat, Jyothi; Sambandamurthy, Vasan K.; Björkelid, Christofer; Jones, T. Alwyn; Das, Kaveri; Bandodkar, Balachandra; Malolanarasimhan, Krishnan; Mukherjee, Kakoli

    2014-01-01

    Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis. PMID:24687493

  5. Knockdown of PKM2 Suppresses Tumor Growth and Invasion in Lung Adenocarcinoma.

    PubMed

    Sun, Hong; Zhu, Anyou; Zhang, Lunjun; Zhang, Jie; Zhong, Zhengrong; Wang, Fengchao

    2015-01-01

    Accumulating evidence shows that activity of the pyruvate kinase M2 (PKM2) isoform is closely related to tumorigenesis. In this study, we investigated the relationship between PKM2 expression, tumor invasion, and the prognosis of patients with lung adenocarcinoma. We retrospectively analyzed 65 cases of patients with lung adenocarcinoma who were divided into low and a high expression groups based on PKM2 immunohistochemical staining. High PKM2 expression was significantly associated with reduced patient survival. We used small interfering RNA (siRNA) technology to investigate the effect of targeted PKM2-knockout on tumor growth at the cellular level. In vitro, siRNA-mediated PKM2-knockdown significantly inhibited the proliferation, glucose uptake (25%), ATP generation (20%) and fatty acid synthesis of A549 cells, while the mitochondrial respiratory capacity of the cells increased (13%).Western blotting analysis showed that PKM2-knockout significantly inhibited the expression of the glucose transporter GLUT1 and ATP citrate lyase, which is critical for fatty acid synthesis. Further Western blotting analysis showed that PKM2-knockdown inhibited the expression of matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF), which are important in degradation of the extracellular matrix and angiogenesis, respectively. These observations show that PKM2 activates both glycolysis and lipid synthesis, thereby regulating cell proliferation and invasion. This information is important in elucidating the mechanisms by which PKM2 influences the growth and metastasis of lung adenocarcinoma at the cellular and molecular level, thereby providing the basic data required for the development of PKM2-targeted gene therapy.

  6. Beta tubulin isoforms are not interchangeable for rescuing impaired radial migration due to Tubb3 knockdown.

    PubMed

    Saillour, Yoann; Broix, Loïc; Bruel-Jungerman, Elodie; Lebrun, Nicolas; Muraca, Giuseppe; Rucci, Julien; Poirier, Karine; Belvindrah, Richard; Francis, Fiona; Chelly, Jamel

    2014-03-15

    Over the last years, the critical role of cytoskeletal proteins in cortical development including neuronal migration as well as in neuronal morphology has been well established. Inputs from genetic studies were provided through the identification of several mutated genes encoding either proteins associated with microtubules (DCX, LIS1, KIF2A, KIF5C, DYNC1H1) or tubulin subunits (TUBA1A, TUBB2B, TUBB5 and TUBG1), in malformations of cortical development (MCD). We also reported the identification of missense mutations in TUBB3, the postmitotic neuronal specific tubulin, in six different families presenting either polymicrogyria or gyral disorganization in combination with cerebellar and basal ganglial abnormalities. Here, we investigate further the association between TUBB3 mutations and MCDs by analyzing the consequences of Tubb3 knockdown on cortical development in mice. Using the in utero-electroporation approach, we demonstrate that Tubb3 knockdown leads to delayed bipolar morphology and radial migration with evidence, suggesting that the neuronal arrest is a transient phenomenon overcome after birth. Silenced blocked cells display a round-shape and decreased number of processes and a delay in the acquisition of the bipolar morphology. Also, more Tbr2 positive cells are observed, although less cells express the proliferation marker Ki67, suggesting that Tubb3 inactivation might have an indirect effect on intermediate progenitor proliferation. Furthermore, we show by rescue experiments the non-interchangeability of other beta-tubulins which are unable to rescue the phenotype. Our study highlights the critical and specific role of Tubb3 on the stereotyped morphological changes and polarization processes that are required for initiating radial migration to the cortical plate. PMID:24179174

  7. SIRT2 knockdown increases basal autophagy and prevents postslippage death by abnormally prolonging the mitotic arrest that is induced by microtubule inhibitors.

    PubMed

    Inoue, Toshiaki; Nakayama, Yuji; Li, Yanze; Matsumori, Haruka; Takahashi, Haruka; Kojima, Hirotada; Wanibuchi, Hideki; Katoh, Motonobu; Oshimura, Mitsuo

    2014-06-01

    Mitotic catastrophe, a form of cell death that occurs during mitosis and after mitotic slippage to a tetraploid state, plays important roles in the efficacy of cancer cell killing by microtubule inhibitors (MTIs). Prolonged mitotic arrest by the spindle assembly checkpoint is a well-known requirement for mitotic catastrophe, and thus for conferring sensitivity to MTIs. We previously reported that turning off spindle assembly checkpoint activation after a defined period of time is another requirement for efficient postslippage death from a tetraploid state, and we identified SIRT2, a member of the sirtuin protein family, as a regulator of this process. Here, we investigated whether SIRT2 regulates basal autophagy and whether, in that case, autophagy regulation by SIRT2 is required for postslippage death, by analogy with previous insights into SIRT1 functions in autophagy. We show, by combined knockdown of autophagy genes and SIRT2, that SIRT2 serves this function at least partially by suppressing basal autophagy levels. Notably, increased autophagy induced by rapamycin and mild starvation caused mitotic arrest for an abnormally long period of time in the presence of MTIs, and this was followed by delayed postslippage death, which was also observed in cells with SIRT2 knockdown. These results underscore a causal association among increased autophagy levels, mitotic arrest for an abnormally long period of time after exposure to MTIs, and resistance to MTIs. Although autophagy acts as a tumor suppressor mechanism, this study highlights its negative aspects, as increased autophagy may cause mitotic catastrophe malfunction. Thus, SIRT2 offers a novel target for tumor therapy. PMID:24712640

  8. N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification.

    PubMed

    Hossain, Md Motarab; Banik, Naren L; Ray, Swapan K

    2013-10-15

    Malignant neuroblastomas mostly occur in children and are frequently associated with N-Myc amplification. Oncogene amplification, which is selective increase in copy number of the oncogene, provides survival advantages in solid tumors including malignant neuroblastoma. We have decreased expression of N-Myc oncogene using short hairpin RNA (shRNA) plasmid to increase anti-tumor efficacy of the isoflavonoid apigenin (APG) in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines that harbor N-Myc amplification. N-Myc knockdown induced morphological and biochemical features of neuronal differentiation. Combination of N-Myc knockdown and APG most effectively induced morphological and biochemical features of apoptotic death. This combination therapy also prevented cell migration and decreased N-Myc driven survival, angiogenic, and invasive factors. Collectively, N-Myc knockdown and APG treatment is a promising strategy for controlling the growth of human malignant neuroblastoma cell lines that harbor N-Myc amplification.

  9. Knockdown of malic enzyme 2 suppresses lung tumor growth, induces differentiation and impacts PI3K/AKT signaling.

    PubMed

    Ren, Jian-Guo; Seth, Pankaj; Clish, Clary B; Lorkiewicz, Pawel K; Higashi, Richard M; Lane, Andrew N; Fan, Teresa W-M; Sukhatme, Vikas P

    2014-01-01

    Mitochondrial malic enzyme 2 (ME2) catalyzes the oxidative decarboxylation of malate to yield CO2 and pyruvate, with concomitant reduction of dinucleotide cofactor NAD(+) or NADP(+). We find that ME2 is highly expressed in many solid tumors. In the A549 non-small cell lung cancer (NSCLC) cell line, ME2 depletion inhibits cell proliferation and induces cell death and differentiation, accompanied by increased reactive oxygen species (ROS) and NADP(+)/NADPH ratio, a drop in ATP, and increased sensitivity to cisplatin. ME2 knockdown impacts phosphoinositide-dependent protein kinase 1 (PDK1) and phosphatase and tensin homolog (PTEN) expression, leading to AKT inhibition. Depletion of ME2 leads to malate accumulation and pyruvate decrease, and exogenous cell permeable dimethyl-malate (DMM) mimics the ME2 knockdown phenotype. Both ME2 knockdown and DMM treatment reduce A549 cell growth in vivo. Collectively, our data suggest that ME2 is a potential target for cancer therapy. PMID:24957098

  10. Knockdown of Nuclear-Located Enhancer RNAs and Long ncRNAs Using Locked Nucleic Acid GapmeRs.

    PubMed

    Roux, Benoit T; Lindsay, Mark A; Heward, James A

    2017-01-01

    The human genome is widely transcribed outside of protein-coding genes, producing thousands of noncoding RNAs from different subfamilies including enhancer RNAs. Functional studies to determine the role of individual genes are challenging with noncoding RNAs appearing to be more difficult to knockdown than mRNAs. One factor that may have hindered progress is that the majority of noncoding RNAs are thought to be located within the nucleus, where the efficiency of traditional RNA interference techniques is debatable. Here we present an alternative RNA interference technique utilizing Locked Nucleic Acids, which is able to efficiently knockdown noncoding RNAs irrespective of intracellular location. PMID:27662866

  11. NLRC5 knockdown in chicken macrophages alters response to LPS and poly (I:C) stimulation

    PubMed Central

    2012-01-01

    Background NLRC5 is a member of the CARD domain containing, nucleotide-binding oligomerization (NOD)-like receptor (NLR) family, which recognizes pathogen-associated molecular patterns (PAMPs) and initiates an innate immune response leading to inflammation and/or cell death. However, the specific role of NLRC5 as a modulator of the inflammatory immune response remains controversial. It has been reported to be a mediator of type I IFNs, NF-kB, and MHC class I gene. But no study on NLRC5 function has been reported to date in chickens. In the current study, we investigated the role of NLRC5 in the regulation of IFNA, IFNB, IL-6, and MHC class I in the chicken HD11 macrophage cell line, by using RNAi technology. HD11 cells were transfected with one of five siRNAs (s1, s2, s3, negative-siRNA, or a mixture of s1, s2, s3-siRNAs). After 24 hours, cells were exposed to LPS or poly (I:C) or a vehicle control. Gene expression of NLRC5, IFNA, IFNB, IL-6, and MHC class I at 2, 4, 6, and 8 hours post stimulation (hps) was quantified by qPCR. Results The expression of NLRC5, IFNA, IFNB, and IL-6 genes in negative irrelevant transfection controls was up-regulated at 2 hps after LPS treatment compared to the vehicle controls. S3-siRNA effectively knocked down NLRC5 expression at 4 hps, and the expression of IFNA and IFNB (but not IL-6 and MHC class I) was also down-regulated at 4 hps in s3-siRNA transfected cells, compared to negative irrelevant transfection controls. Stimulation by LPS appeared to relatively restore the decrease in NLRC5, IFNA, and IFNB expression, but the difference is not significant. Conclusions Functional characterization of chicken NLRC5 in an in vitro system demonstrated its importance in regulating intracellular molecules involved in inflammatory response. The knockdown of NLRC5 expression negatively mediates gene expression of IFNA and IFNB in the chicken HD11 cell line; therefore, NLRC5 likely has a role in positive regulation of IFNA and IFNB expression

  12. RNAi-mediated knockdown of IKK1 in transgenic mice using a transgenic construct containing the human H1 promoter.

    PubMed

    Moreno-Maldonado, Rodolfo; Murillas, Rodolfo; Navarro, Manuel; Page, Angustias; Suarez-Cabrera, Cristian; Alameda, Josefa P; Bravo, Ana; Casanova, M Llanos; Ramirez, Angel

    2014-01-01

    Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models. PMID:24523631

  13. Persistence of RNAi-Mediated Knockdown in Drosophila Complicates Mosaic Analysis Yet Enables Highly Sensitive Lineage Tracing.

    PubMed

    Bosch, Justin A; Sumabat, Taryn M; Hariharan, Iswar K

    2016-05-01

    RNA interference (RNAi) has emerged as a powerful way of reducing gene function in Drosophila melanogaster tissues. By expressing synthetic short hairpin RNAs (shRNAs) using the Gal4/UAS system, knockdown is efficiently achieved in specific tissues or in clones of marked cells. Here we show that knockdown by shRNAs is so potent and persistent that even transient exposure of cells to shRNAs can reduce gene function in their descendants. When using the FLP-out Gal4 method, in some instances we observed unmarked "shadow RNAi" clones adjacent to Gal4-expressing clones, which may have resulted from brief Gal4 expression following recombination but prior to cell division. Similarly, Gal4 driver lines with dynamic expression patterns can generate shadow RNAi cells after their activity has ceased in those cells. Importantly, these effects can lead to erroneous conclusions regarding the cell autonomy of knockdown phenotypes. We have investigated the basis of this phenomenon and suggested experimental designs for eliminating ambiguities in interpretation. We have also exploited the persistence of shRNA-mediated knockdown to design a sensitive lineage-tracing method, i-TRACE, which is capable of detecting even low levels of past reporter expression. Using i-TRACE, we demonstrate transient infidelities in the expression of some cell-identity markers near compartment boundaries in the wing imaginal disc. PMID:26984059

  14. Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana

    DOE PAGES

    Czarnecki, Olaf; Bryan, Anthony C.; Jawdy, Sara S.; Yang, Xiaohan; Cheng, Zong-Ming; Chen, Jin-Gui; Tuskan, Gerald A.

    2016-02-17

    Genetic engineering of plants that results in successful establishment of new biochemical or regulatory pathways requires stable introduction of one or more genes into the plant genome. It might also be necessary to down-regulate or turn off expression of endogenous genes in order to reduce activity of competing pathways. An established way to knockdown gene expression in plants is expressing a hairpin-RNAi construct, eventually leading to degradation of a specifically targeted mRNA. Knockdown of multiple genes that do not share homologous sequences is still challenging and involves either sophisticated cloning strategies to create vectors with different serial expression constructs ormore » multiple transformation events that is often restricted by a lack of available transformation markers. Synthetic RNAi fragments were assembled in yeast carrying homologous sequences to six or seven non-family genes and introduced into pAGRIKOLA. Transformation of Arabidopsis thaliana and subsequent expression analysis of targeted genes proved efficient knockdown of all target genes. In conclusion, we present a simple and cost-effective method to create constructs to simultaneously knockdown multiple non-family genes or genes that do not share sequence homology. The presented method can be applied in plant and animal synthetic biology as well as traditional plant and animal genetic engineering.« less

  15. Rescue of Impaired Long-Term Facilitation at Sensorimotor Synapses of Aplysia following siRNA Knockdown of CREB1

    PubMed Central

    Zhou, Lian; Zhang, Yili; Liu, Rong-Yu; Smolen, Paul; Cleary, Leonard J.

    2015-01-01

    Memory impairment is often associated with disrupted regulation of gene induction. For example, deficits in cAMP response element-binding protein (CREB) binding protein (CBP; an essential cofactor for activation of transcription by CREB) impair long-term synaptic plasticity and memory. Previously, we showed that small interfering RNA (siRNA)-induced knockdown of CBP in individual sensory neurons significantly reduced levels of CBP and impaired 5-HT-induced long-term facilitation (LTF) in sensorimotor cocultures from Aplysia. Moreover, computational simulations of the biochemical cascades underlying LTF successfully predicted training protocols that restored LTF following CBP knockdown. We examined whether simulations could also predict a training protocol that restores LTF impaired by siRNA-induced knockdown of the transcription factor CREB1. Simulations based on a previously described model predicted rescue protocols that were specific to CREB1 knockdown. Empirical studies demonstrated that one of these rescue protocols partially restored impaired LTF. In addition, the effectiveness of the rescue protocol was enhanced by pretreatment with rolipram, a selective cAMP phosphodiesterase inhibitor. These results provide further evidence that computational methods can help rescue disruptions in signaling cascades underlying memory formation. Moreover, the study demonstrates that the effectiveness of computationally designed training protocols can be enhanced with complementary pharmacological approaches. PMID:25632137

  16. miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction

    PubMed Central

    Guda, Swaroopa; Brendel, Christian; Renella, Raffaele; Du, Peng; Bauer, Daniel E; Canver, Matthew C; Grenier, Jennifer K; Grimson, Andrew W; Kamran, Sophia C; Thornton, James; de Boer, Helen; Root, David E; Milsom, Michael D; Orkin, Stuart H; Gregory, Richard I; Williams, David A

    2015-01-01

    RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNAmiR) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNAmiRs resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNAmiRs for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences. PMID:26080908

  17. Rescue of impaired long-term facilitation at sensorimotor synapses of Aplysia following siRNA knockdown of CREB1.

    PubMed

    Zhou, Lian; Zhang, Yili; Liu, Rong-Yu; Smolen, Paul; Cleary, Leonard J; Byrne, John H

    2015-01-28

    Memory impairment is often associated with disrupted regulation of gene induction. For example, deficits in cAMP response element-binding protein (CREB) binding protein (CBP; an essential cofactor for activation of transcription by CREB) impair long-term synaptic plasticity and memory. Previously, we showed that small interfering RNA (siRNA)-induced knockdown of CBP in individual sensory neurons significantly reduced levels of CBP and impaired 5-HT-induced long-term facilitation (LTF) in sensorimotor cocultures from Aplysia. Moreover, computational simulations of the biochemical cascades underlying LTF successfully predicted training protocols that restored LTF following CBP knockdown. We examined whether simulations could also predict a training protocol that restores LTF impaired by siRNA-induced knockdown of the transcription factor CREB1. Simulations based on a previously described model predicted rescue protocols that were specific to CREB1 knockdown. Empirical studies demonstrated that one of these rescue protocols partially restored impaired LTF. In addition, the effectiveness of the rescue protocol was enhanced by pretreatment with rolipram, a selective cAMP phosphodiesterase inhibitor. These results provide further evidence that computational methods can help rescue disruptions in signaling cascades underlying memory formation. Moreover, the study demonstrates that the effectiveness of computationally designed training protocols can be enhanced with complementary pharmacological approaches. PMID:25632137

  18. Combined antisense knockdown of type 1 and type 2 iodothyronine deiodinases disrupts embryonic development in zebrafish (Danio rerio).

    PubMed

    Walpita, Chaminda N; Crawford, Alexander D; Darras, Veerle M

    2010-03-01

    Thyroid hormones (THs) are important regulators of gene expression during vertebrate development. In teleosts, early embryos rely on the maternal TH deposit in the egg yolk, consisting predominantly of T(4). Activation of T(4) to T(3) by iodothyronine deiodinases (Ds) may therefore be an important factor in determining T(3)-dependent development. In zebrafish, both Ds capable of T(3) production, D1 and D2, are first expressed very early during embryonic development. We sought to determine their relative importance for zebrafish embryonic development by inhibiting their expression via antisense oligonucleotides against D1 and D2, and by a combined knockdown of both deiodinases. The impact of these treatments on the rate of embryonic development was estimated via three morphological indices: otic vesicle length, head-trunk angle and pigmentation index. Knockdown of D1 alone seemed not to affect developmental progression. In contrast, D2 knockdown resulted in a clear developmental delay in all parameters scored, suggesting that D2 is the major contributor to TH activation in developing zebrafish embryos. Importantly, combined knockdown of D1 and D2 caused not only a more pronounced developmental delay than D2 knockdown alone but also the appearance of dysmorphologies in a substantial minority of treated embryos. This shows that although D1 may not be essential in euthyroid conditions, it may be crucial under depleted thyroid status as is the case when T(3) production by D2 is inhibited. These results indicate that zebrafish embryos are dependent on T(4) uptake and its subsequent activation to T(3), and suggest that substantial inhibition of embryonic T(4) to T(3) conversion reduces intracellular T(3) availability below the threshold level necessary for normal development. PMID:19800339

  19. Knockdown of H19 enhances differentiation capacity to epidermis of parthenogenetic embryonic stem cells.

    PubMed

    Yin, Y; Wang, H; Liu, K; Wang, F; Ye, X; Liu, M; Xiang, R; Liu, N; Liu, L

    2014-01-01

    Parthenogenetic embryonic stem (pES) cells are pluripotent stem cells derived from artificially activated oocytes without embryo destruction, thus eliciting less ethic concerns, and have been demonstrated promising for autologous stem cell therapy. However, pES cells could carry inappropriate imprinting such as relatively high expression of H19, a paternal imprinted gene, and may negatively influence their lineage differentiation. We show that knockdown of H19 by shRNA in mouse pES cells does not alter self-renewal and expression of genes associated with pluripotency. We find that down-regulation of H19 promotes differentiation of pES cells to epidermis. In addition, H19 depletion also facilitates differentiation of pES cells to cardiomyocytes and strong heart-like beating. Our data support the notion that reduction of H19 improves pES cell differentiation in the lineages of ectoderm and mesoderm, and provide further evidence suggesting that defective imprinting can be manipulated to allow potential application of pES cells for stem cell therapy.

  20. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    PubMed

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  1. Knockdown of Unconventional Myosin ID Expression Induced Morphological Change in Oligodendrocytes

    PubMed Central

    Yamazaki, Reiji; Ishibashi, Tomoko; Baba, Hiroko

    2016-01-01

    Myelin is a special multilamellar structure involved in various functions in the nervous system. In the central nervous system, the oligodendrocyte (OL) produces myelin and has a unique morphology. OLs have a dynamic membrane sorting system associated with cytoskeletal organization, which aids in the production of myelin. Recently, it was reported that the assembly and disassembly of actin filaments is crucial for myelination. However, the partner myosin molecule which associates with actin filaments during the myelination process has not yet been identified. One candidate myosin is unconventional myosin ID (Myo1d) which is distributed throughout central nervous system myelin; however, its function is still unclear. We report here that Myo1d is expressed during later stages of OL differentiation, together with myelin proteolipid protein (PLP). In addition, Myo1d is distributed at the leading edge of the myelin-like membrane in cultured OL, colocalizing mainly with actin filaments, 2′,3′-cyclic nucleotide phosphodiesterase and partially with PLP. Myo1d-knockdown with specific siRNA induces significant morphological changes such as the retraction of processes and degeneration of myelin-like membrane, and finally apoptosis. Furthermore, loss of Myo1d by siRNA results in the impairment of intracellular PLP transport. Together, these results suggest that Myo1d may contribute to membrane dynamics either in wrapping or transporting of myelin membrane proteins during formation and maintenance of myelin. PMID:27655972

  2. Genetic architecture of a hormonal response to gene knockdown in honey bees.

    PubMed

    Ihle, Kate E; Rueppell, Olav; Huang, Zachary Y; Wang, Ying; Fondrk, M Kim; Page, Robert E; Amdam, Gro V

    2015-01-01

    Variation in endocrine signaling is proposed to underlie the evolution and regulation of social life histories, but the genetic architecture of endocrine signaling is still poorly understood. An excellent example of a hormonally influenced set of social traits is found in the honey bee (Apis mellifera): a dynamic and mutually suppressive relationship between juvenile hormone (JH) and the yolk precursor protein vitellogenin (Vg) regulates behavioral maturation and foraging of workers. Several other traits cosegregate with these behavioral phenotypes, comprising the pollen hoarding syndrome (PHS) one of the best-described animal behavioral syndromes. Genotype differences in responsiveness of JH to Vg are a potential mechanistic basis for the PHS. Here, we reduced Vg expression via RNA interference in progeny from a backcross between 2 selected lines of honey bees that differ in JH responsiveness to Vg reduction and measured JH response and ovary size, which represents another key aspect of the PHS. Genetic mapping based on restriction site-associated DNA tag sequencing identified suggestive quantitative trait loci (QTL) for ovary size and JH responsiveness. We confirmed genetic effects on both traits near many QTL that had been identified previously for their effect on various PHS traits. Thus, our results support a role for endocrine control of complex traits at a genetic level. Furthermore, this first example of a genetic map of a hormonal response to gene knockdown in a social insect helps to refine the genetic understanding of complex behaviors and the physiology that may underlie behavioral control in general.

  3. In vivo knockdown of Piccolino disrupts presynaptic ribbon morphology in mouse photoreceptor synapses

    PubMed Central

    Regus-Leidig, Hanna; Fuchs, Michaela; Löhner, Martina; Leist, Sarah R.; Leal-Ortiz, Sergio; Chiodo, Vince A.; Hauswirth, William W.; Garner, Craig C.; Brandstätter, Johann H.

    2014-01-01

    Piccolo is the largest known cytomatrix protein at active zones of chemical synapses. A growing number of studies on conventional chemical synapses assign Piccolo a role in the recruitment and integration of molecules relevant for both endo- and exocytosis of synaptic vesicles, the dynamic assembly of presynaptic F-actin, as well as the proteostasis of presynaptic proteins, yet a direct function in the structural organization of the active zone has not been uncovered in part due to the expression of multiple alternatively spliced isoforms. We recently identified Piccolino, a Piccolo splice variant specifically expressed in sensory ribbon synapses of the eye and ear. Here we down regulated Piccolino in vivo via an adeno-associated virus-based RNA interference approach and explored the impact on the presynaptic structure of mouse photoreceptor ribbon synapses. Detailed immunocytochemical light and electron microscopical analysis of Piccolino knockdown in photoreceptors revealed a hitherto undescribed photoreceptor ribbon synaptic phenotype with striking morphological changes of synaptic ribbon ultrastructure. PMID:25232303

  4. Genetic Architecture of a Hormonal Response to Gene Knockdown in Honey Bees

    PubMed Central

    Rueppell, Olav; Huang, Zachary Y.; Wang, Ying; Fondrk, M. Kim; Page, Robert E.; Amdam, Gro V.

    2015-01-01

    Variation in endocrine signaling is proposed to underlie the evolution and regulation of social life histories, but the genetic architecture of endocrine signaling is still poorly understood. An excellent example of a hormonally influenced set of social traits is found in the honey bee (Apis mellifera): a dynamic and mutually suppressive relationship between juvenile hormone (JH) and the yolk precursor protein vitellogenin (Vg) regulates behavioral maturation and foraging of workers. Several other traits cosegregate with these behavioral phenotypes, comprising the pollen hoarding syndrome (PHS) one of the best-described animal behavioral syndromes. Genotype differences in responsiveness of JH to Vg are a potential mechanistic basis for the PHS. Here, we reduced Vg expression via RNA interference in progeny from a backcross between 2 selected lines of honey bees that differ in JH responsiveness to Vg reduction and measured JH response and ovary size, which represents another key aspect of the PHS. Genetic mapping based on restriction site-associated DNA tag sequencing identified suggestive quantitative trait loci (QTL) for ovary size and JH responsiveness. We confirmed genetic effects on both traits near many QTL that had been identified previously for their effect on various PHS traits. Thus, our results support a role for endocrine control of complex traits at a genetic level. Furthermore, this first example of a genetic map of a hormonal response to gene knockdown in a social insect helps to refine the genetic understanding of complex behaviors and the physiology that may underlie behavioral control in general. PMID:25596612

  5. Peroxynitrite induced mitochondrial biogenesis following MnSOD knockdown in normal rat kidney (NRK) cells.

    PubMed

    Marine, Akira; Krager, Kimberly J; Aykin-Burns, Nukhet; Macmillan-Crow, Lee Ann

    2014-01-01

    Superoxide is widely regarded as the primary reactive oxygen species (ROS) which initiates downstream oxidative stress. Increased oxidative stress contributes, in part, to many disease conditions such as cancer, atherosclerosis, ischemia/reperfusion, diabetes, aging, and neurodegeneration. Manganese superoxide dismutase (MnSOD) catalyzes the dismutation of superoxide into hydrogen peroxide which can then be further detoxified by other antioxidant enzymes. MnSOD is critical in maintaining the normal function of mitochondria, thus its inactivation is thought to lead to compromised mitochondria. Previously, our laboratory observed increased mitochondrial biogenesis in a novel kidney-specific MnSOD knockout mouse. The current study used transient siRNA mediated MnSOD knockdown of normal rat kidney (NRK) cells as the in vitro model, and confirmed functional mitochondrial biogenesis evidenced by increased PGC1α expression, mitochondrial DNA copy numbers and integrity, electron transport chain protein CORE II, mitochondrial mass, oxygen consumption rate, and overall ATP production. Further mechanistic studies using mitoquinone (MitoQ), a mitochondria-targeted antioxidant and L-NAME, a nitric oxide synthase (NOS) inhibitor demonstrated that peroxynitrite (at low micromolar levels) induced mitochondrial biogenesis. These findings provide the first evidence that low levels of peroxynitrite can initiate a protective signaling cascade involving mitochondrial biogenesis which may help to restore mitochondrial function following transient MnSOD inactivation. PMID:24563852

  6. Dynamic knockdown of E. coli central metabolism for redirecting fluxes of primary metabolites

    PubMed Central

    Brockman, Irene M.; Prather, Kristala L. J.

    2015-01-01

    Control of native enzyme levels is important when optimizing strains for overproduction of heterologous compounds. However, for many central metabolic enzymes, static knockdown results in poor growth and protein expression. We have developed a strategy for dynamically modulating the abundance of native enzymes within the host cell and applied this to a model system for myo-inositol production from glucose. This system relies on controlled degradation of a key glycolytic enzyme, phosphofructokinase-I (Pfk-I). Through tuning Pfk-I levels, we have been able to develop an E. coli strain with a growth mode close to wild type and a production mode with an increased glucose-6-phosphate pool available for conversion into myo-inositol. The switch to production mode is trigged by inducer addition, allowing yield, titer, and productivity to be managed through induction time. By varying the time of Pfk-I degradation, we were able to achieve a two-fold improvement in yield and titers of myo-inositol. PMID:25542851

  7. Mitofusin-2 knockdown increases ER-mitochondria contact and decreases amyloid β-peptide production.

    PubMed

    Leal, Nuno Santos; Schreiner, Bernadette; Pinho, Catarina Moreira; Filadi, Riccardo; Wiehager, Birgitta; Karlström, Helena; Pizzo, Paola; Ankarcrona, Maria

    2016-09-01

    Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria-associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM-associated proteins and enhanced ER to mitochondria Ca(2+) transfer from ER to mitochondria in Alzheimer's disease (AD) and amyloid β-peptide (Aβ)-related neuronal models. Here, we report that siRNA knockdown of mitofusin-2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased Ca(2+) transfer from ER to mitchondria and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra- and extracellular Aβ40 and Aβ42 . Analysis of γ-secretase protein expression, maturation and activity revealed that the low Aβ concentrations were a result of impaired γ-secretase complex function. Amyloid-β precursor protein (APP), β-site APP-cleaving enzyme 1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data shows that modulation of ER-mitochondria contact affects γ-secretase activity and Aβ generation. Increased ER-mitochondria contact results in lower γ-secretase activity suggesting a new mechanism by which Aβ generation can be controlled. PMID:27203684

  8. Establishment of bovine embryonic stem cells after knockdown of CDX2

    PubMed Central

    Wu, Xia; Song, Miao; Yang, Xi; Liu, Xin; Liu, Kun; Jiao, Cuihua; Wang, Jinze; Bai, Chunling; Su, Guanghua; Liu, Xuefei; Li, Guangpeng

    2016-01-01

    Bovine embryonic stem cells (bESCs) have not been successfully established yet. One reason could be that CDX2, as the trophectoderm regulator, expresses in bovine inner cell mass (ICM), which probably becomes a technical barrier for maintaining the pluripotency of bESCs in vitro. We hypothesized that CDX2 knockdown (CDX2-KD) could remove such negative effort, which will be helpful for capturing complete and permanent capacity of pluripotency. Expression and localization of pluripotent genes were not affected in CDX2-KD blastocysts. The CDX2-KD bESCs grew into monolayers on feeder layer. Pluripotent genes expressed at an improved levels and lasted longer time in CDX2-KD bESCs, along with down-regulation of DNA methylation on promoters of both OCT4 and SOX2. The cystic structure typical for trophoblast cells did not show during culturing CDX2-KD bESCs. CDX2-KD bESC-derived Embryoid bodies showed with compact morphology and with the improved levels of differentiations in three germ layers. CDX2-KD bESCs still carried the capacity of forming teratomas with three germ layers after long-term culture. In summary, CDX2 in bovine ICM was inducer of trophoblast lineage with negative effect on maintenance of pluripotency of bESCs. Precise regulation CDX2 expression to switch on/off will be studied next for application on establishment of bESCs. PMID:27320776

  9. Proteomics analysis of E-cadherin knockdown in epithelial breast cancer cells.

    PubMed

    Vergara, Daniele; Simeone, Pasquale; Latorre, Dominga; Cascione, Francesca; Leporatti, Stefano; Trerotola, Marco; Giudetti, Anna Maria; Capobianco, Loredana; Lunetti, Paola; Rizzello, Antonia; Rinaldi, Rosaria; Alberti, Saverio; Maffia, Michele

    2015-05-20

    E-cadherin is the core protein of the epithelial adherens junction. Through its cytoplasmic domain, E-cadherin interacts with several signaling proteins; among them, α- and β-catenins mediate the link of E-cadherin to the actin cytoskeleton. Loss of E-cadherin expression is a crucial step of epithelial-mesenchymal transition (EMT) and is involved in cancer invasion and metastatization. In human tumors, down-regulation of E-cadherin is frequently associated with poor prognosis. Despite the critical role of E-cadherin in cancer progression, little is known about proteome alterations linked with its down-regulation. To address this point, we investigated proteomics, biophysical and functional changes of epithelial breast cancer cell lines upon shRNA-mediated stable knockdown of E-cadherin expression (shEcad). shEcad cells showed a distinct proteomic signature including altered expression of enzymes and proteins involved in cytoskeletal dynamic and migration. Moreover, these results suggest that, besides their role in mechanical adhesion, loss of E-cadherin expression may contribute to cancer progression by modifying a complex network of pathways that tightly regulate fundamental processes as oxidative stress, immune evasion and cell metabolism. Altogether, these results extend our knowledge on the cellular modifications associated with E-cadherin down-regulation in breast cancer cells.

  10. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  11. Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference

    PubMed Central

    Ghosh, Sanjay; Tibbit, Charlotte; Liu, Ji-Long

    2016-01-01

    Long non-coding RNAs (lncRNAs) have emerged as regulators of gene expression across metazoa. Interestingly, some lncRNAs function independently of their transcripts – the transcription of the lncRNA locus itself affects target genes. However, current methods of loss-of-function analysis are insufficient to address the role of lncRNA transcription from the transcript which has impeded analysis of their function. Using the minimal CRISPR interference (CRISPRi) system, we show that coexpression of the catalytically inactive Cas9 (dCas9) and guide RNAs targeting the endogenous roX locus in the Drosophila cells results in a robust and specific knockdown of roX1 and roX2 RNAs, thus eliminating the need for recruiting chromatin modifying proteins for effective gene silencing. Additionally, we find that the human and Drosophila codon optimized dCas9 genes are functional and show similar transcription repressive activity. Finally, we demonstrate that the minimal CRISPRi system suppresses roX transcription efficiently in vivo resulting in loss-of-function phenotype, thus validating the method for the first time in a multicelluar organism. Our analysis expands the genetic toolkit available for interrogating lncRNA function in situ and is adaptable for targeting multiple genes across model organisms. PMID:26850642

  12. The Knockdown of αkap Alters the Postsynaptic Apparatus of Neuromuscular Junctions in Living Mice

    PubMed Central

    Martinez-Pena y Valenzuela, Isabel; Aittaleb, Mohamed; Chen, Po-Ju

    2015-01-01

    A muscle-specific nonkinase anchoring protein (αkap), encoded within the calcium/calmodulin kinase II (camk2) α gene, was recently found to control the stability of acetylcholine receptor (AChR) clusters on the surface of cultured myotubes. However, it remains unknown whether this protein has any effect on receptor stability and the maintenance of the structural integrity of neuromuscular synapses in vivo. By knocking down the endogenous expression of αkap in mouse sternomastoid muscles with shRNA, we found that the postsynaptic receptor density was dramatically reduced, the turnover rate of receptors at synaptic sites was significantly increased, and the insertion rates of both newly synthesized and recycled receptors into the postsynaptic membrane were depressed. Moreover, we found that αkap shRNA knockdown impaired synaptic structure as postsynaptic AChR clusters and their associated postsynaptic scaffold proteins within the neuromuscular junction were completely eliminated. These results provide new mechanistic insight into the role of αkap in regulating the stability of the postsynaptic apparatus of neuromuscular synapses. PMID:25834039

  13. Artificial mirtron-mediated gene knockdown: Functional DMPK silencing in mammalian cells

    PubMed Central

    Seow, Yiqi; Sibley, Christopher R.; Wood, Matthew J.A.

    2012-01-01

    Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs. Here we present a design algorithm for artificial mirtrons and demonstrate, for the first time, efficient gene knockdown of myotonic dystrophy protein kinase (DMPK) target sequences in Renilla luciferase 3′ UTR and subsequently pathogenic DMPK mRNA, causative of Type I myotonic dystrophy, using artificial mirtrons cloned as eGFP introns. Deep sequencing of artificial mirtrons suggests that functional mature transcripts corresponding to the designed sequence were produced in high abundance. They were further shown to be splicing-dependent, Drosha-independent, and partially dependent on exportin-5, resulting in the precise generation of pre-miRNAs. In a murine myoblast line containing a pathogenic copy of human DMPK with more than 500 CUG repeats, the DMPK artificial mirtron corrected DM1-associated splicing abnormalities of the Serca-1 mRNA, demonstrating the therapeutic potential of mirtron-mediated RNAi. Thus, further development and exploitation of the unique properties of mirtrons will benefit future research and therapeutic RNAi applications as an alternative to conventional RNAi strategies. PMID:22647847

  14. Investigation of micronucleus induction in MTH1 knockdown cells exposed to UVA, UVB or UVC.

    PubMed

    Fotouhi, Asal; Cornella, Nicola; Ramezani, Mehrafarin; Wojcik, Andrzej; Haghdoost, Siamak

    2015-11-01

    The longer wave parts of UVR can increase the production of reactive oxygen species (ROS) which can oxidize nucleotides in the DNA or in the nucleotide pool leading to mutations. Oxidized bases in the DNA are repaired mainly by the DNA base excision repair system and incorporation of oxidized nucleotides into newly synthesized DNA can be prevented by the enzyme MTH1. Here we hypothesize that the formation of several oxidized base damages (from pool and DNA) in close proximity, would cause a high number of base excision repair events, leading to DNA double strand breaks (DSB) and therefore giving rise to cytogenetic damage. If this hypothesis is true, cells with low levels of MTH1 will show higher cytogenetic damage after the longer wave parts of UVR. We analyzed micronuclei induction (MN) as an endpoint for cytogenetic damage in the human lymphoblastoid cell line, TK6, with a normal and a reduced level of MTH1 exposed to UVR. The results indicate a higher level of micronuclei at all incubation times after exposure to the longer wave parts of UVR. There is no significant difference between wildtype and MTH1-knockdown TK6 cells, indicating that MTH1 has no protective role in UVR-induced cytogenetic damage. This indicates that DSBs induced by UV arise from damage forms by direct interaction of UV or ROS with the DNA rather than through oxidation of dNTP. PMID:26520386

  15. Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma

    PubMed Central

    Deng, Liang; Yang, Jihong; Chen, Hongwu; Ma, Bo; Pan, Kangming; Su, Caikun; Xu, Fengfeng; Zhang, Jihong

    2016-01-01

    TMEM16A plays an important role in cell proliferation in various cancers. However, less was known about the expression and role of TMEM16A in hepatocellular carcinoma. We screened the expression of TMEM16A in patients’ hepatocellular carcinoma tissues, and also analyzed the biological function of hepatocellular carcinoma cells by knockdown of TMEM16A, as well as the expression of MAPK signaling proteins, including p38, p-p38, ERK1/2, p-ERK1/2, JNK, and p-JNK, and cell cycle regulatory protein cyclin D1 in TMEM16A siRNA-transfected SMMC-7721 cells by Western blot. Our results showed that TMEM16A was overexpressed in hepatocellular carcinoma tissues. Inhibition of TMEM16A suppressed the cell proliferation, migration, and invasion, and cell cycle progression but did not influence the cell apoptosis. TMEM16A siRNA-suppressed cancer cell proliferation and tumor growth were accompanied by a reduction of p38 and ERK1/2 activation and cyclin D1 induction, and were not influenced by other tested MAPK signaling proteins. In addition, inhibition of TMEM16A suppressed tumorigenicity in vivo. TMEM16A is overexpressed in hepatocellular carcinoma, and that inhibition of TMEM16A suppressed MAPK and growth of hepatocellular carcinoma. TMEM16A could be a potentially novel therapeutic target for human cancers, including hepatocellular carcinoma. PMID:26834491

  16. p27 Nuclear localization and growth arrest caused by perlecan knockdown in human endothelial cells

    SciTech Connect

    Sakai, Katsuya; Oka, Kiyomasa; Matsumoto, Kunio; Nakamura, Toshikazu

    2010-02-12

    Perlecan, a secreted heparan sulfate proteoglycan, is a major component of the vascular basement membrane and participates in angiogenesis. Here, we used small interference RNA-mediated knockdown of perlecan expression to investigate the regulatory function of perlecan in the growth of human vascular endothelial cells. Basic fibroblast growth factor (bFGF)-induced ERK phosphorylation and cyclin D1 expression were unchanged by perlecan deficiency in endothelial cells; however, perlecan deficiency inhibited the Rb protein phosphorylation and DNA synthesis induced by bFGF. By contrast to cytoplasmic localization of the cyclin-dependent kinase inhibitor p27 in control endothelial cells, p27 was localized in the nucleus and its expression increased in perlecan-deficient cells, which suggests that p27 mediates inhibition of Rb phosphorylation. In addition to the well-characterized function of perlecan as a co-receptor for heparin-binding growth factors such as bFGF, our results suggest that perlecan plays an indispensible role in endothelial cell proliferation and acts through a mechanism that involves subcellular localization of p27.

  17. Knockdown of long noncoding RNA H19 sensitizes human glioma cells to temozolomide therapy.

    PubMed

    Jiang, Pengfei; Wang, Ping; Sun, Xiaoling; Yuan, Zhongshun; Zhan, Rucai; Ma, Xiangyu; Li, Weiguo

    2016-01-01

    Temozolomide (TMZ) is commonly used in glioma chemotherapy. However, a great clinical challenge for TMZ is chemoresistance. H19 transcripts are recognized as long noncoding RNAs, which potentially interact with chromatin-modifying complexes to regulate gene expression via epigenetic changes. Our data based on glioma patients showed that the expression of H19 was significantly upregulated in TMZ-resistant tumors compared with the TMZ-sensitive tumors. To determine the function of H19 in glioma, cell lines U87 and U251 were exposed to TMZ to establish TMZ-resistant clones U87(TMZ) and U251(TMZ). In U87(TMZ) and U251(TMZ), the expression level of H19 transcripts was increased compared to wild-type or nonresistant clones, as determined by real-time quantitative reverse transcription polymerase chain reaction. Concomitant treatment with small interfering RNA specifically targeting H19 and TMZ in resistant glioma clones resulted in decreased IC50 values for TMZ, and increased apoptotic rates than control small interfering RNA-treated cells. This was also evident by the increased PARP cleavage in resistant cells exposed to TMZ + si-H19. Furthermore, the reduced expression of H19 altered major drug resistance genes, such as MDR, MRP, and ABCG2, both at the mRNA and protein levels. Taken together, these findings suggest that H19 plays an important role in the development of TMZ resistance, and may represent a novel therapeutic target for TMZ-resistant gliomas. PMID:27366087

  18. Knockdown of long noncoding RNA H19 sensitizes human glioma cells to temozolomide therapy

    PubMed Central

    Jiang, Pengfei; Wang, Ping; Sun, Xiaoling; Yuan, Zhongshun; Zhan, Rucai; Ma, Xiangyu; Li, Weiguo

    2016-01-01

    Temozolomide (TMZ) is commonly used in glioma chemotherapy. However, a great clinical challenge for TMZ is chemoresistance. H19 transcripts are recognized as long noncoding RNAs, which potentially interact with chromatin-modifying complexes to regulate gene expression via epigenetic changes. Our data based on glioma patients showed that the expression of H19 was significantly upregulated in TMZ-resistant tumors compared with the TMZ-sensitive tumors. To determine the function of H19 in glioma, cell lines U87 and U251 were exposed to TMZ to establish TMZ-resistant clones U87TMZ and U251TMZ. In U87TMZ and U251TMZ, the expression level of H19 transcripts was increased compared to wild-type or nonresistant clones, as determined by real-time quantitative reverse transcription polymerase chain reaction. Concomitant treatment with small interfering RNA specifically targeting H19 and TMZ in resistant glioma clones resulted in decreased IC50 values for TMZ, and increased apoptotic rates than control small interfering RNA-treated cells. This was also evident by the increased PARP cleavage in resistant cells exposed to TMZ + si-H19. Furthermore, the reduced expression of H19 altered major drug resistance genes, such as MDR, MRP, and ABCG2, both at the mRNA and protein levels. Taken together, these findings suggest that H19 plays an important role in the development of TMZ resistance, and may represent a novel therapeutic target for TMZ-resistant gliomas. PMID:27366087

  19. Resistance-resistant antibiotics.

    PubMed

    Oldfield, Eric; Feng, Xinxin

    2014-12-01

    New antibiotics are needed because drug resistance is increasing while the introduction of new antibiotics is decreasing. We discuss here six possible approaches to develop 'resistance-resistant' antibiotics. First, multitarget inhibitors in which a single compound inhibits more than one target may be easier to develop than conventional combination therapies with two new drugs. Second, inhibiting multiple targets in the same metabolic pathway is expected to be an effective strategy owing to synergy. Third, discovering multiple-target inhibitors should be possible by using sequential virtual screening. Fourth, repurposing existing drugs can lead to combinations of multitarget therapeutics. Fifth, targets need not be proteins. Sixth, inhibiting virulence factor formation and boosting innate immunity may also lead to decreased susceptibility to resistance. Although it is not possible to eliminate resistance, the approaches reviewed here offer several possibilities for reducing the effects of mutations and, in some cases, suggest that sensitivity to existing antibiotics may be restored in otherwise drug-resistant organisms.

  20. Stable Toll-Like Receptor 10 Knockdown in THP-1 Cells Reduces TLR-Ligand-Induced Proinflammatory Cytokine Expression

    PubMed Central

    Le, Hai Van; Kim, Jae Young

    2016-01-01

    Toll-like receptor 10 (TLR10) is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1), lipopolysaccharide (LPS), and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8), Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha (TNF-α) and Chemokine (C–C Motif) Ligand 20 (CCL20) expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10. PMID:27258267

  1. Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown

    NASA Astrophysics Data System (ADS)

    Fakhoury, Johans J.; Edwardson, Thomas G.; Conway, Justin W.; Trinh, Tuan; Khan, Farhad; Barłóg, Maciej; Bazzi, Hassan S.; Sleiman, Hanadi F.

