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Sample records for leu-phe knockdown resistance

  1. Formyl Met-Leu-Phe-Stimulated FPR1 Phosphorylation in Plate-Adherent Human Neutrophils: Enhanced Proteolysis but Lack of Inhibition by Platelet-Activating Factor

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    Algirdas J. Jesaitis

    2018-01-01

    Full Text Available N-formyl-Met-Leu-Phe (fMLF is a model PAMP/DAMP driving human PMN to sites of injury/infection utilizing the GPCR, FPR1. We examined a microtiter plate format for measurement of FPR1 phosphorylation in adherent PMN at high densities and found that a new phosphosensitive FPR1 fragment, 25K-FPR1, accumulates in SDS-PAGE extracts. 25K-FPR1 is fully inhibited by diisopropylfluorophosphate PMN pretreatment but is not physiologic, as its formation failed to be significantly perturbed by ATP depletion, time and temperature of adherence, or adherence mechanism. 25K-FPR1 was minimized by extracting fMLF-exposed PMN in lithium dodecylsulfate at 4°C prior to reduction/alkylation. After exposure of adherent PMN to a 5 log range of PAF before or after fMLF, unlike in suspension PMN, no inhibition of fMLF-induced FPR1 phosphorylation was observed. However, PAF induced the release of 40% of PMN lactate dehydrogenase, implying significant cell lysis. We infer that PAF-induced inhibition of fMLF-dependent FPR1 phosphorylation observed in suspension PMN does not occur in the unlysed adherent PMN. We speculate that although the conditions of the assay may induce PAF-stimulated necrosis, the cell densities on the plates may approach levels observed in inflamed tissues and provide for an explanation of PAF’s divergent effects on FPR1 phosphorylation as well as PMN function.

  2. Multiple origins of knockdown resistance mutations in the Afrotropical mosquito vector Anopheles gambiae.

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    João Pinto

    2007-11-01

    Full Text Available How often insecticide resistance mutations arise in natural insect populations is a fundamental question for understanding the evolution of resistance and also for modeling its spread. Moreover, the development of resistance is regarded as a favored model to study the molecular evolution of adaptive traits. In the malaria vector Anopheles gambiae two point mutations (L1014F and L1014S in the voltage-gated sodium channel gene, that confer knockdown resistance (kdr to DDT and pyrethroid insecticides, have been described. In order to determine whether resistance alleles result from single or multiple mutation events, genotyping of the kdr locus and partial sequencing of the upstream intron-1 was performed on a total of 288 A. gambiae S-form collected from 28 localities in 15 countries. Knockdown resistance alleles were found to be widespread in West Africa with co-occurrence of both 1014S and 1014F in West-Central localities. Differences in intron-1 haplotype composition suggest that kdr alleles may have arisen from at least four independent mutation events. Neutrality tests provided evidence for a selective sweep acting on this genomic region, particularly in West Africa. The frequency and distribution of these kdr haplotypes varied geographically, being influenced by an interplay between different mutational occurrences, gene flow and local selection. This has important practical implications for the management and sustainability of malaria vector control programs.

  3. Contrasting patterns of insecticide resistance and knockdown resistance (kdr) in the dengue vectors Aedes aegypti and Aedes albopictus from Malaysia.

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    Ishak, Intan H; Jaal, Zairi; Ranson, Hilary; Wondji, Charles S

    2015-03-25

    Knowledge on the extent, distribution and mechanisms of insecticide resistance is essential for successful insecticide-based dengue control interventions. Here, we report an extensive resistance profiling of the dengue vectors Aedes aegypti and Aedes albopictus across Malaysia and establish the contribution of knockdown resistance mechanism revealing significant contrast between both species. Aedes mosquitoes were collected from four states in Malaysia in 2010 using ovitraps and tested against six major insecticides using WHO bioassays. Knockdown resistance (kdr) was investigated in both species. A moderate resistance to temephos was detected from samples collected in 2010 in Penang, Kuala Lumpur, Johor Bharu and Kota Bharu (1.5 Malaysia but neither of these mutations were found in Ae. albopictus. Additionally, signatures of selection were detected on the Voltage-gated sodium channel gene in Ae. aegypti but not in Ae. albopictus. The presence of the 1534C allele was significantly associated with pyrethroid resistance and an additive effect to pyrethroid resistance was observed in individuals containing both kdr alleles. Findings from this study will help to design and implement successful insecticide-based interventions against Ae. aegypti and Ae. albopictus to improve dengue control across Malaysia.

  4. Investigating knockdown resistance (kdr) mechanism against pyrethroids/DDT in the malaria vector Anopheles funestus across Africa.

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    Irving, Helen; Wondji, Charles S

    2017-08-09

    Understanding the molecular basis of insecticide resistance is key to improve the surveillance and monitoring of malaria vector populations under control. In the major malaria vector Anopheles funestus, little is currently known about the role of the knockdown resistance (kdr) mechanism. Here, we investigated the presence and contribution of knockdown resistance (kdr) to pyrethroids/DDT resistance observed in Anopheles funestus across Africa. Pyrosequencing genotyping and sequencing of the voltage gated sodium channel (VGSC) gene did not detect the common L1014F mutation in field collected An. funestus across Africa. Amplification and cloning of the full-length of the sodium channel gene in pyrethroid resistant mosquitoes revealed evidences of alternative splicing events with three transcripts of 2092, 2061 and 2117 amino acids (93% average similarity to An. gambiae). Several amino acid changes were detected close to the domain II of the protein such as L928R, F938 W, I939S, L802S and T1008 M. However, all these mutations are found at low frequency and their role in pyrethroid resistance could not be established. The presence of the exclusive alternative splicing at exon 19 was not associated with resistance phenotype. Analysis of patterns of genetic diversity of the VGSC gene revealed a high polymorphism level of this gene across Africa with no evidence of directional selection suggesting a limited role for knockdown resistance in pyrethroid resistance in An. funestus. Patterns of genetic differentiation correlate with previous observations of the existence of barriers to gene flow Africa-wide with southern population significantly differentiated from other regions. Despite an apparent limited role of knockdown resistance in An. funestus, it is necessary to continue to monitor the contribution of the mutations detected here as increasing selection from insecticide-based interventions may change the dynamic in field populations as previously observed in other

  5. Knockdown of UbcH10 Enhances the Chemosensitivity of Dual Drug Resistant Breast Cancer Cells to Epirubicin and Docetaxel

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    Cheng Wang

    2015-03-01

    Full Text Available Breast cancer is one of the most common and lethal cancers in women. As a hub gene involved in a diversity of tumors, the ubiquitin-conjugating enzyme H10 (UbcH10, may also play some roles in the genesis and development of breast cancer. In the current study, we found that the expression of UbcH10 was up-regulated in some breast cancer tissues and five cell lines. We established a dual drug resistant cell line MCF-7/EPB (epirubicin/TXT (docetaxel and a lentiviral system expressing UbcH10 shRNA to investigate the effects of UbcH10 knockdown on the chemosensitivity of MCF-7/EPB/TXT cells to epirubicin and docetaxel. The knockdown of UbcH10 inhibited the proliferation of both MCF-7 and MCF-7/EPB/TXT cells, due to the G1 phase arrest in cell cycle. Furthermore, UbcH10 knockdown increased the sensitivity of MCF-7/EPB/TXT cells to epirubicin and docetaxel and promoted the apoptosis induced by these two drugs. Protein detection showed that, in addition to inhibiting the expression of Ki67 and cyclin D1, UbcH10 RNAi also impaired the increased BCL-2 and MDR-1 expression levels in MCF-7/EPB/TXT cells, which may contribute to abating the drug resistance in the breast cancer cells. Our research in the current study demonstrated that up-regulation of UbcH10 was involved in breast cancer and its knockdown can inhibit the growth of cancer cells and increase the chemosensitivity of the dual drug resistant breast cancer cells to epirubicin and docetaxel, suggesting that UbcH10 may be a promising target for the therapy of breast cancer.

  6. Knockdown resistance in pyrethroid-resistant horn fly (Diptera: Muscidae populations in Brazil Resistência Knockdown em populações de mosca-dos-chifres do Brasil resistentes aos piretróides

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    Gustavo A. Sabatini

    2009-09-01

    Full Text Available To investigate the kdr (knockdown resistance resistance-associated gene mutation and determine its frequency in pyrethroid-resistant horn fly (Haematobia irritans populations, a total of 1,804 horn flies of 37 different populations from all Brazilian regions (North, Northeast, Central-West, Southeast, and South were molecular screened through polymerase chain reaction (PCR. The kdr gene was not detected in 87.08% of the flies. However, the gene was amplified in 12.92% of the flies, of which 11.70% were resistant heterozygous and 1.22% were resistant homozygous. Deviation from Hardy-Weinberg equilibrium (HWE was found only in 1 ranch with an excess of heterozygous. When populations were grouped by region, three metapopulations showed significant deviations of HWE (Central-West population, South population and Southeast population. This indicates that populations are isolated one from another and kdr occurrence seems to be an independent effect probably reflecting the insecticide strategy used by each ranch. Although resistance to pyrethroids is disseminated throughout Brazil, only 48% of resistant populations had kdr flies, and the frequency of kdr individuals in each of these resistant populations was quite low. But this study shows that, with the apparent exception of the Northeast region, the kdr mechanism associated with pyrethroid resistance occurs all over Brazil.Com o objetivo de verificar a ocorrência e determinar a frequência da mutação kdr (knock down resistance em populações de Haematobia irritans (mosca-dos-chifres resistentes aos piretróides, foram analisados 1.804 indivíduos de 37 populações de todas as Regiões do Brasil. Com exceção da Região Nordeste, o kdr (knock down resistance gene foi encontrado em populações de todas as regiões. A mutação não foi detectada em 87,08% dos indivíduos. Entretanto, o gene foi amplificado de 12,92% das moscas, das quais 11,70% se mostraram heterozigotas resistentes e 1

  7. Knockdown resistance, Rdl alleles, and the annual entomological Inoculation rate of wild mosquito populations from Lower Moshi, Northern Tanzania

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    Aneth M Mahande

    2012-01-01

    Full Text Available Aim: Understanding vector behavioral response due to ecological factors is important in the control of disease vectors. This study was conducted to determine the knockdown resistance (kdr alleles, dieldrin resistance alleles, and entomological inoculation rates (EIRs of malaria vectors in lower Moshi irrigation schemes for the mitigation of disease transmission. Materials and Methods: The study was longitudinal design conducted for 14 months. Mosquitoes were collected fortnightly by using a CDC miniature light trap in 20 houses. Mosquitoes were identified morphologically in the field, of which 10% of this population was identified to species level by using molecular techniques. Samples from this study population were taken for kdr and resistance to dieldrin (rdl genes detection. Results: A total of 6220 mosquitoes were collected by using a light trap, of which 86.0% (n=5350 were Anopheles gambiae sensu lato and 14.0% (n=870 were Culex quinquefasciatus. Ten percent of the An. gambiae s.l. (n=535 collected were taken for species identification, of which 99.8% (n=534 were identified as An. arabiensis while 0.2% (n=1 were An. gambiae sensu stricto. Of the selected mosquitoes, 3.5% (n=19 were sporozoite positive. None of the mosquitoes tested had the kdr gene. The rdl resistant allele was detected at a frequency of 0.48 throughout the year. EIR was determined to be 0.54 ib/trap/year. Conclusion: The findings of this study suggest that the homozygous and the heterozygous resistance present in rdl genes demonstrated the effect of pesticide residues on resistance selection pressure in mosquitoes. A better insecticide usage protocol needs to be developed for farmers to use in order to avoid excessive use of pesticides. Key words: An. arabiensis, EIR, Knockdown mutation, Moshi, rdl locus, Tanzania

  8. Knockdown resistance (kdr) of the voltage-gated sodium channel gene of Aedes aegypti population in Denpasar, Bali, Indonesia.

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    Hamid, Penny Humaidah; Prastowo, Joko; Widyasari, Anis; Taubert, Anja; Hermosilla, Carlos

    2017-06-05

    Aedes aegypti is the main vector of several arthropod-borne viral infections in the tropics profoundly affecting humans, such as dengue fever (DF), West Nile (WN), chikungunya and more recently Zika. Eradication of Aedes still largely depends on insecticides, which is the most cost-effective strategy, and often inefficient due to resistance development in exposed Aedes populations. We here conducted a study of Ae. aegypti resistance towards several insecticides regularly used in the city of Denpasar, Bali, Indonesia. Aedes aegypti egg samples were collected with ovitraps and thereafter hatched in the insectary of the Gadjah Mada University. The F0 generation was used for all bioassay-related experiments and knockdown resistance (kdr) assays. Results clearly showed resistance development of Ae. aegypti against tested insecticides. Mortalities of Ae. aegypti were less than 90% with highest resistance observed against 0.75% permethrin. Mosquitoes from the southern parts of Denpasar presented high level of resistance pattern in comparison to those from the western and northern parts of Denpasar. Kdr analysis of voltage-gated sodium channel (Vgsc) gene showed significant association to S989P and V1016G mutations linked to resistance phenotypes against 0.75% permethrin. Conversely, Ae. aegypti F1534C gene mutation did not result in any significant correlation to resistance development. Periodically surveillance of insecticide resistances in Ae. aegypti mosquitoes will help local public health authorities to set better goals and allow proper evaluation of on-going mosquito control strategies. Initial detection of insecticide resistance will contribute to conduct proper actions in delaying mosquito resistance development such as insecticide rotation or combination of compounds in order to prolong chemical efficacy in combating Ae. aegypti vectors in Indonesia.

  9. Liraglutide increases FGF-21 activity and insulin sensitivity in high fat diet and adiponectin knockdown induced insulin resistance.

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    Mengliu Yang

    Full Text Available BACKGROUND: Liraglutide is a glucagon-like peptide-1 analogue that stimulates insulin secretion and improves β-cell function. However, it is not clear whether liraglutide achieves its glucose lowering effect only by its known effects or whether other as yet unknown mechanisms are involved. The aim of this study was to examine the effects of liraglutide on Fibroblast growth factor-21 (FGF-21 activity in High-fat diet (HFD fed ApoE(-/- mice with adiponectin (Acrp30 knockdown. METHOD: HFD-fed ApoE(-/- mice were treated with adenovirus vectors expressing shAcrp30 to produce insulin resistance. Hyperinsulinemic-euglycemic clamp studies were performed to evaluate insulin sensitivity of the mouse model. QRT-PCR and Western blot were used to measure the mRNA and protein expression of the target genes. RESULTS: The combination of HFD, ApoE deficiency, and hypoadiponectinemia resulted in an additive effect on insulin resistance. FGF-21 mRNA expressions in both liver and adipose tissues were significantly increased while FGF-21 receptor 1 (FGFR-1 and β-Klotho mRNA levels in adipose tissue, as well as FGFR-1-3 and β-Klotho mRNA levels in liver were significantly decreased in this model. Liraglutide treatment markedly improved insulin resistance and increased FGF-21 expression in liver and FGFR-3 in adipose tissue, restored β-Klotho mRNA expression in adipose tissue as well as FGFR-1-3, β-Klotho levels and phosphorylation of FGFR1 up to the levels observed in control mice in liver. Liraglutide treatment also further increased FGF-21 proteins in liver and plasma. In addition, as shown by hyperinsulinemic-euglycemic clamp, liraglutide treatment also markedly improved glucose metabolism and insulin sensitivity in these animals. CONCLUSION: These findings demonstrate an additive effect of HFD, ApoE deficiency, and adiponectin knockdown on insulin resistance and unveil that the regulation of glucose metabolism and insulin sensitivity by liraglutide may be

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

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    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. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Knockdown of long non-coding RNA Taurine Up-Regulated 1 inhibited doxorubicin resistance of bladder urothelial carcinoma via Wnt/β-catenin pathway.

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    Xie, Dalong; Zhang, Hui; Hu, Xuanhao; Shang, Chao

    2017-10-24

    In genitourinary system, bladder cancer (BC) is the most common and lethal malignant tumor, which most common type is bladder urothelial carcinoma (BUC). Long non-coding RNA (lncRNA) Taurine Up-Regulated 1 (TUG1) gene is high-expressed in several malignant tumors, including BC. In this study, over-expression of TUG1 was found in BUC tissues and cell line resistant to doxorubicin (Dox). Knockdown of TUG1 inhibited the Dox resistance and promoted the cytotoxicity induced by Dox in T24/Dox cells. TUG1 knockdown also depressed the Wnt/β-catenin pathway, and the activation the Wnt/β-catenin pathway partly reversed the inhibitory effects of TUG1 knockdown on Dox resistance in T24/Dox cells. In conclusion, up-regulation of lncRNA TUG1 was related with the poor response of BUC patients to Dox chemotherapy, knockdown of TUG1 inhibited the Dox resistance of BUC cells via Wnt/β-catenin pathway. These findings might assist in the discovery of novel potential diagnostic and therapeutic target for BUC, thereby improve the effects of clinical treatment in patients.

  12. Distribution of knock-down resistance mutations in Anopheles gambiae molecular forms in west and west-central Africa

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    Caccone Adalgisa

    2008-04-01

    Full Text Available Abstract Background Knock-down resistance (kdr to DDT and pyrethroids in the major Afrotropical vector species, Anopheles gambiae sensu stricto, is associated with two alternative point mutations at amino acid position 1014 of the voltage-gated sodium channel gene, resulting in either a leucine-phenylalanine (L1014F, or a leucine-serine (L1014S substitution. In An. gambiae S-form populations, the former mutation appears to be widespread in west Africa and has been recently reported from Uganda, while the latter, originally recorded in Kenya, has been recently found in Gabon, Cameroon and Equatorial Guinea. In M-form populations surveyed to date, only the L1014F mutation has been found, although less widespread and at lower frequencies than in sympatric S-form populations. Methods Anopheles gambiae M- and S-form specimens from 19 sites from 11 west and west-central African countries were identified to molecular form and genotyped at the kdr locus either by Hot Oligonucleotide Ligation Assay (HOLA or allele-specific PCR (AS-PCR. Results The kdr genotype was determined for about 1,000 An. gambiae specimens. The L1014F allele was found at frequencies ranging from 6% to 100% in all S-form samples (N = 628, with the exception of two samples from Angola, where it was absent, and coexisted with the L1014S allele in samples from Cameroon, Gabon and north-western Angola. The L1014F allele was present in M-form samples (N = 354 from Benin, Nigeria, and Cameroon, where both M- and S-forms were sympatric. Conclusion The results represent the most comprehensive effort to analyse the overall distribution of the L1014F and L1014S mutations in An. gambiae molecular forms, and will serve as baseline data for resistance monitoring. The overall picture shows that the emergence and spread of kdr alleles in An. gambiae is a dynamic process and that there is marked intra- and inter-form heterogeneity in resistance allele frequencies. Further studies are needed to

  13. Characterizing the insecticide resistance of Anopheles gambiae in Mali.

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    Cisse, Moussa B M; Keita, Chitan; Dicko, Abdourhamane; Dengela, Dereje; Coleman, Jane; Lucas, Bradford; Mihigo, Jules; Sadou, Aboubacar; Belemvire, Allison; George, Kristen; Fornadel, Christen; Beach, Raymond

    2015-08-22

    The impact of indoor residual spraying (IRS) and long-lasting insecticide nets (LLINs), key components of the national malaria control strategy of Mali, is threatened by vector insecticide resistance. The objective of this study was to assess the level of insecticide resistance in Anopheles gambiae sensu lato populations from Mali against four classes of insecticide recommended for IRS: organochlorines (OCs), pyrethroids (PYs), carbamates (CAs) and organophosphates (OPs). Characterization of resistance was done in 13 sites across southern Mali and assessed presence and distribution of physiological mechanisms that included target-site modifications: knockdown resistance (kdr) and altered acetycholinesterase (AChE), and/or metabolic mechanisms: elevated esterases, glutathione S-transferases (GSTs), and monooxygenases. The World Health Organization (WHO) tube test was used to determine phenotypic resistance of An. gambiae s.l. to: dichlorodiphenyltrichloroethane (DDT) (OC), deltamethrin (PY), lambda-cyhalothrin (PY), bendiocarb (CA), and fenitrothion (OP). Identification of sibling species and presence of the ace-1 (R) and Leu-Phe kdr, resistance-associated mutations, were determined using polymerase chain reaction (PCR) technology. Biochemical assays were conducted to detect increased activity of GSTs, oxidases and esterases. Populations tested showed high levels of resistance to DDT in all 13 sites, as well as increased resistance to deltamethrin and lambda-cyhalothrin in 12 out of 13 sites. Resistance to fenitrothion and bendiocarb was detected in 1 and 4 out of 13 sites, respectively. Anopheles coluzzii, An. gambiae sensu stricto and Anopheles arabiensis were identified with high allelic frequencies of kdr in all sites where each of the species were found (13, 12 and 10 sites, respectively). Relatively low allelic frequencies of ace-1 (R) were detected in four sites where this assessment was conducted. Evidence of elevated insecticide metabolism, based on oxidase

  14. A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae

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    Lynd, Amy; Ranson, Hilary; McCall, P J; Randle, Nadine P; Black, William C; Walker, Edward D; Donnelly, Martin J

    2005-01-01

    Background A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation. Methods A Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique. Results and Discussion The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous. Conclusion The method is capable of detecting both the East and West African kdr alleles in the homozygous and

  15. A simplified high-throughput method for pyrethroid knock-down resistance (kdr detection in Anopheles gambiae

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    Walker Edward D

    2005-03-01

    Full Text Available Abstract Background A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation. Methods A Hot Ligation Oligonucleotide Assay (HOLA was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique. Results and Discussion The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous. Conclusion The method is capable of detecting both the East and West African kdr alleles

  16. A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.

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    Saingamsook, Jassada; Saeung, Atiporn; Yanola, Jintana; Lumjuan, Nongkran; Walton, Catherine; Somboon, Pradya

    2017-10-10

    Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale. A multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR). The results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory. Our multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.

  17. RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells

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    L. Zhao

    2014-01-01

    Full Text Available Fanconi anemia complementation group F protein (FANCF is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.

  18. RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells

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    Zhao, L.; Li, N.; Yu, J.K.; Tang, H.T.; Li, Y.L.; He, M.; Yu, Z.J.; Bai, X.F.; Zheng, Z.H.; Wang, E.H.; Wei, M.J.

    2013-01-01

    Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer

  19. RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells

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    Zhao, L.; Li, N.; Yu, J.K.; Tang, H.T.; Li, Y.L.; He, M.; Yu, Z.J.; Bai, X.F. [Department of Pharmacology, School of Pharmacy, China Medical University, Heping Ward, Shenyang City, Liaoning (China); Zheng, Z.H.; Wang, E.H. [Institute of Pathology and Pathophysiology, China Medical University, Heping Ward, Shenyang City, Liaoning (China); Wei, M.J. [Department of Pharmacology, School of Pharmacy, China Medical University, Heping Ward, Shenyang City, Liaoning (China)

    2013-12-12

    Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.

  20. TRAF1 knockdown alleviates palmitate-induced insulin resistance in HepG2 cells through NF-κB pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wanlu [Department of Pathogen Biology, Medical College, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province (China); Tang, Zhuqi; Zhu, Xiaohui [Department of Endocrinology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province (China); Xia, Nana; Zhao, Yun; Wang, Suxin [Department of Pathogen Biology, Medical College, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu Province (China); Cui, Shiwei, E-mail: neifenmicui@163.com [Department of Endocrinology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province (China); Wang, Cuifang, E-mail: binghuodinghuo@163.com [Department of Endocrinology, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu Province (China)

    2015-11-20

    High-fat diet (HFD) and inflammation are key contributors to insulin resistance (IR) and Type 2 diabetes mellitus (T2DM). With HFD, plasma free fatty acids (FFAs) can activate the nuclear factor-κB (NF-κB) in target tissues, then initiate negative crosstalk between FFAs and insulin signaling. However, the molecular link between IR and inflammation remains to be identified. We here reported that tumor necrosis factor receptor-associated factor 1 (TRAF1), an adapter in signal transduction, was involved in the onset of IR in hepatocytes. TRAF1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAF1 led to inhibition of the activity of NF-κB. Given the fact that the activation of NF-κB played a facilitating role in IR, the phosphorylation of Akt and GSK3β was also analyzed. We found that depletion of TRAF1 markedly reversed PA-induced attenuation of the phosphorylation of Akt and GSK3β in the cells. The accumulation of lipid droplets in hepatocyte and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt and GSK3β. Glucose uptake assay indicated that knocking down TRAF1 blocked the effect of PA on the suppression of glucose uptake. These data implicated that TRAF1 knockdown might alleviate PA-induced IR in HepG2 cells through NF-κB pathway. - Highlights: • TRAF1 accelerated PA-induced IR in HepG2 cells mediated through NF-κB signaling. • Knockdown of TRAF1 alleviated PA-induced IR in HepG2 cells. • Knockdown of TRAF1 alleviated PA-induced lipid accumulation in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced suppression of glucose uptake in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced gluconeogenesis in HepG2 cells.

  1. TRAF1 knockdown alleviates palmitate-induced insulin resistance in HepG2 cells through NF-κB pathway

    International Nuclear Information System (INIS)

    Zhang, Wanlu; Tang, Zhuqi; Zhu, Xiaohui; Xia, Nana; Zhao, Yun; Wang, Suxin; Cui, Shiwei; Wang, Cuifang

    2015-01-01

    High-fat diet (HFD) and inflammation are key contributors to insulin resistance (IR) and Type 2 diabetes mellitus (T2DM). With HFD, plasma free fatty acids (FFAs) can activate the nuclear factor-κB (NF-κB) in target tissues, then initiate negative crosstalk between FFAs and insulin signaling. However, the molecular link between IR and inflammation remains to be identified. We here reported that tumor necrosis factor receptor-associated factor 1 (TRAF1), an adapter in signal transduction, was involved in the onset of IR in hepatocytes. TRAF1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAF1 led to inhibition of the activity of NF-κB. Given the fact that the activation of NF-κB played a facilitating role in IR, the phosphorylation of Akt and GSK3β was also analyzed. We found that depletion of TRAF1 markedly reversed PA-induced attenuation of the phosphorylation of Akt and GSK3β in the cells. The accumulation of lipid droplets in hepatocyte and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt and GSK3β. Glucose uptake assay indicated that knocking down TRAF1 blocked the effect of PA on the suppression of glucose uptake. These data implicated that TRAF1 knockdown might alleviate PA-induced IR in HepG2 cells through NF-κB pathway. - Highlights: • TRAF1 accelerated PA-induced IR in HepG2 cells mediated through NF-κB signaling. • Knockdown of TRAF1 alleviated PA-induced IR in HepG2 cells. • Knockdown of TRAF1 alleviated PA-induced lipid accumulation in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced suppression of glucose uptake in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced gluconeogenesis in HepG2 cells.

  2. Dynamics of multiple insecticide resistance in the malaria vector Anopheles gambiae in a rice growing area in South-Western Burkina Faso

    Directory of Open Access Journals (Sweden)

    Ouédraogo Jean-Bosco

    2008-09-01

    Full Text Available Abstract Background Insecticide resistance of the main malaria vector, Anopheles gambiae, has been reported in south-western Burkina Faso, West Africa. Cross-resistance to DDT and pyrethroids was conferred by alterations at site of action in the sodium channel, the Leu-Phe kdr mutation; resistance to organophosphates and carbamates resulted from a single point mutation in the oxyanion hole of the acetylcholinesterase enzyme designed as ace-1R. Methods An entomological survey was carried out during the rainy season of 2005 at Vallée du Kou, a rice growing area in south-western Burkina Faso. At the Vallée du Kou, both insecticide resistance mechanisms have been previously described in the M and S molecular forms of An. gambiae. This survey aimed i to update the temporal dynamics and the circumsporozoite infection rate of the two molecular forms M and S of An. gambiae ii to update the frequency of the Leu-Phe kdr mutation within these forms and finally iii to investigate the occurrence of the ace-1R mutation. Mosquitoes collected by indoor residual collection and by human landing catches were counted and morphologically identified. Species and molecular forms of An. gambiae, ace-1R and Leu-Phe kdr mutations were determined using PCR techniques. The presence of the circumsporozoite protein of Plasmodium falciparum was determined using ELISA. Results Anopheles gambiae populations were dominated by the M form. However the S form occurred in relative important proportion towards the end of the rainy season with a maximum peak in October at 51%. Sporozoite rates were similar in both forms. The frequency of the Leu-Phe kdr mutation in the S form reached a fixation level while it is still spreading in the M form. Furthermore, the ace-1R mutation prevailed predominately in the S form and has just started spreading in the M form. The two mutations occurred concomitantly both in M and S populations. Conclusion These results showed that the Vallée du Kou

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

    Science.gov (United States)

    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.

  4. A novel amino acid substitution in a voltage-gated sodium channel is associated with knockdown resistance to permethrin in Aedes aegypti.

    Science.gov (United States)

    Chang, Cheng; Shen, Wen-Kai; Wang, Tzu-Ting; Lin, Ying-Hsi; Hsu, Err-Lieh; Dai, Shu-Mei

    2009-04-01

    To identify pertinent mutations associated with knockdown resistance to permethrin, the entire coding sequence of the voltage-gated sodium channel gene Aa-para was sequenced and analyzed from a Per-R strain with 190-fold resistance to permethrin and two susceptible strains of Aedes aegypti. The longest transcript, a 6441bp open reading frame, encodes 2147 amino acid residues with an estimated molecular mass of 241kDa. A total of 33 exons were found in the Aa-para gene over 293kb of genomic DNA. Three previously unreported optional exons were identified. The first two exons, m and n, were located within the intracellular domain I/II, and the third, f', was found within the II/III linkers. The two mutually exclusive exons, d and l, were the only alternative exons in all the cDNA clones sequenced in this study. The most distinct finding was a novel amino acid substitution mutation, D1794Y, located within the extracellular linker between IVS5 and IVS6, which is concurrent with the known V1023G mutation in Aa-para of the Per-R strain. The high frequency and coexistence of the two mutations in the Per-R strain suggest that they might exert a synergistic effect to provide the knockdown resistance to permethrin. Furthermore, both cDNA and genomic DNA data from the same individual mosquitoes have demonstrated that RNA editing was not involved in amino acid substitutions of the Per-R strain.

  5. Overcoming drug resistance of MCF-7/ADR cells by altering intracellular distribution of doxorubicin via MVP knockdown with a novel siRNA polyamidoamine-hyaluronic acid complex.

    Science.gov (United States)

    Han, Min; Lv, Qing; Tang, Xin-Jiang; Hu, Yu-Lan; Xu, Dong-Hang; Li, Fan-Zhu; Liang, Wen-Quan; Gao, Jian-Qing

    2012-10-28

    Drug resistance is one of the critical reasons leading to failure in chemotherapy. Enormous studies have been focused on increasing intracellular drug accumulation through inhibiting P-glycoprotein (Pgp). Meanwhile, we found that major vault protein (MVP) may be also involved in drug resistance of human breast cancer MCF-7/ADR cells by transporting doxorubicin (DOX) from the action target (i.e. nucleus) to cytoplasma. Herein polyamidoamine (PAMAM) dendrimers was functionalized by a polysaccharide hyaluronic acid (HA) to effectively deliver DOX as well as MVP targeted small-interfering RNA (MVP-siRNA) to down regulate MVP expression and improve DOX chemotherapy in MCF-7/ADR cells. In comparison with DOX solution (IC50=48.5 μM), an enhanced cytotoxicity could be observed for DOX PAMAM-HA (IC50=11.3 μM) as well as enhanced tumor target, higher intracellular accumulation, increased blood circulating time and less in vivo toxicity. Furthermore, codelivery of siRNA and DOX by PAMAM-HA exhibited satisfactory gene silencing effect as well as enhanced stability and efficient intracellular delivery of siRNA, which allowed DOX access to nucleus and induced subsequent much more cytotoxicity than siRNA absent case as a result of MVP knockdown. This observation highlights a promising application of novel nanocarrier PAMAM-HA, which could co-deliver anticancer drug and siRNA, in reversing drug resistance by altering intracellular drug distribution. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Lol, Juan C; Castellanos, María E; Liebman, Kelly A; Lenhart, Audrey; Pennington, Pamela M; Padilla, Norma R

    2013-09-18

    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. 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). 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). 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.

  7. Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection.

    Science.gov (United States)

    Singh, Om P; Dykes, Cherry L; Lather, Manila; Agrawal, Om P; Adak, Tridibes

    2011-03-14

    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 pyrethroid-group of insecticides. The most common mutation(s) associated with knockdown resistance in insects, including anophelines, has been reported to be present at residue Leu1014 in the IIS6 transmembrane segment of the vgsc gene. This study reports the presence of two alternative kdr-like mutations, L1014S and L1014F, at this residue in a major malaria vector Anopheles stephensi and describes new PCR assays for their detection. Part of the vgsc (IIS4-S5 linker-to-IIS6 transmembrane segment) of An. stephensi collected from Alwar (Rajasthan, India) was PCR-amplified from genomic DNA, sequenced and analysed for the presence of deduced amino acid substitution(s). Analysis of DNA sequences revealed the presence of two alternative non-synonymous point mutations at L1014 residue in the IIS6 transmembrane segment of vgsc, i.e., T>C mutation on the second position and A>T mutation on the third position of the codon, leading to Leu (TTA)-to-Ser (TCA) and -Phe (TTT) amino acid substitutions, respectively. Polymerase chain reaction (PCR) assays were developed for identification of each of these two point mutations. Genotyping of An. stephensi mosquitoes from Alwar by PCR assays revealed the presence of both mutations, with a high frequency of L1014S. The PCR assays developed for detection of the kdr mutations were specific as confirmed by DNA sequencing of PCR-genotyped samples. Two alternative kdr-like mutations, L1014S and L1014F, were detected in An. stephensi with a high allelic frequency of L1014S. The occurrence of L1014S is being reported for the first time in An. stephensi. Two specific PCR assays were developed for detection of two kdr-like mutations in An. stephensi.

  8. First detection of multiple knockdown resistance (kdr)-like mutations in voltage-gated sodium channel using three new genotyping methods in Anopheles sinensis from Guangxi Province, China.

    Science.gov (United States)

    Tan, Wei L; Li, Chun X; Wang, Zhong M; Liu, Mei D; Dong, Yan D; Feng, Xiang Y; Wu, Zhi M; Guo, Xiao X; Xing, Dan; Zhang, Ying M; Wang, Zhong C; Zhao, Tong Y

    2012-09-01

    To investigate knockdown resistance (kdr)-like mutations associated with pyrethroid resistance in Anopheles sinensis (Wiedemann, 1828), from Guangxi province, southwest China, a segment of a sodium channel gene was sequenced and genotyped using three new genotyping assays. Direct sequencing revealed the presence of TTG-to-TCG and TG-to-TTT mutations at allele position L1014, which led to L1014S and L1014F substitutions in a few individual and two novel substitutions of N1013S and L1014W in two DNA templates. A low frequency of the kdr allele mostly in the heterozygous state of L1014S and L1014F was observed in this mosquito population. In this study, the genotyping of An. sinensis using three polymerase chain reaction-based methods generated consistent results, which agreed with the results of DNA sequencing. In total, 52 mosquitoes were genotyped using a direct sequencing assay. The number of mosquitoes and their genotypes were as follows: L/L = 24, L/S = 19, L/F = 8, and F/W = 1. The allelic frequency of L1014, 1014S, and 1014F were 72, 18, and 9%, respectively.

  9. Identification of TCT, a novel knockdown resistance allele mutation and analysis of resistance detection methods in the voltage-gated Na⁺ channel of Culex pipiens pallens from Shandong Province, China.

    Science.gov (United States)

    Liu, Hong-Mei; Cheng, Peng; Huang, Xiaodan; Dai, Yu-Hua; Wang, Hai-Fang; Liu, Li-Juan; Zhao, Yu-Qiang; Wang, Huai-Wei; Gong, Mao-Qing

    2013-02-01

    The present study aimed to investigate deltamethrin resistance in Culex pipiens pallens (C. pipiens pallens) mosquitoes and its correlation with knockdown resistance (kdr) mutations. In addition, mosquito‑resistance testing methods were analyzed. Using specific primers in polymerase chain reaction (PCR) and allele-specific (AS)-PCR, kdr gene sequences isolated from wild C. pipiens pallens mosquitoes were sequenced. Linear regression analysis was used to determine the correlation between the mutations and deltamethrin resistance. A kdr allelic gene was cloned and sequenced. Analysis of the DNA sequences revealed the presence of two point mutations at the L1014 residue in the IIS6 transmembrane segment of the voltage‑gated sodium channel (VGSC): L1014F, TTA→TTT, replacing a leucine (L) with a phenylalanine (F); L1014S, TTA→TCA, replacing leucine (L) with serine (S). Two alternative kdr-like mutations, L1014F and L1014S, were identified to be positively correlated with the deltamethrin-resistant phenotype. In addition a novel mutation, TCT, was identified in the VGSC of C. pipiens pallens. PCR and AS-PCR yielded consistent results with respect to mosquito resistance. However, the detection rate of PCR was higher than that of AS-PCR. Further studies are required to determine the specific resistance mechanism. PCR and AS-PCR demonstrated suitability for mosquito resistance field tests, however, the former method may be superior to the latter.

  10. HIV-1 Resistant CDK2-Knockdown Macrophage-Like Cells Generated from 293T Cell-Derived Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Kuan-Teh Jeang

    2012-07-01

    Full Text Available A major challenge in studies of human diseases involving macrophages is low yield and heterogeneity of the primary cells and limited ability of these cells for transfections and genetic manipulations. To address this issue, we developed a simple and efficient three steps method for somatic 293T cells reprogramming into monocytes and macrophage-like cells. First, 293T cells were reprogrammed into induced pluripotent stem cells (iPSCs through a transfection-mediated expression of two factors, Oct-4 and Sox2, resulting in a high yield of iPSC. Second, the obtained iPSC were differentiated into monocytes using IL-3 and M-CSF treatment. And third, monocytes were differentiated into macrophage-like cells in the presence of M-CSF. As an example, we developed HIV-1-resistant macrophage-like cells from 293T cells with knockdown of CDK2, a factor critical for HIV-1 transcription. Our study provides a proof-of-principle approach that can be used to study the role of host cell factors in HIV-1 infection of human macrophages.

  11. Polymorphism of intron-1 in the voltage-gated sodium channel gene of Anopheles gambiae s.s. populations from Cameroon with emphasis on insecticide knockdown resistance mutations.

    Science.gov (United States)

    Etang, Josiane; Vicente, Jose L; Nwane, Philippe; Chouaibou, Mouhamadou; Morlais, Isabelle; Do Rosario, Virgilio E; Simard, Frederic; Awono-Ambene, Parfait; Toto, Jean Claude; Pinto, Joao

    2009-07-01

    Sequence variation at the intron-1 of the voltage-gated sodium channel gene in Anopheles gambiae M- and S-forms from Cameroon was assessed to explore the number of mutational events originating knockdown resistance (kdr) alleles. Mosquitoes were sampled between December 2005 and June 2006 from three geographical areas: (i) Magba in the western region; (ii) Loum, Tiko, Douala, Kribi, and Campo along the Atlantic coast; and (iii) Bertoua, in the eastern continental plateau. Both 1014S and 1014F kdr alleles were found in the S-form with overall frequencies of 14% and 42% respectively. Only the 1014F allele was found in the M-form at lower frequency (11%). Analysis of a 455 bp region of intron-1 upstream the kdr locus revealed four independent mutation events originating kdr alleles, here named MS1 -1014F, S1-1014S and S2-1014S kdr-intron-1 haplotypes in S-form and MS3-1014F kdr-intron-1 haplotype in the M-form. Furthermore, there was evidence for mutual introgression of kdr 1014F allele between the two molecular forms, MS1 and MS3 being widely shared by them. Although no M/S hybrid was observed in analysed samples, this wide distribution of haplotypes MS1 and MS3 suggests inter-form hybridizing at significant level and emphasizes the rapid diffusion of the kdr alleles in Africa. The mosaic of genetic events found in Cameroon is representative of the situation in the West-Central African region and highlights the importance of evaluating the spatial and temporal evolution of kdr alleles for a better management of insecticide resistance.

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

    Science.gov (United States)

    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

  13. Shell Buckling Knockdown Factors

    Data.gov (United States)

    National Aeronautics and Space Administration — The Shell Buckling Knockdown Factor (SBKF) Project, NASA Engineering and Safety Center (NESC) Assessment #: 07-010-E, was established in March of 2007 by the NESC in...

  14. [Knockdown of DNA-PKcs inhibits cell cycle and its mechanism of drug-resistant Bel7402/5-Fu hepatocellular carcinoma cells].

    Science.gov (United States)

    Li, Dayu; Liu, Yun; Yu, Chunbo; Liu, Xiping; Fan, Fang

    2017-12-01

    Objective To study the effect of the knock-down of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) on the cell cycle of the multidrug-resistant (MDR) Bel7402/5-Fu hepatocellular carcinoma cells and its MDR mechanism. Methods After cationic liposome-mediated siDNA-PKcs oligonucleotide transfection, the drug sensitivity of Bel7402/5-Fu cells to 5-fluorouracil (5-Fu) and adriamycin (ADM) was determined by MTT assay; the cell cycle were detected by flow cytometry; meanwhile, the protein expressions of cell cycle-related proteins P21, cell cycle protein B1 (cyclin B1), cell cycle division protein 2 (CDC2) were tested by Western blotting; the expressions of ataxia telangiectasia mutated (ATM) and p53 at both mRNA and protein levels were detected by real-time PCR and Western blot analysis. Results The MTT results showed siDNA-PKcs increased the chemotherapeutic sensitivity of Bel7402/5-Fu cells to 5-Fu and ADM. The flow cytometric analysis showed siDNA-PKcs decreased the percentage of S-phase cells but increased the percentage of G2/M phase cells. Western blotting showed siDNA-PKcs increased the protein expression of P21 but decreased cyclinB1 and CDC2 proteins. In addition, siDNA-PKcs also increased the expressions of ATM and p53. Conclusion DNA-PKcs silencing increases P21 while decreases cyclin B1 and CDC2 expressions, and finally induces G2/M phase arrest in Bel7402/5-Fu cells, which may be related to ATM-p53 signaling pathway.

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

    Science.gov (United States)

    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 protocolo estándar de la OMS. Mediante un diagnóstico molecular basado en la PCR se genotiparon los mosquitos y se detectaron los genotipos kdr. Resultados Los An. gambiae evaluados eran resistentes a lambdacialotrina en Muheza, Arumeru y Muleba. De 350 An. gambiae s.l. genotipados, 35% eran An. gambiae s.s. y 65% eran An. arabiensis. Se detectaron mutaciones L1014S y L1014F tanto en An. gambiae s.s. como en An. arabiensis. La mutación puntual L1014S se encontró con una frecuencia alélica de 4-33%, mientras que L1014F tenía una frecuencia alélica de 6-14%. La mutación L1014S estaba ampliamente asociada a An. gambiae s.s. (Chi-Cuadrado = 23.41; P < 0.0001) y la L1014F estaba asociada con An. arabiensis (Chi-Square = 11.21; P = 0.0008). El alelo L1014S estaba significativamente asociado con mosquitos resistentes a la lambdacialotrina (P < 0.001). Conclusión La

  16. Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

    Science.gov (United States)

    Bass, Chris; Nikou, Dimitra; Donnelly, Martin J; Williamson, Martin S; Ranson, Hilary; Ball, Amanda; Vontas, John; Field, Linda M

    2007-01-01

    Background Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals). Results The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot

  17. Identification of an alternative knockdown resistance (kdr)-like mutation, M918L, and a novel mutation, V1010A, in the Thrips tabaci voltage-gated sodium channel gene.

    Science.gov (United States)

    Wu, Meixiang; Gotoh, Hiroki; Waters, Timothy; Walsh, Douglas B; Lavine, Laura Corley

    2014-06-01

    Knockdown resistance (kdr) has been identified as a main mechanism against pyrethroid insecticides in many arthropod pests including in the onion thrips, Thrips tabaci. To characterize and identify pyrethroid-resistance in onion thrips in Washington state, we conducted insecticide bioassays and sequenced a region of the voltage gated sodium channel gene from several different T. tabaci populations. Field collected Thrips tabaci were found to have large variations in resistance to the pyrethroid insecticide lambda-cyhalothrin. We identified two single nucleotide substitutions in our analysis of a partial sequence of the T. tabaci voltage-gated sodium channel gene. One mutation resulted in the non-synonymous substitution of methionine with leucine (M918L), which is well known to be responsible for super knockdown resistance in some pest species. Another non-synonymous substitution, a valine (GTT) to alanine (GCT) replacement at amino acid 1010 (V1010A) was identified in our study and was associated with lambda-cyhalothrin resistance. We have characterized a known kdr mutation and identified a novel mutation in the voltage-gated sodium channel gene of Thrips tabaci associated with resistance to lambda-cyhalothrin. This gene region and these mutations are expected to be useful in the development of a diagnostic test to detect kdr resistance in many onion thrips populations. © 2013 Society of Chemical Industry.

  18. Detection of knockdown resistance (kdr mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

    Directory of Open Access Journals (Sweden)

    Ball Amanda

    2007-08-01

    Full Text Available Abstract Background Knockdown resistance (kdr is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods Fluorescence-based assays based on 1 TaqMan probes and 2 high resolution melt (HRM analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR, Heated Oligonucleotide Ligation Assay (HOLA, Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost, and safety (requirement for hazardous chemicals. Results The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions and the most specific (with the lowest number of incorrect scores. Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS

  19. 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.

    Science.gov (United States)

    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

  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.

    Science.gov (United States)

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

    2015-07-23

    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.

  1. CERKL knockdown causes retinal degeneration in zebrafish.

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    Marina Riera

    Full Text Available 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.

  2. Dual knockdown of N-ras and epiregulin synergistically suppressed the growth of human hepatoma cells

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    Zhao, Meng; He, Hong-wei; Sun, Huan-xing; Ren, Kai-huan [Department of Oncology, Institute of Medicinal Biotechnology, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100050 (China); Shao, Rong-guang, E-mail: shaor@bbn.cn [Department of Oncology, Institute of Medicinal Biotechnology, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100050 (China)

    2009-09-18

    Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, we evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.

  3. Alpha2,3-sialyltransferase III knockdown sensitized ovarian cancer cells to cisplatin-induced apoptosis.

    Science.gov (United States)

    Wang, Xiaoyu; Zhang, Yiting; Lin, Haiyingjie; Liu, Yan; Tan, Yi; Lin, Jie; Gao, Fenze; Lin, Shaoqiang

    2017-01-22

    Emerging evidence indicates that β-galactoside-α2,3-sialyltransferase III (ST3Gal3) involves in development, inflammation, neoplastic transformation, and metastasis. However, the role of ST3Gal3 in regulating cancer chemoresistance remains elusive. Herein, we investigated the functional effects of ST3Gal3 in cisplatin-resistant ovarian cancer cells. We found that the levels of ST3Gal3 mRNA differed significantly among ovarian cancer cell lines. HO8910PM cells that have high invasive and metastatic capacity express elevated ST3Gal3 mRNA and are resistant to cisplatin, comparing to SKOV3 cells that have a lower level of ST3Gal3 expression and are more chemosensitive to cisplatin. We found that the expression of ST3Gal3 has reverse correlation with the dosage of cisplatin used in both SKOV3 and HO8910PM cells, and high dose of cisplatin could down-regulate ST3Gal3 expression. We then examined the functional effects of ST3Gal3 knockdown in cancer cell lines using FACS analysis. The number of apoptotic cells was much higher in cells if ST3Gal3 expression was knocked down by siRNA and/or by treating cells with higher dosage of cisplatin in comparison to control cells. Interestingly, in HO8910PM cells with ST3Gal3 knockdown, the levels of caspase 8 and caspase 3 proteins increased, which was more obvious in cells treated with both ST3Gal3 knockdown and cisplatin, suggesting that ST3Gal3 knockdown synergistically enhanced cisplatin-induced apoptosis in ovarian cancer cells. Taken together, these results uncover an alternative mechanism of cisplatin-resistance through ST3Gal3 and open a window for effective prevention of chemoresistance and relapse of ovarian cancer by targeting ST3Gal3. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Differentiation of breast cancer stem cells by knockdown of CD44: promising differentiation therapy

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    Pham Phuc V

    2011-12-01

    Full Text Available Abstract Background Breast cancer stem cells (BCSCs are the source of breast tumors. Compared with other cancer cells, cancer stem cells show high resistance to both chemotherapy and radiotherapy. Targeting of BCSCs is thus a potentially promising and effective strategy for breast cancer treatment. Differentiation therapy represents one type of cancer stem-cell-targeting therapy, aimed at attacking the stemness of cancer stem cells, thus reducing their chemo- and radioresistance. In a previous study, we showed that down-regulation of CD44 sensitized BCSCs to the anti-tumor agent doxorubicin. This study aimed to determine if CD44 knockdown caused BCSCs to differentiate into breast cancer non-stem cells (non-BCSCs. Methods We isolated a breast cancer cell population (CD44+CD24- cells from primary cultures of malignant breast tumors. These cells were sorted into four sub-populations based on their expression of CD44 and CD24 surface markers. CD44 knockdown in the BCSC population was achieved using small hairpin RNA lentivirus particles. The differentiated status of CD44 knock-down BCSCs was evaluated on the basis of changes in CD44+CD24- phenotype, tumorigenesis in NOD/SCID mice, and gene expression in relation to renewal status, metastasis, and cell cycle in comparison with BCSCs and non-BCSCs. Results Knockdown of CD44 caused BCSCs to differentiate into non-BCSCs with lower tumorigenic potential, and altered the cell cycle and expression profiles of some stem cell-related genes, making them more similar to those seen in non-BCSCs. Conclusions Knockdown of CD44 is an effective strategy for attacking the stemness of BCSCs, resulting in a loss of stemness and an increase in susceptibility to chemotherapy or radiation. The results of this study highlight a potential new strategy for breast cancer treatment through the targeting of BCSCs.

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

    Science.gov (United States)

    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

  6. Enhanced toxic cloud knockdown spray system for decontamination applications

    Science.gov (United States)

    Betty, Rita G [Rio Rancho, NM; Tucker, Mark D [Albuquerque, NM; Brockmann, John E [Albuquerque, NM; Lucero, Daniel A [Albuquerque, NM; Levin, Bruce L [Tijeras, NM; Leonard, Jonathan [Albuquerque, NM

    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.

  7. Knockdown of angiopoietin-like 2 mimics the benefits of intermittent fasting on insulin responsiveness and weight loss.

    Science.gov (United States)

    Martel, Cécile; Pinçon, Anthony; Bélanger, Alexandre Maxime; Luo, Xiaoyan; Gillis, Marc-Antoine; de Montgolfier, Olivia; Thorin-Trescases, Nathalie; Thorin, Éric

    2018-01-01

    Angiopoietin-like 2 (ANGPTL2) is an inflammatory adipokine linking obesity to insulin resistance. Intermittent fasting, on the other hand, is a lifestyle intervention able to prevent obesity and diabetes but difficult to implement and maintain. Our objectives were to characterize a link between ANGPTL2 and intermittent fasting and to investigate whether the knockdown of ANGPTL2 reproduces the benefits of intermittent fasting on weight gain and insulin responsiveness in knockdown and wild-type littermates mice. Intermittent fasting, access to food ad libitum once every other day, was initiated at the age of three months and maintained for four months. Intermittent fasting decreased by 63% (p < 0.05) gene expression of angptl2 in adipose tissue of wild-type mice. As expected, intermittent fasting improved insulin sensitivity (p < 0.05) and limited weight gain (p < 0.05) in wild-type mice. Knockdown mice fed ad libitum, however, were comparable to wild-type mice following the intermittent fasting regimen: insulin sensitivity and weight gain were identical, while intermittent fasting had no additional impact on these parameters in knockdown mice. Energy intake was similar between both wild-type fed intermittent fasting and ANGPTL2 knockdown mice fed ad libitum, suggesting that intermittent fasting and knockdown of ANGPTL2 equally lower feeding efficiency. These results suggest that the reduction of ANGPTL2 could be a useful and promising strategy to prevent obesity and insulin resistance, although further investigation of the mechanisms linking ANGPTL2 and intermittent fasting is warranted. Impact statement Intermittent fasting is an efficient diet pattern to prevent weight gain and improve insulin sensitivity. It is, however, a difficult regimen to follow and compliance is expected to be very low. In this work, we demonstrate that knockdown of ANGPTL2 in mice fed ad libitum mimics the beneficial effects of intermittent fasting on weight gain and insulin

  8. Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction

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    Renier Myburgh

    2014-01-01

    Full Text Available Gene knockdown using micro RNA (miRNA-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knockdown through optimizing the structure of miRNA mimics. Knockdown of two target genes was analyzed: CCR5 and green fluorescent protein. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs (bp, positioning of the targeting strand on the 5′ side of the miRNA, together with nucleotide mismatches in upper stem positions 1 and 12 placed on the passenger strand. Our mirGE proved superior to miR-30 in four aspects: yield of targeting strand incorporation into RNA-induced silencing complex (RISC; incorporation into RISC of correct targeting strand; precision of cleavage by Drosha; and ratio of targeting strand over passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knockdown with an efficiency of over 90% upon single-copy transduction. Importantly, single-copy expression of this construct rendered transduced target cells, including primary human macrophages, resistant to infection with a CCR5-tropic strain of HIV. Our results provide new insights for a better knockdown efficiency of constructs containing miRNA. Our results also provide the proof-of-principle that cells can be rendered HIV resistant through single-copy vector transduction, rendering this approach more compatible with clinical applications.

  9. Insecticidal potency of RNAi-based catalase knockdown in Rhynchophorus ferrugineus (Oliver) (Coleoptera: Curculionidae).