    2015-12-01

    Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into `gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable

  2. Knockdown and Mortality of Five Stored Product Beetle Species After Short Exposures of Thiamethoxam.

    PubMed

    Tsaganou, Fotoula C; Vassilakos, Thomas N; Athanassiou, Christos G

    2014-12-01

    Laboratory bioassays were conducted to evaluate the effectiveness of thiamethoxam, against five major stored-grain beetle species, the lesser grain borer, Rhyzopertha dominica (F.), the rice weevil, Sitophilus oryzae (L.), the confused flour beetle, Tribolium confusum Jacquelin du Val, the larger grain borer, Prostephanus truncatus (Horn), and the sawtoothed grain beetle, Oryzaephilus surinamensis (L.). Adults of the above species were exposed on wheat (or maize in the case of P. truncatus) treated with thiamethoxam at 0.1, 1, and 10 ppm for 0, 2, 4, 6, 8, 16, 40, 72, and 96 h. After each of these intervals, mortality was recorded (immediate mortality) and the surviving individuals were transferred in untreated wheat (or maize), where mortality was recorded again 7 d later (delayed mortality). During both immediate and delayed mortality counts, the number of adults that were knocked down was also recorded. Immediate mortality was low in all exposures, with the exception of the highest dose rate and after 72-96 h. At these conditions, during this interval, most of the surviving individuals were knocked down. Delayed mortality was further increased with the increase of dose and the initial exposure, but knockdown was extremely low, with the exception of P. truncatus. The results of the present work show that O. surinamensis was the least susceptible species, while P. truncatus was the most susceptible. These findings show that, despite the increased mortality, recovery after short exposures is likely for all species tested here. In this regard, partially treated areas on which the insects are exposed only for short intervals may reduce thiamethoxam efficacy. PMID:26470089

  3. Pheophytinase Knockdown Impacts Carbon Metabolism and Nutraceutical Content Under Normal Growth Conditions in Tomato.

    PubMed

    Lira, Bruno Silvestre; Rosado, Daniele; Almeida, Juliana; de Souza, Amanda Pereira; Buckeridge, Marcos Silveira; Purgatto, Eduardo; Guyer, Luzia; Hörtensteiner, Stefan; Freschi, Luciano; Rossi, Magdalena

    2016-03-01

    Although chlorophyll (Chl) degradation is an essential biochemical pathway for plant physiology, our knowledge regarding this process still has unfilled gaps. Pheophytinase (PPH) was shown to be essential for Chl breakdown in dark-induced senescent leaves. However, the catalyzing enzymes involved in pigment turnover and fruit ripening-associated degreening are still controversial. Chl metabolism is closely linked to the biosynthesis of other isoprenoid-derived compounds, such as carotenoids and tocopherols, which are also components of the photosynthetic machinery. Chls, carotenoids and tocopherols share a common precursor, geranylgeranyl diphosphate, produced by the plastidial methylerythritol 4-phosphate (MEP) pathway. Additionally, the Chl degradation-derived phytol can be incorporated into tocopherol biosynthesis. In this context, tomato turns out to be an interesting model to address isoprenoid-metabolic cross-talk since fruit ripening combines degreening and an intensely active MEP leading to carotenoid accumulation. Here, we investigate the impact of PPH deficiency beyond senescence by the comprehensive phenotyping of SlPPH-knockdown tomato plants. In leaves, photosynthetic parameters indicate altered energy usage of excited Chl. As a mitigatory effect, photosynthesis-associated carotenoids increased while tocopherol content remained constant. Additionally, starch and soluble sugar profiles revealed a distinct pattern of carbon allocation in leaves that suggests enhanced sucrose exportation. The higher levels of carbohydrates in sink organs down-regulated carotenoid biosynthesis. Additionally, the reduction in Chl-derived phytol recycling resulted in decreased tocopherol content in transgenic ripe fruits. Summing up, tocopherol and carotenoid metabolism, together with the antioxidant capacity of the hydrophilic and hydrophobic fractions, were differentially affected in leaves and fruits of the transgenic plants. Thus, in tomato, PPH plays a role beyond

  4. RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

    PubMed Central

    M’BAYE, Mohamed; HUA, Guohua; KHAN, Hamid Ali; YANG, Liguo

    2015-01-01

    Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse. PMID:26063610

  5. Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity

    PubMed Central

    Kraus, Daniel; Yang, Qin; Kong, Dong; Banks, Alexander S.; Zhang, Lin; Rodgers, Joseph T.; Pirinen, Eija; Pulinilkunnil, Thomas C.; Gong, Fengying; Wang, Ya-chin; Cen, Yana; Sauve, Anthony A.; Asara, John M.; Peroni, Odile D.; Monia, Brett P.; Bhanot, Sanjay; Alhonen, Leena; Puigserver, Pere; Kahn, Barbara B.

    2014-01-01

    In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes1. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity2. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S-adenosylmethionine (SAM) as a methyl donor3,4. Nicotinamide is a precursor of NAD+, an important cofactor linking cellular redox states with energy metabolism5. SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation6. Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine–spermine N1-acetyltransferase (SSAT; encoded by Sat1) and by polyamine oxidase (PAO), and has a major role in energy metabolism7,8. We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD+ levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD+-dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes. PMID

  6. CDK5 knockdown in astrocytes provide neuroprotection as a trophic source via Rac1.

    PubMed

    Posada-Duque, Rafael Andrés; Palacio-Castañeda, Valentina; Cardona-Gómez, Gloria Patricia

    2015-09-01

    Astrocytes perform metabolic and structural support functions in the brain and contribute to the integrity of the blood-brain barrier. Astrocytes influence neuronal survival and prevent gliotoxicity by capturing glutamate (Glu), reactive oxygen species, and nutrients. During these processes, astrocytic morphological changes are supported by actin cytoskeleton remodeling and require the involvement of Rho GTPases, such as Rac1. The protein cyclin-dependent kinase 5 (CDK5) may have a dual effect on astrocytes because it has been shown to be involved in migration, senescence, and the dysfunction of glutamate recapture; however, its role in astrocytes remains unclear. Treating a possible deregulation of CDK5 with RNAi is a strategy that has been proposed as a therapy for neurodegenerative diseases. Models of glutamate gliotoxicity in the C6 astroglioma cell line, primary cultures of astrocytes, and co-cultures with neurons were used to analyze the effects of CDK5 RNAi in astrocytes and the role of Rac1 in neuronal viability. In C6 cells and primary astrocytes, CDK5 RNAi prevented the cell death generated by glutamate-induced gliotoxicity, and this finding was corroborated by pharmacological inhibition with roscovitine. This effect was associated with the appearance of lamellipodia, protrusions, increased cell area, stellation, Rac1 activation, BDNF release, and astrocytic protection in neurons that were exposed to glutamate excitotoxicity. Interestingly, Rac1 inhibition in astrocytes blocked BDNF upregulation and the astrocyte-mediated neuroprotection. Actin cytoskeleton remodeling and stellation may be a functional phenotype for BDNF release that promotes neuroprotection. In summary, our findings suggest that CDK5- knockdown in astrocytes acts as a trophic source for neuronal protection in a Rac1-dependent manner. PMID:26160434

  7. Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity.

    PubMed

    Kraus, Daniel; Yang, Qin; Kong, Dong; Banks, Alexander S; Zhang, Lin; Rodgers, Joseph T; Pirinen, Eija; Pulinilkunnil, Thomas C; Gong, Fengying; Wang, Ya-chin; Cen, Yana; Sauve, Anthony A; Asara, John M; Peroni, Odile D; Monia, Brett P; Bhanot, Sanjay; Alhonen, Leena; Puigserver, Pere; Kahn, Barbara B

    2014-04-10

    In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S-adenosylmethionine (SAM) as a methyl donor. Nicotinamide is a precursor of NAD(+), an important cofactor linking cellular redox states with energy metabolism. SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation. Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine-spermine N(1)-acetyltransferase (SSAT; encoded by Sat1) and by polyamine oxidase (PAO), and has a major role in energy metabolism. We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD(+) levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD(+)-dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes.

  8. Knockdown of LRP/LR Induces Apoptosis in Breast and Oesophageal Cancer Cells.

    PubMed

    Khumalo, Thandokuhle; Ferreira, Eloise; Jovanovic, Katarina; Veale, Rob B; Weiss, Stefan F T

    2015-01-01

    Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting LRP mRNA using siRNA-LAMR1. This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay. Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1. This reduction in cellular viability is as a consequence of apoptosis induction as indicated by the exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells, respectively, detected by an Annexin-V/FITC assay as well as nuclear morphological changes observed post-staining with Hoechst. These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment. PMID:26427016

  9. Knockdown of LRP/LR Induces Apoptosis in Breast and Oesophageal Cancer Cells

    PubMed Central

    Jovanovic, Katarina; Veale, Rob B.; Weiss, Stefan F. T.

    2015-01-01

    Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting LRP mRNA using siRNA-LAMR1. This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay. Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1. This reduction in cellular viability is as a consequence of apoptosis induction as indicated by the exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells, respectively, detected by an Annexin-V/FITC assay as well as nuclear morphological changes observed post-staining with Hoechst. These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment. PMID:26427016

  10. STAT3 Knockdown Reduces Pancreatic Cancer Cell Invasiveness and Matrix Metalloproteinase-7 Expression in Nude Mice

    PubMed Central

    Huang, Ke jian; Wu, Wei dong; Jiang, Tao; Cao, Jun; Feng, Zhen zhong; Qiu, Zheng jun

    2011-01-01

    Aims Transducer and activator of transcription-3 (STAT3) plays an important role in tumor cell invasion and metastasis. The aim of the present study was to investigate the effects of STAT3 knockdown in nude mouse xenografts of pancreatic cancer cells and underlying gene expression. Methods A STAT3 shRNA lentiviral vector was constructed and infected into SW1990 cells. qRT-PCR and western immunoblot were performed to detect gene expression. Nude mouse xenograft assays were used to assess changes in phenotypes of these stable cells in vivo. HE staining was utilized to evaluate tumor cell invasion and immunohistochemistry was performed to analyze gene expression. Results STAT3 shRNA successfully silenced expression of STAT3 mRNA and protein in SW1990 cells compared to control cells. Growth rate of the STAT3-silenced tumor cells in nude mice was significantly reduced compared to in the control vector tumors and parental cells-generated tumors. Tumor invasion into the vessel and muscle were also suppressed in the STAT3-silenced tumors compared to controls. Collagen IV expression was complete and continuous surrounding the tumors of STAT3-silenced SW1990 cells, whereas collagen IV expression was incomplete and discontinuous surrounding the control tumors. Moreover, microvessel density was significantly lower in STAT3-silenced tumors than parental or control tumors of SW1990 cells. In addition, MMP-7 expression was reduced in STAT3-silenced tumors compared to parental SW1990 xenografts and controls. In contrast, expression of IL-1β and IgT7α was not altered. Conclusion These data clearly demonstrate that STAT3 plays an important role in regulation of tumor growth, invasion, and angiogenesis, which could be act by reducing MMP-7 expression in pancreatic cancer cells. PMID:21991388

  11. TCRP1 contributes to cisplatin resistance by preventing Pol β degradation in lung cancer cells.

    PubMed

    Liu, Xiaorong; Wang, Chengkun; Gu, Yixue; Zhang, Zhijie; Zheng, Guopei; He, Zhimin

    2015-01-01

    Cisplatin (DDP) is the first-line chemotherapy drug widely used for the treatment of lung cancer patients, whereas the majority of cancer patients will eventually show resistance to DDP. The mechanisms responsible for DDP resistance are not fully understood. Tongue cancer resistance-associated protein 1 (TCRP1) gene was recently cloned and reported to specially mediate DDP resistance in human oral squamous cell carcinoma (OSCC) cells. However, the mechanisms of TCRP1-mediated DDP resistance are far from clear, and whether TCRP1 participates in DDP resistance in lung cancer cells remains unknown. Here, we show that TCRP1 contributes to DDP resistance in lung cancer cells. Knockdown of TCRP1 sensitizes the cells to DDP and increases the DDP-induced DNA damage. We have identified that Pol β is associated with DDP resistance, and Pol β knockdown delays the repair of DDP-induced DNA damage in A549/DDP cells. We find TCRP1 interacts with Pol β in lung cancer cells. Moreover, TCRP1 knockdown decreases the level of Pol β and increases the level of its ubiquitination. These results suggest that TCRP1 contributes to DDP resistance through the prevention of Pol β degradation in lung cancer cells. These findings provide new insights into chemoresistance and may contribute to prevention and reversal of DDP resistance in treatment of lung cancer in the future.

  12. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    DOE PAGES

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L.; et al

    2015-03-31

    Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstratemore » reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.« less

  13. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    SciTech Connect

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L.; Booth, Benjamin W.; Evans-Holm, Martha; Venken, Koen J.T.; Levis, Robert W.; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.

    2015-03-31

    Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

  14. Inwardly rectifying Kir3.1 subunit knockdown impairs learning and memory in an olfactory associative task in rat.

    PubMed

    Kourrich, Saïd; Masmejean, Frédérique; Martin-Eauclaire, Marie France; Soumireu-Mourat, Bernard; Mourre, Christiane

    2003-05-12

    Inward-rectifier potassium channels gated by the direct action of G proteins are activated or inhibited by numerous neurotransmitters and they modulate neuronal excitability. Using an olfactory associative task, the effect of Kir3.1 subunit knockdown was tested on learning and memory. Repeated intracerebroventricular injections of antisense oligodeoxyribonucleotide to the Kir3.1 subunit significantly reduced hippocampal expression of its mRNA target determined by Western blotting. The antisense knockdown had no effect on locomotor and drinking activity or on attention processes. The reduction in Kir3.1 subunit impaired the learning of the odor associations and the procedural side of the task. This reduction correlated with the performance impairment. The results suggest that Kir3.1 channel activity is implicated in the memory processes. PMID:12750011

  15. The effects of knockdown of rho-associated kinase 1 and zipper-interacting protein kinase on gene expression and function in cultured human arterial smooth muscle cells.

    PubMed

    Deng, Jing-Ti; Wang, Xiu-Ling; Chen, Yong-Xiang; O'Brien, Edward R; Gui, Yu; Walsh, Michael P

    2015-01-01

    Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and

  16. The effects of knockdown of rho-associated kinase 1 and zipper-interacting protein kinase on gene expression and function in cultured human arterial smooth muscle cells.

    PubMed

    Deng, Jing-Ti; Wang, Xiu-Ling; Chen, Yong-Xiang; O'Brien, Edward R; Gui, Yu; Walsh, Michael P

    2015-01-01

    Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and

  17. Knockdown of kinesin KIF11 abrogates directed migration in response to epidermal growth factor-mediated chemotaxis.

    PubMed

    Wang, Fang; Lin, Stanley Li

    2014-09-26

    Establishment of microtubule polarity is critical for directional cell migration involved in morphogenesis, differentiation, cell division, and metastasis. Current models, involving iterative microtubule capture and inactivation of microtubule depolymerizing mechanisms at the leading edge, cannot account for the biased migration exhibited by cells in culture in the absence of directional cues, suggesting central mechanisms governing microtubule polarity remain unknown. We engineered two human MDA-MB-231/IMP1 breast carcinoma cell lines, denoted kdKIF11-1 and kdKIF11-2, in which the kinesin KIF11 (also known as Eg5) was stably knocked down by two different shRNAs. Western blot analysis showed knockdown by each shRNA decreased KIF11 expression by 58% and 79% for kdKIF11-1 and kdKIF11-2, respectively, whereas Rac1 expression was unaffected. All cell lines retained a well-defined microtubule structure. Compared to cells infected with the control viral vector, both KIF11 knockdown cell lines displayed a 14-45% increase in cell motility in a scratch wound healing assay. In contrast, KIF11 knockdown decreased invasion by 70%, compared to the control, as measured by invasion through Matrigel-coated transwells. To determine whether the reduction in invasion was due to reduced chemotaxis, we substituted collagen for Matrigel in the transwell assay and similarly observed a 44-54% reduction in migration, using EGF as the chemoattractant. However, when including EGF in both the upper and lower chambers of the transwell to stimulate migration but eliminate chemotaxis, transwell migration decreased for the control cell line only, indicating that KIF11 knockdown did not impair migration, but severely impaired chemotaxis. We conclude KIF11 is a key downstream molecule that responds to directional cues in chemotaxis to govern the direction of migration.

  18. G-protein Coupled Receptor 34 Knockdown Impairs the Proliferation and Migration of HGC-27 Gastric Cancer Cells In Vitro

    PubMed Central

    Jin, Zhong-Tian; Li, Kun; Li, Mei; Ren, Zhi-Gang; Wang, Fu-Shun; Zhu, Ji-Ye; Leng, Xi-Sheng; Yu, Wei-Dong

    2015-01-01

    Background: Overexpression of G-protein coupled receptor 34 (GPR34) affects the progression and prognosis of human gastric adenocarcinoma, however, the role of GPR34 in gastric cancer development and progression has not been well-determined. The current study aimed to investigate the effect of GPR34 knockdown on the proliferation, migration, and apoptosis of HGC-27 gastric cancer cells and the underlying mechanisms. Methods: The expression of GPR34 in gastric cancer cell line HGC-27 was detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. HGC-27 cells were employed to construct the stable GPR34 knockdown cell model in this study. Real-time RT-PCR and Western blotting were applied to validate the effect of short hairpin RNA (ShRNA) on the expression of GPR34 in HGC-27 gastric cells. The proliferation, migration of these cells were examined by Cell Counting Kit-8 and transwell. We also measured expression profile of PI3K/PDK1/AKT and ERK using Western blotting. Results: The ShRNA directed against GPR34 effectively inhibited both endogenous mRNA and protein expression levels of GPR34, and significantly down-regulated the expression of PIK3CB (P < 0.01), PIK3CD (P < 0.01), PDK1 (P < 0.01), phosphorylation of PDK1 (P < 0.01), Akt (P < 0.01), and ERK (P < 0.01). Furthermore, GPR34 knockdown resulted in an obvious reduction in HGC-27 cancer cell proliferation and migration activity (P < 0.01). Conclusions: GPR34 knockdown impairs the proliferation and migration of HGC-27 gastric cancer cells in vitro and provides a potential implication for therapy of gastric cancer. PMID:25673461

  19. Knockdown of a Zebrafish Aryl Hydrocarbon Receptor Repressor (AHRRa) Affects Expression of Genes Related to Photoreceptor Development and Hematopoiesis

    PubMed Central

    Aluru, Neelakanteswar; Jenny, Matthew J.; Hahn, Mark E.

    2014-01-01

    The aryl hydrocarbon receptor repressor (AHRR) is a transcriptional repressor of aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor (HIF) and is regulated by an AHR-dependent mechanism. Zebrafish (Danio rerio) possess two AHRR paralogs; AHRRa regulates constitutive AHR signaling during development, whereas AHRRb regulates polyaromatic hydrocarbon-induced gene expression. However, little is known about the endogenous roles and targets of AHRRs. The objective of this study was to elucidate the role of AHRRs during zebrafish development using a loss-of-function approach followed by gene expression analysis. Zebrafish embryos were microinjected with morpholino oligonucleotides against AHRRa or AHRRb to knockdown AHRR protein expression. At 72 h postfertilization (hpf), microarray analysis revealed that the expression of 279 and 116 genes was altered by knockdown of AHRRa and AHRRb, respectively. In AHRRa-morphant embryos, 97 genes were up-regulated and 182 genes were down-regulated. Among the down-regulated genes were several related to photoreceptor function, including cone-specific genes such as several opsins (opn1sw1, opn1sw2, opn1mw1, and opn1lw2), phosphodiesterases (pde6H and pde6C), retinol binding protein (rbp4l), phosducin, and arrestins. Down-regulation was confirmed by RT-PCR and with samples from an independent experiment. The four genes tested (opn1sw1, pde6H, pde6C, and arr3b) were not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. AHRRa knockdown also caused up-regulation of embryonic hemoglobin (hbbe3), suggesting a role for AHRR in regulating hematopoiesis. Knockdown of AHRRb caused up-regulation of 31 genes and down-regulation of 85 genes, without enrichment for any specific biological process. Overall, these results suggest that AHRRs may have important roles in development, in addition to their roles in regulating xenobiotic signaling. PMID:24675095

  20. Knockdown of PU.1 AS lncRNA inhibits adipogenesis through enhancing PU.1 mRNA translation.

    PubMed

    Pang, Wei-Jun; Lin, Li-Gen; Xiong, Yan; Wei, Ning; Wang, Yu; Shen, Qing-Wu; Yang, Gong-She

    2013-11-01

    PU.1 is an Ets family transcription factor involved in the myelo-lymphoid differentiation. We have previously demonstrated that PU.1 is also expressed in the adipocyte lineage. However, the expression levels of PU.1 mRNA and protein in preadipocytes do not match the levels in mature adipocytes. PU.1 mRNA level is higher in preadipocytes, whereas its protein is expressed in the adipocytes but not in the preadipocytes. The underlying mechanism remains elusive. Here, we find that miR-155 knockdown or overexpression has no effect on the levels of PU.1 mRNA and protein in preadipocytes or adipocytes. MiR-155 regulates adipogenesis not through PU.1, but via C/EBPβ which is another target of miR-155. We also checked the expression levels of PU.1 mRNA and antisense long non-coding RNA (AS lncRNA). Interestingly, compared with the level of PU.1 mRNA, the level of PU.1 AS lncRNA is much higher in preadipocytes, whereas it is opposite in the adipocytes. We further discover that PU.1 AS lncRNA binds to its mRNA forming an mRNA/AS lncRNA compound. The knockdown of PU.1 AS by siRNA inhibits adipogenesis and promotes PU.1 protein expression in both preadipocytes and adipocytes. Furthermore, the repression of PU.1 AS decreases the expression and secretion of adiponectin. We also find that the effect of retroviral-mediated PU.1 AS knockdown on adipogenesis is consistent with that of PU.1 AS knockdown by siRNA. Taken together, our results suggest that PU.1 AS lncRNA promotes adipogenesis through preventing PU.1 mRNA translation via binding to PU.1 mRNA to form mRNA/AS lncRNA duplex in preadipocytes.

  1. A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

    PubMed

    Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

    2014-12-05

    Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.

  2. RNAi-mediated knock-down of Dab and Numb attenuate Aβ levels via γ-secretase mediated APP processing

    PubMed Central

    2012-01-01

    Amyloid-β-protein (Aβ), the key component of senile plaques in Alzheimer's disease (AD) brain, is produced from amyloid precursor protein (APP) by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB) domains, including Dab (gene: DAB) and Numb (gene: NUMB), can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells) or APP-C99 (H4-APP-C99 cells) increased levels of APP-C-terminal fragments (APP-CTFs) and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided. PMID:23211096

  3. RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum.

    PubMed

    Abd El Halim, Hesham M; Alshukri, Baida M H; Ahmad, Munawar S; Nakasu, Erich Y T; Awwad, Mohammed H; Salama, Elham M; Gatehouse, Angharad M R; Edwards, Martin G

    2016-01-01

    The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30-60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control. PMID:27411529

  4. Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum.

    PubMed

    Tabunoki, Hiroko; Gorman, Maureen J; Dittmer, Neal T; Kanost, Michael R

    2016-01-01

    Insects can rapidly adapt to environmental changes through physiological responses. The red flour beetle Tribolium castaneum is widely used as a model insect species. However, the stress-response system of this species remains unclear. Superoxide dismutase 2 (SOD2) is a crucial antioxidative enzyme that is found in mitochondria. T. castaneum SOD2 (TcSOD2) is composed of 215 amino acids, and has an iron/manganese superoxide dismutase domain. qRT-PCR experiments revealed that TcSOD2 was present through all developmental stages. To evaluate TcSOD2 function in T. castaneum, we performed RNAi and also assessed the phenotype and antioxidative tolerance of the knockdown of TcSOD2 by exposing larvae to paraquat. The administration of paraquat resulted in significantly higher 24-h mortality in TcSOD2 knockdown larval groups than in the control groups. The TcSOD2 knockdown adults moved significantly more slowly, had lower ATP content, and exhibited a different body color from the control groups. We found that TcSOD2 dsRNA treatment in larvae resulted in increased expression of tyrosinase and laccase2 mRNA after 10 days. This is the first report showing that TcSOD2 has an antioxidative function and demonstrates that T. castaneum may use an alternative antioxidative system when the SOD2-based system fails. PMID:27387523

  5. Knockdown of PARP-1 Inhibits Proliferation and ERK Signals, Increasing Drug Sensitivity in Osteosarcoma U2OS Cells.

    PubMed

    Li, Sheng; Cui, Zhengli; Meng, Xianfeng

    2016-01-01

    Poly(ADP-ribose) polymerase 1 (PARP-1) is reported to be involved in DNA repair and is now recognized as a key regulator in carcinogenesis. However, the potential role and the molecular mechanism underlying the effect of PARP-1 on osteosarcoma (OS) cells have not been elucidated. In this study, the results showed that knockdown of PARP-1 resulted in decreased cell proliferation, increased cell apoptosis, and G0/G1 phase arrest in U2OS cells. In addition, increased expression of active caspase 3 and Bax, but reduced Bcl-2, cyclin D1, and phosphorylated extracellular signal regulated kinase 1/2 (pERK1/2) were observed in PARP-1 knockdown in U2OS cells. Moreover, knockdown of PARP-1 correlated with elevated chemosensitivity of U2OS cells to cisplatin through inactivation of the ERK1/2 signaling pathway. In conclusion, our findings demonstrated that PARP-1 plays an important role in regulating OS growth, combining PARP-1 gene therapy with traditional chemotherapy, and may serve as a promising approach to OS therapy. PMID:27656839

  6. Knockdown of heme oxygenase-1 promotes apoptosis and autophagy and enhances the cytotoxicity of doxorubicin in breast cancer cells

    PubMed Central

    ZHU, XIAO-FENG; LI, WEN; MA, JIE-YI; SHAO, NAN; ZHANG, YUN-JIAN; LIU, RUI-MING; WU, WEI-BIN; LIN, YING; WANG, SHEN-MING

    2015-01-01

    Heme oxygenase-1 (HMOX-1) is a microsomal enzyme that exerts anti-apoptotic and cytoprotective effects. In the present study, HMOX-1 was demonstrated to be overexpressed and able to be induced by doxorubicin in breast cancer cell lines. Knockdown of HMOX-1 using short interfering (si)RNA enhanced the cytotoxicity of doxorubicin in MDA-MB-231 and BT549 cells. Knockdown of HMOX-1 downregulated B cell lymphoma (Bcl)-2 and Bcl-extra large expression, and significantly enhanced doxorubicin-induced apoptosis in MDA-MB-231 and BT549 cells. Additionally, knockdown of HMOX-1 upregulated light chain 3B expression and markedly increased the accumulation of autophagic vacuoles in MDA-MB-231 and BT549 cells treated with doxorubicin. These results indicated that HMOX-1 may be involved in conferring the chemoresistance of breast cancer cells, by preventing apoptosis and autophagy. Therefore, HMOX-1 may represent a potential therapeutic target for enhancing the cytotoxicity and efficacy of doxorubicin during the treatment of breast cancer. PMID:26722274

  7. Knockdown of CMTM3 promotes metastasis of gastric cancer via the STAT3/Twist1/EMT signaling pathway

    PubMed Central

    Yuan, Wanqiong; Li, Ting; Mo, Xiaoning; Wang, Xiaolin; Liu, Baocai; Wang, Wenyan; Su, Yu; Xu, Lan; Han, Wenling

    2016-01-01

    CMTM3 (CKLF-like MARVEL transmembrane domain containing 3) possesses tumor suppressor properties in multiple types of malignancies. Restoration of CMTM3 significantly inhibits the metastasis of gastric cancer, and its expression level is correlated with prognosis. However, the physiological effects and the mechanism of CMTM3 remain unknown. Here, we suppress CMTM3 expression by shRNA to explore its endogenous effects and its mechanism of action in gastric cancer. Stable knockdown of CMTM3 promotes cell migration, invasion and tumor metastasis, increases MMP2 expression and enhances MMP2 activity. CMTM3 inhibits EMT along with the upregulation of E-cadherin and the downregulation of N-cadherin, Vimentin and Twist1. It has no obvious effects on Zeb1 and Snail. CMTM3 suppresses the phosphorylation of STAT3 but not Akt. More importantly, the EMT phenotype and cell migration induced by CMTM3 knockdown can be reversed by the Jak2/STAT3 inhibitor JSI-124 or by siRNA against STAT3 or Twist1. Overall, this study demonstrates that knockdown of CMTM3 promotes the metastasis of gastric cancer through the STAT3/Twist1/EMT pathway. PMID:27121055

  8. RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum

    PubMed Central

    Abd El Halim, Hesham M.; Alshukri, Baida M. H.; Ahmad, Munawar S.; Nakasu, Erich Y. T.; Awwad, Mohammed H.; Salama, Elham M.; Gatehouse, Angharad M. R.; Edwards, Martin G.

    2016-01-01

    The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30–60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control. PMID:27411529

  9. Superoxide dismutase 2 knockdown leads to defects in locomotor activity, sensitivity to paraquat, and increased cuticle pigmentation in Tribolium castaneum

    PubMed Central

    Tabunoki, Hiroko; Gorman, Maureen J.; Dittmer, Neal T.; Kanost, Michael R.

    2016-01-01

    Insects can rapidly adapt to environmental changes through physiological responses. The red flour beetle Tribolium castaneum is widely used as a model insect species. However, the stress–response system of this species remains unclear. Superoxide dismutase 2 (SOD2) is a crucial antioxidative enzyme that is found in mitochondria. T. castaneum SOD2 (TcSOD2) is composed of 215 amino acids, and has an iron/manganese superoxide dismutase domain. qRT-PCR experiments revealed that TcSOD2 was present through all developmental stages. To evaluate TcSOD2 function in T. castaneum, we performed RNAi and also assessed the phenotype and antioxidative tolerance of the knockdown of TcSOD2 by exposing larvae to paraquat. The administration of paraquat resulted in significantly higher 24-h mortality in TcSOD2 knockdown larval groups than in the control groups. The TcSOD2 knockdown adults moved significantly more slowly, had lower ATP content, and exhibited a different body color from the control groups. We found that TcSOD2 dsRNA treatment in larvae resulted in increased expression of tyrosinase and laccase2 mRNA after 10 days. This is the first report showing that TcSOD2 has an antioxidative function and demonstrates that T. castaneum may use an alternative antioxidative system when the SOD2-based system fails. PMID:27387523

  10. A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets

    PubMed Central

    Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

    2014-01-01

    Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways. PMID:25475013

  11. Knockdown of PFTAIRE Protein Kinase 1 (PFTK1) Inhibits Proliferation, Invasion, and EMT in Colon Cancer Cells.

    PubMed

    Zhu, Jiankang; Liu, Chongzhong; Liu, Fengyue; Wang, Yadong; Zhu, Min

    2016-01-01

    PFTK1 is a member of the cyclin-dependent kinase (CDK) family and is upregulated in many types of tumors. However, its expression and role in colon cancer remain unclear. In this study, we aimed to investigate the expression and function of PFTK1 in colon cancer. Our results showed that PFTK1 was highly expressed in colon cancer cell lines. The in vitro experiments demonstrated that knockdown of PFTK1 inhibited the proliferation, migration, and invasion of colon cancer cells as well as the epithelial-to-mesenchymal transition (EMT) progress. Furthermore, knockdown of PFTK1 suppressed the expression of Shh as well as Smo, Ptc, and Gli-1 in colon cancer cells. Taken together, these results suggest that knockdown of PFTK1 inhibited the proliferation and invasion of colon cancer cells as well as the EMT progress by suppressing the Sonic hedgehog signaling pathway. Therefore, these findings reveal that PFTK1 may be a potential therapeutic target for the treatment of colon cancer. PMID:27458094

  12. Effect of siRNA-mediated knockdown of eIF3c gene on survival of colon cancer cells

    PubMed Central

    Song, Ning; Wang, Yan; Gu, Xiao-dong; Chen, Zong-you; Shi, Liu-bin

    2013-01-01

    Eukaryotic initiation factor subunit c (eIF3c) has been identified as an oncogene that is over-expressed in tumor cells and, therefore, is a potential therapeutic target for gene-based cancer treatment. This study was focused on investigating the effect of small interfering RNA (siRNA)-mediated eIF3c gene knockdown on colon cancer cell survival. The eIF3c gene was observed to be highly expressed in colon cancer cell models. The expression levels of the gene in eIF3c siRNA infected and control siRNA infected cells were compared via real-time polymerase chain reaction (PCR) and western blotting analysis. Cell proliferation levels were analyzed employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays. Furthermore, the effects of eIF3c gene knockdown on the cell cycle and apoptosis were analyzed using flow cytometry. The results showed that suppression of eIF3c expression significantly (P<0.001) reduced cell proliferation and colony formation of RKO colon cancer cells. The cell cycle was arrested by decreasing the number of cells entering S phase. Further, apoptosis was induced as a result of eIF3c knockdown. Collectively, eIF3c deletion effectively reduced the survival of colon cancer cells and could be used as a therapeutic tool for colon cancer therapy. PMID:23733421

  13. Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro.

    PubMed

    Sato, Yuki; Sato, Shiori; Kikuchi, Takahiro; Nonaka, Asumi; Kumagai, Yuki; Sasaki, Akira; Kobayashi, Masayuki

    2016-09-15

    Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro. PMID:27381842

  14. Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene.

    PubMed

    Linhartová, Zuzana; Saito, Taiju; Kašpar, Vojtěch; Rodina, Marek; Prášková, Eva; Hagihara, Seishi; Pšenička, Martin

    2015-10-15

    Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-μM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants

  15. Progranulin modulates zebrafish motoneuron development in vivo and rescues truncation defects associated with knockdown of Survival motor neuron 1

    PubMed Central

    2010-01-01

    Background Progranulin (PGRN) encoded by the GRN gene, is a secreted glycoprotein growth factor that has been implicated in many physiological and pathophysiological processes. PGRN haploinsufficiency caused by autosomal dominant mutations within the GRN gene leads to progressive neuronal atrophy in the form of frontotemporal lobar degeneration (FTLD). This form of the disease is associated with neuronal inclusions that bear the ubiquitinated TAR DNA Binding Protein-43 (TDP-43) molecular signature (FTLD-U). The neurotrophic properties of PGRN in vitro have recently been reported but the role of PGRN in neurons is not well understood. Here we document the neuronal expression and functions of PGRN in spinal cord motoneuron (MN) maturation and branching in vivo using zebrafish, a well established model of vertebrate embryonic development. Results Whole-mount in situ hybridization and immunohistochemical analyses of zebrafish embryos revealed that zfPGRN-A is expressed within the peripheral and central nervous systems including the caudal primary (CaP) MNs within the spinal cord. Knockdown of zfPGRN-A mRNA translation mediated by antisense morpholino oligonucleotides disrupted normal CaP MN development resulting in both truncated MNs and inappropriate early branching. Ectopic over-expression of zfPGRN-A mRNA resulted in increased MN branching and rescued the truncation defects brought about by knockdown of zfPGRN-A expression. The ability of PGRN to interact with established MN developmental pathways was tested. PGRN over-expression was found to reverse the truncation defect resulting from knockdown of Survival of motor neuron 1 (smn1). This is involved in small ribonucleoprotein biogenesis RNA processing, mutations of which cause Spinal Muscular Atrophy (SMA) in humans. It did not reverse the MN defects caused by interfering with the neuronal guidance pathway by knockdown of expression of NRP-1, a semaphorin co-receptor. Conclusions Expression of PGRN within MNs and

  16. Anesthetic-resistant spontaneous mutant of Drosophila melanogaster: intensified response to /sup 60/Cobalt radiation damage

    SciTech Connect

    Gamo, S.; Nakashima-Tanaka, E.; Megumi, T.; Ueda, I.

    1985-02-25

    Accumulating evidence suggests that the extent of acute damage by ionizing irradiation is closely related to the state of membrane orderliness. Decreased orderliness apparently protects organisms from ionizing irradiation. Because anesthetics decrease membrane orderliness, anesthesia is expected to affect damages caused by ionizing irradiation. The present study compared the effects of /sup 60/Co irradiation on Drosophila melanogaster between an anesthetic-resistant spontaneous mutant and an anesthetic-sensitive strain. An anesthetic-resistant mutant strain, Eth-29, of Drosophila melanogaster has previously been established. Eth-29 is resistant to diethyl-ether, chloroform and halothane. The anesthetic-resistant strain was found to be radiosensitive when evaluated by survival at the eighth day after irradiation or by dyskinesia (knock-down) at the second day. The results indicate that anesthetic resistance may be related to an increase in orderliness. The findings in reciprocal crosses between Eth-29 and the control strain indicate that the mechanism of survival is different from that of knock-down. Presumably, knock-down is the direct sequela of irradiation, and the present result suggests that membrane damage may be involved in inducing knock-down. 18 references, 3 figures.

  17. Resistance-Resistant Antibiotics

    PubMed Central

    Oldfield, Eric; Feng, Xinxin

    2014-01-01

    New antibiotics are needed because as drug resistance is increasing, the introduction of new antibiotics is decreasing. Here, we discuss six possible approaches to develop ‘resistance-resistant’ antibiotics. First, multi-target inhibitors in which a single compound inhibits more than one target may be easier to develop than conventional combination therapies with two new drugs. Second, inhibiting multiple targets in the same metabolic pathway is expected to be an effective strategy due to synergy. Third, discovering multiple-target inhibitors should be possible by using sequential virtual screening. Fourth, re-purposing existing drugs can lead to combinations of multi-target therapeutics. Fifth, targets need not be proteins. Sixth, inhibiting virulence factor formation and boosting innate immunity may also lead to decreased susceptibility to resistance. Although it is not possible to eliminate resistance, the approaches reviewed here offer several possibilities for reducing the effects of mutations and in some cases suggest that sensitivity to existing antibiotics may be restored, in otherwise drug resistant organisms. PMID:25458541

  18. Targeting COX-2/PGE2 Pathway in HIPK2 Knockdown Cancer Cells: Impact on Dendritic Cell Maturation

    PubMed Central

    Garufi, Alessia; Pistritto, Giuseppa; Ceci, Claudia; Di Renzo, Livia; Santarelli, Roberta; Faggioni, Alberto; Cirone, Mara; D’Orazi, Gabriella

    2012-01-01

    Background Homeodomain-interacting protein kinase 2 (HIPK2) is a multifunctional protein that exploits its kinase activity to modulate key molecular pathways in cancer to restrain tumor growth and induce response to therapies. For instance, HIPK2 knockdown induces upregulation of oncogenic hypoxia-inducible factor-1 (HIF-1) activity leading to a constitutive hypoxic and angiogenic phenotype with increased tumor growth in vivo. HIPK2 inhibition, therefore, releases pathways leading to production of pro-inflammatory molecules such as vascular endothelial growth factor (VEGF) or prostaglandin E2 (PGE2). Tumor-produced inflammatory mediators other than promote tumour growth and vascular development may permit evasion of anti-tumour immune responses. Thus, dendritic cells (DCs) dysfunction induced by tumor-produced molecules, may allow tumor cells to escape immunosurveillance. Here we evaluated the molecular mechanism of PGE2 production after HIPK2 depletion and how to modulate it. Methodology/Principal findings We show that HIPK2 knockdown in colon cancer cells resulted in cyclooxygenase-2 (COX-2) upregulation and COX-2-derived PGE2 generation. At molecular level, COX-2 upregulation depended on HIF-1 activity. We previously reported that zinc treatment inhibits HIF-1 activity. Here, zinc supplementation to HIPK2 depleted cells inhibited HIF-1-induced COX-2 expression and PGE2/VEGF production. At translational level, while conditioned media of both siRNA control and HIPK2 depleted cells inhibited DCs maturation, conditioned media of only zinc-treated HIPK2 depleted cells efficiently restored DCs maturation, seen as the expression of co-stimulatory molecules CD80 and CD86, cytokine IL-10 release, and STAT3 phosphorylation. Conclusion/Significance These findings show that: 1) HIPK2 knockdown induced COX-2 upregulation, mostly depending on HIF-1 activity; 2) zinc treatment downregulated HIF-1-induced COX-2 and inhibited PGE2/VEGF production; and 3) zinc treatment of HIPK2

  19. Knockdown of SVCT2 impairs in-vitro cell attachment, migration and wound healing in bone marrow stromal cells.

    PubMed

    Sangani, Rajnikumar; Pandya, Chirayu D; Bhattacharyya, Maryka H; Periyasamy-Thandavan, Sudharsan; Chutkan, Norman; Markand, Shanu; Hill, William D; Hamrick, Mark; Isales, Carlos; Fulzele, Sadanand

    2014-03-01

    Bone marrow stromal cell (BMSC) adhesion and migration are fundamental to a number of pathophysiologic processes, including fracture and wound healing. Vitamin C is beneficial for bone formation, fracture repair and wound healing. However, the role of the vitamin C transporter in BMSC adhesion, migration and wound healing is not known. In this study, we knocked-down the sodium-dependent vitamin C transporter, SVCT2, the only known transporter of vitamin C in BMSCs, and performed cell adhesion, migration, in-vitro scratch wound healing and F-actin re-arrangement studies. We also investigated the role of oxidative stress on the above processes. Our results demonstrate that both oxidative stress and down-regulation of SVCT2 decreased cell attachment and spreading. A trans-well cell migration assay showed that vitamin C helped in BMSC migration and that knockdown of SVCT2 decreased cell migration. In the in-vitro scratch wound healing studies, we established that oxidative stress dose-dependently impairs wound healing. Furthermore, the supplementation of vitamin C significantly rescued the BMSCs from oxidative stress and increased wound closing. The knockdown of SVCT2 in BMSCs strikingly decreased wound healing, and supplementing with vitamin C failed to rescue cells efficiently. The knockdown of SVCT2 and induction of oxidative stress in cells produced an alteration in cytoskeletal dynamics. Signaling studies showed that oxidative stress phosphorylated members of the MAP kinase family (p38) and that vitamin C inhibited their phosphorylation. Taken together, these results indicate that both the SVCT2 transporter and oxidative stress play a vital role in BMSC attachment, migration and cytoskeletal re-arrangement. BMSC-based cell therapy and modulation of SVCT2 could lead to a novel therapeutic approach that enhances bone remodeling, fracture repair and wound healing in chronic disease conditions.

  20. Knockdown of TACC3 inhibits trophoblast cell migration and invasion through the PI3K/Akt signaling pathway.

    PubMed

    Zhu, Xiaojun; Cao, Qianqian; Li, Xia; Wang, Zhengping

    2016-10-01

    The insufficient invasion of trophoblasts is known to be correlated with the development of preeclampsia. Transforming acidic coiled‑coil protein 3 (TACC3), a member of the TACC domain family, is important in the regulation of cell differentiation, migration and invasion. However, the role of TACC3 in trophoblast function during placental development remains to be fully elucidated. The present study aimed to determine the expression and function of TACC3 in human placenta and to examine the underlying mechanisms. TACC3 expression was analyzed in preeclamptic placentas using reverse transcription‑quantitative polymerase chain reaction and western blotting. Cell proliferation was determined by the MTT assay, and cell migration and invasion were measured using Transwell assays. The expression levels of TACC3, matrix metalloproteinase (MMP)‑2, MMP‑9, tissue inhibitor of metalloproteinase (TIMP)‑1, TIMP‑2, phosphoinositide 3‑kinase (PI3K), phosphorylated (p)‑PI3K, AKT and p‑AKT were detected by western blotting. The results showed that the expression of TACC3 was downregulated in preeclamptic placentas. The knockdown of TACC3 significantly inhibited HTR8/SVneo cell proliferation, migration and invasion, and inhibited the expression of matrix metalloproteinases. In addition, the knockdown of TACC3 significantly reduced the levels of p‑PI3K and Akt in the HTR8/SVneo cells. Taken together, the results of the present study demonstrated that the knockdown of TACC3 inhibited the migration and invasion of HTR8/SVneo cells through suppression of the PI3K/Akt signaling pathway. Therefore, TACC3 may serve as a novel potential target for treating preeclampsia. PMID:27572091

  1. Reactivation of maternal SNORD116 cluster via SETDB1 knockdown in Prader-Willi syndrome iPSCs

    PubMed Central

    Cruvinel, Estela; Budinetz, Tara; Germain, Noelle; Chamberlain, Stormy; Lalande, Marc; Martins-Taylor, Kristen

    2014-01-01

    Prader-Willi syndrome (PWS), a disorder of genomic imprinting, is characterized by neonatal hypotonia, hypogonadism, small hands and feet, hyperphagia and obesity in adulthood. PWS results from the loss of paternal copies of the cluster of SNORD116 C/D box snoRNAs and their host transcript, 116HG, on human chromosome 15q11-q13. We have investigated the mechanism of repression of the maternal SNORD116 cluster and 116HG. Here, we report that the zinc-finger protein ZNF274, in association with the histone H3 lysine 9 (H3K9) methyltransferase SETDB1, is part of a complex that binds to the silent maternal but not the active paternal alleles. Knockdown of SETDB1 in PWS-specific induced pluripotent cells (iPSCs) causes a decrease in the accumulation of H3K9 trimethylation (H3K9me3) at 116HG and corresponding accumulation of the active chromatin mark histone H3 lysine 4 dimethylation (H3K4me2). We also show that upon knockdown of SETDB1 in PWS-specific iPSCs, expression of maternally silenced 116HG RNA is partially restored. SETDB1 knockdown in PWS iPSCs also disrupts DNA methylation at the PWS-IC where a decrease in 5-methylcytosine is observed in association with a concomitant increase in 5-hydroxymethylcytosine. This observation suggests that the ZNF274/SETDB1 complex bound to the SNORD116 cluster may protect the PWS-IC from DNA demethylation during early development. Our findings reveal novel epigenetic mechanisms that function to repress the maternal 15q11-q13 region. PMID:24760766

  2. Reactivation of maternal SNORD116 cluster via SETDB1 knockdown in Prader-Willi syndrome iPSCs.

    PubMed

    Cruvinel, Estela; Budinetz, Tara; Germain, Noelle; Chamberlain, Stormy; Lalande, Marc; Martins-Taylor, Kristen

    2014-09-01

    Prader-Willi syndrome (PWS), a disorder of genomic imprinting, is characterized by neonatal hypotonia, hypogonadism, small hands and feet, hyperphagia and obesity in adulthood. PWS results from the loss of paternal copies of the cluster of SNORD116 C/D box snoRNAs and their host transcript, 116HG, on human chromosome 15q11-q13. We have investigated the mechanism of repression of the maternal SNORD116 cluster and 116HG. Here, we report that the zinc-finger protein ZNF274, in association with the histone H3 lysine 9 (H3K9) methyltransferase SETDB1, is part of a complex that binds to the silent maternal but not the active paternal alleles. Knockdown of SETDB1 in PWS-specific induced pluripotent cells (iPSCs) causes a decrease in the accumulation of H3K9 trimethylation (H3K9me3) at 116HG and corresponding accumulation of the active chromatin mark histone H3 lysine 4 dimethylation (H3K4me2). We also show that upon knockdown of SETDB1 in PWS-specific iPSCs, expression of maternally silenced 116HG RNA is partially restored. SETDB1 knockdown in PWS iPSCs also disrupts DNA methylation at the PWS-IC where a decrease in 5-methylcytosine is observed in association with a concomitant increase in 5-hydroxymethylcytosine. This observation suggests that the ZNF274/SETDB1 complex bound to the SNORD116 cluster may protect the PWS-IC from DNA demethylation during early development. Our findings reveal novel epigenetic mechanisms that function to repress the maternal 15q11-q13 region. PMID:24760766

  3. Effects of TET1 knockdown on gene expression and DNA methylation in porcine induced pluripotent stem cells.

    PubMed

    Fan, Anran; Ma, Kuiying; An, Xinglan; Ding, Yu; An, Peipei; Song, Guangqi; Tang, Lina; Zhang, Sheng; Zhang, Peng; Tan, Wentao; Tang, Bo; Zhang, Xueming; Li, Ziyi

    2013-12-01

    TET1 is implicated in maintaining the pluripotency of embryonic stem cells. However, its precise effects on induced pluripotent stem cells (iPSCs), and particularly on porcine iPSCs (piPSCs), are not well defined. To investigate the role of TET1 in the pluripotency and differentiation of piPSCs, piPSCs were induced from porcine embryonic fibroblasts by overexpression of POU5F1 (OCT4), SOX2, KLF4, and MYC (C-MYC). siRNAs targeting to TET1 were used to transiently knockdown the expression of TET1 in piPSCs. Morphological abnormalities and loss of the undifferentiated state of piPSCs were observed in the piPSCs after the downregulation of TET1. The effects of TET1 knockdown on the expression of key stem cell factors and differentiation markers were analyzed to gain insights into the molecular mechanisms underlying the phenomenon. The results revealed that knockdown of TET1 resulted in the downregulated expression of pluripotency-related genes, such as LEFTY2, KLF2, and SOX2, and the upregulated expression of differentiation-related genes including PITX2, HAND1, GATA6, and LEF1. However, POU5F1, MYC, KLF4, and NANOG were actually not downregulated. Further analysis showed that the methylation levels of the promoters for POU5F1 and MYC increased significantly after TET1 downregulation, whereas there were no obvious changes in the promoters of SOX2, KLF4, and NANOG. The methylation of the whole genome increased, while hydroxymethylation slightly declined. Taken together, these results suggest that TET1 may play important roles in the self-renewal of piPSCs and the maintenance of their characteristics by regulating the expression of genes and the DNA methylation. PMID:24051058

  4. Targeted siRNA Delivery and mRNA Knockdown Mediated by Bispecific Digoxigenin-binding Antibodies

    PubMed Central

    Schneider, Britta; Grote, Michael; John, Matthias; Haas, Alexander; Bramlage, Birgit; lckenstein, Ludger M; Jahn-Hofmann, Kerstin; Bauss, Frieder; Cheng, Weijun; Croasdale, Rebecca; Daub, Karin; Dill, Simone; Hoffmann, Eike; Lau, Wilma; Burtscher, Helmut; Ludtke, James L; Metz, Silke; Mundigl, Olaf; Neal, Zane C; Scheuer, Werner; Stracke, Jan; Herweijer, Hans; Brinkmann, Ulrjch

    2012-01-01

    Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3′end was bound in a 2:1 ratio to the bsAbs. These bsAb–siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC50 siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature. PMID:23344238

  5. CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA

    PubMed Central

    Miura, Hiromi; Gurumurthy, Channabasavaiah B; Sato, Takehito; Sato, Masahiro; Ohtsuka, Masato

    2015-01-01

    Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method. PMID:26242611

  6. CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA.

    PubMed

    Miura, Hiromi; Gurumurthy, Channabasavaiah B; Sato, Takehito; Sato, Masahiro; Ohtsuka, Masato

    2015-08-05

    Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.

  7. Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

    PubMed Central

    Maher, Kathryn N.; Catanese, Mary; Chase, Daniel L.

    2013-01-01

    RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab. PMID:24121477

  8. Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi

    PubMed Central

    Fowler, Daniel K.; Williams, Carly; Gerritsen, Alida T.; Washbourne, Philip

    2016-01-01

    Artificial microRNA (amiRNA) sequences embedded in natural microRNA (miRNA) backbones have proven to be useful tools for RNA interference (RNAi). amiRNAs have reduced off-target and toxic effects compared to other RNAi-based methods such as short-hairpin RNAs (shRNA). amiRNAs are often less effective for knockdown, however, compared to their shRNA counterparts. We screened a large empirically-designed amiRNA set in the synthetic inhibitory BIC/miR-155 RNA (SIBR) scaffold and show common structural and sequence-specific features associated with effective amiRNAs. We then introduced exogenous motifs into the basal stem region which increase amiRNA biogenesis and knockdown potency. We call this modified backbone the enhanced SIBR (eSIBR) scaffold. Using chained amiRNAs for multi-gene knockdown, we show that concatenation of miRNAs targeting different genes is itself sufficient for increased knockdown efficacy. Further, we show that eSIBR outperforms wild-type SIBR (wtSIBR) when amiRNAs are chained. Finally, we use a lentiviral expression system in cultured neurons, where we again find that eSIBR amiRNAs are more potent for multi-target knockdown of endogenous genes. eSIBR will be a valuable tool for RNAi approaches, especially for studies where knockdown of multiple targets is desired. PMID:26582923

  9. Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis.

    PubMed

    Al Nakouzi, Nader; Wang, Chris Kedong; Beraldi, Eliana; Jager, Wolfgang; Ettinger, Susan; Fazli, Ladan; Nappi, Lucia; Bishop, Jennifer; Zhang, Fan; Chauchereau, Anne; Loriot, Yohann; Gleave, Martin

    2016-01-01

    Clusterin (CLU) is a stress-activated molecular chaperone that confers treatment resistance to taxanes when highly expressed. While CLU inhibition potentiates activity of taxanes and other anti-cancer therapies in preclinical models, progression to treatment-resistant disease still occurs implicating additional compensatory survival mechanisms. Taxanes are believed to selectively target cells in mitosis, a complex mechanism controlled in part by balancing antagonistic roles of Cdc25C and Wee1 in mitosis progression. Our data indicate that CLU silencing induces a constitutive activation of Cdc25C, which delays mitotic exit and hence sensitizes cancer cells to mitotic-targeting agents such as taxanes. Unchecked Cdc25C activation leads to mitotic catastrophe and cell death unless cells up-regulate protective mechanisms mediated through the cell cycle regulators Wee1 and Cdk1. In this study, we show that CLU silencing induces a constitutive activation of Cdc25C via the phosphatase PP2A leading to relief of negative feedback inhibition and activation of Wee1-Cdk1 to promote survival and limit therapeutic efficacy. Simultaneous inhibition of CLU-regulated cell cycle effector Wee1 may improve synergistic responses of biologically rational combinatorial regimens using taxanes and CLU inhibitors. PMID:27198502

  10. Antibiotic Resistance

    MedlinePlus

    ... lives. But there is a growing problem of antibiotic resistance. It happens when bacteria change and become able ... resistant to several common antibiotics. To help prevent antibiotic resistance Don't use antibiotics for viruses like colds ...

  11. Drug Resistance

    MedlinePlus

    HIV Treatment Drug Resistance (Last updated 3/1/2016; last reviewed 3/1/2016) Key Points As HIV multiplies in the ... the risk of drug resistance. What is HIV drug resistance? Once a person becomes infected with HIV, ...

  12. Inhibition or knockdown of ABC transporters enhances susceptibility of adult and juvenile schistosomes to Praziquantel.

    PubMed

    Kasinathan, Ravi S; Sharma, Lalit Kumar; Cunningham, Charles; Webb, Thomas R; Greenberg, Robert M

    2014-10-01

    Parasitic flatworms of the genus Schistosoma cause schistosomiasis, a neglected tropical disease that affects hundreds of millions. Treatment of schistosomiasis depends almost entirely on the drug praziquantel (PZQ). Though essential to treating and controlling schistosomiasis, a major limitation of PZQ is that it is not active against immature mammalian-stage schistosomes. Furthermore, there are reports of field isolates with heritable reductions in PZQ susceptibility, and researchers have selected for PZQ-resistant schistosomes in the laboratory. P-glycoprotein (Pgp; ABCB1) and other ATP binding cassette (ABC) transporters remove a wide variety of toxins and xenobiotics from cells, and have been implicated in multidrug resistance (MDR). Changes in ABC transporter structure or expression levels are also associated with reduced drug susceptibility in parasitic helminths, including schistosomes. Here, we show that the activity of PZQ against schistosome adults and juveniles ex vivo is potentiated by co-administration of either the highly potent Pgp inhibitor tariquidar or combinations of inhibitors targeting multiple ABC multidrug transporters. Adult worms exposed to sublethal PZQ concentrations remain active, but co-administration of ABC transporter inhibitors results in complete loss of motility and disruption of the tegument. Notably, juvenile schistosomes (3-4 weeks post infection), normally refractory to 2 µM PZQ, become paralyzed when transporter inhibitors are added in combination with the PZQ. Experiments using the fluorescent PZQ derivative (R)-PZQ-BODIPY are consistent with the transporter inhibitors increasing effective intraworm concentrations of PZQ. Adult worms in which expression of ABC transporters has been suppressed by RNA interference show increased responsiveness to PZQ and increased retention of (R)-PZQ-BODIPY consistent with an important role for these proteins in setting levels of PZQ susceptibility. These results indicate that parasite ABC

  13. Knock-down of CD44 regulates endothelial cell differentiation via NFκB-mediated chemokine production.

    PubMed

    Olofsson, Berit; Porsch, Helena; Heldin, Paraskevi

    2014-01-01

    A striking feature of microvascular endothelial cells is their capacity to fuse and differentiate into tubular structures when grown in three-dimensional (3D) extracellular matrices, in collagen or Matrigel, mimicking the in vivo blood vessel formation. In this study we demonstrate that human telomerase-immortalised foreskin microvascular endothelial (TIME) cells express high levels of the hyaluronan receptor CD44 and the hyaluronidase HYAL2. Knock-down of CD44 or HYAL2 resulted in an inability of TIME cells to form a tubular network, suggesting a key regulatory role of hyaluronan in controlling TIME cell tubulogenesis in 3D matrices. Knock-down of CD44 resulted in an upregulation of mRNA expression of the chemokines CXCL9 and CXCL12, as well as their receptors CXCR3 and CXCR4. This was accompanied by a defect maturation of the tubular structure network and increased phosphorylation of the inhibitor of NFκB kinase (IKK) complex and thus translocation of NFκB into the nucleus and activation of chemokine targed genes. Furthermore, the interaction between CD44 and hyaluronan determines the adhesion of breast cancer cells. In summary, our observations support the notion that the interaction between CD44 and hyaluronan regulates microvascular endothelial cell tubulogenesis by affecting the expression of cytokines and their receptors, as well as breast cancer dissemination. PMID:24614402

  14. Tissue Inhibitor of Matrix Metalloproteinases-1 Knockdown Suppresses the Proliferation of Human Adipose-Derived Stem Cells

    PubMed Central

    Zhang, Peihua; Li, Jin; Qi, Yawei; Tang, Xudong; Duan, Jianfeng; Liu, Li; Wu, Zeyong; Liang, Jie; Li, Jiangfeng; Wang, Xian; Zeng, Guofang; Liu, Hongwei

    2016-01-01

    Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional matrix metalloproteinase, and it is involved in the regulation of cell proliferation and apoptosis in various cell types. However, little is known about the effect of TIMP-1 expression on the proliferation of adipose-derived stem cells (ADSCs). Therefore, TIMP-1 expression in the ADSCs was firstly detected by western blotting, and TIMP-1 gene was knocked down by lentivirus-mediated shRNA. Cell proliferation was then evaluated by MTT assay and Ki67 staining, respectively. Cell cycle progression was determined by flow cytometry. The changes of p51, p21, cyclin E, cyclin-dependent kinase 2 (CDK2), and P-CDK2 caused by TIMP-1 knockdown were detected by western blotting. The results indicated that ADSCs highly expressed TIMP-1 protein, and the knockdown of TIMP-1 inhibited cell proliferation and arrested cell cycle progression at G1 phase in the ADSCs possibly through the upregulation of p53, p21, and P-CDK2 protein levels and concurrent downregulation of cyclin E and CDK2 protein levels. These findings suggest that TIMP-1 works as a positive regulator of cell proliferation in ADSCs. PMID:27239203

  15. Design of 8-ft-Diameter Barrel Test Article Attachment Rings for Shell Buckling Knockdown Factor Project

    NASA Technical Reports Server (NTRS)

    Lovejoy, Andrew E.; Hilburger, Mark W.

    2010-01-01

    The Shell Buckling Knockdown Factor (SBKF) project includes the testing of sub-scale cylinders to validate new shell buckling knockdown factors for use in the design of the Ares-I and Ares-V launch vehicles. Test article cylinders represent various barrel segments of the Ares-I and Ares-V vehicles, and also include checkout test articles. Testing will be conducted at Marshall Space Flight Center (MSFC) for test articles having an eight-foot diameter outer mold line (OML) and having lengths that range from three to ten feet long. Both ends of the test articles will be connected to the test apparatus using attachment rings. Three multiple-piece and one single-piece design for the attachment rings were developed and analyzed. The single-piece design was chosen and will be fabricated from either steel or aluminum (Al) depending on the required safety factors (SF) for test hardware. This report summarizes the design and analysis of these attachment ring concepts.

  16. Knockdown of RhoA expression alters ovarian cancer biological behavior in vitro and in nude mice.

    PubMed

    Wang, Xiaoxia; Jiang, Wenyan; Kang, Jiali; Liu, Qicai; Nie, Miaoling

    2015-08-01

    RhoA regulates cell proliferation, migration, angiogenesis and gene expression. Altered RhoA activity contributes to cancer progression. The present study investigated the effects of RhoA knockdown on the regulation of ovarian cancer biological behavior in vitro and in nude mice. The expression of RhoA was knocked down using a lentivirus carrying RhoA short hairpin RNA (shRNA) in ovarian cancer cells and was confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The altered ovarian cancer biological behaviors were assayed by cell viability, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL), migration, invasion, and nude mice tumorigenicity assays, while the altered gene expression was detected by RT-qPCR and western blot analysis. The results showed that lentivirus-carrying RhoA shRNA significantly suppressed RhoA expression in ovarian cancer cells, which suppressed tumor cell viability, migration, invasion and adhesion in vitro. RhoA silencing also inhibited the tumorigenicity of ovarian cancer cells in nude mice, which was characterized by the suppression of tumor xenograft formation and growth and induction of tumor cell apoptosis. The results of the present study demonstrated that knockdown of RhoA expression had a significant antitumor effect on ovarian cancer cells in vitro and in nude mice, suggesting that RhoA may be a target for the development of a novel therapeutic strategy in the control of ovarian cancer.

  17. Knockdown of ApoL1 in Zebrafish Larvae Affects the Glomerular Filtration Barrier and the Expression of Nephrin

    PubMed Central

    Kotb, Ahmed M.; Simon, Ole; Blumenthal, Antje; Vogelgesang, Silke; Dombrowski, Frank; Amann, Kerstin; Zimmermann, Uwe; Endlich, Karlhans; Endlich, Nicole

    2016-01-01

    APOL1, a secreted high-density lipoprotein, is expressed in different human tissues. Genetic variants of APOL1 are described to be associated with the development of end stage renal diseases in African Americans. In human kidney, APOL1 is mainly expressed in podocytes that are responsible for proper blood filtration. Since mice do not express ApoL1, the zebrafish is an ideal model to study the role of ApoL1. Injection of morpholinos against zApoL1 into zebrafish eggs and larvae, respectively, induces severe edema indicating a leakage of the filtration barrier. This was demonstrated in zApoL1 knockdown larvae by intravascular injection of fluorescently-labeled 10- and 500-kDa dextrans and by clearance of the vitamin D-binding protein from the circulation. Immunohistochemistry and RT-PCR revealed the reduction of nephrin, a podocyte-specific protein essential for blood filtration. Coinjection of human nephrin mRNA rescued the zApoL1 knockdown induced phenotype. Reduced APOL1 and nephrin levels were also found in biopsies of patients suffering from end stage renal diseases. Our results demonstrate that zApoL1 is essential for proper blood filtration in the zebrafish glomerulus and that zApoL1 affects the expression of nephrin. PMID:27138898

  18. Knockdown of the coenzyme Q synthesis gene Smed-dlp1 affects planarian regeneration and tissue homeostasis.

    PubMed

    Shiobara, Yumiko; Harada, Chiaki; Shiota, Takeshi; Sakamoto, Kimitoshi; Kita, Kiyoshi; Tanaka, Saeko; Tabata, Kenta; Sekie, Kiyoteru; Yamamoto, Yorihiro; Sugiyama, Tomoyasu

    2015-12-01

    The freshwater planarian is a model organism used to study tissue regeneration that occupies an important position among multicellular organisms. Planarian genomic databases have led to the identification of genes that are required for regeneration, with implications for their roles in its underlying mechanism. Coenzyme Q (CoQ) is a fundamental lipophilic molecule that is synthesized and expressed in every cell of every organism. Furthermore, CoQ levels affect development, life span, disease and aging in nematodes and mice. Because CoQ can be ingested in food, it has been used in preventive nutrition. In this study, we investigated the role of CoQ in planarian regeneration. Planarians synthesize both CoQ9 and rhodoquinone 9 (RQ9). Knockdown of Smed-dlp1, a trans-prenyltransferase gene that encodes an enzyme that synthesizes the CoQ side chain, led to a decrease in CoQ9 and RQ9 levels. However, ATP levels did not consistently decrease in these animals. Knockdown animals exhibited tissue regression and curling. The number of mitotic cells decreased in Smed-dlp1 (RNAi) animals. These results suggested a failure in physiological cell turnover and stem cell function. Accordingly, regenerating planarians died from lysis or exhibited delayed regeneration. Interestingly, the observed phenotypes were partially rescued by ingesting food supplemented with α-tocopherol. Taken together, our results suggest that oxidative stress induced by reduced CoQ9 levels affects planarian regeneration and tissue homeostasis.

  19. Targeted knockdown of insulin-like growth factor binding protein-2 disrupts cardiovascular development in zebrafish embryos.

    PubMed

    Wood, Antony W; Schlueter, Peter J; Duan, Cunming

    2005-04-01

    IGF binding protein-2 (IGFBP-2) is an evolutionarily conserved protein that binds IGFs and modulates their biological activities. Although the actions of IGFBP-2 have been well studied in vitro, we have a poor understanding of its in vivo functions, particularly during early development. Using the transparent zebrafish embryo as a model, we show that IGFBP-2 mRNA is expressed in lens epithelium and cranial boundary regions during early embryonic development and becomes localized to the liver by the completion of embryogenesis. Targeted knock-down of IGFBP-2 by antisense morpholino-modified oligonucleotides resulted in delayed development, reduced body growth, reduced IGF-I mRNA levels, and disruptions to cardiovascular development, including reduced blood cell number, reduced blood circulation, cardiac dysfunction, and brain ventricle edema. Detailed examination of vascular tissues, using a stable transgenic line of zebrafish expressing green fluorescent protein in vascular endothelial cells, revealed specific angiogenic (vessel sprouting) defects in IGFBP-2 knockdown embryos, with effects being localized in regions associated with IGFBP-2 mRNA expression. These findings suggest that IGFBP-2 is required for general embryonic development and growth and plays a local role in regulating vascular development in a model vertebrate organism.

  20. Stable SREBP-1a knockdown decreases the cell proliferation rate in human preadipocyte cells without inducing senescence

    SciTech Connect

    Alvarez, María Soledad; Fernandez-Alvarez, Ana; Cucarella, Carme; Casado, Marta

    2014-04-25

    Highlights: • SGBS cells mostly expressed SREBP-1a variant. • SREBP-1a knockdown decreased the proliferation of SGBS cells without inducing senescence. • We have identified RBBP8 and CDKN3 genes as potential SREBP-1a targets. - Abstract: Sterol regulatory element binding proteins (SREBP), encoded by the Srebf1 and Srebf2 genes, are important regulators of genes involved in cholesterol and fatty acid metabolism. Whereas SREBP-2 controls the cholesterol synthesis, SREBP-1 proteins (-1a and -1c) function as the central hubs in lipid metabolism. Despite the key function of these transcription factors to promote adipocyte differentiation, the roles of SREBP-1 proteins during the preadipocyte state remain unknown. Here, we evaluate the role of SREBP-1 in preadipocyte proliferation using RNA interference technology. Knockdown of the SREBP-1a gene decreased the proliferation rate in human SGBS preadipocyte cell strain without inducing senescence. Furthermore, our data identified retinoblastoma binding protein 8 and cyclin-dependent kinase inhibitor 3 genes as new potential SREBP-1 targets, in addition to cyclin-dependent kinase inhibitor 1A which had already been described as a gene regulated by SREBP-1a. These data suggested a new role of SREBP-1 in adipogenesis via regulation of preadipocyte proliferation.

  1. Knockdown of the pericellular matrix molecule perlecan lowers in situ cell and matrix stiffness in developing cartilage.

    PubMed

    Xu, Xin; Li, Zhiyu; Leng, Yue; Neu, Corey P; Calve, Sarah

    2016-10-15

    The pericellular matrix (PCM) is a component of the extracellular matrix that is found immediately surrounding individual chondrocytes in developing and adult cartilage, and is rich in the proteoglycan perlecan. Mutations in perlecan are the basis of several developmental disorders, which are thought to arise from disruptions in the mechanical stability of the PCM. We tested the hypothesis that defects in PCM organization will reduce the stiffness of chondrocytes in developing cartilage by combining a murine model of Schwartz-Jampel syndrome, in which perlecan is knocked down, with our novel atomic force microscopy technique that can measure the stiffness of living cells and surrounding matrix in embryonic and postnatal tissues in situ. Perlecan knockdown altered matrix organization and significantly decreased the stiffness of both chondrocytes and interstitial matrix as a function of age and genotype. Our results demonstrate that the knockdown of a spatially restricted matrix molecule can have a profound influence on cell and tissue stiffness, implicating a role for outside-in mechanical signals from the PCM in regulating the intracellular mechanisms required for the overall development of cartilage.

  2. MiRNA-Embedded ShRNAs for Radiation-Inducible LGMN Knockdown and the Antitumor Effects on Breast Cancer

    PubMed Central

    Zhang, Zhi-Qiang; Cao, Zhi; Liu, Cong; Li, Rong; Wang, Wei-Dong; Wang, Xing-Yong

    2016-01-01

    Legumain (LGMN) is highly expressed in breast cancer (BC) and other solid tumors and is a potential anticancer target. Here we investigate the anti-tumor effects of short hairpin RNAs (shRNAs) targeting LGMN embedded in a microRNA-155 (miR-155) architecture, which is driven by a radiation-inducible chimeric RNA polymerase II (Pol II) promoter. Lentiviral vectors were generated with the chimeric promoter which controlled the expression of downstream shRNA-miR-155 cassette. Fluorescence was observed by using confocal microscopy. Real-time quantitative PCR and Western blotting were used to determine the expression level of LGMN, MMP2, and MMP9. Furthermore, the proliferation and invasive ability of BC cells was analyzed via plate colony formation and invasion assays. Here we demonstrated that the chimeric promoter could be effectively induced by radiation treatment. Furthermore, the shRNA-miR-155 cassette targeting LGMN could be effectively activated by the chimeric promoter. Radiation plus knockdown of LGMN impairs colony formation and dampens cell migration and invasion in BC cells. Inhibition of LGMN downregulates MMP2 and MMP9 expression in BC cells. Pol II-driven shRNA-miR-155 could effectively suppress the growth and invasiveness of BC cells, and that the interference effects could be regulated by radiation doses. Moreover, knockdown of LGMN alleviates the aggressive phenotype of BC cells through modulating MMPs expression. PMID:27656894

  3. Knock-down of BCL6 / STAT6 sensitizes primary B cell lymphoma cells for treatment with current therapeutic agents

    PubMed Central

    Häberle, Marie-Therese; Kelsch, Elena; Dorsch, Karola; Möller, Peter; Ritz, Olga

    2014-01-01

    Primary mediastinal B cell lymphoma (PMBL) is characterized by specific molecular hallmarks including the expression of B Cell Lymphoma factor 6 (BCL6) and the presence of the activated Signal Transducers and Activators of Transcription factor 6 (STAT6). Recently we have shown that combined targeting of BCL6 and activated STAT6 by specific chemical inhibitors in PMBL resulted in additive efficacy regarding their negative effects on cell viability. Given that despite general efficient immuno-chemotherapy in PMBL the delayed treatment-related sequelae remains still a main challenge, we analyzed the role of BCL6 and activated STAT6 in the sensitivity of PMBL cells to the current treatment components. We found that the knock-down of BCL6 / STAT6 by siRNA sensitized the PMBL cells to the effects of R-CHOP components in two of three PMBL cell lines. In one cell line, MedB-1, which is marked by less expression of BCL6 and mutated STAT6, the knock-down of BCL6 / STAT6 did not enhance the efficiency of Doxorubicin, Rituximab, and Vincristin. Thus, the targeting of BCL6 and STAT6 in addition or prior to the treatment with components of the current immuno-chemotherapy may sensitize the PMBL tumor cells for drug effects, at least in parts of PMBL cases. PMID:25594020

  4. Histone Methylase MLL1 plays critical roles in tumor growth and angiogenesis and its knockdown suppresses tumor growth in vivo

    PubMed Central

    Ansari, Khairul I.; Kasiri, Sahba; Mandal, Subhrangsu S.

    2012-01-01

    Mixed lineage leukemias (MLL) are human histone H3 lysine-4 specific methyl transferases that play critical roles in gene expression, epigenetics, and cancer. Herein, we demonstrated that antisense-mediated knockdown of MLL1 induced cell cycle arrest and apoptosis in cultured cells. Intriguingly, application of MLL1-antisense specifically knocked down MLL1 in vivo and suppressed the growth of xenografted cervical tumor implanted in nude mouse. MLL1-knockdown downregulated various growth and angiogenic factors such as HIF1α, VEGF and CD31 in tumor tissue affecting tumor growth. MLL1 is overexpressed along the line of vascular network and localized adjacent to endothelial cell layer expressing CD31, indicating potential roles of MLL1 in vasculogenesis. MLL1 is also overexpressed in the hypoxic regions along with HIF1α. Overall, our studies demonstrated that MLL1 is a key player in hypoxia signaling, vasculogenesis, and tumor growth, and its depletion suppresses tumor growth in vivo, indicating its potential in novel cancer therapy. PMID:22926525

  5. Knock-down of BCL6 / STAT6 sensitizes primary B cell lymphoma cells for treatment with current therapeutic agents.

    PubMed

    Häberle, Marie-Therese; Kelsch, Elena; Dorsch, Karola; Möller, Peter; Ritz, Olga

    2014-01-01

    Primary mediastinal B cell lymphoma (PMBL) is characterized by specific molecular hallmarks including the expression of B Cell Lymphoma factor 6 (BCL6) and the presence of the activated Signal Transducers and Activators of Transcription factor 6 (STAT6). Recently we have shown that combined targeting of BCL6 and activated STAT6 by specific chemical inhibitors in PMBL resulted in additive efficacy regarding their negative effects on cell viability. Given that despite general efficient immuno-chemotherapy in PMBL the delayed treatment-related sequelae remains still a main challenge, we analyzed the role of BCL6 and activated STAT6 in the sensitivity of PMBL cells to the current treatment components. We found that the knock-down of BCL6 / STAT6 by siRNA sensitized the PMBL cells to the effects of R-CHOP components in two of three PMBL cell lines. In one cell line, MedB-1, which is marked by less expression of BCL6 and mutated STAT6, the knock-down of BCL6 / STAT6 did not enhance the efficiency of Doxorubicin, Rituximab, and Vincristin. Thus, the targeting of BCL6 and STAT6 in addition or prior to the treatment with components of the current immuno-chemotherapy may sensitize the PMBL tumor cells for drug effects, at least in parts of PMBL cases. PMID:25594020

  6. Knockdown of CUL4B Suppresses the Proliferation and Invasion in Non-Small Cell Lung Cancer Cells.

    PubMed

    Wang, Xuguang; Chen, Zhe

    2016-01-01

    Cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, was found to be overexpressed in many types of tumors. However, the expression pattern and role of CUL4B in non-small cell lung cancer (NSCLC) remain largely unknown. Therefore, in the present study, we investigated the role of CUL4B in NSCLC, and the underlying mechanism was also explored. Our results showed that CUL4B was highly expressed in NSCLC cell lines. Silencing CUL4B obviously inhibited proliferation and migration/invasion of NSCLC cells, and it also suppressed the epithelial-mesenchymal transition (EMT) progress in NSCLC cells. Furthermore, knockdown of CUL4B significantly inhibited the expression of β-catenin, cyclin D1, and c-Myc in NSCLC cells. Taken together, these results suggest that knockdown of CUL4B inhibited the proliferation and invasion through suppressing the Wnt/β-catenin signaling pathway in NSCLC cells. Therefore, CUL4B may represent a novel therapeutic target for the treatment of NSCLC. PMID:27656838

  7. Knockdown of the pericellular matrix molecule perlecan lowers in situ cell and matrix stiffness in developing cartilage.

    PubMed

    Xu, Xin; Li, Zhiyu; Leng, Yue; Neu, Corey P; Calve, Sarah

    2016-10-15

    The pericellular matrix (PCM) is a component of the extracellular matrix that is found immediately surrounding individual chondrocytes in developing and adult cartilage, and is rich in the proteoglycan perlecan. Mutations in perlecan are the basis of several developmental disorders, which are thought to arise from disruptions in the mechanical stability of the PCM. We tested the hypothesis that defects in PCM organization will reduce the stiffness of chondrocytes in developing cartilage by combining a murine model of Schwartz-Jampel syndrome, in which perlecan is knocked down, with our novel atomic force microscopy technique that can measure the stiffness of living cells and surrounding matrix in embryonic and postnatal tissues in situ. Perlecan knockdown altered matrix organization and significantly decreased the stiffness of both chondrocytes and interstitial matrix as a function of age and genotype. Our results demonstrate that the knockdown of a spatially restricted matrix molecule can have a profound influence on cell and tissue stiffness, implicating a role for outside-in mechanical signals from the PCM in regulating the intracellular mechanisms required for the overall development of cartilage. PMID:27578148

  8. RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation.

    PubMed

    Walsh, Naomi; Larkin, AnneMarie; Swan, Niall; Conlon, Kevin; Dowling, Paul; McDermott, Ray; Clynes, Martin

    2011-07-28

    We previously identified Hop as over expressed in invasive pancreatic cancer cell lines and malignant tissues of pancreatic cancer patients, suggesting an important role for Hop in the biology of invasive pancreatic cancer. Hop is a co-chaperone protein that binds to both Hsp70/Hsp90. We hypothesised that by targeting Hop, signalling pathways modulating invasion and client protein stabilisation involving Hsp90-dependent complexes may be altered. In this study, we show that Hop knockdown by small interfering (si)RNA reduces the invasion of pancreatic cancer cells, resulting in decreased expression of the downstream target gene, matrix metalloproteinases-2 (MMP-2). Hop in conditioned media co-immunoprecipitates with MMP-2, implicating a possible extracellular function for Hop. Knockdown of Hop expression also reduced expression levels of Hsp90 client proteins, HER2, Bcr-Abl, c-MET and v-Src. Furthermore, Hop is strongly expressed in high grade PanINs compared to lower PanIN grades, displaying differential localisation in invasive ductal pancreatic cancer, indicating that the localisation of Hop is an important factor in pancreatic tumours. Our data suggests that the attenuation of Hop expression inactivates key signal transduction proteins which may decrease the invasiveness of pancreatic cancer cells possibly through the modulation of Hsp90 activity. Therefore, targeting Hop in pancreatic cancer may constitute a viable strategy for targeted cancer therapy.

  9. Knockdown of triglyceride synthesis does not enhance palmitate lipotoxicity or prevent oleate-mediated rescue in rat hepatocytes.

    PubMed

    Leamy, Alexandra K; Hasenour, Clinton M; Egnatchik, Robert A; Trenary, Irina A; Yao, Cong-Hui; Patti, Gary J; Shiota, Masakazu; Young, Jamey D

    2016-09-01

    Experiments in a variety of cell types, including hepatocytes, consistently demonstrate the acutely lipotoxic effects of saturated fatty acids, such as palmitate (PA), but not unsaturated fatty acids, such as oleate (OA). PA+OA co-treatment fully prevents PA lipotoxicity through mechanisms that are not well defined but which have been previously attributed to more efficient esterification and sequestration of PA into triglycerides (TGs) when OA is abundant. However, this hypothesis has never been directly tested by experimentally modulating the relative partitioning of PA/OA between TGs and other lipid fates in hepatocytes. In this study, we found that addition of OA to PA-treated hepatocytes enhanced TG synthesis, reduced total PA uptake and PA lipid incorporation, decreased phospholipid saturation and rescued PA-induced ER stress and lipoapoptosis. Knockdown of diacylglycerol acyltransferase (DGAT), the rate-limiting step in TG synthesis, significantly reduced TG accumulation without impairing OA-mediated rescue of PA lipotoxicity. In both wild-type and DGAT-knockdown hepatocytes, OA co-treatment significantly reduced PA lipid incorporation and overall phospholipid saturation compared to PA-treated hepatocytes. These data indicate that OA's protective effects do not require increased conversion of PA into inert TGs, but instead may be due to OA's ability to compete against PA for cellular uptake and/or esterification and, thereby, normalize the composition of cellular lipids in the presence of a toxic PA load. PMID:27249207

  10. Knockdown of EphB1 receptor decreases medulloblastoma cell growth and migration and increases cellular radiosensitization

    PubMed Central

    Timofeeva, Olga; Pasquale, Elena B.; Hirsch, Kellen; MacDonald, Tobey J.; Dritschilo, Anatoly; Lee, Yi Chien; Henkemeyer, Mark; Rood, Brian; Jung, Mira; Wang, Xiao-Jing; Kool, Marcel

    2015-01-01

    The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target. PMID:25879388

  11. Knockdown of the coenzyme Q synthesis gene Smed-dlp1 affects planarian regeneration and tissue homeostasis

    PubMed Central

    Shiobara, Yumiko; Harada, Chiaki; Shiota, Takeshi; Sakamoto, Kimitoshi; Kita, Kiyoshi; Tanaka, Saeko; Tabata, Kenta; Sekie, Kiyoteru; Yamamoto, Yorihiro; Sugiyama, Tomoyasu

    2015-01-01

    The freshwater planarian is a model organism used to study tissue regeneration that occupies an important position among multicellular organisms. Planarian genomic databases have led to the identification of genes that are required for regeneration, with implications for their roles in its underlying mechanism. Coenzyme Q (CoQ) is a fundamental lipophilic molecule that is synthesized and expressed in every cell of every organism. Furthermore, CoQ levels affect development, life span, disease and aging in nematodes and mice. Because CoQ can be ingested in food, it has been used in preventive nutrition. In this study, we investigated the role of CoQ in planarian regeneration. Planarians synthesize both CoQ9 and rhodoquinone 9 (RQ9). Knockdown of Smed-dlp1, a trans-prenyltransferase gene that encodes an enzyme that synthesizes the CoQ side chain, led to a decrease in CoQ9 and RQ9 levels. However, ATP levels did not consistently decrease in these animals. Knockdown animals exhibited tissue regression and curling. The number of mitotic cells decreased in Smed-dlp1 (RNAi) animals. These results suggested a failure in physiological cell turnover and stem cell function. Accordingly, regenerating planarians died from lysis or exhibited delayed regeneration. Interestingly, the observed phenotypes were partially rescued by ingesting food supplemented with α-tocopherol. Taken together, our results suggest that oxidative stress induced by reduced CoQ9 levels affects planarian regeneration and tissue homeostasis. PMID:26516985

  12. Knockdown of ribosomal protein S7 causes developmental abnormalities via p53 dependent and independent pathways in zebrafish.

    PubMed

    Duan, Juan; Ba, Qian; Wang, Ziliang; Hao, Miao; Li, Xiaoguang; Hu, Pingting; Zhang, Deyi; Zhang, Ruiwen; Wang, Hui

    2011-08-01

    Ribosomal proteins (RPs), structural components of the ribosome involved in protein synthesis, are of significant importance in all organisms. Previous studies have suggested that some RPs may have other functions in addition to assembly of the ribosome. The small ribosomal subunits RPS7, has been reported to modulate the mdm2-p53 interaction. To further investigate the biological functions of RPS7, we used morpholino antisense oligonucleotides (MO) to specifically knockdown RPS7 in zebrafish. In RPS7-deficient embryos, p53 was activated, and its downstream target genes and biological events were induced, including apoptosis and cell cycle arrest. Hematopoiesis was also impaired seriously in RPS7-deficient embryos, which was confirmed by the hemoglobin O-dianisidine staining of blood cells, and the expression of scl, gata1 and α-E1 globin were abnormal. The matrix metalloproteinase (mmp) family genes were also activated in RPS7 morphants, indicating that improper cell migration might also cause development defects. Furthermore, simultaneously knockdown of the p53 protein by co-injecting a p53 MO could partially reverse the abnormal phenotype in the morphants. These results strengthen the hypothesis that specific ribosomal proteins regulate p53 and that their deficiency affects hematopoiesis. Moreover, our data implicate that RPS7 is a regulator of matrix metalloproteinase (mmp) family in zebrafish system. These specific functions of RPS7 may provide helpful clues to study the roles of RPs in human disease.

  13. The analysis of an Arabidopsis triple knock-down mutant reveals functions for MBF1 genes under oxidative stress conditions.

    PubMed

    Arce, Débora Pamela; Godoy, Andrea Verónica; Tsuda, Kenichi; Yamazaki, Ken-Ichi; Valle, Estela Marta; Iglesias, María José; Di Mauro, María Florencia; Casalongué, Claudia Anahí

    2010-02-15

    Transcriptional co-activators of the multiprotein bridging factor 1 (MBF1) type belong to a small multigenic family that controls gene expression by connecting transcription factors and the basal transcription machinery. In this report, a triple knock-down mutant (abc-) for the Arabidopsis thaliana MBF1 genes AtMBF1a, AtMBF1b and AtMBF1c was generated. The phenotypic characterization using oxidative agents such as hydrogen peroxide and methyl viologen revealed that the abc- mutant was more sensitive to oxidative stress. The triple knock-down mutant, abc- was also sensitive to osmotic stress mediated by high concentrations of sorbitol. Furthermore, the abc- phenotype was partially or completely rescued by AtMBF1c cDNA over-expression (abc- +c) depending on physiological and developmental conditions. AtMBF1s regulate the expression of ABR1, which is a member of the ethylene-response factor family and acts as ABA repressor. Thus, we conclude that AtMBF1 gene family may function as a regulatory component of the cross-talk node between ethylene, ABA and stress signal pathways. Furthermore, higher levels of a HSP70 mRNA and an immunoreactive HSP70 protein were detected in the abc- mutant. The participation of MBF1c as a possible negative regulator of HSP genes was discussed.

  14. Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein

    PubMed Central

    Perrot-Rechenmann, Catherine; Friml, Jiří

    2016-01-01

    The Auxin Binding Protein 1 (ABP1) is one of the most studied proteins in plants. Since decades ago, it has been the prime receptor candidate for the plant hormone auxin with a plethora of described functions in auxin signaling and development. The developmental importance of ABP1 has recently been questioned by identification of Arabidopsis thaliana abp1 knock-out alleles that show no obvious phenotypes under normal growth conditions. In this study, we examined the contradiction between the normal growth and development of the abp1 knock-outs and the strong morphological defects observed in three different ethanol-inducible abp1 knock-down mutants ( abp1-AS, SS12K, SS12S). By analyzing segregating populations of abp1 knock-out vs. abp1 knock-down crosses we show that the strong morphological defects that were believed to be the result of conditional down-regulation of ABP1 can be reproduced also in the absence of the functional ABP1 protein. This data suggests that the phenotypes in  abp1 knock-down lines are due to the off-target effects and asks for further reflections on the biological function of ABP1 or alternative explanations for the missing phenotypic defects in the abp1 loss-of-function alleles. PMID:26925228

  15. Knockdown of ApoL1 in Zebrafish Larvae Affects the Glomerular Filtration Barrier and the Expression of Nephrin.

    PubMed

    Kotb, Ahmed M; Simon, Ole; Blumenthal, Antje; Vogelgesang, Silke; Dombrowski, Frank; Amann, Kerstin; Zimmermann, Uwe; Endlich, Karlhans; Endlich, Nicole

    2016-01-01

    APOL1, a secreted high-density lipoprotein, is expressed in different human tissues. Genetic variants of APOL1 are described to be associated with the development of end stage renal diseases in African Americans. In human kidney, APOL1 is mainly expressed in podocytes that are responsible for proper blood filtration. Since mice do not express ApoL1, the zebrafish is an ideal model to study the role of ApoL1. Injection of morpholinos against zApoL1 into zebrafish eggs and larvae, respectively, induces severe edema indicating a leakage of the filtration barrier. This was demonstrated in zApoL1 knockdown larvae by intravascular injection of fluorescently-labeled 10- and 500-kDa dextrans and by clearance of the vitamin D-binding protein from the circulation. Immunohistochemistry and RT-PCR revealed the reduction of nephrin, a podocyte-specific protein essential for blood filtration. Coinjection of human nephrin mRNA rescued the zApoL1 knockdown induced phenotype. Reduced APOL1 and nephrin levels were also found in biopsies of patients suffering from end stage renal diseases. Our results demonstrate that zApoL1 is essential for proper blood filtration in the zebrafish glomerulus and that zApoL1 affects the expression of nephrin. PMID:27138898

  16. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    PubMed Central

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L; Booth, Benjamin W; Evans-Holm, Martha; Venken, Koen JT; Levis, Robert W; Spradling, Allan C; Hoskins, Roger A; Bellen, Hugo J

    2015-01-01

    Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates. DOI: http://dx.doi.org/10.7554/eLife.05338.001 PMID:25824290

  17. Knockdown of Oncogenic KRAS in Non-Small Cell Lung Cancers Suppresses Tumor Growth and Sensitizes Tumor Cells to Targeted Therapy

    PubMed Central

    Sunaga, Noriaki; Shames, David S.; Girard, Luc; Peyton, Michael; Larsen, Jill E.; Imai, Hisao; Soh, Junichi; Sato, Mitsuo; Yanagitani, Noriko; Kaira, Kyoichi; Xie, Yang; Gazdar, Adi F.; Mori, Masatomo; Minna, John D.

    2011-01-01

    Oncogenic KRAS is found in >25% of lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC), and is an important target for drug development. To this end, we generated four NSCLC lines with stable knockdown selective for oncogenic KRAS. As expected, stable knockdown of oncogenic KRAS led to inhibition of in vitro and in vivo tumor growth in the KRAS mutant NSCLC cells, but not in NSCLC cells that have wild-type KRAS (but mutant NRAS). Surprisingly, we did not see large-scale induction of cell death and the growth inhibitory effect was not complete. To further understand the ability of NSCLCs to grow despite selective removal of mutant KRAS expression, we performed microarray expression profiling of NSCLC cell lines with or without mutant KRAS knockdown and isogenic human bronchial epithelial cell lines (HBECs) with and without oncogenic KRAS. We found that while the MAPK pathway is significantly down-regulated after mutant KRAS knockdown, these NSCLCs showed increased levels of phospho-STAT3 and phospho-EGFR, and variable changes in phospho-Akt. In addition, mutant KRAS knockdown sensitized the NSCLCs to p38 and EGFR inhibitors. Our findings suggest that targeting oncogenic KRAS by itself will not be sufficient treatment but may offer possibilities of combining anti-KRAS strategies with other targeted drugs. PMID:21306997

  18. Pre-Test Analysis Predictions for the Shell Buckling Knockdown Factor Checkout Tests - TA01 and TA02

    NASA Technical Reports Server (NTRS)

    Thornburgh, Robert P.; Hilburger, Mark W.

    2011-01-01

    This report summarizes the pre-test analysis predictions for the SBKF-P2-CYL-TA01 and SBKF-P2-CYL-TA02 shell buckling tests conducted at the Marshall Space Flight Center (MSFC) in support of the Shell Buckling Knockdown Factor (SBKF) Project, NASA Engineering and Safety Center (NESC) Assessment. The test article (TA) is an 8-foot-diameter aluminum-lithium (Al-Li) orthogrid cylindrical shell with similar design features as that of the proposed Ares-I and Ares-V barrel structures. In support of the testing effort, detailed structural analyses were conducted and the results were used to monitor the behavior of the TA during the testing. A summary of predicted results for each of the five load sequences is presented herein.

  19. Knockdown of the small conductance Ca2+‐activated K+ channels is potently cytotoxic in breast cancer cell lines

    PubMed Central

    Abdulkareem, Zana Azeez; Gee, Julia MW

    2015-01-01

    Background and Purpose Small conductance calcium‐activated potassium (KCa2.x) channels have a widely accepted canonical function in regulating cellular excitability. In this study, we address a potential non‐canonical function of KCa2.x channels in breast cancer cell survival, using in vitro models. Experimental Approach The expression of all KCa2.x channel isoforms was initially probed using RT‐PCR, Western blotting and microarray analysis in five widely studied breast cancer cell lines. In order to assess the effect of pharmacological blockade and siRNA‐mediated knockdown of KCa2.x channels on these cell lines, we utilized MTS proliferation assays and also followed the corresponding expression of apoptotic markers. Key Results All of the breast cancer cell lines, regardless of their lineage or endocrine responsiveness, were highly sensitive to KCa2.x channel blockade. UCL1684 caused cytotoxicity, with LD50 values in the low nanomolar range, in all cell lines. The role of KCa2.x channels was confirmed using pharmacological inhibition and siRNA‐mediated knockdown. This reduced cell viability and also reduced expression of Bcl‐2 but increased expression of active caspase‐7 and caspase‐9. Complementary to these results, a variety of cell lines can be protected from apoptosis induced by staurosporine using the KCa2.x channel activator CyPPA. Conclusions and Implications In addition to a well‐established role for KCa2.x channels in migration, blockade of these channels was potently cytotoxic in breast cancer cell lines, pointing to modulation of KCa2.x channels as a potential therapeutic approach to breast cancer. PMID:26454020

  20. Mammary Gland Specific Knockdown of the Physiological Surge in Cx26 during Lactation Retains Normal Mammary Gland Development and Function

    PubMed Central

    Stewart, Michael K. G.; Plante, Isabelle; Bechberger, John F.; Naus, Christian C.; Laird, Dale W.