    Science.gov (United States)

    Al-Ayedh, Hassan; Rizwan-Ul-Haq, Muhammad; Hussain, Abid; Aljabr, Ahmed M

    2016-11-01

    Palm trees around the world are prone to notorious Rhynchophorus ferrugineus, which causes heavy losses of palm plantations. In Middle Eastern countries, this pest is a major threat to date palm orchards. Conventional pest control measures with the major share of synthetic insecticides have resulted in insect resistance and environmental issues. Therefore, in order to explore better alternatives, the RNAi approach was employed to knock down the catalase gene in fifth and tenth larval instars with different dsRNA application methods, and their insecticidal potency was studied. dsRNA of 444 bp was prepared to knock down catalase in R. ferrugineus. Out of the three dsRNA application methods, dsRNA injection into larvae was the most effective, followed by dsRNA application by artificial feeding. Both methods resulted in significant catalase knockdown in various tissues, especially the midgut. As a result, the highest growth inhibition of 123.49 and 103.47% and larval mortality of 80 and 40% were observed in fifth-instar larvae, whereas larval growth inhibition remained at 86.83 and 69.08% with larval mortality at 30 and 10% in tenth-instar larvae after dsRNA injection and artificial diet treatment. The topical application method was the least efficient, with the lowest larval growth inhibition of 57.23 and 45.61% and 0% mortality in fifth- and tenth-instar larvae. Generally, better results were noted at the high dsRNA dose of 5 µL. Catalase enzyme is found in most insect body tissues, and thus its dsRNA can cause broad-scale gene knockdown within the insect body, depending upon the application method. Significant larval mortality and growth inhibition after catalase knockdown in R. ferrugineus confirms its insecticidal potency and suggests a bright future for RNAi-based bioinsecticides in pest control. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  10. Ribosomal protein gene knockdown causes developmental defects in zebrafish.

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    Tamayo Uechi

    Full Text Available 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.

  11. DJ-1 KNOCK-DOWN IMPAIRS ASTROCYTE MITOCHONDRIAL FUNCTION

    Science.gov (United States)

    LARSEN, N. J.; AMBROSI, G.; MULLETT, S. J.; BERMAN, S. B.; HINKLE, D. A.

    2012-01-01

    Mitochondrial dysfunction has long been implicated in the pathogenesis of Parkinson’s disease (PD). PD brain tissues show evidence for mitochondrial respiratory chain Complex I deficiency. Pharmacological inhibitors of Complex I, such as rotenone, cause experimental parkinsonism. The cytoprotective protein DJ-1, whose deletion is sufficient to cause genetic PD, is also known to have mitochondria-stabilizing properties. We have previously shown that DJ-1 is over-expressed in PD astrocytes, and that DJ-1 deficiency impairs the capacity of astrocytes to protect co-cultured neurons against rotenone. Since DJ-1 modulated, astrocyte-mediated neuroprotection against rotenone may depend upon proper astrocytic mitochondrial functioning, we hypothesized that DJ-1 deficiency would impair astrocyte mitochondrial motility, fission/fusion dynamics, membrane potential maintenance, and respiration, both at baseline and as an enhancement of rotenone-induced mitochondrial dysfunction. In astrocyte-enriched cultures, we observed that DJ-1 knock-down reduced mitochondrial motility primarily in the cellular processes of both untreated and rotenone treated cells. In these same cultures, DJ-1 knock-down did not appreciably affect mitochondrial fission, fusion, or respiration, but did enhance rotenone-induced reductions in the mitochondrial membrane potential. In neuron–astrocyte co-cultures, astrocytic DJ-1 knock-down reduced astrocyte process mitochondrial motility in untreated cells, but this effect was not maintained in the presence of rotenone. In the same co-cultures, astrocytic DJ-1 knock-down significantly reduced mitochondrial fusion in the astrocyte cell bodies, but not the processes, under the same conditions of rotenone treatment in which DJ-1 deficiency is known to impair astrocyte-mediated neuroprotection. Our studies therefore demonstrated the following new findings: (i) DJ-1 deficiency can impair astrocyte mitochondrial physiology at multiple levels, (ii) astrocyte

  12. Heritable and lineage-specific gene knockdown in zebrafish embryo.

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    Mei Dong

    Full Text Available BACKGROUND: Reduced expression of developmentally important genes and tumor suppressors due to haploinsufficiency or epigenetic suppression has been shown to contribute to the pathogenesis of various malignancies. However, methodology that allows spatio-temporally knockdown of gene expression in various model organisms such as zebrafish has not been well established, which largely limits the potential of zebrafish as a vertebrate model of human malignant disorders. PRINCIPAL FINDING: Here, we report that multiple copies of small hairpin RNA (shRNA are expressed from a single transcript that mimics the natural microRNA-30e precursor (mir-shRNA. The mir-shRNA, when microinjected into zebrafish embryos, induced an efficient knockdown of two developmentally essential genes chordin and alpha-catenin in a dose-controllable fashion. Furthermore, we designed a novel cassette vector to simultaneously express an intronic mir-shRNA and a chimeric red fluorescent protein driven by lineage-specific promoter, which efficiently reduced the expression of a chromosomally integrated reporter gene and an endogenously expressed gata-1 gene in the developing erythroid progenitors and hemangioblasts, respectively. SIGNIFICANCE: This methodology provides an invaluable tool to knockdown developmental important genes in a tissue-specific manner or to establish animal models, in which the gene dosage is critically important in the pathogenesis of human disorders. The strategy should be also applicable to other model organisms.

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

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    Tagami, Mizuki [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Kusuhara, Sentaro, E-mail: kusu@med.kobe-u.ac.jp [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Imai, Hisanori [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Uemura, Akiyoshi [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Department of Vascular Biology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Honda, Shigeru; Tsukahara, Yasutomo; Negi, Akira [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan)

    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.

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

    Science.gov (United States)

    Lieberman, Judy

    2015-08-01

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Expression profile of CREB knockdown in myeloid leukemia cells

    International Nuclear Information System (INIS)

    Pellegrini, Matteo; Cheng, Jerry C; Voutila, Jon; Judelson, Dejah; Taylor, Julie; Nelson, Stanley F; Sakamoto, Kathleen M

    2008-01-01

    The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution. To understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line transduced with CREB shRNA. By combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. Decreased expression of specific histone genes was validated in K562, TF-1, and primary AML cells transduced with CREB shRNA. We have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. We speculate that regulation of histone genes may play an important role by possibly altering the regulation of DNA replication during the cell cycle

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

    Science.gov (United States)

    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. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  17. A protein knockdown strategy to study the function of β-catenin in tumorigenesis

    Directory of Open Access Journals (Sweden)

    Zhou Pengbo

    2003-09-01

    Full Text Available Abstract Background The Wnt signaling pathway plays critical roles in cell proliferation and cell fate determination at many stages of development. A critical downstream target of Wnt signaling is the cytosolic β-catenin, which is stabilized upon Wnt activation and promotes transcription of a variety of target genes including c-myc and cyclin D. Aberrant Wnt signaling, which results from mutations of either β-catenin or adenomatous polyposis coli (APC, renders β-catenin resistant to degradation, and has been associated with multiple types of human cancers. Results A protein knockdown strategy was designed to reduce the cytosolic β-catenin levels through accelerating its turnover rate. By engineering a chimeric protein with the β-catenin binding domain of E-cadherin fused to βTrCP ubiquitin-protein ligase, the stable β-catenin mutant was recruited to the cellular SCF (Skp1, Cullin 1, and F-box-containing substrate receptor ubiquitination machinery for ubiquitination and degradation. The DLD1 colon cancer cells express wild type β-catenin at abnormally high levels due to loss of APC. Remarkably, conditional expression of βTrCP-E-cadherin under the control of a tetracycline-repressive promoter in DLD1 cells selectively knocked down the cytosolic, but not membrane-associated subpopulation of β-catenin. As a result, DLD1 cells were impaired in their growth and clonogenic ability in vitro, and lost their tumorigenic potential in nude mice. Conclusion We have designed a novel approach to induce degradation of stabilized/mutated β-catenin. Our results suggest that a high concentration of cytoplasmic β-catenin is critical for the growth of colorectal tumor cells. The protein knockdown strategy can be utilized not only as a novel method to dissect the role of oncoproteins in tumorigenesis, but also as a unique tool to delineate the function of a subpopulation of proteins localized to a specific subcellular compartment.

  18. Interaction of Hb Grey Lynn (Vientiane) [α91(FG3)Leu>Phe (α1)] with Hb E [β26(B8) Glu>Lys] and α(+)-thalassemia: Molecular and Hematological Analysis.

    Science.gov (United States)

    Singha, Kritsada; Fucharoen, Goonnapa; Fucharoen, Supan

    2015-01-01

    Hemoglobin (Hb) Grey Lynn is a Hb variant caused by a mutation at codon 91 of α1-globin gene whereas Hb E is a common β-globin chain variant among Southeast Asian population. We report two hitherto undescribed conditions of Hb Grey Lynn found in Thai individuals. The study was done on two unrelated Thai subjects. Hematological parameters were recorded and Hb analysis was carried out using automated Hb analyzers. Mutations were identified by DNA analysis. Hematological features of the patients were compared with those of various forms of Hb Grey Lynn documented previously. Hb and DNA analyses identified a heterozygous Hb Grey Lynn in one patient and a double heterozygous Hb Grey Lynn and Hb E with α(+)-thalassemia in another. Interaction of α(Grey Lynn) with β(E) chains leads to the formation of a new Hb variant, namely the Hb Grey Lynn E (α(GL)2β(E)2), detectable by liquid chromatography (10.3%) but masked by Hb E on capillary electrophoresis. Interaction of these multiple globin gene defects could lead to complex hemoglobinopathies requiring combined analysis with multiple Hb analyzers followed by DNA testing to provide accurate diagnosis of the cases.

  19. RNCR3 knockdown inhibits diabetes mellitus-induced retinal reactive gliosis

    International Nuclear Information System (INIS)

    Liu, Chang; Li, Chao-peng; Wang, Jia-Jian; Shan, Kun; Liu, Xin; Yan, Biao

    2016-01-01

    Retinal reactive gliosis is an important pathological feature of diabetic retinopathy. Identifying the underlying mechanisms causing reactive gliosis will be important for developing new therapeutic strategies for treating diabetic retinopathy. Herein, we show that long noncoding RNA-RNCR3 knockdown significantly inhibits retinal reactive gliosis. RNCR3 knockdown leads to a marked reduction in the release of several cytokines. RNCR3 knockdown alleviates diabetes mellitus-induced retinal neurodegeneration, as shown by less apoptotic retinal cells and ameliorative visual function. RNCR3 knockdown could also decrease Müller glial cell viability and proliferation, and reduce the expression of glial reactivity-related genes including GFAP and vimentin in vitro. Collectively, this study shows that RNCR3 knockdown may be a promising strategy for the prevention of diabetes mellitus-induced retinal neurodegeneration. - Highlights: • RNCR3 knockdown inhibits retinal reactive gliosis. • RNCR3 knockdown causes a significant change in cytokine profile. • RNCR3 knockdown alleviates diabetes mellitus-induced retinal neurodegeneration. • RNCR3 knockdown affects Müller glial cell function in vitro.

  20. Knockdown of BAG3 sensitizes bladder cancer cells to treatment with the BH3 mimetic ABT-737.

    Science.gov (United States)

    Mani, Jens; Antonietti, Patrick; Rakel, Stefanie; Blaheta, Roman; Bartsch, Georg; Haferkamp, Axel; Kögel, Donat

    2016-02-01

    BAG3 is overexpressed in several malignancies and mediates a non-canonical, selective form of (macro)autophagy. By stabilizing pro-survival Bcl-2 proteins in complex with HSP70, BAG3 can also exert an apoptosis-antagonizing function. ABT-737 is a high affinity Bcl-2 inhibitor that fails to target Mcl-1. This failure may confer resistance in various cancers. Urothelial cancer cells were treated with the BH3 mimetics ABT-737 and (-)-gossypol, a pan-Bcl-2 inhibitor which inhibits also Mcl-1. To clarify the importance of the core autophagy regulator ATG5 and BAG3 in ABT-737 treatment, cell lines carrying a stable lentiviral knockdown of ATG5 and BAG3 were created. The synergistic effect of ABT-737 and pharmaceutical inhibition of BAG3 with the HSF1 inhibitor KRIBB11 or sorafenib was also evaluated. Total cell death and apoptosis were quantified by FACS analysis of propidium iodide, annexin. Target protein analysis was conducted by Western blotting. Knockdown of BAG3 significantly downregulated Mcl-1 protein levels and sensitized urothelial cancer cells to apoptotic cell death induced by ABT-737, while inhibition of bulk autophagy through depletion of ATG5 had no discernible effect on cell death. Similar to knockdown of BAG3, pharmacological targeting of the BAG3/Mcl-1 pathway with KRIBB11 was capable to sensitize both cell lines to treatment with ABT-737. Our results show that BAG3, but not bulk autophagy has a major role in the response of bladder cancer cells to BH3 mimetics. They also suggest that BAG3 is a suitable target for combined therapies aimed at synergistically inducing apoptosis in bladder cancer.

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

    Science.gov (United States)

    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.

  2. Knockdown of miR-27a sensitizes colorectal cancer stem cells to TRAIL by promoting the formation of Apaf-1-caspase-9 complex.

    Science.gov (United States)

    Zhang, Rui; Xu, Jian; Zhao, Jian; Bai, Jinghui

    2017-07-11

    MicroRNAs have been proved to participate in multiple biological processes in cancers. For developing resistance to cytotoxic drug, cancer cells, especially the cancer stem cells, usually change their microRNA expression profile to survive in hostile environments. In the present study, we found that expression of microRNA-27a was increased in colorectal cancer stem cells. High level of microRNA-27a was indicated to induce the resistance to TNF-related apoptosis-inducing ligand (TRAIL). Knockdown of microRNA-27a resensitized colorectal cancer stem cells to TRAIL-induced cell death. Mechanically, the gene of Apaf-1, which is associated with the mitochondrial apoptosis, was demonstrated to be the target of microRNA-27a in colorectal cancer stem cells. Knockdown of microRNA-27a increased the expression level of Apaf-1, thus enhancing the formation of Apaf-1-caspase-9 complex and subsequently promoting the TRAIL-induced apoptosis in colorectal cancer stem cells. These findings suggested that knockdown of microRNA-27a in colorectal cancer stem cells by the specific antioligonucleotides was potential to reverse the chemoresistance to TRAIL. It may represent a novel therapeutic strategy for treating the colorectal cancer more effectively.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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

  4. Knockdown of Pnpla6 protein results in motor neuron defects in zebrafish

    Directory of Open Access Journals (Sweden)

    Yang Song

    2013-03-01

    Mutations in patatin-like phospholipase domain containing 6 (PNPLA6, also known as neuropathy target esterase (NTE or SPG39, cause hereditary spastic paraplegia (HSP. Although studies on animal models, including mice and Drosophila, have extended our understanding of PNPLA6, its roles in neural development and in HSP are not clearly understood. Here, we describe the generation of a vertebrate model of PNPLA6 insufficiency using morpholino oligonucleotide knockdown in zebrafish (Danio rerio. Pnpla6 knockdown resulted in developmental abnormalities and motor neuron defects, including axon truncation and branching. The phenotypes in pnpla6 knockdown morphants were rescued by the introduction of wild-type, but not mutant, human PNPLA6 mRNA. Our results also revealed the involvement of BMP signaling in pnpla6 knockdown phenotypes. Taken together, these results demonstrate an important role of PNPLA6 in motor neuron development and implicate overexpression of BMP signaling as a possible mechanism underlying the developmental defects in pnpla6 morphants.

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

    OpenAIRE

    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 medi...

  6. [Knockdown of ATG5 enhances the sensitivity of human renal carcinoma cells to sunitinib].

    Science.gov (United States)

    Li, Peng; Han, Qi; Tang, Ming; Zhang, Keqin

    2017-03-01

    Objective To investigate the expression levels of autophagy-related gene 5 (ATG5) and microtubule-associated protein 1 light chain 3 (LC3) and their effects on sunitinib resistance in human renal carcinoma cells. Methods After clinic-pathologic feature and survival analysis, 99 renal clear cell carcinoma tissues with different histological grades were used to detect the expression of ATG5 and LC3 by immunohistochemistry. Renal carcinoma cell line A-498 was infected with lentivirus-mediated ATG5 shRNA. Western blot analysis was performed to confirm the efficiency of ATG5 knockdown. Proliferation rate of A-498 cells in control group and ATG5 low expression group was determined by flow cytometry. Finally, the survival rate was detected by MTT assay after A-498 cells were treated with different concentrations of sunitinib. Results The expression levels of ATG5 and LC3 in renal clear cell carcinoma tissues were significantly higher than those in para-tumor tissues. The expression levels of ATG5 and LC3 were associated with classification, histological grade, TNM stage and survival rate, rather than gender, age, location, tumor size. Compared with the control group, the protein expressions of ATG5 and LC3 significantly decreased in A-498 cells with ATG5 low expression. The cell proliferation rate in ATG5 downregulation group was lower than that in the control group. Compared with control group, the survival rate in ATG5 low expression group were significantly reduced in a dose-dependent manner after sunitinib treatment. Conclusion Autophagy is active in renal clear cell carcinoma, and the drug sensitivity to sunitinib in renal cancer cells can be enhanced by the downregulation of ATG5.

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

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Sung-Keum; Kim, Jae-Hee; Choi, Ha-Na [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Choe, Tae-Boo [Department of Microbiological Engineering, Kon-Kuk University, Gwangjin-gu, Seoul (Korea, Republic of); Hong, Seok-Il [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Yi, Jae-Youn [Laboratory of Modulation of Radiobiological Responses, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Hwang, Sang-Gu [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Lee, Hyun-Gyu [Department of Microbiology and Immunology, College of Medicine, Yonsei University, 250 Seongsan-no, Seodaemun-gu, Seoul (Korea, Republic of); Lee, Yun-Han, E-mail: yhlee87@yuhs.ac [Department of Radiation Oncology, College of Medicine, Yonsei University, 250 Seongsan-no, Seodaemun-gu, Seoul (Korea, Republic of); Park, In-Chul, E-mail: parkic@kcch.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Sujun [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China); Southern Medical University, Guangzhou, Guangdong 510515 (China); Wu, Binwen, E-mail: wubinwengd@aliyun.com [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China); Li, Dongfeng; Zhou, Weihong; Deng, Gang; Zhang, Kaijun; Li, Youjia [East Department of Gastroenterology, Institute of Geriatrics, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080 (China)

    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.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    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 ∗ , -193b and -193a, and inversely inhibit miR-31 and -9 ∗ . 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

  10. Knockdown of ZFR suppresses cell proliferation and invasion of human pancreatic cancer

    Directory of Open Access Journals (Sweden)

    Xiaolan Zhao

    Full Text Available BACKGROUND: Zinc finger RNA binding protein (ZFR is involved in the regulation of growth and cancer development. However, little is known about ZFR function in pancreatic cancer. METHODS: Herein, to investigate whether ZFR is involved in tumor growth, Oncomine microarray data was firstly used to evaluate ZFR gene expression in human pancreatic tumors. Then short hairpin RNA (shRNA targeting ZFR was designed and delivered into PANC-1 pancreatic cancer cells to knock down ZFR expression. Cell viability, cell proliferation and cell cycle analysis after ZFR knockdown were determined by MTT, colony forming and FACS, respectively. In addition, cell migration and invasion were assessed using the Transwell system. RESULTS: The expression of ZFR was significantly higher in pancreatic tumors than normal pancreas tissues by Oncomine database analysis. Knockdown of ZFR by shRNA-expressing lentivirus significantly decreased the viability and invasion ability of pancreatic cancer cells. Moreover, FACS analysis showed that knockdown of ZFR in PANC-1 cells caused a significant cell cycle arrest at G0/G1 phase. Furthermore, knockdown of ZFR decreased the levels of CDK2, CDK4, CyclinA and CyclinD1 and enhanced the expression of p27, which has evidenced by qRT-PCR and Western blot analysis. CONCLUSIONS: Knockdown of ZFR might provide a novel alternative to targeted therapy of pancreatic cancer and deserves further investigation.

  11. Transcription factor Runx2 knockdown regulates colon cancer transplantation tumor growth in vitro: an experimental study

    Directory of Open Access Journals (Sweden)

    Bin Xu1

    2017-05-01

    Full Text Available Objective: To study the effect of transcription factor Runx2 knockdown on colon cancer transplantation tumor growth in vitro. Methods: Colon cancer cell lines HT29 were cultured and transfected with negative control (NC - shRNA plasmids and Runx2-shRNA plasmids respectively, the colon cancer cells transfected with shRNA were subcutaneously injected into C57 nude mice, and they were included in NC group and Runx2 knockdown group respectively. 1 week, 2 weeks and 3 weeks after model establishment, serum was collected to determine the contents of tumor markers, and tumor lesions were collected to determine proliferation and apoptosis gene expression. Results: CCSA-2, CEA and CA19-9 levels in serum as well as Rac1, Wnt3a, PLD2 and FAM96B protein expression in transplantation tumor lesions of Runx2 knockdown group were significantly lower than those of NC group while MS4A12, ASPP2 and Fas protein expression in transplantation tumor lesions of Runx2 knockdown group were significantly higher than those of NC group. Conclusion: Transcription factor Runx2 knockdown could inhibit the colon cancer transplantation tumor growth in vitro.

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

    Energy Technology Data Exchange (ETDEWEB)

    Abdelalim, Essam Mohamed, E-mail: emohamed@qf.org.qa [Qatar Biomedical Research Institute, Qatar Foundation, Doha 5825 (Qatar); Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan); Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia (Egypt); Tooyama, Ikuo [Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jie [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou (China); Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Yang, Xi-fei [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Ren, Xiao-hu [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou (China); Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Meng, Xiao-jing [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou (China); Huang, Hai-yan [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Zhao, Qiong-hui [Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen (China); Yuan, Jian-hui; Hong, Wen-xu; Xia, Bo; Huang, Xin-feng; Zhou, Li [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Liu, Jian-jun, E-mail: bio-research@hotmail.com [Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen (China); Zou, Fei, E-mail: zoufei616@163.com [Department of Occupational Health and Occupational Medicine, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou (China)

    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.

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

    International Nuclear Information System (INIS)

    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

    2014-01-01

    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

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

    Directory of Open Access Journals (Sweden)

    Quan He

    2014-01-01

    Full Text Available 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.

  16. Colorectal cancer cell lines made resistant to SN38-and Oxaliplatin: Roles of altered ion transporter function in resistance?

    DEFF Research Database (Denmark)

    Sandra, Christensen; Jensen, Niels Frank; Stoeckel, Johanne Danmark

    2013-01-01

    , respectively. Studies are ongoing to assess glutamate uptake in parental and resistant CRC cells and the effect of inhibition/knockdown of SLC1A1 and -3 on SN38- and Oxp resistance. In conclusion, SN38-and Oxp-resistance in CRC cells is associated with SLC1A1 and -3 dysregulation. As these transporters have...

  17. Knockdown of platinum-induced growth differentiation factor 15 abrogates p27-mediated tumor growth delay in the chemoresistant ovarian cancer model A2780cis

    International Nuclear Information System (INIS)

    Meier, Julia C; Haendler, Bernard; Seidel, Henrik; Groth, Philip; Adams, Robert; Ziegelbauer, Karl; Kreft, Bertolt; Beckmann, Georg; Sommer, Anette; Kopitz, Charlotte

    2015-01-01

    Molecular mechanisms underlying the development of resistance to platinum-based treatment in patients with ovarian cancer remain poorly understood. This is mainly due to the lack of appropriate in vivo models allowing the identification of resistance-related factors. In this study, we used human whole-genome microarrays and linear model analysis to identify potential resistance-related genes by comparing the expression profiles of the parental human ovarian cancer model A2780 and its platinum-resistant variant A2780cis before and after carboplatin treatment in vivo. Growth differentiation factor 15 (GDF15) was identified as one of five potential resistance-related genes in the A2780cis tumor model. Although A2780-bearing mice showed a strong carboplatin-induced increase of GDF15 plasma levels, the basal higher GDF15 plasma levels of A2780cis-bearing mice showed no further increase after short-term or long-term carboplatin treatment. This correlated with a decreased DNA damage response, enhanced AKT survival signaling and abrogated cell cycle arrest in the carboplatin-treated A2780cis tumors. Furthermore, knockdown of GDF15 in A2780cis cells did not alter cell proliferation but enhanced cell migration and colony size in vitro. Interestingly, in vivo knockdown of GDF15 in the A2780cis model led to a basal-enhanced tumor growth, but increased sensitivity to carboplatin treatment as compared to the control-transduced A2780cis tumors. This was associated with larger necrotic areas, a lobular tumor structure and increased p53 and p16 expression of the carboplatin-treated shGDF15-A2780cis tumors. Furthermore, shRNA-mediated GDF15 knockdown abrogated p27 expression as compared to control-transduced A2780cis tumors. In conclusion, these data show that GDF15 may contribute to carboplatin resistance by suppressing tumor growth through p27. These data show that GDF15 might serve as a novel treatment target in women with platinum-resistant ovarian cancer

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

    Directory of Open Access Journals (Sweden)

    Rita Marcia Cardoso de Paiva

    2015-12-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  20. Neurobasal media facilitates increased specificity of siRNA-mediated knockdown in primary cerebellar cultures

    DEFF Research Database (Denmark)

    Gustafsson, Julie Ry; Katsioudi, Georgia; Issazadeh-Navikas, Shohreh

    2016-01-01

    be effectively grown in Neurobasal™ media. NEW METHOD: We tested the efficiency of siRNA from the Accell range from Dharmacon™ when delivered in Neurobasal™ media in contrast to the recommended Accell Delivery media provided by the manufacturer. RESULTS: We observed a more specific knockdown of target...... in Neurobasal™ media, than in Accell Delivery media when using cerebellar granule neurons. Transfection efficiency and cell viability was comparable between the two media. COMPARISON WITH EXISTING METHODS: Delivery of siRNA in Neurobasal™ media facilitates increased specificity of the knockdown compared...... to delivery in Accell Delivery media. The off-target effect observed in Accell Delivery media was not a secondary biological response to downregulation of target, but rather a mixture of specific and non-specific off-target effects. CONCLUSIONS: Specific knockdown of target can be achieved in primary...

  1. Thioredoxin reductase 1 knockdown enhances selenazolidine cytotoxicity in human lung cancer cells via mitochondrial dysfunction

    Science.gov (United States)

    Poerschke, Robyn L.; Moos, Philip J.

    2010-01-01

    Thioredoxin reductase (TR1) is a selenoprotein that is involved in cellular redox status control and deoxyribonucleotide biosynthesis. Many cancers, including lung, overexpress TR1, making it a potential cancer therapy target. Previous work has shown that TR1 knockdown enhances the sensitivity of cancer cells to anticancer treatments, as well as certain selenocompounds. However, it is unknown if TR1 knockdown produces similar effect on the sensitivity of human lung cancer cells. To further elucidate the role of TR1 in the mechanism of selenocompounds in lung cancer, a lentiviral microRNA delivery system to knockdown TR1 expression in A549 human lung adenocarcinoma cells was utilized. Cell viability was assessed after 48 hr treatment with the selenocysteine prodrug selenazolidines 2-butylselenazolidine-4(R)-carboxylic acid (BSCA) and 2-cyclohexylselenazolidine-4-(R)-carboxylic acid (ChSCA), selenocystine (SECY), methylseleninic acid (MSA), 1,4-phenylenebis(methylene)selenocyanate (p-XSC), and selenomethionine (SEM). TR1 knockdown increased the cytotoxicity of BSCA, ChSCA, and SECY but did not sensitize cells to MSA, SEM, or p-XSC. GSH and TR1 depletion together decreased cell viability, while no change was observed with GSH depletion alone. Reactive oxygen species generation was induced only in TR1 knockdown cells treated with the selenazolidines or SECY. These three compounds also decreased total intracellular glutathione levels and oxidized thioredoxin, but in a TR1 independent manner. TR1 knockdown increased selenazolidine and SECY-induced mitochondrial membrane depolarization, as well as DNA strand breaks and AIF translocation from the mitochondria. These results indicate the ability of TR1 to modulate the cytotoxic effects of BSCA, ChSCA and SECY in human lung cancer cells through mitochondrial dysfunction. PMID:20920480

  2. Brain gene expression changes elicited by peripheral vitellogenin knockdown in the honey bee.

    Science.gov (United States)

    Wheeler, M M; Ament, S A; Rodriguez-Zas, S L; Robinson, G E

    2013-10-01

    Vitellogenin (Vg) is best known as a yolk protein precursor. Vg also functions to regulate behavioural maturation in adult honey bee workers, but the underlying molecular mechanisms by which it exerts this novel effect are largely unknown. We used abdominal vitellogenin (vg) knockdown with RNA interference (RNAi) and brain transcriptomic profiling to gain insights into how Vg influences honey bee behavioural maturation. We found that vg knockdown caused extensive gene expression changes in the bee brain, with much of this transcriptional response involving changes in central biological functions such as energy metabolism. vg knockdown targeted many of the same genes that show natural, maturation-related differences, but the direction of change for the genes in these two contrasts was not correlated. By contrast, vg knockdown targeted many of the same genes that are regulated by juvenile hormone (JH) and there was a significant correlation for the direction of change for the genes in these two contrasts. These results indicate that the tight coregulatory relationship that exists between JH and Vg in the regulation of honey bee behavioural maturation is manifest at the genomic level and suggest that these two physiological factors act through common pathways to regulate brain gene expression and behaviour. © 2013 Royal Entomological Society.

  3. Gene knockdown of CENPA reduces sphere forming ability and stemness of glioblastoma initiating cells

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    Jinan Behnan

    2016-09-01

    Knockdown of CENPA reduced sphere forming ability, proliferation and cell viability of GICs. We also detected significant reduction in the expression of stemness marker SOX2 and the proliferation marker Ki67. These results indicate that CENPA might represent a promising therapeutic target for GBM treatment.

  4. Genetic and chemical knockdown: a complementary strategy for evaluating an anti-infective target

    Directory of Open Access Journals (Sweden)

    Ramachandran V

    2013-02-01

    Full Text Available Vasanthi Ramachandran,1,* Ragini Singh,2,* Xiaoyu Yang,1 Ragadeepthi Tunduguru,1 Subrat Mohapatra,2 Swati Khandelwal,2 Sanjana Patel,2 Santanu Datta21AstraZeneca India R&D, Bangalore, India; 2Cellworks India, Bangalore, India *These authors contributed equally to this workAbstract: The equity of a drug target is principally evaluated by its genetic vulnerability with tools ranging from antisense- and microRNA-driven knockdowns to induced expression of the target protein. In order to upgrade the process of antibacterial target identification and discern its most effective type of inhibition, an in silico toolbox that evaluates its genetic and chemical vulnerability leading either to stasis or cidal outcome was constructed and validated. By precise simulation and careful experimentation using enolpyruvyl shikimate-3-phosphate synthase and its specific inhibitor glyphosate, it was shown that genetic knockdown is distinct from chemical knockdown. It was also observed that depending on the particular mechanism of inhibition, viz competitive, uncompetitive, and noncompetitive, the antimicrobial potency of an inhibitor could be orders of magnitude different. Susceptibility of Escherichia coli to glyphosate and the lack of it in Mycobacterium tuberculosis could be predicted by the in silico platform. Finally, as predicted and simulated in the in silico platform, the translation of growth inhibition to a cidal effect was able to be demonstrated experimentally by altering the carbon source from sorbitol to glucose.Keywords: knockdown, inhibition, in silico, vulnerability

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

    Science.gov (United States)

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

    2016-06-30

    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.

  6. Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells.

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    Tuoen Liu

    Full Text Available Myelodysplastic syndromes (MDS are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q compared to non-del(5q MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q-associated MDS.

  7. Knockdown of Heparanase Suppresses Invasion of Human Trophoblasts by Activating p38 MAPK Signaling Pathway

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    Guanglu Che

    2018-01-01

    Full Text Available Preeclampsia is a pregnancy-related disease with increasing maternal and perinatal morbidity and mortality worldwide. Defective trophoblast invasion is considered to be a major factor in the pathophysiological mechanism of preeclampsia. Heparanase, the only endo-β-glucuronidase in mammalian cells, has been shown to be abnormally expressed in the placenta of preeclampsia patients in our previous study. The biological role and potential mechanism of heparanase in trophoblasts remain unclear. In the present study, stably transfected HTR8/SVneo cell lines with heparanase overexpression or knockdown were constructed. The effect of heparanase on cellular proliferation, apoptosis, invasion, tube formation, and potential pathways in trophoblasts was explored. Our results showed that overexpression of heparanase promoted proliferation and invasion. Knockdown of heparanase suppressed proliferation, invasion, and tube formation but induced apoptosis. These findings reveal that downregulation of heparanase may contribute to defective placentation and plays a crucial role in the pathogenesis of preeclampsia. Furthermore, increased activation of p38 MAPK in heparanase-knockdown HTR8/SVneo cell was shown by MAPK pathway phosphorylation array and Western blotting assay. After pretreatment with 3 specific p38 MAPK inhibitors (BMS582949, SB203580, or BIRB796, inadequate invasion in heparanase-knockdown HTR8/SVneo cell was rescued. That indicates that knockdown of heparanase decreases HTR8/SVneo cell invasion through excessive activation of the p38 MAPK signaling pathway. Our study suggests that heparanase can be a potential predictive biomarker for preeclampsia at an early stage of pregnancy and represents a promising therapeutic target for the treatment of preeclampsia.

  8. Knockdown of TFIIS by RNA silencing inhibits cancer cell proliferation and induces apoptosis

    International Nuclear Information System (INIS)

    Hubbard, Kyle; Catalano, Jennifer; Puri, Raj K; Gnatt, Averell

    2008-01-01

    A common element among cancer cells is the presence of improperly controlled transcription. In these cells, the degree of specific activation of some genes is abnormal, and altering the aberrant transcription may therefore directly target cancer. TFIIS is a transcription elongation factor, which directly binds the transcription motor, RNA Polymerase II and allows it to read through various transcription arrest sites. We report on RNA interference of TFIIS, a transcription elongation factor, and its affect on proliferation of cancer cells in culture. RNA interference was performed by transfecting siRNA to specifically knock down TFIIS expression in MCF7, MCF10A, PL45 and A549 cells. Levels of TFIIS expression were determined by the Quantigene method, and relative protein levels of TFIIS, c-myc and p53 were determined by C-ELISA. Induction of apoptosis was determined by an enzymatic Caspase 3/7 assay, as well as a non-enzymatic assay detecting cytoplasmic mono- and oligonucleosomes. A gene array analysis was conducted for effects of TFIIS siRNA on MCF7 and MCF10A cell lines. Knockdown of TFIIS reduced cancer cell proliferation in breast, lung and pancreatic cancer cell lines. More specifically, TFIIS knockdown in the MCF7 breast cancer cell line induced cancer cell death and increased c-myc and p53 expression whereas TFIIS knockdown in the non-cancerous breast cell line MCF10A was less affected. Differential effects of TFIIS knockdown in MCF7 and MCF10A cells included the estrogenic, c-myc and p53 pathways, as observed by C-ELISA and gene array, and were likely involved in MCF7 cell-death. Although transcription is a fundamental process, targeting select core transcription factors may provide for a new and potent avenue for cancer therapeutics. In the present study, knockdown of TFIIS inhibited cancer cell proliferation, suggesting that TFIIS could be studied as a potential cancer target within the transcription machinery

  9. Knockdown of CDK2AP1 in human embryonic stem cells reduces the threshold of differentiation.

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    Khaled N Alsayegh

    Full Text Available Recent studies have suggested a role for the Cyclin Dependent Kinase-2 Associated Protein 1 (CDK2AP1 in stem cell differentiation and self-renewal. In studies with mouse embryonic stem cells (mESCs derived from generated mice embryos with targeted deletion of the Cdk2ap1 gene, CDK2AP1 was shown to be required for epigenetic silencing of Oct4 during differentiation, with deletion resulting in persistent self-renewal and reduced differentiation potential. Differentiation capacity was restored in these cells following the introduction of a non-phosphorylatible form of the retinoblastoma protein (pRb or exogenous Cdk2ap1. In this study, we investigated the role of CDK2AP1 in human embryonic stem cells (hESCs. Using a shRNA to reduce its expression in hESCs, we found that CDK2AP1 knockdown resulted in a significant reduction in the expression of the pluripotency genes, OCT4 and NANOG. We also found that CDK2AP1 knockdown increased the number of embryoid bodies (EBs formed when differentiation was induced. In addition, the generated EBs had significantly higher expression of markers of all three germ layers, indicating that CDK2AP1 knockdown enhanced differentiation. CDK2AP1 knockdown also resulted in reduced proliferation and reduced the percentage of cells in the S phase and increased cells in the G2/M phase of the cell cycle. Further investigation revealed that a higher level of p53 protein was present in the CDK2AP1 knockdown hESCs. In hESCs in which p53 and CDK2AP1 were simultaneously downregulated, OCT4 and NANOG expression was not affected and percentage of cells in the S phase of the cell cycle was not reduced. Taken together, our results indicate that the knockdown of CDK2AP1 in hESCs results in increased p53 and enhances differentiation and favors it over a self-renewal fate.

  10. Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles.

    Science.gov (United States)

    Mueller, Christian; Tang, Qiushi; Gruntman, Alisha; Blomenkamp, Keith; Teckman, Jeffery; Song, Lina; Zamore, Phillip D; Flotte, Terence R

    2012-03-01

    α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

  11. Knockdown of the placental growth factor gene inhibits laser induced choroidal neovascularization in a murine model.

    Science.gov (United States)

    Nourinia, Ramin; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Akrami, Hassan; Rezaei Kanavi, Mozhgan; Samiei, Shahram

    2013-01-01

    To evaluate the effect of placental growth factor (PlGF) gene knockdown in a murine model of laser-induced choroidal neovascularization. Choroidal neovascularization was induced in the left eyes of 11 mice by infrared laser. Small interfering RNA (siRNA, 20 picomoles/10 μl) corresponding to PlGF mRNA was administered intravitreally by Hamilton syringe in all subjects. One month later, fluorescein angiography and histolologic examination were performed. No leakage was apparent in the 11 eyes treated with siRNA cognate to PlGF. The results of histological evaluation were consistent with angiographic findings showing absence of choroidal neovascularization. Knockdown of the PlGF gene can inhibit the growth of laser-induced choroidal neovascularization in mice.

  12. Knockdown of the Placental Growth Factor Gene Inhibits Laser Induced Choroidal Neovascularization in a Murine Model

    Directory of Open Access Journals (Sweden)

    Ramin Nourinia

    2013-01-01

    Full Text Available Purpose: To evaluate the effect of placental growth factor (PlGF gene knockdown in a murine model of laser-induced choroidal neovascularization. Methods: Choroidal neovascularization was induced in the left eyes of 11 mice by infrared laser. Small interfering RNA (siRNA, 20 picomoles/10 μl corresponding to PlGF mRNA was administered intravitreally by Hamilton syringe in all subjects. One month later, fluorescein angiography and histolologic examination were performed. Results: No leakage was apparent in the 11 eyes treated with siRNA cognate to PlGF. The results of histological evaluation were consistent with angiographic findings showing absence of choroidal neovascularization. Conclusion: Knockdown of the PlGF gene can inhibit the growth of laser-induced choroidal neovascularization in mice.

  13. "Behaviour changes in Permethrin-resistant strain of Anopheles Stephensi "

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    Vatandoost H

    2000-09-01

    Full Text Available Behaviour studies indicated that the permethrin resistant strin of An. Stephensi was 3-fold resistant to knock-down compared with the susceptible strain. The resistant strain was however 3-fold less irritable to permethrin and less responsive than the susceptible strain to the movement of an aspirator. If reduced irritability and reduced responsiveness to catch are consequences of the changes in the nervous system, then such a form of resistance may be disadvantageous to mosquitoes in natural populations.

  14. Goat activin receptor type IIB knockdown by muscle specific promoter driven artificial microRNAs.

    Science.gov (United States)

    Patel, Amrutlal K; Shah, Ravi K; Patel, Utsav A; Tripathi, Ajai K; Joshi, Chaitanya G

    2014-10-10

    Activin receptor type IIB (ACVR2B) is a transmembrane receptor which mediates signaling of TGF beta superfamily ligands known to function in regulation of muscle mass, embryonic development and reproduction. ACVR2B antagonism has shown to enhance the muscle growth in several disease and transgenic models. Here, we show ACVR2B knockdown by RNA interference using muscle creatine kinase (MCK) promoter driven artificial microRNAs (amiRNAs). Among the various promoter elements tested, the ∼1.26 kb MCK promoter region showed maximum transcriptional activity in goat myoblasts cells. We observed up to 20% silencing in non-myogenic 293T cells and up to 32% silencing in myogenic goat myoblasts by MCK directed amiRNAs by transient transfection. Goat myoblasts stably integrated with MCK directed amiRNAs showed merely 8% silencing in proliferating myoblasts which was increased to 34% upon induction of differentiation at transcript level whereas up to 57% silencing at protein level. Knockdown of ACVR2B by 5'-UTR derived amiRNAs resulted in decreased SMAD2/3 signaling, increased expression of myogenic regulatory factors (MRFs) and enhanced proliferation and differentiation of myoblasts. Unexpectedly, knockdown of ACVR2B by 3'-UTR derived amiRNAs resulted in increased SMAD2/3 signaling, reduced expression of MRFs and suppression of myogenesis. Our study offers muscle specific knockdown of ACVR2B as a potential strategy to enhance muscle mass in the farm animal species. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Cui, Ju, E-mail: juzi.cui@gmail.com [The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing (China); Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); Jin, Guoxiang; Yu, Bin [Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); Wang, Zai [Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing (China); Lin, Raozhou [Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); Huang, Jian-Dong, E-mail: jdhuang@hku.hk [Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); The Centre for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Shenzhen (China)

    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.

  18. Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown

    Science.gov (United States)

    Moore, Chris B.; Guthrie, Elizabeth H.; Huang, Max Tze-Han; Taxman, Debra J.

    2013-01-01

    Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery. PMID:20387148

  19. CD147 knockdown improves the antitumor efficacy of trastuzumab in HER2-positive breast cancer cells.

    Science.gov (United States)

    Xiong, Lijuan; Ding, Li; Ning, Haoyong; Wu, Chenglin; Fu, Kaifei; Wang, Yuxiao; Zhang, Yan; Liu, Yan; Zhou, Lijun

    2016-09-06

    Trastuzumab is widely used in the clinical treatment of human epidermal growth factor receptor-2 (HER2)-positive breast cancer, but the patient response rate is low. CD147 stimulates cancer cell proliferation, migration, metastasis and differentiation and is involved in chemoresistance in many types of cancer cells. Whether CD147 alters the effect of trastuzumab on HER2-positive breast cancer cells has not been previously reported. Our study confirmed that CD147 suppression enhances the effects of trastuzumab both in vitro and in vivo. CD147 suppression increased the inhibitory rate of trastuzumab and cell apoptosis in SKBR3, BT474, HCC1954 and MDA-MB453 cells compared with the controls. Furthermore, CD147 knockdown increased expression of cleaved Caspase-3/9 and poly (ADP-ribose) polymerase (PARP) and decreased both mitogen-activated protein kinase (MAPK) and Akt phosphorylation in the four cell lines. In an HCC1954 xenograft model, trastuzumab achieved greater suppression of tumor growth in the CD147-knockdown group than in the shRNA negative control (NC) group. These data indicated that enhancement of the effect of trastuzumab on HER2-positive cells following CD147 knockdown might be attributed to increased apoptosis and decreased phosphorylation of signaling proteins. CD147 may be a key protein for enhancing the clinical efficacy of trastuzumab.

  20. In Vivo Testing of MicroRNA-Mediated Gene Knockdown in Zebrafish

    Directory of Open Access Journals (Sweden)

    Ivone Un San Leong

    2012-01-01

    Full Text Available The zebrafish (Danio rerio has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis. The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.

  1. MADD knock-down enhances doxorubicin and TRAIL induced apoptosis in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Andrea Turner

    Full Text Available The Map kinase Activating Death Domain containing protein (MADD isoform of the IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.

  2. ETV4 and Myeov knockdown impairs colon cancer cell line proliferation and invasion

    International Nuclear Information System (INIS)

    Moss, Alan C.; Lawlor, Garrett; Murray, David; Tighe, Donal; Madden, Stephen F.; Mulligan, Anne-Marie; Keane, Conor O.; Brady, Hugh R.; Doran, Peter P.; MacMathuna, Padraic

    2006-01-01

    We have identified novel colorectal cancer-associated genes using NCBI's UNIGENE cDNA libraries. Colon cancer libraries were examined using Digital Differential Display and disease-associated genes were selected. Among these were ETV4 and MYEOV, novel colorectal cancer-associated genes. Samples of matched normal and neoplastic colon were obtained from human subjects and gene expression was quantified using real-time PCR. ETV4 gene expression was significantly increased in colonic neoplasia in comparison to matched normal colonic tissue (p < 0.05). Myeov expression was also increased in colon neoplasia in comparison to matched normal tissue. The effect of siRNA-mediated knockdown of ETV4 and Myeov on cell proliferation and invasion was assessed. ETV4 knockdown resulted in a 90% decrease in cell proliferation (p < 0.05) and a 67% decrease in cell invasion. Myeov knockdown resulted in a 48% decrease in cell proliferation (p < 0.05) and a 36% decrease in cell invasion. These data suggest that ETV4 and Myeov may provide novel targets for therapeutic intervention

  3. Promising Noninvasive Cellular Phenotype in Prostate Cancer Cells Knockdown of Matrix Metalloproteinase 9

    Directory of Open Access Journals (Sweden)

    Aditi Gupta

    2013-01-01

    Full Text Available Cell surface interaction of CD44 and MMP9 increases migration and invasion of PC3 cells. We show here that stable knockdown of MMP9 in PC3 cells switches CD44 isoform expression from CD44s to CD44v6 which is more glycosylated. These cells showed highly adhesive morphology with extensive cell spreading which is due to the formation of focal adhesions and well organized actin-stress fibers. MMP9 knockdown blocks invadopodia formation and matrix degradation activity as well. However, CD44 knockdown PC3 cells failed to develop focal adhesions and stress fibers; hence these cells make unstable adhesions. A part of the reason for these changes could be caused by silencing of CD44v6 as well. Immunostaining of prostate tissue microarray sections illustrated significantly lower levels of CD44v6 in adenocarcinoma than normal tissue. Our results suggest that interaction between CD44 and MMP9 is a potential mechanism of invadopodia formation. CD44v6 expression may be essential for the protection of non-invasive cellular phenotype. CD44v6 decrease may be a potential marker for prognosis and therapeutics.

  4. Effects of cIAP-1, cIAP-2 and XIAP triple knockdown on prostate cancer cell susceptibility to apoptosis, cell survival and proliferation

    Directory of Open Access Journals (Sweden)

    Dowling Catherine

    2009-06-01

    Full Text Available Abstract Background Manipulating apoptotic resistance represents an important strategy for the treatment of hormone refractory prostate cancer. We hypothesised that the Inhibitor of Apoptosis (IAP Proteins may be mediating this resistance and knockdown of cIAP-1, cIAP-2 and XIAP would increase sensitivity to apoptosis. Methods cIAP-1, cIAP-2 and XIAP where knocked down either individually or in combination using siRNA in androgen independent prostate cancer PC-3 cells as confirmed by real-time PCR and western blotting. Cells were then treated with TRAIL, Etoposide, or Tunicamycin, and apoptosis assessed by PI DNA staining. Apoptosis was confirmed with Annexin V labelling and measurement of PARP cleavage, and was inhibited using the pan-caspase inhibitor, zVAD.fmk. Clonogenic assays and assessment of ID-1 expression by western blotting were used to measure recovery and proliferation. Results PC-3 are resistant to TRAIL induced apoptosis and have elevated expression of cIAP-1, cIAP-2 and XIAP. Combined knockdown sensitised PC-3 to TRAIL induced apoptosis, but not to Etoposide or Tunicmycin, with corresponding increases in caspase activity and PARP cleavage which was inhibited by ZVAD.fmk. Triple knock down decreased proliferation which was confirmed by decreased ID-1 expression. Conclusion Simultaneous knock down of the IAPs not only sensitised the PC-3 to TRAIL but also inhibited their proliferation rates and clonogenic survival. The inability to alter sensitivity to other triggers of apoptosis suggests that this effect is specific for death receptor pathways and knock down might facilitate immune-surveillance mechanisms to counter cancer progression and, in combination with therapeutic approaches using TRAIL, could represent an important treatment strategy.

  5. Effects of cIAP-1, cIAP-2 and XIAP triple knockdown on prostate cancer cell susceptibility to apoptosis, cell survival and proliferation.

    LENUS (Irish Health Repository)

    Gill, Catherine

    2009-01-01

    BACKGROUND: Manipulating apoptotic resistance represents an important strategy for the treatment of hormone refractory prostate cancer. We hypothesised that the Inhibitor of Apoptosis (IAP) Proteins may be mediating this resistance and knockdown of cIAP-1, cIAP-2 and XIAP would increase sensitivity to apoptosis. METHODS: cIAP-1, cIAP-2 and XIAP where knocked down either individually or in combination using siRNA in androgen independent prostate cancer PC-3 cells as confirmed by real-time PCR and western blotting. Cells were then treated with TRAIL, Etoposide, or Tunicamycin, and apoptosis assessed by PI DNA staining. Apoptosis was confirmed with Annexin V labelling and measurement of PARP cleavage, and was inhibited using the pan-caspase inhibitor, zVAD.fmk. Clonogenic assays and assessment of ID-1 expression by western blotting were used to measure recovery and proliferation. RESULTS: PC-3 are resistant to TRAIL induced apoptosis and have elevated expression of cIAP-1, cIAP-2 and XIAP. Combined knockdown sensitised PC-3 to TRAIL induced apoptosis, but not to Etoposide or Tunicmycin, with corresponding increases in caspase activity and PARP cleavage which was inhibited by ZVAD.fmk. Triple knock down decreased proliferation which was confirmed by decreased ID-1 expression. CONCLUSION: Simultaneous knock down of the IAPs not only sensitised the PC-3 to TRAIL but also inhibited their proliferation rates and clonogenic survival. The inability to alter sensitivity to other triggers of apoptosis suggests that this effect is specific for death receptor pathways and knock down might facilitate immune-surveillance mechanisms to counter cancer progression and, in combination with therapeutic approaches using TRAIL, could represent an important treatment strategy.

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

    International Nuclear Information System (INIS)

    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

    2009-01-01

    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 + ) are capable of self-renewal and express high levels of embryonic stem cell genes and SirT1 compared to GBM-CD133 - cells. To evaluate the role of SirT1 in GBM-CD133 + , we used a lentiviral vector expressing shRNA to knock-down SirT1 expression (sh-SirT1) in GBM-CD133 + . Silencing of SirT1 significantly enhanced the sensitivity of GBM-CD133 + 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 + . Kaplan-Meier survival analysis indicated that the mean survival rate of GBM-CD133 + 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.

  7. USP22 knockdown enhanced chemosensitivity of hepatocellular carcinoma cells to 5-Fu by up-regulation of Smad4 and suppression of Akt.

    Science.gov (United States)

    Zhang, Jing; Luo, Nan; Tian, Yu; Li, Jiazhi; Yang, Xiaozhou; Yin, Huimin; Xiao, Congshu; Sheng, Jie; Li, Yang; Tang, Bo; Li, Rongkuan

    2017-04-11

    USP22, a member of the deubiquitinases (DUBs) family, is known to be a key subunit of the human Spt-Ada-Gcn5 acetyltransferase (hSAGA) transcriptional cofactor complex. Within hSAGA, USP22 removes ubiquitin from histone proteins, thus regulating the transcription and expression of downstream genes. USP22 plays important roles in many cancers; however, its effect and the mechanism underlying HCC chemoresistance remain unclear. In the present study, we found that USP22 was highly expressed in chemoresistant HCC tissues and cells and was correlated with the prognosis of HCC patients who received chemotherapy. Silencing USP22 in chemoresistant HCC Bel/Fu cells dramatically inhibited proliferation, migration, invasion and epithelial-mesenchymal transition in vitro; suppressed tumorigenic and metastatic capacities in vivo; and inhibited drug resistance-related proteins (MDR1, LRP, MRP1). Mechanistically, we found that USP22 knockdown exerts its function through down-regulating PI3K and activating Smad4, which inhibited phosphorylation of Akt. Silencing Smad4 blocked USP22 knockdown-induced Akt inhibition in Bel/Fu cells. Our results, for the first time, provide evidence that USP22 plays a critical role in the development of chemoresistant HCC cells and that high USP22 expression serves as a molecular marker for the prognosis of HCC patients who undergo chemotherapy.

  8. Consistent effects of a major QTL for thermal resistance in field-released Drosophila melanogaster

    DEFF Research Database (Denmark)

    Loeschcke, Volker; Kristensen, Torsten Nygård; Norry, Fabian M

    2011-01-01

    Molecular genetic markers can be used to identify quantitative trait loci (QTL) for thermal resistance and this has allowed characterization of a major QTL for knockdown resistance to high temperature in Drosophila melanogaster. The QTL showed trade-off associations with cold resistance under lab...... of field fitness at different environmental temperatures with genotypic variation in a QTL for thermal tolerance. Graphical abstract...