    2014-01-01

    Connexin26 (Cx26) is the major Cx protein expressed in the human mammary gland and is up-regulated during pregnancy while remaining elevated throughout lactation. It is currently unknown if patients with loss-of-function Cx26 mutations that result in hearing loss and skin diseases have a greater susceptibility to impaired breast development. To investigate if Cx26 plays a critical role in mammary gland development and differentiation, a novel Cx26 conditional knockout mouse model was generated by crossing Cx26fl/fl mice with mice expressing Cre under the β-Lactoglobulin promoter. Conditional knockdown of Cx26 from the mammary gland resulted in a dramatic reduction in detectable gap junction plaques confirmed by a significant ∼65-70% reduction in Cx26 mRNA and protein throughout parturition and lactation. Interestingly, this reduction was accompanied by a decrease in mammary gland Cx30 gap junction plaques at parturition, while no change was observed for Cx32 or Cx43. Whole mount, histological and immunofluorescent assessment of breast tissue revealed comparatively normal lobuloalveolar development following pregnancy in the conditionally knockdown mice compared to control mice. In addition, glands from genetically-modified mice were capable of producing milk proteins that were evident in the lumen of alveoli and ducts at similar levels as controls, suggesting normal gland function. Together, our results suggest that low levels of Cx26 expression throughout pregnancy and lactation, and not the physiological surge in Cx26, is sufficient for normal gland development and function. PMID:24988191

  1. Characterization of PTPRG in Knockdown and Phosphatase-Inactive Mutant Mice and Substrate Trapping Analysis of PTPRG in Mammalian Cells

    PubMed Central

    Zhang, Wandong; Savelieva, Katerina V.; Tran, David T.; Pogorelov, Vladimir M.; Cullinan, Emily B.; Baker, Kevin B.; Platt, Kenneth A.; Hu, Sean; Rajan, Indrani; Xu, Nianhua; Lanthorn, Thomas H.

    2012-01-01

    Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98–99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, “substrate trapping” mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better

  2. Characterization of PTPRG in knockdown and phosphatase-inactive mutant mice and substrate trapping analysis of PTPRG in mammalian cells.

    PubMed

    Zhang, Wandong; Savelieva, Katerina V; Tran, David T; Pogorelov, Vladimir M; Cullinan, Emily B; Baker, Kevin B; Platt, Kenneth A; Hu, Sean; Rajan, Indrani; Xu, Nianhua; Lanthorn, Thomas H

    2012-01-01

    Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98-99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, "substrate trapping" mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding

  3. Aire knockdown in medullary thymic epithelial cells affects Aire protein, deregulates cell adhesion genes and decreases thymocyte interaction.

    PubMed

    Pezzi, Nicole; Assis, Amanda Freire; Cotrim-Sousa, Larissa Cotrim; Lopes, Gabriel Sarti; Mosella, Maritza Salas; Lima, Djalma Sousa; Bombonato-Prado, Karina F; Passos, Geraldo Aleixo

    2016-09-01

    We demonstrate that even a partial reduction of Aire mRNA levels by siRNA-induced Aire knockdown (Aire KD) has important consequences to medullary thymic epithelial cells (mTECs). Aire knockdown is sufficient to reduce Aire protein levels, impair its nuclear location, and cause an imbalance in large-scale gene expression, including genes that encode cell adhesion molecules. These genes drew our attention because adhesion molecules are implicated in the process of mTEC-thymocyte adhesion, which is critical for T cell development and the establishment of central self-tolerance. Accordingly, we consider the following: 1) mTECs contribute to the elimination of self-reactive thymocytes through adhesion; 2) Adhesion molecules play a crucial role during physical contact between these cells; and 3) Aire is an important transcriptional regulator in mTECs. However, its role in controlling mTEC-thymocyte adhesion remains unclear. Because Aire controls adhesion molecule genes, we hypothesized that the disruption of its expression could influence mTEC-thymocyte interaction. To test this hypothesis, we used a murine Aire(+) mTEC cell line as a model system to reproduce mTEC-thymocyte adhesion in vitro. Transcriptome analysis of the mTEC cell line revealed that Aire KD led to the down-modulation of more than 800 genes, including those encoding for proteins involved in cell adhesion, i.e., the extracellular matrix constituent Lama1, the CAM family adhesion molecules Vcam1 and Icam4, and those that encode peripheral tissue antigens. Thymocytes co-cultured with Aire KD mTECs had a significantly reduced capacity to adhere to these cells. This finding is the first direct evidence that Aire also plays a role in controlling mTEC-thymocyte adhesion. PMID:27505711

  4. CRM-1 knockdown inhibits extrahepatic cholangiocarcinoma tumor growth by blocking the nuclear export of p27Kip1.

    PubMed

    Luo, Jian; Chen, Yongjun; Li, Qiang; Wang, Bing; Zhou, Yanqiong; Lan, Hongzhen

    2016-08-01

    Cholangiocarcinoma is a deadly disease which responds poorly to surgery and conventional chemotherapy or radiotherapy. Early diagnosis is difficult due to the anatomical and biological characteristics of cholangiocarcinoma. Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cyclin‑dependent kinase inhibitor and in the present study, we found that p27Kip1 expression was suppressed in the nucleus and increased in the cytoplasm in 53 samples of cholangiocarcinoma from patients with highly malignant tumors (poorly-differentiated and tumor-node-metastsis (TNM) stage III-IV) compared with that in samples from 10 patients with chronic cholangitis. The expression of phosphorylated (p-)p27Kip1 (Ser10), one of the phosphorylated forms of p27Kip1, was increased in the patient samples with increasing malignancy and clinical stage. Coincidentally, chromosome region maintenance 1 (CRM-1; also referred to as exportin 1 or Xpo1), a critical protein responsible for protein translocation from the nucleus to the cytoplasm, was also overexpressed in the tumor samples which were poorly differentiated and of a higher clinical stage. Through specific short hairpin RNA (shRNA)-mediated knockdown of CRM-1 in the cholangiocarcinoma cell line QBC939, we identified an elevation of cytoplasmic p27Kip1 and a decrease of nuclear p27Kip1. Furthermore, the viability and colony formation ability of QBC939 cells was largely reduced with G1 arrest. Consistent with the findings of the in vitro experiments, in a xenograft mouse model, the tumors formed in the CRM-1 knockdown group were markedly smaller and weighed less than those in the control group in vivo. Taken together, these findings demonstrated that the interplay between CRM-1 and p27Kip1 may provide potentially potent biomarkers and functional targets for the development of future cholangiocarcinoma treatments.

  5. Knockdown of PRKAR1A, the gene responsible for Carney complex, interferes with differentiation in osteoblastic cells.

    PubMed

    Zhang, Mei; Manchanda, Parmeet K; Wu, Dayong; Wang, Qianben; Kirschner, Lawrence S

    2014-03-01

    PRKAR1A is the gene encoding the type 1A regulatory subunit of protein kinase A, and it is the cause of the inherited human tumor syndrome Carney complex. Data from our laboratory has demonstrated that Prkar1a loss causes tumors in multiple cell lineages, including neural crest cells and osteoblasts. We have proposed that one mechanism by which tumorigenesis occurs is through the failure of terminal differentiation. In the present study, we directly test the effects of Prkar1a reduction on osteogenic differentiation in mouse and human cells in vitro. We found that Prkar1a levels noticeably increased during osteoblastic differentiation, indicating a positive correlation between the expression of Prkar1a and osteogenic potential. To validate this hypothesis, we generated stable Prkar1a knockdown in both mouse and human cells. These cells displayed significantly suppressed bone nodule formation and decreased expression of osteoblast markers such as osteocalcin and osteopontin. These observations imply that the antiosteogenic effect of Prkar1a ablation is not species or cell line specific. Furthermore, because Runt-related transcription factor-2 (Runx2) is a key mediator of osteoblast differentiation, we reasoned that the function of this transcription factor may be inhibited by Prkar1a knockdown. Chromatin immunoprecipitation and luciferase assays demonstrated that Prkar1a ablation repressed DNA binding and function of Runx2 at its target genes. Additionally, we determined that this effect is likely due to reductions in the Runx2-cooperating transcription factors forkhead box O1 and activating transcription factor 4. Taken together, this study provides direct evidence that ablation of Prkar1a interferes with signaling pathways necessary for osteoblast differentiation. PMID:24506536

  6. CRM-1 knockdown inhibits extrahepatic cholangiocarcinoma tumor growth by blocking the nuclear export of p27Kip1

    PubMed Central

    Luo, Jian; Chen, Yongjun; Li, Qiang; Wang, Bing; Zhou, Yanqiong; Lan, Hongzhen

    2016-01-01

    Cholangiocarcinoma is a deadly disease which responds poorly to surgery and conventional chemotherapy or radiotherapy. Early diagnosis is difficult due to the anatomical and biological characteristics of cholangiocarcinoma. Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cyclin-dependent kinase inhibitor and in the present study, we found that p27Kip1 expression was suppressed in the nucleus and increased in the cytoplasm in 53 samples of cholangiocarcinoma from patients with highly malignant tumors (poorly-differentiated and tumor-node-metastsis (TNM) stage III–IV) compared with that in samples from 10 patients with chronic cholangitis. The expression of phosphorylated (p-)p27Kip1 (Ser10), one of the phosphorylated forms of p27Kip1, was increased in the patient samples with increasing malignancy and clinical stage. Coincidentally, chromosome region maintenance 1 (CRM-1; also referred to as exportin 1 or Xpo1), a critical protein responsible for protein translocation from the nucleus to the cytoplasm, was also overexpressed in the tumor samples which were poorly differentiated and of a higher clinical stage. Through specific short hairpin RNA (shRNA)-mediated knockdown of CRM-1 in the cholangiocarcinoma cell line QBC939, we identified an elevation of cytoplasmic p27Kip1 and a decrease of nuclear p27Kip1. Furthermore, the viability and colony formation ability of QBC939 cells was largely reduced with G1 arrest. Consistent with the findings of the in vitro experiments, in a xenograft mouse model, the tumors formed in the CRM-1 knockdown group were markedly smaller and weighed less than those in the control group in vivo. Taken together, these findings demonstrated that the interplay between CRM-1 and p27Kip1 may provide potentially potent biomarkers and functional targets for the development of future cholangiocarcinoma treatments. PMID:27279267

  7. In vitro and intrathecal siRNA mediated KV1.1 knock-down in primary sensory neurons

    PubMed Central

    Baker, Mark D.; Chen, Ya-Chun; Shah, Syed U.; Okuse, Kenji

    2011-01-01

    KV1.1 is a Shaker homologue K+ channel that contributes to the juxta-paranodal membrane conductance in myelinated axons, and is blocked by fampridine (4-aminopyridine), used to treat the symptoms of multiple sclerosis. The present experiments investigate KV1.1 function in primary sensory neurons and A-fibres, and help define its characteristics as a drug-target using sequence specific small-interfering RNAs (siRNAs). siRNA (71 nM) was used to knock-down functional expression of KV1.1 in sensory neurons (> 25 μm in apparent diameter) in culture, and was also delivered intrathecally in vivo (9.3 μg). K+ channel knock-down in sensory neurons was found to make the voltage-threshold for action potential generation significantly more negative than in control (p = 0.02), led to the breakdown of accommodation and promoted spontaneous action potential firing. Exposure to dendrotoxin-K (DTX-K, 10–100 nM) also selectively abolished K+ currents at negative potentials and made voltage-threshold more negative, consistent with KV1.1 controlling excitability close to the nominal resting potential of the neuron cell body, near − 60 mV. Introduction of one working siRNA sequence into the intrathecal space in vivo was associated with a small increase in the amplitude of the depolarising after-potential in sacral spinal roots (p < 0.02), suggesting a reduction in the number of working K+ channels in internodal axon membrane. Our study provides evidence that KV1.1 contributes to the control of peripheral sensory nerve excitability, and suggests that its characteristics as a putative drug target can be assessed by siRNA transfection in primary sensory neurons in vitro and in vivo. PMID:21903165

  8. RNAi KNOCKDOWN OF BmRab3 LED TO LARVA AND PUPA LETHALITY IN SILKWORM Bombyx mori L.

    PubMed

    Singh, Chabungbam Orville; Xin, Hu-hu; Chen, Rui-ting; Wang, Mei-xian; Liang, Shuang; Lu, Yan; Cai, Zi-zheng; Zhang, Deng-pan; Miao, Yun-gen

    2015-06-01

    Rab3 GTPases are known to play key a role in vesicular trafficking, and express highest in brain and endocrine tissues. In mammals, Rab3 GTPases are paralogs unlike in insect. In this study, we cloned Rab3 from the silk gland tissue of silkworm Bombyx mori, and identified it as BmRab3. Our in silico analysis indicated that BmRab3 is an isoform with a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa, respectively. Further, BmRab3 showed the C-terminal hypervariability for GGT2 site but having two other putative guanine nucleotide exchange factor/GDP dissociation inhibitor interaction sites. Multiple alignment sequence indicated high similarities of BmRab3 with Rab3 isoforms of other species. The phylogeny tree showed BmRab3 clustered between the species of Tribolium castaneum and Aedes aegypti. Meanwhile, the expression analysis of BmRab3 showed the highest expression in middle silk glands (MSGs) than all other tissues in the third day of fifth-instar larva. Simultaneously, we showed the differential expression of BmRab3 in the early instar larva development, followed by higher expression in male than female pupae. In vivo dsRNA interference of BmRab3 reduced the expression of BmRab3 by 75% compared to the control in the MSGs in the first day. But as the worm grew to the third day, the difference of BmRab3 between knockdown and control was only about 10%. The knockdown later witnessed underdevelopment of the larvae and pharate pupae lethality in the overall development of silkworm B. mori L.

  9. Selective knockdown of mutant SOD1 in Schwann cells ameliorates disease in G85R mutant SOD1 transgenic mice.

    PubMed

    Wang, Lijun; Pytel, Peter; Feltri, M Laura; Wrabetz, Lawrence; Roos, Raymond P

    2012-10-01

    Mutants of superoxide dismutase type 1 (mtSOD1) that have full dismutase activity (e.g., G37R) as well as none (e.g., G85R) cause familial amyotrophic lateral sclerosis (FALS), indicating that mtSOD1-induced FALS results from a toxicity rather than loss in SOD1 enzymatic activity. Still, it has remained unclear whether mtSOD1 dismutase activity can influence disease. A previous study demonstrated that Cre-mediated knockdown of G37R expression in Schwann cells (SCs) of G37R transgenic mice shortened the late phase of disease and survival. These results suggested that the neuroprotective effect of G37R expressed in SCs was greater than its toxicity, presumably because its dismutase activity counteracted reactive oxygen species (ROS). In order to further investigate this, we knocked down G85R in SCs by crossing G85R(flox) mice with myelin-protein-zero (P(0)):Cre mice, which express Cre recombinase in SCs. Knockdown of G85R in SCs of G85R mice delayed disease onset and extended survival indicating that G85R expression in SCs is neurotoxic. These results demonstrate differences in the effect on disease of dismutase active vs. inactive mtSOD1 suggesting that both a loss as well as gain in function of mtSOD1 influence FALS pathogenesis. The results suggest that mtSOD1-induced FALS treatment may have to be adjusted depending on the cell type targeted and particular mtSOD1 involved.

  10. New insights for Ets2 function in trophoblast using lentivirus-mediated gene knockdown in trophoblast stem cells.

    PubMed

    Odiatis, C; Georgiades, P

    2010-07-01

    Mouse trophoblast stem (TS) cells represent a unique in vitro system that provides an unlimited supply of TS cells for the study of trophoblast differentiation and TS cell self-renewal. Although the mouse transcription factor Ets2 is required for TS cell self-renewal, its role in this and in TS cell differentiation has not been explored fully, partly due to the early lethality of Ets2 null mice. To address this, we developed a novel lentivirus-based system that resulted in efficient Ets2 knockdown in the overwhelming majority of TS cells. This system enables functional studies in TS cells, especially for genes required for TS cell self-renewal because TS cell derivation using gene-knockout embryos for such genes depends on TS cell self-renewal. Using morphological/morphometric criteria and gene expression analysis, we show that the requirement for Ets2 in self-renewal of TS cells cultured in 'stem cell medium' (SCM) involves maintenance of the expression of genes that inhibit TS cell differentiation in SCM, such as Cdx2 and Esrrb, and preservation of the undifferentiated TS cell morphology. During TS cell differentiation caused by Cdx2/Esrrb downregulation, due to either Ets2 knockdown in SCM or culture in differentiation medium (DM), Ets2 is also required for the promotion of trophoblast giant cell (TGC) and junctional zone trophoblast (JZT) differentiation. This TGC differentiation involves Ets2-dependent expression of Hand1, a gene required for the differentiation of all TGC types. This study uncovers new roles for Ets2 in TS cell self-renewal and differentiation and demonstrates the usefulness of this lentivirus system for gene function studies in TS cells.

  11. High Yield Production of Influenza Virus in Madin Darby Canine Kidney (MDCK) Cells with Stable Knockdown of IRF7

    PubMed Central

    Hamamoto, Itsuki; Takaku, Hiroshi; Tashiro, Masato; Yamamoto, Norio

    2013-01-01

    Influenza is a serious public health problem that causes a contagious respiratory disease. Vaccination is the most effective strategy to reduce transmission and prevent influenza. In recent years, cell-based vaccines have been developed with continuous cell lines such as Madin-Darby canine kidney (MDCK) and Vero. However, wild-type influenza and egg-based vaccine seed viruses will not grow efficiently in these cell lines. Therefore, improvement of virus growth is strongly required for development of vaccine seed viruses and cell-based influenza vaccine production. The aim of our research is to develop novel MDCK cells supporting highly efficient propagation of influenza virus in order to expand the capacity of vaccine production. In this study, we screened a human siRNA library that involves 78 target molecules relating to three major type I interferon (IFN) pathways to identify genes that when knocked down by siRNA lead to enhanced production of influenza virus A/Puerto Rico/8/1934 in A549 cells. The siRNAs targeting 23 candidate genes were selected to undergo a second screening pass in MDCK cells. We examined the effects of knockdown of target genes on the viral production using newly designed siRNAs based on sequence analyses. Knockdown of the expression of a canine gene corresponding to human IRF7 by siRNA increased the efficiency of viral production in MDCK cells through an unknown process that includes the mechanisms other than inhibition of IFN-α/β induction. Furthermore, the viral yield greatly increased in MDCK cells stably transduced with the lentiviral vector for expression of short hairpin RNA against IRF7 compared with that in control MDCK cells. Therefore, we propose that modified MDCK cells with lower expression level of IRF7 could be useful not only for increasing the capacity of vaccine production but also facilitating the process of seed virus isolation from clinical specimens for manufacturing of vaccines. PMID:23555825

  12. Genetic knockdown of estrogen receptor-alpha in the subfornical organ augments ANG II-induced hypertension in female mice.

    PubMed

    Xue, Baojian; Zhang, Zhongming; Beltz, Terry G; Guo, Fang; Hay, Meredith; Johnson, Alan Kim

    2015-03-15

    The present study tested the hypotheses that 1) ERα in the brain plays a key role in the estrogen-protective effects against ANG II-induced hypertension, and 2) that the subfornical organ (SFO) is a key site where ERα mediates these protective actions. In this study, a "floxed" ERα transgenic mouse line (ERα(flox)) was used to create models in which ERα was knocked down in the brain or just in the SFO. Female mice with ERα ablated in the nervous system (Nestin-ERα(-) mice) showed greater increases in blood pressure (BP) in response to ANG II. Furthermore, females with ERα knockdown specifically in the SFO [SFO adenovirus-Cre (Ad-Cre) injected ERα(flox) mice] also showed an enhanced pressor response to ANG II. Immunohistochemical (IHC), RT-PCR, and Western blot analyses revealed a marked reduction in the expression of ERα in nervous tissues and, in particular, in the SFO. These changes were not present in peripheral tissues in Nestin-ERα(-) mice or Ad-Cre-injected ERα(flox) mice. mRNA expression of components of the renin-angiotensin system in the lamina terminalis were upregulated in Nestin-ERα(-) mice. Moreover, ganglionic blockade on day 7 after ANG II infusions resulted in a greater reduction of BP in Nestin-ERα(-) mice or SFO Ad-Cre-injected mice, suggesting that knockdown of ERα in the nervous system or the SFO alone augments central ANG II-induced increase in sympathetic tone. The results indicate that interfering with the action of estrogen on SFO ERα is sufficient to abolish the protective effects of estrogen against ANG II-induced hypertension.

  13. Targeted gene knockdown in zebrafish reveals distinct intraembryonic functions for insulin-like growth factor II signaling.

    PubMed

    White, Yvonne A R; Kyle, Joshua T; Wood, Antony W

    2009-09-01

    IGF-II is the predominant IGF ligand regulating prenatal growth in all vertebrates, including humans, but its central role in placental development has confounded efforts to fully elucidate its functions within the embryo. Here we use a nonplacental model vertebrate (zebrafish) to interrogate the intraembryonic functions of IGF-II signaling. The zebrafish genome contains two coorthologs of mammalian IGF2 (igf2a, igf2b), which exhibit distinct patterns of expression during embryogenesis. Expression of igf2a mRNA is restricted to the notochord, primarily during segmentation/neurulation. By contrast, igf2b mRNA is expressed in midline tissues adjacent to the notochord, with additional sites of expression in the ventral forebrain, and the pronephros. To identify their intraembryonic functions, we suppressed the expression of each gene with morpholino oligonucleotides. Knockdown of igf2a led to defects in dorsal midline development, characterized by delayed segmentation, notochord undulations, and ventral curvature. Similarly, suppression of igf2b led to defects in dorsal midline development but also induced ectopic fusion of the nephron primordia, and defects in ventral forebrain development. Subsequent onset of severe body edema in igf2b, but not igf2a morphants, further suggested a distinct role for igf2b in development of the embryonic kidney. Simultaneous knockdown of both genes increased the severity of dorsal midline defects, confirming a conserved role for both genes in dorsal midline development. Collectively, these data provide evidence that the zebrafish orthologs of IGF2 function in dorsal midline development during segmentation/neurulation, whereas one paralog, igf2b, has evolved additional, distinct functions during subsequent organogenesis.

  14. Aire knockdown in medullary thymic epithelial cells affects Aire protein, deregulates cell adhesion genes and decreases thymocyte interaction.

    PubMed

    Pezzi, Nicole; Assis, Amanda Freire; Cotrim-Sousa, Larissa Cotrim; Lopes, Gabriel Sarti; Mosella, Maritza Salas; Lima, Djalma Sousa; Bombonato-Prado, Karina F; Passos, Geraldo Aleixo

    2016-09-01

    We demonstrate that even a partial reduction of Aire mRNA levels by siRNA-induced Aire knockdown (Aire KD) has important consequences to medullary thymic epithelial cells (mTECs). Aire knockdown is sufficient to reduce Aire protein levels, impair its nuclear location, and cause an imbalance in large-scale gene expression, including genes that encode cell adhesion molecules. These genes drew our attention because adhesion molecules are implicated in the process of mTEC-thymocyte adhesion, which is critical for T cell development and the establishment of central self-tolerance. Accordingly, we consider the following: 1) mTECs contribute to the elimination of self-reactive thymocytes through adhesion; 2) Adhesion molecules play a crucial role during physical contact between these cells; and 3) Aire is an important transcriptional regulator in mTECs. However, its role in controlling mTEC-thymocyte adhesion remains unclear. Because Aire controls adhesion molecule genes, we hypothesized that the disruption of its expression could influence mTEC-thymocyte interaction. To test this hypothesis, we used a murine Aire(+) mTEC cell line as a model system to reproduce mTEC-thymocyte adhesion in vitro. Transcriptome analysis of the mTEC cell line revealed that Aire KD led to the down-modulation of more than 800 genes, including those encoding for proteins involved in cell adhesion, i.e., the extracellular matrix constituent Lama1, the CAM family adhesion molecules Vcam1 and Icam4, and those that encode peripheral tissue antigens. Thymocytes co-cultured with Aire KD mTECs had a significantly reduced capacity to adhere to these cells. This finding is the first direct evidence that Aire also plays a role in controlling mTEC-thymocyte adhesion.

  15. Knockdown of zebrafish Lgi1a results in abnormal development, brain defects and a seizure-like behavioral phenotype

    PubMed Central

    Teng, Yong; Xie, Xiayang; Walker, Steven; Rempala, Grzegorz; Kozlowski, David J.; Mumm, Jeff S.; Cowell, John K.

    2010-01-01

    Epilepsy is a common disorder, typified by recurrent seizures with underlying neurological disorders or disease. Approximately one-third of patients are unresponsive to currently available therapies. Thus, a deeper understanding of the genetics and etiology of epilepsy is needed to advance the development of new therapies. Previously, treatment of zebrafish with epilepsy-inducing pharmacological agents was shown to result in a seizure-like phenotype, suggesting that fish provide a tractable model to understand the function of epilepsy-predisposing genes. Here, we report the first model of genetically linked epilepsy in zebrafish and provide an initial characterization of the behavioral and neurological phenotypes associated with morpholino (MO) knockdown of leucine-rich, glioma-inactivated 1a (lgi1a) expression. Mutations in the LGI1 gene in humans have been shown to predispose to a subtype of autosomal dominant epilepsy. Low-dose Lgi1a MO knockdown fish (morphants) appear morphologically normal but are sensitized to epilepsy-inducing drugs. High-dose Lgi1a morphants have morphological defects which persist into adult stages that are typified by smaller brains and eyes and abnormalities in tail shape, and display hyperactive swimming behaviors. Increased apoptosis was observed throughout the central nervous system of high-dose morphant fish, accounting for the size reduction of neural tissues. These observations demonstrate that zebrafish can be exploited to dissect the embryonic function(s) of genes known to predispose to seizure-like behavior in humans, and offer potential insight into the relationship between developmental neurobiological abnormalities and seizure. PMID:20819949

  16. Effects of siRNA-Mediated Knockdown of HDAC1 on the Biological Behavior of Esophageal Carcinoma Cell Lines

    PubMed Central

    Wang, Xing; Guo, Haisheng; Liu, Weixin; Yang, Chunmei; Yang, Lei; Wang, Dongguan; Wang, Xunguo

    2016-01-01

    Background HDAC1 has been shown to be closely associated with the occurrence of tumors. We aimed to investigate the effects of siRNA-mediated HDAC1 knockdown on the biological behavior of esophageal carcinoma cell lines. Material/Methods HDAC1 expression in esophageal cancer cell lines TE-1, Eca109, and EC9706 was compared by Western blot analysis. These cells were transfected with siRNA-HDAC1 and cell proliferation was evaluated by MTT assay to select the optimum cell line for subsequent experiments. The effects of siRNA-HDAC1 on the migration and invasion of the selected cell line were assessed by transwell assay. The expression of cell cycle-related proteins cyclinD1, p21 and p27, and epithelial-mesenchymal transition (EMT)-related protein zonula occludens-1 (ZO-1), E-cadherin and vimentin was determined by Western blot analysis. Results HDAC1 expression in TE-1, Eca109 and EC9706 cells was significantly higher compared with normal esophageal cell line HEEC (P<0.01). MTT assay, Western blot and RT-PCR analyses demonstrated that the inhibitory effects of siRNA on HDAC1 expression and cell viability in TE-1 cells were the highest among all cell lines, which was therefore used in subsequent experiments. After TE-1 cells were transfected with siRNA-HDAC1, their migration and invasion were significantly lower compared with the controls (P<0.01). CyclinD1 and vimentin expression was significantly lower compared with the controls (P<0.01), whereas the expression of p21, p27, ZO-1 and E-cadherin was significantly higher (P<0.01). Conclusions The siRNA-mediated HDAC1 knockdown significantly inhibited the proliferation, migration and invasion of TE-1 cells probably by regulating the expression of cell cycle- and EMT-related proteins. PMID:27086779

  17. Cuticle Thickening in a Pyrethroid-Resistant Strain of the Common Bed Bug, Cimex lectularius L. (Hemiptera: Cimicidae).

    PubMed

    Lilly, David G; Latham, Sharissa L; Webb, Cameron E; Doggett, Stephen L

    2016-01-01

    Thickening of the integument as a mechanism of resistance to insecticides is a well recognised phenomenon in the insect world and, in recent times, has been found in insects exhibiting pyrethroid-resistance. Resistance to pyrethroid insecticides in the common bed bug, Cimex lectularius L., is widespread and has been frequently inferred as a reason for the pest's resurgence. Overexpression of cuticle depositing proteins has been demonstrated in pyrethroid-resistant bed bugs although, to date, no morphological analysis of the cuticle has been undertaken in order to confirm a phenotypic link. This paper describes examination of the cuticle thickness of a highly pyrethroid-resistant field strain collected in Sydney, Australia, in response to time-to-knockdown upon forced exposure to a pyrethroid insecticide. Mean cuticle thickness was positively correlated to time-to-knockdown, with significant differences observed between bugs knocked-down at 2 hours, 4 hours, and those still unaffected at 24 hours. Further analysis also demonstrated that the 24 hours survivors possessed a statistically significantly thicker cuticle when compared to a pyrethroid-susceptible strain of C. lectularius. This study demonstrates that cuticle thickening is present within a pyrethroid-resistant strain of C. lectularius and that, even within a stable resistant strain, cuticle thickness will vary according to time-to-knockdown upon exposure to an insecticide. This response should thus be considered in future studies on the cuticle of insecticide-resistant bed bugs and, potentially, other insects. PMID:27073871

  18. Cuticle Thickening in a Pyrethroid-Resistant Strain of the Common Bed Bug, Cimex lectularius L. (Hemiptera: Cimicidae)

    PubMed Central

    Lilly, David G.; Latham, Sharissa L.; Webb, Cameron E.; Doggett, Stephen L.

    2016-01-01

    Thickening of the integument as a mechanism of resistance to insecticides is a well recognised phenomenon in the insect world and, in recent times, has been found in insects exhibiting pyrethroid-resistance. Resistance to pyrethroid insecticides in the common bed bug, Cimex lectularius L., is widespread and has been frequently inferred as a reason for the pest’s resurgence. Overexpression of cuticle depositing proteins has been demonstrated in pyrethroid-resistant bed bugs although, to date, no morphological analysis of the cuticle has been undertaken in order to confirm a phenotypic link. This paper describes examination of the cuticle thickness of a highly pyrethroid-resistant field strain collected in Sydney, Australia, in response to time-to-knockdown upon forced exposure to a pyrethroid insecticide. Mean cuticle thickness was positively correlated to time-to-knockdown, with significant differences observed between bugs knocked-down at 2 hours, 4 hours, and those still unaffected at 24 hours. Further analysis also demonstrated that the 24 hours survivors possessed a statistically significantly thicker cuticle when compared to a pyrethroid-susceptible strain of C. lectularius. This study demonstrates that cuticle thickening is present within a pyrethroid-resistant strain of C. lectularius and that, even within a stable resistant strain, cuticle thickness will vary according to time-to-knockdown upon exposure to an insecticide. This response should thus be considered in future studies on the cuticle of insecticide-resistant bed bugs and, potentially, other insects. PMID:27073871

  19. The knockdown of chloroplastic ascorbate peroxidases reveals its regulatory role in the photosynthesis and protection under photo-oxidative stress in rice.

    PubMed

    Caverzan, Andréia; Bonifacio, Aurenivia; Carvalho, Fabricio E L; Andrade, Claudia M B; Passaia, Gisele; Schünemann, Mariana; Maraschin, Felipe Dos Santos; Martins, Marcio O; Teixeira, Felipe K; Rauber, Rafael; Margis, Rogério; Silveira, Joaquim Albenisio Gomes; Margis-Pinheiro, Márcia

    2014-01-01

    The inactivation of the chloroplast ascorbate peroxidases (chlAPXs) has been thought to limit the efficiency of the water-water cycle and photo-oxidative protection under stress conditions. In this study, we have generated double knockdown rice (Oryza sativa L.) plants in both OsAPX7 (sAPX) and OsAPX8 (tAPX) genes, which encode chloroplastic APXs (chlAPXs). By employing an integrated approach involving gene expression, proteomics, biochemical and physiological analyses of photosynthesis, we have assessed the role of chlAPXs in the regulation of the protection of the photosystem II (PSII) activity and CO2 assimilation in rice plants exposed to high light (HL) and methyl violagen (MV). The chlAPX knockdown plants were affected more severely than the non-transformed (NT) plants in the activity and structure of PSII and CO2 assimilation in the presence of MV. Although MV induced significant increases in pigment content in the knockdown plants, the increases were apparently not sufficient for protection. Treatment with HL also caused generalized damage in PSII in both types of plants. The knockdown and NT plants exhibited differences in photosynthetic parameters related to efficiency of utilization of light and CO2. The knockdown plants overexpressed other antioxidant enzymes in response to the stresses and increased the GPX activity in the chloroplast-enriched fraction. Our data suggest that a partial deficiency of chlAPX expression modulate the PSII activity and integrity, reflecting the overall photosynthesis when rice plants are subjected to acute oxidative stress. However, under normal growth conditions, the knockdown plants exhibit normal phenotype, biochemical and physiological performance.

  20. Antibiotic Resistance

    MedlinePlus

    ... For Consumers Consumer Information by Audience For Women Antibiotic Resistance Share Tweet Linkedin Pin it More sharing options ... these products really help. To Learn More about Antibiotic Resistance Get Smart About Antibiotics (Video) Fact Sheets and ...

  1. RESISTIVITY METHODS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Resistivity methods were among the first geophysical techniques developed. The basic concept originated with Conrad Schlumberger, who conducted the initial resistivity field tests in Normandy, France during 1912. The resistivity method, employed in its earliest and most conventional form, uses an ex...

  2. L1-CAM knock-down radiosensitizes neuroblastoma IMR-32 cells by simultaneously decreasing MycN, but increasing PTEN protein expression.

    PubMed

    Rached, Johnny; Nasr, Zeina; Abdallah, Jad; Abou-Antoun, Tamara

    2016-10-01

    Childhood neuroblastoma is one of the most malignant types of cancers leading to a high mortality rate. These cancerous cells can be highly metastatic and malignant giving rise to disease recurrence and poor prognosis. The proto-oncogene myelocytomatosis neuroblastoma (MycN) is known to be amplified in this type of cancer, thus, promoting high malignancy and resistance. The L1 cell adhesion molecule (L1-CAM) cleavage has been found upregulated in many types of malignant cancers. In the present study, we explored the interplay between L1-CAM, MycN and PTEN as well as the role played by PDGFR and VEGFR on tumorigenicity in neuroblastoma cells. We investigated the effect of L1-CAM knock-down (KD) and PDGFR/VEGFR inhibition with sunitinib malate (Sutent®) treatment on subsequent tumorsphere formation and cellular proliferation and migration in the MycN-amplified IMR-32 neuroblastoma cells. We further examined the effect of combined L1-CAM KD with Sutent treatment or radiotherapy on these cellular functions in our cells. Tumorsphere formation is one of the indicators of aggressiveness in malignant cancers, which was significantly inhibited in IMR-32 cells after L1-CAM KD or Sutent treatment, however, no synergistic effect was observed with dual treatments, rather L1-CAM KD alone showed a greater inhibition on tumorsphere formation compared to Sutent treatment alone. In addition, cellular proliferation and migration were significantly inhibited after L1-CAM KD in the IMR-32 cells with no synergistic effect observed on the rate of cell proliferation when combined with Sutent treatment. Again, L1-CAM KD alone exhibited greater inhibitory effect than Sutent treatment on cell proliferation. L1-CAM KD led to the simultaneous downregulation of MycN, but the upregulation of PTEN protein expression. Notably, radiotherapy (2 Gy) of the IMR-32 cells led to significant upregulation of both L1-CAM and MycN, which was abrogated with L1-CAM KD in our cells. In addition, L1-CAM KD

  3. L1-CAM knock-down radiosensitizes neuroblastoma IMR-32 cells by simultaneously decreasing MycN, but increasing PTEN protein expression.

    PubMed

    Rached, Johnny; Nasr, Zeina; Abdallah, Jad; Abou-Antoun, Tamara

    2016-10-01

    Childhood neuroblastoma is one of the most malignant types of cancers leading to a high mortality rate. These cancerous cells can be highly metastatic and malignant giving rise to disease recurrence and poor prognosis. The proto-oncogene myelocytomatosis neuroblastoma (MycN) is known to be amplified in this type of cancer, thus, promoting high malignancy and resistance. The L1 cell adhesion molecule (L1-CAM) cleavage has been found upregulated in many types of malignant cancers. In the present study, we explored the interplay between L1-CAM, MycN and PTEN as well as the role played by PDGFR and VEGFR on tumorigenicity in neuroblastoma cells. We investigated the effect of L1-CAM knock-down (KD) and PDGFR/VEGFR inhibition with sunitinib malate (Sutent®) treatment on subsequent tumorsphere formation and cellular proliferation and migration in the MycN-amplified IMR-32 neuroblastoma cells. We further examined the effect of combined L1-CAM KD with Sutent treatment or radiotherapy on these cellular functions in our cells. Tumorsphere formation is one of the indicators of aggressiveness in malignant cancers, which was significantly inhibited in IMR-32 cells after L1-CAM KD or Sutent treatment, however, no synergistic effect was observed with dual treatments, rather L1-CAM KD alone showed a greater inhibition on tumorsphere formation compared to Sutent treatment alone. In addition, cellular proliferation and migration were significantly inhibited after L1-CAM KD in the IMR-32 cells with no synergistic effect observed on the rate of cell proliferation when combined with Sutent treatment. Again, L1-CAM KD alone exhibited greater inhibitory effect than Sutent treatment on cell proliferation. L1-CAM KD led to the simultaneous downregulation of MycN, but the upregulation of PTEN protein expression. Notably, radiotherapy (2 Gy) of the IMR-32 cells led to significant upregulation of both L1-CAM and MycN, which was abrogated with L1-CAM KD in our cells. In addition, L1-CAM KD

  4. Antimicrobial (Drug) Resistance

    MedlinePlus

    ... Antimicrobial (Drug) Resistance Antibiotic-Resistant Mycobacterium tuberculosis (TB) Methicillin-Resistant Staphylococcus aureus (MRSA) Vancomycin-Resistant Enterococci (VRE) Multidrug-Resistant Neisseria ...

  5. Insulin-like growth factor 2 silencing restores taxol sensitivity in drug resistant ovarian cancer.

    PubMed

    Brouwer-Visser, Jurriaan; Lee, Jiyeon; McCullagh, KellyAnne; Cossio, Maria J; Wang, Yanhua; Huang, Gloria S

    2014-01-01

    Drug resistance is an obstacle to the effective treatment of ovarian cancer. We and others have shown that the insulin-like growth factor (IGF) signaling pathway is a novel potential target to overcome drug resistance. The purpose of this study was to validate IGF2 as a potential therapeutic target in drug resistant ovarian cancer and to determine the efficacy of targeting IGF2 in vivo. An analysis of The Cancer Genome Atlas (TCGA) data in the serous ovarian cancer cohort showed that high IGF2 mRNA expression is significantly associated with shortened interval to disease progression and death, clinical indicators of drug resistance. In a genetically diverse panel of ovarian cancer cell lines, the IGF2 mRNA levels measured in cell lines resistant to various microtubule-stabilizing agents including Taxol were found to be significantly elevated compared to the drug sensitive cell lines. The effect of IGF2 knockdown on Taxol resistance was investigated in vitro and in vivo. Transient IGF2 knockdown significantly sensitized drug resistant cells to Taxol treatment. A Taxol-resistant ovarian cancer xenograft model, developed from HEY-T30 cells, exhibited extreme drug resistance, wherein the maximal tolerated dose of Taxol did not delay tumor growth in mice. Blocking the IGF1R (a transmembrane receptor that transmits signals from IGF1 and IGF2) using a monoclonal antibody did not alter the response to Taxol. However, stable IGF2 knockdown using short-hairpin RNA in HEY-T30 effectively restored Taxol sensitivity. These findings validate IGF2 as a potential therapeutic target in drug resistant ovarian cancer and show that directly targeting IGF2 may be a preferable strategy compared with targeting IGF1R alone.

  6. Insulin-Like Growth Factor 2 Silencing Restores Taxol Sensitivity in Drug Resistant Ovarian Cancer

    PubMed Central

    Brouwer-Visser, Jurriaan; Lee, Jiyeon; McCullagh, KellyAnne; Cossio, Maria J.; Wang, Yanhua; Huang, Gloria S.

    2014-01-01

    Drug resistance is an obstacle to the effective treatment of ovarian cancer. We and others have shown that the insulin-like growth factor (IGF) signaling pathway is a novel potential target to overcome drug resistance. The purpose of this study was to validate IGF2 as a potential therapeutic target in drug resistant ovarian cancer and to determine the efficacy of targeting IGF2 in vivo. An analysis of The Cancer Genome Atlas (TCGA) data in the serous ovarian cancer cohort showed that high IGF2 mRNA expression is significantly associated with shortened interval to disease progression and death, clinical indicators of drug resistance. In a genetically diverse panel of ovarian cancer cell lines, the IGF2 mRNA levels measured in cell lines resistant to various microtubule-stabilizing agents including Taxol were found to be significantly elevated compared to the drug sensitive cell lines. The effect of IGF2 knockdown on Taxol resistance was investigated in vitro and in vivo. Transient IGF2 knockdown significantly sensitized drug resistant cells to Taxol treatment. A Taxol-resistant ovarian cancer xenograft model, developed from HEY-T30 cells, exhibited extreme drug resistance, wherein the maximal tolerated dose of Taxol did not delay tumor growth in mice. Blocking the IGF1R (a transmembrane receptor that transmits signals from IGF1 and IGF2) using a monoclonal antibody did not alter the response to Taxol. However, stable IGF2 knockdown using short-hairpin RNA in HEY-T30 effectively restored Taxol sensitivity. These findings validate IGF2 as a potential therapeutic target in drug resistant ovarian cancer and show that directly targeting IGF2 may be a preferable strategy compared with targeting IGF1R alone. PMID:24932685

  7. SPINK5 knockdown in organotypic human skin culture as a model system for Netherton syndrome: effect of genetic inhibition of serine proteases kallikrein 5 and kallikrein 7.

    PubMed

    Wang, Shirley; Olt, Sabine; Schoefmann, Nicole; Stuetz, Anton; Winiski, Anthony; Wolff-Winiski, Barbara

    2014-07-01

    Netherton syndrome (NS; OMIM 256500) is a genetic skin disease resulting from defects in the serine protease inhibitor Kazal-type 5 (SPINK5) gene, which encodes the protease inhibitor lympho-epithelial Kazal type inhibitor (LEKTI). We established a SPINK5 knockdown skin model by transfecting SPINK5 small interfering RNA (siRNA) into normal human epidermal keratinocytes, which were used together with fibroblast-populated collagen gels to generate organotypic skin cultures. This model recapitulates some of the NS skin morphology: thicker, parakeratotic stratum corneum frequently detached from the underlying epidermis and loss of corneodesmosomes. As enhanced serine protease activity has been implicated in the disease pathogenesis, we investigated the impact of the kallikreins KLK5 [stratum corneum trypsin-like enzyme (SCTE)] and KLK7 [stratum corneum chymotrypsin-like enzyme (SCCE)] on the SPINK5 knockdown phenotype by generating double knockdowns in the organotypic model. Knockdown of KLK5 or KLK7 partially ameliorated the epidermal architecture: increased epidermal thickness and expression of desmocollin 1 (DSC1), desmoglein 1 (DSG1) and (pro)filaggrin. Thus, inhibition of serine proteases KLK5 and KLK7 could be therapeutically beneficial in NS.

  8. Lentiviral-Mediated Short Hairpin RNA Knockdown of MTDH Inhibits Cell Growth and Induces Apoptosis by Regulating the PTEN/AKT Pathway in Hepatocellular Carcinoma

    PubMed Central

    Li, Wen-Fang; Ou, Qin; Dai, Hang; Liu, Chang-An

    2015-01-01

    The activation of oncogenes and the loss of tumor suppressor genes are believed to play critical roles in the pathogenesis of human hepatocellular carcinoma (HCC). Metaherin (MTDH), also called astrocyte elevated gene-1 (AEG-1), is frequently amplified in a variety of cancers, but the roles of MTDH with regard to growth and apoptosis in HCC have not yet been studied. In the present study, we first analyzed the expression of MTDH in HCC samples. We found that MTDH protein levels are higher in most HCC cancerous tissues compared with their matched adjacent non-tumor tissues. Additionally, the MTDH mRNA was also higher in HCC tissues compared to their matched adjacent non-tumor tissues. Knockdown of the endogenous MTDH using small interfering RNA further showed that deficiency of MTDH suppressed cell growth and caused apoptosis in HCC cells. Knockdown MTDH promoted PTEN and p53 expression in HCC cells and inhibited AKT phosphorylation. Knockdown MTDH also inhibited tumor growth in vivo. All these results indicated that MTDH protein levels in most HCC tissues are higher than non-tumor tissues, and knockdown of MTDH inhibited growth and induced apoptosis in HCC cells through the activation of PTEN. Therefore, MTDH might be an effective targeted therapy gene for HCC. PMID:26287185

  9. RNAi-mediated knockdown of the Halloween gene spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ecdysteroids play a critical role in coordinating insect growth, development, and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNAi-mediated knockdown of the CYP3...

  10. Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

    SciTech Connect

    Marchissio, Maria Julia; Francés, Daniel Eleazar Antonio; Carnovale, Cristina Ester; Marinelli, Raúl Alberto

    2012-10-15

    Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H{sub 2}O{sub 2} across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H{sub 2}O{sub 2} release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72 h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p < 0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H{sub 2}O{sub 2} release, assessed by Amplex Red, was reduced by about 45% (p < 0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+ 120%, p < 0.05) and loss of mitochondrial membrane potential (− 80%, p < 0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H{sub 2}O{sub 2} release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death. -- Highlights: ► Aquaporin-8 is expressed in mitochondria of human hepatoma HepG2 cells. ► Aquaporin-8 knockdown impairs mitochondrial H{sub 2}O{sub 2} release and increases ROS. ► Aquaporin

  11. Reduced 64Cu Uptake and Tumor Growth Inhibition by Knockdown of Human Copper Transporter 1 in Xenograft Mouse Model of Prostate Cancer

    PubMed Central

    Cai, Huawei; Wu, Jiu-sheng; Muzik, Otto; Hsieh, Jer-Tsong; Lee, Robert J.; Peng, Fangyu

    2015-01-01

    Copper is an element required for cell proliferation and angiogenesis. Human prostate cancer xenografts with increased 64Cu radioactivity were visualized previously by PET using 64CuCl2 as a radiotracer (64CuCl2 PET). This study aimed to determine whether the increased tumor 64Cu radioactivity was due to increased cellular uptake of 64Cu mediated by human copper transporter 1 (hCtr1) or simply due to nonspecific binding of ionic 64CuCl2 to tumor tissue. In addition, the functional role of hCtr1 in proliferation of prostate cancer cells and tumor growth was also assessed. Methods A lentiviral vector encoding short-hairpin RNA specific for hCtr1 (Lenti-hCtr1-shRNA) was constructed for RNA interference–mediated knockdown of hCtr1 expression in prostate cancer cells. The degree of hCtr1 knockdown was determined by Western blot, and the effect of hCtr1 knockdown on copper uptake and proliferation were examined in vitro by cellular 64Cu uptake and cell proliferation assays. The effects of hCtr1 knockdown on tumor uptake of 64Cu were determined by PET quantification and tissue radioactivity assay. The effects of hCtr1 knockdown on tumor growth were assessed by PET/CT and tumor size measurement with a caliper. Results RNA interference–mediated knockdown of hCtr1 was associated with the reduced cellular uptake of 64Cu and the suppression of prostate cancer cell proliferation in vitro. At 24 h after intravenous injection of the tracer 64CuCl2, the 64Cu uptake by the tumors with knockdown of hCtr1 (4.02 ± 0.31 percentage injected dose per gram [%ID/g] in Lenti-hCtr1-shRNA-PC-3 and 2.30 ± 0.59 %ID/g in Lenti-hCtr1-shRNA-DU-145) was significantly lower than the 64Cu uptake by the control tumors without knockdown of hCtr1 (7.21 ± 1.48 %ID/g in Lenti-SCR-shRNA-PC-3 and 5.57 ± 1.20 % ID/g in Lenti-SCR-shRNA-DU-145, P < 0.001) by PET quantification. Moreover, the volumes of prostate cancer xenograft tumors with knockdown of hCtr1 (179 ± 111 mm3 for Lenti-hCtr1-shRNA-PC-3

  12. Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability.