  9. OAZ1 knockdown enhances viability and inhibits ER and LHR transcriptions of granulosa cells in geese.

    Directory of Open Access Journals (Sweden)

    Bo Kang

    Full Text Available An increasing number of studies suggest that ornithine decarboxylase antizyme 1 (OAZ1, which is regarded as a tumor suppressor gene, regulates follicular development, ovulation, and steroidogenesis. The granulosa cells in the ovary play a critical role in these ovarian functions. However, the action of OAZ1 mediating physiological functions of granulosa cells is obscure. OAZ1 knockdown in granulosa cells of geese was carried out in the current study. The effect of OAZ1 knockdown on polyamine metabolism, cell proliferation, apoptosis, and hormone receptor transcription of primary granulosa cells in geese was measured. The viability of granulosa cells transfected with the shRNA OAZ1 at 48 h was significantly higher than the control (p<0.05. The level of putrescine and spermidine in granulosa cells down-regulating OAZ1 was 7.04- and 2.11- fold higher compared with the control, respectively (p<0.05. The CCND1, SMAD1, and BCL-2 mRNA expression levels in granulosa cells down-regulating OAZ1 were each significantly higher than the control, respectively (p<0.05, whereas the PCNA and CASPASE 3 expression levels were significantly lower than the control (p<0.05. The estradiol concentration, ER and LHR mRNA expression levels were significantly lower in granulosa cells down-regulating OAZ1 compared with the control (p<0.05. Taken together, our results indicated that OAZ1 knockdown elevated the putrescine and spermidine contents and enhanced granulosa cell viability and inhibited ER and LHR transcriptions of granulosa cells in geese.

  10. Shp2 knockdown and Noonan/LEOPARD mutant Shp2-induced gastrulation defects.

    Directory of Open Access Journals (Sweden)

    Chris Jopling

    2007-12-01

    Full Text Available Shp2 is a cytoplasmic protein-tyrosine phosphatase that is essential for normal development. Activating and inactivating mutations have been identified in humans to cause the related Noonan and LEOPARD syndromes, respectively. The cell biological cause of these syndromes remains to be determined. We have used the zebrafish to assess the role of Shp2 in early development. Here, we report that morpholino-mediated knockdown of Shp2 in zebrafish resulted in defects during gastrulation. Cell tracing experiments demonstrated that Shp2 knockdown induced defects in convergence and extension cell movements. In situ hybridization using a panel of markers indicated that cell fate was not affected by Shp2 knock down. The Shp2 knockdown-induced defects were rescued by active Fyn and Yes and by active RhoA. We generated mutants of Shp2 with mutations that were identified in human patients with Noonan or LEOPARD Syndrome and established that Noonan Shp2 was activated and LEOPARD Shp2 lacked catalytic protein-tyrosine phosphatase activity. Expression of Noonan or LEOPARD mutant Shp2 in zebrafish embryos induced convergence and extension cell movement defects without affecting cell fate. Moreover, these embryos displayed craniofacial and cardiac defects, reminiscent of human symptoms. Noonan and LEOPARD mutant Shp2s were not additive nor synergistic, consistent with the mutant Shp2s having activating and inactivating roles in the same signaling pathway. Our results demonstrate that Shp2 is required for normal convergence and extension cell movements during gastrulation and that Src family kinases and RhoA were downstream of Shp2. Expression of Noonan or LEOPARD Shp2 phenocopied the craniofacial and cardiac defects of human patients. The finding that defective Shp2 signaling induced cell movement defects as early as gastrulation may have implications for the monitoring and diagnosis of Noonan and LEOPARD syndrome.

  11. Aldolase B knockdown prevents high glucose-induced methylglyoxal overproduction and cellular dysfunction in endothelial cells.

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    Jianghai Liu

    Full Text Available We used cultured endothelial cells as a model to examine whether up-regulation of aldolase B and enhanced methylglyoxal (MG formation play an important role in high glucose-induced overproduction of advanced glycosylation endproducts (AGEs, oxidative stress and cellular dysfunction. High glucose (25 mM incubation up-regulated mRNA levels of aldose reductase (an enzyme converting glucose to fructose and aldolase B (a key enzyme that catalyzes MG formation from fructose and enhanced MG formation in human umbilical vein endothelial cells (HUVECs and HUVEC-derived EA. hy926 cells. High glucose-increased MG production in EA. hy926 cells was completely prevented by siRNA knockdown of aldolase B, but unaffected by siRNA knockdown of aldolase A, an enzyme responsible for MG formation during glycolysis. In addition, inhibition of cytochrome P450 2E1 or semicarbazide-sensitive amine oxidase which produces MG during the metabolism of lipid and proteins, respectively, did not alter MG production. Both high glucose (25 mM and MG (30, 100 µM increased the formation of N(ε-carboxyethyl-lysine (CEL, a MG-induced AGE, oxidative stress (determined by the generation of oxidized DCF, H(2O(2, protein carbonyls and 8-oxo-dG, O-GlcNAc modification (product of the hexosamine pathway, membrane protein kinase C activity and nuclear translocation of NF-κB in EA. hy926 cells. However, the above metabolic and signaling alterations induced by high glucose were completely prevented by knockdown of aldolase B and partially by application of aminoguanidine (a MG scavenger or alagebrium (an AGEs breaker. In conclusion, efficient inhibition of aldolase B can prevent high glucose-induced overproduction of MG and related cellular dysfunction in endothelial cells.

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

    Science.gov (United States)

    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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Attenuated food anticipatory activity and abnormal circadian locomotor rhythms in Rgs16 knockdown mice.

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    Naoto Hayasaka

    Full Text Available Regulators of G protein signaling (RGS are a multi-functional protein family, which functions in part as GTPase-activating proteins (GAPs of G protein α-subunits to terminate G protein signaling. Previous studies have demonstrated that the Rgs16 transcripts exhibit robust circadian rhythms both in the suprachiasmatic nucleus (SCN, the master circadian light-entrainable oscillator (LEO of the hypothalamus, and in the liver. To investigate the role of RGS16 in the circadian clock in vivo, we generated two independent transgenic mouse lines using lentiviral vectors expressing short hairpin RNA (shRNA targeting the Rgs16 mRNA. The knockdown mice demonstrated significantly shorter free-running period of locomotor activity rhythms and reduced total activity as compared to the wild-type siblings. In addition, when feeding was restricted during the daytime, food-entrainable oscillator (FEO-driven elevated food-anticipatory activity (FAA observed prior to the scheduled feeding time was significantly attenuated in the knockdown mice. Whereas the restricted feeding phase-advanced the rhythmic expression of the Per2 clock gene in liver and thalamus in the wild-type animals, the above phase shift was not observed in the knockdown mice. This is the first in vivo demonstration that a common regulator of G protein signaling is involved in the two separate, but interactive circadian timing systems, LEO and FEO. The present study also suggests that liver and/or thalamus regulate the food-entrained circadian behavior through G protein-mediated signal transduction pathway(s.

  14. Vivo-morpholinos induced transient knockdown of physical activity related proteins.

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    David P Ferguson

    Full Text Available Physical activity is associated with disease prevention and overall wellbeing. Additionally there has been evidence that physical activity level is a result of genetic influence. However, there has not been a reliable method to silence candidate genes in vivo to determine causal mechanisms of physical activity regulation. Vivo-morpholinos are a potential method to transiently silence specific genes. Thus, the aim of this study was to validate the use of Vivo-morpholinos in a mouse model for voluntary physical activity with several sub-objectives. We observed that Vivo-morpholinos achieved between 60-97% knockdown of Drd1-, Vmat2-, and Glut4-protein in skeletal muscle, the delivery moiety of Vivo-morpholinos (scramble did not influence physical activity and that a cocktail of multiple Vivo-morpholinos can be given in a single treatment to achieve protein knockdown of two different targeted proteins in skeletal muscle simultaneously. Knocking down Drd1, Vmat2, or Glut4 protein in skeletal muscle did not affect physical activity. Vivo-morpholinos injected intravenously alone did not significantly knockdown Vmat2-protein expression in the brain (p = 0.28. However, the use of a bradykinin analog to increase blood-brain-barrier permeability in conjunction with the Vivo-morpholinos significantly (p = 0.0001 decreased Vmat2-protein in the brain with a corresponding later over-expression of Vmat2 coincident with a significant (p = 0.0016 increase in physical activity. We conclude that Vivo-morpholinos can be a valuable tool in determining causal gene-phenotype relationships in whole animal models.

  15. A Simple Retroelement Based Knock-Down System in Dictyostelium: Further Insights into RNA Interference Mechanisms.

    Science.gov (United States)

    Friedrich, Michael; Meier, Doreen; Schuster, Isabelle; Nellen, Wolfgang

    2015-01-01

    We have previously shown that the most abundant Dictyostelium discoideum retroelement DIRS-1 is suppressed by RNAi mechanisms. Here we provide evidence that both inverted terminal repeats have strong promoter activity and that bidirectional expression apparently generates a substrate for Dicer. A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene. Surprisingly, no transitivity was observed on the endogene. This was in contrast to previous observations, where endogenous siRNAs caused spreading on an artificial transgene. Knock-down was successful on seven target genes that we examined. In three cases a phenotypic analysis proved the efficiency of the approach. One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression. ADVANTAGES OF THE DIRS-1–RNAI SYSTEM: The knock-down system required a short DNA fragment (~400 bp) of the target gene as an initial trigger. Further siRNAs were generated by RdRPs since we have shown some siRNAs with a 5'-triphosphate group. Extrachromosomal vectors facilitate the procedure and allowed for molecular and phenotypic analysis within one week. The system provides an efficient and rapid method to reduce protein levels including those of essential genes.

  16. [Effect of NOR1 gene knockdown on the biological behavior of HeLa cells].

    Science.gov (United States)

    Tan, Yixin; Li, Wenjuan; Yi, Mei; Wang, Wei; Zheng, Pan; Zhang, Haijing; Xiang, Bo; Li, Guiyuan

    2014-08-01

    To explore the effect of the oxidored nitro domain containing protein 1 (NOR1) gene knockdown on the biological behavior of HeLa cells in cervical carcinoma. The recombinant plasmids pSUPER-shNOR1-1, pSUPER-shNOR1-2 and pSUPERscramble, which targeted to NOR1 gene, were constructed by pSUPER.neo+GFP vector, transfected into HeLa cells respectively using Lipofectamine 2000 reagent, and followed by G418 selection. The expression level of NOR1 mRNA and protein were determined by RT-PCR and Western blotting, respectively. Methyl thiazolyl tetrazolium (MTT) assay was performed to determine the growth curve of cell viability. The stable transfectants were treated with H₂O₂ and cell apoptosis was determined by Hoechst 33258 staining and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay. The expression levels of Bcl-2, cleaved caspase 9 and poly ADP-ribose polymerase (PARP) were measured by Western blot. NOR1- knockdown HeLa cells were successfully constructed by transfection of pSUPER-shNOR1-1 or pSUPER-shNOR1-2 plasmids into HeLa cells. MTT assay showed that the silence of endogenous NOR1 in HeLa cells could lead to the increase in cell viability and proliferation, and the inhibition of H₂O₂-induced apoptosis compared with the negative control. Western blot showed that the expression level of active caspase 9 and cleaved PARP was inhibited in NOR1-knockdown cells when they were treated with H₂O₂ while the expression level of Bcl-2 protein increased. Silence of endogenous NOR1 facilitates the cell viability and growth of HeLa cells, and attenuates HeLa cells apoptosis induced by H₂O₂, which might be mediated by up-regulation of Bcl-2 level and down-regulation of the cleaved caspase 9 cascade.

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

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    Enise Bagci

    Full Text Available 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

  18. Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism[S

    OpenAIRE

    Mullen, Thomas D.; Spassieva, Stefka; Jenkins, Russell W.; Kitatani, Kazuyuki; Bielawski, Jacek; Hannun, Yusuf A.; Obeid, Lina M.

    2011-01-01

    Mammalian ceramide synthases 1 to 6 (CerS1–6) generate Cer in an acyl-CoA-dependent manner, and expression of individual CerS has been shown to enhance the synthesis of ceramides with particular acyl chain lengths. However, the contribution of each CerS to steady-state levels of specific Cer species has not been evaluated. We investigated the knockdown of individual CerS in the MCF-7 human breast adenocarcinoma cell line by using small-interfering RNA (siRNA). We found that siRNA-induced down...

  19. Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

    DEFF Research Database (Denmark)

    Klauber, Thomas Christopher Bogh; Søndergaard, Rikke Vicki; Sawant, Rupa R.

    2016-01-01

    capability, but are very different regarding siRNA decondensation, cellular internalization and induction of reporter gene knockdown. Lipid conjugation of bPEI 1.8. kDa improves the siRNA delivery properties, but with markedly different formulation requirements and mechanisms of action compared...... today.A major reason for the lack of progress is insufficient understanding of cell-polyplex interaction. We investigate siRNA delivery using polyethyleneimine (PEI) based vectors and examine how crucial formulation parameters determine the challenges associated with PEI as a delivery vector. We further...

  20. Hypersensitivity of mouse NEIL1-knockdown cells to hydrogen peroxide during S phase

    International Nuclear Information System (INIS)

    Yamamoto, Ryohei; Ohshiro, Yukari; Shimotani, Tatsuhiko; Yamamoto, Mizuki; Matsuyama, Satoshi; Ide, Hiroshi; Kubo, Kihei

    2014-01-01

    Oxidative base damage occurs spontaneously due to reactive oxygen species generated as byproducts of respiration and other pathological processes in mammalian cells. Many oxidized bases are mutagenic and/or toxic, and most are repaired through the base excision repair pathway. Human endonuclease VIII-like protein 1 (hNEIL1) is thought to play an important role during the S phase of the cell cycle by removing oxidized bases in DNA replication fork-like (bubble) structures, and the protein level of hNEIL1 is increased in S phase. Compared with hNEIL1, there is relatively little information on the properties of the mouse ortholog mNEIL1. Since mouse cell nuclei lack endonuclease III-like protein (NTH) activity, in contrast to human cell nuclei, mNEIL1 is a major DNA glycosylase for repair of oxidized pyrimidines in mouse nuclei. In this study, we made mNEIL1-knockdown cells using an shRNA expression vector and examined the cell cycle-related variation in hydrogen peroxide (H 2 O 2 ) sensitivity. Hypersensitivity to H 2 O 2 caused by mNEIL1 knockdown was more significant in S phase than in G1 phase, suggesting that mNEIL1 has an important role during S phase, similarly to hNEIL1

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

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    Kaushali Thakore-Shah

    Full Text Available 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.

  2. Knockdown of ARK5 Expression Suppresses Invasion and Metastasis of Gastric Cancer

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    Dehu Chen

    2017-06-01

    Full Text Available Background/Aims: Gastric cancer (GC is a common and lethal malignancy, and AMP-activated protein kinase-related kinase 5 (ARK5 has been discovered to promote cancer metastasis in certain types of cancer. In this study, we explored the role of ARK5 in GC invasion and metastasis. Methods: ARK5 and epithelial-mesenchymal transition (EMT-related markers were determined by immunohistochemistry and western blot in GC specimens. Other methods including stably transfected against ARK5 into SGC7901 and AGS cells, western blot, migration and invasion assays in vitro and nude mice tumorigenicity in vivo were also employed. Results: The results demonstrated that ARK5 expression was increased and positively correlated with metastasis, EMT-related markers and poor prognosis in patients with GC. Knockdown of ARK5 expression remarkably suppressed GC cells invasion and metastasis via regulating EMT, rather than proliferation in vitro and in vivo. And knockdown of ARK5 expression in GC cells resulted in the down-regulation of the mTOR/p70S6k signals, Slug and SIP1. Conclusion: The elevated ARK5 expression was closely associated with cancer metastasis and patient survival, and it seemed to function in GC cells migration and invasion via EMT alteration, together with the alteration of the mTOR/p70S6k signals, Slug and SIP1, thus providing a potential therapeutic target for GC.

  3. The knockdown of OsVIT2 and MIT affects iron localization in rice seed.

    Science.gov (United States)

    Bashir, Khurram; Takahashi, Ryuichi; Akhtar, Shamim; Ishimaru, Yasuhiro; Nakanishi, Hiromi; Nishizawa, Naoko K

    2013-11-20

    The mechanism of iron (Fe) uptake in plants has been extensively characterized, but little is known about how Fe transport to different subcellular compartments affects Fe localization in rice seed. Here, we discuss the characterization of a rice vacuolar Fe transporter 2 (OsVIT2) T-DNA insertion line (osvit2) and report that the knockdown of OsVIT2 and mitochondrial Fe transporter (MIT) expression affects seed Fe localization. osvit2 plants accumulated less Fe in their shoots when grown under normal or excess Fe conditions, while the accumulation of Fe was comparable to that in wild-type (WT) plants under Fe-deficient conditions. The accumulation of zinc, copper, and manganese also changed significantly in the shoots of osvit2 plants. The growth of osvit2 plants was also slow compared to that of WT plants. The concentration of Fe increased in osvit2 polished seeds. Previously, we reported that the expression of OsVIT2 was higher in MIT knockdown (mit-2) plants, and in this study, the accumulation of Fe in mit-2 seeds decreased significantly. These results suggest that vacuolar Fe trafficking is important for plant Fe homeostasis and distribution, especially in plants grown in the presence of excess Fe. Moreover, changes in the expression of OsVIT2 and MIT affect the concentration and localization of metals in brown rice as well as in polished rice seeds.

  4. Knockdown of autophagy enhances innate immune response in hepatitis C virus infected hepatocytes

    Science.gov (United States)

    Shrivastava, Shubham; Raychoudhuri, Amit; Steele, Robert; Ray, Ranjit; Ray, Ratna B.

    2010-01-01

    The role of autophagy in disease pathogenesis following viral infection is beginning to be elucidated. We have previously reported that hepatitis C virus (HCV) infection in hepatocytes induces autophagy. However, the biological significance of HCV induced autophagy has not been clarified. Autophagy has recently been identified as a novel component of innate immune system against viral infection. In the present study, we have shown that knockdown of autophagy related protein Beclin1 or ATG7 in immortalized human hepatocytes (IHH) inhibited HCV growth. Beclin1 or ATG7 knockdown IHH when infected with HCV exhibited an increased expression of IFN-β, OAS-1, IFN-α and IFI27 mRNAs of the interferon signaling pathways as compared to infection of control IHH. Subsequent study demonstrated that HCV infection in autophagy impaired IHH displayed caspase activation, PARP cleavage and apoptotic cell death. Conclusion The disruption of autophagy machinery in HCV infected hepatocytes activated IFN signaling pathway, and induced apoptosis. Together, these results suggest that HCV induced autophagy impairs innate immune response. PMID:21274862

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

    Directory of Open Access Journals (Sweden)

    Stephanie M Marshall

    Full Text Available 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.

  6. Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma.

    Science.gov (United States)

    Kundu, Anup K; Iyer, Swathi V; Chandra, Sruti; Adhikari, Amit S; Iwakuma, Tomoo; Mandal, Tarun K

    2017-01-01

    The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318-1, a murine osteosarcoma cell line. The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP) and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency. Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent. This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.

  7. Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma.

    Directory of Open Access Journals (Sweden)

    Anup K Kundu

    Full Text Available The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318-1, a murine osteosarcoma cell line.The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency.Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent.This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.

  8. Knockdown of RMI1 impairs DNA repair under DNA replication stress.

    Science.gov (United States)

    Xu, Chang; Fang, Lianying; Kong, Yangyang; Xiao, Changyan; Yang, Mengmeng; Du, Li-Qing; Liu, Qiang

    2017-12-09

    RMI1 (RecQ-mediated genome instability protein 1) forms a conserved BTR complex with BLM, Topo IIIα, and RMI2, and its absence causes genome instability. It has been revealed that RMI1 localizes to nuclear foci with BLM and Topo IIIα in response to replication stress, and that RMI1 functions downstream of BLM in promoting replication elongation. However, the precise functions of RMI1 during replication stress are not completely understood. Here we report that RMI1 knockdown cells are hypersensitive to hydroxyurea (HU). Using comet assay, we show that RMI1 knockdown cells exhibit accumulation of broken DNAs after being released from HU treatment. Moreover, we demonstrate that RMI1 facilitates the recovery from activated checkpoint and resuming the cell cycle after replicative stress. Surprisingly, loss of RMI1 results in a failure of RAD51 loading onto DNA damage sites. These findings reveal the importance of RMI1 in response to replication stress, which could explain the molecular basis for its function in maintaining genome integrity. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Knockdown of versican 1 blocks cigarette-induced loss of insoluble elastin in human lung fibroblasts.

    Science.gov (United States)

    Xu, Lu-lu; Lu, Yun-tao; Zhang, Jing; Wu, Lian; Merrilees, Mervyn J; Qu, Jie-ming

    2015-08-15

    COPD lung is characterized by loss of alveolar elastic fibers and an increase in the chondroitin sulfate (CS) matrix proteoglycan versican V1 (V1). V1 is a known inhibitor of elastic fiber deposition and this study investigates the effects of knockdown of V1, and add-back of CS, on CCL-210 lung fibroblasts treated with cigarette smoke extract (CSE) as a model for COPD. CSE inhibited fibroblast proliferation, viability, tropoelastin synthesis, and elastin deposition, and increased V1 synthesis and secretion. V1 siRNA decreased V1 and constituent CS, did not affect tropoelastin production, but blocked the CSE-induced loss in insoluble elastin. Exogenous CS reduced insoluble elastin, even in the presence of V1 siRNA. These findings confirm that V1 and CS impair the assembly of tropoelastin monomers into insoluble fibers, and further demonstrate that specific knockdown of V1 alleviates the impaired assembly of elastin seen in cultures of pulmonary fibroblasts exposed to CSE, indicating a regulatory role for this protein in the pathophysiology of COPD. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    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 [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Madden, Stephen L., E-mail: steve.madden@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Jiang, Yide, E-mail: yide.jiang@genzyme.com [Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States)

    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.

  11. Knockdown of Zebrafish Blood Vessel Epicardial Substance Results in Incomplete Retinal Lamination

    Directory of Open Access Journals (Sweden)

    Yu-Ching Wu

    2014-01-01

    Full Text Available Cell polarity during eye development determines the normal retinal lamination and differentiation of photoreceptor cells in the retina. In vertebrates, blood vessel epicardial substance (Bves is known to play an important role in the formation and maintenance of the tight junctions essential for epithelial cell polarity. In the current study, we generated a transgenic zebrafish Bves (zbves promoter-EGFP zebrafish line to investigate the expression pattern of Bves in the retina and to study the role of zbves in retinal lamination. Immunostaining with different specific antibodies from retinal cells and transmission electron microscopy were used to identify the morphological defects in normal and Bves knockdown zebrafish. In normal zebrafish, Bves is located at the apical junctions of embryonic retinal neuroepithelia during retinogenesis; later, it is strongly expressed around inner plexiform layer (IPL and retinal pigment epithelium (RPE. In contrast, a loss of normal retinal lamination and cellular polarity was found with undifferentiated photoreceptor cells in Bves knockdown zebrafish. Herein, our results indicated that disruption of Bves will result in a loss of normal retinal lamination.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Jiajia [Department of Laboratory Medicine, Peking University Third Hospital, Beijing (China); Zhu, Xi [Department of Urology, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing (China); Zhang, Jie, E-mail: zhangjiebjmu@163.com [Department of Laboratory Medicine, Peking University Third Hospital, Beijing (China)

    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.

  13. Use of zebrafish and knockdown technology to define proprotein convertase activity.

    Science.gov (United States)

    Chitramuthu, Babykumari P; Bennett, Hugh P J

    2011-01-01

    The Zebrafish (Danio rerio) is a powerful and well-established tool used extensively for the study of early vertebrate development and as a model of human diseases. Zebrafish genes orthologous to their mammalian counterparts generally share conserved biological function. Protein knockdown or overexpression can be effectively achieved by microinjection of morpholino antisense oligonucleotides (MOs) or mRNA, respectively, into developing embryos at the one- to two-cell stage. Correlating gene expression patterns with the characterizing of phenotypes resulting from over- or underexpression can reveal the function of a particular protein. The microinjection technique is simple and results are reproducible. We defined the expression pattern of the proprotein convertase PCSK5 within the lateral line neuromasts and various organs including the liver, gut and otic vesicle by whole-mount in situ hybridization (ISH) and immunofluorescence (IF). MO-mediated knockdown of zebrafish PCSK5 expression generated embryos that display abnormal neuromast deposition within the lateral line system resulting in uncoordinated patterns of swimming.

  14. TREX1 Knockdown Induces an Interferon Response to HIV that Delays Viral Infection in Humanized Mice

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    Lee Adam Wheeler

    2016-05-01

    Full Text Available Despite their antiviral effect, the in vivo effect of interferons on HIV transmission is difficult to predict, because interferons also activate and recruit HIV-susceptible cells to sites of infection. HIV does not normally induce type I interferons in infected cells, but does if TREX1 is knocked down. Here, we investigated the effect of topical TREX1 knockdown and local interferon production on HIV transmission in human cervicovaginal explants and humanized mice. In explants in which TREX1 was knocked down, HIV induced interferons, which blocked infection. In humanized mice, even though TREX1 knockdown increased infiltrating immune cells, it delayed viral replication for 3–4 weeks. Similarly intravaginal application of type I interferons the day before HIV infection induced interferon responsive genes, reduced inflammation, and decreased viral replication. However, intravenous interferon enhanced inflammation and infection. Thus, in models of human sexual transmission, a localized interferon response inhibits HIV transmission but systemic interferons do not.

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

    Zheng, Jiajia; Zhu, Xi; Zhang, Jie

    2014-01-01

    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

  17. [Knockdown of NEDD9 inhibits the proliferation, invasion and migration of esophageal carcinoma EC109 cells].

    Science.gov (United States)

    Zhang, Wen; Li, Shaojun; Zhao, Yunlong; Guo, Nannan; Li, Yingjie

    2016-12-01

    Objective To observe the expression of the neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) in esophageal cancer, to investigate the impact of decreased expression of NEDD9 on invasion and migration, and to explicit the function of NEDD9 in EC109 human esophageal cancer cell line. Methods Immunohistochemical staining was used to detect the expression of NEDD9 in human esophageal cancer tissues and paracancerous normal tissues. RNA interfering (RNAi) was used to knockdown NEDD9 in EC109 cells. The interference efficiency was detected by reverse transcription PCR (RT-PCR) and Western blot analysis. Cell proliferation was determined by MTT assay and the invasion and migration abilities of EC109 cells were monitored by Transwell TM assay. The protein levels of proliferating cell nuclear antigen (PCNA), Bax and Bcl-2 were tested by Western blotting. Results The positive expression rate of NEDD9 in esophageal carcinoma tissues was significantly higher compared with that in the paracancerous tissues. After NEDD9 expression was successfully downregulated in EC109 cells by siRNA, the proliferation, invasion and migration rates in transfection group were significantly lower than those in control group; meanwhile, the expression of Bcl-2 was reduced and Bax expression was enhanced. Conclusion The protein expression level of NEDD9 is higher in esophageal carcinoma tissues than that in adjacent normal tissues. Knockdown of NEDD9 expression can restrain the proliferation, invasion and migration of EC109 cells.

  18. [Knockdown of InR gene in ventral nephrocytes promotes resistance to toxic stress in Drosophila melanogaster females].

    Science.gov (United States)

    Andreenkova, O V; Karpova, E K; Menshanov, P N; Rauschenbach, I Yu

    2015-02-01

    Hemolymph filtration in insects is performed by nephrocytes, additional cells of the circulatory system that are not connected to Malpighian vessels. Drosophila has two types of nephrocytes: the ventral ("garland"), which are situated around the connection site of the esophagus and proventriculus, and the pericardial, which are localized around the heart. In this study, we examined the role of the of insulin-like receptor (InR)gene in regulation of the function of ventral nephrocytes (VNC) in D. melanogaster females. Immunofluorescent analysis of female VNC with anti-InR antibodies revealed for the first time that the InR gene is expressed in VNC cells. To determine whether a change in the level of InR expression has an effect on VNC function in Drosophila females, we implemented an antisense suppressor of the InR gene, together with a driver that is expressed specifically in VNC. VNC function was evaluated by survival of the females exposed to toxic stress (treatment with AgNO3). This study has shown for the first time that suppression of InR expression in VNC leads to a rise in the survival of flies under conditions of toxic stress.

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

    Energy Technology Data Exchange (ETDEWEB)

    Massarsky, Andrey, E-mail: andrey.massarsky@duke.edu [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Bone, Audrey J. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Dong, Wu [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); School of Animal Science and Technology, Inner Mongolia Provincial Key Laboratory for Toxicants and Animal Disease, Inner Mongolia University for the Nationalities, Tongliao, Inner Mongolia 028000 (China); Hinton, David E. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Prasad, G.L. [RAI Services Company, Winston-Salem, NC 27101 (United States); Di Giulio, Richard T. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States)

    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 6 h post fertilization (hpf) until 96 hpf to TPM{sub 0.5} and TPM{sub 1.0} (corresponding to 0.5 and 1.0 μg/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 96 hpf 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. - Highlights: • Total particulate matter (TPM) is the particulate phase of cigarette smoke. • Zebrafish is proposed as a ‘bridge model’ to study the effects of TPM. • We investigate the roles of antioxidant and aryl hydrocarbon receptor (AHR) pathways.

  20. Simultaneous analysis of multiple Mycobacterium tuberculosis knockdown mutants in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Antje Blumenthal

    2010-12-01

    Full Text Available Mycobacterium tuberculosis (Mtb represents one of the most persistent bacterial threats to human health and new drugs are needed to limit its impact. Conditional knockdown mutants can help validate new drug targets, but the analysis of individual mutants is laborious and time consuming. Here, we describe quantitative DNA tags (qTags and their use to simultaneously analyze conditional Mtb knockdown mutants that allowed silencing the glyoxylate and methylcitrate cycles (via depletion of isocitrate lyase, ICL, the serine protease Rv3671c, and the core subunits of the mycobacterial proteasome, PrcB and PrcA. The impact of gene silencing in multi-strain cultures was determined by measuring the relative abundance of mutant-specific qTags with real-time PCR. This achieved accurate quantification over a broad range of qTag abundances and depletion of ICL, Rv3671c, or PrcBA resulted in the expected impairment of growth of Mtb with butyrate as the primary carbon source, survival during oxidative stress, acid stress and starvation. The impact of depleting ICL, Rv3671c, or PrcBA in multi-strain mouse infections was analyzed with two approaches. We first measured the relative abundance of mutant-specific qTags in total chromosomal DNA isolated from bacteria that were recovered from infected lungs on agar plates. We then developed a two-step amplification procedure, which allowed us to measure the abundances of individual mutants directly in infected lung tissue. Both strategies confirmed that inactivation of Rv3671c and PrcBA severely reduced persistence of Mtb in mice. The multi-strain infections furthermore suggested that silencing ICL not only prevented growth of Mtb during acute infections but also prevented survival of Mtb during chronic infections. Analyses of the ICL knockdown mutant in single-strain infections confirmed this and demonstrated that silencing of ICL during chronic infections impaired persistence of Mtb to the extent that the pathogen

  1. Diminished exercise capacity and mitochondrial bc1 complex deficiency in tafazzin-knockdown mice.

    Directory of Open Access Journals (Sweden)

    Corey ePowers

    2013-04-01

    Full Text Available The phospholipid, cardiolipin, is essential for maintaining mitochondrial structure and optimal function. Cardiolipin-deficiency in humans, Barth syndrome, is characterized by exercise intolerance, dilated cardiomyopathy, neutropenia and 3-methyl-glutaconic aciduria. The causative gene is the mitochondrial acyl-transferase, tafazzin that is essential for remodeling acyl chains of cardiolipin. We sought to determine metabolic rates in tafazzin-deficient mice during resting and exercise, and investigate the impact of cardiolipin deficiency on mitochondrial respiratory chain activities. Tafazzin knockdown in mice markedly impaired oxygen consumption rates during an exercise, without any significant effect on resting metabolic rates. CL-deficiency resulted in significant reduction of mitochondrial respiratory reserve capacity in neonatal cardiomyocytes that is likely to be caused by diminished activity of complex-III, which requires CL for its assembly and optimal activity. Our results may provide mechanistic insights of Barth syndrome pathogenesis.

  2. Knockdown of cytosolic NADP(+) -dependent isocitrate dehydrogenase enhances MPP(+) -induced oxidative injury in PC12 cells.

    Science.gov (United States)

    Yang, Eun Sun; Park, Jeen-Woo

    2011-05-01

    1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridium ion (MPP(+)) have been shown to induce Parkinson's disease-like symptoms as well as neurotoxicity in humans and animal species. Recently, we reported that maintenance of redox balance and cellular defense against oxidative damage are primary functions of the novel antioxidant enzyme cytosolic NADP(+) -dependent isocitrate dehydrogenase (IDPc). In this study, we examined the role of IDPc in cellular defense against MPP(+) -induced oxidative injury using PC12 cells transfected with IDPc small interfering RNA (siRNA). Our results demonstrate that MPP(+) -mediated disruption of cellular redox status, oxidative damage to cells, and apoptotic cell death were significantly enhanced by knockdown of IDPc.

  3. Novel liposomal combination treatments using dual genes knockdown in oral cancer treatment

    Science.gov (United States)

    Wu, Jyun-Sian; Yeh, Chia-Hsien; Huang, Leaf; Hsu, Yih-Chih

    2018-02-01

    Small interfering RNA (siRNA) can be used to treat tumor because it can effectively knockdown target oncoprotein expression and it leads to cancer cell death and apoptosis. Hypoxia-inducible factors-1 (HIF-1) is a transcription factor gene. Its high expression of tumor hypoxia cells, activation of transcription factor HIF-1α and angiogenesis found in most cancerous tissues. HIF-1α protein in cancer cells are critical to cell survival, tumor growth and proliferation. Epidermal growth factor receptor (EGFR) gene is another common head and neck oncogene. The dual self-designed siRNA sequences were encapsulated in the lipid-calcium-phosphate (LCP) and targeted to sigma receptors on the surface of cancer cells via binding to amino ethyl anisamide (AEAA). We used human oral cancer cells to establish the xenograft animal model to study the combination therapy for therapeutic results.

  4. Lentivirus-mediated knockdown of NLK inhibits small-cell lung cancer growth and metastasis

    Directory of Open Access Journals (Sweden)

    Lv MT

    2016-11-01

    Full Text Available Mutian Lv,1 Yaming Li,1 Xin Tian,2 Shundong Dai,3,4 Jing Sun,5 Guojiang Jin,6 Shenyi Jiang7 1Department of Nuclear Medicine, 2Molecular Oncology Laboratory of Cancer Research Institute, The First Affiliated Hospital of China Medical University, 3Department of Pathology, The First Affiliated Hospital, College of Basic Medical Sciences of China Medical University, 4Department of Pathology, Institute of Pathology and Pathophysiology, 5Department of Immunology and Biotherapy, Liaoning Cancer Hospital and Institute, 6Department of Laboratory Medicine, 7Department of Rheumatology, The First Affiliated Hospital of China Medical University, Shenyang, People’s Republic of China Abstract: Nemo-like kinase (NLK, an evolutionarily conserved serine/threonine kinase, has been recognized as a critical regulator of various cancers. In this study, we investigated the role of NLK in human small-cell lung cancer (SCLC, which is the most aggressive form of lung cancer. NLK expression was evaluated by quantitative real-time polymerase chain reaction in 20 paired fresh SCLC tissue samples and found to be noticeably elevated in tumor tissues. Lentivirus-mediated RNAi efficiently suppressed NLK expression in NCI-H446 cells, resulting in a significant reduction in cell viability and proliferation in vitro. Moreover, knockdown of NLK led to cell cycle arrest at the S-phase via suppression of Cyclin A, CDK2, and CDC25A, which could contribute to cell growth inhibition. Furthermore, knockdown of NLK decreased the migration of NCI-H446 cells and downregulated matrix metalloproteinase 9. Treatment with NLK short hairpin RNA significantly reduced SCLC tumor growth in vivo. In conclusion, this study suggests that NLK plays an important role in the growth and metastasis of SCLC and may serve as a potential therapeutic target for the treatment of SCLC. Keywords: NLK, SCLC, RNAi, proliferation, migration

  5. Knockdown of hypocretin attenuates extended access of cocaine self-administration in rats.

    Science.gov (United States)

    Schmeichel, Brooke E; Matzeu, Alessandra; Koebel, Pascale; Vendruscolo, Leandro F; Sidhu, Harpreet; Shahryari, Roxana; Kieffer, Brigitte L; Koob, George F; Martin-Fardon, Rémi; Contet, Candice

    2018-04-06

    The hypocretin/orexin (HCRT) neuropeptide system regulates feeding, arousal state, stress responses, and reward, especially under conditions of enhanced motivational relevance. In particular, HCRT neurotransmission facilitates drug-seeking behavior in circumstances that demand increased effort and/or motivation to take the drug. The present study used a shRNA-encoding adeno-associated viral vector to knockdown Hcrt expression throughout the dorsal hypothalamus in adult rats and determine the role of HCRT in cocaine self-administration. Chronic Hcrt silencing did not impact cocaine self-administration under short-access conditions, but robustly attenuated cocaine intake under extended access conditions, a model that mimics key features of compulsive cocaine taking. In addition, Hcrt silencing decreased motivation for both cocaine and a highly palatable food reward (i.e., sweetened condensed milk; SCM) under a progressive ratio schedule of reinforcement, but did not alter responding for SCM under a fixed ratio schedule. Importantly, Hcrt silencing did not affect food or water consumption, and had no consequence for general measures of arousal and stress reactivity. At the molecular level, chronic Hcrt knockdown reduced the number of neurons expressing dynorphin (DYN), and to a smaller extent melanin-concentrating hormone (MCH), in the dorsal hypothalamus. These original findings support the hypothesis that HCRT neurotransmission promotes operant responding for both drug and non-drug rewards, preferentially under conditions requiring a high degree of motivation. Furthermore, the current study provides compelling evidence for the involvement of the HCRT system in cocaine self-administration also under low-effort conditions in rats allowed extended access, possibly via functional interactions with DYN and MCH signaling.

  6. Keap1 knockdown increases markers of metabolic syndrome after long-term high fat diet feeding.

    Science.gov (United States)

    More, Vijay R; Xu, Jialin; Shimpi, Prajakta C; Belgrave, Clyde; Luyendyk, James P; Yamamoto, Masayuki; Slitt, Angela L

    2013-08-01

    The nuclear factor E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway upregulates antioxidant and biotransformation enzyme expression to counter cellular oxidative stress. The contributions of Nrf2 to other cellular functions, such as lipid homeostasis, are emerging. This study was conducted to determine how enhanced Nrf2 activity influences the progression of metabolic syndrome with long-term high-fat diet (HFD) feeding. C57BL/6 and Keap1-knockdown (Keap1-KD) mice, which exhibit enhanced Nrf2 activity, were fed a HFD for 24 weeks. Keap1-KD mice had higher body weight and white adipose tissue mass compared to C57BL/6 mice on HFD, along with increased inflammation and lipogenic gene expression. HFD feeding increased hepatic steatosis and inflammation to a greater extent in Keap1-KD mice compared to C57BL/6 mice, which was associated with increased liver Cd36, fatty acid-binding protein 4, and monocyte chemoattractant protein 1 mRNA expression, as well as increased acetyl-CoA carboxylase 1 and stearoyl-CoA desaturase-1 protein expression. The HFD altered short-term glucose homeostasis to a greater degree in Keap-KD mice compared to C57BL/6 mice, which was accompanied by downregulation of insulin receptor substrate 1 mRNA expression in skeletal muscle. Together, the results indicate that Keap1 knockdown, on treatment with HFD, increases certain markers of metabolic syndrome. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Expression and knockdown of zebrafish folliculin suggests requirement for embryonic brain morphogenesis.

    Science.gov (United States)

    Kenyon, Emma J; Luijten, Monique N H; Gill, Harmeet; Li, Nan; Rawlings, Matthew; Bull, James C; Hadzhiev, Yavor; van Steensel, Maurice A M; Maher, Eamonn; Mueller, Ferenc

    2016-07-08

    Birt-Hogg-Dubé syndrome (BHD) is a dominantly inherited familial cancer syndrome characterised by the development of benign skin fibrofolliculomas, multiple lung and kidney cysts, spontaneous pneumothorax and susceptibility to renal cell carcinoma. BHD is caused by mutations in the gene encoding Folliculin (FLCN). Little is known about what FLCN does in a healthy individual and how best to treat those with BHD. As a first approach to developing a vertebrate model for BHD we aimed to identify the temporal and spatial expression of flcn transcripts in the developing zebrafish embryo. To gain insights into the function of flcn in a whole organism system we generated a loss of function model of flcn by the use of morpholino knockdown in zebrafish. flcn is expressed broadly and upregulated in the fin bud, somites, eye and proliferative regions of the brain of the Long-pec stage zebrafish embryos. Together with knockdown phenotypes, expression analysis suggest involvement of flcn in zebrafish embryonic brain development. We have utilised the zFucci system, an in vivo, whole organism cell cycle assay to study the potential role of flcn in brain development. We found that at the 18 somite stage there was a significant drop in cells in the S-M phase of the cell cycle in flcn morpholino injected embryos with a corresponding increase of cells in the G1 phase. This was particularly evident in the brain, retina and somites of the embryo. Timelapse analysis of the head region of flcn morpholino injected and mismatch control embryos shows the temporal dynamics of cell cycle misregulation during development. In conclusion we show that zebrafish flcn is expressed in a non-uniform manner and is likely required for the maintenance of correct cell cycle regulation during embryonic development. We demonstrate the utilisation of the zFucci system in testing the role of flcn in cell proliferation and suggest a function for flcn in regulating cell proliferation in vertebrate embryonic

  8. Quantification of Functionalised Gold Nanoparticle-Targeted Knockdown of Gene Expression in HeLa Cells

    Science.gov (United States)

    Jiwaji, Meesbah; Sandison, Mairi E.; Reboud, Julien; Stevenson, Ross; Daly, Rónán; Barkess, Gráinne; Faulds, Karen; Kolch, Walter; Graham, Duncan; Girolami, Mark A.; Cooper, Jonathan M.; Pitt, Andrew R.

    2014-01-01

    Introduction Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. PMID:24926959

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

    Science.gov (United States)

    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.

  10. Keratin23 (KRT23) knockdown decreases proliferation and affects the DNA damage response of colon cancer cells

    DEFF Research Database (Denmark)

    Birkenkamp-Demtröder, Karin; Hahn, Stephan; Mansilla, Francisco

    2013-01-01

    correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression in vitro. Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed...... response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced......Keratin 23 (KRT23) is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation...

  11. shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

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    Qin Wang

    2014-01-01

    Full Text Available Colon cancer is one of the most common tumors of the digestive tract. Resistance to ionizing radiation (IR decreased therapeutic efficiency in these patients’ radiotherapy. XRCC2 is the key protein of DNA homologous recombination repair, and its high expression is associated with enhanced resistance to DNA damage induced by IR. Here, we investigated the effect of XRCC2 silencing on colon tumor cells’ growth and sensitivity to X-radiation in vitro and in vivo. Colon tumor cells (T84 cell line were cultivated in vitro and tumors originated from the cell line were propagated as xenografts in nude mice. The suppression of XRCC2 expression was achieved by using vector-based short hairpin RNA (shRNA in T84 cells. We found that the knockdown of XRCC2 expression effectively decreased T84 cellular proliferation and colony formation, and led to cell apoptosis and cell cycle arrested in G2/M phase induced by X-radiation in vitro. In addition, tumor xenograft studies suggested that XRCC2 silencing inhibited tumorigenicity after radiation treatment in vivo. Our data suggest that the suppression of XRCC2 expression rendered colon tumor cells more sensitive to radiation therapy in vitro and in vivo, implying XRCC2 as a promising therapeutic target for the treatment of radioresistant human colon cancer.

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

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    Lian Ling

    2012-03-01

    Full Text Available Abstract 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

  13. Insecticide resistance and resistance mechanisms in bed bugs, Cimex spp. (Hemiptera: Cimicidae).

    Science.gov (United States)

    Dang, Kai; Doggett, Stephen L; Veera Singham, G; Lee, Chow-Yang

    2017-06-29

    The worldwide resurgence of bed bugs [both Cimex lectularius L. and Cimex hemipterus (F.)] over the past two decades is believed in large part to be due to the development of insecticide resistance. The transcriptomic and genomic studies since 2010, as well as morphological, biochemical and behavioral studies, have helped insecticide resistance research on bed bugs. Multiple resistance mechanisms, including penetration resistance through thickening or remodelling of the cuticle, metabolic resistance by increased activities of detoxification enzymes (e.g. cytochrome P450 monooxygenases and esterases), and knockdown resistance by kdr mutations, have been experimentally identified as conferring insecticide resistance in bed bugs. Other candidate resistance mechanisms, including behavioral resistance, some types of physiological resistance (e.g. increasing activities of esterases by point mutations, glutathione S-transferase, target site insensitivity including altered AChEs, GABA receptor insensitivity and altered nAChRs), symbiont-mediated resistance and other potential, yet undiscovered mechanisms may exist. This article reviews recent studies of resistance mechanisms and the genes governing insecticide resistance, potential candidate resistance mechanisms, and methods of monitoring insecticide resistance in bed bugs. This article provides an insight into the knowledge essential for the development of both insecticide resistance management (IRM) and integrated pest management (IPM) strategies for successful bed bug management.

  14. Knockdown of HIF-1α and IL-8 induced apoptosis of hepatocellular carcinoma triggers apoptosis of vascular endothelial cells.

    Science.gov (United States)

    Choi, Sung Hoon; Park, Jun Yong; Kang, Wonseok; Kim, Seung Up; Kim, Do Young; Ahn, Sang Hoon; Ro, Simon Wonsang; Han, Kwang-Hyub

    2016-01-01

    A local hypoxic microenvironment is one of the most important characteristics of solid tumors. Hypoxia inducible factor-1α (HIF-1α) and Interleukin-8 (IL-8) activate tumor survival from hypoxic-induced apoptosis in each pathway. This study aimed to evaluate whether knockdown of HIF-1α and IL-8 induced apoptosis of the hepatocellular carcinoma (HCC) and endothelial cell lines. HCC cell lines were infected with adenovirus-expressing shRNA for HIF-1α and IL-8 and maintained under hypoxic conditions (1% O2, 24 h). The expression levels of HIF-1α and both apoptotic and growth factors were examined by real-time quantitative PCR and western blot. We also investigated apoptosis by TUNEL assay (FACS and Immunofluorescence) and measured the concentration of cytochrome C. Inhibition of HIF-1α and IL-8 up-regulated the expression of apoptotic factors while downregulating anti-apoptotic factors simultaneously. Knockdown of HIF-1α and IL-8 increased the concentration of cytochrome C and enhanced DNA fragmentation in HCC cell lines. Moreover, culture supernatant collected from the knockdown of HIF-1α and IL-8 in HCC cell lines induced apoptosis in human umbilical vein endothelial cells under hypoxia, and the expression of variable apoptotic ligand increased from HCC cell lines, time-dependently. These data suggest that adenovirus-mediated knockdown of HIF-1α and IL-8 induced apoptosis in HCC cells and triggered apoptosis of vascular endothelial cells.

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

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    Akira Marine

    2014-01-01

    Full Text Available 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.

  16. Effects of vivo morpholino knockdown of lateral hypothalamus orexin/hypocretin on renewal of alcohol seeking.

    Science.gov (United States)

    Prasad, Asheeta A; McNally, Gavan P

    2014-01-01

    Two experiments used vivo morpholinos to assess the role of orexin/hypocretin in ABA renewal of extinguished alcohol seeking. Rats were trained to respond for alcoholic beer in a distinctive context, A, and then extinguished in a second distinctive context, B. When rats were tested in the extinction context, ABB, responding was low but when they were tested in the training context, ABA, responding was significantly higher. Microinjection of an orexin/hypocretin antisense vivo morpholino into LH significantly reduced orexin/hypocretin protein expression but had no effect on the ABA renewal of alcohol seeking (Experiment 1). Microinjection of a higher dose of the antisense vivo morpholino into LH also significantly reduced orexin/hypocretin protein expression but this was not selective and yielded significant reduction in melanin-concentrating hormone (MCH) protein expression. This non-selective knockdown did significantly reduce ABA renewal as well as reduce the reacquisition of alcohol seeking. Taken together, these findings show an important role for LH in the ABA renewal of alcohol seeking but that orexin/hypocretin is not necessary for this renewal.

  17. Effects of vivo morpholino knockdown of lateral hypothalamus orexin/hypocretin on renewal of alcohol seeking.

    Directory of Open Access Journals (Sweden)

    Asheeta A Prasad

    Full Text Available Two experiments used vivo morpholinos to assess the role of orexin/hypocretin in ABA renewal of extinguished alcohol seeking. Rats were trained to respond for alcoholic beer in a distinctive context, A, and then extinguished in a second distinctive context, B. When rats were tested in the extinction context, ABB, responding was low but when they were tested in the training context, ABA, responding was significantly higher. Microinjection of an orexin/hypocretin antisense vivo morpholino into LH significantly reduced orexin/hypocretin protein expression but had no effect on the ABA renewal of alcohol seeking (Experiment 1. Microinjection of a higher dose of the antisense vivo morpholino into LH also significantly reduced orexin/hypocretin protein expression but this was not selective and yielded significant reduction in melanin-concentrating hormone (MCH protein expression. This non-selective knockdown did significantly reduce ABA renewal as well as reduce the reacquisition of alcohol seeking. Taken together, these findings show an important role for LH in the ABA renewal of alcohol seeking but that orexin/hypocretin is not necessary for this renewal.

  18. Sex determination in beetles: Production of all male progeny by Parental RNAi knockdown of transformer

    Science.gov (United States)

    Shukla, Jayendra Nath; Palli, Subba Reddy

    2012-01-01

    Sex in insects is determined by a cascade of regulators ultimately controlling sex-specific splicing of a transcription factor, Doublesex (Dsx). We recently identified homolog of dsx in the red flour beetle, Tribolium castaneum (Tcdsx). Here, we report on the identification and characterization of a regulator of Tcdsx splicing in T. castaneum. Two male-specific and one female-specific isoforms of T. castaneum transformer (Tctra) were identified. RNA interference-aided knockdown of Tctra in pupa or adults caused a change in sex from females to males by diverting the splicing of Tcdsx pre-mRNA to male-specific isoform. All the pupa and adults developed from Tctra dsRNA injected final instar larvae showed male-specific sexually dimorphic structures. Tctra parental RNAi caused an elimination of females from the progeny resulting in production of all male progeny. Transformer parental RNAi could be used to produce all male population for use in pest control though sterile male release methods. PMID:22924109

  19. Analysis of changes to mRNA levels and CTCF occupancy upon TFII-I knockdown

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    Maud Marques

    2015-06-01

    Full Text Available CTCF is a key regulator of nuclear chromatin structure, chromatin organization and gene regulation. The impact of CTCF on transcriptional output is quite varied, ranging from repression, to transcriptional pausing and transactivation. The multifunctional nature of CTCF is mediated, in part, through differential association with protein partners having unique properties. We identified the general transcription factor TFII-I as an interacting partner of CTCF. To gain an understanding of the function of TFII-I in regulating gene expression and CTCF binding genome wide, we conducted microarray experiments following TFII-I knockdown and chromatin immunoprecipitation of CTCF followed by next generation sequencing (ChIP-seq from the same TFII-I depleted cells. Here, we described the experimental design and the quality control and analysis that were performed on the dataset. The data is publicly available through the GEO database with accession number GSE60918. The interpretation and description of these data are included in a manuscript in revision (1.

  20. Efficient and specific gene knockdown by small interfering RNAs produced in bacteria

    Science.gov (United States)

    Huang, Linfeng; Jin, Jingmin; Deighan, Padraig; Kiner, Evgeny; McReynolds, Larry; Lieberman, Judy

    2013-01-01

    Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells1,2 and may be used for therapeutic purposes to knockdown genes implicated in disease3. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in E. coli. This method relies on ectopic expression of p19, a siRNA-binding protein found in a plant RNA virus4, 5. When expressed in E. coli, p19 stabilizes ~21 nt siRNA-like species produced by bacterial RNase III. Transfection of mammalian cells with siRNAs, generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene, at low nanomolar concentrations reproducibly knocks down gene expression by ~90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes. PMID:23475073

  1. U6 snRNA expression prevents toxicity in TDP-43-knockdown cells.

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    Masao Yahara

    Full Text Available Depletion of amyotrophic lateral sclerosis (ALS-associated transactivation response (TAR RNA/DNA-binding protein 43 kDa (TDP-43 alters splicing efficiency of multiple transcripts and results in neuronal cell death. TDP-43 depletion can also disturb expression levels of small nuclear RNAs (snRNAs as spliceosomal components. Despite this knowledge, the relationship between cell death and alteration of snRNA expression during TDP-43 depletion remains unclear. Here, we knocked down TDP-43 in murine neuroblastoma Neuro2A cells and found a time lag between efficient TDP-43 depletion and appearance of cell death, suggesting that several mechanisms mediate between these two events. The amount of U6 snRNA was significantly decreased during TDP-43 depletion prior to increase of cell death, whereas that of U1, U2, and U4 snRNAs was not. Downregulation of U6 snRNA led to cell death, whereas transient exogenous expression of U6 snRNA counteracted the effect of TDP-43 knockdown on cell death, and slightly decreased the mis-splicing rate of Dnajc5 and Sortilin 1 transcripts, which are assisted by TDP-43. These results suggest that regulation of the U6 snRNA expression level by TDP-43 is a key factor in the increase in cell death upon TDP-43 loss-of-function.

  2. Cofilin Knockdown Attenuates Hemorrhagic Brain Injury-induced Oxidative Stress and Microglial Activation in Mice.

    Science.gov (United States)

    Alhadidi, Qasim; Nash, Kevin M; Alaqel, Saleh; Sayeed, Muhammad Shahdaat Bin; Shah, Zahoor A

    2018-05-08

    Intracerebral hemorrhage (ICH) resulting from the rupture of the blood vessels in the brain is associated with significantly higher mortality and morbidity. Clinical studies focused on alleviating the primary injury, hematoma formation and expansion, were largely ineffective, suggesting that secondary injury-induced inflammation and the formation of reactive species also contribute to the overall injury process. In this study, we explored the effects of cofilin knockdown in a mouse model of ICH. Animals given stereotaxic injections of cofilin siRNA, 72-h prior to induction of ICH by collagenase injection within the area of siRNA administration showed significantly decreased cofilin expression levels and lower hemorrhage volume and edema, and the animals performed significantly better in neurobehavioral tasks i.e., rotarod, grip strength and neurologic deficit scores. Cofilin siRNA knocked-down mice had reduced ICH-induced DNA fragmentation, blood-brain barrier disruption and microglial activation, with a concomitant increase in astrocyte activation. Increased expression of pro-survival proteins and decreased markers of oxidative stress were also observed in cofilin siRNA-treated mice possibly due to the reduced levels of cofilin. Our results suggest that cofilin plays a major role in ICH-induced secondary injury, and could become a potential therapeutic target. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Hai Van Le

    2016-06-01

    Full Text Available 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.

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

    Science.gov (United States)

    Le, Hai Van; Kim, Jae Young

    2016-06-01

    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.

  5. Depdc5 knockdown causes mTOR-dependent motor hyperactivity in zebrafish.

    Science.gov (United States)

    de Calbiac, Hortense; Dabacan, Adriana; Marsan, Elise; Tostivint, Hervé; Devienne, Gabrielle; Ishida, Saeko; Leguern, Eric; Baulac, Stéphanie; Muresan, Raul C; Kabashi, Edor; Ciura, Sorana

    2018-05-01

    DEPDC5 was identified as a major genetic cause of focal epilepsy with deleterious mutations found in a wide range of inherited forms of focal epilepsy, associated with malformation of cortical development in certain cases. Identification of frameshift, truncation, and deletion mutations implicates haploinsufficiency of DEPDC5 in the etiology of focal epilepsy. DEPDC5 is a component of the GATOR1 complex, acting as a negative regulator of mTOR signaling. Zebrafish represents a vertebrate model suitable for genetic analysis and drug screening in epilepsy-related disorders. In this study, we defined the expression of depdc5 during development and established an epilepsy model with reduced Depdc5 expression. Here we report a zebrafish model of Depdc5 loss-of-function that displays a measurable behavioral phenotype, including hyperkinesia, circular swimming, and increased neuronal activity. These phenotypic features persisted throughout embryonic development and were significantly reduced upon treatment with the mTORC1 inhibitor, rapamycin, as well as overexpression of human WT DEPDC5 transcript. No phenotypic rescue was obtained upon expression of epilepsy-associated DEPDC5 mutations (p.Arg487* and p.Arg485Gln), indicating that these mutations cause a loss of function of the protein. This study demonstrates that Depdc5 knockdown leads to early-onset phenotypic features related to motor and neuronal hyperactivity. Restoration of phenotypic features by WT but not epilepsy-associated Depdc5 mutants, as well as by mTORC1 inhibition confirm the role of Depdc5 in the mTORC1-dependent molecular cascades, defining this pathway as a potential therapeutic target for DEPDC5 -inherited forms of focal epilepsy.

  6. Knockdown of Progesterone Receptor (PGR) in Macaque Granulosa Cells Disrupts Ovulation and Progesterone Production.

    Science.gov (United States)

    Bishop, Cecily V; Hennebold, Jon D; Kahl, Christoph A; Stouffer, Richard L

    2016-05-01

    Adenoviral vectors (vectors) expressing short-hairpin RNAs complementary to macaque nuclear progesterone (P) receptor PGR mRNA (shPGR) or a nontargeting scrambled control (shScram) were used to determine the role PGR plays in ovulation/luteinization in rhesus monkeys. Nonluteinized granulosa cells collected from monkeys (n = 4) undergoing controlled ovarian stimulation protocols were exposed to either shPGR, shScram, or no virus for 24 h; human chorionic gonadotropin (hCG) was then added to half of the wells to induce luteinization (luteinized granulosa cells [LGCs]; n = 4-6 wells/treatment/monkey). Cells/media were collected 48, 72, and 120 h postvector for evaluation of PGR mRNA and P levels. Addition of hCG increased (P < 0.05) PGR mRNA and medium P levels in controls. However, a time-dependent decline (P < 0.05) in PGR mRNA and P occurred in shPGR vector groups. Injection of shPGR, but not shScram, vector into the preovulatory follicle 20 h before hCG administration during controlled ovulation protocols prevented follicle rupture in five of six monkeys as determined by laparoscopic evaluation, with a trapped oocyte confirmed in three of four follicles of excised ovaries. Injection of shPGR also prevented the rise in serum P levels following the hCG bolus compared to shScram (P < 0.05). Nuclear PGR immunostaining was undetectable in granulosa cells from shPGR-injected follicles, compared to intense staining in shScram controls. Thus, the nuclear PGR appears to mediate P action in the dominant follicle promoting ovulation in primates. In vitro and in vivo effects of PGR knockdown in LGCs also support the hypothesis that P enhances its own synthesis in the primate corpus luteum by promoting luteinization. © 2016 by the Society for the Study of Reproduction, Inc.

  7. PlGF gene knockdown in human retinal pigment epithelial cells.

    Science.gov (United States)

    Akrami, Hassan; Soheili, Zahra-Soheila; Sadeghizadeh, Majid; Ahmadieh, Hamid; Rezaeikanavi, Mozhgan; Samiei, Shahram; Khalooghi, Keynoush

    2011-04-01

    To evaluate the knockdown of placental growth factor (PlGF) gene expression in human retinal pigment epithelium (RPE) cells and its effect on cell proliferation, apoptosis and angiogenic potential of RPE cells. Human RPE cells were isolated by dispase I solution and cultured in DMEM/F12 supplemented with 10% fetal calf serum (FCS). A small interfering RNA (siRNA) corresponding to PlGF mRNA and a scrambled siRNA (scRNA) were introduced into the cells. Cell proliferation and cell death were examined by ELISA. PlGF mRNA and protein were quantified by real-time polymerase chain reaction (PCR) and western blot. The levels of gene expression for human retinal pigment epithelium-specific protein 65 kDa (RPE65), cellular retinaldehyde-binding protein (CRALBP) and tyrosinase were examined by real-time PCR. The angiogenic activity of RPE cell-derived conditioned media was assayed by a tube formation assay using human umbilical vein endothelial cells (HUVECs). At a final siRNA concentration of 20 pmol/ml, the transfection efficiency was about 80%. The amount of PlGF transcripts was reduced to 10% after 36 h of incubation, and the amount of PlGF protein in culture supernatant was significantly decreased. Suppression of PlGF gene had no effect on RPE cell proliferation and survival, and there were no notable changes in the transcript levels of RPE65, CRALBP or tyrosinase for the cultures treated by siRNA cognate to PlGF. Vascular tube formation was efficiently reduced in HUVECs. Our findings present PlGF as a key modulator of angiogenic potential in RPE cells of the human retina.

  8. Functional characterization of Pol III U6 promoters for gene knockdown and knockout in Plutella xylostella.

    Science.gov (United States)

    Huang, Yuping; Wang, Yajun; Zeng, Baosheng; Liu, Zhaoxia; Xu, Xuejiao; Meng, Qian; Huang, Yongping; Yang, Guang; Vasseur, Liette; Gurr, Geoff M; You, Minsheng

    2017-10-01

    RNA polymerase type III (Pol-III) promoters such as U6 are commonly used to express small RNAs, including short hairpin RNAs (shRNAs) and single guide RNAs (sgRNAs). Functional U6 promoters are widely used in CRISPR systems, and their characterization can facilitate genome editing of non-model organisms. In the present study, six U6 small nuclear RNA (snRNA) promoters containing two conserved elements of a proximal sequence element (PSEA) and a TATA box, were identified and characterized in the diamondback moth (Plutella xylostella) genome. Relative efficiency of the U6 promoters to express shRNA induced EGFP knockdown was tested in a P. xylostella cell line, revealing that the PxU6:3 promoter had the strongest expression effect. Further work with the PxU6:3 promoter showed its efficacy in EGFP knockout using CRISPR/Cas9 system in the cells. The expression plasmids with versatile Pxabd-A gene specific sgRNA driven by the PxU6:3 promoter, combined with Cas9 mRNA, could induce mutagenesis at specific genomic loci in vivo. The phenotypes induced by sgRNA expression plasmids were similar to those done in vitro transcription sgRNAs. A plasmid with two tandem arranged PxU6:3:sgRNA expression cassettes targeting Pxabd-A loci was generated, which caused a 28,856 bp fragment deletion, suggesting that the multi-sgRNA expression plasmid can be used for multi-targeting. Our work indicates that U6 snRNA promoters can be used for functional studies of genes with the approach of reverse genetics in P. xylostella. These essential promoters also provide valuable potential for CRISPR-derived gene drive as a tactic for population control in this globally significant pest. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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. © The American Genetic Association. 2015.

  10. Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish.

    Science.gov (United States)

    Viringipurampeer, I A; Shan, X; Gregory-Evans, K; Zhang, J P; Mohammadi, Z; Gregory-Evans, C Y

    2014-05-01

    Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c(w59) mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c(w59) embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c(w59) mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.

  11. Knockdown of IL-8 Provoked Premature Senescence of Placenta-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Li, Juan-Juan; Ma, Feng-Xia; Wang, You-Wei; Chen, Fang; Lu, Shi-Hong; Chi, Ying; Du, Wen-Jing; Song, Bao-Quan; Hu, Liang-Ding; Chen, Hu; Han, Zhong-Chao

    2017-06-15

    Mesenchymal stem cells (MSCs) have shown promise for use in cell therapy, and due to their tumor tropism can serve as vehicles for delivering therapeutic agents to tumor sites. Because interleukin-8 (IL-8) is known to mediate the protumor effect of MSCs, elimination of IL-8 secretion by MSCs may enhance their safety for use in cancer gene therapy. However, little is known concerning the effect of endogenously secreted IL-8 on MSCs. We performed studies using placenta-derived MSCs (PMSCs) to determine whether knockdown of IL-8 would influence their biological activity. We first verified that IL-8 and its membrane receptor CXCR2, but not CXCR1, were highly expressed in PMSCs. We then employed lentivirus-mediated small hairpin RNA interference to generate stable IL-8-silenced PMSCs, which displayed a variety of characteristic senescent phenotypes. We observed that at day 9 post-transfection, IL-8-silenced PMSCs had become larger and displayed a more flattened appearance when compared with their controls. Moreover, their proliferation, colony forming unit-fibroblast formation, adipogenic and osteogenic differentiation, and immunosuppressive potentials were significantly impaired. Enhanced senescence-associated β-galactosidase (SA-β-gal) activity and specific global gene expression profiles confirmed that IL-8 silencing evoked the senescence process in PMSCs. Increased levels of p-Akt and decreased levels of FOXO3a protein expression suggested that reactive oxygen species played a role in the initiation and maintenance of senescence in IL-8-silenced PMSCs. Notably, the majority of CXCR2 ligands were downregulated in presenescent IL-8-silenced PMSCs but upregulated in senescent cells, indicating an antagonistic pleiotropy of the IL-8/CXCR2 signaling pathway in PMSCs. This effect may promote the proliferation of young cells and accelerate senescence of old cells.

  12. [Knock-down of ZEB1 inhibits the proliferation, invasion and migration of gastric cancer cells].

    Science.gov (United States)

    Chen, Dengyu; Chu, Yifan; Zheng, Qingwei; Xu, Zhiben; Zhou, Ping; Li, Sheng

    2017-08-01

    Objective To down-regulate the expression of zinc-finger E-box binding homeobox 1 (ZEB1) gene by shRNA, and investigate its effect on invasion, migration and proliferation, as well as the related gene expressions of lncRNA HOTAIR and E-cadherin in human gastric cancer BGC823 cells. Methods RNA interfering (RNAi) was used to knock down ZEB1 in gastric cancer BGC823 cells. The recombinant plasmid shZEB1 was constructed and transfected into the gastric cancer BGC823 cells by Lipofectamine TM 2000, and the stably transfected cells were isolated by G418 selection and limited dilution. The expression of ZEB1 mRNA and protein was detected by real-time quantitative PCR and Western blot analysis. Cell proliferation was determined by MTT assay, and the invasion and migration abilities of BGC823 cells were monitored by Transwell TM invasion assay and wound healing assay, respectively. The expressions of lncRNA HOTAIR and E-cadherin mRNA were detected by real-time quantitative PCR. Results After ZEB1 expression was successfully down-regulated in BGC823 cells by siRNA, the proliferation, invasion and migration rates in shZEB1 transfection group were significantly lower than those in control group; meanwhile, the expression of lncRNA HOTAIR was reduced and E-cadherin expression was enhanced. Conclusion Knock-down of ZEB1 expression by RNA interference can decease lncRNA HOTAIR expression and restrain cell proliferation, invasion and migration in gastric cancer BGC823 cells.

  13. A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown

    DEFF Research Database (Denmark)

    Higgins, Geoff S; Prevo, Remko; Lee, Yin-Fai

    2010-01-01

    The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor...... radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B......) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA...

  14. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC

    DEFF Research Database (Denmark)

    Garbarino, J.; Pan, M. H.; Chin, H. F.

    2012-01-01

    small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT...... synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein...... membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well. -Garbarino, J., M. Pan, H.F. Chin, F.W. Lund, F.R. Maxfield, and J.L. Breslow. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma...

  15. Can RNAi-mediated hsp90α knockdown in combination with 17-AAG be a therapy for glioma?

    Science.gov (United States)

    Mehta, Adi; Shervington, Amal; Howl, John; Jones, Sarah; Shervington, Leroy

    2013-01-01

    Heat shock protein 90 promotes tumor progression and survival and has emerged as a vital therapeutic target. Previously we reported that the combinatorial treatment of 17AAG/sihsp90α significantly downregulated Hsp90α mRNA and protein levels in Glioblastoma Multiforme (GBM). Here we investigated the ability of cell penetrating peptide (Tat48-60 CPP)-mediated siRNA-induced hsp90α knockdown as a single agent and in combination with 17-allylamino-17-demethoxygeldanamycin (17-AAG) to induce tumor growth inhibition in GBM and whether it possessed therapeutic implications. GBM and non-tumorigenic cells exposed to siRNA and/or 17-AAG were subsequently assessed by qRT-PCR, immunofluorescence, FACS analysis, quantitative Akt, LDH leakage and cell viability assays. PAGE was performed for serum stability assessment. A combination of siRNA/17-AAG treatment significantly induced Hsp90α gene and protein knockdown by 95% and 98%, respectively, concomitant to 84% Akt kinase activity attenuation, induced cell cycle arrest and tumor-specific cytotoxicity by 88%. Efficient complex formation between CPP and siRNA exhibited improved serum stability of the siRNA with minimal intrinsic toxicity in vitro. The preliminary in vivo results showed that combination therapy induced hsp90α knockdown and attenuated Akt kinase activity in intracranial glioblastoma mouse models. The results imply that RNAi-mediated hsp90α knockdown increases 17-AAG treatment efficacy in GBM. In addition, the cytotoxic response observed was the consequence of downregulation of hsp90α gene expression, reduced Akt kinase activity and S-G2/M cell cycle arrest. These results are novel and highlight the ability of Tat to efficiently deliver siRNA in GBM and suggest that the dual inhibition of Hsp90 has therapeutic potentials.

  16. Knockdown of MAGEA6 Activates AMP-Activated Protein Kinase (AMPK) Signaling to Inhibit Human Renal Cell Carcinoma Cells.

    Science.gov (United States)

    Ye, Xueting; Xie, Jing; Huang, Hang; Deng, Zhexian

    2018-01-01

    Melanoma antigen A6 (MAGEA6) is a cancer-specific ubiquitin ligase of AMP-activated protein kinase (AMPK). The current study tested MAGEA6 expression and potential function in renal cell carcinoma (RCC). MAGEA6 and AMPK expression in human RCC tissues and RCC cells were tested by Western blotting assay and qRT-PCR assay. shRNA method was applied to knockdown MAGEA6 in human RCC cells. Cell survival and proliferation were tested by MTT assay and BrdU ELISA assay, respectively. Cell apoptosis was tested by the TUNEL assay and single strand DNA ELISA assay. The 786-O xenograft in nude mouse model was established to test RCC cell growth in vivo. MAGEA6 is specifically expressed in RCC tissues as well as in the established (786-O and A498) and primary human RCC cells. MAGEA6 expression is correlated with AMPKα1 downregulation in RCC tissues and cells. It is not detected in normal renal tissues nor in the HK-2 renal epithelial cells. MAGEA6 knockdown by targeted-shRNA induced AMPK stabilization and activation, which led to mTOR complex 1 (mTORC1) in-activation and RCC cell death/apoptosis. AMPK inhibition, by AMPKα1 shRNA or the dominant negative AMPKα1 (T172A), almost reversed MAGEA6 knockdown-induced RCC cell apoptosis. Conversely, expression of the constitutive-active AMPKα1 (T172D) mimicked the actions by MAGEA6 shRNA. In vivo, MAGEA6 shRNA-bearing 786-O tumors grew significantly slower in nude mice than the control tumors. AMPKα1 stabilization and activation as well as mTORC1 in-activation were detected in MAGEA6 shRNA tumor tissues. MAGEA6 knockdown inhibits human RCC cells via activating AMPK signaling. © 2018 The Author(s). Published by S. Karger AG, Basel.

  17. Acute Podocyte Vascular Endothelial Growth Factor (VEGF-A) Knockdown Disrupts alphaVbeta3 Integrin Signaling in the Glomerulus

    Science.gov (United States)

    Veron, Delma; Villegas, Guillermo; Aggarwal, Pardeep Kumar; Bertuccio, Claudia; Jimenez, Juan; Velazquez, Heino; Reidy, Kimberly; Abrahamson, Dale R.; Moeckel, Gilbert; Kashgarian, Michael; Tufro, Alda

    2012-01-01

    Podocyte or endothelial cell VEGF-A knockout causes thrombotic microangiopathy in adult mice. To study the mechanism involved in acute and local injury caused by low podocyte VEGF-A we developed an inducible, podocyte-specific VEGF-A knockdown mouse, and we generated an immortalized podocyte cell line (VEGFKD) that downregulates VEGF-A upon doxycycline exposure. Tet-O-siVEGF:podocin-rtTA mice express VEGF shRNA in podocytes in a doxycycline-regulated manner, decreasing VEGF-A mRNA and VEGF-A protein levels in isolated glomeruli to ∼20% of non-induced controls and urine VEGF-A to ∼30% of control values a week after doxycycline induction. Induced tet-O-siVEGF:podocin-rtTA mice developed acute renal failure and proteinuria, associated with mesangiolysis and microaneurisms. Glomerular ultrastructure revealed endothelial cell swelling, GBM lamination and podocyte effacement. VEGF knockdown decreased podocyte fibronectin and glomerular endothelial alphaVbeta3 integrin in vivo. VEGF receptor-2 (VEGFR2) interacts with beta3 integrin and neuropilin-1 in the kidney in vivo and in VEGFKD podocytes. Podocyte VEGF knockdown disrupts alphaVbeta3 integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGFKD podocytes downregulates fibronectin and disrupts alphaVbeta3 integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alphaVbeta3 integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alphaVbeta3 integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure. PMID:22808199

  18. Signal Transducer and Activator of Transcription 1 (STAT1) Knock-down Induces Apoptosis in Malignant Pleural Mesothelioma.

    Science.gov (United States)

    Arzt, Lisa; Halbwedl, Iris; Gogg-Kamerer, Margit; Popper, Helmut H

    2017-07-01

    Malignant pleural mesothelioma (MPM) is the most common primary tumor of the pleura. Its incidence is still increasing in Europe and the prognosis remains poor. We investigated the oncogenic function of signal transducer and activator of transcription 1 (STAT1) in MPM in more detail. A miRNA profiling was performed on 52 MPM tissue samples. Upregulated miRNAs (targeting SOCS1/3) were knocked-down using miRNA inhibitors. mRNA expression levels of STAT1/3, SOCS1/3 were detected in MPM cell lines. STAT1 has been knocked-down using siRNA and qPCR was used to detect mRNA expression levels of all JAK/STAT family members and genes that regulate them. An immunohistochemical staining was performed to detect the expression of caspases. STAT1 was upregulated and STAT3 was downregulated, SOCS1/3 protein was not detected but it was possible to detect SOCS1/3 mRNA in MPM cell lines. The upregulated miRNAs were successfully knocked-down, however the expected effect on SOCS1 expression was not detected. STAT1 knock-down had different effects on STAT3/5 expression. Caspase 3a and 8 expression was found to be increased after STAT1 knock-down. The physiologic regulation of STAT1 via SOCS1 is completely lost in MPM and it does not seem that the miRNAs identified by now, do inhibit the expression of SOCS1. MPM cell lines compensate STAT1 knock-down by increasing the expression of STAT3 or STAT5a, two genes which are generally considered to be oncogenes. And much more important, STAT1 knock-down induces apoptosis in MPM cell lines and STAT1 might therefore be a target for therapeutic intervention.

  19. Knockdown of asporin affects transforming growth factor-β1-induced matrix synthesis in human intervertebral annulus cells

    Directory of Open Access Journals (Sweden)

    Xu Jiang

    2016-10-01

    Conclusion: Our results have verified a functional feedback loop between TGF-β1 and asporin in human intervertebral annulus cells indicating that TGF-β1-induced annulus matrix biosynthesis can be significantly upregulated by knockdown of asporin. Therefore, asporin could be a potential new therapeutic target and inhibition of asporin could be adopted to enhance the anabolic effect of TGF-β1 in human intervertebral annulus cells in degenerative IVD diseases.

  20. Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD.

    Science.gov (United States)

    Lattante, Serena; de Calbiac, Hortense; Le Ber, Isabelle; Brice, Alexis; Ciura, Sorana; Kabashi, Edor

    2015-03-15

    Mutations in SQSTM1, encoding for the protein SQSTM1/p62, have been recently reported in 1-3.5% of patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS/FTLD). Inclusions positive for SQSTM1/p62 have been detected in patients with neurodegenerative disorders, including ALS/FTLD. In order to investigate the pathogenic mechanisms induced by SQSTM1 mutations in ALS/FTLD, we developed a zebrafish model. Knock-down of the sqstm1 zebrafish ortholog, as well as impairment of its splicing, led to a specific phenotype, consisting of behavioral and axonal anomalies. Here, we report swimming deficits associated with shorter motor neuronal axons that could be rescued by the overexpression of wild-type human SQSTM1. Interestingly, no rescue of the loss-of-function phenotype was observed when overexpressing human SQSTM1 constructs carrying ALS/FTLD-related mutations. Consistent with its role in autophagy regulation, we found increased mTOR levels upon knock-down of sqstm1. Furthermore, treatment of zebrafish embryos with rapamycin, a known inhibitor of the mTOR pathway, yielded an amelioration of the locomotor phenotype in the sqstm1 knock-down model. Our results suggest that loss-of-function of SQSTM1 causes phenotypic features characterized by locomotor deficits and motor neuron axonal defects that are associated with a misregulation of autophagic processes. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

    Science.gov (United States)

    Xie, Zhongcong; Dong, Yuanlin; Maeda, Uta; Xia, Weiming; Tanzi, Rudolph E

    2012-03-22

    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.

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

    Directory of Open Access Journals (Sweden)

    Xie Zhongcong

    2012-03-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

    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

  4. Keratin23 (KRT23 knockdown decreases proliferation and affects the DNA damage response of colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Karin Birkenkamp-Demtröder

    Full Text Available Keratin 23 (KRT23 is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression in vitro. Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed that KRT23 depletion affected molecules of the cell cycle and DNA replication, recombination and repair. In vitro analyses confirmed that KRT23 depletion significantly decreased the cellular proliferation of SW948 and LS1034 cells and markedly decreased the expression of genes involved in DNA damage response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced proliferation of the KRT23 depleted cells compared to irradiated control cells.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. © 2013 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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

    2016-07-14

    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.

  8. Synergistic effect of some essential oils on toxicity and knockdown effects, against mosquitos, cockroaches and housefly

    Directory of Open Access Journals (Sweden)

    Idin Zibaee

    2015-12-01

    Full Text Available The toxicity and knockdown effect of Eucalyptus globulus, Rosmarinus officinalis essential oils and their mixed formulation on Periplaneta Americana (L., Blattella germanica (L., Supella longipalpa, Culex pipiens, Anopheles stephensi and Musca domestica were evaluated in a series of laboratory experiments. In all bioassay five different doses (0.625, 1.25, 2.5, 5 and 10% were used by filter paper (cm2 and aerosol (cm3 bioassay methods, all essential oils was toxic to cockroaches, mosquitos and housefly species the lowest and the highest LC50 belong to mixed formulation on B. germanica (LC50 6.1 and E. globulus on P. americana (LC50 27.7 respectively. In continuous exposure experiments, Mortality (LT50 values for cockroaches ranged from 1403.3 min with 0.625% E. globulus (for P. americana to 2.2 min with 10% mixed formulation for A. stephensi. The KT50 values ranged from 0.1 to 1090.8 min for 10% and 0.625 for mixed formulation and R. officinalis respectively. The mortality after 24 h for mixed formulation was 100% but for single essential oils ranged from 81.5 to 98.3 for P. americana treated with R. officinalis and A. stephensi treated with E. globulus respectively. Studies on persistence of essential oils on impregnated paper revealed that it has more adulticidal activity for longer period at low storage temperature. Gas chromatographic-mass spectrometric analysis of essential oil showed 14 and 16 peaks for E. globules and R. officinalis respectively. α-Pinene (39.8%, 1, 8-Cineole (13.2%, Camphene (9.1% and Borneol (3.7% were present in major amounts for R. officinalis and 1,8-Cineole (31.4%, α-Pinene (15.3%, d-Limonene (9.7% and α-Terpinolen (5.3% were present in major amounts for E. globulus respectively. Our results showed that two surveyed essential oils has compatible with synergistic effect on various insect species, furthermore it is useful for applying as integrated pest management tool for studied insects management, especially in

  9. AAV-mediated knock-down of HRC exacerbates transverse aorta constriction-induced heart failure.

    Directory of Open Access Journals (Sweden)

    Chang Sik Park

    Full Text Available Histidine-rich calcium binding protein (HRC is located in the lumen of sarcoplasmic reticulum (SR that binds to both triadin (TRN and SERCA affecting Ca(2+ cycling in the SR. Chronic overexpression of HRC that may disrupt intracellular Ca(2+ homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca(2+ cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV-mediated HRC knock-down (KD systems, respectively.AAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH to examine whether HRC-KD could enhance cardiac function in failing heart (FH. Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH, since predesigned siRNA-mediated HRC-KD enhanced Ca(2+ cycling and increased activities of RyR2 and SERCA2 without change in SR Ca(2+ load in neonatal rat ventricular cells (NRVCs and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay.Increased Ca(2+ leak and cytosolic Ca(2+ concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through

  10. Suppressor of cytokine signaling 3 knockdown in the mediobasal hypothalamus: counterintuitive effects on energy balance

    NARCIS (Netherlands)

    de Backer, M. W. A.; Brans, M. A. D.; van Rozen, A. J.; van der Zwaal, E. M.; Luijendijk, M. C. M.; Garner, K. G.; de Krom, M.; van Beekum, O.; La Fleur, S. E.; Adan, R. A. H.

    2010-01-01

    An increase in brain suppressor of cytokine signaling 3 (SOCS3) has been implicated in the development of both leptin and insulin resistance. Socs3 mRNA is localized throughout the brain, and it remains unclear which brain areas are involved in the effect of SOCS3 levels on energy balance. We

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

    Directory of Open Access Journals (Sweden)

    Bateman Andrew

    2010-10-01

    Full Text Available Abstract 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

  12. Mosquito knock-down and adulticidal activities of essential oils by vaporizer, impregnated filter paper and aerosol methods

    Directory of Open Access Journals (Sweden)

    M. Ramar

    2014-09-01

    Full Text Available Essential oils from 12 medicinal plants were evaluated by three different bioassay methods (Vaporizer, Filter paper and Aerosol for Knock-down and adulticidal efficacy on the filarial vector mosquito, Culex quinquefasciatus. Based on screening results the effective plants were selected for investigating Knock-down and adulticidal potential against adult female of the laboratory-reared mosquito species, Cx. quinquefasciatus. In vaporizer bioassay method four different doses (1.25, 2.5, 5 and 10% were used. Four different doses (0.625, 1.25, 2.5 and 10% were used both filter paper (cm2 and aerosol (cm3 bioassay methods. Five essential oils (calamus, camphor, citronella, clove and eucalyptus were identified as potential treatments in vaporizer bioassay. The result showed that the knock down time decreased with increased concentration in clove oil treatment; the Knock-down time (KT 50 = 46.1 ± 0.1, 38.5 ± 0.1, 30.7 ± 0.2, and 20.1 ± 0.1 minutes was recorded at 1.25, 2.5, 5 and 10% /cm3 respectively. In filter paper method nine essential oils were identified as potential treatments. After 1 hr exposure period clove oil recorded the lowest median Knock-down time (KT50 which was calculated as 9.15 ± 0.1min/cm2. Followed by citronella (KT50 =11.4 ± 0.1 min and eucalyptus (KT50 =11.4 ±0.1min oils since they recorded lower median Knock-down time. All the twelve essential oils were identified as potential treatments in aerosol activity. The lethal time decreased when the concentration increased. At 5 % concentration the median lethal time (LT50 for clove oil was calculated as (LT50=3.80 ± 0.1minutes. The Cinnamon oil was effective which recorded (LT50 = 1.99 mins as median lethal time. Camphor (LT50 =19.6± 0.1 min oil were found to be less toxic by aerosol method. These results suggest that clove oil and cinnamon oil have the potential to be used as a eco-friendly approach for the control of the major important filaria vector Cx. quinquefasciatus

  13. Insecticide resistance and intracellular proteases.

    Science.gov (United States)

    Wilkins, Richard M

    2017-12-01

    Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  14. PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

    Science.gov (United States)

    Singh, Om P; Bali, Prerna; Hemingway, Janet; Subbarao, Sarala K; Dash, Aditya P; Adak, Tridibes

    2009-01-01

    Background Anopheles culicifacies s.l., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (kdr) is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of kdr mutation (L1014F) in a field population of An. culicifacies s.l. and three new PCR-based methods for kdr genotyping. Methods The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant An. culicifacies s.l. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F) in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR), an Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR) were developed and tested for kdr genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped. Results The genotyping of this An. culicifacies s.l. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the kdr allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium. Conclusion The Leu-Phe mutation, which generates the kdr phenotype in many insects, was detected in a pyrethroid and DDT resistant An. culicifacies s.l. population. Three PCR-based methods were developed for kdr genotyping. All the three assays were specific. The ARMS method was refractory to non

  15. PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

    Directory of Open Access Journals (Sweden)

    Dash Aditya P

    2009-07-01

    Full Text Available Abstract Background Anopheles culicifacies s.l., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (kdr is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of kdr mutation (L1014F in a field population of An. culicifacies s.l. and three new PCR-based methods for kdr genotyping. Methods The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant An. culicifacies s.l. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR, an Amplification Refractory Mutation System (ARMS and Primer Introduced Restriction Analysis-PCR (PIRA-PCR were developed and tested for kdr genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped. Results The genotyping of this An. culicifacies s.l. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the kdr allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium. Conclusion The Leu-Phe mutation, which generates the kdr phenotype in many insects, was detected in a pyrethroid and DDT resistant An. culicifacies s.l. population. Three PCR-based methods were developed for kdr genotyping. All the three assays were specific. The ARMS method

  16. Sertoli cell specific knockdown of RAR-related orphan receptor (ROR) alpha at puberty reduces sperm count in rats.

    Science.gov (United States)

    Mandal, Kamal; Sarkar, Rajesh K; Sen Sharma, Souvik; Jain, Ayushi; Majumdar, Subeer S

    2018-01-30

    Globally, there is an alarming decline in sperm count. Very often hormonal supplementation fails to restore normal sperm count. Sertoli cells (Sc) present within seminiferous tubules provide appropriate niche and factors required for the differentiation of germ cells (Gc) into mature sperm (spermatogenesis). Functionally compromised Sc may be one of the reasons for failure of hormones to facilitate normal spermatogenesis. Although role of secretory proteins and signaling molecules of Sc has been studied well, role of transcription factors regulating sperm count has not been addressed appropriately. Retinoic acid receptor-related orphan receptor (ROR)-alpha is one of such transcription factors reported in testis but its role in testicular function is not yet known. In a separate study, we found abundant ROR-alpha binding sites on promoter regions of several genes upregulated in pubertal rat Sc as compared to infant Sc. Immunostaining studies also revealed presence of ROR alpha in nucleus of pubertal Sc. We generated a transgenic knockdown rat model expressing shRNA targeted to ROR-alpha under Sc specific promoter, which is transcriptionally active only at and after puberty. ROR-alpha knockdown animals were found to have abnormal association of Sc and Gc, including Gc sloughing and restricted release of sperm. The knockdown animals displayed compromised spermatogenesis leading to significant reduction in sperm count. This is the first report describing the Sc specific role of ROR-alpha in maintaining quantitatively normal sperm output. Identification of various such molecules can generate avenues to limit or reverse an alarmingly declining sperm count witnessed globally in men. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Rajnikumar Sangani

    2014-03-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Britta Schneider

    2012-01-01

    Full Text Available 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.

  19. HOXB7 mRNA is overexpressed in pancreatic ductal adenocarcinomas and its knockdown induces cell cycle arrest and apoptosis

    International Nuclear Information System (INIS)

    Chile, Thais; Bacchella, Telésforo; Giorgi, Ricardo Rodrigues; Fortes, Maria Angela Henriques Zanella; Corrêa-Giannella, Maria Lúcia Cardillo; Brentani, Helena Paula; Maria, Durvanei Augusto; Puga, Renato David; Paula, Vanessa de Jesus R de; Kubrusly, Marcia Saldanha; Novak, Estela Maria

    2013-01-01

    Human homeobox genes encode nuclear proteins that act as transcription factors involved in the control of differentiation and proliferation. Currently, the role of these genes in development and tumor progression has been extensively studied. Recently, increased expression of HOXB7 homeobox gene (HOXB7) in pancreatic ductal adenocarcinomas (PDAC) was shown to correlate with an invasive phenotype, lymph node metastasis and worse survival outcomes, but no influence on cell proliferation or viability was detected. In the present study, the effects arising from the knockdown of HOXB7 in PDAC cell lines was investigated. Real time quantitative PCR (qRT-PCR) (Taqman) was employed to assess HOXB7 mRNA expression in 29 PDAC, 6 metastatic tissues, 24 peritumoral tissues and two PDAC cell lines. siRNA was used to knockdown HOXB7 mRNA in the cell lines and its consequences on apoptosis rate and cell proliferation were measured by flow cytometry and MTT assay respectively. Overexpression of HOXB7 mRNA was observed in the tumoral tissues and in the cell lines MIA PaCa-2 and Capan-1. HOXB7 knockdown elicited (1) an increase in the expression of the pro-apoptotic proteins BAX and BAD in both cell lines; (2) a decrease in the expression of the anti-apoptotic protein BCL-2 and in cyclin D1 and an increase in the number of apoptotic cells in the MIA PaCa-2 cell line; (3) accumulation of cell in sub-G1 phase in both cell lines; (4) the modulation of several biological processes, especially in MIA PaCa-2, such as proteasomal ubiquitin-dependent catabolic process and cell cycle. The present study confirms the overexpression of HOXB7 mRNA expression in PDAC and demonstrates that decreasing its protein level by siRNA could significantly increase apoptosis and modulate several biological processes. HOXB7 might be a promising target for future therapies

  20. Knockdown of the fat mass and obesity gene disrupts cellular energy balance in a cell-type specific manner.

    Directory of Open Access Journals (Sweden)

    Ryan T Pitman

    Full Text Available Recent studies suggest that FTO variants strongly correlate with obesity and mainly influence energy intake with little effect on the basal metabolic rate. We suggest that FTO influences eating behavior by modulating intracellular energy levels and downstream signaling mechanisms which control energy intake and metabolism. Since FTO plays a particularly important role in adipocytes and in hypothalamic neurons, SH-SY5Y neuronal cells and 3T3-L1 adipocytes were used to understand how siRNA mediated knockdown of FTO expression alters cellular energy homeostasis. Cellular energy status was evaluated by measuring ATP levels using a luminescence assay and uptake of fluorescent glucose. FTO siRNA in SH-SY5Y cells mediated mRNA knockdown (-82%, increased ATP concentrations by up to 46% (P = 0.013 compared to controls, and decreased phosphorylation of AMPk and Akt in SH-SY5Y by -52% and -46% respectively as seen by immunoblotting. In contrast, FTO siRNA in 3T3-L1 cells decreased ATP concentration by -93% (p<0.0005, and increased AMPk and Akt phosphorylation by 204% and 70%, respectively suggesting that FTO mediates control of energy levels in a cell-type specific manner. Furthermore, glucose uptake was decreased in both SH-SY5Y (-51% p = 0.015 and 3T3-L1 cells (-30%, p = 0.0002. We also show that FTO knockdown decreases NPY mRNA expression in SH-SY5Y cells (-21% through upregulation of pSTAT3 (118%. These results provide important evidence that FTO-variant linked obesity may be associated with altered metabolic functions through activation of downstream metabolic mediators including AMPk.

  1. Bortezomib resistance in mantle cell lymphoma is associated with plasmacytic differentiation

    DEFF Research Database (Denmark)

    Pérez-Galán, Patricia; Mora-Jensen, Helena; Weniger, Marc A

    2011-01-01

    bortezomib-resistant MCL cell lines and primary tumor cells from MCL patients with inferior clinical response to bortezomib also expressed plasmacytic features. Knockdown of IRF4 was toxic for the subset of MCL cells with plasmacytic differentiation, but only slightly sensitized cells to bortezomib. We...

  2. RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation.

    LENUS (Irish Health Repository)

    Walsh, Naomi

    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.

  3. Human equilibrative nucleoside transporter-1 knockdown tunes cellular mechanics through epithelial-mesenchymal transition in pancreatic cancer cells.

    Directory of Open Access Journals (Sweden)

    Yeonju Lee

    Full Text Available We report cell mechanical changes in response to alteration of expression of the human equilibrative nucleoside transporter-1 (hENT1, a most abundant and widely distributed plasma membrane nucleoside transporter in human cells and/or tissues. Modulation of hENT1 expression level altered the stiffness of pancreatic cancer Capan-1 and Panc 03.27 cells, which was analyzed by atomic force microscopy (AFM and correlated to microfluidic platform. The hENT1 knockdown induced reduction of cellular stiffness in both of cells up to 70%. In addition, cellular phenotypic changes such as cell morphology, migration, and expression level of epithelial-mesenchymal transition (EMT markers were observed after hENT1 knockdown. Cells with suppressed hENT1 became elongated, migrated faster, and had reduced E-cadherin and elevated N-cadherin compared to parental cells which are consistent with epithelial-mesenchymal transition (EMT. Those cellular phenotypic changes closely correlated with changes in cellular stiffness. This study suggests that hENT1 expression level affects cellular phenotype and cell elastic behavior can be a physical biomarker for quantify hENT1 expression and detect phenotypic shift. Furthermore, cell mechanics can be a critical tool in detecting disease progression and response to therapy.

  4. RNAi Knockdown of Hypoxia-Inducible Factor-1α Decreased the Proliferation, Migration, and Invasion of Hypoxic Hepatocellular Carcinoma Cells.

    Science.gov (United States)

    Chen, ChengShi; Liu, Rong; Wang, JianHua; Yan, ZhiPing; Qian, Sheng; Zhang, Wei

    2015-04-01

    The obstruction of hepatic arterial blood flow results in tumor tissue hypoxia and elevated expression of hypoxia-inducible factor-1alpha (HIF-1α). Our study evaluated whether lentivirus-mediated short interference RNA against HIF-1α inhibits proliferation, invasion, and migration of hepatocellular carcinoma (HCC) cells under hypoxia. RNA interference knockdown of HIF-1α was achieved by HIF-1α-directed lentiviral shRNA, in a rat HCC cell line cultured under hypoxia condition for varying length of times. The expression levels of HIF-1α and vascular endothelial growth factor were examined using reverse transcription polymerase chain reaction and western blot analyses. Cell proliferation, migration, and invasion were measured by cell viability, transwell migration, and invasion assays, respectively. Inhibition of HIF-1α expression by shRNA suppressed vascular endothelial growth factor mRNA and protein levels under both normoxia and hypoxia. It also suppressed cell migration and invasion, which were enhanced under hypoxic conditions. RNAi knockdown of HIF-1α further suppressed hypoxia-mediated inhibition of the cell proliferation. These data suggest that shRNA of HIF-1α could antagonize the hypoxia-mediated increase in hepatic cancer cell migration and invasion, and synergize with hypoxia to inhibit the cell proliferation in HCC cells.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

    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

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

    Science.gov (United States)

    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.

  7. Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein.

    Science.gov (United States)

    Michalko, Jaroslav; Glanc, Matouš; 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, María Soledad [Instituto de Biomedicina de Valencia (IBV-CSIC), Jaime Roig 11, E-46010 Valencia (Spain); Fernandez-Alvarez, Ana [Fundación Instituto Leloir, IIBBA-CONICET, Av. Patricias Argentinas 435, Ciudad Autónoma de Buenos Aires C1405BWE (Argentina); Cucarella, Carme [Instituto de Biomedicina de Valencia (IBV-CSIC), Jaime Roig 11, E-46010 Valencia (Spain); Casado, Marta, E-mail: mcasado@ibv.csic.es [Instituto de Biomedicina de Valencia (IBV-CSIC), Jaime Roig 11, E-46010 Valencia (Spain)

    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.

  9. Knockdown of XBP1 by RNAi in Mouse Granulosa Cells Promotes Apoptosis, Inhibits Cell Cycle, and Decreases Estradiol Synthesis

    Directory of Open Access Journals (Sweden)

    Nan Wang

    2017-05-01

    Full Text Available Granulosa cells are crucial for follicular growth, development, and follicular atresia. X-box binding protein 1 (XBP1, a basic region-leucine zipper protein, is widely involved in cell differentiation, proliferation, apoptosis, cellular stress response, and other signaling pathways. In this study, RNA interference, flow cytometry, western blot, real-time PCR, Cell Counting Kit (CCK8, and ELISA were used to investigate the effect of XBP1 on steroidogenesis, apoptosis, cell cycle, and proliferation of mouse granulosa cells. ELISA analysis showed that XBP1 depletion significantly decreased the concentrations of estradiol (E2. Additionally, the expression of estrogen synthesis enzyme Cyp19a1 was sharply downregulated. Moreover, flow cytometry showed that knockdown of XBP1 increased the apoptosis rate and arrests the cell cycle in S-phase in granulosa cells (GCs. Further study confirmed these results. The expression of CCAAT-enhancer-binding protein homologous protein (CHOP, cysteinyl aspartate specific proteases-3 (caspase-3, cleaved caspase-3, and Cyclin E was upregulated, while that of Bcl-2, Cyclin A1, and Cyclin B1 was downregulated. Simultaneously, CCK8 analysis indicated that XBP1 disruption inhibited cell proliferation. In addition, XBP1 knockdown also alters the expression of Has2 and Ptgs2, two essential genes for folliculogenesis. Collectively, these data reveal a novel critical role of XBP1 in folliculogenesis by regulating the cell cycle, apoptosis, and steroid synthesis of mouse granulosa cells.

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

    Science.gov (United States)

    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

  11. Knockdown of astrocyte elevated gene-1 inhibits proliferation and enhancing chemo-sensitivity to cisplatin or doxorubicin in neuroblastoma cells

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    Xie Li

    2009-02-01

    Full Text Available Abstract Background Astrocyte elevated gene-1 (AEG-1 was originally characterized as a HIV-1-inducible gene in primary human fetal astrocyte. Recent studies highlight a potential role of AEG-1 in promoting tumor progression and metastasis. The aim of this study was to investigate if AEG-1 serves as a potential therapeutic target of human neuroblastoma. Methods We employed RNA interference to reduce AEG-1 expression in human neuroblastoma cell lines and analyzed their phenotypic changes. Results We found that the knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibited cell proliferation and apoptosis. The specific downregulation induced cell arrest in the G0/G1 phase of cell cycle. In the present study, we also observed a significant enhancement of chemo-sensitivity to cisplatin and doxorubicin by knockdown of AEG-1. Conclusion Our study suggests that overexpressed AEG-1 enhance the tumorogenic properties of neuroblastoma cells. The inhibition of AEG-1 expression could be a new adjuvant therapy for neuroblastoma.

  12. Knockdown of BAG3 induces epithelial–mesenchymal transition in thyroid cancer cells through ZEB1 activation

    Science.gov (United States)

    Meng, X; Kong, D-H; Li, N; Zong, Z-H; Liu, B-Q; Du, Z-X; Guan, Y; Cao, L; Wang, H-Q

    2014-01-01

    The process by which epithelial features are lost in favor of a mesenchymal phenotype is referred to as epithelial–mesenchymal transition (EMT). Most carcinomas use this mechanism to evade into neighboring tissues. Reduction or a loss of E-cadherin expression is a well-established hallmark of EMT. As a potent suppressor of E-cadherin, transcription factor ZEB1 is one of the key inducers of EMT, whose expression promotes tumorigenesis and metastasis of carcinomas. Bcl-2-associated athanogene 3 (BAG3) affects multifaceted cellular functions, including proliferation, apoptosis, cell adhesion and invasion, viral infection, and autophagy. Recently, we have reported a novel role of BAG3 implicated in EMT, while the mechanisms are poorly elucidated. The current study demonstrated that knockdown of BAG3 induced EMT, and increased cell migratory and invasiveness in thyroid cancer cells via transcriptional activation of ZEB1. We also found that BAG3 knockdown led to nuclear accumulation of β-catenin, which was responsible for the transcriptional activation of ZEB1. These results indicate BAG3 as a regulator of ZEB1 expression in EMT and as a regulator of metastasis in thyroid cancer cells, providing potential targets to prevent and/or treat thyroid cancer cell invasion and metastasis. PMID:24577090

  13. Knockdown of BAG3 induces epithelial-mesenchymal transition in thyroid cancer cells through ZEB1 activation.

    Science.gov (United States)

    Meng, X; Kong, D-H; Li, N; Zong, Z-H; Liu, B-Q; Du, Z-X; Guan, Y; Cao, L; Wang, H-Q

    2014-02-27

    The process by which epithelial features are lost in favor of a mesenchymal phenotype is referred to as epithelial-mesenchymal transition (EMT). Most carcinomas use this mechanism to evade into neighboring tissues. Reduction or a loss of E-cadherin expression is a well-established hallmark of EMT. As a potent suppressor of E-cadherin, transcription factor ZEB1 is one of the key inducers of EMT, whose expression promotes tumorigenesis and metastasis of carcinomas. Bcl-2-associated athanogene 3 (BAG3) affects multifaceted cellular functions, including proliferation, apoptosis, cell adhesion and invasion, viral infection, and autophagy. Recently, we have reported a novel role of BAG3 implicated in EMT, while the mechanisms are poorly elucidated. The current study demonstrated that knockdown of BAG3 induced EMT, and increased cell migratory and invasiveness in thyroid cancer cells via transcriptional activation of ZEB1. We also found that BAG3 knockdown led to nuclear accumulation of β-catenin, which was responsible for the transcriptional activation of ZEB1. These results indicate BAG3 as a regulator of ZEB1 expression in EMT and as a regulator of metastasis in thyroid cancer cells, providing potential targets to prevent and/or treat thyroid cancer cell invasion and metastasis.

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

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    Ravi S Kasinathan

    2014-10-01

    Full Text Available 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

  15. PIK3CA expression in diffuse large B cell lymphoma tissue and the effect of its knockdown in vitro

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    Cui W

    2017-04-01

    Full Text Available Wenli Cui,1–4,* Shutao Zheng,5,6,* Zebing Liu,1–3 Weige Wang,1–3 Ying Cai,1–3 Rui Bi,1–3 Bing Cao,1–3 Xiaoyan Zhou1–3 1Department of Pathology, Shanghai Cancer Center, Fudan University, 2Department of Oncology, Shanghai Medical College, Fudan University, 3Institute of Pathology, Fudan University, Shanghai, 4Department of Pathology, The First Affiliated Hospital of Xinjiang Medical University, 5Clinical Medical Research Institute, First Affiliated Hospital of Xinjiang Medical University, 6State Key Lab Incubation Base of Xinjiang Major Diseases Research, First Affiliated Hospital of Xinjiang Medical University, Xinjiang Uygur Autonomous Region, Urumqi, People’s Republic of China *These authors contributed equally to this work Abstract: PIK3CA has been extensively investigated from its molecular mechanism perspective and epidemiological association with its mutations in different types of cancers. However, little has been reported regarding the clinicopathological significance of PIK3CA expression in diffuse large B cell lymphoma (DLBCL. In the present study, we investigated the clinicopathological significance of PIK3CA in DLBCL by performing immunohistochemical evaluation of PIK3CA in tissue microarrays consisting of 199 cases of DLBCL. Kaplan–Meier survival analysis was performed to analyze the association between PIK3CA expression and overall prognosis. To further investigate the role of PIK3CA mediated in the proliferation, cell cycle and apoptosis of DLBCL cells, Cell Counting Kit-8 (CCK-8 and flow cytometry assays were carried out in DLBCL cell lines after successful, stable knockdown of PIK3CA using lentiviral short hairpin RNA inference. Our results indicated that although PIK3CA was shown to be extensively expressed in DLBCL, no significant association was observed between PIK3CA expression and clinical outcome or between PIK3CA expression and other clinicopathological parameters, except between performance state (PS

  16. PRX1 knockdown potentiates vitamin K3 toxicity in cancer cells: a potential new therapeutic perspective for an old drug.

    Science.gov (United States)

    He, Tiantian; Hatem, Elie; Vernis, Laurence; Lei, Ming; Huang, Meng-Er

    2015-12-21

    Many promising anticancer molecules are abandoned during the course from bench to bedside due to lack of clear-cut efficiency and/or severe side effects. Vitamin K3 (vitK3) is a synthetic naphthoquinone exhibiting significant in vitro and in vivo anticancer activity against multiple human cancers, and has therapeutic potential when combined with other anticancer molecules. The major mechanism for the anticancer activity of vitK3 is the generation of cytotoxic reactive oxygen species (ROS). We thus reasoned that a rational redox modulation of cancer cells could enhance vitK3 anticancer efficiency. Cancer cell lines with peroxiredoxin 1 (PRX1) gene transiently or stably knocked-down and corresponding controls were exposed to vitK3 as well as a set of anticancer molecules, including vinblastine, taxol, doxorubicin, daunorubicin, actinomycin D and 5-fluorouracil. Cytotoxic effects and cell death events were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay, cell clonogenic assay, measurement of mitochondrial membrane potential and annexin V/propidium iodide double staining. Global ROS accumulation and compartment-specific H2O2 generation were determined respectively by a redox-sensitive chemical probe and H2O2-sensitive sensor HyPer. Oxidation of endogenous antioxidant proteins including TRX1, TRX2 and PRX3 was monitored by redox western blot. We observed that the PRX1 knockdown in HeLa and A549 cells conferred enhanced sensitivity to vitK3, reducing substantially the necessary doses to kill cancer cells. The same conditions (combination of vitK3 and PRX1 knockdown) caused little cytotoxicity in non-cancerous cells, suggesting a cancer-cell-selective property. Increased ROS accumulation had a crucial role in vitK3-induced cell death in PRX1 knockdown cells. The use of H2O2-specific sensors HyPer revealed that vitK3 lead to immediate accumulation of H2O2 in the cytosol, nucleus, and mitochondrial matrix. PRX1 silencing

  17. [Knockdown of STAT3 inhibits proliferation and migration of HepG2 hepatoma cells induced by IFN1].

    Science.gov (United States)

    Li, Xiaofang; Wang, Yuqi; Yan, Ben; Fang, Peipei; Ma, Chao; Xu, Ning; Fu, Xiaoyan; Liang, Shujuan

    2018-02-01

    Objective To prepare lentiviruses expressing shRNA sequences targeting human signal transducer and activator of transcription 3 (STAT3) and detect the effect of STAT3 knockdown on type I interferon (IFN1)-induced proliferation and migration in HepG2 cells. Methods Four STAT3-targeting shRNA sequences (shRNA1-shRNA4) and one control sequence (Ctrl shRNA) were selected and cloned respectively into pLKO.1-sp6-pgk-GFP to construct shRNA-expressing vectors. Along with backbone psPAX2 and pMD2.G vectors, they were separately transfected into HEK293T cells to prepare lentiviruses. HepG2 cells were infected with the lentiviruses. Cytoplastic STAT3 level was detected by Western blotting to screen effective shRNA sequence(s) targeting STAT3. Proliferation and migration of HepG2 cells were analyzed by CCK-8 assay and Transwell TM migration and scratching assay, respectively. To detect the effect of IFN1 on cell proliferation and migration of HepG2 cells, the cells were treated with 2000 U/mL IFNα2b for indicated time and the activation of IFN-triggered STAT1 signal transduction was assayed by Western blotting. Results Two most effective STAT3-targeting shRNA sequences shRNA1 and shRNA2 were selected, and the expression of both STAT3 shRNA significantly decreased proliferation and migration of HepG2 cells. When treated with IFNα2b, 2000 U/mL of IFN1 showed more competent in attenuating growth and migration of HepG2 cells. Our data further proved that knockdown of STAT3 increased the phosphorylation of STAT1, and IFNα2b further enhanced the activation of STAT1 signaling in HepG2 cells. Conclusion Knockdown of STAT3 inhibits cell migration and growth, and rescues IFN response through up-regulating STAT1 signal transduction in HepG2 hepatoma cells.