    PubMed

    Marchissio, Maria Julia; Francés, Daniel Eleazar Antonio; Carnovale, Cristina Ester; Marinelli, Raúl Alberto

    2012-10-15

    Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H(2)O(2) across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H(2)O(2) release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p<0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H(2)O(2) release, assessed by Amplex Red, was reduced by about 45% (p<0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+120%, p<0.05) and loss of mitochondrial membrane potential (-80%, p<0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H(2)O(2) release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death. PMID:22910329

  13. Atg7 Knockdown Augments Concanavalin A-Induced Acute Hepatitis through an ROS-Mediated p38/MAPK Pathway.

    PubMed

    Zhuang, Yan; Li, Yi; Li, Xuefeng; Xie, Qing; Wu, Min

    2016-01-01

    Concanavalin A (ConA), a T-cell mitogen that induces acute autoimmune hepatitis, is widely used to model pathophysiological processes of human acute autoimmune liver disease. Although autophagy has been extensively studied in the past decade, little is known about its molecular mechanism underlying the regulation of ConA-induced acute hepatitis. In this study, we used a Cre-conditional atg7 KO mouse to investigate the effects of Atg7-associated autophagy on ConA-induced murine hepatitis. Our results demonstrated that atg7 deficiency in mice enhanced macrophage activation and increased pro-inflammatory cytokines upon ConA stimulation. Atg7 silencing resulted in accumulation of dysfunctional mitochondria, disruption of reactive oxygen species (ROS) degradation, and increase in pro-inflammatory cytokines in Raw264.7 cells. p38/MAPK and NF-κB levels were increased upon ConA induction due to Atg7 deficiency. Blocking ROS production inhibited ConA-induced p38/IκB phosphorylation and subsequent intracellular inflammatory responses. Hence, this study demonstrated that atg7 knockout in mice or Atg7 knockdown in cell culture augmented ConA-induced acute hepatitis and related cellular malfunction, indicating protective effects of Atg7 on regulating mitochondrial ROS via a p38/MAPK-mediated pathway. Collectively, our findings reveal that autophagy may attenuate macrophage-mediated inflammatory response to ConA and may be the potential therapeutic targets for acute liver injury. PMID:26939081

  14. Dentate gyrus-specific knockdown of adult neurogenesis impairs spatial and object recognition memory in adult rats

    PubMed Central

    Jessberger, Sebastian; Clark, Robert E.; Broadbent, Nicola J.; Clemenson, Gregory D.; Consiglio, Antonella; Lie, D. Chichung; Squire, Larry R.; Gage, Fred H.

    2009-01-01

    New granule cells are born throughout life in the dentate gyrus of the hippocampal formation. Given the fundamental role of the hippocampus in processes underlying certain forms of learning and memory, it has been speculated that newborn granule cells contribute to cognition. However, previous strategies aiming to causally link newborn neurons with hippocampal function used ablation strategies that were not exclusive to the hippocampus or that were associated with substantial side effects, such as inflammation. We here used a lentiviral approach to specifically block neurogenesis in the dentate gyrus of adult male rats by inhibiting WNT signaling, which is critically involved in the generation of newborn neurons, using a dominant-negative WNT (dnWNT). We found a level-dependent effect of adult neurogenesis on the long-term retention of spatial memory in the water maze task, as rats with substantially reduced levels of newborn neurons showed less preference for the target zone in probe trials >2 wk after acquisition compared with control rats. Furthermore, animals with strongly reduced levels of neurogenesis were impaired in a hippocampus-dependent object recognition task. Social transmission of food preference, a behavioral test that also depends on hippocampal function, was not affected by knockdown of neurogenesis. Here we identified a role for newborn neurons in distinct aspects of hippocampal function that will set the ground to further elucidate, using experimental and computational strategies, the mechanism by which newborn neurons contribute to behavior. PMID:19181621

  15. Knockdown of microRNA-195 contributes to protein phosphatase-2A inactivation in rats with chronic brain hypoperfusion.

    PubMed

    Liu, Cheng-Di; Wang, Qin; Zong, De-Kang; Pei, Shuang-Chao; Yan, Yan; Yan, Mei-Ling; Sun, Lin-Lin; Hao, Yang-Yang; Mao, Meng; Xing, Wen-Jing; Ren, Huan; Ai, Jing

    2016-09-01

    Reduction of protein phosphatase-2A (PP2A) activity is a common clinical feature of Alzheimer's disease and vascular dementia. In this study, we observed that chronic brain hypoperfusion induced by bilateral common carotid artery occlusion of rats led to PP2A inactivation based on the increase in tyrosine-307 phosphorylation and leucine-309 demethylation of PP2AC and the depression in PP2ABα. Knockdown of miR-195 using overexpression of its antisense molecule oligonucleotide (pre-AMO-miR-195) delivered by a lentivirus (lenti-pre-AMO-miR-195) increased tyrosine-307 phosphorylation and decreased both PP2ABα expression and leucine-309 methylation; these effects were prevented by the overexpression of miR-195 using lenti-pre-miR-195 and controlled by an increase in methylesterase (PME-1) and a decrease in leucine carboxyl methyltransferase-1. In vitro studies demonstrated that miR-195 regulated PME-1 expression by binding to the Ppme1 gene 3'-untranslated region (3'UTR) domain. Masking the miR-195 binding sites in the amyloid precursor protein (APP) and β-site APP cleaving enzyme 1 genes prevented miR-195-induced leucine carboxyl methyltransferase-1 elevation. We concluded that the miR-195 downregulation in chronic brain hypoperfusion involved PP2A inactivity, which was mediated by the post-transcriptional regulation PME-1, APP, and β-site APP cleaving enzyme 1 expression. PMID:27459928

  16. Salt and acid-base metabolism in claudin-16 knockdown mice: impact for the pathophysiology of FHHNC patients.

    PubMed

    Himmerkus, Nina; Shan, Qixian; Goerke, Boeren; Hou, Jianghui; Goodenough, Daniel A; Bleich, Markus

    2008-12-01

    Claudin-16 is defective in familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). Claudin-16 knockdown (CLDN16 KD) mice show reduced cation selectivity in the thick ascending limb. The defect leads to a collapse of the lumen-positive diffusion voltage, which drives Ca(2+) and Mg(2+) absorption. Because of the reduced tight junction permeability ratio for Na(+) over Cl(-), we proposed a backleak of NaCl into the lumen. Systemic analysis had revealed lower blood pressure and a moderately increased plasma aldosterone concentration. In this study, we measured the amiloride-sensitive equivalent short-circuit current in isolated, perfused collecting ducts and found it increased by fivefold in CLDN16 KD mice compared with wild-type (WT) mice. Amiloride treatment unmasked renal Na(+) loss in the thick ascending limb of the nephron. Under amiloride treatment, CLDN16 KD mice developed hyponatremia and the renal fractional excretion of Na(+) was twofold higher in CLDN16 KD compared with WT mice. The loss of claudin-16 also resulted in increased urinary flow, reduced HCO(3)(-) excretion, and lower urine pH. We conclude that perturbation in salt and acid-base metabolism in CLDN16 KD mice has its origin in the defective cation permselectivity of the thick ascending limb of the nephron. This study has contributed to the still incomplete understanding of the symptoms of FHHNC patients.

  17. TDP-43 aggregation mirrors TDP-43 knockdown, affecting the expression levels of a common set of proteins

    PubMed Central

    Prpar Mihevc, S.; Baralle, Marco; Buratti, Emanuele; Rogelj, Boris

    2016-01-01

    TDP-43 protein plays an important role in regulating transcriptional repression, RNA metabolism, and splicing. Typically it shuttles between the nucleus and the cytoplasm to perform its functions, while abnormal cytoplasmic aggregation of TDP-43 has been associated with neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). For the purpose of this study we selected a set of proteins that were misregulated following silencing of TDP-43 and analysed their expression in a TDP-43-aggregation model cell line HEK293 Flp-in Flag-TDP-43-12x-Q/N F4L. Following TDP-43 sequestration in insoluble aggregates, we observed higher nuclear levels of EIF4A3, and POLDIP3β, whereas nuclear levels of DNMT3A, HNRNPA3, PABPC1 and POLDIP3α dropped, and cytoplasmic levels of RANBP1 dropped. In addition, immunofluorescence signal intensity quantifications showed increased nuclear expression of HNRNPL and YARS, and downregulation of cytoplasmic DPCD. Furthermore, cytoplasmic levels of predominantly nuclear protein ALYREF increased. In conclusion, by identifying a common set of proteins that are differentially expressed in a similar manner in these two different conditions, we show that TDP-43 aggregation has a comparable effect to TDP-43 knockdown. PMID:27665936

  18. Knockdown of Nogo gene by short hairpin RNA interference promotes functional recovery of spinal cord injury in a rat model.

    PubMed

    Liu, Guo-Min; Luo, Yun-Gang; Li, Juan; Xu, Kun

    2016-05-01

    The specific myelin component Nogo protein is one of the major inhibitory molecules of spinal cord axonal outgrowth following spinal cord injury. The present study aimed to investigate the effects of silencing Nogo protein with shRNA interference on the promotion of functional recovery in a rat model with spinal cord hemisection. Nogo-A short hairpin RNAs (Nogo shRNAs) were constructed and transfected into rats with spinal cord hemisection by adenovirus-mediated transfection. Reverse transcription‑polymerase chain reaction and western blotting were performed to analyze the expression of Nogo-A and Growth Associated Protein 43 (GAP-43). In addition, Basso Beattie Bresnahan (BBB) scores were used to assess the functional recovery of rats following spinal cord injury. The results demonstrated that expression of the Nogo‑A gene was observed to be downregulated following transfection and GAP‑43 expression was observed to increase. The BBB scores were increased following treatment with Nogo shRNAs, indicating functional recovery of the injured nerves. Thus, Nogo-A shRNA interference can knockdown Nogo gene expression and upregulate GAP-43 to promote the functional recovery of spinal cord injury in rats. This finding may advance progress toward assisting the regeneration of injured neurons through the use of Nogo-A shRNA. PMID:27035338

  19. RNAi knockdown of oxytocin receptor in the nucleus accumbens inhibits social attachment and parental care in monogamous female prairie voles

    PubMed Central

    Keebaugh, Alaine C.; Barrett, Catherine E.; LaPrairie, Jamie L.; Jenkins, Jasmine J.; Young, Larry J.

    2015-01-01

    Oxytocin modulates many aspects of social cognition and behaviors, including maternal nurturing, social recognition and bonding. Natural variation in oxytocin receptor (OXTR) density in the nucleus accumbens (NAcc) is associated with variation in alloparental behavior, and artificially enhancing OXTR expression in the NAcc enhances alloparental behavior and pair bonding in socially monogamous prairie voles. Furthermore, infusion of an OXTR antagonist into the nucleus accumbens (NAcc) inhibits alloparental behavior and partner preference formation. However, antagonists can promiscuously interact with other neuropeptide receptors. To directly examine the role of OXTR signaling in social bonding, we used RNA interference to selectively knockdown, but not eliminate, OXTR in the NAcc of female prairie voles and examined the impact on social behaviors. Using an adeno-associated viral vector expressing a short hairpin RNA (shRNA) targeting Oxtr mRNA, we reduced accumbal OXTR density in female prairie voles from juvenile age through adulthood. Females receiving the shRNA vector displayed a significant reduction in alloparental behavior and disrupted partner preference formation. These are the first direct demonstrations that OXTR plays a critical role in alloparental behavior and adult social attachment, and suggest that natural variation in OXTR expression in this region alone can create variation in social behavior. PMID:25874849

  20. Phosphatase and tensin homolog reconstruction and vascular endothelial growth factor knockdown synergistically inhibit the growth of glioblastoma.

    PubMed

    Chen, Hongbo; Shen, Xiaomeng; Guo, Caiping; Zhu, Huijun; Zhou, Lanzhen; Zhu, Yongqiang; Wang, Huixia; Zheng, Yi; Huang, Laiqiang

    2010-12-01

    Glioblastoma (GBM) is a highly malignant tumor with poor prognosis. Two hallmarks of this disease are a high expression of vascular endothelial growth factor (VEGF) and a depletion of the phosphatase and tensin homolog (PTEN). In the present study, combined gene therapy using wild-type PTEN reconstruction and VEGF siRNA was examined for its effectiveness in inhibiting tumor growth and tumorigenicity of PTEN-null GBM cells. In U251 GBM cells, PTEN restoration reduced proliferation, arrested the cell cycle at G0/G1 stage, and promoted apoptosis via inhibition of PIK/AKT signaling pathway. Unexpectedly, anchorage-dependent and -independent colony formation ability and the capacity for wound-healing migration of U251 cells with stable expression of VEGF siRNA were significantly inhibited, suggesting that VEGF also appeared to function as an autocrine growth factor in addition to its well-known pro-angiogenic paracrine function. Further, a combined treatment of PTEN restoration and VEGF siRNA had the best tumor suppression effect. In a xenograft study in null mice, both the restoration of PTEN and the expression of VEGF siRNA could significantly inhibit the growth of U251 GBMs, whereas tumor growth was entirely suppressed by a combination of the two treatments. Therefore, the combination of PTEN expression and VEGF knockdown represents an effective gene therapy strategy for malignant gliomas.

  1. Transcriptome analysis of the synganglion from the honey bee mite, Varroa destructor and RNAi knockdown of neural peptide targets.

    PubMed

    Campbell, Ewan M; Budge, Giles E; Watkins, Max; Bowman, Alan S

    2016-03-01

    Varroa mites (Varroa destructor) and the viruses that they transmit are one of the major contributing factors to the global honey bee crisis. Gene products within the nervous system are the targets of all the insecticides currently used to control Varroa but there is a paucity of transcriptomic data available for Varroa neural tissues. A cDNA library from the synganglia ("brains") of adult female Varroa was constructed and 600 ESTs sequenced and analysed revealing several current and potential druggable targets. Contigs coding for the deformed wing virus (DWV) variants V. destructor virus-1 (VDV-1) and the recombinant (VDV-1DVD) were present in the synganglion library. Negative-sense RNA-specific PCR indicated that VDV-1 replicates in the Varroa synganglion and all other tissues tested, but we could not detect DWV replicating in any Varroa tissue. Two neuropeptides were identified in the synganlion EST library: a B-type allatostatin and a member of the crustacean hyperglycaemic hormone (CHH) superfamily. Knockdown of the allatostatin or the CHH-like gene by double-stranded RNA-interference (dsRNAi) resulted in 85% and 55% mortality, respectively, of Varroa. Here, we present the first transcriptomic survey in Varroa and demonstrate that neural genes can be targeted by dsRNAi either for genetic validation of putative targets during drug discovery programmes or as a potential control measure in itself.

  2. A combined optogenetic-knockdown strategy reveals a major role of tomosyn in mossy fiber synaptic plasticity

    PubMed Central

    Ben-Simon, Yoav; Rodenas-Ruano, Alma; Alviña, Karina; Lam, Alice D.; Stuenkel, Edward L.; Castillo, Pablo E.; Ashery, Uri

    2015-01-01

    Summary Neurotransmitter release probability (Pr) largely determines the dynamic properties of synapses. While much is known on the role of presynaptic proteins in transmitter release, their specific contribution to synaptic plasticity is unclear. One such protein, tomosyn, is believed to reduce Pr by interfering with the SNARE complex formation. Tomosyn is enriched at hippocampal mossy fiber-to-CA3 pyramidal cell synapses (MF-CA3), which characteristically exhibit low Pr, strong synaptic facilitation and pre-synaptic PKA-dependent LTP. To evaluate tomosyn's role in MF-CA3 function, we used a combined knockdown (KD)-optogenetic strategy whereby presynaptic neurons with reduced tomosyn levels were selectively activated by light. Using this approach in mouse hippocampal slices we found that facilitation, LTP, and PKA-induced potentiation were significantly impaired at tomosyn-deficient synapses. These findings not only indicate that tomosyn is a key regulator of MF-CA3 plasticity, but also highlight the power of a combined KD-optogenetic approach to determine the role of presynaptic proteins. PMID:26166572

  3. Knockdown of Histone Methyltransferase hSETD1A Inhibits Progression, Migration, and Invasion in Human Hepatocellular Carcinoma.

    PubMed

    Cheng, Xin-Sheng; Sun, Shi-Bo; Zhong, Feng; He, Kun; Zhou, Jie

    2016-01-01

    Our aim was to study the expression of human SET domain containing protein 1A (hSETD1A) in hepatocellular carcinoma patients and its relationship with human hepatocellular carcinoma cell function. A total of 30 patients with hepatocellular carcinoma were enrolled in this study. The expression of hSETD1A was detected by real-time polymerase chain reaction (PCR) and Western blotting. The immortalized normal human liver cell line including SMMC-7721 was subjected to real-time PCR for hSETD1A mRNA. Furthermore, hSETD1A-small hairpin RNA (shRNA) was used to knock down hSETD1A expression in SMMC-7721 cells. Cell proliferation, cell apoptosis, and cell migration were determined by CCK8, flow cytometry, and Transwell assays. The positive expression rate level of hSETD1A mRNA and protein in liver carcinoma tissues was 73.33%. hSETD1A knockdown using a specific hSETD1A-shRNA inhibited cell proliferation and promoted cell apoptosis in SMMC-7721 cells. It was also found that downregulation of hSETD1A inhibited cell migration ability but did not affect cell invasion. In conclusion, the expression of hSETD1A occurs at a high rate in hepatocellular carcinoma patients. The expression state of hSETD1A may be a prognostic factor in hepatocellular carcinoma. PMID:27656834

  4. Armigeres subalbatus (Diptera: Culicidae) prophenoloxidase III is required for mosquito cuticle formation: ultrastructural study on dsRNA-knockdown mosquitoes.

    PubMed

    Tsao, I Y; Christensen, B M; Chen, C C

    2010-07-01

    We previously suggested that Armigeres subalbatus (Coquillett) prophenoloxidase III (As-pro-PO III) might be associated with morphogenesis of larvae and pupae. Because PO and its activation system are present in the insect cuticle, and cuticle formation is a major event during pupal morphogenesis, we used ultrastructural analysis to examine the effects of As-pro-PO III knockdown on the formation of pupal and adult cuticle. Inoculation of As-pro-PO III dsRNA resulted in the incomplete formation of nascent pupal endocuticle and pharate adult cuticle, i.e., significantly fewer cuticular lamellae were deposited, the helicoidal pattern of chitin microfibrils was disorganized, and numerous electron-lucent spaces were present in the cuticular protein matrix. Similar disruptions were observed in the cuticle of adults derived from As-pro-PO III dsRNA-inoculated pupae. It has long been suggested that the quinines, generated by PO-catalyzed oxidation reactions, function as cross-linking agents; therefore, it seems reasonable to suggest that the loss of As-pro-PO III-mediated protein-protein linkages causes morphological abnormalities in the protein matrix. Our findings suggest that As-pro-PO III plays a role in cuticle formation in mosquitoes, a novel function for phenol-oxidizing enzymes.

  5. Knockdown, residual, and antifeedant activity of pyrethroids and home landscape bioinsecticides against Japanese beetles (Coleoptera: Scarabaeidae) on Linden foliage.

    PubMed

    Baumler, Rebecca E; Potter, Daniel A

    2007-04-01

    Residual toxicity and leaf protection capability of five pyrethroids, professional and home garden azadirachtin formulations, and six other bioinsecticides for the home landscape were evaluated against the Japanese beetle, Popillia japonica Newman (Coleoptera: Scarabaeidae), on linden, Tilia cordata L. Capacity of intoxicated beetles to recover and subsequently feed and disperse also was evaluated to provide insight on activity characteristics of the different compounds. Intact shoots were sprayed and left in the field for varying intervals before being challenged with beetles in no-choice and choice tests. All pyrethroids except permethrin gave greater leaf protection, knockdown, and kill than did carbaryl, the standard, after 14 d of weathering. Deltamethrin, cyfluthrin, bifenthrin, and lamda-cyhalothrin gave a high level of protection for at least 19 d, and azadirachtin (Azatin XL) deterred feeding in choice tests for as long as 14 d. Home garden formulations containing pyrethrins in canola oil (Pyola) or azadiractin (Neem-Away) gave good short-term (< 3-d) protection. Formulations of capsaicin, rotenone + pyrethrins, kaolin particle film, D-limonene, or garlic extract were ineffective, the latter two formulations being highly phytotoxic to linden. Results of this study should help support updating of guidelines for insecticidal control of Japanese beetles. PMID:17461070

  6. Osteopontin knockdown in the kidneys of hyperoxaluric rats leads to reduction in renal calcium oxalate crystal deposition.

    PubMed

    Tsuji, Hidenori; Shimizu, Nobutaka; Nozawa, Masahiro; Umekawa, Tohru; Yoshimura, Kazuhiro; De Velasco, Marco A; Uemura, Hirotsugu; Khan, Saeed R

    2014-06-01

    Osteopontin (OPN) expression is increased in kidneys of rats with ethylene glycol (EG) induced hyperoxaluria and calcium oxalate (CaOx) nephrolithiasis. The aim of this study is to clarify the effect of OPN knockdown by in vivo transfection of OPN siRNA on deposition of CaOx crystals in the kidneys. Hyperoxaluria was induced in 6-week-old male Sprague-Dawley rats by administering 1.5% EG in drinking water for 2 weeks. Four groups of six rats each were studied: Group A, untreated animals (tap water); Group B, administering 1.5% EG; Group C, 1.5% EG with in vivo transfection of OPN siRNA; Group D, 1.5% EG with in vivo transfection of negative control siRNA. OPN siRNA transfections were performed on day 1 and 8 by renal sub-capsular injection. Rats were killed at day 15 and kidneys were removed. Extent of crystal deposition was determined by measuring renal calcium concentrations and counting renal crystal deposits. OPN siRNA transfection resulted in significant reduction in expression of OPN mRNA as well as protein in group C compared to group B. Reduction in OPN expression was associated with significant decrease in crystal deposition in group C compared to group B. Specific suppression of OPN mRNA expression in kidneys of hyperoxaluric rats leads to a decrease in OPN production and simultaneously inhibits renal crystal deposition.

  7. Impacts of CD44 knockdown in cancer cells on tumor and host metabolic systems revealed by quantitative imaging mass spectrometry.

    PubMed

    Ohmura, Mitsuyo; Hishiki, Takako; Yamamoto, Takehiro; Nakanishi, Tsuyoshi; Kubo, Akiko; Tsuchihashi, Kenji; Tamada, Mayumi; Toue, Sakino; Kabe, Yasuaki; Saya, Hideyuki; Suematsu, Makoto

    2015-04-30

    CD44 expressed in cancer cells was shown to stabilize cystine transporter (xCT) that uptakes cystine and excretes glutamate to supply cysteine as a substrate for reduced glutathione (GSH) for survival. While targeting CD44 serves as a potentially therapeutic stratagem to attack cancer growth and chemoresistance, the impact of CD44 targeting in cancer cells on metabolic systems of tumors and host tissues in vivo remains to be fully determined. This study aimed to reveal effects of CD44 silencing on alterations in energy metabolism and sulfur-containing metabolites in vitro and in vivo using capillary electrophoresis-mass spectrometry and quantitative imaging mass spectrometry (Q-IMS), respectively. In an experimental model of xenograft transplantation of human colon cancer HCT116 cells in superimmunodeficient NOG mice, snap-frozen liver tissues containing metastatic tumors were examined by Q-IMS. As reported previously, short hairpin CD44 RNA interference (shCD44) in cancer cells caused significant regression of tumor growth in the host liver. Under these circumstances, the CD44 knockdown suppressed polyamines, GSH and energy charges not only in metastatic tumors but also in the host liver. In culture, HCT116 cells treated with shCD44 decreased total amounts of methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides, suggesting roles of these metabolites for cancer growth. Collectively, these results suggest that CD44 expressed in cancer accounts for a key regulator of metabolic interplay between tumor and the host tissue. PMID:25461272

  8. Genomic Instability Associated with p53 Knockdown in the Generation of Huntington’s Disease Human Induced Pluripotent Stem Cells

    PubMed Central

    Tidball, Andrew M.; Neely, M. Diana; Chamberlin, Reed; Aboud, Asad A.; Kumar, Kevin K.; Han, Bingying; Bryan, Miles R.; Aschner, Michael; Ess, Kevin C.; Bowman, Aaron B.

    2016-01-01

    Alterations in DNA damage response and repair have been observed in Huntington’s disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown. PMID:26982737

  9. Vpx-Independent Lentiviral Transduction and shRNA-Mediated Protein Knock-Down in Monocyte-Derived Dendritic Cells.

    PubMed

    Witkowski, Wojciech; Vermeire, Jolien; Landi, Alessia; Naessens, Evelien; Vanderstraeten, Hanne; Nauwynck, Hans; Favoreel, Herman; Verhasselt, Bruno

    2015-01-01

    The function of dendritic cells (DCs) in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. To date, efficient lentiviral transduction of DCs or their monocyte derived counterparts (MDDCs) required high multiplicity of infection (MOI) or the exposure to the HIV-2/SIV protein Vpx to degrade viral restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1). Here we present a Vpx-independent method for efficient (>95%) transduction of MDDCs at lower MOI. The protocol can be used both for ectopic gene expression and knock-down. Introducing shRNA targeting viral entry receptor CD4 and restriction factor SAMHD1 into MDDCs resulted in down-regulation of targeted proteins and, consequently, expected impact on HIV infection. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting. PMID:26208151

  10. Vpx-Independent Lentiviral Transduction and shRNA-Mediated Protein Knock-Down in Monocyte-Derived Dendritic Cells

    PubMed Central

    Witkowski, Wojciech; Vermeire, Jolien; Landi, Alessia; Naessens, Evelien; Vanderstraeten, Hanne; Nauwynck, Hans; Favoreel, Herman; Verhasselt, Bruno

    2015-01-01

    The function of dendritic cells (DCs) in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. To date, efficient lentiviral transduction of DCs or their monocyte derived counterparts (MDDCs) required high multiplicity of infection (MOI) or the exposure to the HIV-2/SIV protein Vpx to degrade viral restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1). Here we present a Vpx-independent method for efficient (>95%) transduction of MDDCs at lower MOI. The protocol can be used both for ectopic gene expression and knock-down. Introducing shRNA targeting viral entry receptor CD4 and restriction factor SAMHD1 into MDDCs resulted in down-regulation of targeted proteins and, consequently, expected impact on HIV infection. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting. PMID:26208151

  11. Knockdown of the sodium-dependent phosphate co-transporter 2b (NPT2b) suppresses lung tumorigenesis.

    PubMed

    Hong, Seong-Ho; Minai-Tehrani, Arash; Chang, Seung-Hee; Jiang, Hu-Lin; Lee, Somin; Lee, Ah-Young; Seo, Hwi Won; Chae, Chanhee; Beck, George R; Cho, Myung-Haing

    2013-01-01

    The sodium-dependent phosphate co-transporter 2b (NPT2b) plays an important role in maintaining phosphate homeostasis. In previous studies, we have shown that high dietary inorganic phosphate (Pi) consumption in mice stimulated lung tumorigenesis and increased NPT2b expression. NPT2b has also been found to be highly expressed in human lung cancer tissues. The association of high expression of NPT2b in the lung with poor prognosis in oncogenic lung diseases prompted us to test whether knockdown of NPT2b may regulate lung cancer growth. To address this issue, aerosols that contained small interfering RNA (siRNA) directed against NPT2b (siNPT2b) were delivered into the lungs of K-ras (LA1) mice, which constitute a murine model reflecting human lung cancer. Our results clearly showed that repeated aerosol delivery of siNPT2b successfully suppressed lung cancer growth and decreased cancer cell proliferation and angiogenesis, while facilitating apoptosis. These results strongly suggest that NPT2b plays a role lung tumorigenesis and represents a novel target for lung cancer therapy. PMID:24194864

  12. siRNA knockdown of tissue inhibitor of metalloproteinase-1 in keloid fibroblasts leads to degradation of collagen type I.

    PubMed

    Aoki, Masayo; Miyake, Koichi; Ogawa, Rei; Dohi, Teruyuki; Akaishi, Satoshi; Hyakusoku, Hiko; Shimada, Takashi

    2014-03-01

    Keloids are defined as overgrowths of scar tissue resulting from abnormal wound healing. They are characterized by excessive dermal deposition of thick, hyalinized collagen bundles resulting from an imbalance between the production and degradation of extracellular matrix (ECM) components. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are two important regulators of ECM degradation and remodeling. To evaluate the role played by knockdown of TIMPs in keloid formation, we transduced human keloid-derived fibroblasts (KFs) with small interfering RNAs targeting TIMP-1 or -2 (siTIMP-1 or siTIMP-2) using a lentiviral vector and assessed the biological effects. We found that MMP-1/TIMP-1 and MMP-1/TIMP-2 complexes were suppressed and that MMP-2 activity was upregulated in KFs expressing siTIMP-1 or siTIMP-2. In addition, increased degradation of collagen type I was observed in the supernatant of KFs expressing siTIMP-1, but not siTIMP-2, with the suppression of cell viability and induction of apoptosis. These results suggest that targeting TIMP-1 using small interfering RNA has significant therapeutic potential as an approach to treating keloids through degradation of their thick collagen bundles. PMID:24042342

  13. The differentiation effect of low-dose cytosine arabinoside is disturbed in PU.1-knockdown K562 cells.

    PubMed

    Nakano, Hiroko; Yanagita, Akane; Takahashi, Shinichiro

    2014-07-01

    We recently demonstrated by using PU.1-knockdown K562 (K562 PU.1KD) cells stably expressing PU.1 short inhibitory RNAs and PU.1-overexpressing K562 (K562 PU.1OE) cells, that therapeutic concentrations of 5-aza-2'-deoxycytidine (5-azadC) induce erythroid differentiation of these cells and that the PU.1 expression level is closely associated with the differentiating and apoptotic effects of 5-azadC on K562 cells. In this study, we investigated whether the effects of low-dose cytosine arabinoside (Ara-C), which is another erythroid differentiation inducer in K562 cells, is associated with the expression level of PU.1 in these cells. As a result, we demonstrated that the effect of Ara-C on cell viability and differentiation, as determined by the WST-8 assay and β-globin mRNA expression analysis, respectively, was suppressed in K562 PU.1KD cells compared to their controls. Collectively, these findings suggest that sufficient expression of PU.1 is indispensable for the erythroid differentiation of K562 cells.

  14. Immunoregulation of autocrine prolactin: suppressing the expression of costimulatory molecules and cytokines in T lymphocytes by prolactin receptor knockdown.

    PubMed

    Xu, Dongming; Lin, Ling; Lin, Xiahong; Huang, Ziyang; Lei, Zhenmin

    2010-01-01

    Ample evidence indicates that prolactin (PRL) secreted from the pituitary gland plays an important role in a variety of human immune responses. However, the immunoregulation of autocrine PRL in T lymphocytes is not fully understood. To evaluate the role of autocrine PRL in T lymphocyte activation, PRL receptor (PRLR) in Jurkat cells was silenced by lentivirus-mediated stable expression of PRLR shRNAi. Knockdown of PRLR resulted in a considerable reduction of phytohemagglutinin (PHA)-induced T cell proliferation. Moreover, the synthesis and secretion of CD137, CD154, IL-2 and IL-4 were significantly decreased, while the production of CD28, IFN-gamma and IL-10 was not affected in PHA-primed PRLR-deficient cells. These results demonstrate the importance of autocrine regulation of the PRL signaling in T lymphocyte growth and activation, and support a mechanism by which autocrine PRL participates in the immunoregulation through selectively influencing the expression of certain critical costimulatory molecules and cytokines.

  15. Hypomorphic Smn knockdown C2C12 myoblasts reveal intrinsic defects in myoblast fusion and myotube morphology

    SciTech Connect

    Shafey, Dina; Cote, Patrice D.; Kothary, Rashmi . E-mail: rkothary@ohri.ca

    2005-11-15

    Dosage of the survival motor neuron (SMN) protein has been directly correlated with the severity of disease in patients diagnosed with spinal muscular atrophy (SMA). It is also clear that SMA is a neurodegenerative disorder characterized by the degeneration of the {alpha}-motor neurons in the anterior horn of the spinal cord and atrophy of the associated skeletal muscle. What is more controversial is whether it is neuronal and/or muscle-cell-autonomous defects that are responsible for the disease per se. Although motor neuron degeneration is generally accepted as the primary event in SMA, intrinsic muscle defects in this disease have not been ruled out. To gain a better understanding of the influence of SMN protein dosage in muscle, we have generated a hypomorphic series of myoblast (C2C12) stable cell lines with variable Smn knockdown. We show that depletion of Smn in these cells resulted in a decrease in the number of nuclear 'gems' (gemini of coiled bodies), reduced proliferation with no increase in cell death, defects in myoblast fusion, and malformed myotubes. Importantly, the severity of these abnormalities is directly correlated with the decrease in Smn dosage. Taken together, our work supports the view that there is an intrinsic defect in skeletal muscle cells of SMA patients and that this defect contributes to the overall pathogenesis in this devastating disease.

  16. Transgenic resistance.

    PubMed

    Cillo, Fabrizio; Palukaitis, Peter

    2014-01-01

    Transgenic resistance to plant viruses is an important technology for control of plant virus infection, which has been demonstrated for many model systems, as well as for the most important plant viruses, in terms of the costs of crop losses to disease, and also for many other plant viruses infecting various fruits and vegetables. Different approaches have been used over the last 28 years to confer resistance, to ascertain whether particular genes or RNAs are more efficient at generating resistance, and to take advantage of advances in the biology of RNA interference to generate more efficient and environmentally safer, novel "resistance genes." The approaches used have been based on expression of various viral proteins (mostly capsid protein but also replicase proteins, movement proteins, and to a much lesser extent, other viral proteins), RNAs [sense RNAs (translatable or not), antisense RNAs, satellite RNAs, defective-interfering RNAs, hairpin RNAs, and artificial microRNAs], nonviral genes (nucleases, antiviral inhibitors, and plantibodies), and host-derived resistance genes (dominant resistance genes and recessive resistance genes), and various factors involved in host defense responses. This review examines the above range of approaches used, the viruses that were tested, and the host species that have been examined for resistance, in many cases describing differences in results that were obtained for various systems developed in the last 20 years. We hope this compilation of experiences will aid those who are seeking to use this technology to provide resistance in yet other crops, where nature has not provided such.

  17. Resistance mechanisms

    PubMed Central

    Cag, Yasemin; Caskurlu, Hulya; Fan, Yanyan; Cao, Bin

    2016-01-01

    By definition, the terms sepsis and septic shock refer to a potentially fatal infectious state in which the early administration of an effective antibiotic is the most significant determinant of the outcome. Because of the global spread of resistant bacteria, the efficacy of antibiotics has been severely compromised. S. pneumonia, Escherichia coli (E. coli), Klebsiella, Acinetobacter, and Pseudomonas are the predominant pathogens of sepsis and septic shock. It is common for E. coli, Klebsiella, Acinetobacter and Pseudomonas to be resistant to multiple drugs. Multiple drug resistance is caused by the interplay of multiple resistance mechanisms those emerge via the acquisition of extraneous resistance determinants or spontaneous mutations. Extended-spectrum beta-lactamases (ESBLs), carbapenemases, aminoglycoside-modifying enzymes (AMEs) and quinolone resistance determinants are typically external and disseminate on mobile genetic elements, while porin-efflux mechanisms are activated by spontaneous modifications of inherited structures. Porin and efflux mechanisms are frequent companions of multiple drug resistance in Acinetobacter and P. aeruginosa, but only occasionally detected among E. coli and Klebsiella. Antibiotic resistance became a global health threat. This review examines the major resistance mechanisms of the leading microorganisms of sepsis. PMID:27713884

  18. Transgenic resistance.

    PubMed

    Cillo, Fabrizio; Palukaitis, Peter

    2014-01-01

    Transgenic resistance to plant viruses is an important technology for control of plant virus infection, which has been demonstrated for many model systems, as well as for the most important plant viruses, in terms of the costs of crop losses to disease, and also for many other plant viruses infecting various fruits and vegetables. Different approaches have been used over the last 28 years to confer resistance, to ascertain whether particular genes or RNAs are more efficient at generating resistance, and to take advantage of advances in the biology of RNA interference to generate more efficient and environmentally safer, novel "resistance genes." The approaches used have been based on expression of various viral proteins (mostly capsid protein but also replicase proteins, movement proteins, and to a much lesser extent, other viral proteins), RNAs [sense RNAs (translatable or not), antisense RNAs, satellite RNAs, defective-interfering RNAs, hairpin RNAs, and artificial microRNAs], nonviral genes (nucleases, antiviral inhibitors, and plantibodies), and host-derived resistance genes (dominant resistance genes and recessive resistance genes), and various factors involved in host defense responses. This review examines the above range of approaches used, the viruses that were tested, and the host species that have been examined for resistance, in many cases describing differences in results that were obtained for various systems developed in the last 20 years. We hope this compilation of experiences will aid those who are seeking to use this technology to provide resistance in yet other crops, where nature has not provided such. PMID:25410101

  19. Plant Translation Factors and Virus Resistance

    PubMed Central

    Sanfaçon, Hélène

    2015-01-01

    Plant viruses recruit cellular translation factors not only to translate their viral RNAs but also to regulate their replication and potentiate their local and systemic movement. Because of the virus dependence on cellular translation factors, it is perhaps not surprising that many natural plant recessive resistance genes have been mapped to mutations of translation initiation factors eIF4E and eIF4G or their isoforms, eIFiso4E and eIFiso4G. The partial functional redundancy of these isoforms allows specific mutation or knock-down of one isoform to provide virus resistance without hindering the general health of the plant. New possible targets for antiviral strategies have also been identified following the characterization of other plant translation factors (eIF4A-like helicases, eIF3, eEF1A and eEF1B) that specifically interact with viral RNAs and proteins and regulate various aspects of the infection cycle. Emerging evidence that translation repression operates as an alternative antiviral RNA silencing mechanism is also discussed. Understanding the mechanisms that control the development of natural viral resistance and the emergence of virulent isolates in response to these plant defense responses will provide the basis for the selection of new sources of resistance and for the intelligent design of engineered resistance that is broad-spectrum and durable. PMID:26114476

  20. Resistant Hypertension.

    PubMed

    Doroszko, Adrian; Janus, Agnieszka; Szahidewicz-Krupska, Ewa; Mazur, Grzegorz; Derkacz, Arkadiusz

    2016-01-01

    Resistant hypertension is a severe medical condition which is estimated to appear in 9-18% of hypertensive patients. Due to higher cardiovascular risk, this disorder requires special diagnosis and treatment. The heterogeneous etiology, risk factors and comorbidities of resistant hypertension stand in need of sophisticated evaluation to confirm the diagnosis and select the best therapeutic options, which should consider lifestyle modifications as well as pharmacological and interventional treatment. After having excluded pseudohypertension, inappropriate blood pressure measurement and control as well as the white coat effect, suspicion of resistant hypertension requires an analysis of drugs which the hypertensive patient is treated with. According to one definition - ineffective treatment with 3 or more antihypertensive drugs including diuretics makes it possible to diagnose resistant hypertension. A multidrug therapy including angiotensin - converting enzyme inhibitors, angiotensin II receptor blockers, beta blockers, diuretics, long-acting calcium channel blockers and mineralocorticoid receptor antagonists has been demonstrated to be effective in resistant hypertension treatment. Nevertheless, optional, innovative therapies, e.g. a renal denervation or baroreflex activation, may create a novel pathway of blood pressure lowering procedures. The right diagnosis of this disease needs to eliminate the secondary causes of resistant hypertension e.g. obstructive sleep apnea, atherosclerosis and renal or hormonal disorders. This paper briefly summarizes the identification of the causes of resistant hypertension and therapeutic strategies, which may contribute to the proper diagnosis and an improvement of the long term management of resistant hypertension.

  1. Managing Resistance.

    ERIC Educational Resources Information Center

    Maag, John W.

    2000-01-01

    This article presents some considerations and ideas for managing students' resistance. They are organized around four topics: the impact of context on behavior, the importance of being comprehensive and nonrestrictive in behavior, the adaptive function of resistant behavior, and the benefit of joining children in their frame of reference.…

  2. Gene-knockdown in the honey bee mite Varroa destructor by a non-invasive approach: studies on a glutathione S-transferase

    PubMed Central

    2010-01-01

    Background The parasitic mite Varroa destructor is considered the major pest of the European honey bee (Apis mellifera) and responsible for declines in honey bee populations worldwide. Exploiting the full potential of gene sequences becoming available for V. destructor requires adaptation of modern molecular biology approaches to this non-model organism. Using a mu-class glutathione S-transferase (VdGST-mu1) as a candidate gene we investigated the feasibility of gene knockdown in V. destructor by double-stranded RNA-interference (dsRNAi). Results Intra-haemocoelic injection of dsRNA-VdGST-mu1 resulted in 97% reduction in VdGST-mu1 transcript levels 48 h post-injection compared to mites injected with a bolus of irrelevant dsRNA (LacZ). This gene suppression was maintained to, at least, 72 h. Total GST catalytic activity was reduced by 54% in VdGST-mu1 gene knockdown mites demonstrating the knockdown was effective at the translation step as well as the transcription steps. Although near total gene knockdown was achieved by intra-haemocoelic injection, only half of such treated mites survived this traumatic method of dsRNA administration and less invasive methods were assessed. V. destructor immersed overnight in 0.9% NaCl solution containing dsRNA exhibited excellent reduction in VdGST-mu1 transcript levels (87% compared to mites immersed in dsRNA-LacZ). Importantly, mites undergoing the immersion approach had greatly improved survival (75-80%) over 72 h, approaching that of mites not undergoing any treatment. Conclusions Our findings on V. destructor are the first report of gene knockdown in any mite species and demonstrate that the small size of such organisms is not a major impediment to applying gene knockdown approaches to the study of such parasitic pests. The immersion in dsRNA solution method provides an easy, inexpensive, relatively high throughput method of gene silencing suitable for studies in V. destructor, other small mites and immature stages of ticks

  3. HOXA/PBX3 knockdown impairs growth and sensitizes cytogenetically normal acute myeloid leukemia cells to chemotherapy

    PubMed Central

    Dickson, Glenda J.; Liberante, Fabio G.; Kettyle, Laura M; O’Hagan, Kathleen A.; Finnegan, Damian P. J.; Bullinger, Lars; Geerts, Dirk; McMullin, Mary Frances; Lappin, Terry R. J.; Mills, Ken I.; Thompson, Alexander

    2013-01-01

    The cytogenetically normal subtype of acute myeloid leukemia is associated with an intermediate risk which complicates therapeutic options. Lower overall HOX/TALE expression appears to correlate with more favorable prognosis/better response to treatment in some leukemias and solid cancer. The functional significance of the associated gene expression and response to chemotherapy is not known. Three independent microarray datasets obtained from large cohorts of patients along with quantitative polymerase chain reaction validation were used to identify a four-gene HOXA/TALE signature capable of prognostic stratification. Biochemical analysis was used to identify interactions between the four encoded proteins and targeted knockdown used to examine the functional importance of sustained expression of the signature in leukemia maintenance and response to chemotherapy. An 11 HOXA/TALE code identified in an intermediate-risk group of patients (n=315) compared to a group with a favorable risk (n=105) was reduced to a four-gene signature of HOXA6, HOXA9, PBX3 and MEIS1 by iterative analysis of independent platforms. This signature maintained the favorable/intermediate risk partition and where applicable, correlated with overall survival in cytogenetically normal acute myeloid leukemia. We further showed that cell growth and function are dependent on maintained levels of these core genes and that direct targeting of HOXA/PBX3 sensitizes cytogenetically normal acute myeloid leukemia cells to standard chemotherapy. Together the data support a key role for HOXA/TALE in cytogenetically normal acute myeloid leukemia and demonstrate that targeting of clinically significant HOXA/PBX3 elements may provide therapeutic benefit to patients with this subtype of leukemia. PMID:23539541

  4. CaT1 knock-down strategies fail to affect CRAC channels in mucosal-type mast cells.

    PubMed

    Kahr, Heike; Schindl, Rainer; Fritsch, Reinhard; Heinze, Barbara; Hofbauer, Michael; Hack, Marlene E; Mörtelmaier, Manuel A; Groschner, Klaus; Peng, Ji-Bin; Takanaga, Hitomi; Hediger, Matthias A; Romanin, Christoph

    2004-05-15

    CaT1, the calcium transport protein 1 encoded by TRPV6, is able to generate a Ca(2+) conductance similar but not identical to the classical CRAC current in mucosal-type mast cells. Here we show that CaT1-derived Ca(2+) entry into HEK293 cells is effectively inhibited either by expression of various dominant negative N-terminal fragments of CaT1 (N(334)-CaT1, N(198)-CaT1 and N(154)-CaT1) or by antisense suppression. By contrast, the endogenous CRAC current of the mast cells was unaffected by CaT1 antisense and siRNA knockdown but markedly suppressed by two (N(334)-CaT1, N(198)-CaT1) of the dominant negative N-CaT1 fragments. Inhibition of CRAC current was not an unspecific, toxic effect, as inward rectifier K(+) and MagNuM currents of the mast cells were not significantly affected by these N-CaT1 fragments. The shortest N(154)-CaT1 fragment inhibited CaT1-derived currents in mast cells, but failed to inhibit CRAC currents. Thus, the structural requirements of rCaT N-terminal fragments for inhibition of rCaT1 and CRAC channels are different. These results together with the lack of CaT1 antisense and siRNA effects on currents render it unlikely that CaT1 is a component of native CRAC channels in mast cells. The data further demonstrate a novel strategy for CRAC current inhibition by an N-terminal structure of CaT1.