  18. Pre-Test Analysis Predictions for the Shell Buckling Knockdown Factor Checkout Tests - TA01 and TA02

    Science.gov (United States)

    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. Knock-down of ELMO1 in Paediatric Rhabdomyosarcoma Cells by Nanoparticle Mediated siRNA Delivery

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    Xinyue Huang

    2016-03-01

    Full Text Available Rhabdomyosarcoma (RMS is the most common soft tissue sarcoma that is found in children and has a poor outcome for those with metastatic disease. Two histological groups have been distinguished - embryonal (ERMS and alveolar (ARMS forms. The ARMS subtype has higher rates of metastasis, as well as higher levels of ELMO1, which is thought to be involved in cell migration. Therefore, the knock-down of ELMO1 by targeted siRNA could provide a mechanism to prevent the metastatic behaviour of ARMS cells. However, challenges still lie in the delivery of nucleotides to a tumour site. Herein, we have described the use of a variety of mesoporous silica nanoparticles as a delivery system for siRNA that is specific for ELMO1 and shown the effective reduction in cell invasive behaviour in these cells.

  20. CRM-1 knockdown inhibits extrahepatic cholangiocarcinoma tumor growth by blocking the nuclear export of p27Kip1.

    Science.gov (United States)

    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.

  1. Peripheral-specific y2 receptor knockdown protects mice from high-fat diet-induced obesity.

    Science.gov (United States)

    Shi, Yan-Chuan; Lin, Shu; Castillo, Lesley; Aljanova, Aygul; Enriquez, Ronaldo F; Nguyen, Amy D; Baldock, Paul A; Zhang, Lei; Bijker, Martijn S; Macia, Laurence; Yulyaningsih, Ernie; Zhang, Hui; Lau, Jackie; Sainsbury, Amanda; Herzog, Herbert

    2011-11-01

    Y2 receptors, particularly those in the brain, have been implicated in neuropeptide Y (NPY)-mediated effects on energy homeostasis and bone mass. Recent evidence also indicates a role for Y2 receptors in peripheral tissues in this process by promoting adipose tissue accretion; however their effects on energy balance remain unclear. Here, we show that adult-onset conditional knockdown of Y2 receptors predominantly in peripheral tissues results in protection against diet-induced obesity accompanied by significantly reduced weight gain, marked reduction in adiposity and improvements in glucose tolerance without any adverse effect on lean mass or bone. These changes occur in association with significant increases in energy expenditure, respiratory exchange ratio, and physical activity and despite concurrent hyperphagia. On a chow diet, knockdown of peripheral Y2 receptors results in increased respiratory exchange ratio and physical activity with no effect on lean or bone mass, but decreases energy expenditure without effecting body weight or food intake. These results suggest that peripheral Y2 receptor signaling is critical in the regulation of oxidative fuel selection and physical activity and protects against the diet-induced obesity. The lack of effects on bone mass seen in this model further indicates that bone mass is primarily controlled by non-peripheral Y2 receptors. This study provides evidence that novel drugs that target peripheral rather than central Y2 receptors could provide benefits for the treatment of obesity and glucose intolerance without adverse effects on lean and bone mass, with the additional benefit of avoiding side effects often associated with pharmaceuticals that act on the central nervous system.

  2. Network Analysis for the Identification of Differentially Expressed Hub Genes Using Myogenin Knock-down Muscle Satellite Cells.

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    Adeel Malik

    Full Text Available Muscle, a multinucleate syncytium formed by the fusion of mononuclear myoblasts, arises from quiescent progenitors (satellite cells via activation of muscle-specific transcription factors (MyoD, Myf5, myogenin: MYOG, and MRF4. Subsequent to a decline in Pax7, induction in the expression of MYOG is a hallmark of myoblasts that have entered the differentiation phase following cell cycle withdrawal. It is evident that MYOG function cannot be compensated by any other myogenic regulatory factors (MRFs. Despite a plethora of information available regarding MYOG, the mechanism by which MYOG regulates muscle cell differentiation has not yet been identified. Using an RNA-Seq approach, analysis of MYOG knock-down muscle satellite cells (MSCs have shown that genes associated with cell cycle and division, DNA replication, and phosphate metabolism are differentially expressed. By constructing an interaction network of differentially expressed genes (DEGs using GeneMANIA, cadherin-associated protein (CTNNA2 was identified as the main hub gene in the network with highest node degree. Four functional clusters (modules or communities were identified in the network and the functional enrichment analysis revealed that genes included in these clusters significantly contribute to skeletal muscle development. To confirm this finding, in vitro studies revealed increased expression of CTNNA2 in MSCs on day 12 compared to day 10. Expression of CTNNA2 was decreased in MYOG knock-down cells. However, knocking down CTNNA2, which leads to increased expression of extracellular matrix (ECM genes (type I collagen α1 and type I collagen α2 along with myostatin (MSTN, was not found significantly affecting the expression of MYOG in C2C12 cells. We therefore propose that MYOG exerts its regulatory effects by acting upstream of CTNNA2, which in turn regulates the differentiation of C2C12 cells via interaction with ECM genes. Taken together, these findings highlight a new

  3. Knock-down of hypoxia-induced carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular acidosis

    Energy Technology Data Exchange (ETDEWEB)

    Doyen, Jérome [Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis,, Nice (France); Department of Radiation Oncology, Centre Antoine-Lacassagne, Nice (France); Parks, Scott K. [Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis,, Nice (France); Marcié, Serge [Department of Radiation Oncology, Centre Antoine-Lacassagne, Nice (France); Pouysségur, Jacques [Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis,, Nice (France); Centre Scientifique de Monaco (Monaco); Chiche, Johanna, E-mail: chiche@unice.fr [Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis,, Nice (France)

    2013-01-07

    The relationship between acidosis within the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases (CA) IX and CAXII constitute a robust intracellular pH (pH{sub i})-regulating system that confers a survival advantage on hypoxic human colon carcinoma LS174Tr cells in acidic microenvironments. Here we investigate the role of acidosis, CAIX and CAXII knock-down in combination with ionizing radiation. Fibroblasts cells (-/+ CAIX) and LS174Tr cells (inducible knock-down for ca9/ca12) were analyzed for cell cycle phase distribution and survival after irradiation in extracellular pH{sub o} manipulations and hypoxia (1% O{sub 2}) exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pH{sub i}-regulating capacity of fibroblasts through inhibition of Na{sup +}/H{sup +} exchanger 1 sensitize cells to radiation-induced cell death. Secondly, the pH{sub i}-regulating function of CAIX plays a key protective role in irradiated fibroblasts in an acidic environment as accompanied by a reduced number of cells in the radiosensitive phases of the cell cycle. Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50 and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pH{sub i} regulation. Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo. These findings highlight the combinatory use of radiotherapy with targeting of the pH{sub i}-regulating CAs as an anti-cancer strategy.

  4. Effects of DCK knockdown on proliferation, apoptosis and tumorigenicity in vivo of cervical cancer HeLa cells.

    Science.gov (United States)

    Shang, Q-Y; Wu, C-S; Gao, H-R

    2017-09-01

    The present study explored the effect that deoxycytidine kinase (DCK) knockdown had on proliferation, apoptosis and tumorigenicity in vivo of cervical cancer HeLa cells. Human cervical cancer HeLa cells that had received no prior treatment were selected from the HeLa group. The HeLa-negative control (NC) group consisted of cells that had undergone an empty vector treatment, and finally the HeLa-short hairpin RNA (shRNA) group included cells that were treated by means of shRNA-DCK expression. DCK expressions were evaluated by quantitative real-time polymerase chain reaction in addition to western blotting assays. Cell proliferation was estimated using the Cell Counting Kit-8 (CCK-8) assay and cell cycle progression. Cell apoptosis was determined by flow cytometry. BALB/c nude mice (n=24) were selected to establish transplanted tumor models, with gross tumor volume measured every 3 days. The results in vitro were as follows: compared with the HeLa group, the HeLa-shRNA group exhibited downregulation of DCK expression and inhibition of cell proliferation at 48, 72 and 96 h. Additionally, more cells in the HeLa-shRNA group were arrested in G0/G1 stage and less in S and G2/M stages, as well as in promotion of cell apoptosis. In vivo results are as follows: when comparing the HeLa and HeLa-NC groups, the gross tumor volume of the transplanted tumor in nude mice in the HeLa-shRNA group was found to have decreased in 13, 16, 19 and 22 days. Based on these findings, our study suggests that DCK knockdown facilitates apoptosis while inhibiting proliferation and tumorigenicity in vivo of cervical cancer HeLa cells.

  5. Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

    Directory of Open Access Journals (Sweden)

    Motoharu Ono

    Full Text Available We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

  6. RNAi-mediated knockdown of MTNR1B without disrupting the effects of melatonin on apoptosis and cell cycle in bovine granulose cells

    Directory of Open Access Journals (Sweden)

    Wenju Liu

    2018-04-01

    Full Text Available Melatonin is well known as a powerful free radical scavenger and exhibits the ability to prevent cell apoptosis. In the present study, we investigated the role of melatonin and its receptor MTNR1B in regulating the function of bovine granulosa cells (GCs and hypothesized the involvement of MTNR1B in mediating the effect of melatonin on GCs. Our results showed that MTNR1B knockdown significantly promoted GCs apoptosis but did not affect the cell cycle. These results were further verified by increasing the expression of pro-apoptosis genes (BAX and CASP3, decreasing expression of the anti-apoptosis genes (BCL2 and BCL-XL and anti-oxidant genes (SOD1 and GPX4 without affecting cell cycle factors (CCND1, CCNE1 and CDKN1A and TP53. In addition, MTNR1B knockdown did not disrupt the effects of melatonin in suppressing the GCs apoptosis or blocking the cell cycle. Moreover, MTNR1B knockdown did not affect the role of melatonin in increasing BCL2, BCL-XL, and CDKN1A expression, or decreasing BAX, CASP3, TP53, CCND1 and CCNE1 expression. The expression of MTNR1A was upregulated after MTNR1B knockdown, and melatonin promoted MTNR1A expression with or without MTNR1B knockdown. However, despite melatonin supplementation, the expression of SOD1 and GPX4 was still suppressed after MTNR1B knockdown. In conclusion, these findings indicate that melatonin and MTNR1B are involved in BCL2 family and CASP3-dependent apoptotic pathways in bovine GCs. MTNR1A and MTNR1B may coordinate the work of medicating the appropriate melatonin responses to GCs.

  7. Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

    Science.gov (United States)

    Ono, Motoharu; Yamada, Kayo; Avolio, Fabio; Afzal, Vackar; Bensaddek, Dalila; Lamond, Angus I

    2015-01-01

    We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

  8. RNAi-mediated knockdown of pituitary tumor-transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

    International Nuclear Information System (INIS)

    Huang, S.Q.; Liao, Q.J.; Wang, X.W.; Xin, D.Q.; Chen, S.X.; Wu, Q.J.; Ye, G.

    2012-01-01

    Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy

  9. Sodium Channel Mutations and Pyrethroid Resistance in Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Yuzhe Du

    2016-10-01

    Full Text Available Pyrethroid insecticides are widely used to control insect pests and human disease vectors. Voltage-gated sodium channels are the primary targets of pyrethroid insecticides. Mutations in the sodium channel have been shown to be responsible for pyrethroid resistance, known as knockdown resistance (kdr, in various insects including mosquitoes. In Aedes aegypti mosquitoes, the principal urban vectors of dengue, zika, and yellow fever viruses, multiple single nucleotide polymorphisms in the sodium channel gene have been found in pyrethroid-resistant populations and some of them have been functionally confirmed to be responsible for kdr in an in vitro expression system, Xenopus oocytes. This mini-review aims to provide an update on the identification and functional characterization of pyrethroid resistance-associated sodium channel mutations from Aedes aegypti. The collection of kdr mutations not only helped us develop molecular markers for resistance monitoring, but also provided valuable information for computational molecular modeling of pyrethroid receptor sites on the sodium channel.

  10. A botanical containing freeze dried açai pulp promotes healthy aging and reduces oxidative damage in sod1 knockdown flies

    OpenAIRE

    Laslo, Mara; Sun, Xiaoping; Hsiao, Cheng-Te; Wu, Wells W.; Shen, Rong-Fong; Zou, Sige

    2012-01-01

    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 Aça...

  11. Antimicrobial Resistance

    Science.gov (United States)

    ... least 10 countries (Australia, Austria, Canada, France, Japan, Norway, Slovenia, South Africa, Sweden and the United Kingdom ... plan Global report on surveillance Country situation analysis Policy to combat antimicrobial resistance More on antimicrobial resistance ...

  12. Antimicrobial Resistance

    Science.gov (United States)

    ... can prevent and manage antimicrobial resistance. It is collaborating with partners to strengthen the evidence base and ... on the global action plan. WHO has been leading multiple initiatives to address antimicrobial resistance: World Antibiotic ...

  13. Effects of 60Co gamma radiation on defense function of human polymorphonuclear leukocytes

    International Nuclear Information System (INIS)

    Sasagawa, Sumiko; Suzuki, Kazuo; Sakatani, Tatsuichiro; Brooks, G.T.; Fujikura, Toshio.

    1986-06-01

    The effects of radiation on defense function of polymorphonuclear leukocytes (PMN) were studied following irradiation with 60 Co γ radiation (30 - 3,000 rad) using PMN separated from the peripheral blood of healthy volunteers. The migration distances for all three measures of chemotaxis to fMet-Leu-Phe (10 -8 M), chemokinesis induced by fMet-Leu-Phe, and random migration tended to decrease with increasing dose, showing 0.0054 μm/rad (p -5 M) in conjunction with cytochalasin B (CB, 5 μg/ml) there was a significant dose trend, showing the dose effects of decreasing 0.0022 % release/rad for BGL and 0.0030 % release/rad for LYZ with increasing dose. In superoxide anion (O 2 - ) production, a slight and marginally significant linear dose trend was found. These results suggest that the defense function of PMN is not so resistant to radiation as predicted from the fact that PMN in the peripheral blood are differentiated and mature. It is thought that radiation inflicts substantially harmful effects on the defense function of peripheral PMN. (author)

  14. Antibiotic resistance

    Directory of Open Access Journals (Sweden)

    Marianne Frieri

    2017-07-01

    Full Text Available Summary: Antimicrobial resistance in bacterial pathogens is a challenge that is associated with high morbidity and mortality. Multidrug resistance patterns in Gram-positive and -negative bacteria are difficult to treat and may even be untreatable with conventional antibiotics. There is currently a shortage of effective therapies, lack of successful prevention measures, and only a few new antibiotics, which require development of novel treatment options and alternative antimicrobial therapies. Biofilms are involved in multidrug resistance and can present challenges for infection control. Virulence, Staphylococcus aureus, Clostridium difficile infection, vancomycin-resistant enterococci, and control in the Emergency Department are also discussed. Keywords: Antibiotic resistance, Biofilms, Infections, Public health, Emergency Department

  15. Neuronal markers are expressed in human gliomas and NSE knockdown sensitizes glioblastoma cells to radiotherapy and temozolomide

    International Nuclear Information System (INIS)

    Yan, Tao; Skaftnesmo, Kai Ove; Leiss, Lina; Sleire, Linda; Wang, Jian; Li, Xingang; Enger, Per Øyvind

    2011-01-01

    Expression of neuronal elements has been identified in various glial tumors, and glioblastomas (GBMs) with neuronal differentiation patterns have reportedly been associated with longer survival. However, the neuronal class III β-tubulin has been linked to increasing malignancy in astrocytomas. Thus, the significance of neuronal markers in gliomas is not established. The expressions of class III β-tubulin, neurofilament protein (NFP), microtubule-associated protein 2 (MAP2) and neuron-specific enolase (NSE) were investigated in five GBM cell lines and two GBM biopsies with immunocytochemistry and Western blot. Moreover, the expression levels were quantified by real-time qPCR under different culture conditions. Following NSE siRNA treatment we used Electric cell-substrate impedance sensing (ECIS) to monitor cell growth and migration and MTS assays to study viability after irradiation and temozolomide treatment. Finally, we quantitated NSE expression in a series of human glioma biopsies with immunohistochemistry using a morphometry software, and collected survival data for the corresponding patients. The biopsies were then grouped according to expression in two halves which were compared by survival analysis. Immunocytochemistry and Western blotting showed that all markers except NFP were expressed both in GBM cell lines and biopsies. Notably, qPCR demonstrated that NSE was upregulated in cellular stress conditions, such as serum-starvation and hypoxia, while we found no uniform pattern for the other markers. NSE knockdown reduced the migration of glioma cells, sensitized them to hypoxia, radio- and chemotherapy. Furthermore, we found that GBM patients in the group with the highest NSE expression lived significantly shorter than patients in the low-expression group. Neuronal markers are aberrantly expressed in human GBMs, and NSE is consistently upregulated in different cellular stress conditions. Knockdown of NSE reduces the migration of GBM cells and sensitizes

  16. Impact of Subolesin and Cystatin Knockdown by RNA Interference in Adult Female Haemaphysalis longicornis (Acari: Ixodidae on Blood Engorgement and Reproduction

    Directory of Open Access Journals (Sweden)

    Md. Khalesur Rahman

    2018-04-01

    Full Text Available Currently, multi-antigenic vaccine use is the method of choice for the strategic control of ticks. Therefore, determining the efficacy of combined antigens is a promising avenue of research in the development of anti-tick vaccines. The antigen responsible for blood intake and reproduction has proven suitable as a vaccine antigen. It has been shown to silence Haemaphysalis longicornis salivary cystatin (HlSC-1 and subolesin by RNA interference. Adult unfed female ticks were injected with double-stranded RNA of (A subolesin, (B cystatin, (C subolesin plus cystatin, and (D injection buffer, then fed alongside normal unfed males up to spontaneous drop-down. The percentage of knockdowns was determined by real-time polymerase chain reaction. Sixty-three percent and 53% knockdown rates were observed in subolesin and cystatin double-stranded RNA-injected ticks respectively, while 32 and 26% knockdown rates of subolesin and cystatin transcript were observed in subolesin plus cystatin double-stranded RNA-injected ticks. Subolesin and/or cystatin knockdown causes a significant (p < 0.05 reduction in tick engorgement, egg mass weight, and egg conversion ratio. Most importantly, combined silencing did not act synergistically, but caused a similarly significant (p < 0.05 reduction in tick engorgement, egg mass weight, and egg conversion ratio. Therefore, the elucidation of multiple antigens may be helpful in the future of vaccines.

  17. Partial Correction of Psoriasis upon Genetic Knock-Down of Human TNF-α by Lentivirus-Encoded shRNAs in a Xenograft Mouse Model

    DEFF Research Database (Denmark)

    Jakobsen, Maria; Stenderup, Karin; Rosada, Cecilia

    samples treated with irrelevant shRNAs, were selected and cloned into lentiviral vectors. The lentiviral vectors expressing TNF- shRNAs were used to transduce HEK-293 cells and verify vector-derived knock-down of stable TNF- expression in vitro. The most efficient TNF- -directed shRNA, which in cell lines...

  18. Salt Sensitive Tet-Off-Like Systems to Knockdown Primordial Germ Cell Genes for Repressible Transgenic Sterilization in Channel Catfish, Ictalurus punctatus.

    Science.gov (United States)

    Li, Hanbo; Su, Baofeng; Qin, Guyu; Ye, Zhi; Alsaqufi, Ahmed; Perera, Dayan A; Shang, Mei; Odin, Ramjie; Vo, Khoi; Drescher, David; Robinson, Dalton; Zhang, Dan; Abass, Nermeen; Dunham, Rex A

    2017-05-31

    Repressible knockdown approaches were investigated for transgenic sterilization in channel catfish, Ictalurus punctatus . Two primordial germ cell (PGC) marker genes, nanos and dead end , were targeted for knockdown, and an off-target gene, vasa , was monitored. Two potentially salt sensitive repressible promoters, zebrafish adenylosuccinate synthase 2 (ADSS) and zebrafish racemase (Rm), were each coupled with four knockdown strategies: ds-sh RNA targeting the 5' end (N1) or 3' end (N2) of channel catfish nanos , full-length cDNA sequence of channel catfish nanos for overexpression (cDNA) and ds-sh RNA targeting channel catfish dead end (DND). Each construct had an untreated group and treated group with sodium chloride as the repressor compound. Spawning rates of full-sibling P₁ fish exposed or not exposed to the constructs as treated and untreated embryos were 93% and 59%, respectively, indicating potential sterilization of fish and repression of the constructs. Although the mRNA expression data of PGC marker genes were inconsistent in P₁ fish, most F₁ individuals were able to downregulate the target genes in untreated groups and repress the knockdown process in treated groups. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but more data from F₂ or F₃ are needed for evaluation.

  19. Salt Sensitive Tet-Off-Like Systems to Knockdown Primordial Germ Cell Genes for Repressible Transgenic Sterilization in Channel Catfish, Ictalurus punctatus

    Directory of Open Access Journals (Sweden)

    Hanbo Li

    2017-05-01

    Full Text Available Repressible knockdown approaches were investigated for transgenic sterilization in channel catfish, Ictalurus punctatus. Two primordial germ cell (PGC marker genes, nanos and dead end, were targeted for knockdown, and an off-target gene, vasa, was monitored. Two potentially salt sensitive repressible promoters, zebrafish adenylosuccinate synthase 2 (ADSS and zebrafish racemase (Rm, were each coupled with four knockdown strategies: ds-sh RNA targeting the 5′ end (N1 or 3′ end (N2 of channel catfish nanos, full-length cDNA sequence of channel catfish nanos for overexpression (cDNA and ds-sh RNA targeting channel catfish dead end (DND. Each construct had an untreated group and treated group with sodium chloride as the repressor compound. Spawning rates of full-sibling P1 fish exposed or not exposed to the constructs as treated and untreated embryos were 93% and 59%, respectively, indicating potential sterilization of fish and repression of the constructs. Although the mRNA expression data of PGC marker genes were inconsistent in P1 fish, most F1 individuals were able to downregulate the target genes in untreated groups and repress the knockdown process in treated groups. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but more data from F2 or F3 are needed for evaluation.

  20. Knockdown of long noncoding RNA linc-ITGB1 suppresses migration, invasion of hepatocellular carcinoma via regulating ZEB1.

    Science.gov (United States)

    Yu, W-W; Wang, K; Liao, G-J

    2017-11-01

    This research focuses on the influence of linc-ITGB1 on the metastasis of hepatocellular carcinoma and further explores its underlying mechanism. A total of 70 hepatocellular carcinoma patients were chosen for our study. RT-qPCR was used for detecting the expression level of linc-ITGB1 in their cancer tissues. Moreover, the expression level of linc-ITGB1 was also detected in hepatocellular carcinoma cell lines. Furthermore, whether linc-ITGB1 could affect the migrated and invaded ability of hepatocellular carcinoma cells was determined by wound healing assay and transwell assay. We further explored the potential mechanism by RT-qPCR and Western blot assay. Linc-ITGB1 expression level in hepatocellular carcinoma tissues was remarkably higher than that in adjacent tissues. Moreover, migrated and invaded ability of hepatocellular carcinoma cells was inhibited through knockdown of linc-ITGB1. Further study revealed that silenced linc-ITGB1 inhibited the expression of ZEB1 and then suppressed epithelial to mesenchymal transition (EMT), which was important during the metastasis of hepatocellular carcinoma. Moreover, the inhibition of cell invasion by silenced linc-ITGB1 could be rescued through overexpression of ZEB1 in hepatocellular carcinoma. The results indicate that linc-ITGB1, a novel oncogene in tumorigenesis, could promote the metastasis and EMT via ZEB1, which may offer a possible therapeutic target in hepatocellular carcinoma.

  1. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC.

    Science.gov (United States)

    Garbarino, Jeanne; Pan, Meihui; Chin, Harvey F; Lund, Frederik W; Maxfield, Frederick R; Breslow, Jan L

    2012-12-01

    STARD4, a member of the evolutionarily conserved START gene family, has been implicated in the nonvesicular intracellular transport of cholesterol. However, the direction of transport and the membranes with which this protein interacts are not clear. We present studies of STARD4 function using small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein receptor (LDLR) levels were increased and decreased, respectively. We also observed a decrease in NPC1 protein expression, suggesting the induction of compensatory pathways to maintain cholesterol balance. These data indicate a role for STARD4 in nonvesicular transport of cholesterol from the plasma membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well.

  2. Hypomorphic Smn knockdown C2C12 myoblasts reveal intrinsic defects in myoblast fusion and myotube morphology

    International Nuclear Information System (INIS)

    Shafey, Dina; Cote, Patrice D.; Kothary, Rashmi

    2005-01-01

    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 α-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

  3. A Combined Optogenetic-Knockdown Strategy Reveals a Major Role of Tomosyn in Mossy Fiber Synaptic Plasticity

    Directory of Open Access Journals (Sweden)

    Yoav Ben-Simon

    2015-07-01

    Full Text Available Neurotransmitter release probability (Pr largely determines the dynamic properties of synapses. While much is known about 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 protein kinase A (PKA-dependent long-term potentiation (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.

  4. CaMKII knockdown affects both early and late phases of olfactory long-term memory in the honeybee.

    Science.gov (United States)

    Scholl, Christina; Kübert, Natalie; Muenz, Thomas S; Rössler, Wolfgang

    2015-12-01

    Honeybees are able to solve complex learning tasks and memorize learned information for long time periods. The molecular mechanisms mediating long-term memory (LTM) in the honeybee Apis mellifera are, to a large part, still unknown. We approached this question by investigating the potential function of the calcium/calmodulin-dependent protein kinase II (CaMKII), an enzyme known as a 'molecular memory switch' in vertebrates. CaMKII is able to switch to a calcium-independent constitutively active state, providing a mechanism for a molecular memory and has further been shown to play an essential role in structural synaptic plasticity. Using a combination of knockdown by RNA interference and pharmacological manipulation, we disrupted the function of CaMKII during olfactory learning and memory formation. We found that learning, memory acquisition and mid-term memory were not affected, but all manipulations consistently resulted in an impaired LTM. Both early LTM (24 h after learning) and late LTM (72 h after learning) were significantly disrupted, indicating the necessity of CaMKII in two successive stages of LTM formation in the honeybee. © 2015. Published by The Company of Biologists Ltd.

  5. Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage.

    Science.gov (United States)

    Tulpule, Asmin; Lensch, M William; Miller, Justine D; Austin, Karyn; D'Andrea, Alan; Schlaeger, Thorsten M; Shimamura, Akiko; Daley, George Q

    2010-04-29

    Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by pediatric bone marrow failure and congenital anomalies. The effect of FA gene deficiency on hematopoietic development in utero remains poorly described as mouse models of FA do not develop hematopoietic failure and such studies cannot be performed on patients. We have created a human-specific in vitro system to study early hematopoietic development in FA using a lentiviral RNA interference (RNAi) strategy in human embryonic stem cells (hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a reduction in hematopoietic fates and progenitor numbers that can be rescued by FA gene complementation. Our data indicate that hematopoiesis is impaired in FA from the earliest stages of development, suggesting that deficiencies in embryonic hematopoiesis may underlie the progression to bone marrow failure in FA. This work illustrates how hESCs can provide unique insights into human development and further our understanding of genetic disease.

  6. The TetO rat as a new translational model for type 2 diabetic retinopathy by inducible insulin receptor knockdown.

    Science.gov (United States)

    Reichhart, Nadine; Crespo-Garcia, Sergio; Haase, Nadine; Golic, Michaela; Skosyrski, Sergej; Rübsam, Anne; Herrspiegel, Christina; Kociok, Norbert; Alenina, Natalia; Bader, Michael; Dechend, Ralf; Strauss, Olaf; Joussen, Antonia M

    2017-01-01

    Although the renin-angiotensin system plays an important role in the progression of diabetic retinopathy, its influence therein has not been systematically evaluated. Here we test the suitability of a new translational model of diabetic retinopathy, the TetO rat, for addressing the role of angiotensin-II receptor 1 (AT1) blockade in experimental diabetic retinopathy. Diabetes was induced by tetracycline-inducible small hairpin RNA (shRNA) knockdown of the insulin receptor in rats, generating TetO rats. Systemic treatment consisted of an AT1 blocker (ARB) at the onset of diabetes, following which, 4-5 weeks later the retina was analysed in vivo and ex vivo. Retinal function was assessed by Ganzfeld electroretinography (ERG). Retinal vessels in TetO rats showed differences in vessel calibre, together with gliosis. The total number and the proportion of activated mononuclear phagocytes was increased. TetO rats presented with loss of retinal ganglion cells (RGC) and ERG indicated photoreceptor malfunction. Both the inner and outer blood-retina barriers were affected. The ARB treated group showed reduced gliosis and an overall amelioration of retinal function, alongside RGC recovery, whilst no statistically significant differences in vascular and inflammatory features were detected. The TetO rat represents a promising translational model for the early neurovascular changes associated with type 2 diabetic retinopathy. ARB treatment had an effect on the neuronal component of the retina but not on the vasculature.

  7. Increased cell motility and invasion upon knockdown of lipolysis stimulated lipoprotein receptor (LSR in SW780 bladder cancer cells

    Directory of Open Access Journals (Sweden)

    Ørntoft Torben F

    2008-07-01

    Full Text Available Abstract Background Mechanisms underlying the malignant development in bladder cancer are still not well understood. Lipolysis stimulated lipoprotein receptor (LSR has previously been found to be upregulated by P53. Furthermore, we have previously found LSR to be differentially expressed in bladder cancer. Here we investigated the role of LSR in bladder cancer. Methods A time course siRNA knock down experiment was performed to investigate the functional role of LSR in SW780 bladder cancer cells. Since LSR was previously shown to be regulated by P53, siRNA against TP53 was included in the experimental setup. We used Affymetrix GeneChips for measuring gene expression changes and we used Ingenuity Pathway Analysis to investigate the relationship among differentially expressed genes upon siRNA knockdown. Results By Ingenuity Pathway analysis of the microarray data from the different timepoints we identified six gene networks containing genes mainly related to the functional categories "cancer", "cell death", and "cellular movement". We determined that genes annotated to the functional category "cellular movement" including "invasion" and "cell motility" were highly significantly overrepresented. A matrigel assay showed that 24 h after transfection the invasion capacity was significantly increased 3-fold (p Conclusion We conclude that LSR may impair bladder cancer cells from gaining invasive properties.

  8. [Improvement and the mechanism of cardiac function by knockdown of ADAM10 in adriamycin-induced cardiomyopathy rats].

    Science.gov (United States)

    Li, Xiaoou; Xie, Lili; He, Bing; Huang, Wei

    2018-01-01

    Objective To study the role of a disintegrin and metalloproteinase10 (ADAM10) in shedding neural cadherin (N-cadherin) and develop an approach to interfere the process of ventricular remodeling in adriamycin-induced cardiomyopathy (ACM) rats. Methods In a rat model of ACM, the effects of intraperitoneal injection of the lentiviral RNAi vector of ADAM10 on the morphology of cardiomyocytes and contractile function were observed by HE staining and color Doppler echocardiography. The expressions of N-cadherin and C-terminal fragment 1 (CTF1) were detected by Western blotting and immunohistochemistry. Results In the in vivo experiment, a large amount of fluorescence was seen in the isolated primary cardiomyocytes, which indicated that the transfection in the rat model was successful. In the treatment group, the morphology of cardiomyocytes and function of the heart were evidently improved, N-cadherin protein expression was remarkably up-regulated and CTF1 protein was obviously down-regulated compared with the model group. Conclusion Knock-down of ADAM10 increases N-cadherin expression and decreases CTF1 expression, thus improves cardiac function in the rat model of ACM.

  9. [Knockdown of dopamine receptor D2 upregulates the expression of adiogenic genes in mouse primary mesencephalic neurons].

    Science.gov (United States)

    Ding, Jiaqi; Chen, Xiaoli; Lin, Jiaji; Zhu, Junling; Li, Zhuyi

    2018-01-01

    Objective To study the effects of dopamine receptor D2 (DRD2) on the adipogenesis genes in mouse primary mesencephalic neurons. Methods The lentiviral vectors which expressed specific shRNA targeting DRD2 were constructed to decrease DRD2 expression in mouse primary mesencephalic neurons. High throughput sequencing (HTS) analysis was used to investigate gene expression changes between the DRD2 knock-down group and the negative control group. Real-time quantitative PCR (qRT-PCR) and Western blot analysis were applied to verify the differently expressed genes. Fatty acids were measured by fatty acid detection kit. Results DRD2 expression was effectively down-regulated in mouse primary mesencephalic neurons by lentiviral vectors. HTS revealed adipogenesis genes were significantly up-regulated after DRD2 down-regulation, mainly including delta(14)-sterol reductase, acetyl-coenzyme A synthetase, insulin-induced gene 1 protein and especially stearoyl-coenzyme A desaturase 1 (SCD1, 4-fold upregulated). The qRT-PCR and Western blot analysis verified that SCD1 was upregulated 2.6 folds and 2 folds respectively by lentiviral DRD2-shRNA vectors. Moreover, the SCD1-related free fatty acids were significantly more increased than the negative control group. Conclusion DRD2 in primary mesencephalic neurons had a significant regulative effect on the adipogenesis genes. The up-regulation of SCD1 can accelerate the conversion of saturated fatty acids to monounsaturated fatty acids and prevent the damage of lipid toxicity to cells.

  10. Proteomic Analysis to Identify Functional Molecules in Drug Resistance Caused by E-Cadherin Knockdown in 3D-Cultured Colorectal Cancer Models

    Science.gov (United States)

    2013-09-01

    of human MCF10A cells. Proteomics 2011, 11 (10), 2019−26. (23) Siu, S. O.; Lam , M. P.; Lau , E.; Kong, R. P.; Lee, S. M.; Chu, I. K. Fully automatable...signaling. J. Proteome Res. 2011, 10 (12), 5383−97. (17) Kettenbach, A. N .; Gerber, S. A. Rapid and reproducible single- stage phosphopeptide enrichment of...McGowan, T.; Bandhakavi, S.; Cheng, B.; Rhodus, N . L.; Griffin, T. J. Large-scale phosphoproteomics Journal of Proteome Research Article dx.doi.org

  11. Plant Translation Factors and Virus Resistance

    Directory of Open Access Journals (Sweden)

    Hélène Sanfaçon

    2015-06-01

    Full Text Available 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.

  12. Gene-knockdown in the honey bee mite Varroa destructor by a non-invasive approach: studies on a glutathione S-transferase

    Directory of Open Access Journals (Sweden)

    Campbell Ewan M

    2010-08-01

    Full Text Available Abstract 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

  13. Knock-down and speed of kill of a combination of fipronil and permethrin for the prevention of Ctenocephalides felis flea infestation in dogs.

    Science.gov (United States)

    Halos, Lénaïg; Fourie, Josephus J; Fankhauser, Becky; Beugnet, Frederic

    2016-02-02

    A topical combination of fipronil + permethrin (Frontline Tri-Act/Frontect, Merial) has recently been developed to control fleas, ticks, mosquitoes, sandflies and stable flies on dogs. Two studies were conducted to assess its speed of kill and knock-down effect on Ctenocephalides felis fleas. The combination was compared to either fipronil alone or to a combination of permethrin, dinotefuran, and pyriproxyfen, In each study, 18 dogs were randomly allocated to one of three groups: (Group 1: untreated dog; Group 2: treated once on D0 with the combination of fipronil and permethrin; Group 3: treated once on D0 either with fipronil alone (study 1) or with a combination of permethrin, dinetofuran and pyriproxyfen (study 2)). Each dog was infested with 100 unfed adult C. felis fleas on Days 2 (study 2), 7, 14, 21 and 28. Fleas were collected from dogs at 1 h and 12 h post- infestations (PI) (study 1) or at 2 h and 6 h PI (study 2) to assess efficacy and from collection pans underneath cages 1 h (study 1) or 5 min (study 2) PI to assess knock-down effect. All treated dogs had significantly (p ≤ 0.01) lower flea counts than untreated dogs at every time point in both studies. For a whole month, a significant knock-down effect against infesting fleas is obtained in five minutes PI with the combination of permethrin and fipronil. Complete efficacy (>95%) was achieved in 1 h (study 1) or 2 h (study 2) PI for 14 days and by 6 h PI for all challenges conducted throughout the month. Efficacy remains >85% at 2 h PI for the whole month. A significantly higher efficacy of the fipronil + permethrin combination compared to other treatments was demonstrated at the earliest time points for the month (1 h knock-down effect and insecticidal efficacy compared to fipronil alone; 5 min knock-down effect compared to the combination of permethrin + dinetofuran + pyriproxyfen). The rapid flea knock-down effect and speed of kill demonstrated by the spot on combination of

  14. Resistant Hypertension.

    Science.gov (United States)

    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.

  15. Camptothecin resistance

    DEFF Research Database (Denmark)

    Brangi, M; Litman, Thomas; Ciotti, M

    1999-01-01

    . Glucuronides were found at equal levels in both parental and resistant colon cancer cell lines for epirubicin and to a lesser extent for SN-38 and mitoxantrone. Low levels of glucuronidation could also be detected in the resistant breast cancer cells. These results were confirmed by analysis of the UGT1A...

  16. Parthenogenetic embryonic stem cells with H19 siRNA-mediated knockdown as a potential resource for cell therapy.

    Science.gov (United States)

    Kwak, Minhye; Hong, Su; Yu, Seong-Lan; Sim, Bo-Woong; Seo, Jeong-Sun; Kang, Jaeku

    2012-02-01

    Embryonic stem (ES) cells are used in cell therapy and tissue engineering due to their ability to produce different cells types. However, studies of ES cells that are derived from fertilized embryos have raised concerns about the limitations imposed by ethical and political considerations. Therefore, many studies of stem cells use the stem cells that are derived from unfertilized oocytes and adult tissue. Although parthenogenetic embryonic stem (ESP) cells also avoid ethical and political dilemmas and can be used in cell-based therapy, the ESP cells exhibit growth retardation problems. Therefore, to investigate the potential for muscle growth from genetically modified ESP cells, we established four ES cell types, including normal embryonic stem (ESN) cells, ESP cells, ESP cells that overexpress the insulin-like growth factor 2 (Igf2) gene (ESI) and ESP cells with down-regulated H19 gene expression (ESH). Using these cells, we examined the expression profiles of genes that were related to imprinting and muscle using microarrays. The gene expression patterns of ESI and ESH cells were similar and were more closely related to the ESN pattern than that of the ESP cells. Differentiated ESH cells exhibited increased expression of bone morphologic protein 4 (BMP4), which is a mesoderm marker, compared with the differentiated ESI cells. We showed that Igf2 expression was induced by H19 silencing in the ESP cells via hypermethylation of the H19 imprinting control region 1 (ICR1). Moreover, the proportion of ESH-derived chimera was slightly higher than those produced from the ESP cells. In addition, we detected increased cell proliferation in the MEF cells following H19 knock-down. These results indicate that the ESH cells may be a source of cell-based therapy for conditions such as muscular atrophy.

  17. γ-Catenin at Adherens Junctions: Mechanism and Biologic Implications in Hepatocellular Cancer after β-Catenin Knockdown

    Directory of Open Access Journals (Sweden)

    Emily Diane Wickline

    2013-04-01

    Full Text Available β-Catenin is important in liver homeostasis as a part of Wnt signaling and adherens junctions (AJs, while its aberrant activation is observed in hepatocellular carcinoma (HCC. We have reported hepatocyte-specific β-catenin knockout (KO mice to lack adhesive defects as γ-catenin compensated at AJ. Because γ-catenin is a desmosomal protein, we asked if its increase in KO might deregulate desmosomes. No changes in desmosomal proteins or ultrastructure other than increased plakophilin-3 were observed. To further elucidate the role and regulation of γ-catenin, we contemplate an in vitro model and show γ-catenin increase in HCC cells upon β-catenin knockdown (KD. Here, γ-catenin is unable to rescue β-catenin/T cell factor (TCF reporter activity; however, it sufficiently compensates at AJs as assessed by scratch wound assay, centrifugal assay for cell adhesion (CAFCA, and hanging drop assays. γ-Catenin increase is observed only after β-catenin protein decrease and not after blockade of its transactivation. γ-Catenin increase is associated with enhanced serine/threonine phosphorylation and abrogated by protein kinase A (PKA inhibition. In fact, several PKA-binding sites were detected in γ-catenin by in silico analysis. Intriguingly γ-catenin KD led to increased β-catenin levels and transactivation. Thus, γ-catenin compensates for β-catenin loss at AJ without affecting desmosomes but is unable to fulfill functions in Wnt signaling. γ-Catenin stabilization after β-catenin loss is brought about by PKA. Catenin-sensing mechanism may depend on absolute β-catenin levels and not its activity. Anti-β-catenin therapies for HCC affecting total β-catenin may target aberrant Wnt signaling without negatively impacting intercellular adhesion, provided mechanisms leading to γ-catenin stabilization are spared.

  18. Knockdown of TC-1 enhances radiosensitivity of non-small cell lung cancer via the Wnt/β-catenin pathway.

    Science.gov (United States)

    Wu, Dapeng; Li, Lei; Yan, Wei

    2016-04-15

    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 blotin vitroandin 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 fromin vitroandin vivostudies indicated that TC-1 silencing sensitized NSCLC cell lines to radiotherapy through the Wnt/β-catenin signaling pathway. © 2016. Published by The Company of Biologists Ltd.

  19. Knock-down of miR-221 and miR-222 in the radiosensitization of breast cancer cells

    International Nuclear Information System (INIS)

    Zhang Chunzhi; Kang Chunsheng; Cao Yongzhen; Pu Peiyu; Lu Zhonghong; Du Yue

    2009-01-01

    Objective: To investigate the radiosensitizing effect of knock-down of miR-221 miR-222 on MCF-7 human breast cancer cells and explore the possible mechanism. Methods: Antisense oligonucleotides of miR-221 and miR-222 (AS-miR-221 and AS-miR-222), mediated by lipofectamine, were transfected to MCF-7 cells to knock down miR-221 and miR-222, Northern blotting was conducted to detect the expression of miR-221 and miR-222 in transfected cells. The cell apoptosis was detected by flow cytometry and Caspase-3 and Caspase-7 activity assay. Clonogenic assay was used to measure the sensitizing enhancement ratio. Target genes of miR-221 and miR-222 relevant to radio-sensitivity were searched using bioinformatics analysis. The targeted protein expression was determined by Western blot analysis. Results: The expression of miR-221 and miR-222 in the AS-miR-221/222 cells determined by Northern blotting was significantly reduced. Compared with the control group, the cell apoptosis and mitotic cell death after the radiation were significantly higher in AS-miR-221/222 cells. The sensitizing enhancement ratio was 1.87. Based on bioinformatics analysis, PTEN was a target gene of miR-221 and miR-222 which could enhance the radiosensitivity of MCF-7 cells. In AS-miR-221/222 cells, the expression of PTEN was up-regulated while pAkt down-regulated. Conclusions: AS-miR-221 and AS-miR-222 may enhance the radiosensitivity of MCF-7 breast cancer cells by up-regulating the expression of PTEN. (authors)

  20. RNAi-mediated knockdown of serine protease inhibitor genes increases the mortality of Plutella xylostella challenged by destruxin A.

    Science.gov (United States)

    Han, Pengfei; Fan, Jiqiao; Liu, Yu; Cuthbertson, Andrew G S; Yan, Shaoqiao; Qiu, Bao-Li; Ren, Shunxiang

    2014-01-01

    Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides.

  1. Knockdown of Hsc70-5/mortalin induces loss of synaptic mitochondria in a Drosophila Parkinson's disease model.

    Directory of Open Access Journals (Sweden)

    Jun-Yi Zhu

    Full Text Available Mortalin is an essential component of the molecular machinery that imports nuclear-encoded proteins into mitochondria, assists in their folding, and protects against damage upon accumulation of dysfunctional, unfolded proteins in aging mitochondria. Mortalin dysfunction associated with Parkinson's disease (PD increases the vulnerability of cultured cells to proteolytic stress and leads to changes in mitochondrial function and morphology. To date, Drosophila melanogaster has been successfully used to investigate pathogenesis following the loss of several other PD-associated genes. We generated the first loss-of-Hsc70-5/mortalin-function Drosophila model. The reduction of Mortalin expression recapitulates some of the defects observed in the existing Drosophila PD-models, which include reduced ATP levels, abnormal wing posture, shortened life span, and reduced spontaneous locomotor and climbing ability. Dopaminergic neurons seem to be more sensitive to the loss of mortalin than other neuronal sub-types and non-neuronal tissues. The loss of synaptic mitochondria is an early pathological change that might cause later degenerative events. It precedes both behavioral abnormalities and structural changes at the neuromuscular junction (NMJ of mortalin-knockdown larvae that exhibit increased mitochondrial fragmentation. Autophagy is concomitantly up-regulated, suggesting that mitochondria are degraded via mitophagy. Ex vivo data from human fibroblasts identifies increased mitophagy as an early pathological change that precedes apoptosis. Given the specificity of the observed defects, we are confident that the loss-of-mortalin model presented in this study will be useful for further dissection of the complex network of pathways that underlie the development of mitochondrial parkinsonism.

  2. RNAi-Mediated Knockdown of Serine Protease Inhibitor Genes Increases the Mortality of Plutella xylostella Challenged by Destruxin A

    Science.gov (United States)

    Han, Pengfei; Fan, Jiqiao; Liu, Yu; Cuthbertson, Andrew G. S.; Yan, Shaoqiao; Qiu, Bao-Li; Ren, Shunxiang

    2014-01-01

    Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides. PMID:24837592

  3. Pulmonary oxidative stress is increased in cyclooxygenase-2 knockdown mice with mild pulmonary hypertension induced by monocrotaline.

    Directory of Open Access Journals (Sweden)

    Francesca Seta

    Full Text Available The aim of this study was to examine the role of cyclooxygenase-2 (COX-2 and downstream signaling of prostanoids in the pathogenesis of pulmonary hypertension (PH using mice with genetically manipulated COX-2 expression. COX-2 knockdown (KD mice, characterized by 80-90% suppression of COX-2, and wild-type (WT control mice were treated weekly with monocrotaline (MCT over 10 weeks. Mice were examined for cardiac hypertrophy/function and right ventricular pressure. Lung histopathological analysis was performed and various assays were carried out to examine oxidative stress, as well as gene, protein, cytokine and prostanoid expression. We found that MCT increased right ventricular systolic and pulmonary arterial pressures in comparison to saline-treated mice, with no evidence of cardiac remodeling. Gene expression of endothelin receptor A and thromboxane synthesis, regulators of vasoconstriction, were increased in MCT-treated lungs. Bronchoalveolar lavage fluid and lung sections demonstrated mild inflammation and perivascular edema but activation of inflammatory cells was not predominant under the experimental conditions. Heme oxygenase-1 (HO-1 expression and indicators of oxidative stress in lungs were significantly increased, especially in COX-2 KD MCT-treated mice. Gene expression of NOX-4, but not NOX-2, two NADPH oxidase subunits crucial for superoxide generation, was induced by ∼4-fold in both groups of mice by MCT. Vasodilatory and anti-aggregatory prostacyclin was reduced by ∼85% only in MCT-treated COX-2 KD mice. This study suggests that increased oxidative stress-derived endothelial dysfunction, vasoconstriction and mild inflammation, exacerbated by the lack of COX-2, contribute to the pathogenesis of early stages of PH when mild hemodynamic changes are evident and not yet accompanied by vascular and cardiac remodeling.

  4. Inducible knockdown of pregnancy-associated plasma protein-A gene expression in adult female mice extends life span.

    Science.gov (United States)

    Bale, Laurie K; West, Sally A; Conover, Cheryl A

    2017-08-01

    Pregnancy-associated plasma protein-A (PAPP-A) knockout (KO) mice, generated through homologous recombination in embryonic stem cells, have a significantly increased lifespan compared to wild-type littermates. However, it is unknown whether this longevity advantage would pertain to PAPP-A gene deletion in adult animals. In the present study, we used tamoxifen (Tam)-inducible Cre recombinase-mediated excision of the floxed PAPP-A (fPAPP-A) gene in mice at 5 months of age. fPAPP-A mice, which were either positive (pos) or negative (neg) for Tam-Cre, received Tam treatment with quarterly boosters. Only female mice could be used with this experimental design. fPAPP-A/neg and fPAPP-A/pos mice had similar weights at the start of the experiment and showed equivalent weight gain. We found that fPAPP-A/pos mice had a significant extension of life span (P = 0.005). The median life span was increased by 21% for fPAPP-A/pos compared to fPAPP-A/neg mice. Analysis of mortality in life span quartiles indicated that the proportion of deaths of fPAPP-A/pos mice were lower than fPAPP-A/neg mice at young adult ages (P = 0.002 for 601-800 days) and higher than fPAPP-A/neg mice at older ages (P = 0.004 for >1000 days). Thus, survival curves and age-specific mortality indicate that female mice with knockdown of PAPP-A gene expression as adults have an extended healthy life span. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  5. RNAi-mediated knockdown of SPOOK reduces ecdysteroid titers and causes precocious metamorphosis in the desert locust Schistocerca gregaria.

    Science.gov (United States)

    Sugahara, Ryohei; Tanaka, Seiji; Shiotsuki, Takahiro

    2017-09-01

    The Halloween gene SPOOK (SPO) is involved in the production of the active metabolite of ecdysteroid, 20-hydroxyecdysone (20E), in insects. A previous study showed that RNAi-mediated knockdown of SPO in Schistocerca gregaria last instar nymphs markedly reduced the hemolymph 20E titer, but did not affect metamorphosis. In the present study, the effects of SPO interference on development were re-examined in this locust. Injections of SPO double-stranded RNA (dsSPO) into nymphs at mid and late instars significantly delayed nymphal development and interfered with molting. The 20E levels of dsSPO-treated nymphs were generally low, with a delayed, small peak, suggesting that disturbance of the 20E levels caused the above developmental abnormalities. A small proportion of the dsSPO-injected nymphs metamorphosed precociously, producing adults and adultoids. Precocious adults were characterized by small body size, short wings with abbreviated venation, and normal reproductive activity. Fourth instar nymphs that precociously metamorphosed at the following instar exhibited temporal expression patterns of ecdysone-induced protein 93F and the juvenile hormone (JH) early-inducible gene Krüppel homolog 1 similar to those observed at the last instar in normal nymphs. Adultoids displayed mating behavior and adultoid females developed eggs, but never laid eggs. JH injection around the expected time of the 20E peak in the dsSPO-injected nymphs completely inhibited the appearance of adultoids, suggesting that appearance of adultoids might be due to a reduced titer of JH rather than of 20E. These results suggest that SPO plays an important role in controlling morphogenesis, metamorphosis, and reproduction in S. gregaria. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. [Knock-down of BCL11A expression in breast cancer cells promotes MDA-MB-231 cell apoptosis].

    Science.gov (United States)

    Li, Hongli; Gui, Chen; Yan, Lijun

    2016-11-01

    Objective To detect the expression and pathological significance of B-cell CLL/lymphoma 11A (BCL11A) in breast cancer and investigate the effect of its silencing on the apoptosis of human MDA-MB-231 breast cancer cells. MethodsImmunohistochemistry was used to detect the expression of BCL11A in 62 cases of human breast cancer tissues and 8 cases of normal tissues. We synthesized siRNA targeting BCL11A, and then siRNA was transfected into MDA-MB-231 cells. Forty-eight hours later, the suppression effect of siRNA on BCL11A was determined by quantitative real-time PCR and Western blotting. The apoptosis of MDA-MB-231 cells was detected by flow cytometry. Results The BCL11A protein was mainly expressed in cytoplasm. The expression level of BCL11A in breast cancer tissues was higher than that in paracancerous tissues. The expression had correlations with tumor grade, tumor stage, while it had no correlations with the patients' age and tumor size. BCL11A-siRNA significantly suppressed the expression of BCL11A mRNA and protein as compared with the control group. MDA-MB-231 cells transfected with BCL11A-siRNA had higher apoptosis rate compared with the control group. Conclusion The BCL11A protein is highly expressed in breast cancer and knock-down of BCL11A promotes the apoptosis of MDA-MB-231 cells.

  7. Antibiotic Resistance

    DEFF Research Database (Denmark)

    Munck, Christian

    morbidity and mortality as well as an increase in the cost of treatment. Understanding how bacteria respond to antibiotic exposure gives the foundations for a rational approach to counteract antimicrobial resistance. In the work presented in this thesis, I explore the two fundamental sources...... of antimicrobial resistance: (1) adaptive mutations and (2) horizontal acquisition of resistance genes from antibiotic gene reservoirs. By studying the geno- and phenotypic changes of E. coli in response to single and drug-pair exposures, I uncover the evolutionary trajectories leading to adaptive resistance. I...... to rationally design drug combinations that limit the evolution of antibiotic resistance due to counteracting evolutionary trajectories. My results highlight that an in-depth knowledge about the genetic responses to the individual antimicrobial compounds enables the prediction of responses to drug combinations...

  8. Go-6976 Reverses Hyperglycemia-Induced Insulin Resistance Independently of cPKC Inhibition in Adipocytes

    Science.gov (United States)

    Robinson, Katherine A.; Hegyi, Krisztina; Hannun, Yusuf A.; Buse, Maria G.; Sethi, Jaswinder K.