  5. Knockdown of GSK3β increases basal autophagy and AMPK signalling in nutrient-laden human aortic endothelial cells

    PubMed Central

    Weikel, Karen A.; Cacicedo, José M.; Ruderman, Neil B.; Ido, Yasuo

    2016-01-01

    High concentrations of glucose and palmitate increase endothelial cell inflammation and apoptosis, events that often precede atherogenesis. They may do so by decreasing basal autophagy and AMP-activated protein kinase (AMPK) activity, although the mechanisms by which this occurs are not clear. Decreased function of the lysosome, an organelle required for autophagy and AMPK, have been associated with hyperactivity of glycogen synthase kinase 3β (GSK3β). To determine whether GSK3β affects nutrient-induced changes in autophagy and AMPK activity, we used a primary human aortic endothelial cell (HAEC) model of type 2 diabetes that we had previously characterized with impaired AMPK activity and autophagy [Weikel et al. (2015) Am. J. Phys. Cell Physiol. 308, C249–C263]. Presently, we found that incubation of HAECs with excess nutrients (25 mM glucose and 0.4 mM palmitate) increased GSK3β activity and impaired lysosome acidification. Suppression of GSK3β in these cells by treatment with a chemical inhibitor or overexpression of kinase-dead GSK3β attenuated these lysosomal changes. Under control and excess nutrient conditions, knockdown of GSK3β increased autophagosome formation, forkhead box protein O1 (FOXO1) activity and AMPK signalling and decreased Akt signalling. Similar changes in autophagy, AMPK and Akt signalling were observed in aortas from mice treated with the GSK3β inhibitor CHIR 99021. Thus, increasing basal autophagy and AMPK activity by inhibiting GSK3β may be an effective strategy in the setting of hyperglycaemia and dyslipidaemia for restoring endothelial cell health and reducing atherogenesis. PMID:27534430

  6. Lentivirus-mediated knockdown of eukaryotic translation initiation factor 3 subunit D inhibits proliferation of HCT116 colon cancer cells.

    PubMed

    Yu, Xiaojun; Zheng, Bo'an; Chai, Rui

    2014-12-12

    Dysregulation of protein synthesis is emerging as a major contributory factor in cancer development. eIF3D (eukaryotic translation initiation factor 3 subunit D) is one member of the eIF3 (eukaryotic translation initiation factor 3) family, which is essential for initiation of protein synthesis in eukaryotic cells. Acquaintance with eIF3D is little since it has been identified as a dispensable subunit of eIF3 complex. Recently, eIF3D was found to embed somatic mutations in human colorectal cancers, indicating its importance for tumour progression. To further probe into its action in colon cancer, we utilized lentivirus-mediated RNA interference to knock down eIF3D expression in one colon cancer cell line HCT116. Knockdown of eIF3D in HCT116 cells significantly inhibited cell proliferation and colony formation in vitro. Flow cytometry analysis indicated that depletion of eIF3D led to cell-cycle arrest in the G2/M phase, and induced an excess accumulation of HCT116 cells in the sub-G1 phase representing apoptotic cells. Signalling pathways responsible for cell growth and apoptosis have also been found altered after eIF3D silencing, such as AMPKα (AMP-activated protein kinase alpha), Bad, PRAS40 [proline-rich Akt (PKB) substrate of 40 kDa], SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase), GSK3β and PARP [poly(ADP-ribose) polymerase]. Taken together, these findings suggest that eIF3D might play an important role in colon cancer progression.

  7. Knockdown of fbxl10/kdm2bb rescues chd7 morphant phenotype in a zebrafish model of CHARGE syndrome.

    PubMed

    Balow, Stephanie A; Pierce, Lain X; Zentner, Gabriel E; Conrad, Patricia A; Davis, Stephani; Sabaawy, Hatem E; McDermott, Brian M; Scacheri, Peter C

    2013-10-01

    CHARGE syndrome is a sporadic autosomal-dominant genetic disorder characterized by a complex array of birth defects so named for its cardinal features of ocular coloboma, heart defects, choanal atresia, growth retardation, genital abnormalities, and ear abnormalities. Approximately two-thirds of individuals clinically diagnosed with CHARGE syndrome have heterozygous loss-of-function mutations in the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7), an ATP-dependent chromatin remodeler. To examine the role of Chd7 in development, a zebrafish model was generated through morpholino (MO)-mediated targeting of the zebrafish chd7 transcript. High doses of chd7 MO induce lethality early in embryonic development. However, low dose-injected embryos are viable, and by 4 days post-fertilization, morphant fish display multiple defects in organ systems analogous to those affected in humans with CHARGE syndrome. The chd7 morphants show elevated expression of several potent cell-cycle inhibitors including ink4ab (p16/p15), p21 and p27, accompanied by reduced cell proliferation. We also show that Chd7 is required for proper organization of neural crest-derived craniofacial cartilage structures. Strikingly, MO-mediated knockdown of the jumonji domain-containing histone demethylase fbxl10/kdm2bb, a repressor of ribosomal RNA (rRNA) genes, rescues cell proliferation and cartilage defects in chd7 morphant embryos and can lead to complete rescue of the CHARGE syndrome phenotype. These results indicate that CHARGE-like phenotypes in zebrafish can be mitigated through modulation of fbxl10 levels and implicate FBXL10 as a possible therapeutic target in CHARGE syndrome.

  8. Knockdown of the corazonin gene reveals its critical role in the control of gregarious characteristics in the desert locust.

    PubMed

    Sugahara, Ryohei; Saeki, Shinjiro; Jouraku, Akiya; Shiotsuki, Takahiro; Tanaka, Seiji

    2015-08-01

    The two plague locusts, Schistocerca gregaria and Locusta migratoria, exhibit density-dependent phase polyphenism. Nymphs occurring at low population densities (solitarious forms) are uniformly colored and match their body color to the background color of their habitat, whereas those occurring at high population densities (gregarious) develop black patterns. An injection of the neuropeptide, corazonin (Crz) has been shown to induce black patterns in locusts and affect the classical morphometric ratio, F/C (F, hind femur length; C, maximum head width). We herein identified and cloned the CRZ genes from S. gregaria (SgCRZ) and L. migratoria. A comparative analysis of prepro-Crz sequences among insects showed that the functional peptide was well conserved; its conservation was limited to the peptide region. Silencing of the identified SgCRZ gene in gregarious S. gregaria nymphs markedly lightened their body color and shifted the adult F/C ratio toward the value typical of solitarious forms. In addition, knockdown of the gene in solitarious nymphs strongly inhibited darkening even after a transfer to crowded conditions; however, these individuals developed black patterns after being injected with the Crz as a rescue treatment. SgCRZ was constitutively expressed in the brains of S. gregaria during nymphal development in both phases. This gene was highly expressed not only in the brain in both phases, but also in the corpora allata in the gregarious phase. This conspicuous phase-dependent difference in SgCRZ gene expression may indicate a functional role in the control of phase polyphenism in this locust. PMID:26092175

  9. Knockdown of TC-1 enhances radiosensitivity of non-small cell lung cancer via the Wnt/β-catenin pathway

    PubMed Central

    Wu, Dapeng; Li, Lei; Yan, Wei

    2016-01-01

    ABSTRACT Thyroid cancer 1 (TC-1, C8ofr4) is widely expressed in vertebrates and associated with many kinds of tumors. Previous studies indicated that TC-1 functions as a positive regulator in the Wnt/β-catenin signaling pathway in non-small cell lung cancer (NSCLC). However, its exact role and regulation mechanism in radiosensitivity of NSCLC are still unclear. The expression level of TC-1 was measured by qRT-PCR and western blot in NSCLC cell lines. Proliferation and apoptosis of NSCLC cells in response to TC-1 knockdown or/and radiation were determined by MTT assay and flow cytometry, respectively. The activation of the Wnt/β-catenin signaling pathway was further examined by western blot in vitro and in vivo. Compared to TC-1 siRNA or radiotherapy alone, TC-1 silencing combined with radiation inhibited cell proliferation and induced apoptosis in NSCLC cell lines by inactivating of the Wnt/β-catenin signaling pathway. Furthermore, inhibition of the Wnt/β-catenin signaling pathway by XAV939, a Wnt/β-catenin signaling inhibitor, contributed to proliferation inhibition and apoptosis induction in NSCLC A549 cells. Combinative treatment of A549 xenografts with TC-1 siRNA and radiation caused significant tumor regression and inactivation of the Wnt/β-catenin signaling pathway relative to TC-1 siRNA or radiotherapy alone. The results from in vitro and in vivo studies indicated that TC-1 silencing sensitized NSCLC cell lines to radiotherapy through the Wnt/β-catenin signaling pathway. PMID:27029901

  10. Knockdown of ASIC1 and epithelial sodium channel subunits inhibits glioblastoma whole cell current and cell migration.

    PubMed

    Kapoor, Niren; Bartoszewski, Rafal; Qadri, Yawar J; Bebok, Zsuzsanna; Bubien, James K; Fuller, Catherine M; Benos, Dale J

    2009-09-01

    High grade gliomas such as glioblastoma multiforme express multiple members of the epithelial sodium channel (ENaC)/Degenerin family, characteristically displaying a basally active amiloride-sensitive cation current not seen in normal human astrocytes or lower grade gliomas. Using quantitative real time PCR, we have shown higher expression of ASIC1, alphaENaC, and gammaENaC in D54-MG human glioblastoma multiforme cells compared with primary human astrocytes. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, alphaENaC, gammaENaC, and deltaENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, alphaENaC, or gammaENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down alphaENaC or gammaENaC also abolished the high P(K)(+)/P(Na)(+) of D54-MG cells. Knocking down deltaENaC in D54-MG cells reduced deltaENaC protein expression but had no effect on either the whole cell current or K(+) permeability. Using co-immunoprecipitation we show interactions between ASIC1, alphaENaC, and gammaENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. We also found a significant inhibition of D54-MG cell migration after ASIC1, alphaENaC, or gammaENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology. PMID:19561078

  11. Dicer Knockdown Inhibits Endothelial Cell Tumor Growth via MicroRNA 21a-3p Targeting of Nox-4*

    PubMed Central

    Gordillo, Gayle M.; Biswas, Ayan; Khanna, Savita; Pan, Xueliang; Sinha, Mithun; Roy, Sashwati; Sen, Chandan K.

    2014-01-01

    MicroRNAs (miR) are emerging as biomarkers and potential therapeutic targets in tumor management. Endothelial cell tumors are the most common soft tissue tumors in infants, yet little is known about the significance of miR in regulating their growth. A validated mouse endothelial cell (EOMA) tumor model was used to demonstrate that post-transcriptional gene silencing of dicer, the enzyme that converts pre-miR to mature miR, can prevent tumor formation in vivo. Tumors were formed in eight of eight mice injected with EOMA cells transfected with control shRNA but formed in only four of ten mice injected with EOMA cells transfected with dicer shRNA. Tumors that formed in the dicer shRNA group were significantly smaller than tumors in the control group. This response to dicer knockdown was mediated by up-regulated miR 21a-3p activity targeting the nox-4 3′-UTR. EOMA cells were transfected with miR 21a-3p mimic and luciferase reporter plasmids containing either intact nox-4 3′-UTR or with mutation of the proposed 3′-UTR miR21a-3p binding sites. Mean luciferase activity was decreased by 85% in the intact compared with the site mutated vectors (p < 0.01). Attenuated Nox-4 activity resulted in decreased cellular hydrogen peroxide production and decreased production of oxidant-inducible monocyte chemoattractant protein-1, which we have previously shown to be critically required for endothelial cell tumor formation. These findings provide the first evidence establishing the significance of dicer and microRNA in promoting endothelial cell tumor growth in vivo. PMID:24497637

  12. Ectopic expression and knockdown of a zebrafish sox21 reveal its role as a transcriptional repressor in early development.

    PubMed

    Argenton, Francesco; Giudici, Simona; Deflorian, Gianluca; Cimbro, Simona; Cotelli, Franco; Beltrame, Monica

    2004-02-01

    Sox proteins are DNA-binding proteins belonging to the HMG box superfamily and they play key roles in animal embryonic development. Zebrafish Sox21a is part of group B Sox proteins and its chicken and mouse orthologs have been described as transcriptional repressor and activator, respectively, in two different target gene contexts. Zebrafish sox21a is present as a maternal transcript in the oocyte and is mainly expressed at the developing midbrain-hindbrain boundary from the onset of neurulation. In order to understand its role in vivo, we ectopically expressed sox21a by microinjection. Ectopic expression of full length sox21a leads to dorsalization of the embryos. A subset of the dorsalized embryos shows a partial axis splitting, and hence an ectopic neural tube, as an additional phenotype. At gastrulation, injected embryos show expansion of the expression domains of organizer-specific genes, such as chordin and goosecoid. Molecular markers used in somitogenesis highlight that sox21a-injected embryos have shortened AP axis, undulating axial structures, enlarged or even radialized paraxial territory. The developmental abnormalities caused by ectopic expression of sox21a are suggestive of defects in convergence-extension morphogenetic movements. Antisense morpholino oligonucleotides, designed to functionally knockdown sox21a, cause ventralization of the embryos. Moreover, gain-of-function experiments with chimeric constructs, where Sox21a DNA-binding domain is fused to a transcriptional activator (VP16) or repressor (EnR) domain, suggests that zebrafish Sox21a acts as a repressor in dorso-ventral patterning.

  13. Both IGF1R and INSR Knockdown Exert Antitumorigenic Effects in Prostate Cancer In Vitro and In Vivo.

    PubMed

    Ofer, Philipp; Heidegger, Isabel; Eder, Iris E; Schöpf, Bernd; Neuwirt, Hannes; Geley, Stephan; Klocker, Helmut; Massoner, Petra

    2015-12-01

    The IGF network with its main receptors IGF receptor 1 (IGF1R) and insulin receptor (INSR) is of major importance for cancer initiation and progression. To date, clinical studies targeting this network were disappointing and call for thorough analysis of the IGF network in cancer models. We highlight the oncogenic effects controlled by IGF1R and INSR in prostate cancer cells and show similarities as well as differences after receptor knockdown (KD). In PC3 prostate cancer cells stably transduced with inducible short hairpin RNAs, targeting IGF1R or INSR attenuated cell growth and proliferation ultimately driving cells into apoptosis. IGF1R KD triggered rapid and strong antiproliferative and proapoptotic responses, whereas these effects were less pronounced and delayed after INSR KD. Down-regulation of the antiapoptotic proteins myeloid cell leukemia-1 and survivin was observed in both KDs, whereas IGF1R KD also attenuated expression of prosurvival proteins B cell lymphoma-2 and B cell lymphoma-xL. Receptor KD induced cell death involved autophagy in particular upon IGF1R KD; however, no difference in mitochondrial energy metabolism was observed. In a mouse xenograft model, induction of IGF1R or INSR KD after tumor establishment eradicated most of the tumors. After 20 days of receptor KD, tumor cells were found only in 1/14 IGF1R and 3/14 INSR KD tumor remnants. Collectively, our data underline the oncogenic functions of IGF1R and INSR in prostate cancer namely growth, proliferation, and survival in vitro as well as in vivo and identify myeloid cell leukemia-1 and survivin as important mediators of inhibitory and apoptotic effects.

  14. Both IGF1R and INSR Knockdown Exert Antitumorigenic Effects in Prostate Cancer In Vitro and In Vivo

    PubMed Central

    Ofer, Philipp; Heidegger, Isabel; Eder, Iris E.; Schöpf, Bernd; Neuwirt, Hannes; Geley, Stephan; Massoner, Petra

    2015-01-01

    The IGF network with its main receptors IGF receptor 1 (IGF1R) and insulin receptor (INSR) is of major importance for cancer initiation and progression. To date, clinical studies targeting this network were disappointing and call for thorough analysis of the IGF network in cancer models. We highlight the oncogenic effects controlled by IGF1R and INSR in prostate cancer cells and show similarities as well as differences after receptor knockdown (KD). In PC3 prostate cancer cells stably transduced with inducible short hairpin RNAs, targeting IGF1R or INSR attenuated cell growth and proliferation ultimately driving cells into apoptosis. IGF1R KD triggered rapid and strong antiproliferative and proapoptotic responses, whereas these effects were less pronounced and delayed after INSR KD. Down-regulation of the antiapoptotic proteins myeloid cell leukemia-1 and survivin was observed in both KDs, whereas IGF1R KD also attenuated expression of prosurvival proteins B cell lymphoma-2 and B cell lymphoma-xL. Receptor KD induced cell death involved autophagy in particular upon IGF1R KD; however, no difference in mitochondrial energy metabolism was observed. In a mouse xenograft model, induction of IGF1R or INSR KD after tumor establishment eradicated most of the tumors. After 20 days of receptor KD, tumor cells were found only in 1/14 IGF1R and 3/14 INSR KD tumor remnants. Collectively, our data underline the oncogenic functions of IGF1R and INSR in prostate cancer namely growth, proliferation, and survival in vitro as well as in vivo and identify myeloid cell leukemia-1 and survivin as important mediators of inhibitory and apoptotic effects. PMID:26452103

  15. Knockdown of Expression of Cdk5 or p35 (a Cdk5 Activator) Results in Podocyte Apoptosis

    PubMed Central

    Zheng, Ya-Li; Zhang, Xia; Fu, Hai-Xia; Guo, Mei; Shukla, Varsha; Amin, Niranjana D.; E, Jing; Bao, Li; Luo, Hong-Yan; Li, Bo; Lu, Xiao-Hua; Gao, Yong-Cai

    2016-01-01

    Podocytes are terminally differentiated glomerular epithelial cells. Podocyte loss has been found in many renal diseases. Cdk5 is a cyclin-dependent protein kinase which is predominantly regulated by p35. To study the role of Cdk5/p35 in podocyte survival, we first applied western blotting (WB) analysis to confirm the time-course expression of Cdk5 and p35 during kidney development and in cultured immortalized mouse podocytes. We also demonstrated that p35 plays an important role in promoting podocyte differentiation by overexpression of p35 in podocytes. To deregulate the expression of Cdk5 or p35 in mouse podocytes, we used RNAi and analyzed cell function and apoptosis assaying for podocyte specific marker Wilms Tumor 1 (WT1) and cleaved caspase 3, respectively. We also counted viable cells using cell counting kit-8. We found that depletion of Cdk5 causes decreased expression of WT1 and apoptosis. It is noteworthy, however, that downregulation of p35 reduced Cdk5 activity, but had no effect on cleaved caspase 3 expression. It did, however, reduce expression of WT1, a transcription factor, and produced podocyte dysmorphism. On the other hand increased apoptosis could be detected in p35-deregulated podocytes using the TUNEL analysis and immunofluorescent staining with cleaved caspase3 antibody. Viability of podocytes was decreased in both Cdk5 and p35 knockdown cells. Knocking down Cdk5 or p35 gene by RNAi does not affect the cycline I expression, another Cdk5 activator in podocyes. We conclude that Cdk5 and p35 play a crucial role in maintaining podocyte differentiation and survival, and suggest these proteins as targets for therapeutic intervention in podocyte-damaged kidney diseases. PMID:27479491

  16. The neuropeptide Y Y1 receptor knockdown modulates activator protein 1-involved feeding behavior in amphetamine-treated rats

    PubMed Central

    2013-01-01

    Background Hypothalamic neuropeptide Y (NPY) and two immediate early genes, c-fos and c-jun, have been found to be involved in regulating the appetite-suppressing effect of amphetamine (AMPH). The present study investigated whether cerebral catecholamine (CA) might regulate NPY and POMC expression and whether NPY Y1 receptor (Y1R) participated in activator protein-1 (AP-1)–mediated feeding. Methods Rats were given AMPH daily for 4 days. Changes in the expression of NPY, Y1R, c-Fos, c-Jun, and AP-1 were assessed and compared. Results Decreased CA could modulate NPY and melanocortin receptor 4 (MC4R) expressions. NPY and food intake decreased the most on Day 2, but Y1R, c-Fos, and c-Jun increased by approximately 350%, 280%, and 300%, respectively, on Day 2. Similarly, AP-1/DNA binding activity was increased by about 180% on Day 2. The expression patterns in Y1R, c-Fos, c-Jun, and AP-1/DNA binding were opposite to those in NPY during AMPH treatment. Y1R knockdown was found to modulate the opposite regulation between NPY and AP-1, revealing an involvement of Y1R in regulating NPY/AP-1–mediated feeding. Conclusions These results point to a molecular mechanism of CA/NPY/Y1R/AP-1 signaling in the control of AMPH-mediated anorexia and may advance the medical research of anorectic and anti-obesity drugs. PMID:24225225

  17. CHIP buffers heterogeneous Bcl-2 expression levels to prevent augmentation of anticancer drug-resistant cell population.

    PubMed

    Tsuchiya, M; Nakajima, Y; Waku, T; Hiyoshi, H; Morishita, T; Furumai, R; Hayashi, Y; Kishimoto, H; Kimura, K; Yanagisawa, J

    2015-08-27

    Many types of cancer display heterogeneity in various features, including gene expression and malignant potential. This heterogeneity is associated with drug resistance and cancer progression. Recent studies have shown that the expression of a major protein quality control ubiquitin ligase, carboxyl terminus of Hsc70-interacting protein (CHIP), is negatively correlated with breast cancer clinicopathological stages and poor overall survival. Here we show that CHIP acts as a capacitor of heterogeneous Bcl-2 expression levels and prevents an increase in the anticancer drug-resistant population in breast cancer cells. CHIP knockdown in breast cancer cells increased variation in Bcl-2 expression levels, an antiapoptotic protein, among the cells. Our results also showed that CHIP knockdown increased the proportion of anticancer drug-resistant cells. These findings suggest that CHIP buffers variation in gene expression levels, affecting resistance to anticancer drugs. In single-cell clones derived from breast cancer cell lines, CHIP knockdown did not alter the variation in Bcl-2 expression levels and the proportion of anticancer drug-resistant cells. In contrast, when clonal cells were treated with a mutagen, the variation in Bcl-2 expression levels and proportion of anticancer drug-resistant cells were altered by CHIP knockdown. These results suggest that CHIP masks genetic variations to suppress heterogeneous Bcl-2 expression levels and prevents augmentation of the anticancer drug-resistant population of breast cancer cells. Because genetic variation is a major driver of heterogeneity, our results suggest that the degree of heterogeneity in expression levels is decided by a balance between genetic variation and the buffering capacity of CHIP.

  18. Suppression of a phospholipase D gene, OsPLDbeta1, activates defense responses and increases disease resistance in rice.

    PubMed

    Yamaguchi, Takeshi; Kuroda, Masaharu; Yamakawa, Hiromoto; Ashizawa, Taketo; Hirayae, Kazuyuki; Kurimoto, Leona; Shinya, Tomonori; Shibuya, Naoto

    2009-05-01

    Phospholipase D (PLD) plays an important role in plants, including responses to abiotic as well as biotic stresses. A survey of the rice (Oryza sativa) genome database indicated the presence of 17 PLD genes in the genome, among which OsPLDalpha1, OsPLDalpha5, and OsPLDbeta1 were highly expressed in most tissues studied. To examine the physiological function of PLD in rice, we made knockdown plants for each PLD isoform by introducing gene-specific RNA interference constructs. One of them, OsPLDbeta1-knockdown plants, showed the accumulation of reactive oxygen species in the absence of pathogen infection. Reverse transcription-polymerase chain reaction and DNA microarray analyses revealed that the knockdown of OsPLDbeta1 resulted in the up-/down-regulation of more than 1,400 genes, including the induction of defense-related genes such as pathogenesis-related protein genes and WRKY/ERF family transcription factor genes. Hypersensitive response-like cell death and phytoalexin production were also observed at a later phase of growth in the OsPLDbeta1-knockdown plants. These results indicated that the OsPLDbeta1-knockdown plants spontaneously activated the defense responses in the absence of pathogen infection. Furthermore, the OsPLDbeta1-knockdown plants exhibited increased resistance to the infection of major pathogens of rice, Pyricularia grisea and Xanthomonas oryzae pv oryzae. These results suggested that OsPLDbeta1 functions as a negative regulator of defense responses and disease resistance in rice. PMID:19286937

  19. Resisting HRD's Resistance to Diversity

    ERIC Educational Resources Information Center

    Bierema, Laura L.

    2010-01-01

    Purpose: The purpose of this paper is to empirically illustrate how human resource development (HRD) resists and omits issues of diversity in academic programs, textbooks, and research; analyze the research on HRD and diversity over a ten-year period; discuss HRD's resistance to diversity; and offer some recommendations for a more authentic…

  20. Knockdown of the FoxM1 enhances the sensitivity of gastric cancer cells to cisplatin by targeting Mcl-1.

    PubMed

    Li, Xiaomei; Liang, Jun; Liu, Ying-Xun; Wang, Yuming; Yang, Xiao-Hui; Bao-Hongluan; Zhang, Gui-Lling; Du, Juan; Wu, Xia-Hong

    2016-06-01

    Resistance to chemotherapy is a main obstacle for effective treatment of gastric cancer, the mechanism of which is still poorly understood. Forkhead box M1 (FoxM1) plays an important role in chemo-resistance of various tumors. This study aimed to explore whether FoxM1 mediated resistance of the gastric cancer cell line SGC7901 to the chemotherapy agent cisplatin (DDP). In the study, we detected FoxM1 and Mcl-1 expression via real time-PCR and western blot and demonstrated that FoxM1 is overexpressed in cisplatin-resistance GC cells and Mcl-1 expression is regulated by FoxM1. We examined SGC7901/DDP cell viability by MTT assay, which revealed that suppression of the FoxM1/Mcl-1 pathway impaired cell viability and thus increased sensitivity to cisplatin in gastric cancer cells. Taken together, the study implied that the FoxM1/Mcl-1 pathway may overcome cispaltin resistance of gastric cancer and provide a new therapeutic target for the treatment of gastric cancer. PMID:27455555

  1. RNAi validation of resistance genes and their interactions in the highly DDT-resistant 91-R strain of Drosophila melanogaster.

    PubMed

    Gellatly, Kyle J; Yoon, Kyong Sup; Doherty, Jeffery J; Sun, Weilin; Pittendrigh, Barry R; Clark, J Marshall

    2015-06-01

    4,4'-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will increase in terms of level and extent. Drosophila melanogaster is a model dipteran that has many available genetic tools, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold), however, there is no mechanistic scheme that accounts for this level of resistance. Recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms in the 91-R strain. Their interactions, however, remain unclear. Use of UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has validated the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (Mdr50, Mdr65, and Mrp1) involved in DDT resistance. Further, increased sensitivity to DDT in the 91-R strain after intra-abdominal dsRNA injection for Mdr50, Mdr65, and Mrp1 was determined by a DDT contact bioassay, directly implicating these genes in DDT efflux and resistance. PMID:26047118

  2. RNAi validation of resistance genes and their interactions in the highly DDT-resistant 91-R strain of Drosophila melanogaster.

    PubMed

    Gellatly, Kyle J; Yoon, Kyong Sup; Doherty, Jeffery J; Sun, Weilin; Pittendrigh, Barry R; Clark, J Marshall

    2015-06-01

    4,4'-dichlorodiphenyltrichloroethane (DDT) has been re-recommended by the World Health Organization for malaria mosquito control. Previous DDT use has resulted in resistance, and with continued use resistance will increase in terms of level and extent. Drosophila melanogaster is a model dipteran that has many available genetic tools, numerous studies done on insecticide resistance mechanisms, and is related to malaria mosquitoes allowing for extrapolation. The 91-R strain of D. melanogaster is highly resistant to DDT (>1500-fold), however, there is no mechanistic scheme that accounts for this level of resistance. Recently, reduced penetration, increased detoxification, and direct excretion have been identified as resistance mechanisms in the 91-R strain. Their interactions, however, remain unclear. Use of UAS-RNAi transgenic lines of D. melanogaster allowed for the targeted knockdown of genes putatively involved in DDT resistance and has validated the role of several cuticular proteins (Cyp4g1 and Lcp1), cytochrome P450 monooxygenases (Cyp6g1 and Cyp12d1), and ATP binding cassette transporters (Mdr50, Mdr65, and Mrp1) involved in DDT resistance. Further, increased sensitivity to DDT in the 91-R strain after intra-abdominal dsRNA injection for Mdr50, Mdr65, and Mrp1 was determined by a DDT contact bioassay, directly implicating these genes in DDT efflux and resistance.

  3. Resistivity analysis

    DOEpatents

    Bruce, Michael R.; Bruce, Victoria J.; Ring, Rosalinda M.; Cole, Edward Jr. I.; Hawkins, Charles F.; Tangyungong, Paiboon

    2006-06-13

    According to an example embodiment of the present invention a semiconductor die having a resistive electrical connection is analyzed. Heat is directed to the die as the die is undergoing a state-changing operation to cause a failure due to suspect circuitry. The die is monitored, and a circuit path that electrically changes in response to the heat is detected and used to detect that a particular portion therein of the circuit is resistive. In this manner, the detection and localization of a semiconductor die defect that includes a resistive portion of a circuit path is enhanced.

  4. Knockdown of MAP4 and DNAL1 produces a post-fusion and pre-nuclear translocation impairment in HIV-1 replication

    SciTech Connect

    Gallo, Daniel E. Hope, Thomas J.

    2012-01-05

    DNAL1 and MAP4 are both microtubule-associated proteins. These proteins were identified as HIV-1 dependency factors in a screen with wild-type HIV-1. In this study we demonstrate that knockdown using DNAL1 and MAP4 siRNAs and shRNAs inhibits HIV-1 infection regardless of envelope. Using a fusion assay, we show that DNAL1 and MAP4 do not impact fusion. By assaying for late reverse transcripts and 2-LTR circles, we show that DNAL1 and MAP4 inhibit both by approximately 50%. These results demonstrate that DNAL1 and MAP4 impact reverse transcription but not nuclear translocation. DNAL1 and MAP4 knockdown cells do not display cytoskeletal defects. Together these experiments indicate that DNAL1 and MAP4 may exert their functions in the HIV life cycle at reverse transcription, prior to nuclear translocation.

  5. Knockdown of MAP4 and DNAL1 produces a post-fusion and pre-nuclear translocation impairment in HIV-1 replication.

    PubMed

    Gallo, Daniel E; Hope, Thomas J

    2012-01-01

    DNAL1 and MAP4 are both microtubule-associated proteins. These proteins were identified as HIV-1 dependency factors in a screen with wild-type HIV-1. In this study we demonstrate that knockdown using DNAL1 and MAP4 siRNAs and shRNAs inhibits HIV-1 infection regardless of envelope. Using a fusion assay, we show that DNAL1 and MAP4 do not impact fusion. By assaying for late reverse transcripts and 2-LTR circles, we show that DNAL1 and MAP4 inhibit both by approximately 50%. These results demonstrate that DNAL1 and MAP4 impact reverse transcription but not nuclear translocation. DNAL1 and MAP4 knockdown cells do not display cytoskeletal defects. Together these experiments indicate that DNAL1 and MAP4 may exert their functions in the HIV life cycle at reverse transcription, prior to nuclear translocation.

  6. siRNA-mediated knockdown against CDCA1 and KNTC2, both frequently overexpressed in colorectal and gastric cancers, suppresses cell proliferation and induces apoptosis

    SciTech Connect

    Kaneko, Naoyuki; Miura, Koh; Gu, Zhaodi; Karasawa, Hideaki; Ohnuma, Shinobu; Sasaki, Hiroyuki; Tsukamoto, Nobukazu; Yokoyama, Satoru; Yamamura, Akihiro; Nagase, Hiroki; Shibata, Chikashi; Sasaki, Iwao; Horii, Akira

    2009-12-25

    Ndc80 has been shown to play an important role in stable microtubule-kinetochore attachment, chromosome alignment, and spindle checkpoint activation in mitosis. It is composed of two heterodimers, CDCA1-KNTC2 and SPC24-SPC25. Overexpression of CDCA1 and KNTC2 is reported to be associated with poor prognosis in non-small cell lung cancers (NSCLC), and siRNA-mediated knockdown against CDCA1 or KNTC2 has been found to inhibit cell proliferation and induction of apoptosis in NSCLC, ovarian cancer, cervical cancer and glioma. Therefore, CDCA1 and KNTC2 can be considered good candidates for molecular target therapy as well as diagnosis in some cancers. However, the role of the Ndc80 complex in colorectal and gastric cancers (CRC and GC) still remains unclear. In the present study, we used qRT-PCR to evaluate the expression levels of CDCA1, KNTC2, SPC24 and SPC25 in CRC and GC and employed siRNA-mediated knockdown to examine cell proliferation and apoptosis. mRNA overexpression of these four genes was observed in CRCs and GCs when compared with the corresponding normal mucosae. Additionally, the expression levels of tumor/normal ratios of CDCA1, KNTC2, SPC24 and SPC25 correlated with each other in CRCs. MTT assays revealed that cell growths after the siRNA-mediated knockdown of either CDCA1 or KNTC2 were significantly suppressed, and flow cytometry analyses revealed significant increases of the subG1 fractions after knockdown against both genes. Our present results suggest that expressional control of component molecules of Ndc80 can be utilized for molecular target therapy of patients with CRC and GC.

  7. Antimicrobial Resistance

    MedlinePlus

    ... and health professionals can play their part; rewarding innovation and development of new treatment options and other ... and industry can help tackle resistance by: fostering innovation and research and development of new vaccines, diagnostics, ...

  8. Antimicrobial Resistance

    MedlinePlus

    ... antibiotic are known as methicillin-resistant S. aureus or MRSA. Antibiotics and other antimicrobial drugs first became widely ... factors for infection are known as community-associated MRSA (CA-MRSA). Recently, several cases overseas and in ...

  9. Knockdown of the Drosophila FIG4 induces deficient locomotive behavior, shortening of motor neuron, axonal targeting aberration, reduction of life span and defects in eye development.

    PubMed

    Kyotani, Akane; Azuma, Yumiko; Yamamoto, Itaru; Yoshida, Hideki; Mizuta, Ikuko; Mizuno, Toshiki; Nakagawa, Masanori; Tokuda, Takahiko; Yamaguchi, Masamitsu

    2016-03-01

    Mutations in Factor-Induced-Gene 4 (FIG4) gene have been identified in Charcot-Marie-Tooth disease type 4J (CMT4J), Yunis-Varon syndrome and epilepsy with polymicrogyria. FIG4 protein regulates a cellular abundance of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a signaling lipid on the cytosolic surface of membranes of the late endosomal compartment. PI(3,5)P2 is required for retrograde membrane trafficking from lysosomal and late endosomal compartments to the Golgi. However, it is still unknown how the neurodegeneration that occurs in these diseases is related to the loss of FIG4 function. Drosophila has CG17840 (dFIG4) as a human FIG4 homolog. Here we specifically knocked down dFIG4 in various tissues, and investigated their phenotypes. Neuron-specific knockdown of dFIG4 resulted in axonal targeting aberrations of photoreceptor neurons, shortened presynaptic terminals of motor neurons in 3rd instar larvae and reduced climbing ability in adulthood and life span. Fat body-specific knockdown of dFIG4 resulted in enlarged lysosomes in cells that were detected by staining with LysoTracker. In addition, eye imaginal disk-specific knockdown of dFIG4 disrupted differentiation of pupal ommatidial cell types, such as cone cells and pigment cells, suggesting an additional role of dFIG4 during eye development.

  10. Knockdown of AMPKα2 Promotes Pulmonary Arterial Smooth Muscle Cells Proliferation via mTOR/Skp2/p27Kip1 Signaling Pathway

    PubMed Central

    Ke, Rui; Liu, Lu; Zhu, Yanting; Li, Shaojun; Xie, Xinming; Li, Fangwei; Song, Yang; Yang, Lan; Gao, Li; Li, Manxiang

    2016-01-01

    It has been shown that activation of adenosine monophosphate-activated protein kinase (AMPK) suppresses proliferation of a variety of tumor cells as well as nonmalignant cells. In this study, we used post-transcriptional gene silencing with small interfering RNA (siRNA) to specifically examine the effect of AMPK on pulmonary arterial smooth muscle cells (PASMCs) proliferation and to further elucidate its underlying molecular mechanisms. Our results showed that knockdown of AMPKα2 promoted primary cultured PASMCs proliferation; this was accompanied with the elevation of phosphorylation of mammalian target of rapamycin (mTOR) and S-phase kinase-associated protein 2 (Skp2) protein level and reduction of p27Kip1. Importantly, prior silencing of mTOR with siRNA abolished AMPKα2 knockdown-induced Skp2 upregulation, p27Kip1 reduction as well as PASMCs proliferation. Furthermore, pre-depletion of Skp2 by siRNA also eliminated p27Kip1 downregulation and PASMCs proliferation caused by AMPKα2 knockdown. Taken together, our study indicates that AMPKα2 isoform plays an important role in regulation of PASMCs proliferation by modulating mTOR/Skp2/p27Kip1 axis, and suggests that activation of AMPKα2 might have potential value in the prevention and treatment of pulmonary arterial hypertension. PMID:27258250

  11. Knockdown of long non-coding RNA HOTAIR inhibits malignant biological behaviors of human glioma cells via modulation of miR-326.

    PubMed

    Ke, Jing; Yao, Yi-long; Zheng, Jian; Wang, Ping; Liu, Yun-hui; Ma, Jun; Li, Zhen; Liu, Xiao-bai; Li, Zhi-qing; Wang, Zhen-hua; Xue, Yi-xue

    2015-09-01

    Glioma is the most common and aggressive primary adult brain tumor. Long non-coding RNAs (lncRNAs) have important roles in a variety of biological properties of cancers. Here, we elucidated the function and the possible molecular mechanisms of lncRNA HOTAIR in human glioma U87 and U251 cell lines. Quantitative RT-PCR demonstrated that HOTAIR expression was up-regulated in glioma tissues and cell lines. Knockdown of HOTAIR exerted tumor-suppressive function in glioma cells. Further, HOTAIR was confirmed to be the target of miR-326 and miR-326 mediated the tumor-suppressive effects of HOTAIR knockdown on glioma cell lines. Moreover, over-expressed miR-326 reduced the FGF1 expression which played an oncogenic role in glioma by activating PI3K/AKT and MEK 1/2 pathways. In addition, the in vivo studies also supported the above findings. Taken together, knockdown of HOTAIR up-regulated miR-326 expression, and further inducing the decreased expression of FGF1, these results provided a comprehensive analysis of HOTAIR-miR-326-FGF1 axis in human glioma and provided a new potential therapeutic strategy for glioma treatment.

  12. Knockdown of the Drosophila FIG4 induces deficient locomotive behavior, shortening of motor neuron, axonal targeting aberration, reduction of life span and defects in eye development.

    PubMed

    Kyotani, Akane; Azuma, Yumiko; Yamamoto, Itaru; Yoshida, Hideki; Mizuta, Ikuko; Mizuno, Toshiki; Nakagawa, Masanori; Tokuda, Takahiko; Yamaguchi, Masamitsu

    2016-03-01

    Mutations in Factor-Induced-Gene 4 (FIG4) gene have been identified in Charcot-Marie-Tooth disease type 4J (CMT4J), Yunis-Varon syndrome and epilepsy with polymicrogyria. FIG4 protein regulates a cellular abundance of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a signaling lipid on the cytosolic surface of membranes of the late endosomal compartment. PI(3,5)P2 is required for retrograde membrane trafficking from lysosomal and late endosomal compartments to the Golgi. However, it is still unknown how the neurodegeneration that occurs in these diseases is related to the loss of FIG4 function. Drosophila has CG17840 (dFIG4) as a human FIG4 homolog. Here we specifically knocked down dFIG4 in various tissues, and investigated their phenotypes. Neuron-specific knockdown of dFIG4 resulted in axonal targeting aberrations of photoreceptor neurons, shortened presynaptic terminals of motor neurons in 3rd instar larvae and reduced climbing ability in adulthood and life span. Fat body-specific knockdown of dFIG4 resulted in enlarged lysosomes in cells that were detected by staining with LysoTracker. In addition, eye imaginal disk-specific knockdown of dFIG4 disrupted differentiation of pupal ommatidial cell types, such as cone cells and pigment cells, suggesting an additional role of dFIG4 during eye development. PMID:26708557

  13. Simultaneous siRNA-mediated knockdown of antiapoptotic BCL2, Bcl-xL, XIAP and survivin in bladder cancer cells

    PubMed Central

    KUNZE, DOREEN; KRAEMER, KAI; ERDMANN, KATI; FROEHNER, MICHAEL; WIRTH, MANFRED P.; FUESSEL, SUSANNE

    2012-01-01

    Bladder cancer (BCa) represents the ninth most common malignancy worldwide. Despite intensive treatment with surgery and chemotherapy the prognosis for BCa patients particularly at advanced stages is poor. The ability to evade apoptosis is a hallmark of cancer cells. Since the antiapoptotic genes BCL2, Bcl-xL, XIAP and survivin are frequently upregulated in BCa tissues, their combined siRNA-mediated knockdown might be more potent in decreasing BCa growth than the single inhibition of one target. Against each target two siRNAs were selected that specifically reduced the mRNA and protein levels of their appropriate target in EJ28 and J82 BCa cells. Inhibition of survivin provoked the strongest antiproliferative effect of all single target treatments, for example cell counts decreased by 50%. Simultaneous targeting of all four antiapoptotic genes downregulated expression levels of all targets and mediated significant reductions in cell viability and cell counts as well as induction of apoptosis. In EJ28 cells, combined knockdown of BCL2, Bcl-xL, XIAP and survivin caused a 2.5-fold enhancement in apoptosis rate and reduced cellular viability by 40%. Therefore, simultaneous knockdown of antiapoptotic BCL2, Bcl-xL, XIAP and survivin may represent a promising treatment option for bladder cancer. PMID:22797576

  14. Knockdown of Selenocysteine-Specific Elongation Factor in Amblyomma maculatum Alters the Pathogen Burden of Rickettsia parkeri with Epigenetic Control by the Sin3 Histone Deacetylase Corepressor Complex

    PubMed Central

    Adamson, Steven W.; Browning, Rebecca E.; Budachetri, Khemraj; Ribeiro, José M. C.; Karim, Shahid

    2013-01-01

    Selenocysteine is the 21st naturally-occurring amino acid. Selenoproteins have diverse functions and many remain uncharacterized, but they are typically associated with antioxidant activity. The incorporation of selenocysteine into the nascent polypeptide chain recodes the TGA stop codon and this process depends upon a number of essential factors including the selenocysteine elongation factor (SEF). The transcriptional expression of SEF did not change significantly in tick midguts throughout the blood meal, but decreased in salivary glands to 20% at the end of the fast feeding phase. Since selenoprotein translation requires this specialized elongation factor, we targeted this gene for knockdown by RNAi to gain a global view of the role selenoproteins play in tick physiology. We found no significant differences in tick engorgement and embryogenesis but detected no antioxidant capacity in tick saliva. The transcriptional profile of selenoproteins in R. parkeri-infected Amblyomma maculatum revealed declined activity of selenoprotein M and catalase and increased activity of selenoprotein O, selenoprotein S, and selenoprotein T. Furthermore, the pathogen burden was significantly altered in SEF-knockdowns. We then determined the global impact of SEF-knockdown by RNA-seq, and mapped huge shifts in secretory gene expression that could be the result of downregulation of the Sin3 histone deacetylase corepressor complex. PMID:24282621

  15. Dmp53, basket and drICE gene knockdown and polyphenol gallic acid increase life span and locomotor activity in a Drosophila Parkinson’s disease model

    PubMed Central

    Ortega-Arellano, Hector Flavio; Jimenez-Del-Rio, Marlene; Velez-Pardo, Carlos

    2013-01-01

    Understanding the mechanism(s) by which dopaminergic (DAergic) neurons are eroded in Parkinson’s disease (PD) is critical for effective therapeutic strategies. By using the binary tyrosine hydroxylase (TH)-Gal4/UAS-X RNAi Drosophila melanogaster system, we report that Dmp53, basket and drICE gene knockdown in dopaminergic neurons prolong life span (p < 0.05; log-rank test) and locomotor activity (p < 0.05; χ2 test) in D. melanogaster lines chronically exposed to (1 mM) paraquat (PQ, oxidative stress (OS) generator) compared to untreated transgenic fly lines. Likewise, knockdown flies displayed higher climbing performance than control flies. Amazingly, gallic acid (GA) significantly protected DAergic neurons, ameliorated life span, and climbing abilities in knockdown fly lines treated with PQ compared to flies treated with PQ only. Therefore, silencing specific gene(s) involved in neuronal death might constitute an excellent tool to study the response of DAergic neurons to OS stimuli. We propose that a therapy with antioxidants and selectively “switching off” death genes in DAergic neurons could provide a means for pre-clinical PD individuals to significantly ameliorate their disease condition. PMID:24385865

  16. A Modular Lentiviral and Retroviral Construction System to Rapidly Generate Vectors for Gene Expression and Gene Knockdown In Vitro and In Vivo

    PubMed Central

    Geiling, Benjamin; Vandal, Guillaume; Posner, Ada R.; de Bruyns, Angeline; Dutchak, Kendall L.; Garnett, Samantha; Dankort, David

    2013-01-01

    The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems. PMID:24146852

  17. Adeno-associated virus-mediated knockdown of melanocortin-4 receptor in the paraventricular nucleus of the hypothalamus promotes high-fat diet-induced hyperphagia and obesity

    PubMed Central

    Garza, Jacob C; Kim, Chung Sub; Liu, Jing; Zhang, Wei; Lu, Xin-Yun

    2013-01-01

    Pharmacological and genetic studies have suggested that melanocortin-4 receptor (MC4R) signaling in the paraventricular nucleus of hypothalamus (PVN) regulates appetite and energy balance. However, the specific role of MC4R signaling in PVN neurons in these processes remains to be further elucidated in normally developed animals. In the present study, we employed RNA interference to determine whether MC4R knockdown in the PVN modulates food intake and body weight in adult rats. Adeno-associated viral (AAV) vectors encoding short hairpin RNAs targeting MC4R (AAV-shRNA-MC4R) were generated to induce MC4R knockdown in the PVN. By in situ hybridization, we detected a high-level expression of Dicer, a key enzyme required for shRNA-mediated gene silencing, along the entire rostrocaudal extent of the PVN. Bilateral injection of AAV-shRNA-MC4R vectors into the PVN of the adult rat resulted in significant and specific reduction of MC4R mRNA expression. Animals with MC4R knockdown exhibited an increase in food intake and excessive body weight gain when exposed to a high-fat diet. Our results provide evidence that AAV-mediated silencing of MC4R on PVN neurons promotes hyperphagia and obesity in response to the dietary challenge in the adult animal. PMID:18492813

  18. Knockdown of long non-coding RNA HOTAIR inhibits malignant biological behaviors of human glioma cells via modulation of miR-326

    PubMed Central

    Ke, Jing; Yao, Yi-long; Zheng, Jian; Wang, Ping; Liu, Yun-hui; Ma, Jun; Li, Zhen; Liu, Xiao-bai; Li, Zhi-qing; Wang, Zhen-hua; Xue, Yi-xue

    2015-01-01

    Glioma is the most common and aggressive primary adult brain tumor. Long non-coding RNAs (lncRNAs) have important roles in a variety of biological properties of cancers. Here, we elucidated the function and the possible molecular mechanisms of lncRNA HOTAIR in human glioma U87 and U251 cell lines. Quantitative RT-PCR demonstrated that HOTAIR expression was up-regulated in glioma tissues and cell lines. Knockdown of HOTAIR exerted tumor-suppressive function in glioma cells. Further, HOTAIR was confirmed to be the target of miR-326 and miR-326 mediated the tumor-suppressive effects of HOTAIR knockdown on glioma cell lines. Moreover, over-expressed miR-326 reduced the FGF1 expression which played an oncogenic role in glioma by activating PI3K/AKT and MEK 1/2 pathways. In addition, the in vivo studies also supported the above findings. Taken together, knockdown of HOTAIR up-regulated miR-326 expression, and further inducing the decreased expression of FGF1, these results provided a comprehensive analysis of HOTAIR-miR-326-FGF1 axis in human glioma and provided a new potential therapeutic strategy for glioma treatment. PMID:26183397

  19. Developmental acclimation to low or high humidity conditions affect starvation and heat resistance of Drosophila melanogaster.