    2014-01-01

    Chronic hyperglycemia induces insulin resistance by mechanisms that are incompletely understood. One model of hyperglycemia-induced insulin resistance involves chronic preincubation of adipocytes in the presence of high glucose and low insulin concentrations. We have previously shown that the mTOR complex 1 (mTORC1) plays a partial role in the development of insulin resistance in this model. Here, we demonstrate that treatment with Go-6976, a widely used “specific” inhibitor of cPKCs, alleviates hyperglycemia-induced insulin resistance. However, the effects of mTOR inhibitor, rapamycin and Go-6976 were not additive and only rapamycin restored impaired insulin-stimulated AKT activation. Although, PKCα, (but not –β) was abundantly expressed in these adipocytes, our studies indicate cPKCs do not play a major role in causing insulin-resistance in this model. There was no evidence of changes in the expression or phosphorylation of PKCα, and PKCα knock-down did not prevent the reduction of insulin-stimulated glucose transport. This was also consistent with lack of IRS-1 phosphorylation on Ser-24 in hyperglycemia-induced insulin-resistant adipocytes. Treatment with Go-6976 did inhibit a component of the mTORC1 pathway, as evidenced by decreased phosphorylation of S6 ribosomal protein. Raptor knock-down enhanced the effect of insulin on glucose transport in insulin resistant adipocytes. Go-6976 had the same effect in control cells, but was ineffective in cells with Raptor knock-down. Taken together these findings suggest that Go-6976 exerts its effect in alleviating hyperglycemia-induced insulin-resistance independently of cPKC inhibition and may target components of the mTORC1 signaling pathway. PMID:25330241

  9. Knockdown of MAP4 and DNAL1 produces a post-fusion and pre-nuclear translocation impairment in HIV-1 replication

    International Nuclear Information System (INIS)

    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.

  10. Role of G-protein-coupled receptor-related genes in insecticide resistance of the mosquito, Culex quinquefasciatus.

    Science.gov (United States)

    Li, Ting; Liu, Lena; Zhang, Lee; Liu, Nannan

    2014-09-29

    G-protein-coupled receptors regulate signal transduction pathways and play diverse and pivotal roles in the physiology of insects, however, the precise function of GPCRs in insecticide resistance remains unclear. Using quantitative RT-PCR and functional genomic methods, we, for the first time, explored the function of GPCRs and GPCR-related genes in insecticide resistance of mosquitoes, Culex quinquefasciatus. A comparison of the expression of 115 GPCR-related genes at a whole genome level between resistant and susceptible Culex mosquitoes identified one and three GPCR-related genes that were up-regulated in highly resistant Culex mosquito strains, HAmCq(G8) and MAmCq(G6), respectively. To characterize the function of these up-regulated GPCR-related genes in resistance, the up-regulated GPCR-related genes were knockdown in HAmCq(G8) and MAmCq(G6) using RNAi technique. Knockdown of these four GPCR-related genes not only decreased resistance of the mosquitoes to permethrin but also repressed the expression of four insecticide resistance-related P450 genes, suggesting the role of GPCR-related genes in resistance is involved in the regulation of resistance P450 gene expression. This results help in understanding of molecular regulation of resistance development in Cx. quinquefasciatus.

  11. RNAi validation of resistance genes and their interactions in the highly DDT-resistant 91-R strain of Drosophila melanogaster.

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells

    OpenAIRE

    Lee, Su Jeong; Park, Jeen-Woo

    2014-01-01

    Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocyte...

  13. Comparison of the CAS-POL and IOM samplers for determining the knockdown efficiencies of water sprays on float coal dust.

    Science.gov (United States)

    Seaman, Clara E; Shahan, Michael R; Beck, Timothy W; Mischler, Steven E

    2018-03-01

    Float coal dust, generated by mining operations, is distributed throughout mine airways by ventilating air designed to purge gases and respirable dust. Float coal dust poses an explosion hazard in the event of a methane ignition. Current regulation requires the application of inert rock dust in areas subjected to float coal dust in order to mitigate the hazard. An alternate method using water sprays, which have been effective in controlling respirable dust hazards, has been proposed as a way to control float coal dust generated on longwall faces. However, the knockdown efficiency of the proposed water sprays on float coal dust needs to be verified. This study used gravimetric isokinetic Institute of Occupational Medicine (IOM) samplers alongside a real-time aerosol monitor (Cloud Aerosol Spectrometer with polarization; CAS-POL) to study the effects of spray type, operating pressure, and spray orientation on knockdown efficiencies for seven different water sprays. Because the CAS-POL has not been used to study mining dust, the CAS-POL measurements were validated with respect to the IOM samplers. This study found that the CAS-POL was able to resolve the same trends measured by the IOM samplers, while providing additional knockdown information for specific particle size ranges and locations in the test area. In addition, the CAS-POL data was not prone to the same process errors, which may occur due to the handling of the IOM filter media, and was able to provide a faster analysis of the data after testing. This study also determined that pressure was the leading design criteria influencing spray knockdown efficiency, with spray type also having some effect and orientation having little to no effect. The results of this study will be used to design future full-scale float coal dust capture tests involving multiple sprays, which will be evaluated using the CAS-POL.

  14. Confusion, knock-down and kill of Aedes aegypti using metofluthrin in domestic settings: a powerful tool to prevent dengue transmission?

    Science.gov (United States)

    Ritchie, Scott A; Devine, Gregor J

    2013-09-11

    Dengue control methods are reliant upon control of the vector, primarily Aedes aegypti. Current adulticiding methods in North Queensland include treating premises with residual synthetic pyrethroid insecticides (interior residual spraying; IRS), a laborious, intrusive task. The vapor active synthetic pyrethroid metofluthrin might offer an efficient alternative as some studies indicate that it prevents biting and has strong knockdown effects. However, its expellant and/or irritant effects, longevity, residual activity and the speed with which biting behavior is disrupted have not yet been characterized. We exposed cohorts of Cairns colony (F2-4) Ae. aegypti to rooms (17-24 m3) treated with 5% and 10% AI metofluthrin emanators. Using free-flying and caged populations we measured biting (human landing rate), expulsion through unscreened windows, knockdown and death over periods ranging between a few minutes and 24 hrs. Observations of the behavior of single female Ae. aegypti exposed to metofluthrin were also made. Female Ae. aegypti exposed to 5% or 10% metofluthrin formulations were almost entirely inhibited from biting. This was the result of rapid knockdown and mortality (80-90% in less than one hour) and to the behavioral impacts of exposure that, within minutes, caused female Ae. aegypti to become disoriented, stop landing on hosts, and seek resting sites. Exposed mosquitoes did not exhibit any increased propensity to exit treated rooms and the 10% AI resin remained fully active for at least 20 days. The new, high-dose, resin formulations of metofluthrin act quickly to prevent biting and to knockdown and kill free-flying female Ae. aegypti in our experimental rooms. There was no evidence that metofluthrin induced escape from treated areas. Resin-based metofluthrin emanators show great potential as a replacement for labor intensive IRS for dengue vector control.

  15. Confusion, knock-down and kill of Aedes aegypti using metofluthrin in domestic settings: a powerful tool to prevent dengue transmission?

    OpenAIRE

    Ritchie, Scott A; Devine, Gregor J

    2013-01-01

    Background Dengue control methods are reliant upon control of the vector, primarily Aedes aegypti. Current adulticiding methods in North Queensland include treating premises with residual synthetic pyrethroid insecticides (interior residual spraying; IRS), a laborious, intrusive task. The vapor active synthetic pyrethroid metofluthrin might offer an efficient alternative as some studies indicate that it prevents biting and has strong knockdown effects. However, its expellant and/or irritant e...

  16. EWS Knockdown and Taxifolin Treatment Induced Differentiation and Removed DNA Methylation from p53 Promoter to Promote Expression of Puma and Noxa for Apoptosis in Ewing's Sarcoma.

    Science.gov (United States)

    Hossain, Mohammad Motarab; Ray, Swapan Kumar

    2014-10-01

    Ewing's sarcoma is a pediatric tumor that mainly occurs in soft tissues and bones. Malignant characteristics of Ewing's sarcoma are correlated with expression of EWS oncogene. We achieved knockdown of EWS expression using a plasmid vector encoding EWS short hairpin RNA (shRNA) to increase anti-tumor mechanisms of taxifolin (TFL), a new flavonoid, in human Ewing's sarcoma cells in culture and animal models. Immunofluorescence microscopy and flow cytometric analysis showed high expression of EWS in human Ewing's sarcoma SK-N-MC and RD-ES cell lines. EWS shRNA plus TFL inhibited 80% cell viability and caused the highest decreases in EWS expression at mRNA and protein levels in both cell lines. Knockdown of EWS expression induced morphological features of differentiation. EWS shRNA plus TFL caused more alterations in molecular markers of differentiation than either agent alone. EWS shRNA plus TFL caused the highest decreases in cell migration with inhibition of survival, angiogenic and invasive factors. Knockdown of EWS expression was associated with removal of DNA methylation from p53 promoter, promoting expression of p53, Puma, and Noxa. EWS shRNA plus TFL induced the highest amounts of apoptosis with activation of extrinsic and intrinsic pathways in both cell lines in culture. EWS shRNA plus TFL also inhibited growth of Ewing's sarcoma tumors in animal models due to inhibition of differentiation inhibitors and angiogenic and invasive factors and also induction of activation of caspase-3 for apoptosis. Collectively, knockdown of EWS expression increased various anti-tumor mechanisms of TFL in human Ewing's sarcoma in cell culture and animal models.

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

    DEFF Research Database (Denmark)

    Norry, Fabian M.; Scannapieco, Alejandra C.; Sambucetti, Pablo

    2008-01-01

    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...

  18. Widespread Pyrethroid and DDT Resistance in the Major Malaria Vector Anopheles funestus in East Africa Is Driven by Metabolic Resistance Mechanisms

    Science.gov (United States)

    Mulamba, Charles; Riveron, Jacob M.; Ibrahim, Sulaiman S.; Irving, Helen; Barnes, Kayla G.; Mukwaya, Louis G.; Birungi, Josephine; Wondji, Charles S.

    2014-01-01

    Background Establishing the extent, geographical distribution and mechanisms of insecticide resistance in malaria vectors is a prerequisite for resistance management. Here, we report a widespread distribution of insecticide resistance in the major malaria vector An. funestus across Uganda and western Kenya under the control of metabolic resistance mechanisms. Methodology/Principal Findings Female An. funestus collected throughout Uganda and western Kenya exhibited a Plasmodium infection rate between 4.2 to 10.4%. Widespread resistance against both type I (permethrin) and II (deltamethrin) pyrethroids and DDT was observed across Uganda and western Kenya. All populations remain highly susceptible to carbamate, organophosphate and dieldrin insecticides. Knockdown resistance plays no role in the pyrethroid and DDT resistance as no kdr mutation associated with resistance was detected despite the presence of a F1021C replacement. Additionally, no signature of selection was observed on the sodium channel gene. Synergist assays and qRT-PCR indicated that metabolic resistance plays a major role notably through elevated expression of cytochrome P450s. DDT resistance mechanisms differ from West Africa as the L119F-GSTe2 mutation only explains a small proportion of the genetic variance to DDT resistance. Conclusion The extensive distribution of pyrethroid and DDT resistance in East African An. funestus populations represents a challenge to the control of this vector. However, the observed carbamate and organophosphate susceptibility offers alternative solutions for resistance management. PMID:25333491

  19. Insecticide resistance status of Aedes aegypti (L.) from Colombia.

    Science.gov (United States)

    Fonseca-González, Idalyd; Quiñones, Martha L; Lenhart, Audrey; Brogdon, William G

    2011-04-01

    To evaluate the insecticide susceptibility status of Aedes aegypti (L.) in Colombia, and as part of the National Network of Insecticide Resistance Surveillance, 12 mosquito populations were assessed for resistance to pyrethroids, organophosphates and DDT. Bioassays were performed using WHO and CDC methodologies. The underlying resistance mechanisms were investigated through biochemical assays and RT-PCR. All mosquito populations were susceptible to malathion, deltamethrin and cyfluthrin, and highly resistant to DDT and etofenprox. Resistance to lambda-cyhalothrin, permethrin and fenitrothion ranged from moderate to high in some populations from Chocó and Putumayo states. In Antioquia state, the Santa Fe population was resistant to fenitrothion. Biochemical assays showed high levels of both cytochrome P450 monooxygenases (CYP) and non-specific esterases (NSE) in some of the fenitrothion- and pyrethroid-resistant populations. All populations showed high levels of glutathione-S-transferase (GST) activity. GSTe2 gene was found overexpressed in DDT-resistant populations compared with Rockefeller susceptible strain. Differences in insecticide resistance status were observed between insecticides and localities. Although the biochemical assay results suggest that CYP and NSE could play an important role in the pyrethroid and fenitrothion resistance detected, other mechanisms remain to be investigated, including knockdown resistance. Resistance to DDT was high in all populations, and GST activity is probably the main enzymatic mechanism associated with this resistance. The results of this study provide baseline data on insecticide resistance in Colombian A. aegypti populations, and will allow comparison of changes in susceptibility status in this vector over time. Copyright © 2011 Society of Chemical Industry.

  20. Nanoparticle-mediated knockdown of DNA repair sensitizes cells to radiotherapy and extends survival in a genetic mouse model of glioblastoma.

    Science.gov (United States)

    Kievit, Forrest M; Wang, Kui; Ozawa, Tatsuya; Tarudji, Aria W; Silber, John R; Holland, Eric C; Ellenbogen, Richard G; Zhang, Miqin

    2017-10-01

    Glioblastoma (GBM) remains incurable, and recurrent tumors rarely respond to standard-of-care radiation and chemo-therapies. Therefore, strategies that enhance the effects of these therapies should provide significant benefits to GBM patients. We have developed a nanoparticle delivery vehicle that can stably bind and protect nucleic acids for specific delivery into brain tumor cells. These nanoparticles can deliver therapeutic siRNAs to sensitize GBM cells to radiotherapy and improve GBM treatment via systemic administration. We show that nanoparticle-mediated knockdown of the DNA repair protein apurinic endonuclease 1 (Ape1) sensitizes GBM cells to radiotherapy and extend survival in a genetic mouse model of GBM. Specific knockdown of Ape1 activity by 30% in brain tumor tissue doubled the extended survival achieved with radiotherapy alone. Ape1 is a promising target for increasing the effectiveness of radiotherapy, and nanoparticle-mediated delivery of siRNA is a promising strategy for tumor specific knockdown of Ape1. Copyright © 2017. Published by Elsevier Inc.

  1. Dmp53, basket and drICE gene knockdown and polyphenol gallic acid increase life span and locomotor activity in a Drosophila Parkinson's disease model

    Directory of Open Access Journals (Sweden)

    Hector Flavio Ortega-Arellano

    2013-01-01

    Full Text Available 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.

  2. LOXL4 knockdown enhances tumor growth and lung metastasis through collagen-dependent extracellular matrix changes in triple-negative breast cancer.

    Science.gov (United States)

    Choi, Sul Ki; Kim, Hoe Suk; Jin, Tiefeng; Moon, Woo Kyung

    2017-02-14

    Lysyl oxidase (LOX) family genes catalyze collagen cross-link formation. To determine the effects of lysyl oxidase-like 4 (LOXL4) expression on breast tumor formation and metastasis, we evaluated primary tumor growth and lung metastasis in mice injected with LOXL4-knockdown MDA-MB-231 triple-negative human breast cancer cells. In addition, we analyzed overall survival in breast cancer patients based on LOXL4 expression using a public online database. In the mouse xenograft model, LOXL4 knockdown increased primary tumor growth and lung colonization as well as collagen I and IV, lysine hydroxylase 1 and 2, and prolyl 4-hydroxylase subunit alpha 1 and 2 levels. Second harmonic generation imaging revealed that LOXL4 knockdown resulted in the thickening of collagen bundles within tumors. In addition, weak LOXL4 expression was associated with poor overall survival in breast cancer patients from the BreastMark dataset, and this association was strongest in triple-negative breast cancer patients. These results demonstrate that weak LOXL4 expression leads to remodeling of the extracellular matrix through induction of collagen synthesis, deposition, and structural changes. These alterations in turn promote tumor growth and metastasis and are associated with poor clinical outcomes in triple-negative breast cancer.

  3. Short-hairpin Mediated Myostatin Knockdown Resulted in Altered Expression of Myogenic Regulatory Factors with Enhanced Myoblast Proliferation in Fetal Myoblast Cells of Goats.

    Science.gov (United States)

    Kumar, Rohit; Singh, Satyendra Pal; Mitra, Abhijit

    2018-01-02

    Myostatin (MSTN) is a well-known negative regulator of skeletal muscle development. Reduced expression due to natural mutations in the coding region and knockout as well as knockdown of MSTN results in an increase in the muscle mass. In the present study, we demonstrated as high as 60 and 52% downregulation (p < 0.01) of MSTN mRNA and protein in the primary fetal myoblast cells of goats using synthetic shRNAs (n = 3), without any interferon response. We, for the first time, evaluated the effect of MSTN knockdown on the expression of MRFs (namely, MyoD, Myf5), follistatin (FST), and IGFs (IGF-1 & IGF-2) in goat myoblast cells. MSTN knockdown caused an upregulation (p < 0.05) of MyoD and downregulation (p < 0.01) of MYf5 and FST expression. Moreover, we report up to ∼four fold (p < 0.001) enhanced proliferation in myoblasts after four days of culture. The anti-MSTN shRNA demonstrated in the present study could be used for the production of transgenic goats to increase the muscle mass.

  4. H19 mediates methotrexate resistance in colorectal cancer through activating Wnt/β-catenin pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Ke-feng [Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical University, Zhanjiang, Guangdong (China); Liang, Wei-Cheng [School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong (China); Feng, Lu [Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong (China); Pang, Jian-xin [School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515 (China); Waye, Mary Miu-Yee [School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong (China); Zhang, Jin-Fang [Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong (China); Fu, Wei-Ming, E-mail: fuweiming76@smu.edu.cn [School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515 (China)

    2017-01-15

    Colorectal cancer (CRC) is a common malignancy, most of which remain unresponsive to chemotherapy. As one of the earliest cytotoxic drugs, methotrexate (MTX) serves as an anti-metabolite and anti-folate chemotherapy for various cancers. Unfortunately, MTX resistance prevents its clinical application in cancer therapy. Thereby, overcoming the drug resistance is an alternative strategy to maximize the therapeutic efficacy of MTX in clinics. Long noncoding RNAs (lncRNAs) have gained widespread attention in recent years. More and more emerging evidences have demonstrated that they play important regulatory roles in various biological activities and disease progression including drug resistance. In the present study, a MTX-resistant colorectal cell line HT-29 (HT-29-R) was developed, which displayed the active proliferation and shortened cell cycle. LncRNA H19 was found to be significantly upregulated in this resistant cell line. Further investigation showed that H19 knockdown sensitized the MTX resistance in HT-29-R cells while its overexpression improved the MTX resistance in the parental cells, suggesting that H19 mediate MTX resistance. The Wnt/β-catenin signaling was activated in HT-29-R cells, and H19 knockdown suppressed this signaling in the parental cells. In conclusion, H19 mediated MTX resistance via activating Wnt/β-catenin signaling, which help to develop H19 as a promising therapeutic target for MTX resistant CRC. - Highlights: • A methotrexate (MTX) -resistant colorectal cancer cell line HT-29 (HT-29-R) has been developed. • H19 was upregulated in HT-29-R cells. • H19 mediated MTX resistance in colorectal cancer (CRC). • Wnt/β-catenin pathway was involved in the H19-mediated MTX resistance in CRC cells.

  5. H19 mediates methotrexate resistance in colorectal cancer through activating Wnt/β-catenin pathway

    International Nuclear Information System (INIS)

    Wu, Ke-feng; Liang, Wei-Cheng; Feng, Lu; Pang, Jian-xin; Waye, Mary Miu-Yee; Zhang, Jin-Fang; Fu, Wei-Ming

    2017-01-01

    Colorectal cancer (CRC) is a common malignancy, most of which remain unresponsive to chemotherapy. As one of the earliest cytotoxic drugs, methotrexate (MTX) serves as an anti-metabolite and anti-folate chemotherapy for various cancers. Unfortunately, MTX resistance prevents its clinical application in cancer therapy. Thereby, overcoming the drug resistance is an alternative strategy to maximize the therapeutic efficacy of MTX in clinics. Long noncoding RNAs (lncRNAs) have gained widespread attention in recent years. More and more emerging evidences have demonstrated that they play important regulatory roles in various biological activities and disease progression including drug resistance. In the present study, a MTX-resistant colorectal cell line HT-29 (HT-29-R) was developed, which displayed the active proliferation and shortened cell cycle. LncRNA H19 was found to be significantly upregulated in this resistant cell line. Further investigation showed that H19 knockdown sensitized the MTX resistance in HT-29-R cells while its overexpression improved the MTX resistance in the parental cells, suggesting that H19 mediate MTX resistance. The Wnt/β-catenin signaling was activated in HT-29-R cells, and H19 knockdown suppressed this signaling in the parental cells. In conclusion, H19 mediated MTX resistance via activating Wnt/β-catenin signaling, which help to develop H19 as a promising therapeutic target for MTX resistant CRC. - Highlights: • A methotrexate (MTX) -resistant colorectal cancer cell line HT-29 (HT-29-R) has been developed. • H19 was upregulated in HT-29-R cells. • H19 mediated MTX resistance in colorectal cancer (CRC). • Wnt/β-catenin pathway was involved in the H19-mediated MTX resistance in CRC cells.

  6. Antimicrobial Resistance

    Science.gov (United States)

    ... past two decades due to the increase in immunocompromised and elderly patients, increasing use of invasive indwelling ... aureus developing resistance to vancomycin, a very powerful antibiotic prescribed for the most intractable bacterial infections. In ...

  7. Drug Resistance

    Science.gov (United States)

    ... Drug-resistance testing is also recommended for all pregnant women with HIV before starting HIV medicines and also in some pregnant women already taking HIV medicines. Pregnant women will work with their health ...

  8. Antimicrobial resistance

    DEFF Research Database (Denmark)

    Llor, Carl; Bjerrum, Lars

    2014-01-01

    Antimicrobial resistance is a global public health challenge, which has accelerated by the overuse of antibiotics worldwide. Increased antimicrobial resistance is the cause of severe infections, complications, longer hospital stays and increased mortality. Overprescribing of antibiotics......-the-counter sale of antibiotics, the use of antimicrobial stewardship programmes, the active participation of clinicians in audits, the utilization of valid rapid point-of-care tests, the promotion of delayed antibiotic prescribing strategies, the enhancement of communication skills with patients with the aid...

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

    Directory of Open Access Journals (Sweden)

    Deng L

    2016-01-01

    Full Text Available Liang Deng,1,* Jihong Yang,2,* Hongwu Chen,3 Bo Ma,4 Kangming Pan,1 Caikun Su,1 Fengfeng Xu,1 Jihong Zhang1 1Department of Hepatobiliary Surgery, The Eastern Hospital of the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 2Department of General Surgery, The Affiliated Hospital of Hebei University, Baoding, 3Department of Emergency, 4Department of Gastroenterology, The Eastern Hospital of the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, People’s Republic of China*These authors contributed equally to this workAbstract: 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.Keywords: TMEM16A, cell cycle, proliferation, apoptosis

  10. Knockdown of human serine/threonine kinase 33 suppresses human small cell lung carcinoma by blocking RPS6/BAD signaling transduction.

    Science.gov (United States)

    Sun, E L; Liu, C X; Ma, Z X; Mou, X Y; Mu, X A; Ni, Y H; Li, X L; Zhang, D; Ju, Y R

    2017-01-01

    Small cell lung cancer (SCLC) is characterized by rapid growth rate and a tendency to metastasize to distinct sites of patients' bodies. The human serine/threonine kinase 33 (STK33) gene has shown its potency as a therapeutic target for prevention of lung carcinomas including non-small cell lung cancer (NSCLC), but its function in the oncogenesis and development of SCLC remains unrevealed. In the current study, it was hypothesized that STK33 played a key role in the proliferation, survival, and invasion of SCLC cells. The expression of STK33 in human SCLC cell lines NCI-H466 and DMS153 was inhibited by specific shRNA. The cell proliferation, cell apoptosis, and cell invasion of the cells were assessed with a series of in vitro assays. To explore the mechanism through which STK33 gene exerted its function in the carcinogenesis of SCLC cells, the effect of STK33 knockdown on the activity of S6K1/RPS6/BAD signaling was detected. Then the results were further confirmed with STK33 inhibitor ML281 and in vivo assays. The results demonstrated that inhibition of STK33 in SCLC cells suppressed the cell proliferation and invasion while induced cell apoptosis. Associated with the change in the phenotypic features, knockdown of STK33 also decreased the phosphorylation of RPS6 and BAD while increased the expression of cleaved caspase 9, indicating that apoptosis induced by STK33 suppression was mediated via mitochondrial pathway. Similar to the results of STK33 knockdown, incubating NCI-H466 cells with STK33 inhibitor also reduced the cell viability by suppressing RPS6/BAD pathways. Additionally, STK33 knockdown also inhibited tumor growth and RPS6/BAD activity in mice models. Findings outlined in our study were different from that in NSCLC to some extent: knockdown of STK33 in SCLC cells induced the apoptosis through mitochondrial pathway but independent of S6K1 function, inferring that the function of STK33 might be cancer type specific.

  11. Resistance to BET Inhibitor Leads to Alternative Therapeutic Vulnerabilities in Castration-Resistant Prostate Cancer.

    Science.gov (United States)

    Pawar, Aishwarya; Gollavilli, Paradesi Naidu; Wang, Shaomeng; Asangani, Irfan A

    2018-02-27

    BRD4 plays a major role in the transcription networks orchestrated by androgen receptor (AR) in castration-resistant prostate cancer (CRPC). Several BET inhibitors (BETi) that displace BRD4 from chromatin are being evaluated in clinical trials for CRPC. Here, we describe mechanisms of acquired resistance to BETi that are amenable to targeted therapies in CRPC. BETi-resistant CRPC cells displayed cross-resistance to a variety of BETi in the absence of gatekeeper mutations, exhibited reduced chromatin-bound BRD4, and were less sensitive to BRD4 degraders/knockdown, suggesting a BRD4-independent transcription program. Transcriptomic analysis revealed reactivation of AR signaling due to CDK9-mediated phosphorylation of AR, resulting in sensitivity to CDK9 inhibitors and enzalutamide. Additionally, increased DNA damage associated with PRC2-mediated transcriptional silencing of DDR genes was observed, leading to PARP inhibitor sensitivity. Collectively, our results identify the therapeutic limitation of BETi as a monotherapy; however, our BETi resistance data suggest unique opportunities for combination therapies in treating CRPC. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. Partial ablation of adult Drosophila insulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance.

    Science.gov (United States)

    Haselton, Aaron; Sharmin, Effat; Schrader, Janel; Sah, Megha; Poon, Peter; Fridell, Yih-Woei C

    2010-08-01

    In Drosophila melanogaster (D. melanogaster), neurosecretory insulin-like peptide-producing cells (IPCs), analogous to mammalian pancreatic beta cells are involved in glucose homeostasis. Extending those findings, we have developed in the adult fly an oral glucose tolerance test and demonstrated that IPCs indeed are responsible for executing an acute glucose clearance response. To further develop D. melanogaster as a relevant system for studying age-associated metabolic disorders, we set out to determine the impact of adult-specific partial ablation of IPCs (IPC knockdown) on insulin-like peptide (ILP) action, metabolic outcomes and longevity. Interestingly, while IPC knockdown flies are hyperglycemic and glucose intolerant, these flies remain insulin sensitive as measured by peripheral glucose disposal upon insulin injection and serine phosphorylation of a key insulin-signaling molecule, Akt. Significant increases in stored glycogen and triglyceride levels as well as an elevated level of circulating lipid measured in adult IPC knockdown flies suggest profound modulation in energy metabolism. Additional physiological outcomes measured in those flies include increased resistance to starvation and impaired female fecundity. Finally, increased life span and decreased mortality rates measured in IPC knockdown flies demonstrate that it is possible to modulate ILP action in adult flies to achieve life span extension without insulin resistance. Taken together, we have established and validated an invertebrate genetic system to further investigate insulin action, metabolic homeostasis and regulation of aging regulated by adult IPCs.

  13. Resistant hypertension.

    Science.gov (United States)

    Wei, Fang-Fei; Zhang, Zhen-Yu; Huang, Qi-Fang; Yang, Wen-Yi; Staessen, Jan A

    2018-06-15

    The publication of the first non-randomised proof-of-concept trial of renal denervation as a treatment modality in treatment-resistant hypertension set the stage for a search for novel devices with the expectation that technology would reduce the burden of hypertension by reducing or eliminating the costly and lifelong use of blood pressure lowering medications. As we demonstrate in this review, this idea so attractive to manufacturers and invasive cardiologists and radiologists overlooked decades of careful pathophysiological research in a disease, which still remains enigmatic but remains the major cause of cardiovascular mortality worldwide. To make our point, we first reviewed the prevalence and risks associated with treatment-resistant hypertension. Next, we highlighted the key points required for the diagnosis of treatment-resistant hypertension, including the recording of the ambulatory blood pressure and the assessment of adherence to medication. Finally, we summarised new insights in the management of treatment-resistant hypertension by medication and devices and in the future research. Throughout our review, we focused on new evidence became available since 2013. Our conclusion is that optimising medical treatment based on simple algorithms remains the state of the art in treatment-resistant hypertension.

  14. Anticoagulant Resistance

    DEFF Research Database (Denmark)

    Heiberg, Ann-Charlotte

    Although sewer rat control is carried out in more than 80 % of all Danish municipalities, with usage of large amounts of anticoagulant rodenticides, knowledge on anticoagulant resistance among rats living in the sewers is limited. As rat problems in urban areas are believed to be related to sewer...... problems (70-90 % in UK and DK) unawareness of resistance amongst these populations of Brown rats may constitute a future control problem and knowledge on this issue has become crucial. Rats were captured in sewers from seven different locations in the suburban area of Copenhagen. Locations was chosen...... to represent different sewer rat management strategies i) no anticoagulants for approx. 20 years ii) no anticoagulants for the last 5 years and iii) continuous control for many years. Animals were tested for resistance to bromadiolone by Blood-Clotting Response test, as bromadiolone is the most frequently used...

  15. Reducing Resistance

    DEFF Research Database (Denmark)

    Lindell, Johanna

    care may influence decisions on antibiotic use. Based on video-and audio recordings of physician-patient consultations it is investigated how treatment recommendations are presented, can be changed, are forecast and explained, and finally, how they seemingly meet resistance and how this resistance......Antibiotic resistance is a growing public health problem both nationally and internationally, and efficient strategies are needed to reduce unnecessary use. This dissertation presents four research studies, which examine how communication between general practitioners and patients in Danish primary...... is responded to.The first study in the dissertation suggests that treatment recommendations on antibiotics are often done in a way that encourages patient acceptance. In extension of this, the second study of the dissertation examines a case, where acceptance of such a recommendation is changed into a shared...

  16. Drug resistance

    NARCIS (Netherlands)

    Gorter, J.A.; Potschka, H.; Noebels, J.L.; Avoli, M.; Rogawski, M.A.; Olsen, R.W.; Delgado-Escueta, A.V.

    2012-01-01

    Drug resistance remains to be one of the major challenges in epilepsy therapy. Identification of factors that contribute to therapeutic failure is crucial for future development of novel therapeutic strategies for difficult-to-treat epilepsies. Several clinical studies have shown that high seizure

  17. Knockdown of Laminin gamma-3 (Lamc3 impairs motoneuron guidance in the zebrafish embryo [version 1; referees: 2 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Alexander M. J. Eve

    2017-11-01

    Full Text Available Background: Previous work in the zebrafish embryo has shown that laminin γ-3 (lamc3 is enriched in endothelial cells marked by expression of fli1a, but the role of Lamc3 has been unknown. Methods: We use antisense morpholino oligonucleotides, and CRISPR/Cas9 mutagenesis of F0 embryos, to create zebrafish embryos in which lamc3 expression is compromised. Transgenic imaging, immunofluorescence, and in situ hybridisation reveal that Lamc3 loss-of-function affects the development of muscle pioneers, endothelial cells, and motoneurons. Results: Lamc3 is enriched in endothelial cells during zebrafish development, but it is also expressed by other tissues. Depletion of Lamc3 by use of antisense morpholino oligonucleotides perturbs formation of the parachordal chain and subsequently the thoracic duct, but Lamc3 is not required for sprouting of the cardinal vein. F0 embryos in which lamc3 expression is perturbed by a CRISPR/Cas9 approach also fail to form a parachordal chain, but we were unable to establish a stable lamc3 null line. Lamc3 is dispensable for muscle pioneer specification and for the expression of netrin-1a in these cells. Lamc3 knockdown causes netrin-1a up-regulation in the neural tube and there is increased Netrin-1 protein throughout the trunk of the embryo. Axonal guidance of rostral primary motoneurons is defective in Lamc3 knockdown embryos. Conclusions: We suggest that knockdown of Lamc3 perturbs migration of rostral primary motoneurons at the level of the horizontal myoseptum, indicating that laminin γ3 plays a role in motoneuron guidance.

  18. Virus-mediated shRNA knockdown of prodynorphin in the rat nucleus accumbens attenuates depression-like behavior and cocaine locomotor sensitization.

    Science.gov (United States)

    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.

  19. siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases.

    Science.gov (United States)

    Rebelo, Thalia M; Vania, Leila; Ferreira, Eloise; Weiss, Stefan F T

    2018-07-01

    The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis. Copyright © 2018. Published by Elsevier Inc.

  20. Virus-mediated shRNA knockdown of prodynorphin in the rat nucleus accumbens attenuates depression-like behavior and cocaine locomotor sensitization.

    Directory of Open Access Journals (Sweden)

    Ami Cohen

    Full Text Available 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.

  1. Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Hossain, Md. Motarab [Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC (United States); Banik, Naren L. [Department of Neurosciences, Medical University of South Carolina, Charleston, SC (United States); Ray, Swapan K., E-mail: swapan.ray@uscmed.sc.edu [Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC (United States)

    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

  2. Syndecan-1 knock-down in decidualized human endometrial stromal cells leads to significant changes in cytokine and angiogenic factor expression patterns

    Directory of Open Access Journals (Sweden)

    Krüssel Jan-Steffen

    2010-11-01

    Full Text Available Abstract Background Successful embryonic implantation depends on a synchronized embryo-maternal dialogue. Chemokines, such as chemokine ligand 1 (CXCL1, play essential roles in the maternal reproductive tract leading to morphological changes during decidualization, mediating maternal acceptance towards the semi-allograft embryo and induction of angiogenesis. Chemokine binding to their classical G-protein coupled receptors is essentially supported by the syndecan (Sdc family of heparan sulfate proteoglycans. The aim of this study was to identify the involvement of Sdc-1 at the embryo-maternal interface regarding changes of the chemokine and angiogenic profile of the decidua during the process of decidualization and implantation in human endometrium. Methods A stable Sdc-1 knock-down was generated in the immortalized human endometrial stromal cell line St-T1 and was named KdS1. The ability of KdS1 to decidualize was proven by Insulin-like growth factor binding 1 (IGFBP1 and prolactin (PRL confirmation on mRNA level before further experiments were carried out. Dot blot protein analyses of decidualized knock-down cells vs non-transfected controls were performed. In order to imitate embryonic implantation, decidualized KdS1 were then incubated with IL-1beta, an embryo secretion product, vs controls. Statistical analyses were performed applying the Student's t-test with p Results The induction of the Sdc-1 knock-down revealed significant changes in cytokine and angiogenic factor expression profiles of dKdS1 vs decidualized controls. Incubation with embryonic IL-1beta altered the expression patterns of KdS1 chemokines and angiogenic factors towards inflammatory-associated molecules and factors involved in matrix regulation. Conclusions Sdc-1 knock-down in human endometrial stroma cells led to fulminant changes regarding cytokine and angiogenic factor expression profiles upon decidualization and imitation of embryonic contact. Sdc-1 appears to play an

  3. Knock-Down of the IFR1 Protein Perturbs the Homeostasis of Reactive Electrophile Species and Boosts Photosynthetic Hydrogen Production in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Venkanna, Deepak; Südfeld, Christian; Baier, Thomas; Homburg, Sarah V; Patel, Anant V; Wobbe, Lutz; Kruse, Olaf

    2017-01-01

    The protein superfamily of short-chain dehydrogenases/reductases (SDR), including members of the atypical type (aSDR), covers a huge range of catalyzed reactions and in vivo substrates. This superfamily also comprises isoflavone reductase-like (IRL) proteins, which are aSDRs highly homologous to isoflavone reductases from leguminous plants. The molecular function of IRLs in non-leguminous plants and green microalgae has not been identified as yet, but several lines of evidence point at their implication in reactive oxygen species homeostasis. The Chlamydomonas reinhardtii IRL protein IFR1 was identified in a previous study, analyzing the transcriptomic changes occurring during the acclimation to sulfur deprivation and anaerobiosis, a condition that triggers photobiological hydrogen production in this microalgae. Accumulation of the cytosolic IFR1 protein is induced by sulfur limitation as well as by the exposure of C. reinhardtii cells to reactive electrophile species (RES) such as reactive carbonyls. The latter has not been described for IRL proteins before. Over-accumulation of IFR1 in the singlet oxygen response 1 ( sor1 ) mutant together with the presence of an electrophile response element, known to be required for SOR1-dependent gene activation as a response to RES, in the promoter of IFR1 , indicate that IFR1 expression is controlled by the SOR1-dependent pathway. An implication of IFR1 into RES homeostasis, is further implied by a knock-down of IFR1 , which results in a diminished tolerance toward RES. Intriguingly, IFR1 knock-down has a positive effect on photosystem II (PSII) stability under sulfur-deprived conditions used to trigger photobiological hydrogen production, by reducing PSII-dependent oxygen evolution, in C. reinhardtii . Reduced PSII photoinhibition in IFR1 knock-down strains prolongs the hydrogen production phase resulting in an almost doubled final hydrogen yield compared to the parental strain. Finally, IFR1 knock-down could be

  4. Cetuximab-Induced MET Activation Acts as a Novel Resistance Mechanism in Colon Cancer Cells

    Directory of Open Access Journals (Sweden)

    Na Song

    2014-04-01

    Full Text Available Aberrant MET expression and hepatocyte growth factor (HGF signaling are implicated in promoting resistance to targeted agents; however, the induced MET activation by epidermal growth factor receptor (EGFR inhibitors mediating resistance to targeted therapy remains elusive. In this study, we identified that cetuximab-induced MET activation contributed to cetuximab resistance in Caco-2 colon cancer cells. MET inhibition or knockdown sensitized Caco-2 cells to cetuximab-mediated growth inhibition. Additionally, SRC activation promoted cetuximab resistance by interacting with MET. Pretreatment with SRC inhibitors abolished cetuximab-mediated MET activation and rendered Caco-2 cells sensitive to cetuximab. Notably, cetuximab induced MET/SRC/EGFR complex formation. MET inhibitor or SRC inhibitor suppressed phosphorylation of MET and SRC in the complex, and MET inhibitor singly led to disruption of complex formation. These results implicate alternative targeting of MET or SRC as rational strategies for reversing cetuximab resistance in colon cancer.

  5. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... video) Animation of Antimicrobial Resistance (text version) Arabic Translation of Animation of Antimicrobial Resistance Chinese Translation of Animation of Antimicrobial Resistance French Translation of ...

  6. Neuron-specific knockdown of the Drosophila fat induces reduction of life span, deficient locomotive ability, shortening of motoneuron terminal branches and defects in axonal targeting.

    Science.gov (United States)

    Nakamura, Aya; Tanaka, Ryo; Morishita, Kazushige; Yoshida, Hideki; Higuchi, Yujiro; Takashima, Hiroshi; Yamaguchi, Masamitsu

    2017-07-01

    Mutations in FAT4 gene, one of the human FAT family genes, have been identified in Van Maldergem syndrome (VMS) and Hennekam lymphangiectasia-lymphedema syndrome (HS). The FAT4 gene encodes a large protein with extracellular cadherin repeats, EGF-like domains and Laminin G-like domains. FAT4 plays a role in tumor suppression and planar cell polarity. Drosophila contains a human FAT4 homologue, fat. Drosophila fat has been mainly studied with Drosophila eye and wing systems. Here, we specially knocked down Drosophila fat in nerve system. Neuron-specific knockdown of fat shortened the life span and induced the defect in locomotive abilities of adult flies. In consistent with these phenotypes, defects in synapse structure at neuromuscular junction were observed in neuron-specific fat-knockdown flies. In addition, aberrations in axonal targeting of photoreceptor neuron in third-instar larvae were also observed, suggesting that fat involves in axonal targeting. Taken together, the results indicate that Drosophila fat plays an essential role in formation and/or maintenance of neuron. Both VMS and HS show mental retardation and neuronal defects. We therefore consider that these two rare human diseases could possibly be caused by the defect in FAT4 function in neuronal cells. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  7. Als2 mRNA splicing variants detected in KO mice rescue severe motor dysfunction phenotype in Als2 knock-down zebrafish.

    Science.gov (United States)

    Gros-Louis, Francois; Kriz, Jasna; Kabashi, Edor; McDearmid, Jonathan; Millecamps, Stéphanie; Urushitani, Makoto; Lin, Li; Dion, Patrick; Zhu, Qinzhang; Drapeau, Pierre; Julien, Jean-Pierre; Rouleau, Guy A

    2008-09-01

    Recessive ALS2 mutations are linked to three related but slightly different neurodegenerative disorders: amyotrophic lateral sclerosis, hereditary spastic paraplegia and primary lateral sclerosis. To investigate the function of the ALS2 encoded protein, we generated Als2 knock-out (KO) mice and zAls2 knock-down zebrafish. The Als2(-/-) mice lacking exon 2 and part of exon 3 developed mild signs of neurodegeneration compatible with axonal transport deficiency. In contrast, zAls2 knock-down zebrafish had severe developmental abnormalities, swimming deficits and motor neuron perturbation. We identified, by RT-PCR, northern and western blotting novel Als2 transcripts in mouse central nervous system. These Als2 transcripts were present in Als2 null mice as well as in wild-type littermates and some rescued the zebrafish phenotype. Thus, we speculate that the newly identified Als2 mRNA species prevent the Als2 KO mice from developing severe neurodegenerative disease and might also regulate the severity of the motor neurons phenotype observed in ALS2 patients.

  8. Knockdown of long non-coding RNA HOTAIR increases miR-454-3p by targeting Stat3 and Atg12 to inhibit chondrosarcoma growth.

    Science.gov (United States)

    Bao, Xing; Ren, Tingting; Huang, Yi; Sun, Kunkun; Wang, Shidong; Liu, Kuisheng; Zheng, Bingxin; Guo, Wei

    2017-02-09

    Current practices for the therapy of chondrosarcoma, including wide-margin surgical resection and chemotherapy, are less than satisfactory. Recently, emerging evidence has demonstrated that long non-coding RNAs (lncRNAs) have an essential role in the initiation and progression of tumors. As a typical lncRNA, HOTAIR is significantly overexpressed in various tumors. However, the function and potential biological mechanisms of HOTAIR in human chondrosarcoma remain unknown. Quantitative RT-PCR demonstrated that HOTAIR expression was upregulated in chondrosarcoma tissues and cell lines. High HOTAIR expression is correlated with tumor stage and poor prognosis. Functional experiments reveal that HOTAIR knockdown leads to growth inhibition of human chondrosarcoma cells in vitro and in vivo. In addition to cycle arrest and apoptosis, knockdown of HOTAIR inhibits autophagy, which favors cell death. Mechanistically, we demonstrated that HOTAIR induced DNA methylation of miR-454-3p by recruiting EZH2 and DNMT1 to the miR-454-3p promoter regions, which markedly silences miR-454-3p expression. Further analysis revealed that STAT3 and ATG12 are targets of miR-454-3p, initiate HOTAIR deficiency-induced apoptosis and reduce autophagy. Collectively, our data reveal the roles and functional mechanisms of HOTAIR in human chondrosarcoma and suggest that HOTAIR may act as a prognostic biomarker and potential therapeutic target for chondrosarcoma.

  9. Epstein–Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus

    International Nuclear Information System (INIS)

    Changotra, Harish; Turk, Susan M.; Artigues, Antonio; Thakur, Nagendra; Gore, Mindy; Muggeridge, Martin I.; Hutt-Fletcher, Lindsey M.

    2016-01-01

    The Epstein–Barr virus glycoprotein complex gMgN has been implicated in assembly and release of fully enveloped virus, although the precise role that it plays has not been elucidated. We report here that the long predicted cytoplasmic tail of gM is not required for complex formation and that it interacts with the cellular protein p32, which has been reported to be involved in nuclear egress of human cytomegalovirus and herpes simplex virus. Although redistribution of p32 and colocalization with gM was not observed in virus infected cells, knockdown of p32 expression by siRNA or lentivirus-delivered shRNA recapitulated the phenotype of a virus lacking expression of gNgM. A proportion of virus released from cells sedimented with characteristics of virus lacking an intact envelope and there was an increase in virus trapped in nuclear condensed chromatin. The observations suggest the possibility that p32 may also be involved in nuclear egress of Epstein–Barr virus. - Highlights: • The predicted cytoplasmic tail of gM is not required to complex with gN. • Cellular p32 can interact with the predicted cytoplasmic tail of EBV gM. • Knockdown of p32 recapitulates the phenotype of virus lacking the gNgM complex.

  10. Knockdown of Uba2 inhibits colorectal cancer cell invasion and migration through downregulation of the Wnt/β-catenin signaling pathway.

    Science.gov (United States)

    Cheng, Hongjing; Sun, Xun; Li, Ji; He, Ping; Liu, Wanqi; Meng, Xiangwei

    2018-05-10

    Colorectal cancer is a serious threat to human health, and has a high mortality rate. There is currently no effective therapy for end-stage colorectal cancer. In recent years, molecular targeted therapy has received increasing attention for cancer treatment. In particular, the role of Uba2, a vital component of SUMO-activating enzyme, has been highlighted, which plays important roles in the progression of certain cancers; however, its role in colorectal cancer remains unclear. Accordingly, the aim of this study was to evaluate the relationship between Uba2 and colorectal cancer. Uba2 expression was knocked down in two colorectal cancer cell lines, and gene microarray analysis was conducted, followed by proliferation, migration, and invasion assays. Uba2 knockdown influenced the expression of several genes, and significantly inhibited the proliferation, migration, and invasion of cancer cells. To determine the underlying mechanism, the expression of related signaling pathways and molecules was evaluated in the knockdown cell lines. Overall, the results suggest that Uba2 participates in the progression, invasion, and metastasis of colorectal cancer, and the possible mechanism is via regulating the Wnt signaling pathway and enhancing epithelial-mesenchymal transition behaviors of colorectal cancer cells. Therefore, Uba2 is expected to be an important oncoprotein and potential therapeutic target in colorectal cancer. © 2018 Wiley Periodicals, Inc.

  11. Hypoxia-induced autophagy is inhibited by PADI4 knockdown, which promotes apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis

    Science.gov (United States)

    Fan, Tingting; Zhang, Changsong; Zong, Ming; Fan, Lieying

    2018-01-01

    Impaired apoptosis of rheumatoid arthritis (RA)-fibroblast-like synoviocytes (FLS) is pivotal in the process of RA. Peptidyl arginine deiminase type IV (PADI4) is associated with autoantibody regulation via histone citrullination in RA. The present study aimed to investigate the role of PADI4 in the apoptosis of RA-FLS. FLS were isolated from patients with RA and a rat model. The effects of PADI4 on RA-FLS were investigated in vitro and in vivo. Hypoxia-induced autophagy was induced by 1% O2 and was detected by immunohistochemical and immunofluorescence analysis; in addition, apoptosis was detected by flow cytometry. RA-FLS obtained from RA rat model exhibited significant proliferation under severe hypoxia conditions. Hypoxia also significantly induced autophagy and elevated the expression of PADI4. Subsequently, short hairpin RNA-mediated PADI4 knockdown was demonstrated to significantly inhibit hypoxia-induced autophagy and promote apoptosis in RA-FLS. The results of these in vitro and in vivo studies suggested that PADI4 may be closely associated with hypoxia-induced autophagy, and the inhibition of hypoxia-induced autophagy by PADI4 knockdown may contribute to an increase in the apoptosis of RA-FLS. PMID:29393388

  12. Knockdown of Indian hedgehog protein induces an inhibition of cell growth and differentiation in osteoblast MC3T3-E1 cells

    Science.gov (United States)

    Deng, Ang; Zhang, Hongqi; Hu, Minyu; Liu, Shaohua; Gao, Qile; Wang, Yuxiang; Guo, Chaofeng

    2017-01-01

    Indian hedgehog protein (Ihh) is evolutionarily conserved and serves important roles in controlling the differentiation of progenitor cells into osteoblasts. Ihh null mutant mice exhibit a failure of osteoblast development in endochondral bone. Although studies have demonstrated that Ihh signaling is a potent local factor that regulates osteoblast differentiation, the specific transcription factors that determine osteoblast differentiation remain unclear. Further studies are required to determine the precise mechanism through which Ihh regulates osteoblast differentiation. In the present study, Ihh was knocked down in osteoblast MC3T3-E1 cells using short hairpin RNA, to investigate the function of Ihh in osteoblast proliferation and differentiation and to examine the potential mechanism through which Ihh induces osteoblast apoptosis and cell cycle arrest. It was observed that the knockdown of Ihh induced a marked inhibition of cell growth and increased the apoptosis rate compared with the negative control osteoblasts. Downregulation of Ihh resulted in a cell cycle arrest at the G1 to S phase boundary in osteoblasts. In addition, the knockdown of Ihh decreased the alkaline phosphatase activity and mineral deposition of osteoblasts. The inhibitory roles of Ihh downregulation in osteoblast growth and differentiation may be associated with the transforming growth factor-β/mothers against decapentaplegic homolog and tumor necrosis factor receptor superfamily member 11B/tumor necrosis factor ligand superfamily member 11 signaling pathways. Manipulating either Ihh expression or its signaling components may be of benefit for the treatment of skeletal diseases. PMID:28990069

  13. Knockdown of Indian hedgehog protein induces an inhibition of cell growth and differentiation in osteoblast MC3T3‑E1 cells.

    Science.gov (United States)

    Deng, Ang; Zhang, Hongqi; Hu, Minyu; Liu, Shaohua; Gao, Qile; Wang, Yuxiang; Guo, Chaofeng

    2017-12-01

    Indian hedgehog protein (Ihh) is evolutionarily conserved and serves important roles in controlling the differentiation of progenitor cells into osteoblasts. Ihh null mutant mice exhibit a failure of osteoblast development in endochondral bone. Although studies have demonstrated that Ihh signaling is a potent local factor that regulates osteoblast differentiation, the specific transcription factors that determine osteoblast differentiation remain unclear. Further studies are required to determine the precise mechanism through which Ihh regulates osteoblast differentiation. In the present study, Ihh was knocked down in osteoblast MC3T3‑E1 cells using short hairpin RNA, to investigate the function of Ihh in osteoblast proliferation and differentiation and to examine the potential mechanism through which Ihh induces osteoblast apoptosis and cell cycle arrest. It was observed that the knockdown of Ihh induced a marked inhibition of cell growth and increased the apoptosis rate compared with the negative control osteoblasts. Downregulation of Ihh resulted in a cell cycle arrest at the G1 to S phase boundary in osteoblasts. In addition, the knockdown of Ihh decreased the alkaline phosphatase activity and mineral deposition of osteoblasts. The inhibitory roles of Ihh downregulation in osteoblast growth and differentiation may be associated with the transforming growth factor‑β/mothers against decapentaplegic homolog and tumor necrosis factor receptor superfamily member 11B/tumor necrosis factor ligand superfamily member 11 signaling pathways. Manipulating either Ihh expression or its signaling components may be of benefit for the treatment of skeletal diseases.

  14. CREBBP knockdown enhances RAS/RAF/MEK/ERK signaling in Ras pathway mutated acute lymphoblastic leukemia but does not modulate chemotherapeutic response.

    Science.gov (United States)

    Dixon, Zach A; Nicholson, Lindsay; Zeppetzauer, Martin; Matheson, Elizabeth; Sinclair, Paul; Harrison, Christine J; Irving, Julie A E

    2017-04-01

    Relapsed acute lymphoblastic leukemia is the most common cause of cancer-related mortality in young people and new therapeutic strategies are needed to improve outcome. Recent studies have shown that heterozygous inactivating mutations in the histone acetyl transferase, CREBBP , are particularly frequent in relapsed childhood acute lymphoblastic leukemia and associated with a hyperdiploid karyotype and KRAS mutations. To study the functional impact of CREBBP haploinsufficiency in acute lymphoblastic leukemia, RNA interference was used to knock down expression of CREBBP in acute lymphoblastic leukemia cell lines and various primagraft acute lymphoblastic leukemia cells. We demonstrate that attenuation of CREBBP results in reduced acetylation of histone 3 lysine 18, but has no significant impact on cAMP-dependent target gene expression. Impaired induction of glucocorticoid receptor targets was only seen in 1 of 4 CREBBP knockdown models, and there was no significant difference in glucocorticoid-induced apoptosis, sensitivity to other acute lymphoblastic leukemia chemotherapeutics or histone deacetylase inhibitors. Importantly, we show that CREBBP directly acetylates KRAS and that CREBBP knockdown enhances signaling of the RAS/RAF/MEK/ERK pathway in Ras pathway mutated acute lymphoblastic leukemia cells, which are still sensitive to MEK inhibitors. Thus, CREBBP mutations might assist in enhancing oncogenic RAS signaling in acute lymphoblastic leukemia but do not alter response to MEK inhibitors. Copyright© Ferrata Storti Foundation.