    PubMed

    Parkash, Ravi; Ranga, Poonam; Aggarwal, Dau Dayal

    2014-09-01

    Several Drosophila species originating from tropical humid localities are more resistant to starvation and heat stress than populations from high latitudes but mechanistic bases of such physiological changes are largely unknown. In order to test whether humidity levels affect starvation and heat resistance, we investigated developmental acclimation effects of low to high humidity conditions on the storage and utilization of energy resources, body mass, starvation survival, heat knockdown and heat survival of D. melanogaster. Isofemale lines reared under higher humidity (85% RH) stored significantly higher level of lipids and showed greater starvation survival hours but smaller in body size. In contrast, lines reared at low humidity evidenced reduced levels of body lipids and starvation resistance. Starvation resistance and lipid storage level were higher in females than males. However, the rate of utilization of lipids under starvation stress was lower for lines reared under higher humidity. Adult flies of lines reared at 65% RH and acclimated under high or low humidity condition for 200 hours also showed changes in resistance to starvation and heat but such effects were significantly lower as compared with developmental acclimation. Isofemale lines reared under higher humidity showed greater heat knockdown time and heat-shock survival. These laboratory observations on developmental and adult acclimation effects of low versus high humidity conditions have helped in explaining seasonal changes in resistance to starvation and heat of the wild-caught flies of D. melanogaster. Thus, we may suggest that wet versus drier conditions significantly affect starvation and heat resistance of D. melanogaster.

  20. Lantibiotic Resistance

    PubMed Central

    Draper, Lorraine A.; Ross, R. Paul

    2015-01-01

    SUMMARY The dramatic rise in the incidence of antibiotic resistance demands that new therapeutic options will have to be developed. One potentially interesting class of antimicrobials are the modified bacteriocins termed lantibiotics, which are bacterially produced, posttranslationally modified, lanthionine/methyllanthionine-containing peptides. It is interesting that low levels of resistance have been reported for lantibiotics compared with commercial antibiotics. Given that there are very few examples of naturally occurring lantibiotic resistance, attempts have been made to deliberately induce resistance phenotypes in order to investigate this phenomenon. Mechanisms that hinder the action of lantibiotics are often innate systems that react to the presence of any cationic peptides/proteins or ones which result from cell well damage, rather than being lantibiotic specific. Such resistance mechanisms often arise due to altered gene regulation following detection of antimicrobials/cell wall damage by sensory proteins at the membrane. This facilitates alterations to the cell wall or changes in the composition of the membrane. Other general forms of resistance include the formation of spores or biofilms, which are a common mechanistic response to many classes of antimicrobials. In rare cases, bacteria have been shown to possess specific antilantibiotic mechanisms. These are often species specific and include the nisin lytic protein nisinase and the phenomenon of immune mimicry. PMID:25787977

  1. Lantibiotic resistance.

    PubMed

    Draper, Lorraine A; Cotter, Paul D; Hill, Colin; Ross, R Paul

    2015-06-01

    The dramatic rise in the incidence of antibiotic resistance demands that new therapeutic options will have to be developed. One potentially interesting class of antimicrobials are the modified bacteriocins termed lantibiotics, which are bacterially produced, posttranslationally modified, lanthionine/methyllanthionine-containing peptides. It is interesting that low levels of resistance have been reported for lantibiotics compared with commercial antibiotics. Given that there are very few examples of naturally occurring lantibiotic resistance, attempts have been made to deliberately induce resistance phenotypes in order to investigate this phenomenon. Mechanisms that hinder the action of lantibiotics are often innate systems that react to the presence of any cationic peptides/proteins or ones which result from cell well damage, rather than being lantibiotic specific. Such resistance mechanisms often arise due to altered gene regulation following detection of antimicrobials/cell wall damage by sensory proteins at the membrane. This facilitates alterations to the cell wall or changes in the composition of the membrane. Other general forms of resistance include the formation of spores or biofilms, which are a common mechanistic response to many classes of antimicrobials. In rare cases, bacteria have been shown to possess specific antilantibiotic mechanisms. These are often species specific and include the nisin lytic protein nisinase and the phenomenon of immune mimicry. PMID:25787977

  2. Inhibition of autophagy overcomes glucocorticoid resistance in lymphoid malignant cells.

    PubMed

    Jiang, Lei; Xu, Lingzhi; Xie, Jiajun; Li, Sisi; Guan, Yanchun; Zhang, Yan; Hou, Zhijie; Guo, Tao; Shu, Xin; Wang, Chang; Fan, Wenjun; Si, Yang; Yang, Ya; Kang, Zhijie; Fang, Meiyun; Liu, Quentin

    2015-01-01

    Glucocorticoid (GC) resistance remains a major obstacle to successful treatment of lymphoid malignancies. Till now, the precise mechanism of GC resistance remains unclear. In the present study, dexamethasone (Dex) inhibited cell proliferation, arrested cell cycle in G0/G1-phase, and induced apoptosis in Dex-sensitive acute lymphoblastic leukemia cells. However, Dex failed to cause cell death in Dex-resistant lymphoid malignant cells. Intriguingly, we found that autophagy was induced by Dex in resistant cells, as indicated by autophagosomes formation, LC3-I to LC3-II conversion, p62 degradation, and formation of acidic autophagic vacuoles. Moreover, the results showed that Dex reduced the activity of mTOR pathway, as determined by decreased phosphorylation levels of mTOR, Akt, P70S6K and 4E-BP1 in resistant cells. Inhibition of autophagy by either chloroquine (CQ) or 3-methyladenine (3-MA) overcame Dex-resistance in lymphoid malignant cells by increasing apoptotic cell death in vitro. Consistently, inhibition of autophagy by stably knockdown of Beclin1 sensitized Dex-resistant lymphoid malignant cells to induction of apoptosis in vivo. Thus, inhibition of autophagy has the potential to improve lymphoid malignancy treatment by overcoming GC resistance.

  3. A G-protein-coupled receptor regulation pathway in cytochrome P450-mediated permethrin-resistance in mosquitoes, Culex quinquefasciatus

    PubMed Central

    Li, Ting; Cao, Chuanwang; Yang, Ting; Zhang, Lee; He, Lin; Xi, Zhiyong; Bian, Guowu; Liu, Nannan

    2015-01-01

    Rhodopsin-like G protein-coupled receptors (GPCRs) are known to be involved in the GPCR signal transduction system and regulate many essential physiological processes in organisms. This study, for the first time, revealed that knockdown of the rhodopsin-like GPCR gene in resistant mosquitoes resulted in a reduction of mosquitoes’ resistance to permethrin, simultaneously reducing the expression of two cAMP-dependent protein kinase A genes (PKAs) and four resistance related cytochrome P450 genes. The function of rhodopsin-like GPCR was further confirmed using transgenic lines of Drosophila melanogaster, in which the tolerance to permethrin and the expression of Drosophila resistance P450 genes were both increased. The roles of GPCR signaling pathway second messenger cyclic adenosine monophosphate (cAMP) and downstream effectors PKAs in resistance were investigated using cAMP production inhibitor Bupivacaine HCl and the RNAi technique. Inhibition of cAMP production led to significant decreases in both the expression of four resistance P450 genes and two PKA genes and mosquito resistance to permethrin. Knockdown of the PKA genes had shown the similar effects on permethrin resistance and P450 gene expression. Taken together, our studies revealed, for the first time, the role of the GPCR/cAMP/PKA-mediated regulatory pathway governing P450 gene expression and P450-mediated resistance in Culex mosquitoes. PMID:26656663

  4. Zebrafish Cardiotoxicity: The Effects of CYP1A Inhibition and AHR2 Knockdown Following Exposure to Weak Aryl Hydrocarbon Receptor Agonists

    PubMed Central

    Clark, Bryan William; Van Tiem Garner, Lindsey; Di Giulio, Richard Thomas

    2014-01-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates many of the toxic effects of dioxin-like compounds (DLCs) and some polycyclic aromatic hydrocarbons (PAHs). Strong AHR agonists, such as certain polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause severe cardiac teratogenesis in fish embryos. Moderately strong AHR agonists, such as benzo[a]pyrene and β-naphthoflavone, have been shown to cause similar cardiotoxic effects when coupled with a cytochrome P450 1A (CYP1A) inhibitor, such as fluoranthene (FL). We sought to determine if weak AHR agonists, when combined with a CYP1A inhibitor (FL) or CYP1A morpholino gene knockdown, are capable of causing cardiac deformities similar to moderately strong AHR agonists (Wassenberg and Di Giulio 2004; Wassenberg and Di Giulio 2004; Billiard, Timme-Laragy et al. 2006; Van Tiem and Di Giulio 2011). The weak AHR agonists included the following: carbaryl, phenanthrene, 2-methylindole, 3-methylindole, indigo, and indirubin. The results showed a complex pattern of cardiotoxic response to weak agonist inhibitor exposure and morpholino-knockdown. Danio rerio (zebrafish) embryos were first exposed to weak AHR agonists at equimolar concentrations. The agonists were assessed for their relative potency as inducers of CYP1 enzyme activity, measured by the ethoxyresorufin-o-deethylase (EROD) assay, and cardiac deformities. Carbaryl, 2-methylindole, and 3-methylindole induced the highest CYP1A activity in zebrafish. Experiments were then conducted to determine the individual cardiotoxicity of each compound. Next, zebrafish were co-exposed to each agonist (at concentrations below those determined to be cardiotoxic) and FL in combination to assess if CYP1A inhibition could induce cardiac deformities. Carbaryl, 2-methylindole, 3-methylindole, and phenanthrene significantly increased pericardial edema relative to controls when combined with FL. To further evaluate the

  5. Knockdown and larvicidal activity of six monoterpenes against Aedes aegypti (Diptera: Culicidae) and their structure-activity relationships.

    PubMed

    Lucia, Alejandro; Zerba, Eduardo; Masuh, Hector

    2013-12-01

    The relationships between physicochemical parameters of majority components of Eucalyptus essential oils and their insecticide effect were evaluated on Aedes aegypti (L.) (Diptera: Culicidae). The octanol-water partition coefficients of the monoterpenes were estimated by the atom/fragment contribution method and the vapor pressures were determined by our laboratory in previous studies. The larvicidal activity (LC50 (ppm)) and knockdown effect (KT50 (min)) of each component was determined. The results show that the toxicity of EOs main components of Eucalyptus on adults and larvae of A. aegypti is strongly related to their physicochemical properties (vapor pressure and Log P). However, the interaction of both variables (vapor pressure * Log P) explains the toxicological phenomenon more precisely. The regression models were expressed as follows: KT 50(min) =  - 10.9 + 3.7 * Log P + 1.9 * 1/Pvapor (R(2) = 0.80; F = 42.5) and LC 50(ppm) =  - 94.3 + 438.6 *  1/Log P + 2.8 *  1/Pvapor (F = 57.8; R(2) = 0.85). The six evaluated components present different functional groups. Therefore, it was considered to evaluate the monoterpenes as a group and separated in two groups: oxygenated monoterpenes (α-terpineol, 4-terpineol, and 1,8-cineole) and terpene hydrocarbons (γ-terpinene, p-cymene, and α-pinene). The results show the regression models for each group as follows: (A) oxygenated terpenes: KT 50(min) = - 515.3 + 1613.2 * 1/Log P + 5, 2 * 1/Pvapor (F = 3176.7 R(2) = 0.99) and LC 50(ppm)  =  - 1679.4 + 5402.1 * 1/Log P + 12.7 *  1/Pvapor (F = 282.9; R(2) = 0.99). (B) Hydrocarbons terpenes: KT 50(min) = 18.2 - 58.3 * 1/Log P + 2.7 * 1/Pvapor (F = 171.7;  R(2) = 0.97) and LC 50(ppm) = - 21.1 + 174.9 * 1/Log P - 14.3 * 1/Pvapor (F = 410.0; R(2) = 0.99). The association between

  6. MiRNA‑542‑3p downregulation promotes trastuzumab resistance in breast cancer cells via AKT activation.

    PubMed

    Ma, Tao; Yang, Lu; Zhang, Jin

    2015-03-01

    Trastuzumab (Herceptin) has been widely used in breast cancer treatment. However, the majority of cancers that initially respond to trastuzumab begin to progress again within 1 year. Despite the high resistance rate, the molecular mechanisms underlying this desease are not well understood. In the present study, microRNA (miRNA‑542‑3p modulated trastuzumab resistance in SKBR3 and MCF7/Her2 breast cancer cell lines. Trastuzumab induced miRNA‑542‑3p expression in SKBR3 and MCF7/Her2 cells. Furthermore, knockdown of miRNA‑542‑3p in the two cell lines resulted in decreased drug sensitivity to trastuzumab and cell apoptosis. The blockage of G1/S checkpoint by trastuzumab was rescued as well. miRNA‑542‑3p knockdown also activated the phosphatidylinositol 3‑kinase (PI3K)‑Akt pathway, while LY294002 reversed the effect of miRNA‑542‑3p knockdown. In summary, the results suggested that miRNA‑542‑3p downregulation may contribute to the trastuzumab resistance in breast cancer via, at least in part, the PI3K‑akt pathway. Our findings provide new molecular mechanisms in trastuzumab resistance. PMID:25586125

  7. [Resistant fungi].

    PubMed

    Vehreschild, M J G T; Cornely, O A

    2015-11-01

    Particularly in the area of hematology/oncology and intensive care medicine, infections due to resistant fungi are to be expected. Emergence of resistance in fungi is a less dynamic process than in bacteria; it can, however, have an equally important impact on treatment strategies. In the following article, the most important resistance patterns of yeasts and molds (Candida albicans , Aspergillus fumigatus, the order Mucorales and the genus Fusarium) will be presented and discussed. Their diagnosis mostly being based on blood cultures, resistance testing for yeasts is usually readily available. Culture-based therapeutic adjustments in mold infections are, however, only rarely possible, as most antifungal therapies for these infections are initiated on an empirical basis after identification of typical infiltrates on a CT scan. Response to therapy is then evaluated on the basis of clinical signs and symptoms in combination with follow-up CT scans. In case of therapeutic failure or appearance of suspicious infiltrates under antifungal prophylaxis, an open or CT-guided biopsy is recommended to allow efficient adaptation of antifungal treatment. In individual cases, particularly in patients diagnosed with mucormycosis, resection of the focus of infection may be necessary to achieve a satisfactory treatment response.

  8. Virus-mediated shRNA knockdown of prodynorphin in the rat nucleus accumbens attenuates depression-like behavior and cocaine locomotor sensitization.

    PubMed

    Cohen, Ami; Whitfield, Timothy W; Kreifeldt, Max; Koebel, Pascale; Kieffer, Brigitte L; Contet, Candice; George, Olivier; Koob, George F

    2014-01-01

    Dynorphins, endogenous opioid peptides that arise from the precursor protein prodynorphin (Pdyn), are hypothesized to be involved in the regulation of mood states and the neuroplasticity associated with addiction. The current study tested the hypothesis that dynorphin in the nucleus accumbens (NAcc) mediates such effects. More specifically, we examined whether knockdown of Pdyn within the NAcc in rats would alter the expression of depressive-like and anxiety-like behavior, as well as cocaine locomotor sensitization. Wistar rats were injected with adeno-associated viral (AAV) vectors encoding either a Pdyn-specific short hairpin RNA (AAV-shPdyn) or a scrambled shRNA (AAV-shScr) as control. Four weeks later, rats were tested for anxiety-like behavior in the elevated plus maze test and depressive-like behavior in the forced swim test (FST). Finally, rats received one daily injection of saline or cocaine (20 mg/kg, i.p.), followed by assessment of locomotion for 4 consecutive days. Following 3 days of abstinence, the rats completed 2 additional daily cocaine/saline locomotor trials. Pdyn knockdown in the NAcc led to a significant reduction in depressive-like behavior in the FST, but had no effect on anxiety-like behavior in the elevated plus maze. Pdyn knockdown did not alter baseline locomotor behavior, the locomotor response to acute cocaine, or the initial sensitization of the locomotor response to cocaine over the first 4 cocaine treatment days. However, following 3 days abstinence the locomotor response to the cocaine challenge returned to their original levels in the AAV-shPdyn rats while remaining heightened in the AAV-shScr rats. These results suggest that dynorphin in a very specific area of the nucleus accumbens contributes to depressive-like states and may be involved in neuroadaptations in the NAcc that contribute to the development of cocaine addiction as a persistent and lasting condition.

  9. Knockdown of WHIRLY1 Affects Drought Stress-Induced Leaf Senescence and Histone Modifications of the Senescence-Associated Gene HvS40.

    PubMed

    Janack, Bianka; Sosoi, Paula; Krupinska, Karin; Humbeck, Klaus

    2016-01-01

    The plastid-nucleus located protein WHIRLY1 has been described as an upstream regulator of leaf senescence, binding to the promoter of senescence-associated genes like HvS40. To investigate the impact of WHIRLY1 on drought stress-induced, premature senescence, transgenic barley plants with an RNAi-mediated knockdown of the HvWHIRLY1 gene were grown under normal and drought stress conditions. The course of leaf senescence in these lines was monitored by physiological parameters and studies on the expression of senescence- and drought stress-related genes. Drought treatment accelerated leaf senescence in WT plants, whereas WHIRLY 1 knockdown lines (RNAi-W1) showed a stay-green phenotype. Expression of both senescence-associated and drought stress-responsive genes, was delayed in the transgenic plants. Notably, expression of transcription factors of the WRKY and NAC families, which are known to function in senescence- and stress-related signaling pathways, was affected in plants with impaired accumulation of WHIRLY1, indicating that WHIRLY1 acts as an upstream regulator of drought stress-induced senescence. To reveal the epigenetic indexing of HvS40 at the onset of drought-induced senescence in WT and RNAi-W1 lines, stress-responsive loading with histone modifications of promoter and coding sequences of HvS40 was analyzed by chromatin immunoprecipitation and quantified by qRT-PCR. In the wildtype, the euchromatic mark H3K9ac of the HvS40 gene was low under control conditions and was established in response to drought treatment, indicating the action of epigenetic mechanisms in response to drought stress. However, drought stress caused no significant increase in H3K9ac in plants impaired in accumulation of WHIRLY1. The results show that WHIRLY1 knockdown sets in motion a delay in senescence that involves all aspects of gene expression, including changes in chromatin structure. PMID:27608048

  10. A botanical containing freeze dried açai pulp promotes healthy aging and reduces oxidative damage in sod1 knockdown flies.

    PubMed

    Laslo, Mara; Sun, Xiaoping; Hsiao, Cheng-Te; Wu, Wells W; Shen, Rong-Fong; Zou, Sige

    2013-08-01

    Superoxide dismutase 1 (SOD1), a critical enzyme against oxidative stress, is implicated in aging and degenerative diseases. We previously showed that a nutraceutical containing freeze-dried açai pulp promotes survival of flies fed a high-fat diet or sod1 knockdown flies fed a standard diet. Here, we investigated the effect of açai supplementation initiated at the early or late young adulthood on lifespan, physiological function, and oxidative damage in sod1 knockdown flies. We found that Açai supplementation extended lifespan even when started at the age of 10 days, which is the time shortly before the mortality rate of flies accelerated. Life-long açai supplementation increased lifetime reproductive output in sod1 knockdown flies. Our molecular studies indicate that açai supplementation reduced the protein levels of genes involved in oxidative stress response, cellular growth, and nutrient metabolism. Açai supplementation also affected the protein levels of ribosomal proteins. In addition, açai supplementation decreased the transcript levels of genes involved in oxidative stress response and gluconeogenesis, while increasing the transcript levels of mitochondrial biogenesis genes. Moreover, açai supplementation reduced the level of 4-hydroxynonenal-protein adducts, a lipid peroxidation marker. Our findings suggest that açai supplementation promotes healthy aging in sod1-deficient flies partly through reducing oxidative damage, and modulating nutrient metabolism and oxidative stress response pathways. Our findings provide a foundation to further evaluate the viability of using açai as an effective dietary intervention to promote healthy aging and alleviate symptoms of diseases with a high level of oxidative stress. PMID:22639178

  11. An animal model of PDH deficiency using AAV8-siRNA vector-mediated knockdown of pyruvate dehydrogenase E1α

    PubMed Central

    Ojano-Dirain, Carolyn; Glushakova, Lyudmyla G.; Zhong, Li; Zolotukhin, Sergei; Muzyczka, Nicholas; Srivastava, Arun; Stacpoole, Peter W.

    2010-01-01

    We evaluated the feasibility of self-complementary adeno-associated virus (scAAV) vector-mediated knockdown of the pyruvate dehydrogenase complex using small interfering RNAs directed against the E1α subunit gene (PDHA1). AAV serotype 8 was used to stereotaxically deliver scAAV8-si3-PDHA1-Enhanced Green Fluorescent Protein (knockdown) or scAAV8-EGFP (control) vectors into the right striatum and substantia nigra of rats. Rotational asymmetry was employed to quantify abnormal rotation following neurodegeneration in the nigrostriatal system. By 20 weeks after surgery, the siRNA-injected rats exhibited higher contralateral rotation during the first 10 min following amphetamine administration and lower 90-min total rotations (p≤0.05). Expression of PDC E1α, E1β and E2 subunits in striatum was decreased (p≤0.05) in the siRNA-injected striatum after 14 weeks. By week 25, both PDC activity and expression of E1α were lower (p≤0.05) in siRNA-injected striata compared to controls. E1α expression was associated with PDC activity (R2=0.48; p=0.006) and modestly associated with counterclockwise rotation (R2=0.51;p=0.07). The use of tyrosine-mutant scAAV8 vectors resulted in ~17-fold increase in transduction efficiency of rat striatal neurons in vivo. We conclude that scAAV8-siRNA vector-mediated knockdown of PDC E1α in brain regions typically affected in humans with PDC deficiency results in a reproducible biochemical and clinical phenotype in rats that may be further enhanced with the use of tyrosine-mutant vectors. PMID:20685142

  12. Short Hairpin RNA-Mediated Knockdown of VEGFA in Müller Cells Reduces Intravitreal Neovascularization in a Rat Model of Retinopathy of Prematurity

    PubMed Central

    Wang, Haibo; Smith, George W.; Yang, Zhihong; Jiang, Yanchao; McCloskey, Manabu; Greenberg, Kenneth; Geisen, Pete; Culp, William D.; Flannery, John; Kafri, Tal; Hammond, Scott; Hartnett, M. Elizabeth

    2014-01-01

    Vascular endothelial growth factor (VEGF) A is implicated in aberrant angiogenesis and intravitreous neovascularization (IVNV) in retinopathy of prematurity (ROP). However, VEGFA also regulates retinal vascular development and functions as a retinal neural survival factor. By using a relevant ROP model, the 50/10 oxygen-induced retinopathy (OIR) model, we previously found that broad inhibition of VEGFA bioactivity using a neutralizing antibody to rat VEGF significantly reduced IVNV area compared with control IgG but also significantly reduced body weight gain in the pups, suggesting an adverse effect. Therefore, we propose that knockdown of up-regulated VEGFA in cells that overexpress it under pathological conditions would reduce IVNV without affecting physiological retinal vascular development or overall pup growth. Herein, we determined first that the VEGFA mRNA signal was located within the inner nuclear layer corresponding to CRALBP-labeled Müller cells of pups in the 50/10 OIR model. We then developed a lentiviral-delivered miR-30–embedded shRNA against VEGFA that targeted Müller cells. Reduction of VEGFA by lentivector VEGFA-shRNA–targeting Müller cells efficiently reduced 50/10 OIR up-regulated VEGFA and IVNV in the model, without adversely affecting physiological retinal vascular development or pup weight gain. Knockdown of VEGFA in rat Müller cells by lentivector VEGFA-shRNA significantly reduced VEGFR2 phosphorylation in retinal vascular endothelial cells. Our results suggest that targeted knockdown of overexpressed VEGFA in Müller cells safely reduces IVNV in a relevant ROP model. PMID:23972394

  13. Knockdown of the M2 Isoform of Pyruvate Kinase (PKM2) with shRNA Enhances the Effect of Docetaxel in Human NSCLC Cell Lines In Vitro

    PubMed Central

    Yuan, Sujuan; Zhuang, Xibing; Chen, Wei; Xing, Na; Zhang, Qi

    2016-01-01

    Purpose The aim of our study was to explore the relationships between the M2 isoform of pyruvate kinase (PKM2) and the sensitivity of human non-small cell lung cancer (NSCLC) cells to docetaxel in vitro. Materials and Methods With the method of plasmid transfection, we silenced the expression of PKM2 successfully in A549 and H460 cells. Western blotting and real-time PCR were applied to detect PKM2 expression at protein and gene levels. Cell viability was examined by CCK8 assay. Cell cycle distribution and apoptosis were examined by flow cytometry. P21 and Bax were detected. Results Expression of PKM2 mRNA and protein were significantly decreased by shRNA targeting PKM2. Silencing of PKM2 increased docetaxel sensitivity of human NSCLC A549 and H460 cells in a collaborative manner, resulting in strong suppression of cell viability. The results of flow cytometric assays suggested that knockdown of PKM2 or docetaxel treatment, whether used singly or in combination, blocked the cells in the G2/M phase, which is in consistent with the effect of the two on the expression of p21. Cells with PKM2 silencing were more likely to be induced into apoptosis by docetaxel although knockdown of PKM2 alone can't induce apoptosis significantly, which is in consistent with the effect of the two on Bax expression. Conclusion The results suggest that PKM2 knockdown could serve as a chemosensitizer to docetaxel in non-small lung cancer cells through targeting PKM2, leading to inhibition of cell viability, increase of cell arrest of G2/M phase and apoptosis. PMID:27593857

  14. Knockdown of WHIRLY1 Affects Drought Stress-Induced Leaf Senescence and Histone Modifications of the Senescence-Associated Gene HvS40

    PubMed Central

    Janack, Bianka; Sosoi, Paula; Krupinska, Karin; Humbeck, Klaus

    2016-01-01

    The plastid-nucleus located protein WHIRLY1 has been described as an upstream regulator of leaf senescence, binding to the promoter of senescence-associated genes like HvS40. To investigate the impact of WHIRLY1 on drought stress-induced, premature senescence, transgenic barley plants with an RNAi-mediated knockdown of the HvWHIRLY1 gene were grown under normal and drought stress conditions. The course of leaf senescence in these lines was monitored by physiological parameters and studies on the expression of senescence- and drought stress-related genes. Drought treatment accelerated leaf senescence in WT plants, whereas WHIRLY 1 knockdown lines (RNAi-W1) showed a stay-green phenotype. Expression of both senescence-associated and drought stress-responsive genes, was delayed in the transgenic plants. Notably, expression of transcription factors of the WRKY and NAC families, which are known to function in senescence- and stress-related signaling pathways, was affected in plants with impaired accumulation of WHIRLY1, indicating that WHIRLY1 acts as an upstream regulator of drought stress-induced senescence. To reveal the epigenetic indexing of HvS40 at the onset of drought-induced senescence in WT and RNAi-W1 lines, stress-responsive loading with histone modifications of promoter and coding sequences of HvS40 was analyzed by chromatin immunoprecipitation and quantified by qRT-PCR. In the wildtype, the euchromatic mark H3K9ac of the HvS40 gene was low under control conditions and was established in response to drought treatment, indicating the action of epigenetic mechanisms in response to drought stress. However, drought stress caused no significant increase in H3K9ac in plants impaired in accumulation of WHIRLY1. The results show that WHIRLY1 knockdown sets in motion a delay in senescence that involves all aspects of gene expression, including changes in chromatin structure. PMID:27608048

  15. RNA interference-mediated knockdown of astrocyte elevated gene-1 inhibits growth, induces apoptosis, and increases the chemosensitivity to 5-fluorouracil in renal cancer Caki-1 cells.

    PubMed

    Wang, Peng; Yin, Bo; Shan, Liping; Zhang, Hui; Cui, Jun; Zhang, Mo; Song, Yongsheng

    2014-12-31

    Astrocyte elevated gene-1 (AEG-1) is a recently discovered oncogene that has been reported to be highly expressed in various types of malignant tumors, including renal cell carcinoma. However, the precise role of AEG-1 in renal cancer cell proliferation and apoptosis has not been clarified. In this study, we transfected the renal cancer cell line Caki-1 with a plasmid expressing AEG-1 short hairpin RNA (shRNA) and obtained cell colonies with stable knockdown of AEG-1. We found that AEG-1 down-regulation inhibited cell proliferation and colony formation and arrested cell cycle progression at the sub-G1 and G0/G1 phase. Western blot analysis indicated that the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E were significantly reduced following AEG-1 down-regulation. In addition, AEG-1 knockdown led to the appearance of apoptotic bodies in renal cancer cells, and the ratio of apoptotic cells significantly increased. Expression of the anti-apoptotic factor Bcl-2 was dramatically reduced, whereas the pro-apoptotic factors Bax, caspase-3 and poly (ADP-ribose) polymerase (PARP) were significantly activated. Finally, AEG-1 knockdown in Caki-1 cells remarkably suppressed cell proliferation and enhanced cell apoptosis in response to 5-fluorouracil (5-FU) treatment, suggesting that AEG-1 inhibition sensitizes Caki-1 cells to 5-FU. Taken together, our data suggest that AEG-1 plays an important role in renal cancer formation and development and may be a potential target for future gene therapy for renal cell carcinoma.

  16. Local knockdown of the NaV1.6 sodium channel reduces pain behaviors, sensory neuron excitability, and sympathetic sprouting in rat models of neuropathic pain

    PubMed Central

    Xie, Wenrui; Strong, Judith A.; Zhang, Jun-Ming

    2015-01-01

    In the spinal nerve ligation model of neuropathic pain, as in other pain models, abnormal spontaneous activity of myelinated sensory neurons occurs early and is essential for establishing pain behaviors and other pathologies. Sympathetic sprouting into the dorsal root ganglion (DRG) is observed after spinal nerve ligation, and sympathectomy reduces pain behavior. Sprouting and spontaneous activity may be mutually reinforcing: blocking neuronal activity reduces sympathetic sprouting, and sympathetic spouts functionally increase spontaneous activity in vitro. However, most studies in this field have used nonspecific methods to block spontaneous activity, methods that also block evoked and normal activity. In this study, we injected small inhibitory RNA directed against the NaV1.6 sodium channel isoform into the DRG before spinal nerve ligation. This isoform can mediate high frequency repetitive firing, like that seen in spontaneously active neurons. Local knockdown of NaV1.6 markedly reduced mechanical pain behaviors induced by spinal nerve ligation, reduced sympathetic sprouting into the ligated sensory ganglion, and blocked abnormal spontaneous activity and other measures of hyperexcitability in myelinated neurons in the ligated sensory ganglion. Immunohistochemical experiments showed that sympathetic sprouting preferentially targeted NaV1.6-positive neurons. Under these experimental conditions, NaV1.6 knockdown did not prevent or strongly alter single evoked action potentials, unlike previous less specific methods used to block spontaneous activity. NaV1.6 knockdown also reduced pain behaviors in another pain model, chronic constriction of the sciatic nerve, provided the model was modified so that the lesion site was relatively close to the siRNA-injected lumbar DRGs. The results highlight the relative importance of abnormal spontaneous activity in establishing both pain behaviors and sympathetic sprouting, and suggest that the NaV1.6 isoform may have value as a

  17. In vivo knockdown of GAD67 in the amygdala disrupts fear extinction and the anxiolytic-like effect of diazepam in mice

    PubMed Central

    Heldt, S A; Mou, L; Ressler, K J

    2012-01-01

    In mammals, γ-aminobutyric acid (GABA) transmission in the amygdala is particularly important for controlling levels of fear and anxiety. Most GABA synthesis in the brain is catalyzed in inhibitory neurons from ℒ-glutamic acid by the enzyme glutamic acid decarboxylase 67 (GAD67). In the current study, we sought to examine the acquisition and extinction of conditioned fear in mice with knocked down expression of the GABA synthesizing enzyme GAD67 in the amygdala using a lentiviral-based (LV) RNA interference strategy to locally induce loss-of-function. In vitro experiments revealed that our LV-siRNA-GAD67 construct diminished the expression of GAD67 as determined with western blot and fluorescent immunocytochemical analyses. In vivo experiments, in which male C57BL/6J mice received bilateral amygdala microinjections, revealed that LV-siRNA-GAD67 injections produce significant inhibition of endogenous GAD67 when compared with control injections. In contrast, no significant changes in GAD65 expression were detected in the amygdala, validating the specificity of LV knockdown. Behavioral experiments showed that LV knockdown of GAD67 results in a deficit in the extinction, but not the acquisition or retention, of fear as measured by conditioned freezing. GAD67 knockdown did not affect baseline locomotion or basal measures of anxiety as measured in open field apparatus. However, diminished GAD67 in the amygdala blunted the anxiolytic-like effect of diazepam (1.5 mg kg–1) as measured in the elevated plus maze. Together, these studies suggest that of GABAergic transmission in amygdala mediates the inhibition of conditioned fear and the anxiolytic-like effect of diazepam in adult mice. PMID:23149445

  18. Anthelmintic resistance.

    PubMed

    Waller, P J

    1997-11-01

    Since the first reports of resistance to the broad spectrum anthelmintics were made some three decades ago, this phenomenon has changed from being considered merely as a parasitological curiosity to a state of industry crisis in certain livestock sectors. This extreme situation exists with the small ruminant industry of the tropical/sub-tropical region of southern Latin America where resistance to the entire broad spectrum anthelmintic arsenal now occurs. In contrast, the cattle industry does not appear to be threatened--or so it seems. Although field reports of resistance have been made to the range of broad spectrum anthelmintics in nematode parasites of cattle, it appears that the evolution of resistance in cattle parasites is not as dramatic as for sheep worms. However, one cannot remain confident that this state of affairs will remain static. Concern is shared amongst parasitologists that we have not looked closely enough. In regions of the world where internal parasites are considered a problem in cattle and drenching occurs frequently, no widespread surveys have been carried out. It appears that because of the very high costs and risks associated with taking a new active drug down the development track to marketing, that the pharmaceutical industry has, in general, turned away from this activity. By implication, the international small ruminant industry is too small for these companies to make the necessary investment. This begs two questions: What is the fate of the sheep (and goat) industries in those parts of the world where resistance is rampant and immediate ameliorative parasite control options are required? What will be the response if significant resistance is found in cattle parasites? There is a body of opinion which suggests that if resistance becomes an issue in the control of cattle parasites then the pharmaceutical industry will find it commercially attractive to re-enter the anthelmintic discovery and development business. This is based on the

  19. Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells

    SciTech Connect

    Hossain, Md. Motarab; Banik, Naren L.; Ray, Swapan K.

    2012-08-01

    Neuroblastoma is a solid tumor that mostly occurs in children. Malignant neuroblastomas have poor prognosis because conventional chemotherapeutic agents are hardly effective. Survivin, which is highly expressed in some malignant neuroblastomas, plays a significant role in inhibiting differentiation and apoptosis and promoting cell proliferation, invasion, and angiogenesis. We examined consequences of survivin knockdown by survivin short hairpin RNA (shRNA) plasmid and then treatment with (-)-epigallocatechin-3-gallate (EGCG), a green tea flavonoid, in malignant neuroblastoma cells. Our Western blotting and laser scanning confocal immunofluorescence microscopy showed that survivin was highly expressed in malignant neuroblastoma SK-N-BE2 and SH-SY5Y cell lines and slightly in SK-N-DZ cell line. Expression of survivin was very faint in malignant neuroblastoma IMR32 cell line. We transfected SK-N-BE2 and SH-SY-5Y cells with survivin shRNA, treated with EGCG, and confirmed knockdown of survivin at mRNA and protein levels. Survivin knockdown induced morphological features of neuronal differentiation, as we observed following in situ methylene blue staining. Combination of survivin shRNA and EGCG promoted neuronal differentiation biochemically by increases in the expression of NFP, NSE, and e-cadherin and also decreases in the expression of Notch-1, ID2, hTERT, and PCNA. Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing, respectively, morphological and biochemical features of apoptosis. Apoptosis occurred with activation of caspase-8 and cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and increases in the expression and activity of calpain and caspase-3. Combination therapy decreased migration of cells through matrigel and inhibited proliferative (p-Akt and NF-{kappa}B), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro

  20. AHR2 knockdown prevents PAH-mediated cardiac toxicity and XRE- and ARE-associated gene induction in zebrafish (Danio rerio)

    SciTech Connect

    Van Tiem, Lindsey A.; Di Giulio, Richard T.

    2011-08-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants often present in aquatic systems as complex mixtures. Embryonic fish are sensitive to the developmental toxicity of some PAHs, but the exact mechanisms involved in this toxicity are still unknown. This study explored the role of the aryl hydrocarbon receptor (AHR) in the oxidative stress response of zebrafish to the embryotoxicity of select PAHs. Embryos were exposed to two PAHs, benzo[k]fluoranthene (BkF; a strong AHR agonist) and fluoranthene (FL; a cytochrome P4501A (CYP1A) inhibitor), alone and in combination. CYP1A, CYP1B1, CYP1C1, and redox-responsive genes glutathione s-transferase pi 2 (GSTp2), glutathione peroxidase 1 (GPx1), the glutamate-cysteine ligase catalytic subunit (GCLc), MnSOD and CuZnSOD mRNA expression was examined. CYP1 activity was measured via an in vivo ethoxyresorufin-O-deethlyase (EROD) activity assay, and the area of the pericardium was measured as an index of cardiotoxicity. BkF or FL alone caused no deformities whereas BkF + FL resulted in extreme pericardial effusion. BkF induced CYP activity above controls and co-exposure with FL inhibited this activity. BkF induced expression of all three CYPs, GSTp2, and GCLc. BkF + FL caused greater than additive induction of the three CYPs, GSTp2, GPx1, and GCLc but had no effect on MnSOD or CuZnSOD. AHR2 knockdown protected against the cardiac deformities caused by BkF + FL and significantly inhibited the induction of the CYPs, GSTp2, GPx1, and GCLc after BkF + FL compared to non-injected controls. These results further show the protective role of AHR2 knockdown against cardiotoxic PAHs and the role of AHR2 as a mediator of redox-responsive gene induction. - Research Highlights: > Co-exposure of the PAHs BkF and FL causes cardiotoxicity in zebrafish. > BkF and FL co-exposure upregulates certain XRE- and ARE-associated genes. > AHR2 knockdown prevents the deformities caused by BkF and FL co-exposure. > AHR2

  1. Overcoming cisplatin resistance of ovarian cancer cells by targeting HIF-1-regulated cancer metabolism.

    PubMed

    Ai, Zhihong; Lu, Yang; Qiu, Songbo; Fan, Zhen

    2016-04-01

    Cisplatin is currently one of the most effective chemotherapeutic drugs used for treating ovarian cancer; however, resistance to cisplatin is common. In this study, we explored an experimental strategy for overcoming cisplatin resistance of human ovarian cancer from the new perspective of cancer cell metabolism. By using two pairs of genetically matched cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines, we tested the hypothesis that downregulating hypoxia-inducible factor-1 (HIF-1), which regulates metabolic enzymes involved in glycolysis, is a promising strategy for overcoming cisplatin resistance of human ovarian cancer cells. We found that cisplatin downregulated the level of the regulatable α subunit of HIF-1, HIF-1α, in cisplatin-sensitive ovarian cancer cells through enhancing HIF-1α degradation but did not downregulate HIF-1α in their cisplatin-resistant counterparts. Overexpression of a degradation-resistant HIF-1α (HIF-1α ΔODD) reduced cisplatin-induced apoptosis in cisplatin-sensitive cells, whereas genetic knockdown of HIF-1α or pharmacological promotion of HIF-1α degradation enhanced response to cisplatin in both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. We further demonstrated that knockdown of HIF-1α improved the response of cisplatin-resistant ovarian cancer cells to cisplatin by redirecting the aerobic glycolysis in the resistant cancer cells toward mitochondrial oxidative phosphorylation, leading to cell death through overproduction of reactive oxygen species. Our findings suggest that the HIF-1α-regulated cancer metabolism pathway could be a novel target for overcoming cisplatin resistance in ovarian cancer.

  2. [Resistant hypertension].

    PubMed

    Feldstein, Carlos A

    2008-04-01

    Resistant hypertension, defined as a persistent blood pressure over 140/90 mmHg despite the use of three antihypertensive drugs including a diuretic, is unusual. The diagnosis requires ruling out initially pseudoresistance and a lack of compliance with treatment. Ambulatory blood pressure recording allow the recognition of white coat hypertension. When there is a clinical or laboratory suspicion, secondary causes of hypertension should be discarded. Excessive salt intake, the presence of concomitant diseases such as diabetes mellitus, chronic renal disease, obesity, and psychiatric conditions such as panic attacks, anxiety and depression, should also be sought. The presence of target organ damage requires a more aggressive treatment of hypertension. Recent clinical studies indicate that the administration of aldosterone antagonists as a fourth therapeutic line provides significant additional blood pressure reduction, when added to previous antihypertensive regimens in subjects with resistant hypertension. The possible blood pressure lowering effects of prolonged electrical activation of carotid baroreceptors is under investigation. PMID:18769797

  3. [Resistant hypertension].

    PubMed

    Feldstein, Carlos A

    2008-04-01

    Resistant hypertension, defined as a persistent blood pressure over 140/90 mmHg despite the use of three antihypertensive drugs including a diuretic, is unusual. The diagnosis requires ruling out initially pseudoresistance and a lack of compliance with treatment. Ambulatory blood pressure recording allow the recognition of white coat hypertension. When there is a clinical or laboratory suspicion, secondary causes of hypertension should be discarded. Excessive salt intake, the presence of concomitant diseases such as diabetes mellitus, chronic renal disease, obesity, and psychiatric conditions such as panic attacks, anxiety and depression, should also be sought. The presence of target organ damage requires a more aggressive treatment of hypertension. Recent clinical studies indicate that the administration of aldosterone antagonists as a fourth therapeutic line provides significant additional blood pressure reduction, when added to previous antihypertensive regimens in subjects with resistant hypertension. The possible blood pressure lowering effects of prolonged electrical activation of carotid baroreceptors is under investigation.

  4. Antisense-mediated knockdown of Na(V)1.8, but not Na(V)1.9, generates inhibitory effects on complete Freund's adjuvant-induced inflammatory pain in rat.

    PubMed

    Yu, Yao-Qing; Zhao, Feng; Guan, Su-Min; Chen, Jun

    2011-01-01

    Tetrodotoxin-resistant (TTX-R) sodium channels Na(V)1.8 and Na(V)1.9 in sensory neurons were known as key pain modulators. Comparing with the widely reported Na(V)1.8, roles of Na(V)1.9 on inflammatory pain are poorly studied by antisense-induced specific gene knockdown. Here, we used molecular, electrophysiological and behavioral methods to examine the effects of antisense oligodeoxynucleotide (AS ODN) targeting Na(V)1.8 and Na(V)1.9 on inflammatory pain. Following complete Freund's adjuvant (CFA) inflammation treatment, Na(V)1.8 and Na(V)1.9 in rat dorsal root ganglion (DRG) up-regulated mRNA and protein expressions and increased sodium current densities. Immunohistochemical data demonstrated that Na(V)1.8 mainly localized in medium and small-sized DRG neurons, whereas Na(V)1.9 only expressed in small-sized DRG neurons. Intrathecal (i.t.) delivery of AS ODN was used to down-regulate Na(V)1.8 or Na(V)1.9 expressions confirmed by immunohistochemistry and western blot. Unexpectedly, behavioral tests showed that only Na(V)1.8 AS ODN, but not Na(V)1.9 AS ODN could reverse CFA-induced heat and mechanical hypersensitivity. Our data indicated that TTX-R sodium channels Na(V)1.8 and Na(V)1.9 in primary sensory neurons played distinct roles in CFA-induced inflammatory pain and suggested that antisense oligodeoxynucleotide-mediated blocking of key pain modulator might point toward a potential treatment strategy against certain types of inflammatory pain. PMID:21572961

  5. Pre-resistance-welding resistance check

    DOEpatents

    Destefan, Dennis E.; Stompro, David A.

    1991-01-01

    A preweld resistance check for resistance welding machines uses an open circuited measurement to determine the welding machine resistance, a closed circuit measurement to determine the parallel resistance of a workpiece set and the machine, and a calculation to determine the resistance of the workpiece set. Any variation in workpiece set or machine resistance is an indication that the weld may be different from a control weld.

  6. Annexin A4 induces platinum resistance in a chloride-and calcium-dependent manner.

    PubMed

    Morimoto, Akiko; Serada, Satoshi; Enomoto, Takayuki; Kim, Ayako; Matsuzaki, Shinya; Takahashi, Tsuyoshi; Ueda, Yutaka; Yoshino, Kiyoshi; Fujita, Masami; Fujimoto, Minoru; Kimura, Tadashi; Naka, Tetsuji

    2014-09-15

    Platinum resistance has long been a major issue in the treatment of various cancers. We previously reported that enhanced annexin A4 (ANXA4) expression, a Ca2+-regulated phospholipid-binding protein, induces chemoresistance to platinum-based drugs. In this study, we investigated the role of annexin repeats, a conserved structure of all the annexin family, responsible for platinum-resistance as well as the effect of knockdown of ANXA4. ANXA4 knockdown increased sensitivity to platinum-based drugs both in vitro and in vivo. To identify the domain responsible for chemoresistance, ANXA4 deletion mutants were constructed by deleting annexin repeats one by one from the C terminus. Platinum resistance was induced both in vitro and in vivo in cells expressing either full-length ANXA4 or the deletion mutants, containing at least one intact annexin repeat. However, cells expressing the mutant without any calcium-binding sites in the annexin repeated sequence, which is essential for ANXA4 translocation from the cytosol to plasma membrane, failed to acquire platinum resistance. After cisplatin treatment, the intracellular chloride ion concentration, whose channel is partly regulated by ANXA4, significantly increased in the platinum-resistant cells. These findings indicate that the calcium-binding site in the annexin repeat induces chemoresistance to the platinum-based drug by elevating the intracellular chloride concentration.