  15. In vivo knockdown of antisense non-coding mitochondrial RNAs by a lentiviral-encoded shRNA inhibits melanoma tumor growth and lung colonization.

    Science.gov (United States)

    Varas-Godoy, Manuel; Lladser, Alvaro; Farfan, Nicole; Villota, Claudio; Villegas, Jaime; Tapia, Julio C; Burzio, Luis O; Burzio, Veronica A; Valenzuela, Pablo D T

    2018-01-01

    The family of non-coding mitochondrial RNAs (ncmtRNA) is differentially expressed according to proliferative status. Normal proliferating cells express sense (SncmtRNA) and antisense ncmtRNAs (ASncmtRNAs), whereas tumor cells express SncmtRNA and downregulate ASncmtRNAs. Knockdown of ASncmtRNAs with oligonucleotides induces apoptotic cell death of tumor cells, leaving normal cells unaffected, suggesting a potential application for developing a novel cancer therapy. In this study, we knocked down the ASncmtRNAs in melanoma cell lines with a lentiviral-encoded shRNA approach. Transduction with lentiviral constructs targeted to the ASncmtRNAs induced apoptosis in murine B16F10 and human A375 melanoma cells in vitro and significantly retarded B16F10 primary tumor growth in vivo. Moreover, the treatment drastically reduced the number of lung metastatic foci in a tail vein injection assay, compared to controls. These results provide additional proof of concept to the knockdown of ncmtRNAs for cancer therapy and validate lentiviral-shRNA vectors for gene therapy. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. FLASH knockdown sensitizes cells to Fas-mediated apoptosis via down-regulation of the anti-apoptotic proteins, MCL-1 and Cflip short.

    Directory of Open Access Journals (Sweden)

    Song Chen

    Full Text Available FLASH (FLICE-associated huge protein or CASP8AP2 is a large multifunctional protein that is involved in many cellular processes associated with cell death and survival. It has been reported to promote apoptosis, but we show here that depletion of FLASH in HT1080 cells by siRNA interference can also accelerate the process. As shown previously, depletion of FLASH halts growth by down-regulating histone biosynthesis and arrests the cell cycle in S-phase. FLASH knockdown followed by stimulating the cells with Fas ligand or anti-Fas antibodies was found to be associated with a more rapid cleavage of PARP, accelerated activation of caspase-8 and the executioner caspase-3 and rapid progression to cellular disintegration. As is the case for most anti-apoptotic proteins, FLASH was degraded soon after the onset of apoptosis. Depletion of FLASH also resulted in the reduced intracellular levels of the anti-apoptotic proteins, MCL-1 and the short isoform of cFLIP. FLASH knockdown in HT1080 mutant cells defective in p53 did not significantly accelerate Fas mediated apoptosis indicating that the effect was dependent on functional p53. Collectively, these results suggest that under some circumstances, FLASH suppresses apoptosis.

  17. Neuromedin U receptor 2 knockdown in the paraventricular nucleus modifies behavioral responses to obesogenic high-fat food and leads to increased body weight.

    Science.gov (United States)

    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. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  18. Knockdown of long non-coding RNA MAP3K20 antisense RNA 1 inhibits gastric cancer growth through epigenetically regulating miR-375.

    Science.gov (United States)

    Quan, Yongsheng; Zhang, Yan; Lin, Wei; Shen, Zhaohua; Wu, Shuai; Zhu, Changxin; Wang, Xiaoyan

    2018-03-04

    Emerging evidence has demonstrated that long noncoding RNAs (lncRNAs) play a critical role in tumorigenesis of gastric cancer. LncRNA MAP3K20 antisense RNA 1 (MLK7-AS1) has been identified as one of gastric cancer-specific lncRNAs. However, its precise role in gastric cancer remains unknown. In this study, we found that lncRNA MLK7-AS1 was significantly increased in gastric cancer tissues compared with in adjacent tissues. Gastric cancer patients with high MLK7-AS1 expression had a shorter survival and poorer prognosis. By loss-function assay, we demonstrated that knockdown of MLK7-AS1 inhibited cell proliferation and induced apoptosis in HGC27and MKN-45 cells. Furthermore, we identified miR-375 as a target of MLK7-AS1. MLK7-AS1 interacted with Dnmt1 and recruited it to miR-375 promotor, hyper-methylating miR-375 promotor and repressing miR-375 expression. Taken together, our findings demonstrate that knockdown of MLK7-AS1 by siRNA inhibits gastric cancer growth by epigenetically regulating miR-375. Thus, MLK7-AS1 may be a useful prognostic marker and therapeutic target for gastric cancer patients. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Epstein–Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus

    Energy Technology Data Exchange (ETDEWEB)

    Changotra, Harish; Turk, Susan M. [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA (United States); Artigues, Antonio [Department of Biochemistry, University of Kansas Medical Center, Kansas City, KS (United States); Thakur, Nagendra; Gore, Mindy; Muggeridge, Martin I. [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA (United States); Hutt-Fletcher, Lindsey M., E-mail: lhuttf@lsuhsc.edu [Department of Microbiology and Immunology, Center for Molecular and Tumor Virology and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA (United States)

    2016-02-15

    The Epstein–Barr virus glycoprotein complex gMgN has been implicated in assembly and release of fully enveloped virus, although the precise role that it plays has not been elucidated. We report here that the long predicted cytoplasmic tail of gM is not required for complex formation and that it interacts with the cellular protein p32, which has been reported to be involved in nuclear egress of human cytomegalovirus and herpes simplex virus. Although redistribution of p32 and colocalization with gM was not observed in virus infected cells, knockdown of p32 expression by siRNA or lentivirus-delivered shRNA recapitulated the phenotype of a virus lacking expression of gNgM. A proportion of virus released from cells sedimented with characteristics of virus lacking an intact envelope and there was an increase in virus trapped in nuclear condensed chromatin. The observations suggest the possibility that p32 may also be involved in nuclear egress of Epstein–Barr virus. - Highlights: • The predicted cytoplasmic tail of gM is not required to complex with gN. • Cellular p32 can interact with the predicted cytoplasmic tail of EBV gM. • Knockdown of p32 recapitulates the phenotype of virus lacking the gNgM complex.

  20. Loss of ABCB4 attenuates the caspase-dependent apoptosis regulating resistance to 5-Fu in colorectal cancer.

    Science.gov (United States)

    Hu, Hanqing; Wang, Meng; Guan, Xu; Yuan, Ziming; Liu, Zheng; Zou, Chaoxia; Wang, Guiyu; Gao, Xu; Wang, Xishan

    2018-02-28

    The adenosine triphosphate-binding cassette (ABC) is a large group of proteins involved in material transportation, cellular homeostasis, and closely associated with chemoresistance. ATP-binding cassette protein B4 (ABCB4) is a member of ABCs which has a similar structure to ABCB1, but fewer researches were performed. The present study is aimed to investigate the putative mechanism of ABCB4 in 5-fluorouracil (5-Fu) resistance. Then, we found that ABCB4 was significantly down-regulated in the 5-Fu resistant HCT8 cell lines by polymerase chain reaction (PCR) and Western blot. The knockdown of ABCB4 by small interfering RNA decreased the apoptosis by 5-Fu in resistant HCT8R cell lines without influencing the proliferation. Also, we found a lower expression of cleaved caspase and PARP by Western blot after the knockdown of ABCB4. However, the knockdown of ABCB4 did not influence the proliferation and apoptosis. Furthermore, the histological detection of ABCB4 mRNA level in human colorectal cancer tissues and even in the recurrent tissues after 5-Fu single-agent chemotherapy was employed to provide more concrete evidence that ABCB4 may be a tumor suppressor gene to regulate chemoresistance in colorectal cancer. Moreover, a 109-patient cohort revealed that ABCB4 predicted a poor recurrence-free survival and overall survival. In summary, ABCB4 was down-regulated in the 5-Fu resistant cells and knockdown of ABCB4 alleviated the cell apoptosis and predicts a shorter recurrence-free survival and overall survival. © 2018 The Author(s).

  1. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... Animal & Veterinary Safety & Health Antimicrobial Resistance Animation of Antimicrobial Resistance Share Tweet Linkedin Pin it More sharing options ... of Animation of Antimicrobial Resistance More in Antimicrobial ... Antimicrobial Resistance Monitoring System About NARMS 2015 NARMS Integrated ...

  2. Insulin Resistance

    DEFF Research Database (Denmark)

    Jensen, Benjamin Anderschou Holbech

    Insulin resistance (IR) is escalating with alarming pace and is no longer restricted to westernized countries. As a forerunner for some of the most serious threats to human health including metabolic syndrome, cardiovascular diseases, and type 2-diabetes, the need for new treatment modalities...... interventions. We further show that improving the inflammatory toning, using fish oil as fat source, protects mice against diet induced obesity and -inflammation while preserving insulin sensitivity, even in the absence of free fatty acid receptor 4. Conversely, HFD-induced intestinal dysbiosis is associated...

  3. Knockdown of the dyslexia-associated gene Kiaa0319 impairs temporal responses to speech stimuli in rat primary auditory cortex.

    Science.gov (United States)

    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

    2014-07-01

    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. © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. Ubiquitin ligase RNF123 mediates degradation of heterochromatin protein 1α and β in lamin A/C knock-down cells.

    Directory of Open Access Journals (Sweden)

    Pankaj Chaturvedi

    Full Text Available The nuclear lamina is a key determinant of nuclear architecture, integrity and functionality in metazoan nuclei. Mutations in the human lamin A gene lead to highly debilitating genetic diseases termed as laminopathies. Expression of lamin A mutations or reduction in levels of endogenous A-type lamins leads to nuclear defects such as abnormal nuclear morphology and disorganization of heterochromatin. This is accompanied by increased proteasomal degradation of certain nuclear proteins such as emerin, nesprin-1α, retinoblastoma protein and heterochromatin protein 1 (HP1. However, the pathways of proteasomal degradation have not been well characterized.To investigate the mechanisms underlying the degradation of HP1 proteins upon lamin misexpression, we analyzed the effects of shRNA-mediated knock-down of lamins A and C in HeLa cells. Cells with reduced levels of expression of lamins A and C exhibited proteasomal degradation of HP1α and HP1β but not HP1γ. Since specific ubiquitin ligases are upregulated in lamin A/C knock-down cells, further studies were carried out with one of these ligases, RNF123, which has a putative HP1-binding motif. Ectopic expression of GFP-tagged RNF123 directly resulted in degradation of HP1α and HP1β. Mutational analysis showed that the canonical HP1-binding pentapeptide motif PXVXL in the N-terminus of RNF123 was required for binding to HP1 proteins and targeting them for degradation. The role of endogenous RNF123 in the degradation of HP1 isoforms was confirmed by RNF123 RNAi experiments. Furthermore, FRAP analysis suggested that HP1β was displaced from chromatin in laminopathic cells.Our data support a role for RNF123 ubiquitin ligase in the degradation of HP1α and HP1β upon lamin A/C knock-down. Hence lamin misexpression can cause degradation of mislocalized proteins involved in key nuclear processes by induction of specific components of the ubiquitin-proteasome system.

  5. Knockdown of AMPKα decreases ATM expression and increases radiosensitivity under hypoxia and nutrient starvation in an SV40-transformed human fibroblast cell line, LM217.

    Science.gov (United States)

    Murata, Yasuhiko; Hashimoto, Takuma; Urushihara, Yusuke; Shiga, Soichiro; Takeda, Kazuya; Jingu, Keiichi; Hosoi, Yoshio

    2018-01-22

    Presence of unperfused regions containing cells under hypoxia and nutrient starvation contributes to radioresistance in solid human tumors. It is well known that hypoxia causes cellular radioresistance, but little is known about the effects of nutrient starvation on radiosensitivity. We have reported that nutrient starvation induced decrease of mTORC1 activity and decrease of radiosensitivity in an SV40-transformed human fibroblast cell line, LM217, and that nutrient starvation induced increase of mTORC1 activity and increase of radiosensitivity in human liver cancer cell lines, HepG2 and HuH6 (Murata et al., BBRC 2015). Knockdown of mTOR using small interfering RNA (siRNA) for mTOR suppressed radiosensitivity under nutrient starvation alone in HepG2 cells, which suggests that mTORC1 pathway regulates radiosensitivity under nutrient starvation alone. In the present study, effects of hypoxia and nutrient starvation on radiosensitivity were investigated using the same cell lines. LM217 and HepG2 cells were used to examine the effects of hypoxia and nutrient starvation on cellular radiosensitivity, mTORC1 pathway including AMPK, ATM, and HIF-1α, which are known as regulators of mTORC1 activity, and glycogen storage, which is induced by HIF-1 and HIF-2 under hypoxia and promotes cell survival. Under hypoxia and nutrient starvation, AMPK activity and ATM expression were increased in LM217 cells and decreased in HepG2 cells compared with AMPK activity under nutrient starvation alone or ATM expression under hypoxia alone. Under hypoxia and nutrient starvation, radiosensitivity was decreased in LM217 cells and increased in HepG2 cells compared with radiosensitivity under hypoxia alone. Under hypoxia and nutrient starvation, knockdown of AMPK decreased ATM activity and increased radiation sensitivity in LM217 cells. In both cell lines, mTORC1 activity was decreased under hypoxia and nutrient starvation. Under hypoxia alone, knockdown of mTOR slightly increased ATM

  6. Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells.

    Science.gov (United States)

    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-κB), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro

  7. MicroRNA-181a promotes docetaxel resistance in prostate cancer cells.

    Science.gov (United States)

    Armstrong, Cameron M; Liu, Chengfei; Lou, Wei; Lombard, Alan P; Evans, Christopher P; Gao, Allen C

    2017-06-01

    Docetaxel is one of the primary drugs used for treating castration resistant prostate cancer (CRPC). Unfortunately, over time patients invariably develop resistance to docetaxel therapy and their disease will continue to progress. The mechanisms by which resistance develops are still incompletely understood. This study seeks to determine the involvement of miRNAs, specifically miR-181a, in docetaxel resistance in CRPC. Real-time PCR was used to measure miR-181a expression in parental and docetaxel resistant C4-2B and DU145 cells (TaxR and DU145-DTXR). miR-181a expression was modulated in parental or docetaxel resistant cells by transfecting them with miR-181a mimics or antisense, respectively. Following transfection, cell number was determined after 48 h with or without docetaxel. Cross resistance to cabazitaxel induced by miR-181a was also determined. Western blots were used to determine ABCB1 protein expression and rhodamine assays used to assess activity. Phospho-p53 expression was assessed by Western blot and apoptosis was measured by ELISA in C4-2B TaxR and PC3 cells with inhibited or overexpressed miR-181a expression with or without docetaxel. miR-181a is significantly overexpressed in TaxR and DU145-DTXR cells compared to parental cells. Overexpression of miR-181a in parental cells confers docetaxel and cabazitaxel resistance and knockdown of miR-181a in TaxR cells re-sensitizes them to treatment with both docetaxel and cabazitaxel. miR-181a was not observed to impact ABCB1 expression or activity, a protein which was previously demonstrated to be highly involved in docetaxel resistance. Knockdown of miR-181a in TaxR cells induced phospho-p53 expression. Furthermore, miR-181a knockdown alone induced apoptosis in TaxR cells which could be further enhanced by the addition of DTX. Overexpression of mir-181a in prostate cancer cells contributes to their resistance to docetaxel and cabazitaxel and inhibition of mir-181a expression can restore treatment response

  8. Aedes aegypti resistance development to commonly used insecticides in Jakarta, Indonesia.

    Directory of Open Access Journals (Sweden)

    Penny Humaidah Hamid

    Full Text Available Aedes aegypti is the primary vector of various relevant arthropod-borne viral infectious diseases worldwide. The mosquito control is still mainly performed by using insecticides but their effectiveness is increasingly questioned nowadays. We here conducted a study on Ae. aegypti resistance development towards several commonly used insecticides in the capital city of Jakarta, Indonesia. In order to achieve this goal, Ae. aegypti eggs from Jakarta were collected with ovitraps and hatched in the insectary of the Gadjah Mada University, Indonesia. The F0 generations were used for WHO resistance tests and knockdown resistance (kdr assays. Presented results clearly showed that there was resistance development of Ae. aegypti populations to the here tested pyrethroid insecticides (i. e. permethrin. Observed mortalities were less than 90% with highest resistance against 0.75% permethrin concentrations. Furthermore, a significant association of V1016G gene mutations with resistance phenotypes to 0.75% permethrin was observed. Nevertheless, the F1534C mutation did not show a significant correlation to resistance development. In conclusion, our results show that populations of Ae. aegypti mosquitoes within the city of Jakarta have developed resistance against several routinely used pyrethroid insecticides in local performed control programs. Thus, the regular verification/assessment of resistance development status will hopefully help in the future to assist local public health authorities in their mosquito control programs by recommending and managing the rotation of different routinely used insecticides with diverse effector mechanisms in order to delay Ae. aegypti resistance development.

  9. Knockdown of Long Noncoding RNA TUG1 Inhibits the Proliferation and Cellular Invasion of Osteosarcoma Cells By Sponging MiR-153.

    Science.gov (United States)

    Wang, Heping; Yu, Yanzhang; Fan, Shuxin; Luo, Leifeng

    2017-04-12

    Long noncoding RNA (lncRNA) Taurine-upregulated gene 1 (TUG1) has been confirmed to be involved in the progression of various cancers, however, its mechanism of action in osteosarcoma has not been well addressed. In our study, TUG1 was overexpressed and miR-153 was downregulated in osteosarcoma tissues and cell lines. Loss-of-function assay showed that TUG1 knockdown suppressed the viability, colony formation, and invasion of osteosarcoma cells in vitro. Moreover, TUG1 was confirmed to be a miR-153 sponge. Ectopic expression of TUG1 reversed the inhibitory effect of miR-153 on the proliferation and invasion of osteosarcoma cells. Further transplantation experiment proved the carcinogenesis of TUG1 in osteosarcoma in vivo. Collectively, our study elucidated that TUG1 contributed to the osteosarcoma development by sponging miR-153. These findings may provide a novel lncRNA-targeted therapy for patients with osteosarcoma.

  10. Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

    Science.gov (United States)

    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.

  11. Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells.

    Science.gov (United States)

    Lee, Su Jeong; Park, Jeen-Woo

    2014-04-01

    Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocytes. This effect manifested as DNA fragmentation, changes in cellular redox status, mitochondrial dysfunction, and modulation of apoptotic marker expression. Based on our findings, we suggest that attenuation of IDPc expression may protect skin from UVB-mediated damage, by inducing the apoptosis of UV-damaged cells.

  12. Resistance welding

    DEFF Research Database (Denmark)

    Bay, Niels; Zhang, Wenqi; Rasmussen, Mogens H.

    2003-01-01

    Resistance welding comprises not only the well known spot welding process but also more complex projection welding operations, where excessive plastic deformation of the weld point may occur. This enables the production of complex geometries and material combinations, which are often not possible...... to weld by traditional spot welding operations. Such joining processes are, however, not simple to develop due to the large number of parameters involved. Development has traditionally been carried out by large experimental investigations, but the development of a numerical programme system has changed...... this enabling prediction of the welding performance in details. The paper describes the programme in short and gives examples on industrial applications. Finally investigations of causes for failure in a complex industrial joint of two dissimilar metals are carried out combining numerical modelling...

  13. Antibiotic Resistance

    DEFF Research Database (Denmark)

    Hansen, Malene Plejdrup; Hoffmann, Tammy C; McCullough, Amanda R

    2015-01-01

    hygiene, and possibly vaccination and exercise, may be effective. Also, a large range of complementary and alternative medicines (e.g. zinc, vitamin C and probiotics) are proposed for preventing and treating ARIs, but evidence for efficacy is scarce. General practitioners' (GPs) attitudes towards...... wrong. Shared decision making might be a solution, as it enables clinician and patient to participate jointly in making a health decision, having discussed the options together with the evidence for their harms as well as benefits. Furthermore, GPs' diagnostic uncertainty - often leading...... will greatly improve the use of antibiotics for ARIs. However, used in concert, combinations are likely to enable clinicians and health care systems to implement the strategies that will reduce antimicrobial resistance in the future....

  14. Resisting dehumanization

    DEFF Research Database (Denmark)

    Lassen, Inger Marie

    2018-01-01

    Recent years have seen an increase in the influx of asylum-seekers in Scandinavia, and in Denmark this has led to ever-tighter immigration control. This article discusses emerging practices of refugee solidarity and resistance to hegemonic migration policy in Danish civil society in the wake...... of what has been referred to as the European refugee crisis. It further reflects upon what it means to be a Danish citizen facing the dilemma of law-abiding conduct versus human decency, in line with what Foucault (1983) referred to as ‘ethical self-formation’. This is illustrated through a case study...... of an incident from September 2015, when a member of a Danish City Council offered private shelter to immigrants who were on their way to Norway. The incident led to legal proceedings in August 2016 for what the defendant referred to as ‘the offense of helping fellow human beings in need’. The study is informed...

  15. Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

    Science.gov (United States)

    Li, Ting; Liu, Nannan

    2017-12-01

    This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.

  16. Knockdown of NF-E2-related factor 2 inhibits the proliferation and growth of U251MG human glioma cells in a mouse xenograft model.

    Science.gov (United States)

    Ji, Xiang-Jun; Chen, Sui-Hua; Zhu, Lin; Pan, Hao; Zhou, Yuan; Li, Wei; You, Wan-Chun; Gao, Chao-Chao; Zhu, Jian-Hong; Jiang, Kuan; Wang, Han-Dong

    2013-07-01

    NF-E2-related factor 2 (Nrf2) is a pivotal transcription factor of cellular responses to oxidative stress and recent evidence suggests that Nrf2 plays an important role in cancer pathobiology. However, the underlying mechanism has yet to be elucidated, particularly in glioma. In the present study, we investigated the role of Nrf2 in the clinical prognosis, cell proliferation and tumor growth of human glioblastoma multiforme (GBM). We detected overexpression of Nrf2 protein levels in GBM compared to normal brain tissues. Notably, higher protein levels of Nrf2 were significantly associated with poorer overall survival and 1-year survival for GBM patients. Furthermore, we constructed the plasmid Si-Nrf2 and transduced it into U251MG cells to downregulate the expression of Nrf2 and established stable Nrf2 knockdown cells. The downregulation of Nrf2 suppressed cell proliferation in vitro and tumor growth in mouse xenograft models. We performed immunohistochemistry staining to detect the protein levels of Nrf2, Ki-67, caspase-3 and CD31 in the xenograft tumors and found that the expression levels of Nrf2 and Ki-67 were much lower in the Si-Nrf2 group compared to the Si-control group. In addition, the number of caspase-3-positive cells was significantly increased in the Si-Nrf2 group. By analysis of microvessel density (MVD) assessed by CD31, the MVD value in the Si-Nrf2 group decreased significantly compared to the Si-control group. These findings indicate that the knockdown of Nrf2 may suppress tumor growth by inhibiting cell proliferation, increasing cell apoptosis and inhibiting angiogenesis. These results highlight the potential of Nrf2 as a candidate molecular target to control GBM cell proliferation and tumor growth.

  17. Knockdown of menin affects pre-mRNA processing and promoter fidelity at the interferon-gamma inducible IRF1 gene

    Directory of Open Access Journals (Sweden)

    Auriemma Lauren B

    2012-01-01

    Full Text Available Abstract Background The tumor suppressor menin (MEN1 is mutated in the inherited disease multiple endocrine neoplasia type I, and has several documented cellular roles, including the activation and repression of transcription effected by several transcription factors. As an activator, MEN1 is a component of the Set1-like mixed lineage leukemia (MLL MLL1/MLL2 methyltransferase complex that methylates histone H3 lysine 4 (H3K4. MEN1 is localized to the signal transducer and activator of transcription 1 (STAT1-dependent gene, interferon regulatory factor 1 (IRF1, and is further recruited when IRF1 transcription is triggered by interferon-γ signaling. Results RNAi-mediated knockdown of MEN1 alters the H3K4 dimethylation and H3 acetylation profiles, and the localization of histone deacetylase 3, at IRF1. While MEN1 knockdown does not impact the rate of transcription, IRF1 heteronuclear transcripts become enriched in MEN1-depleted cells. The processed mRNA and translated protein product are concomitantly reduced, and the antiviral state is attenuated. Additionally, the transcription start site at the IRF1 promoter is disrupted in the MEN1-depleted cells. The H3K4 demethylase, lysine specific demethylase 1, is also associated with IRF1, and its inhibition alters H3K4 methylation and disrupts the transcription start site as well. Conclusions Taken together, the data indicate that MEN1 contributes to STAT1-activated gene expression in a novel manner that includes defining the transcription start site and RNA processing.

  18. Variable laterality of corticospinal tract axons that regenerate after spinal cord injury as a result of PTEN deletion or knock-down

    Science.gov (United States)

    Willenberg, Rafer; Zukor, Katherine; Liu, Kai; He, Zhigang; Steward, Oswald

    2016-01-01

    Corticospinal tract (CST) axons from one hemisphere normally extend and terminate predominantly in the contralateral spinal cord. We previously showed that deleting PTEN in the sensorimotor cortex enables CST axons to regenerate after spinal cord injury and that some regenerating axons extend along the “wrong” side. Here, we characterize the degree of specificity of regrowth in terms of laterality. PTEN was selectively deleted via cortical AAV-Cre injections in neonatal PTEN-floxed mice. As adults, mice received dorsal hemisection injuries at T12 or complete crush injuries at T9. CST axons from one hemisphere were traced by unilateral BDA injections in PTEN-deleted mice with spinal cord injury and in non-injured PTEN-floxed mice that had not received AAV-Cre. In non-injured mice, 97.9 ± 0.7% of BDA-labeled axons in white matter and 88.5 ± 1.0% of BDA-labeled axons in grey matter were contralateral to the cortex of origin. In contrast, laterality of CST axons that extended past a lesion due to PTEN deletion varied across animals. In some cases, regenerated axons extended predominantly on the ipsilateral side, in other cases, axons extended predominantly contralaterally, and in others, axons were similar in numbers on both sides. Similar results were seen in analyses of cases from previous studies using shRNA-mediated PTEN knock-down. These results indicate that CST axons that extend past a lesion due to PTEN deletion or knock-down do not maintain the contralateral rule of the non-injured CST, highlighting one aspect for how resultant circuitry from regenerating axons may differ from that of the uninjured CST. PMID:26878190

  19. RNAi knock-down of LHCBM1, 2 and 3 increases photosynthetic H2 production efficiency of the green alga Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Melanie Oey

    Full Text Available Single cell green algae (microalgae are rapidly emerging as a platform for the production of sustainable fuels. Solar-driven H2 production from H2O theoretically provides the highest-efficiency route to fuel production in microalgae. This is because the H2-producing hydrogenase (HYDA is directly coupled to the photosynthetic electron transport chain, thereby eliminating downstream energetic losses associated with the synthesis of carbohydrate and oils (feedstocks for methane, ethanol and oil-based fuels. Here we report the simultaneous knock-down of three light-harvesting complex proteins (LHCMB1, 2 and 3 in the high H2-producing Chlamydomonas reinhardtii mutant Stm6Glc4 using an RNAi triple knock-down strategy. The resultant Stm6Glc4L01 mutant exhibited a light green phenotype, reduced expression of LHCBM1 (20.6% ±0.27%, LHCBM2 (81.2% ±0.037% and LHCBM3 (41.4% ±0.05% compared to 100% control levels, and improved light to H2 (180% and biomass (165% conversion efficiencies. The improved H2 production efficiency was achieved at increased solar flux densities (450 instead of ∼100 µE m(-2 s(-1 and high cell densities which are best suited for microalgae production as light is ideally the limiting factor. Our data suggests that the overall improved photon-to-H2 conversion efficiency is due to: 1 reduced loss of absorbed energy by non-photochemical quenching (fluorescence and heat losses near the photobioreactor surface; 2 improved light distribution in the reactor; 3 reduced photoinhibition; 4 early onset of HYDA expression and 5 reduction of O2-induced inhibition of HYDA. The Stm6Glc4L01 phenotype therefore provides important insights for the development of high-efficiency photobiological H2 production systems.

  20. Contrasting Inducible Knockdown of the Auxiliary PTEX Component PTEX88 in P. falciparum and P. berghei Unmasks a Role in Parasite Virulence.

    Directory of Open Access Journals (Sweden)

    Scott A Chisholm

    Full Text Available 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.

  1. SirT1 knockdown potentiates radiation-induced bystander effect through promoting c-Myc activity and thus facilitating ROS accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yuexia [Institute of Radiation Medicine, Fudan University, Shanghai (China); Central Laboratory, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai (China); Tu, Wenzhi; Zhang, Jianghong; He, Mingyuan; Ye, Shuang; Dong, Chen [Institute of Radiation Medicine, Fudan University, Shanghai (China); Shao, Chunlin, E-mail: clshao@shmu.edu.cn [Institute of Radiation Medicine, Fudan University, Shanghai (China)

    2015-02-15

    Highlights: • γ-Irradiation induced bystander effects between hepatoma cells and hepatocyte cells. • SirT1 played a protective role in regulating this bystander effect. • SirT1 contributed to the protective effects via elimination the accumulation of ROS. • The activity of c-Myc is critical for maintaining the protective role of SirT1. - Abstract: Radiation-induced bystander effect (RIBE) has important implications for secondary cancer risk assessment during cancer radiotherapy, but the bystander signaling processes, especially under hypoxic condition, are still largely unclear. The present study found that micronuclei (MN) formation could be induced in the non-irradiated HL-7702 hepatocyte cells after being treated with the conditioned medium from irradiated hepatoma HepG2 and SK-Hep-1 cells under either normoxia or hypoxia. This bystander response was dramatically diminished or enhanced when the SirT1 gene of irradiated hepatoma cells was overexpressed or knocked down, respectively, especially under hypoxia. Meanwhile, SirT1 knockdown promoted transcriptional activity for c-Myc and facilitated ROS accumulation. But both of the increased bystander responses and ROS generation due to SirT1-knockdown were almost completely suppressed by c-Myc interference. Moreover, ROS scavenger effectively abolished the RIBE triggered by irradiated hepatoma cells even with SirT1 depletion. These findings provide new insights that SirT1 has a profound role in regulating RIBE where a c-Myc-dependent release of ROS may be involved.

  2. SirT1 knockdown potentiates radiation-induced bystander effect through promoting c-Myc activity and thus facilitating ROS accumulation

    International Nuclear Information System (INIS)

    Xie, Yuexia; Tu, Wenzhi; Zhang, Jianghong; He, Mingyuan; Ye, Shuang; Dong, Chen; Shao, Chunlin

    2015-01-01

    Highlights: • γ-Irradiation induced bystander effects between hepatoma cells and hepatocyte cells. • SirT1 played a protective role in regulating this bystander effect. • SirT1 contributed to the protective effects via elimination the accumulation of ROS. • The activity of c-Myc is critical for maintaining the protective role of SirT1. - Abstract: Radiation-induced bystander effect (RIBE) has important implications for secondary cancer risk assessment during cancer radiotherapy, but the bystander signaling processes, especially under hypoxic condition, are still largely unclear. The present study found that micronuclei (MN) formation could be induced in the non-irradiated HL-7702 hepatocyte cells after being treated with the conditioned medium from irradiated hepatoma HepG2 and SK-Hep-1 cells under either normoxia or hypoxia. This bystander response was dramatically diminished or enhanced when the SirT1 gene of irradiated hepatoma cells was overexpressed or knocked down, respectively, especially under hypoxia. Meanwhile, SirT1 knockdown promoted transcriptional activity for c-Myc and facilitated ROS accumulation. But both of the increased bystander responses and ROS generation due to SirT1-knockdown were almost completely suppressed by c-Myc interference. Moreover, ROS scavenger effectively abolished the RIBE triggered by irradiated hepatoma cells even with SirT1 depletion. These findings provide new insights that SirT1 has a profound role in regulating RIBE where a c-Myc-dependent release of ROS may be involved

  3. Improvement of the design and generation of highly specific plant knockdown lines using primary synthetic microRNAs (pri-smiRNAs

    Directory of Open Access Journals (Sweden)

    Alves Leonardo

    2010-03-01

    Full Text Available Abstract Background microRNAs (miRNAs are endogenous small non-coding RNAs that post-transcriptionally regulate gene expression. In plants, they typically show high complementarity to a single sequence motif within their target mRNAs and act by catalyzing specific mRNA cleavage and degradation. miRNAs are processed from much longer primary transcripts via precursor miRNAs containing fold-back structures. Leaving these secondary structures intact, miRNAs can be re-designed experimentally to target mRNAs of choice. Results We designed primary synthetic miRNAs (pri-smiRNAs on the basis of the primary transcript of the Arabidopsis MIR159A gene by replacing the original miR159a and the corresponding miR159a* with novel sequences, keeping the overall secondary structure as predicted by the program RNAfold. We used the program RNAhybrid to optimize smiRNA design and to screen the complete Arabidopsis transcriptome for potential off-targets. To improve the molecular cloning of the pri-smiRNA we inserted restriction sites in the original MIR159A primary transcript to easily accommodate the smiRNA/smiRNA* DNA fragment. As a proof-of-concept, we targeted the single gene encoding chalcone synthase (CHS in Arabidopsis. We demonstrate smiRNA(CHS expression and CHS mRNA cleavage in different transgenic lines. Phenotypic changes in these lines were observed for seed color and flavonol derivatives, and quantified with respect to anthocyanin content. We also tested the effect of mismatches and excess G:U base pairs on knockdown efficiency. Conclusions RNAhybrid-assisted design of smiRNAs and generation of pri-smiRNAs using a novel vector containing restriction sites greatly improves specificity and speed of the generation of stable knockdown lines for functional analyses in plants.

  4. Knock-down of transcript abundance of a family of Kunitz proteinase inhibitor genes in white clover (Trifolium repens) reveals a redundancy and diversity of gene function.

    Science.gov (United States)

    Islam, Afsana; Leung, Susanna; Burgess, Elisabeth P J; Laing, William A; Richardson, Kim A; Hofmann, Rainer W; Dijkwel, Paul P; McManus, Michael T

    2015-12-01

    The transcriptional regulation of four phylogenetically distinct members of a family of Kunitz proteinase inhibitor (KPI) genes isolated from white clover (Trifolium repens; designated Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5) has been investigated to determine their wider functional role. The four genes displayed differential transcription during seed germination, and in different tissues of the mature plant, and transcription was also ontogenetically regulated. Heterologous over-expression of Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5 in Nicotiana tabacum retarded larval growth of the herbivore Spodoptera litura, and an increase in the transcription of the pathogenesis-related genes PR1 and PR4 was observed in the Tr-KPI1 and Tr-KPI4 over-expressing lines. RNA interference (RNAi) knock-down lines in white clover displayed significantly altered vegetative growth phenotypes with inhibition of shoot growth and a stimulation of root growth, while knock-down of Tr-KPI1, Tr-KPI2 and Tr-KPI5 transcript abundance also retarded larval growth of S. litura. Examination of these RNAi lines revealed constitutive stress-associated phenotypes as well as altered transcription of cellular signalling genes. These results reveal a functional redundancy across members of the KPI gene family. Further, the regulation of transcription of at least one member of the family, Tr-KPI2, may occupy a central role in the maintenance of a cellular homeostasis. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  5. Characterization of ibrutinib-sensitive and -resistant mantle lymphoma cells.

    Science.gov (United States)

    Ma, Jiao; Lu, Pin; Guo, Ailin; Cheng, Shuhua; Zong, Hongliang; Martin, Peter; Coleman, Morton; Wang, Y Lynn

    2014-09-01

    Ibrutinib inhibits Bruton tyrosine kinase (BTK), a key component of early B-cell receptor (BCR) signalling pathways. A multicentre phase 2 trial of ibrutinib in patients with relapsed/refractory mantle cell lymphoma (MCL) demonstrated a remarkable response rate. However, approximately one-third of patients have primary resistance to the drug while other patients appear to lose response and develop secondary resistance. Understanding the molecular mechanisms underlying ibrutinib sensitivity is of paramount importance. In this study, we investigated cell lines and primary MCL cells that display differential sensitivity to ibrutinib. We found that the primary cells display a higher BTK activity than normal B cells and MCL cells show differential sensitivity to BTK inhibition. Genetic knockdown of BTK inhibits the growth, survival and proliferation of ibrutinib-sensitive but not resistant MCL cell lines, suggesting that ibrutinib acts through BTK to produce its anti-tumour activities. Interestingly, inhibition of ERK1/2 and AKT, but not BTK phosphorylation per se, correlates well with cellular response to BTK inhibition in cell lines as well as in primary tumours. Our study suggests that, to prevent primary resistance or to overcome secondary resistance to BTK inhibition, a combinatory strategy that targets multiple components or multiple pathways may represent the most effective approach. © 2014 John Wiley & Sons Ltd.

  6. Novel drug-resistance mechanisms of pemetrexed-treated non-small cell lung cancer.

    Science.gov (United States)

    Tanino, Ryosuke; Tsubata, Yukari; Harashima, Nanae; Harada, Mamoru; Isobe, Takeshi

    2018-03-30

    Pemetrexed (PEM) improves the overall survival of patients with advanced non-small cell lung cancer (NSCLC) when administered as maintenance therapy. However, PEM resistance often appears during the therapy. Although thymidylate synthase is known to be responsible for PEM resistance, no other mechanisms have been investigated in detail. In this study, we explored new drug resistance mechanisms of PEM-treated NSCLC using two combinations of parental and PEM-resistant NSCLC cell lines from PC-9 and A549. PEM increased the apoptosis cells in parental PC-9 and the senescent cells in parental A549. However, such changes were not observed in the respective PEM-resistant cell lines. Quantitative RT-PCR analysis revealed that, besides an increased gene expression of thymidylate synthase in PEM-resistant PC-9 cells, the solute carrier family 19 member1 ( SLC19A1) gene expression was markedly decreased in PEM-resistant A549 cells. The siRNA-mediated knockdown of SLC19A1 endowed the parental cell lines with PEM resistance. Conversely, PEM-resistant PC-9 cells carrying an epidermal growth factor receptor (EGFR) mutation acquired resistance to a tyrosine kinase inhibitor erlotinib. Although erlotinib can inhibit the phosphorylation of EGFR and Erk, it is unable to suppress the phosphorylation of Akt in PEM-resistant PC-9 cells. Additionally, PEM-resistant PC-9 cells were less sensitive to the PI3K inhibitor LY294002 than parental PC-9 cells. These results indicate that SLC19A1 negatively regulates PEM resistance in NSCLC, and that EGFR-tyrosine-kinase-inhibitor resistance was acquired with PEM resistance through Akt activation in NSCLC harboring EGFR mutations.

  7. Mapping insecticide resistance and characterization of resistance mechanisms in Anopheles arabiensis (Diptera: Culicidae) in Ethiopia.

    Science.gov (United States)

    Alemayehu, Eba; Asale, Abebe; Eba, Kasahun; Getahun, Kefelegn; Tushune, Kora; Bryon, Astrid; Morou, Evangelia; Vontas, John; Van Leeuwen, Thomas; Duchateau, Luc; Yewhalaw, Delenasaw

    2017-09-02

    The emergence and spread of insecticide resistance in the major African malaria vectors Anopheles gambiae (s.s.) and An. arabiensis may compromise the current vector control interventions and threatens the global malaria control and elimination efforts. Insecticide resistance was monitored in several study sites in Ethiopia from 2013 to 2015 using papers impregnated with discriminating concentrations of DDT, deltamethrin, bendiocarb, propoxur, malathion, fenitrothion and pirimiphos-methyl, following the WHO insecticide susceptibility test procedure. Mosquitoes sampled from different localities for WHO bioassay were morphologically identified as An. gambiae (s.l.) using standard taxonomic keys. Samples were identified to species using species-specific polymerase chain reaction (PCR) and screened for the presence of target site mutations L1014F, L1014S and N1575Y in the voltage gated sodium channel (VGSC) gene and G119S in the acethylcholinesterase (AChE) gene using allele-specific PCR. Biochemical assays were performed to assess elevated levels of acetylcholinesterases, carboxylcholinesterases, glutathione-S-transferases (GSTs) and cytochrome P450s monooxygenases in wild populations of An. arabiensis, compared to the fully susceptible Sekoru An. arabiensis laboratory strain. Populations of An. arabiensis were resistant to DDT and deltamethrin but were susceptible to fenitrothion in all the study sites. Reduced susceptibility to malathion, pirimiphos-methyl, propoxur and bendiocarb was observed in some of the study sites. Knockdown resistance (kdr L1014F) was detected in all mosquito populations with allele frequency ranging from 42 to 91%. Elevated levels of glutathione-S-transferases (GSTs) were detected in some of the mosquito populations. However, no elevated levels of monooxygenases and esterases were detected in any of the populations assessed. Anopheles arabiensis populations from all surveyed sites in Ethiopia exhibited resistance against DDT and pyrethroids

  8. Insecticide resistance is mediated by multiple mechanisms in recently introduced Aedes aegypti from Madeira Island (Portugal).

    Science.gov (United States)

    Seixas, Gonçalo; Grigoraki, Linda; Weetman, David; Vicente, José Luís; Silva, Ana Clara; Pinto, João; Vontas, John; Sousa, Carla Alexandra

    2017-07-01

    Aedes aegypti is a major mosquito vector of arboviruses, including dengue, chikungunya and Zika. In 2005, Ae. aegypti was identified for the first time in Madeira Island. Despite an initial insecticide-based vector control program, the species expanded throughout the Southern coast of the island, suggesting the presence of insecticide resistance. Here, we characterized the insecticide resistance status and the underlying mechanisms of two populations of Ae. aegypti from Madeira Island, Funchal and Paúl do Mar. WHO susceptibility bioassays indicated resistance to cyfluthrin, permethrin, fenitrothion and bendiocarb. Use of synergists significantly increased mortality rates, and biochemical assays indicated elevated activities of detoxification enzymes, suggesting the importance of metabolic resistance. Microarray-based transcriptome analysis detected significant upregulation in both populations of nine cytochrome P450 oxidase genes (including four known pyrethroid metabolizing enzymes), the organophosphate metabolizer CCEae3a, Glutathione-S-transferases, and multiple putative cuticle proteins. Genotyping of knockdown resistance loci linked to pyrethroid resistance revealed fixation of the 1534C mutation, and presence with moderate frequencies of the V1016I mutation in each population. Significant resistance to three major insecticide classes (pyrethroid, carbamate and organophosphate) is present in Ae. aegypti from Madeira Island, and appears to be mediated by multiple mechanisms. Implementation of appropriate resistance management strategies including rotation of insecticides with alternative modes of action, and methods other than chemical-based vector control are strongly advised to delay or reverse the spread of resistance and achieve efficient control.

  9. Electrical resistivity measurements to predict abrasion resistance

    Indian Academy of Sciences (India)

    Home; Journals; Bulletin of Materials Science; Volume 31; Issue 2. Electrical resistivity measurements to predict abrasion resistance of rock aggregates ... It was seen that correlation coefficients were increased for the rock classes. In addition ...

  10. Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line

    Science.gov (United States)

    Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

    2016-04-01

    First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro

  11. Developmental acclimation to low or high humidity conditions affect starvation and heat resistance of Drosophila melanogaster.

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. ABCF2, an Nrf2 target gene, contributes to cisplatin resistance in ovarian cancer cells.

    Science.gov (United States)

    Bao, Lingjie; Wu, Jianfa; Dodson, Matthew; Rojo de la Vega, Elisa Montserrat; Ning, Yan; Zhang, Zhenbo; Yao, Ming; Zhang, Donna D; Xu, Congjian; Yi, Xiaofang

    2017-06-01

    Previously, we have demonstrated that NRF2 plays a key role in mediating cisplatin resistance in ovarian cancer. To further explore the mechanism underlying NRF2-dependent cisplatin resistance, we stably overexpressed or knocked down NRF2 in parental and cisplatin-resistant human ovarian cancer cells, respectively. These two pairs of stable cell lines were then subjected to microarray analysis, where we identified 18 putative NRF2 target genes. Among these genes, ABCF2, a cytosolic member of the ABC superfamily of transporters, has previously been reported to contribute to chemoresistance in clear cell ovarian cancer. A detailed analysis on ABCF2 revealed a functional antioxidant response element (ARE) in its promoter region, establishing ABCF2 as an NRF2 target gene. Next, we investigated the contribution of ABCF2 in NRF2-mediated cisplatin resistance using our stable ovarian cancer cell lines. The NRF2-overexpressing cell line, containing high levels of ABCF2, was more resistant to cisplatin-induced apoptosis compared to its control cell line; whereas the NRF2 knockdown cell line with low levels of ABCF2, was more sensitive to cisplatin treatment than its control cell line. Furthermore, transient overexpression of ABCF2 in the parental cells decreased apoptosis and increased cell viability following cisplatin treatment. Conversely, knockdown of ABCF2 using specific siRNA notably increased apoptosis and decreased cell viability in cisplatin-resistant cells treated with cisplatin. This data indicate that the novel NRF2 target gene, ABCF2, plays a critical role in cisplatin resistance in ovarian cancer, and that targeting ABCF2 may be a new strategy to improve chemotherapeutic efficiency. © 2017 Wiley Periodicals, Inc.

  13. MSH6 mutations arise in glioblastomas during temozolomide therapy and mediate temozolomide resistance

    Science.gov (United States)

    Yip, Stephen; Miao, Jiangyong; Cahill, Daniel P.; Iafrate, A. John; Aldape, Ken; Nutt, Catherine L.; Louis, David N.

    2009-01-01

    Purpose Over the past few years, the alkylating agent temozolomide (TMZ) has become the standard-of-care therapy for patients with glioblastoma, the most common brain tumor. Recently, large-scale cancer genome sequencing efforts have identified a hypermutation phenotype and inactivating MSH6 mismatch repair gene mutations in recurrent, post-TMZ glioblastomas, particularly those growing more rapidly during TMZ treatment. This study aimed to clarify the timing and role of MSH6 mutations in mediating glioblastoma TMZ resistance. Experimental Design MSH6 sequence and microsatellite instability (MSI) status were determined in matched pre- and post-chemotherapy glioblastomas identified by The Cancer Genome Atlas (TCGA) as having post-treatment MSH6 mutations. TMZ-resistant lines were derived in vitro via selective growth under TMZ and the MSH6 gene was sequenced in resistant clones. The role of MSH6 inactivation in mediating resistance was explored using lentiviral shRNA knockdown and MSH6 reconstitution. Results MSH6 mutations were confirmed in post-treatment TCGA glioblastomas but absent in matched pre-treatment tumors. The post-treatment hypermutation phenotype displayed a signature bias toward CpC transitions and was not associated with MSI. In vitro modeling via exposure of an MSH6-wildtype glioblastoma line to TMZ resulted in resistant clones; one clone showed an MSH6 mutation, Thr1219Ile, that had been independently noted in two treated TCGA glioblastomas. Knockdown of MSH6 in the glioblastoma line U251 increased resistance to TMZ cytotoxicity and reconstitution restored cytotoxicity in MSH6-null glioma cells. Conclusions MSH6 mutations are selected for in glioblastomas during TMZ therapy both in vitro and in vivo, and are causally associated with TMZ resistance. PMID:19584161

  14. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... Animal & Veterinary Safety & Health Antimicrobial Resistance Animation of Antimicrobial Resistance Share Tweet Linkedin Pin it More sharing options ... CVM) produced a nine-minute animation explaining how antimicrobial resistance both emerges and proliferates among bacteria. Over time, ...

  15. Combating Antibiotic Resistance

    Science.gov (United States)

    ... Bacteria Phasing Out Certain Antibiotic Use in Farm Animals FDA: Cutting-Edge Technology Sheds Light on Antibiotic Resistance For More Information Antibiotics and Antibiotic Resistance Antimicrobial Resistance Information for Consumers and Health Professionals CDC: ...

  16. Hydraulic resistance of biofilms

    KAUST Repository

    Dreszer, C.; Vrouwenvelder, Johannes S.; Paulitsch-Fuchs, Astrid H.; Zwijnenburg, Arie; Kruithof, Joop C.; Flemming, Hans Curt

    2013-01-01

    resistance is very low compared to the expected biofilm resistance and, thus, biofilm resistance can be determined accurately. Transmembrane pressure drop was monitored. As biofilm parameters, thickness, total cell number, TOC, and extracellular polymeric

  17. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... Veterinary Safety & Health Antimicrobial Resistance Animation of Antimicrobial Resistance Share Tweet Linkedin Pin it More sharing options ... produced a nine-minute animation explaining how antimicrobial resistance both emerges and proliferates among bacteria. Over time, ...

  18. HIV Resistance Testing

    Science.gov (United States)

    ... 14, 2016 Select a Language: Fact Sheet 126 HIV Resistance Testing WHAT IS RESISTANCE? HOW DOES RESISTANCE ... ARVs. If you miss doses of your medications, HIV will multiply more easily. More mutations will occur. ...

  19. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... Animal & Veterinary Safety & Health Antimicrobial Resistance Animation of Antimicrobial Resistance Share Tweet Linkedin Pin it More sharing ... CVM) produced a nine-minute animation explaining how antimicrobial resistance both emerges and proliferates among bacteria. Over ...

  20. Induced resistance: an enhancement of basal resistance?

    NARCIS (Netherlands)

    Vos, M. de; Robben, C.; Pelt, J.A. van; Loon, L.C. van; Pieterse, C.M.J.

    2002-01-01

    Upon primary pathogen attack, plants activate resistance mechanisms at the site of infection. Besides this so-called basal resistance, plants have also the ability to enhance their defensive capacity against future pathogen attack. There are at least two types of biologically induced resistance.

  1. Country-level operational implementation of the Global Plan for Insecticide Resistance Management.

    Science.gov (United States)

    Hemingway, Janet; Vontas, John; Poupardin, Rodolphe; Raman, Jaishree; Lines, Jo; Schwabe, Chris; Matias, Abrahan; Kleinschmidt, Immo

    2013-06-04

    Malaria control is reliant on the use of long-lasting pyrethroid-impregnated nets and/or indoor residual spraying (IRS) of insecticide. The rapid selection and spread of operationally significant pyrethroid resistance in African malaria vectors threatens our ability to sustain malaria control. Establishing whether resistance is operationally significant is technically challenging. Routine monitoring by bioassay is inadequate, and there are limited data linking resistance selection with changes in disease transmission. The default is to switch insecticides when resistance is detected, but limited insecticide options and resistance to multiple insecticides in numerous locations make this approach unsustainable. Detailed analysis of the resistance situation in Anopheles gambiae on Bioko Island after pyrethroid resistance was detected in this species in 2004, and the IRS program switched to carbamate bendiocarb, has now been undertaken. The pyrethroid resistance selected is a target-site knock-down resistance kdr-form, on a background of generally elevated metabolic activity, compared with insecticide-susceptible A. gambiae, but the major cytochrome P450-based metabolic pyrethroid resistance mechanisms are not present. The available evidence from bioassays and infection data suggests that the pyrethroid resistance mechanisms in Bioko malaria vectors are not operationally significant, and on this basis, a different, long-lasting pyrethroid formulation is now being reintroduced for IRS in a rotational insecticide resistance management program. This will allow control efforts to be sustained in a cost-effective manner while reducing the selection pressure for resistance to nonpyrethroid insecticides. The methods used provide a template for evidence-based insecticide resistance management by malaria control programs.

  2. Modulation of neutrophil and monocyte function by recombinant human granulocyte macrophage colony-stimulating factor in patients with lymphoma

    DEFF Research Database (Denmark)

    Kharazmi, A; Nielsen, H; Hovgaard, D

    1991-01-01

    by up to 43-fold. rhGM-CSF treatment did not affect degranulation of the neutrophils as measured by release of vitamin B12 binding protein. Degree of modulation of neutrophil and monocyte function by rhGM-CSF was independent of rhGM-CSF dosages administered. These data suggest that phagocytic defence...... and chemiluminescence responses to f-Met-Leu-Phe, zymosan activated serum (ZAS) and opsonized zymosan (OZ) were determined. It was observed that chemotactic response of neutrophils to f-Met-Leu-Phe and ZAS was reduced, whereas the chemiluminescence response of both cell types to f-Met-Leu-Phe and zymosan was enhanced...

  3. Animation of Antimicrobial Resistance

    Science.gov (United States)

    ... Animal & Veterinary Cosmetics Tobacco Products Animal & Veterinary Home Animal & Veterinary Safety & Health Antimicrobial Resistance Animation of Antimicrobial Resistance Share Tweet Linkedin Pin ...

  4. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... Animal & Veterinary Cosmetics Tobacco Products Animal & Veterinary Home Animal & Veterinary Safety & Health Antimicrobial Resistance Animation of Antimicrobial Resistance Share Tweet Linkedin Pin ...

  5. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... More in Antimicrobial Resistance National Antimicrobial Resistance Monitoring System ... If you need help accessing information in different file formats, see Instructions for Downloading ...