  7. DC-STAMP knock-down deregulates cytokine production and T-cell stimulatory capacity of LPS-matured dendritic cells

    PubMed Central

    2011-01-01

    Background Dendritic cells (DCs) are the highly specialized antigen presenting cells of the immune system that play a key role in regulating immune responses. DCs can efficiently initiate immune responses or induce tolerance. Due to this dual function, DCs are studied in the context of immunotherapy for both cancer and autoimmune diseases. Characterization of DC-specific genes, leading to better understanding of DC immunobiology, will help to guide their use in clinical settings. We previously identified DC-STAMP, a multi-membrane spanning protein preferentially expressed by DCs. DC-STAMP resides in the endoplasmic reticulum (ER) of immature DCs and translocates towards the Golgi compartment upon maturation. In this study we knocked down DC-STAMP in mouse bone marrow-derived DCs (mBMDCs) to determine its function. Results We demonstrate that DC-STAMP knock-down mBMDCs secrete less IL-6, IL-12, TNF-α and IL-10 while IL-1 production is enhanced. Moreover, LPS-matured DC-STAMP knock-down mBMDCs show impaired T cell activation potential and induction of Th1 responses in an alloreaction. Conclusions We show that DC-STAMP plays an important role in cytokine production by mBMDCs following LPS exposure. Our results reveal a novel function of DC-STAMP in regulating DC-initiated immune responses. PMID:21978263

  8. Knock-Down of Cathepsin D Affects the Retinal Pigment Epithelium, Impairs Swim-Bladder Ontogenesis and Causes Premature Death in Zebrafish

    PubMed Central

    Follo, Carlo; Ozzano, Matteo; Mugoni, Vera; Castino, Roberta; Santoro, Massimo; Isidoro, Ciro

    2011-01-01

    The lysosomal aspartic protease Cathepsin D (CD) is ubiquitously expressed in eukaryotic organisms. CD activity is essential to accomplish the acid-dependent extensive or partial proteolysis of protein substrates within endosomal and lysosomal compartments therein delivered via endocytosis, phagocytosis or autophagocytosis. CD may also act at physiological pH on small-size substrates in the cytosol and in the extracellular milieu. Mouse and fruit fly CD knock-out models have highlighted the multi-pathophysiological roles of CD in tissue homeostasis and organ development. Here we report the first phenotypic description of the lack of CD expression during zebrafish (Danio rerio) development obtained by morpholino-mediated knock-down of CD mRNA. Since the un-fertilized eggs were shown to be supplied with maternal CD mRNA, only a morpholino targeting a sequence containing the starting ATG codon was effective. The main phenotypic alterations produced by CD knock-down in zebrafish were: 1. abnormal development of the eye and of retinal pigment epithelium; 2. absence of the swim-bladder; 3. skin hyper-pigmentation; 4. reduced growth and premature death. Rescue experiments confirmed the involvement of CD in the developmental processes leading to these phenotypic alterations. Our findings add to the list of CD functions in organ development and patho-physiology in vertebrates. PMID:21747967

  9. Systemic shRNA mediated knock-down of S100A4 in colorectal cancer xenografted mice reduces metastasis formation

    PubMed Central

    Dahlmann, Mathias; Sack, Ulrike; Herrmann, Pia; Lemm, Margit; Fichtner, Iduna; Schlag, Peter M.; Stein, Ulrike

    2012-01-01

    The metastasis-inducing protein S100A4 was found to be a prognostic indicator for the development of metachronous metastases. S100A4 expression levels correlate with the formation of human colorectal cancer metastases and shorter patients’ survival. Inhibition of S100A4 expression in patients might therefore result in decreased metastasis formation and prolonged survival. In the present study, we used shRNA expression plasmids to inhibit S100A4 expression in the colorectal cancer cell lines HCT116, SW620 and DLD-1. Cell lines with reduced S100A4 expression showed reduced cell migration and invasion in vitro. The knock-down of S100A4 expression also led to significantly diminished formation of liver metastases when intrasplenically transplanted in mice (P = 0.004). We then focused on the therapeutic potential of systemically applied shRNA expression plasmids acting on S100A4 via repeated hydrodynamics-based tail vein injection of plasmid DNA. Mice, intrasplenically transplanted with HCT116 cells and treated systemically with S100A4-shRNA plasmids, showed a decrease of S100A4 and MMP9 expression levels, resulting in significantly reduced liver metastases (P = 0.005). In summary, we show for the first time the intratumoral knock-down of S100A4 via systemic application of S100A4-shRNA plasmid DNA, which restricts metastasis formation in a xenografted mouse model of colorectal cancer. PMID:22878175

  10. Deceleration of liver regeneration by knockdown of augmenter of liver regeneration gene is associated with impairment of mitochondrial DNA synthesis in mice.

    PubMed

    Han, Li-hong; Dong, Ling-yue; Yu, Hao; Sun, Guang-yong; Wu, Yuan; Gao, Jian; Thasler, Wolfgang; An, Wei

    2015-07-15

    Hepatic stimulator substance, also known as augmenter of liver regeneration (ALR), is a novel hepatic mitogen that stimulates liver regeneration after partial hepatectomy (PH). Recent work has indicated that a lack of ALR expression inhibited liver regeneration in rats, and the mechanism seems to be related to increased cell apoptosis. The mitochondria play an important role during liver regeneration. Adequate ATP supply, which is largely dependent on effective mitochondrial biogenesis, is essential for progress of liver regeneration. However, ALR gene expression during liver regeneration, particularly its function with mitochondrial DNA synthesis, remains poorly understood. In this study, ALR expression in hepatocytes of mice was suppressed with ALR short-hairpin RNA interference or ALR deletion (knockout, KO). The ALR-defective mice underwent PH, and the liver was allowed to regenerate for 1 wk. Analysis of liver growth and its correlation with mitochondrial biogenesis showed that both ALR mRNA and protein levels increased robustly in control mice with a maximum at days 3 and 4 post-PH. However, ALR knockdown inhibited hepatic DNA synthesis and decelerated liver regeneration after PH. Furthermore, both in the ALR-knockdown and ALR-KO mice, expression of mitochondrial transcription factor A and peroxisome proliferator-activated receptor-γ coactivator-1α were reduced, resulting in impaired mitochondrial biogenesis. In conclusion, ALR is apparently required to ensure appropriate liver regeneration following PH in mice, and deletion of the ALR gene may delay liver regeneration in part due to impaired mitochondrial biogenesis.

  11. Contrasting Inducible Knockdown of the Auxiliary PTEX Component PTEX88 in P. falciparum and P. berghei Unmasks a Role in Parasite Virulence.

    PubMed

    Chisholm, Scott A; McHugh, Emma; Lundie, Rachel; Dixon, Matthew W A; Ghosh, Sreejoyee; O'Keefe, Meredith; Tilley, Leann; Kalanon, Ming; de Koning-Ward, Tania F

    2016-01-01

    Pathogenesis of malaria infections is linked to remodeling of erythrocytes, a process dependent on the trafficking of hundreds of parasite-derived proteins into the host erythrocyte. Recent studies have demonstrated that the Plasmodium translocon of exported proteins (PTEX) serves as the central gateway for trafficking of these proteins, as inducible knockdown of the core PTEX constituents blocked the trafficking of all classes of cargo into the erythrocyte. However, the role of the auxiliary component PTEX88 in protein export remains less clear. Here we have used inducible knockdown technologies in P. falciparum and P. berghei to assess the role of PTEX88 in parasite development and protein export, which reveal that the in vivo growth of PTEX88-deficient parasites is hindered. Interestingly, we were unable to link this observation to a general defect in export of a variety of known parasite proteins, suggesting that PTEX88 functions in a different fashion to the core PTEX components. Strikingly, PTEX88-deficient P. berghei were incapable of causing cerebral malaria despite a robust pro-inflammatory response from the host. These parasites also exhibited a reduced ability to sequester in peripheral tissues and were removed more readily from the circulation by the spleen. In keeping with these findings, PTEX88-deficient P. falciparum-infected erythrocytes displayed reduced binding to the endothelial cell receptor, CD36. This suggests that PTEX88 likely plays a specific direct or indirect role in mediating parasite sequestration rather than making a universal contribution to the trafficking of all exported proteins.

  12. Knockdown of a nutrient amino acid transporter gene LdNAT1 reduces free neutral amino acid contents and impairs Leptinotarsa decemlineata pupation

    PubMed Central

    Fu, Kai-Yun; Guo, Wen-Chao; Ahmat, Tursun; Li, Guo-Qing

    2015-01-01

    A Leptinotarsa decemlineata SLC6 NAT gene (LdNAT1) was cloned. LdNAT1 was highly expressed in the larval alimentary canal especially midgut. LdNAT1 mRNA levels were high right after the molt and low just before the molt. JH and a JH analog pyriproxyfen activated LdNAT1 expression. RNAi of an allatostatin gene LdAS-C increased JH and upregulated LdNAT1 transcription. Conversely, silencing of a JH biosynthesis gene LdJHAMT decreased JH and reduced LdNAT1 expression. Moreover, 20E and an ecdysteroid agonist halofenozide repressed LdNAT1 expression, whereas a decrease in 20E by RNAi of an ecdysteroidogenesis gene LdSHD and disruption of 20E signaling by knockdown of LdE75 and LdFTZ-F1 activated LdNAT1 expression. Thus, LdNAT1 responded to both 20E and JH. Moreover, knockdown of LdNAT1 reduced the contents of cysteine, histidine, isoleucine, leucine, methionine, phenylalanine and serine in the larval bodies and increased the contents of these amino acids in the larval feces. Furthermore, RNAi of LdNAT1 inhibited insulin/target of rapamycin pathway, lowered 20E and JH titers, reduced 20E and JH signaling, retarded larval growth and impaired pupation. These data showed that LdNAT1 was involved in the absorption of several neutral amino acids critical for larval growth and metamorphosis. PMID:26657797

  13. Hypothalamic nesfatin-1/NUCB2 knockdown augments hepatic gluconeogenesis that is correlated with inhibition of mTOR-STAT3 signaling pathway in rats.

    PubMed

    Wu, Dandong; Yang, Mengliu; Chen, Yang; Jia, Yanjun; Ma, Zhongmin Alex; Boden, Guenther; Li, Ling; Yang, Gangyi

    2014-04-01

    Nesfatin-1, an 82-amino acid neuropeptide, has recently been characterized as a potent metabolic regulator. However, the metabolic mechanisms and signaling steps directly associated with the action of nesfatin-1 have not been well delineated. We established a loss-of-function model of hypothalamic nesfatin-1/NUCB2 signaling in rats through an adenoviral-mediated RNA interference. With this model, we found that inhibition of central nesfatin-1/NUCB2 activity markedly increased food intake and hepatic glucose flux and decreased glucose uptake in peripheral tissue in rats fed either a normal chow diet (NCD) or a high-fat diet (HFD). The change of hepatic glucose fluxes in the hypothalamic nesfatin-1/NUCB2 knockdown rats was accompanied by increased hepatic levels of glucose-6-phosphatase and PEPCK and decreased insulin receptor, insulin receptor substrate 1, and AKT kinase phosphorylation. Furthermore, knockdown of hypothalamic nesfatin-1 led to decreased phosphorylation of mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) and the subsequent suppressor of cytokine signaling 3 levels. These results demonstrate that hypothalamic nesfatin-1/NUCB2 plays an important role in glucose homeostasis and hepatic insulin sensitivity, which is, at least in part, associated with the activation of the mTOR-STAT3 signaling pathway. PMID:24478398

  14. Knock-down of HEXA and HEXB genes correlate with the absence of the immunostimulatory function of HSC-derived dendritic cells.

    PubMed

    Tiribuzi, Roberto; D'Angelo, Francesco; Berardi, Anna C; Martino, Sabata; Orlacchio, Aldo

    2012-01-01

    In an attempt to investigate whether the genetic defect in the HEXA and HEXB genes (which causes the absence of the lysosomal β-N-acetyl-hexosaminidase), are related to the wide inflammation in GM2 gangliosidoses (Tay-Sachs and Sandhoff disease), we have chosen the dendritic cells (DCs) as a study model. Using the RNA interference approach, we generated an in vitro model of HEXs knock-down immunogenic DCs (i-DCs) from CD34(+)-haemopoietic stem cells (CD34(+)-HSCs), thus mimicking the Tay-Sachs (HEXA-/-) and Sandhoff (HEXB-/-) cells. We showed that the absence of β-N-acetyl-hexosaminidase activity does not alter the differentiation of i-DCs from HSCs, but it is critical for the activation of CD4(+)T cells because knock-down of HEXA or HEXB gene causes a loss of function of i-DCs. Notably, the silencing of the HEXA gene had a stronger immune inhibitory effect, thereby indicating a major involvement of β-N-acetyl-hexosaminidase A isoenzyme within this mechanism. PMID:21997228

  15. Mitochondrial delivery of antisense RNA by MITO-Porter results in mitochondrial RNA knockdown, and has a functional impact on mitochondria.

    PubMed

    Furukawa, Ryo; Yamada, Yuma; Kawamura, Eriko; Harashima, Hideyoshi

    2015-07-01

    Mitochondrial genome-targeting nucleic acids are promising therapeutic candidates for treating mitochondrial diseases. To date, a number of systems for delivering genetic information to the cytosol and the nucleus have been reported, and several successful gene therapies involving gene delivery targeted to the cytosol and the nucleus have been reported. However, much less progress has been made concerning mitochondrial gene delivery systems, and mitochondrial gene therapy has never been achieved. Here, we report on the mitochondrial delivery of an antisense RNA oligonucleotide (ASO) to perform mitochondrial RNA knockdown to regulate mitochondrial function. Mitochondrial delivery of the ASO was achieved using a combination of a MITO-Porter system, which contains mitochondrial fusogenic lipid envelopes for mitochondrial delivery via membrane fusion and D-arm, a mitochondrial import signal of tRNA to the matrix. Mitochondrial delivery of the ASO induces the knockdown of the targeted mitochondria-encoded mRNA and protein, namely cytochrome c oxidase subunit II, a component of the mitochondrial respiratory chain. Furthermore, the mitochondrial membrane potential was depolarized by the down regulation of the respiratory chain as the result of the mitochondrial delivery of ASO. This finding constitutes the first report to demonstrate that the nanocarrier-mediated mitochondrial genome targeting of antisense RNA effects mitochondrial function.

  16. CMTM7 knockdown increases tumorigenicity of human non-small cell lung cancer cells and EGFR-AKT signaling by reducing Rab5 activation

    PubMed Central

    Li, Ting; Yuan, Wanqiong; Mo, Xiaoning; Li, Henan; He, Qihua; Ma, Dalong; Han, Wenling

    2015-01-01

    The dysregulation of epidermal growth factor receptor (EGFR) signaling has been well documented to contribute to the progression of non-small cell lung cancer (NSCLC), the leading cause of cancer death in the world. EGF-stimulated EGFR activation induces receptor internalization and degradation, which plays an important role in EGFR signaling. This process is frequently deregulated in cancer cells, leading to enhanced EGFR levels and signaling. Our previous study on CMTM7 is only limited to a brief description of the relationship of overexpressed CMTM7 with EGFR-AKT signaling. The biological functions of endogenous CMTM7 and its molecular mechanism remained unclear. In this study, we show that the stable knockdown of CMTM7 augments the malignant potential of NSCLC cells and enhances EGFR-AKT signaling by decreasing EGFR internalization and degradation. Mechanistically, CMTM7 knockdown reduces the activation of Rab5, a protein known to be required for early endosome fusion. In NSCLC, the loss of CMTM7 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, our findings highlight the role of CMTM7 in the regulation of EGFR signaling in tumor cells, revealing CMTM7 as a novel molecule related to Rab5 activation. PMID:26528697

  17. RNA interference-mediated knockdown of the Halloween gene Spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus.

    PubMed

    Van Ekert, E; Wang, M; Miao, Y-G; Brent, C S; Hull, J J

    2016-10-01

    Ecdysteroids play a critical role in coordinating insect growth, development and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNA interference (RNAi)-mediated knockdown of the CYP307B1 Halloween gene (Spookiest) in the western tarnished plant bug, Lygus hesperus. Transcripts for Ly. hesperus Spookiest (LhSpot) were amplified from all life stages and correlated well with timing of the pre-moult ecdysteroid pulse. In adults, LhSpot was amplified from heads of both genders as well as female reproductive tissues. Heterologous expression of a LhSpot fluorescent chimera in cultured insect cells co-localized with a fluorescent marker of the endoplasmic reticulum/secretory pathway. RNAi-mediated knockdown of LhSpot in fifth instars reduced expression of ecdysone-responsive genes E74 and E75, and prevented adult development. This developmental defect was rescued following application of exogenous 20-hydroxyecdysone but not exogenous 7-dehydrocholesterol. The unequivocal RNAi effects on Ly. hesperus development and the phenotypic rescue by 20-hydroxyecdysone are causal proof of the involvement of LhSpot in ecdysteroid biosynthesis and related developmental processes, and may provide an avenue for development of new control measures against Ly. hesperus. PMID:27189651

  18. Allethrin-Based Mosquito Control Device Causing Knockdown, Morbidity, and Mortality in Four Species of Field-Caught Mosquitoes (Diptera: Culicidae).

    PubMed

    Bibbs, Christopher S; Fulcher, Ali; Xue, Rui-De

    2015-07-01

    A mosquito control device marketed for spatial repellency, the ThermaCELL Mosquito Repellent Appliance, was evaluated in semifield trials against multiple field-caught species of mosquito. Using paper and mesh cages, mosquito test groups of at least 30 mosquitoes were suspended in a 2,337 cubic foot outdoor space while two ThermaCELL repellent devices were active. After 30 min of treatment, cages were moved to the laboratory to observe knockdown, morbidity, and mortality for 24 h. Species tested included Aedes atlanticus Dyar and Knab (98% average mortality), Psorophora ferox Humboldt (97% average mortality), Psorophora columbiae Dyar and Knab (96% average mortality), and Aedes taeniorhynchus Wiedemann (84% average mortality). The repellent devices showed effectiveness with high knockdown and mortality across all species tested. Mosquito control devices like the ThermaCELL Mosquito Repellent Appliance may have further practical applications to help combat viral exposures by limiting host mosquitoes. Such devices may provide a functional alternative to DEET dependence in the current state of mosquito management.

  19. Neuromedin U receptor 2 knockdown in the paraventricular nucleus modifies behavioral responses to obesogenic high-fat food and leads to increased body weight.

    PubMed

    Benzon, C R; Johnson, S B; McCue, D L; Li, D; Green, T A; Hommel, J D

    2014-01-31

    Neuromedin U (NMU) is a highly conserved neuropeptide which regulates food intake and body weight. Transgenic mice lacking NMU are hyperphagic and obese, making NMU a novel target for understanding and treating obesity. Neuromedin U receptor 2 (NMUR2) is a high-affinity receptor for NMU found in discrete regions of the central nervous system, in particular the paraventricular nucleus of the hypothalamus (PVN), where it may be responsible for mediating the anorectic effects of NMU. We hypothesized that selective knock down of NMUR2 in the PVN of rats would increase their sensitivity to the reinforcing properties of food resulting in increased intake and preference for high-fat obesogenic food. To this end, we used viral-mediated RNAi to selectively knock down NMUR2 gene expression in the PVN. In rats fed a standard chow, NMUR2 knockdown produced no significant effect on food intake or body weight. However, when the same rats were fed a high-fat diet (45% fat), they consumed significantly more food, gained more body weight, and had increased feed efficiency relative to controls. Furthermore, NMUR2 knockdown rats demonstrated significantly greater binge-type food consumption of the high-fat diet and showed a greater preference for higher-fat food. These results demonstrate that NMUR2 signaling in the PVN regulates consumption and preference for high-fat foods without disrupting feeding behavior associated with non-obesogenic standard chow.

  20. RNA interference-mediated knockdown of the Halloween gene Spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus.

    PubMed

    Van Ekert, E; Wang, M; Miao, Y-G; Brent, C S; Hull, J J

    2016-10-01

    Ecdysteroids play a critical role in coordinating insect growth, development and reproduction. A suite of cytochrome P450 monooxygenases coded by what are collectively termed Halloween genes mediate ecdysteroid biosynthesis. In this study, we describe cloning and RNA interference (RNAi)-mediated knockdown of the CYP307B1 Halloween gene (Spookiest) in the western tarnished plant bug, Lygus hesperus. Transcripts for Ly. hesperus Spookiest (LhSpot) were amplified from all life stages and correlated well with timing of the pre-moult ecdysteroid pulse. In adults, LhSpot was amplified from heads of both genders as well as female reproductive tissues. Heterologous expression of a LhSpot fluorescent chimera in cultured insect cells co-localized with a fluorescent marker of the endoplasmic reticulum/secretory pathway. RNAi-mediated knockdown of LhSpot in fifth instars reduced expression of ecdysone-responsive genes E74 and E75, and prevented adult development. This developmental defect was rescued following application of exogenous 20-hydroxyecdysone but not exogenous 7-dehydrocholesterol. The unequivocal RNAi effects on Ly. hesperus development and the phenotypic rescue by 20-hydroxyecdysone are causal proof of the involvement of LhSpot in ecdysteroid biosynthesis and related developmental processes, and may provide an avenue for development of new control measures against Ly. hesperus.

  1. Knockdown of the candidate dyslexia susceptibility gene homolog Dyx1c1 in rodents: Effects on auditory processing, visual attention, and cortical and thalamic anatomy

    PubMed Central

    Szalkowski, Caitlin E.; Booker, Anne B.; Truong, Dongnhu T.; Threlkeld, Steven W.; Rosen, Glenn D.; Fitch, Roslyn H.

    2014-01-01

    The current study investigated the behavioral and neuroanatomical effects of embryonic knockdown of the candidate dyslexia susceptibility gene (CDSG) homolog Dyx1c1 through RNA interference in rats. Specifically, we examined long-term effects on visual attention abilities in males, in addition to assessing rapid and complex auditory processing abilities in male and, for the first time, female rats. Results replicated prior evidence of complex acoustic processing deficits in Dyx1c1 male rats, and revealed new evidence of comparable deficits in Dyx1c1 female rats. Moreover, we found new evidence that knocking down Dyx1c1 produced orthogonal impairments in visual attention in the male sub-group. Stereological analyses of male brains from prior RNA interference studies revealed that, despite consistent visible evidence of disruptions in neuronal migration (i.e., heterotopia), knockdown of Dyx1c1 did not significantly alter cortical volume, hippocampal volume, or midsagittal area of the corpus callosum (measured in a separate cohort of like-treated Dyx1c1 male rats). Dyx1c1 transfection did however lead to significant changes in medial geniculate nucleus (MGN) anatomy, with a significant shift to smaller MGN neurons in Dyx1c1 transfected animals. Combined results provide important information about the impact of Dyx1c1 on behavioral functions that parallel domains known to be affected in language impaired populations, as well as information about widespread changes to the brain following early disruption of this candidate dyslexia susceptibility gene. PMID:23594585

  2. Allethrin-Based Mosquito Control Device Causing Knockdown, Morbidity, and Mortality in Four Species of Field-Caught Mosquitoes (Diptera: Culicidae).

    PubMed

    Bibbs, Christopher S; Fulcher, Ali; Xue, Rui-De

    2015-07-01

    A mosquito control device marketed for spatial repellency, the ThermaCELL Mosquito Repellent Appliance, was evaluated in semifield trials against multiple field-caught species of mosquito. Using paper and mesh cages, mosquito test groups of at least 30 mosquitoes were suspended in a 2,337 cubic foot outdoor space while two ThermaCELL repellent devices were active. After 30 min of treatment, cages were moved to the laboratory to observe knockdown, morbidity, and mortality for 24 h. Species tested included Aedes atlanticus Dyar and Knab (98% average mortality), Psorophora ferox Humboldt (97% average mortality), Psorophora columbiae Dyar and Knab (96% average mortality), and Aedes taeniorhynchus Wiedemann (84% average mortality). The repellent devices showed effectiveness with high knockdown and mortality across all species tested. Mosquito control devices like the ThermaCELL Mosquito Repellent Appliance may have further practical applications to help combat viral exposures by limiting host mosquitoes. Such devices may provide a functional alternative to DEET dependence in the current state of mosquito management. PMID:26335485

  3. Long non-coding RNA Linc-ITGB1 knockdown inhibits cell migration and invasion in GBC-SD/M and GBC-SD gallbladder cancer cell lines.

    PubMed

    Wang, Lei; Zhang, Yunjiao; Lv, Wenjie; Lu, Jianhua; Mu, Jiasheng; Liu, Yingbin; Dong, Ping

    2015-11-01

    Gallbladder cancer is a highly aggressive malignancy with a low 5-year survival rate. Despite advances in the molecular understanding of the initiation and progression in gallbladder cancer, treatment modalities such as surgery, radiotherapy, or chemotherapy in advanced cases did not yield promising outcomes. Therefore, it is of great importance to uncover new mechanism underlying gallbladder cancer growth and metastasis. In this study, we identified a differentially expressed long intergenic non-coding RNA, linc-ITGB1, in a pair of higher and lower metastatic gallbladder cancer cell sublines. Then, the potential role of linc-ITGB1 in gallbladder cancer cell proliferation, migration, and invasion was explored using a lentivirus-mediated RNA interference system. Functional analysis showed that knockdown of linc-ITGB1 significantly inhibited gallbladder cancer cell proliferation. Moreover, cell migration and invasion were reduced by over twofold in linc-ITGB1 knockdown cells probably due to upregulation of β-catenin and downregulation of vimentin, slug, and TCF8. In conclusion, linc-ITGB1 potentially promoted gallbladder cancer invasion and metastasis by accelerating the process of epithelial-to-mesenchymal transition, and the application of RNA interference targeting linc-ITGB1 might be a potential form of gallbladder cancer treatment in advanced cases.

  4. Se-methylselenocysteine inhibits apoptosis induced by clusterin knockdown in neuroblastoma N2a and SH-SY5Y cell lines.

    PubMed

    Wang, Chao; Zeng, Zhenyu; Liu, Qiong; Zhang, Renli; Ni, Jiazuan

    2014-11-18

    Apoptosis, as a programmed cell death process, is essential for the maintenance of tissue function in organisms. Alteration of this process is linked to many diseases. Over-expression of clusterin (Clu) can antagonize apoptosis in various cells. Selenium (Se) is an essential trace element for human health. Its biological function is also associated with cell apoptosis. To explore the function of Clu and the impact of Se in the process of apoptosis, several short-hairpin RNAs (shRNA) were designed for the construction of two sets of recombinant plasmids: one set for plasmid-transfection of mouse neuroblastoma N2a cells (N2a cells); and the other set for lentiviral infection of human neuroblastoma SH-SY5Y cells (SH-SY5Y cells). These shRNAs specifically and efficiently interfered with the intracellular expression of Clu at both the mRNA and protein levels. The Clu-knockdown cells showed apoptosis-related features, including down-regulation of antioxidative capacity and the Bcl-2/Bax ratio and up-regulation of caspase-8 activity. Se-methylselenocysteine (MSC) at an optimum concentration of 1 μM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability. The results hereby imply the potentiality of Clu and Se in neuroprotection.

  5. SiRNA-mediated serotonin transporter knockdown in the dorsal raphe nucleus rescues single prolonged stress-induced hippocampal autophagy in rats.

    PubMed

    Wu, Zhong-Min; Zheng, Chun-Hua; Zhu, Zhen-Hua; Wu, Feng-Tian; Ni, Gui-Lian; Liang, Yong

    2016-01-15

    The neurobiological mechanisms underlying the development of post-traumatic stress disorder (PTSD) remain elusive. One of the hypotheses is the dysfunction of serotonin (5-HT) neurotransmission, which is critically regulated by serotonin transporter (SERT). Therefore, we hypothesized that attenuation of SERT gene expression in the hippocampus could prevent hippocampal autophagy and the development of PTSD-like behavior. To this end, we infused SLC6A4 siRNAs into the dorsal raphe nucleus (DRN) to knockdown SERT gene expression after a single prolonged stress (SPS) treatment in rats. Then, we evaluated the effects of SERT gene knockdown on anxiety-related behaviors and extinction of contextual fear memory. We also examined the histological changes and the expression of Beclin-1, LC3-I, and LC3-II in the hippocampus. We found that SPS treatment did not alter anxiety-related behaviors but prolonged the extinction of contextual fear memory, and such a behavioral phenomenon was correlated with increased hippocampal autophagy, decreased 5-HT level, and increased expression of Beclin-1 and LC3-II/LC3-I ratio in the hippocampus. Furthermore, intra-DRN infusion of SLC6A4 siRNAs promoted the extinction of contextual fear memory, prevented hippocampal autophagy, increased 5-HT level, and decreased expression of Beclin-1 and LC3-II/LC3-I ratio. These results indicated that SERT may play a critical role in the pathogenesis of hippocampal autophagy, and is likely involved in the development of PTSD.

  6. The Essential Role of H19 Contributing to Cisplatin Resistance by Regulating Glutathione Metabolism in High-Grade Serous Ovarian Cancer

    PubMed Central

    Zheng, Zhi-Guo; Xu, Hong; Suo, Sha-Sha; Xu, Xiao-Li; Ni, Mao-Wei; Gu, Lin-Hui; Chen, Wei; Wang, Liang-Yan; Zhao, Ye; Tian, Bing; Hua, Yue-Jin

    2016-01-01

    Primary and acquired drug resistance is one of the main obstacles encountered in high-grade serous ovarian cancer (HGSC) chemotherapy. Cisplatin induces DNA damage through cross-linking and long integrated non-coding RNAs (lincRNAs) play an important role in chemical induced DNA-damage response, which suggests that lincRNAs may be also associated with cisplatin resistance. However, the mechanism of long integrated non-coding RNAs (lincRNAs) acting on cisplatin resistance is not well understood. Here, we showed that expression of lin-RECK-3, H19, LUCAT1, LINC00961, and linc-CARS2-2 was enhanced in cisplatin-resistant A2780-DR cells, while transcriptome sequencing showed decreased Linc-TNFRSF19-1 and LINC00515 expression. Additionally, we verified that different H19 expression levels in HGSC tissues showed strong correlation with cancer recurrence. H19 knockdown in A2780-DR cells resulted in recovery of cisplatin sensitivity in vitro and in vivo. Quantitative proteomics analysis indicated that six NRF2-targeted proteins, including NQO1, GSR, G6PD, GCLC, GCLM and GSTP1 involved in the glutathione metabolism pathway, were reduced in H19-knockdown cells. Furthermore, H19-knockdown cells were markedly more sensitive to hydrogen-peroxide treatment and exhibited lower glutathione levels. Our results reveal a previously unknown link between H19 and glutathione metabolism in the regulation of cancer-drug resistance. PMID:27193186

  7. Mixture for Controlling Insecticide-Resistant Malaria Vectors

    PubMed Central

    Costantini, Carlo; Corbel, Vincent; Licciardi, Séverine; Dabiré, Roch K.; Lapied, Bruno; Chandre, Fabrice; Hougard, Jean-Marc

    2008-01-01

    The spread of resistance to pyrethroids in the major Afrotropical malaria vectors Anopheles gambiae s.s. necessitates the development of new strategies to control resistant mosquito populations. To test the efficacy of nets treated with repellent and insecticide against susceptible and insecticide-resistant An. gambiae mosquito populations, we impregnated mosquito bed nets with an insect repellent mixed with a low dose of organophosphorous insecticide and tested them in a rice-growing area near Bobo-Dioulasso, Burkina Faso. During the first 2 weeks posttreatment, the mixture was as effective as deltamethrin alone and was more effective at killing An. gambiae that carried knockdown resistance (kdr) or insensitive acetylcholinesterase resistance (Ace1R) genes. The mixture seemed to not kill more susceptible genotypes for the kdr or Ace1R alleles. Mixing repellents and organophosphates on bed nets could be used to control insecticide-resistant malaria vectors if residual activity of the mixture is extended and safety is verified. PMID:18976553

  8. Reduced efficacy of pyrethroid space sprays for dengue control in an area of Martinique with pyrethroid resistance.

    PubMed

    Marcombe, Sébastien; Carron, Alexandre; Darriet, Frédéric; Etienne, Manuel; Agnew, Philip; Tolosa, Michel; Yp-Tcha, Marie Michèle; Lagneau, Christophe; Yébakima, André; Corbel, Vincent

    2009-05-01

    In the Caribbean, insecticide resistance is widely developed in Aedes aegypti and represents a serious obstacle for dengue vector control. The efficacy of pyrethroid and organophosphate ultra-low volume space sprays was investigated in Martinique where Ae. aegypti has been shown to be resistant to conventional insecticides. In the laboratory, a wild-field caught population showed high levels of resistance to deltamethrin, organophosphate (naled), and pyrethrum. Simulated-field trials showed that this resistance can strongly reduce the knock-down effect and mortality of deltamethrin and synergized pyrethrins when applied by thermal-fogging. Conversely, the efficacy of naled was high against insecticide-resistant mosquitoes. Chemical analyses of nettings exposed to the treatments showed a decrease in residues over distance from release for the pyrethroids, and naled was not detected. This finding has important implications for dengue vector control and emphasizes the need to develop innovative strategies to maintain effective control of resistant Ae. aegypti populations. PMID:19407118

  9. Ephexin4 and EphA2 mediate resistance to anoikis through RhoG and phosphatidylinositol 3-kinase.

    PubMed

    Harada, Kohei; Hiramoto-Yamaki, Nao; Negishi, Manabu; Katoh, Hironori

    2011-07-15

    Disruption of cell-extracellular matrix interaction causes epithelial cells to undergo apoptosis called anoikis, and resistance to anoikis has been suggested to be a critical step for cancer cells to metastasize. EphA2 is frequently overexpressed in a variety of human cancers, and recent studies have found that overexpression of EphA2 contributes to malignant cellular behavior, including resistance to anoikis, in several different types of cancer cells. Here we show that Ephexin4, a guanine nucleotide exchange factor for the small GTPase RhoG that interacts with EphA2, plays an important role in the regulation of anoikis. Knockdown of Ephexin4 promoted anoikis in HeLa cells, and experiments using a knockdown-rescue approach showed that activation of RhoG, phosphatidylinositol 3-kinase (PI3K), and Akt was required for the Ephexin4-mediated suppression of anoikis. Indeed, Ephexin4 knockdown caused a decrease in RhoG activity and Akt phosphorylation in HeLa cells cultured in suspension. In addition, Ephexin4 was involved in the EphA2-mediated suppression of anoikis. Taken together, these results suggest that Ephexin4 mediates resistance to anoikis through activation of RhoG and PI3K downstream of EphA2. PMID:21621533

  10. Knockdown of the Dyslexia-Associated Gene Kiaa0319 Impairs Temporal Responses to Speech Stimuli in Rat Primary Auditory Cortex

    PubMed Central

    Centanni, T. M.; Booker, A. B.; Sloan, A. M.; Chen, F.; Maher, B. J.; Carraway, R. S.; Khodaparast, N.; Rennaker, R.; LoTurco, J. J.; Kilgard, M. P.

    2014-01-01

    One in 15 school age children have dyslexia, which is characterized by phoneme-processing problems and difficulty learning to read. Dyslexia is associated with mutations in the gene KIAA0319. It is not known whether reduced expression of KIAA0319 can degrade the brain's ability to process phonemes. In the current study, we used RNA interference (RNAi) to reduce expression of Kiaa0319 (the rat homolog of the human gene KIAA0319) and evaluate the effect in a rat model of phoneme discrimination. Speech discrimination thresholds in normal rats are nearly identical to human thresholds. We recorded multiunit neural responses to isolated speech sounds in primary auditory cortex (A1) of rats that received in utero RNAi of Kiaa0319. Reduced expression of Kiaa0319 increased the trial-by-trial variability of speech responses and reduced the neural discrimination ability of speech sounds. Intracellular recordings from affected neurons revealed that reduced expression of Kiaa0319 increased neural excitability and input resistance. These results provide the first evidence that decreased expression of the dyslexia-associated gene Kiaa0319 can alter cortical responses and impair phoneme processing in auditory cortex. PMID:23395846

  11. Knockdown of NogoA prevents MPP+‑induced neurotoxicity in PC12 cells via the mTOR/STAT3 signaling pathway.

    PubMed

    Zhong, Jianbin; Li, Xie; Wan, Limei; Chen, Zhibang; Zhong, Simin; Xiao, Songhua; Yan, Zhengwen

    2016-02-01

    NogoA is a myelin‑associated protein, which is important in the inhibition of axonal fiber growth and in regeneration following injury of the mammalian central nervous system. A previous study suggested that NogoA may be key in the process of Parkinson's disease (PD), which is the second most common chronic neurodegenerative disorder worldwide. The regulatory mechanism underlying the effect of NogoA on the process of PD remains to be fully elucidated. The present study aimed to investigate the effect and underlying mechanism of NogoA on cellular viability, apoptosis and autophagy induced by 1-methyl-4-phenylpyridinium (MPP+) in PC12 cells, a commonly used in vitro PD model. PC12 cells were treated with 1 mM MPP+ for 24 h and the cells were harvested for western blotting. The results demonstrated that the protien expression levels of NogoA were increased in the PC12 cells treated with MPP+. Subsequently, NogoA small interfering RNA was synthesized and transfected into PC12 cells to silence the expression of NogoA. NogoA knockdown significantly reduced the MPP+‑induced decrease in cell viability and apoptosis, detected using a cell counting kit‑8 and flow cytometric analysis, respectively. Interference in the expression of NogoA increased the MPP+‑induced decrease in mitochondrial membrane potential, determined quantitatively by flow cytometry using JC-1 dye, and the protein levels of Beclin‑1. In addition, MPP+ treatment activated the mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Knockdown of NogoA significantly inhibited the expression levels of mTOR and STAT3. Furthermore, overexpression of NogoA had similar neurotoxic effects on the PC12 cells as MPP+ treatment. Treatment with rapamycin, an inhibitor of the mTOR/STAT3 signaling pathway had a similar effect to that of NogoA knockdown in the MPP+‑treated PC12 cells. Taken together, the results from the present study demonstrated that

  12. The impact of insecticides management linked with resistance expression in Anopheles spp. populations.

    PubMed

    Silva, Guilherme Liberato da; Pereira, Thiago Nunes; Ferla, Noeli Juarez; Silva, Onilda Santos da

    2016-06-01

    The resistance of some species of Anopheles to chemical insecticides is spreading quickly throughout the world and has hindered the actions of prevention and control of malaria. The main mechanism responsible for resistance in these insects appears to be the target site known as knock-down resistance (kdr), which causes mutations in the sodium channel. Even so, many countries have made significant progress in the prevention of malaria, focusing largely on vector control through long-lasting insecticide nets (LLINs), indoor residual spraying and (IRS) of insecticides. The objective of this review is to contribute with information on the more applied insecticides for the control of the main vectors of malaria, its effects, and the different mechanisms of resistance. Currently it is necessary to look for others alternatives, e.g. biological control and products derived from plants and fungi, by using other organisms as a possible regulator of the populations of malaria vectors in critical outbreaks. PMID:27383351

  13. Inhibition of autophagy induced by PTEN loss promotes intrinsic breast cancer resistance to trastuzumab therapy.

    PubMed

    Ning, Liao; Guo-Chun, Zhang; Sheng-Li, An; Xue-Rui, Li; Kun, Wang; Jian, Zu; Chong-Yang, Ren; Ling-Zhu, Wen; Hai-Tong, Lv

    2016-04-01

    This study aims to explore the effects of the phosphatase and tension homolog (PTEN) expression level on autophagic status and on the resistance of breast cancer to trastuzumab treatment. PTEN and LC3I/II were knocked down with shRNA expression vectors, which were transfected into estrogen receptor (ER)-positive breast cancer cell lines. After trastuzumab treatment, the changes in the autophagy signal transduction pathways and autophagic proteins (LC3I/II, p62, LAMP, and cathepsin B) in these stably transfected cells were detected using western blot. The cells were also orthotopically implanted into nude mice to explore the influence of PTEN knockdown on tumor size, cell viability, and autophagic proteins after trastuzumab treatment. Similar determinations were performed using the LC3I/II overexpressed shPTEN breast cancer cells (LC3I/II-shPTEN). Downregulation of PTEN and autophagic proteins LC3-I and LC3-II was observed in resistant human breast cancer samples. Knockdown of PTEN and PTEN+ LC3I/II with shRNA in breast cancer cells resulted in increased resistance to trastuzumab. Consistently, trastuzumab treatment could not effectively reduce tumor size. Significant decreases in the levels of autophagic proteins LC3I/II, LAMP, p62, cathepsin B, and PI3K-Akt-mTOR and the signaling pathway protein Akt were found in PTEN knockdown cells, compared to the PTEN normal group, after trastuzumab administration, both in vitro and in vivo. However, these findings were reversed with the LC3I/II-shPTEN treatment. Therefore, the loss of PTEN may promote the development of primary resistance to trastuzumab in breast cancer via autophagy defects.

  14. PIAS1 is a crucial factor for prostate cancer cell survival and a valid target in docetaxel resistant cells.

    PubMed

    Puhr, Martin; Hoefer, Julia; Neuwirt, Hannes; Eder, Iris E; Kern, Johann; Schäfer, Georg; Geley, Stephan; Heidegger, Isabel; Klocker, Helmut; Culig, Zoran

    2014-12-15

    Occurrence of an inherent or acquired resistance to the chemotherapeutic drug docetaxel is a major burden for patients suffering from different kinds of malignancies, including castration resistant prostate cancer (PCa). In the present study we address the question whether PIAS1 targeting can be used to establish a basis for improved PCa treatment. The expression status and functional relevance of PIAS1 was evaluated in primary tumors, in metastatic lesions, in tissue of patients after docetaxel chemotherapy, and in docetaxel resistant cells. Patient data were complemented by functional studies on PIAS1 knockdown in vitro as well as in chicken chorioallantoic membrane and mouse xenograft in vivo models. PIAS1 was found to be overexpressed in local and metastatic PCa and its expression was further elevated in tumors after docetaxel treatment as well as in docetaxel resistant cells. Furthermore, PIAS1 knockdown experiments revealed an increased expression of tumor suppressor p21 and declined expression of anti-apoptotic protein Mcl1, which caused diminished cell proliferation and tumor growth in vitro and in vivo. In summary, the presented data indicate that PIAS1 is crucial for parental and docetaxel resistant PCa cell survival and is therefore a promising new target for treatment of primary, metastatic, and chemotherapy resistant PCa.

  15. PIAS1 is a crucial factor for prostate cancer cell survival and a valid target in docetaxel resistant cells

    PubMed Central

    Puhr, Martin; Hoefer, Julia; Neuwirt, Hannes; Eder, Iris E.; Kern, Johann; Schäfer, Georg; Geley, Stephan; Heidegger, Isabel; Klocker, Helmut; Culig, Zoran

    2014-01-01

    Occurrence of an inherent or acquired resistance to the chemotherapeutic drug docetaxel is a major burden for patients suffering from different kinds of malignancies, including castration resistant prostate cancer (PCa). In the present study we address the question whether PIAS1 targeting can be used to establish a basis for improved PCa treatment. The expression status and functional relevance of PIAS1 was evaluated in primary tumors, in metastatic lesions, in tissue of patients after docetaxel chemotherapy, and in docetaxel resistant cells. Patient data were complemented by functional studies on PIAS1 knockdown in vitro as well as in chicken chorioallantoic membrane and mouse xenograft in vivo models. PIAS1 was found to be overexpressed in local and metastatic PCa and its expression was further elevated in tumors after docetaxel treatment as well as in docetaxel resistant cells. Furthermore, PIAS1 knockdown experiments revealed an increased expression of tumor suppressor p21 and declined expression of anti-apoptotic protein Mcl1, which caused diminished cell proliferation and tumor growth in vitro and in vivo. In summary, the presented data indicate that PIAS1 is crucial for parental and docetaxel resistant PCa cell survival and is therefore a promising new target for treatment of primary, metastatic, and chemotherapy resistant PCa. PMID:25474038

  16. Connexin43 knockdown in bone marrow‑derived dendritic cells by small interfering RNA leads to a diminished T-cell stimulation.

    PubMed

    Yu, Fuling; Yan, Hui; Nie, Wencheng; Zhu, Jianhua

    2016-01-01

    Dendritic cells, the most powerful type of antigen‑presenting cells, have the unique ability to induce primary immune responses. Connexin43 expression is upregulated to increase gap junctions when immune cells are exposed to inflammatory factors. The present study applied small‑interfering RNA (siRNA) to decrease connexin43 expression. The results showed that silencing of connexin43 using siRNA resulted in arrest of bone marrow‑derived dendritic cell (BM‑DC) maturation as evidenced by reduced expression of major histocompatibility complex II, CD40, CD80 and CD86. Functionally, connexin43‑silenced BM‑DC showed a markedly decreased capability to induce T-cell stimulation. In conclusion, the present study demonstrated that antigens present on BM‑DCs can be suppressed by connexin43 knockdown in BM‑DCs. The present study therefore presented an effective method to modulate the immunology of BM‑DCs.

  17. Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

    PubMed

    Dubińska-Magiera, Magda; Chmielewska, Magdalena; Kozioł, Katarzyna; Machowska, Magdalena; Hutchison, Christopher J; Goldberg, Martin W; Rzepecki, Ryszard

    2016-05-01

    Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

  18. Expression and knockdown of the PEPC1 gene affect carbon flux in the biosynthesis of triacylglycerols by the green alga Chlamydomonas reinhardtii.

    PubMed

    Deng, Xiaodong; Cai, Jiajia; Li, Yajun; Fei, Xiaowen

    2014-11-01

    The regulation of lipid biosynthesis is important in photosynthetic eukaryotic cells. This regulation is facilitated by the direct synthesis of fatty acids and triacylglycerol (TAG), and by other controls of the main carbon metabolic pathway. In this study, knockdown of the mRNA expression of the Chlamydomonas phosphoenolpyruvate carboxylase isoform 1 (CrPEPC1) gene by RNA interference increased TAG level by 20 % but decreased PEPC activities in the corresponding transgenic algae by 39-50 %. The decrease in CrPEPC1 expression increased the expression of TAG biosynthesis-related genes, such as acyl-CoA:diacylglycerol acyltransferase and phosphatidate phosphatase. Conversely, CrPEPC1 over-expression decreased TAG level by 37 % and increased PEPC activities by 157-184 %. These observations suggest that the lipid content of algal cells can be controlled by regulating the CrPEPC1 gene. PMID:24966045

  19. Hammerhead Ribozyme-Mediated Knockdown of mRNA for Fibrotic Growth Factors: Transforming Growth Factor-Beta 1 and Connective Tissue Growth Factor

    PubMed Central

    Robinson, Paulette M.; Blalock, Timothy D.; Yuan, Rong; Lewin, Alfred S.; Schultz, Gregory S.