  6. TUG1 knockdown ameliorates atherosclerosis via up-regulating the expression of miR-133a target gene FGF1.

    Science.gov (United States)

    Zhang, Lei; Cheng, Hailing; Yue, Yuxia; Li, Shuangzhan; Zhang, Daping; He, Ruili

    Long non-coding RNAs (lncRNAs) have been revealed to participate in the pathological events associated with atherosclerosis. However, the exact role of lncRNA taurine-up-regulated gene 1 (TUG1) and its possible molecular mechanism in atherosclerosis remain unidentified. High-fat diet (HFD)-treated ApoE -/- mice were used as an in vivo model of atherosclerosis. Ox-LDL-induced macrophages and vascular smooth muscle cells (VSMCs) were employed as cell models of atherosclerosis. qRT-PCR was performed to detect the expression of TUG1 and miR-133a. Serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were analyzed by commercially available enzyme kits. Oil red O and hematoxylin and eosin (H&E) staining were conducted to examine atherosclerotic lesion. Luciferase reporter assay combined with RNA immunoprecipitation (RIP) was applied to confirm the interaction between TUG1, miR-133a and FGF1. Cell proliferation ability was determined by Cell Counting Kit-8 (CCK-8) assay and trypan blue dye exclusion test. Cell apoptosis was evaluated with TUNEL assay. Expression and production of inflammatory cytokines was measured with western blot and ELISA analysis. TUG1 expression was up-regulated in HFD-treated ApoE -/- mice, as well as in ox-LDL-induced RAW264.7 and MOVAS cells. TUG1 knockdown inhibited hyperlipidemia, decreased inflammatory response, and attenuated atherosclerotic lesion in HFD-treated ApoE -/- mice. TUG1 could function as a molecular sponge of miR-133a to suppress its expression. TUG1 overexpression accelerated cell growth, improved inflammatory factor expression, and inhibited apoptosis in ox-LDL-stimulated RAW264.7 and MOVAS cells, while this effect was abated after transfection with miR-133 mimic. Moreover, fibroblast growth factor 1 (FGF1) was identified as a direct target of miR-133a. Restored expression of FGF1 overturned the effect of miR-133a on cell

  7. Double transduction of a Cre/LoxP lentiviral vector: a simple method to generate kidney cell-specific knockdown mice.

    Science.gov (United States)

    Nam, Bo Young; Kim, Dong Ki; Park, Jung Tak; Kang, Hye-Young; Paeng, Jisun; Kim, Seonghun; Park, Jimin; Um, Jae Eun; Oh, Hyung Jung; Han, Seung Hyeok; Yoo, Tae-Hyun; Kang, Shin-Wook

    2015-12-15

    In a lentivirus-based gene delivery system, the incorporated gene is continuously expressed for a long time. In this study, we devised a simple way to knock down a specific gene in a kidney cell-specific pattern in adult mice by lentivirus-assisted transfer of short hairpin RNA (shRNA). Kidney collecting duct (CD)-specific aquaporin-3 (AQP3)-knockdown mice were generated by consecutive injection of Hoxb7-Cre-expressing lentivirus (LV-Hoxb7 Cre) and loxP-AQP3 shRNA-expressing lentivirus (LV-loxP shAQP3) in adult C57BL6/J mice. LV-Hoxb7 Cre was designed to express mCherry, while LV-loxP shAQP3 was designed with a floxed enhanced green fluorescent protein (EGFP)-tagged stop sequence, and thus EGFP would be expressed only in the absence of Cre recombination. In mice treated with LV-Hoxb7 Cre alone, mCherry protein expression, which indicates the presence of Cre recombinase, occurred only in CD cells. However, LV-loxP shAQP3 injection alone resulted in an increase in EGFP expression in all kidney cells, indicating the transcription of the floxed region. When LV-Hoxb7 Cre and LV-loxP shAQP3 were sequentially transduced, EGFP expression was attenuated while mCherry expression was sustained in CD cells, demonstrating a CD cell-specific recombination of the floxed region. AQP3 expression in mice injected with LV-Hoxb7 Cre or LV-loxP shAQP3 alone did not differ, but consecutive injection of LV-Hoxb7 Cre and LV-loxP shAQP3 significantly reduced AQP3 expression in CD cells. However, the expression levels of AQP3 were not altered in other cell types. Double transduction of Cre- and loxP-based lentivirus can easily generate kidney cell-specific knockdown mice, and this method might be applicable to other species. Copyright © 2015 the American Physiological Society.

  8. Knockdown of wfs1, a fly homolog of Wolfram syndrome 1, in the nervous system increases susceptibility to age- and stress-induced neuronal dysfunction and degeneration in Drosophila.

    Science.gov (United States)

    Sakakibara, Yasufumi; Sekiya, Michiko; Fujisaki, Naoki; Quan, Xiuming; Iijima, Koichi M

    2018-01-01

    Wolfram syndrome (WS), caused by loss-of-function mutations in the Wolfram syndrome 1 gene (WFS1), is characterized by juvenile-onset diabetes mellitus, bilateral optic atrophy, and a wide spectrum of neurological and psychiatric manifestations. WFS1 encodes an endoplasmic reticulum (ER)-resident transmembrane protein, and mutations in this gene lead to pancreatic β-cell death induced by high levels of ER stress. However, the mechanisms underlying neurodegeneration caused by WFS1 deficiency remain elusive. Here, we investigated the role of WFS1 in the maintenance of neuronal integrity in vivo by knocking down the expression of wfs1, the Drosophila homolog of WFS1, in the central nervous system. Neuronal knockdown of wfs1 caused age-dependent behavioral deficits and neurodegeneration in the fly brain. Knockdown of wfs1 in neurons and glial cells resulted in premature death and significantly exacerbated behavioral deficits in flies, suggesting that wfs1 has important functions in both cell types. Although wfs1 knockdown alone did not promote ER stress, it increased the susceptibility to oxidative stress-, excitotoxicity- or tauopathy-induced behavioral deficits, and neurodegeneration. The glutamate release inhibitor riluzole significantly suppressed premature death phenotypes induced by neuronal and glial knockdown of wfs1. This study highlights the protective role of wfs1 against age-associated neurodegeneration and furthers our understanding of potential disease-modifying factors that determine susceptibility and resilience to age-associated neurodegenerative diseases.

  9. Knockdown of wfs1, a fly homolog of Wolfram syndrome 1, in the nervous system increases susceptibility to age- and stress-induced neuronal dysfunction and degeneration in Drosophila.

    Directory of Open Access Journals (Sweden)

    Yasufumi Sakakibara

    2018-01-01

    Full Text Available Wolfram syndrome (WS, caused by loss-of-function mutations in the Wolfram syndrome 1 gene (WFS1, is characterized by juvenile-onset diabetes mellitus, bilateral optic atrophy, and a wide spectrum of neurological and psychiatric manifestations. WFS1 encodes an endoplasmic reticulum (ER-resident transmembrane protein, and mutations in this gene lead to pancreatic β-cell death induced by high levels of ER stress. However, the mechanisms underlying neurodegeneration caused by WFS1 deficiency remain elusive. Here, we investigated the role of WFS1 in the maintenance of neuronal integrity in vivo by knocking down the expression of wfs1, the Drosophila homolog of WFS1, in the central nervous system. Neuronal knockdown of wfs1 caused age-dependent behavioral deficits and neurodegeneration in the fly brain. Knockdown of wfs1 in neurons and glial cells resulted in premature death and significantly exacerbated behavioral deficits in flies, suggesting that wfs1 has important functions in both cell types. Although wfs1 knockdown alone did not promote ER stress, it increased the susceptibility to oxidative stress-, excitotoxicity- or tauopathy-induced behavioral deficits, and neurodegeneration. The glutamate release inhibitor riluzole significantly suppressed premature death phenotypes induced by neuronal and glial knockdown of wfs1. This study highlights the protective role of wfs1 against age-associated neurodegeneration and furthers our understanding of potential disease-modifying factors that determine susceptibility and resilience to age-associated neurodegenerative diseases.

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

    NARCIS (Netherlands)

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

    2016-01-01

    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

  11. Circadian control of permethrin-resistance in the mosquito Aedes aegypti

    Czech Academy of Sciences Publication Activity Database

    Yang, Y.-Y.; Liu, Y.; Teng, H.-J.; Šauman, Ivo; Sehnal, František; Lee, H.-J.

    2010-01-01

    Roč. 56, č. 9 (2010), s. 1219-1223 ISSN 0022-1910 R&D Projects: GA MŠk LC07032 Grant - others:Centers for Disease Control, Department of Health(TW) DOH96-DC-1206; National Science Council(TW) NSC 95-2313-B-002-084 MY3 Institutional research plan: CEZ:AV0Z50070508 Keywords : insecticide resistence * median knock-down time * clock gene Subject RIV: ED - Physiology Impact factor: 2.310, year: 2010

  12. Global characterization of signalling networks associated with tamoxifen resistance in breast cancer

    DEFF Research Database (Denmark)

    Browne, Brigid C.; Hochgräfe, Falko; Wu, Jianmin

    2013-01-01

    R cells. Phosphorylation of the tyrosine kinase Yes and expression of the actin‐binding protein myristoylated alanine‐rich C‐kinase substrate (MARCKS) were increased two‐ and eightfold in TamR cells respectively, and these proteins were selected for further analysis. Knockdown of either protein in Tam......Acquired resistance to the anti‐estrogen tamoxifen remains a significant challenge in breast cancer management. In this study, we used an integrative approach to characterize global protein expression and tyrosine phosphorylation events in tamoxifen‐resistant MCF7 breast cancer cells (Tam...... was perturbed in TamR cells, together with pathways enriched for proteins associated with growth factor, cell–cell and cell matrix‐initiated signalling. Consistent with known roles for Ras/MAPK and PI3‐kinase signalling in tamoxifen resistance, tyrosine‐phosphorylated MAPK1, SHC1 and PIK3R2 were elevated in Tam...

  13. Autophagy downregulation contributes to insulin resistance mediated injury in insulin receptor knockout podocytes in vitro

    Directory of Open Access Journals (Sweden)

    Ying Xu

    2016-04-01

    Full Text Available It is unknown whether autophagy activity is altered in insulin resistant podocytes and whether autophagy could be a therapeutic target for diabetic nephropathy (DN. Here we used shRNA transfection to knockdown the insulin receptor (IR gene in cultured human immortalized podocytes as an in vitro insulin resistant model. Autophagy related proteins LC3, Beclin, and p62 as well as nephrin, a podocyte injury marker, were assessed using western blot and immunofluorescence staining. Our results show that autophagy is suppressed when podocytes lose insulin sensitivity and that treatment of rapamycin, an mTOR specific inhibitor, could attenuate insulin resistance induced podocytes injury via autophagy activation. The present study deepens our understanding of the role of autophagy in the pathogenesis of DN.

  14. Expression proteomics of UPF1 knockdown in HeLa cells reveals autoregulation of hnRNP A2/B1 mediated by alternative splicing resulting in nonsense-mediated mRNA decay

    Directory of Open Access Journals (Sweden)

    Zavolan Mihaela

    2010-10-01

    Full Text Available Abstract Background In addition to acting as an RNA quality control pathway, nonsense-mediated mRNA decay (NMD plays roles in regulating normal gene expression. In particular, the extent to which alternative splicing is coupled to NMD and the roles of NMD in regulating uORF containing transcripts have been a matter of debate. Results In order to achieve a greater understanding of NMD regulated gene expression we used 2D-DiGE proteomics technology to examine the changes in protein expression induced in HeLa cells by UPF1 knockdown. QPCR based validation of the corresponding mRNAs, in response to both UPF1 knockdown and cycloheximide treatment, identified 17 bona fide NMD targets. Most of these were associated with bioinformatically predicted NMD activating features, predominantly upstream open reading frames (uORFs. Strikingly, however, the majority of transcripts up-regulated by UPF1 knockdown were either insensitive to, or even down-regulated by, cycloheximide treatment. Furthermore, the mRNA abundance of several down-regulated proteins failed to change upon UPF1 knockdown, indicating that UPF1's role in regulating mRNA and protein abundance is more complex than previously appreciated. Among the bona fide NMD targets, we identified a highly conserved AS-NMD event within the 3' UTR of the HNRNPA2B1 gene. Overexpression of GFP tagged hnRNP A2 resulted in a decrease in endogenous hnRNP A2 and B1 mRNA with a concurrent increase in the NMD sensitive isoforms. Conclusions Despite the large number of changes in protein expression upon UPF1 knockdown, a relatively small fraction of them can be directly attributed to the action of NMD on the corresponding mRNA. From amongst these we have identified a conserved AS-NMD event within HNRNPA2B1 that appears to mediate autoregulation of HNRNPA2B1 expression levels.

  15. TUG1 mediates methotrexate resistance in colorectal cancer via miR-186/CPEB2 axis.

    Science.gov (United States)

    Li, Changfeng; Gao, Yongjian; Li, Yongchao; Ding, Dayong

    2017-09-16

    Colorectal cancer (CRC) is a common malignancy, most of which remain unresponsive to chemotherapy. Methotrexate (MTX) is one of the earliest cytotoxic drugs and serves as an anti-metabolite and anti-folate chemotherapy for various types of cancer. However, MTX resistance prevents its clinical application in cancer therapy. Thereby, overcoming the drug resistance is an alternative strategy to maximize the efficacy of MTX therapies in clinics. Long non-coding RNAs (lncRNAs) have gained widespread attention in recent years. More and more evidences have shown that lncRNAs play regulatory roles in various biological activities and disease progression including drug resistance in cancer cells. Here, we observed lncRNA TUG1 was associated to the MTX resistant in colorectal cancer cells. Firstly, quantitative analysis indicated that TUG1 was significantly increased in tumors which were resistant to MTX treatment. TUG1 knockdown re-sensitized the MTX resistance in colorectal cancer cells, which were MTX-resistant colorectal cell line. Furthermore, bioinformatics analysis showed that miR-186 could directly bind to TUG1, suggesting TUG1 might worked as a ceRNA to sponge miR-186. Extensively, our study also showed that CPEB2 was the direct target of miR-186 in colorectal cancer cells. Taken together, our study suggests that lncRNA TUG1 mediates MTX resistance in colorectal cancer via miR-186/CPEB2 axis. Copyright © 2017. Published by Elsevier Inc.

  16. Mapping a Quantitative Trait Locus (QTL conferring pyrethroid resistance in the African malaria vector Anopheles funestus

    Directory of Open Access Journals (Sweden)

    Hunt Richard H

    2007-01-01

    Full Text Available Abstract Background Pyrethroid resistance in Anopheles funestus populations has led to an increase in malaria transmission in southern Africa. Resistance has been attributed to elevated activities of cytochrome P450s but the molecular basis underlying this metabolic resistance is unknown. Microsatellite and SNP markers were used to construct a linkage map and to detect a quantitative trait locus (QTL associated with pyrethroid resistance in the FUMOZ-R strain of An. funestus from Mozambique. Results By genotyping 349 F2 individuals from 11 independent families, a single major QTL, rp1, at the telomeric end of chromosome 2R was identified. The rp1 QTL appears to present a major effect since it accounts for more than 60% of the variance in susceptibility to permethrin. This QTL has a strong additive genetic effect with respect to susceptibility. Candidate genes associated with pyrethroid resistance in other species were physically mapped to An. funestus polytene chromosomes. This showed that rp1 is genetically linked to a cluster of CYP6 cytochrome P450 genes located on division 9 of chromosome 2R and confirmed earlier reports that pyrethroid resistance in this strain is not associated with target site mutations (knockdown resistance. Conclusion We hypothesize that one or more of these CYP6 P450s clustered on chromosome 2R confers pyrethroid resistance in the FUMOZ-R strain of An. funestus.

  17. Knockdown of connexin43-mediated regulation of the zone of polarizing activity in the developing chick limb leads to digit truncation.

    Science.gov (United States)

    Law, Lee Yong; Lin, Jun Sheng; Becker, David L; Green, Colin R

    2002-12-01

    In the developing chick wing, the use of antisense oligodeoxynucleotides to transiently knock down the expression of the gap junction protein, connexin43 (Cx43), results in limb patterning defects, including deletion of the anterior digits. To understand more about how such defects arise, the effects of transient Cx43 knockdown on the expression patterns of several genes known to play pivotal roles in limb formation were examined. Sonic hedgehog (Shh), which is normally expressed in the zone of polarizing activity (ZPA) and is required to maintain both the ZPA and the apical ectodermal ridge (AER), was found to be downregulated in treated limbs within 30 h. Bone morphogenetic protein-2 (Bmp-2), a gene downstream of Shh, was similarly downregulated. Fibroblast growth factor-8 expression, however, was unaltered 30 h after treatment but was greatly reduced at 48 h post-treatment, when the AER begins to regress. Expressions of Bmp-4 and Muscle segment homeobox-like gene (Msx-1) were not affected at any of the time points examined. Cx43 expression is therefore involved in some, but not all patterning cascades, and appears to play a role in the regulation of ZPA activity.

  18. Data on the effect of in vivo knockdown using artificial ErbB3 miRNA on Remak bundle structure

    Directory of Open Access Journals (Sweden)

    Yuki Miyamoto

    2017-06-01

    Full Text Available Mature Schwann cells, the peripheral nervous system (PNS glial cells, have two major roles for neuronal axons (Bunge, 1993 [1]. For large diameter axons, Schwann cells form myelin sheaths with multiple layers. For small diameter axons, they form Remak bundle composed only of single layer of the Schwann cell plasma membrane. In the PNS, ErbB3 forms a dimer with ErbB2 on the Schwann cell plasma membrane. ErbB3 plays a key role in myelination by myelinating Schwann cells, that is to say, its role in myelin thickness. Herein we provide the data regarding the effect of in vivo knockdown of ErbB3 on the thickness between an axon and a neighboring axon in Remak bundle, which is formed by non-myelinating Schwann cells. Since ErbB3 knockout mice are embryonically lethal, Schwann cell lineage-specific transgenic mice transcribing ErbB3 shRNA with an artificial miRNA backbone were generated and used in these experiments (Torii et al., 2014 [2].

  19. GATA4 and GATA6 Knockdown During Luteinization Inhibits Progesterone Production and Gonadotropin Responsiveness in the Corpus Luteum of Female Mice.

    Science.gov (United States)

    Convissar, Scott M; Bennett, Jill; Baumgarten, Sarah C; Lydon, John P; DeMayo, Francesco J; Stocco, Carlos

    2015-12-01

    The surge of luteinizing hormone triggers the genomic reprogramming, cell differentiation, and tissue remodeling of the ovulated follicle, leading to the formation of the corpus luteum. During this process, called luteinization, follicular granulosa cells begin expressing a new set of genes that allow the resulting luteal cells to survive in a vastly different hormonal environment and to produce the extremely high amounts of progesterone (P4) needed to sustain pregnancy. To better understand the molecular mechanisms involved in the regulation of luteal P4 production in vivo, the transcription factors GATA4 and GATA6 were knocked down in the corpus luteum by crossing mice carrying Gata4 and Gata6 floxed genes with mice carrying Cre recombinase fused to the progesterone receptor. This receptor is expressed exclusively in granulosa cells after the luteinizing hormone surge, leading to recombination of floxed genes during follicle luteinization. The findings demonstrated that GATA4 and GATA6 are essential for female fertility, whereas targeting either factor alone causes subfertility. When compared to control mice, serum P4 levels and luteal expression of key steroidogenic genes were significantly lower in conditional knockdown mice. The results also showed that GATA4 and GATA6 are required for the expression of the receptors for prolactin and luteinizing hormone, the main luteotropic hormones in mice. The findings demonstrate that GATA4 and GATA6 are crucial regulators of luteal steroidogenesis and are required for the normal response of luteal cells to luteotropins. © 2015 by the Society for the Study of Reproduction, Inc.

  20. Nuclear shape changes are induced by knockdown of the SWI/SNF ATPase BRG1 and are independent of cytoskeletal connections.

    Directory of Open Access Journals (Sweden)

    Karen M Imbalzano

    Full Text Available Changes in nuclear morphology occur during normal development and have been observed during the progression of several diseases. The shape of a nucleus is governed by the balance of forces exerted by nuclear-cytoskeletal contacts and internal forces created by the structure of the chromatin and nuclear envelope. However, factors that regulate the balance of these forces and determine nuclear shape are poorly understood. The SWI/SNF chromatin remodeling enzyme ATPase, BRG1, has been shown to contribute to the regulation of overall cell size and shape. Here we document that immortalized mammary epithelial cells show BRG1-dependent nuclear shape changes. Specifically, knockdown of BRG1 induced grooves in the nuclear periphery that could be documented by cytological and ultrastructural methods. To test the hypothesis that the observed changes in nuclear morphology resulted from altered tension exerted by the cytoskeleton, we disrupted the major cytoskeletal networks and quantified the frequency of BRG1-dependent changes in nuclear morphology. The results demonstrated that disruption of cytoskeletal networks did not change the frequency of BRG1-induced nuclear shape changes. These findings suggest that BRG1 mediates control of nuclear shape by internal nuclear mechanisms that likely control chromatin dynamics.

  1. Inducible Knock-Down of the Mineralocorticoid Receptor in Mice Disturbs Regulation of the Renin-Angiotensin-Aldosterone System and Attenuates Heart Failure Induced by Pressure Overload.

    Directory of Open Access Journals (Sweden)

    Elena Montes-Cobos

    Full Text Available Mineralocorticoid receptor (MR inactivation in mice results in early postnatal lethality. Therefore we generated mice in which MR expression can be silenced during adulthood by administration of doxycycline (Dox. Using a lentiviral approach, we obtained two lines of transgenic mice harboring a construct that allows for regulatable MR inactivation by RNAi and concomitant expression of eGFP. MR mRNA levels in heart and kidney of inducible MR knock-down mice were unaltered in the absence of Dox, confirming the tightness of the system. In contrast, two weeks after Dox administration MR expression was significantly diminished in a variety of tissues. In the kidney, this resulted in lower mRNA levels of selected target genes, which was accompanied by strongly increased serum aldosterone and plasma renin levels as well as by elevated sodium excretion. In the healthy heart, gene expression and the amount of collagen were unchanged despite MR levels being significantly reduced. After transverse aortic constriction, however, cardiac hypertrophy and progressive heart failure were attenuated by MR silencing, fibrosis was unaffected and mRNA levels of a subset of genes reduced. Taken together, we believe that this mouse model is a useful tool to investigate the role of the MR in pathophysiological processes.

  2. Knock-down of a tonoplast localized low-affinity nitrate transporter OsNPF7.2 affects rice growth under high nitrate ssupply

    Directory of Open Access Journals (Sweden)

    Rui Hu

    2016-10-01

    Full Text Available The large nitrate transporter 1/peptide transporter family (NPF has been shown to transport diverse substrates, including nitrate, amino acids, peptides, phytohormones, and glucosinolates. However, the rice (Oryza sativa root-specific expressed member OsNPF7.2 has not been characterized. Here, our data show that OsNPF7.2 is a tonoplast localized low-affinity nitrate transporter, and affects rice growth under high nitrate supply. The expression analysis showed that OsNPF7.2 was mainly expressed in the elongation and maturation zones of roots, especially in the root sclerenchyma, cortex and stele. It was also induced by high concentrations of nitrate. Subcellular localization analysis showed that OsNPF7.2 was localized on the tonoplast of large and small vacuoles. Heterogenous expression in Xenopus laevis oocytes suggested that OsNPF7.2 was a low-affinity nitrate transporter. Knock-down of OsNPF7.2 retarded rice growth under high concentrations of nitrate. Therefore, we deduce that OsNPF7.2 plays a role in intracellular allocation of nitrate in roots, and thus influences rice growth under high nitrate supply.

  3. Assessing the effects of Aedes aegypti kdr mutations on pyrethroid resistance and its fitness cost.

    Directory of Open Access Journals (Sweden)

    Luiz Paulo Brito

    Full Text Available Pyrethroids are the most used insecticide class worldwide. They target the voltage gated sodium channel (NaV, inducing the knockdown effect. In Aedes aegypti, the main dengue vector, the AaNaV substitutions Val1016Ile and Phe1534Cys are the most important knockdown resistance (kdr mutations. We evaluated the fitness cost of these kdr mutations related to distinct aspects of development and reproduction, in the absence of any other major resistance mechanism. To accomplish this, we initially set up 68 crosses with mosquitoes from a natural population. Allele-specific PCR revealed that one couple, the one originating the CIT-32 strain, had both parents homozygous for both kdr mutations. However, this pyrethroid resistant strain also presented high levels of detoxifying enzymes, which synergistically account for resistance, as revealed by biological and biochemical assays. Therefore, we carried out backcrosses between CIT-32 and Rockefeller (an insecticide susceptible strain for eight generations in order to bring the kdr mutation into a susceptible genetic background. This new strain, named Rock-kdr, was highly resistant to pyrethroid and presented reduced alteration of detoxifying activity. Fitness of the Rock-kdr was then evaluated in comparison with Rockefeller. In this strain, larval development took longer, adults had an increased locomotor activity, fewer females laid eggs, and produced a lower number of eggs. Under an inter-strain competition scenario, the Rock-kdr larvae developed even slower. Moreover, when Rockefeller and Rock-kdr were reared together in population cage experiments during 15 generations in absence of insecticide, the mutant allele decreased in frequency. These results strongly suggest that the Ae. aegypti kdr mutations have a high fitness cost. Therefore, enhanced surveillance for resistance should be priority in localities where the kdr mutation is found before new adaptive alleles can be selected for diminishing the

  4. EHD1 confers resistance to cisplatin in non-small cell lung cancer by regulating intracellular cisplatin concentrations

    International Nuclear Information System (INIS)

    Gao, Jing; Meng, Qingwei; Zhao, Yanbin; Chen, Xuesong; Cai, Li

    2016-01-01

    Non-small cell lung cancer (NSCLC) is one of the most aggressive types of cancer. However, resistance to cisplatin (CDDP) remains a major challenge in NSCLC treatment. The purpose of this study was to investigate the ability of EHD1 [Eps15 homology (EH) domain - containing protein 1] to confer CDDP resistance in NSCLC cells and to investigate mechanisms of this resistance. The associations between EHD1 expression in NSCLC specimens and clinicopathological features, including prognosis, were assessed by immunohistochemistry (IHC). Using DNA microarrays, we performed a genome-wide analysis of cisplatin-resistant NSCLC cells to identify the involvement of the EHD1 gene in this resistance. We overexpressed and knocked down EHD1 in cell lines to investigate the effect of this gene on proliferation and apoptosis. A quantitative analytical method for assessing CDDP in cells was developed. High-performance liquid chromatography was used to measure the concentration of cisplatin in cells. The immunohistochemistry assay showed that adjuvant chemotherapy-treated NSCLC patients expressing EHD1 exhibited reduced OS compared with patients who did not express EHD1 (P = 0.01). Moreover, DNA microarrays indicated that the EHD1 gene was upregulated in CDDP- resistant NSCLC cells. The IC50 value of CDDP in cells that overexpressed EHD1 was 3.3-fold greater than that in the A549-control line, and the IC50 value of EHD1 knockdown cells was at least 5.2-fold lower than that of the control cells, as evidenced by a CCK-8 assay. We found that the percentage of early apoptotic cells was significantly decreased in A549-EHD1 cells, but the rates of early apoptosis were higher in the EHD1 knockdown cell line than in the A549/DDP control line, as indicated by a flow cytometry analysis. High-performance liquid chromatography (HPLC) showed that the total platinum level was lower in A549-EHD1 cells than in control cells, and the concentration of CDDP was higher in the EHD1 knockdown cells than in

  5. Role of PTEN in TNFα induced insulin resistance

    International Nuclear Information System (INIS)

    Bulger, David A.; Conley, Jermaine; Conner, Spencer H.; Majumdar, Gipsy; Solomon, Solomon S.

    2015-01-01

    Aims/hypothesis: PTEN may play a reversible role in TNFα induced insulin resistance, which has been linked to obesity-associated insulin resistance (IR). Methods: Western blots for PTEN and p-Akt were performed on H-411E liver cells incubated with insulin, TNFα, and in selected experiments VO-OHpic vanadium complex in the presence and absence of PTEN siRNA. Total PTEN was compared to β-actin loading control and p-Akt was compared to total Akt. Results: Western blot and Real Time RT-PCR experiments showed increased PTEN after TNFα treatment (p = 0.04); slightly decreased PTEN after insulin treatment; and slightly increased PTEN after insulin + TNFα treatment. PTEN siRNA markedly inhibited the TNFα-induced increase in PTEN (p < 0.01) without significantly changing the p-Akt levels. The vanadium complex, exhibiting insulin-like effects, also significantly prevented the TNFα-induced increase in PTEN. Combining insulin and VO-OHpic was additive, providing both proof of concept and insight into mechanism. Discussion: The PTEN increase due to TNFα treatment was reversible by both PTEN siRNA knockdown and VO-OHpic treatment. Thus, PTEN is identified as a potential new therapeutic target for reducing IR in Type 2 DM. - Highlights: • TNFα treatment induced a significant increase in PTEN in H-411E liver cells. • PTEN siRNA knockdown prevented this effect. • VO-OHpic (vanadium complex) treatment, like insulin, decreased PTEN protein levels. • Thus, PTEN is identified as a potential therapeutic target in DM Type 2

  6. Role of PTEN in TNFα induced insulin resistance

    Energy Technology Data Exchange (ETDEWEB)

    Bulger, David A. [Departments of Medicine and Pharmacology, University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Medicine and Research Services, Veterans Association Medical Center, Memphis, TN 38104 (United States); Wellcome Trust Medical Research Council Institute of Metabolic Science, Cambridge CB2 0QQ (United Kingdom); National Institute of Diabetes & Digestive & Kidney Disease, National Institutes of Health, Bethesda, MD 20892 (United States); Conley, Jermaine [Medicine and Research Services, Veterans Association Medical Center, Memphis, TN 38104 (United States); Conner, Spencer H.; Majumdar, Gipsy [Departments of Medicine and Pharmacology, University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Medicine and Research Services, Veterans Association Medical Center, Memphis, TN 38104 (United States); Solomon, Solomon S., E-mail: ssolomon@uthsc.edu [Departments of Medicine and Pharmacology, University of Tennessee Health Science Center, Memphis, TN 38163 (United States); Medicine and Research Services, Veterans Association Medical Center, Memphis, TN 38104 (United States)

    2015-06-05

    Aims/hypothesis: PTEN may play a reversible role in TNFα induced insulin resistance, which has been linked to obesity-associated insulin resistance (IR). Methods: Western blots for PTEN and p-Akt were performed on H-411E liver cells incubated with insulin, TNFα, and in selected experiments VO-OHpic vanadium complex in the presence and absence of PTEN siRNA. Total PTEN was compared to β-actin loading control and p-Akt was compared to total Akt. Results: Western blot and Real Time RT-PCR experiments showed increased PTEN after TNFα treatment (p = 0.04); slightly decreased PTEN after insulin treatment; and slightly increased PTEN after insulin + TNFα treatment. PTEN siRNA markedly inhibited the TNFα-induced increase in PTEN (p < 0.01) without significantly changing the p-Akt levels. The vanadium complex, exhibiting insulin-like effects, also significantly prevented the TNFα-induced increase in PTEN. Combining insulin and VO-OHpic was additive, providing both proof of concept and insight into mechanism. Discussion: The PTEN increase due to TNFα treatment was reversible by both PTEN siRNA knockdown and VO-OHpic treatment. Thus, PTEN is identified as a potential new therapeutic target for reducing IR in Type 2 DM. - Highlights: • TNFα treatment induced a significant increase in PTEN in H-411E liver cells. • PTEN siRNA knockdown prevented this effect. • VO-OHpic (vanadium complex) treatment, like insulin, decreased PTEN protein levels. • Thus, PTEN is identified as a potential therapeutic target in DM Type 2.

  7. Knockdown manipulator protected against contamination

    International Nuclear Information System (INIS)

    Germond, J.C.; Marchal, Paul; Streif, Gerard; Vertut, Jean; Francois, Daniel.

    1982-01-01

    This device includes protective means comprising a rigid case housing the unit of control motors and not less than one protective sleeve in which is housed an articulated arm. The rigid case includes at least one removable panel on a side opposite that on which the articulated arm is fixed so that the latter and its control unit may be removed from the enclosure after the panel has been opened [fr

  8. Reduction of MLH1 and PMS2 confers temozolomide resistance and is associated with recurrence of glioblastoma.

    Science.gov (United States)

    Shinsato, Yoshinari; Furukawa, Tatsuhiko; Yunoue, Shunji; Yonezawa, Hajime; Minami, Kentarou; Nishizawa, Yukihiko; Ikeda, Ryuji; Kawahara, Kohichi; Yamamoto, Masatatsu; Hirano, Hirofumi; Tokimura, Hiroshi; Arita, Kazunori

    2013-12-01

    Although there is a relationship between DNA repair deficiency and temozolomide (TMZ) resistance in glioblastoma (GBM), it remains unclear which molecule is associated with GBM recurrence. We isolated three TMZ-resistant human GBM cell lines and examined the expression of O6-methylguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR) components. We used immunohistochemical analysis to compare MutL homolog 1 (MLH1), postmeiotic segregation increased 2 (PMS2) and MGMT expression in primary and recurrent GBM specimens obtained from GBM patients during TMZ treatment. We found a reduction in MLH1 expression and a subsequent reduction in PMS2 protein levels in TMZ-resistant cells. Furthermore, MLH1 or PMS2 knockdown confered TMZ resistance. In recurrent GBM tumours, the expression of MLH1 and PMS2 was reduced when compared to primary tumours.

  9. Vorinostat-induced autophagy switches from a death-promoting to a cytoprotective signal to drive acquired resistance.

    Science.gov (United States)

    Dupéré-Richer, D; Kinal, M; Ménasché, V; Nielsen, T H; Del Rincon, S; Pettersson, F; Miller, W H

    2013-02-07

    Histone deacetylase inhibitors (HDACi) have shown promising activity against hematological malignancies in clinical trials and have led to the approval of vorinostat for the treatment of cutaneous T-cell lymphoma. However, de novo or acquired resistance to HDACi therapy is inevitable, and their molecular mechanisms are still unclear. To gain insight into HDACi resistance, we developed vorinostat-resistant clones from the hematological cell lines U937 and SUDHL6. Although cross-resistant to some but not all HDACi, the resistant cell lines exhibit dramatically increased sensitivity toward chloroquine, an inhibitor of autophagy. Consistent with this, resistant cells growing in vorinostat show increased autophagy. Inhibition of autophagy in vorinostat-resistant U937 cells by knockdown of Beclin-1 or Lamp-2 (lysosome-associated membrane protein 2) restores sensitivity to vorinostat. Interestingly, autophagy is also activated in parental U937 cells by de novo treatment with vorinostat. However, in contrast to the resistant cells, inhibition of autophagy decreases sensitivity to vorinostat. These results indicate that autophagy can switch from a proapoptotic signal to a prosurvival function driving acquired resistance. Moreover, inducers of autophagy (such as mammalian target of rapamycin inhibitors) synergize with vorinostat to induce cell death in parental cells, whereas the resistant cells remain insensitive. These data highlight the complexity of the design of combination strategies using modulators of autophagy and HDACi for the treatment of hematological malignancies.

  10. Coxsackie-adenovirus receptor as a novel marker of stem cells in treatment-resistant non-small cell lung cancer

    International Nuclear Information System (INIS)

    Zhang, Xiaochun; Fang, Bingliang; Mohan, Radhe; Chang, Joe Y.

    2012-01-01

    Background: Treatment resistance resulting from the presence of cancer stem cells (CSCs) remains a challenge in cancer treatment. Little is known about possible markers of CSCs in treatment-resistant non-small cell lung cancer (NSCLC). We explored the coxsackie-adenovirus receptor (CAR) as one such marker of CSCs in models of treatment-resistant NSCLC. Materials and methods: Resistant H460 and A549 cell lines were established by repeated exposure to paclitaxel or fractionated radiation. CSC markers were measured by Western blotting and flow cytometry. We also established stable CAR-overexpressing and stable shRNA-CAR-knockdown cell lines and assessed their survival, invasiveness, and tumorigenic capabilities with clonogenic, telomerase, Matrigel, and tumor formation assays. Results: CAR expression was associated with CSC phenotype both in vitro and in vivo. CAR-overexpressing cells were more treatment-resistant, self-renewing, and tumorigenic than were parental cells, and shRNA-mediated knockdown of CAR expression was sufficient to inhibit these functions. CAR expression also correlated with the epithelial–mesenchymal transition. Conclusions: We showed for the first time that CAR is a marker of CSCs and may affect the activities of CSCs in treatment-resistant NSCLC. CAR may prove to be a target for CSC treatment and a predictor of treatment response in patients with NSCLC.

  11. Pyrethroid resistance in an Anopheles funestus population from Uganda.

    Directory of Open Access Journals (Sweden)

    John C Morgan

    2010-07-01

    Full Text Available The susceptibility status of Anopheles funestus to insecticides remains largely unknown in most parts of Africa because of the difficulty in rearing field-caught mosquitoes of this malaria vector. Here we report the susceptibility status of the An. funestus population from Tororo district in Uganda and a preliminary characterisation of the putative resistance mechanisms involved.A new forced egg laying technique used in this study significantly increased the numbers of field-caught females laying eggs and generated more than 4000 F1 adults. WHO bioassays indicated that An. funestus in Tororo is resistant to pyrethroids (62% mortality after 1 h exposure to 0.75% permethrin and 28% mortality to 0.05% deltamethrin. Suspected DDT resistance was also observed with 82% mortality. However this population is fully susceptible to bendiocarb (carbamate, malathion (organophosphate and dieldrin with 100% mortality observed after exposure to each of these insecticides. Sequencing of a fragment of the sodium channel gene containing the 1014 codon conferring pyrethroid/DDT resistance in An. gambiae did not detect the L1014F kdr mutation but a correlation between haplotypes and resistance phenotype was observed indicating that mutations in other exons may be conferring the knockdown resistance in this species. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GSTs, P450s and pNPA compared to a susceptible strain of Anopheles gambiae. RT-PCR further confirmed the involvement of P450s with a 12-fold over-expression of CYP6P9b in the Tororo population compared to the fully susceptible laboratory colony FANG.This study represents the first report of pyrethroid/DDT resistance in An. funestus from East Africa. With resistance already reported in southern and West Africa, this indicates that resistance in An. funestus may be more widespread than previously assumed and therefore this should be taken

  12. Knockdown of hypoxia-inducible factor-1 alpha reduces proliferation, induces apoptosis and attenuates the aggressive phenotype of retinoblastoma WERI-Rb-1 cells under hypoxic conditions.

    Science.gov (United States)

    Xia, Tian; Cheng, Hao; Zhu, Yu

    2014-01-01

    Hypoxia-inducible factor-1 alpha (HIF-1α) plays a critical role in tumor cell adaption to hypoxia by inducing the transcription of numerous genes. The role of HIF-1α in malignant retinoblastoma remains unclear. We analyzed the role of HIF-1α in WERI-Rb-1 retinoblastoma cells under hypoxic conditions. CoCl2 (125 mmol/L) was added to the culture media to mimic hypoxia. HIF-1α was silenced using siRNA. Gene and protein expression were measured by semi-quantitative RT-PCR and Western blotting. Cell cycle and apoptosis were analyzed by flow cytometry. Cell proliferation, adhesion and invasion were assayed using MTT, Transwell invasion, and cell adhesion assays respectively. Hypoxia significantly upregulated HIF-1α protein expression and the HIF-1α target genes VEGF, GLUT1, and Survivin mRNA. HIF-1α mRNA expression was not affected by hypoxia. Transfection of the siRNA expression plasmid pRNAT-CMV3.2/Neo-HIF-1α silenced HIF-1α by approximately 80% in hypoxic WERI-Rb-1 cells. The knockdown of HIF-1α under hypoxic conditions downregulated VEGF, GLUT1, and Survivin mRNA. It also inhibited proliferation, promoted apoptosis, induced the G0/G1 phase cell cycle arrest, and reduced the adhesion and invasion of WERI-Rb-1 cells. HIF-1α plays a major role in the survival and aggressive phenotype of retinoblastoma cells under hypoxic conditions. Targeting HIF-1α may be a promising therapeutic strategy for human malignant retinoblastoma.

  13. Computer modeling of siRNA knockdown effects indicates an essential role of the Ca2+ channel alpha2delta-1 subunit in cardiac excitation-contraction coupling.

    Science.gov (United States)

    Tuluc, Petronel; Kern, Georg; Obermair, Gerald J; Flucher, Bernhard E

    2007-06-26

    L-type Ca(2+) currents determine the shape of cardiac action potentials (AP) and the magnitude of the myoplasmic Ca(2+) signal, which regulates the contraction force. The auxiliary Ca(2+) channel subunits alpha(2)delta-1 and beta(2) are important regulators of membrane expression and current properties of the cardiac Ca(2+) channel (Ca(V)1.2). However, their role in cardiac excitation-contraction coupling is still elusive. Here we addressed this question by combining siRNA knockdown of the alpha(2)delta-1 subunit in a muscle expression system with simulation of APs and Ca(2+) transients by using a quantitative computer model of ventricular myocytes. Reconstitution of dysgenic muscle cells with Ca(V)1.2 (GFP-alpha(1C)) recapitulates key properties of cardiac excitation-contraction coupling. Concomitant depletion of the alpha(2)delta-1 subunit did not perturb membrane expression or targeting of the pore-forming GFP-alpha(1C) subunit into junctions between the outer membrane and the sarcoplasmic reticulum. However, alpha(2)delta-1 depletion shifted the voltage dependence of Ca(2+) current activation by 9 mV to more positive potentials, and it slowed down activation and inactivation kinetics approximately 2-fold. Computer modeling revealed that the altered voltage dependence and current kinetics exert opposing effects on the function of ventricular myocytes that in total cause a 60% prolongation of the AP and a 2-fold increase of the myoplasmic Ca(2+) concentration during each contraction. Thus, the Ca(2+) channel alpha(2)delta-1 subunit is not essential for normal Ca(2+) channel targeting in muscle but is a key determinant of normal excitation and contraction of cardiac muscle cells, and a reduction of alpha(2)delta-1 function is predicted to severely perturb normal heart function.

  14. Gravity-dependent differentiation and root coils in Arabidopsis thaliana wild type and phospholipase-A-I knockdown mutant grown on the International Space Station.

    Science.gov (United States)

    Scherer, G F E; Pietrzyk, P

    2014-01-01

    Arabidopsis roots on 45° tilted agar in 1-g grow in wave-like figures. In addition to waves, formation of root coils is observed in several mutants compromised in gravitropism and/or auxin transport. The knockdown mutant ppla-I-1 of patatin-related phospholipase-A-I is delayed in root gravitropism and forms increased numbers of root coils. Three known factors contribute to waving: circumnutation, gravisensing and negative thigmotropism. In microgravity, deprivation of wild type (WT) and mutant roots of gravisensing and thigmotropism and circumnutation (known to slow down in microgravity, and could potentially lead to fewer waves or increased coiling in both WT and mutant). To resolve this, mutant ppla-I-1 and WT were grown in the BIOLAB facility in the International Space Station. In 1-g, roots of both types only showed waving. In the first experiment in microgravity, the mutant after 9 days formed far more coils than in 1-g but the WT also formed several coils. After 24 days in microgravity, in both types the coils were numerous with slightly more in the mutant. In the second experiment, after 9 days in microgravity only the mutant formed coils and the WT grew arcuated roots. Cell file rotation (CFR) on the mutant root surface in microgravity decreased in comparison to WT, and thus was not important for coiling. Several additional developmental responses (hypocotyl elongation, lateral root formation, cotyledon expansion) were found to be gravity-influenced. We tentatively discuss these in the context of disturbances in auxin transport, which are known to decrease through lack of gravity. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  15. Keap1-knockdown decreases fasting-induced fatty liver via altered lipid metabolism and decreased fatty acid mobilization from adipose tissue.

    Directory of Open Access Journals (Sweden)

    Jialin Xu

    Full Text Available AIMS: The purpose of this study was to determine whether Nrf2 activation, via Keap1-knockdown (Keap1-KD, regulates lipid metabolism and mobilization induced by food deprivation (e.g. fasting. METHODS AND RESULTS: Male C57BL/6 (WT and Keap1-KD mice were either fed ad libitum or food deprived for 24 hours. After fasting, WT mice exhibited a marked increase in hepatic lipid accumulation, but Keap1-KD mice had an attenuated increase of lipid accumulation, along with reduced expression of lipogenic genes (acetyl-coA carboxylase, stearoyl-CoA desaturase-1, and fatty acid synthase and reduced expression of genes related to fatty acid transport, such as fatty acid translocase/CD36 (CD36 and Fatty acid transport protein (FATP 2, which may attribute to the reduced induction of Peroxisome proliferator-activated receptor (Ppar α signaling in the liver. Additionally, enhanced Nrf2 activity by Keap1-KD increased AMP-activated protein kinase (AMPK phosphorylation in liver. In white adipose tissue, enhanced Nrf2 activity did not change the lipolysis rate by fasting, but reduced expression of fatty acid transporters--CD36 and FATP1, via a PPARα-dependent mechanism, which impaired fatty acid transport from white adipose tissue to periphery circulation system, and resulted in increased white adipose tissue fatty acid content. Moreover, enhanced Nrf2 activity increased glucose tolerance and Akt phosphorylation levels upon insulin administration, suggesting Nrf2 signaling pathway plays a key role in regulating insulin signaling and enhanced insulin sensitivity in skeletal muscle. CONCLUSION: Enhanced Nrf2 activity via Keap1-KD decreased fasting-induced steatosis, pointing to an important function of Nrf2 on lipid metabolism under the condition of nutrient deprivation.

  16. A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Marina de Moraes Mourao

    2013-09-01

    Full Text Available Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779, (ii female adult worms (LIBEST_028000: JZ139780 - JZ140379, (iii male adult worms (LIBEST_028001: JZ140380 - JZ141002, (iv eggs (LIBEST_028002: JZ141003 - JZ141497 and (v schistosomula (LIBEST_028003: JZ141498 - JZ141974.

  17. Viral-mediated knockdown of mGluR7 in the nucleus accumbens mediates excessive alcohol drinking and increased ethanol-elicited conditioned place preference in rats.

    Science.gov (United States)

    Bahi, Amine

    2013-10-01

    Whether metabotropic glutamate 7 (mGluR7) -activation enhances or diminishes the reinforcing properties of psychostimulants remains unclear. We have previously shown that systemic mGluR7 activation reduced alcohol consumption and preference as well as locomotor-stimulating and rewarding properties of ethanol. In this study, we further examined the contribution of mGluR7 on the effect of ethanol within the nucleus accumbens (NAcc), a neural target for many drugs of abuse. Using short hairpin RNA (shRNA)-expressing lentiviral vectors (LV) to alter locally the activity of mGluR7 in male rats, we have shown that blocking mGluR7 expression increased ethanol consumption and preference in a two-bottle choice drinking paradigm with no effect either on saccharin or on quinine used for taste discrimination. In addition, mGluR7 knockdown increases preference for environments previously paired with low doses of ethanol in the conditioned place preference (CPP) test, as it shifted the dose-response curve for ethanol CPP to the left, indicating alterations in the rewarding effects of alcohol. More importantly, mGluR7 blockade in the dorsal striatum (DS) neither affected ethanol consumption nor ethanol-elicited CPP. These results show that levels of mGluR7 in the NAcc regulate responsiveness to alcohol. Taken together, these findings clearly demonstrate that mGluR7 signaling within the NAcc is a key modulator of functional responses to ethanol and offer an important target for regulating the addictive effects of alcohol.

  18. Knockdown of a HIF-2α promoter upstream long noncoding RNA impairs colorectal cancer stem cell properties in vitro through HIF-2α downregulation

    Directory of Open Access Journals (Sweden)

    Yao J

    2015-11-01

    Full Text Available Jie Yao,1,* Jianxiong Li,2,* Peiliang Geng,2,* Yi Li,3,* Hong Chen,3 Yunfeng Zhu2 1Department of Oncology, People’s Liberation Army No 161 Hospital, Wuhan, 2Cancer Center, Division of Internal Medicine, Chinese PLA General Hospital, Beijing, 3Department of Oncology, Kunming General Hospital of Chendu Military Command, Kunming, People’s Republic of China *These authors contributed equally to this work Abstract: Currently, various long noncoding RNAs (lncRNAs have been identified as key regulators of multiple cancers. However, cancer stem cell (CSC-related lncRNAs have rarely been reported. In this study, we found an lncRNA that is a promoter upstream transcript of hypoxia-inducible factor-2α (HIF-2α, and we named it “lncRNA-HIF2PUT”. The function of HIF-2α is closely connected with “stem cell-like” properties, and the function of PROMPTs is often associated with the adjacent protein-coding transcripts. Herein, we showed that the expression of lncRNA-HIF2PUT was significantly correlated with HIF-2α in colorectal cancer (CRC tissues. Knockdown of lncRNA-HIF2PUT blocked the HIF-2α expression and inhibited the CSC properties in CRC cell lines DLD-1 and HT29. LncRNA-HIF2PUTsmall interfering RNA transfection resulted in decreased stemness genes expression, impaired colony formation, and spheroid formation ability, retarded migration, and invasion of the cells. These data suggest that lncRNA-HIF2PUT may be a regulator of HIF-2α and a mediator of CSCs in CRC. Keywords: HIF-2α, long noncoding RNA, colorectal cancer, stem cell properties

  19. Network analysis of genes involved in the enhancement of hyperthermia sensitivity by the knockdown of BAG3 in human oral squamous cell carcinoma cells.

    Science.gov (United States)

    Yunoki, Tatsuya; Tabuchi, Yoshiaki; Hayashi, Atsushi; Kondo, Takashi

    2016-07-01

    BCL2-associated athanogene 3 (BAG3), a co-chaperone of the heat shock 70 kDa protein (HSPA) family of proteins, is a cytoprotective protein that acts against various stresses, including heat stress. The aim of the present study was to identify gene networks involved in the enhancement of hyperthermia (HT) sensitivity by the knockdown (KD) of BAG3 in human oral squamous cell carcinoma (OSCC) cells. Although a marked elevation in the protein expression of BAG3 was detected in human the OSCC HSC-3 cells exposed to HT at 44˚C for 90 min, its expression was almost completely suppressed in the cells transfected with small interfering RNA against BAG3 (siBAG) under normal and HT conditions. The silencing of BAG3 also enhanced the cell death that was increased in the HSC-3 cells by exposure to HT. Global gene expression analysis revealed many genes that were differentially expressed by >2-fold in the cells exposed to HT and transfected with siBAG. Moreover, Ingenuity® pathways analysis demonstrated two unique gene networks, designated as Pro-cell death and Anti-cell death, which were obtained from upregulated genes and were mainly associated with the biological functions of induction and the prevention of cell death, respectively. Of note, the expression levels of genes in the Pro-cell death and Anti-cell death gene networks were significantly elevated and reduced in the HT + BAG3-KD group compared to those in the HT control group, respectively. These results provide further insight into the molecular mechanisms involved in the enhancement of HT sensitivity by the silencing of BAG3 in human OSCC cells.

  20. Therapeutic Non-Toxic Doses of TNF Induce Significant Regression in TNFR2-p75 Knockdown Lewis Lung Carcinoma Tumor Implants

    Science.gov (United States)

    Sasi, Sharath P.; Bae, Sanggyu; Song, Jin; Perepletchikov, Aleksandr; Schneider, Douglas; Carrozza, Joseph; Yan, Xinhua; Kishore, Raj; Enderling, Heiko; Goukassian, David A.

    2014-01-01

    Tumor necrosis factor-alpha (TNF) binds to two receptors: TNFR1/p55-cytotoxic and TNFR2/p75-pro-survival. We have shown that tumor growth in p75 knockout (KO) mice was decreased more than 2-fold in Lewis lung carcinoma (LLCs). We hypothesized that selective blocking of TNFR2/p75 LLCs may sensitize them to TNF-induced apoptosis and affect the tumor growth. We implanted intact and p75 knockdown (KD)-LLCs (>90%, using shRNA) into wild type (WT) mice flanks. On day 8 post-inoculation, recombinant murine (rm) TNF-α (12.5 ng/gr of body weight) or saline was injected twice daily for 6 days. Tumor volumes (tV) were measured daily and tumor weights (tW) on day 15, when study was terminated due to large tumors in LLC+TNF group. Tubular bones, spleens and peripheral blood (PB) were examined to determine possible TNF toxicity. There was no significant difference in tV or tW between LLC minus (-) TNF and p75KD/LLC-TNF tumors. Compared to 3 control groups, p75KD/LLC+TNF showed >2-5-fold decreases in tV (ptumors were 100% necrotic, the remaining revealed 40-60% necrosis. No toxicity was detected in bone marrow, spleen and peripheral blood. We concluded that blocking TNFR2/p75 in LLCs combined with intra-tumoral rmTNF injections inhibit LLC tumor growth. This could represent a novel and effective therapy against lung neoplasms and a new paradigm in cancer therapeutics. PMID:24664144

  1. Mechanisms of pyrethroid resistance inHaematobia irritans (Muscidae from Mato Grosso do Sul state, Brazil

    Directory of Open Access Journals (Sweden)

    Antonio Thadeu Medeiros Barros

    Full Text Available Horn fly resistance to pyrethroid insecticides occurs throughout Brazil, but knowledge about the involved mechanisms is still in an incipient stage. This survey was aimed to identify the mechanisms of horn fly resistance to cypermethrin in Mato Grosso do Sul state, Brazil. Impregnated filter paper bioassays using cypermethrin, synergized or not with piperonyl butoxide (PBO and triphenyl phosphate (TPP, were conducted from March 2004 to June 2005 in horn fly populations (n = 33 from all over the state. All populations were highly resistant to cypermethrin, with resistance factors (RF ranging from 89.4 to 1,020.6. Polymerase chain reaction (PCR assays to detect the knockdown resistance (kdr mutation also were performed in 16 samples. The kdr mutation was found in 75% of the tested populations, mostly with relatively low frequencies (<20%, and was absent in some highly resistant populations. Addition of TPP did not significantly reduce the LC50 in any population. However, PBO reduced LC50s above 40-fold in all tested populations, resulting in RFs ≤ 10 in most cases. Horn fly resistance to cypermethrin is widespread in the state, being primarily caused by an enhanced activity of P450 mono-oxygenases and secondarily by reduced target site sensitivity.

  2. Stathmin Mediates Hepatocyte Resistance to Death from Oxidative Stress by down Regulating JNK

    Science.gov (United States)

    Zhao, Enpeng; Amir, Muhammad; Lin, Yu; Czaja, Mark J.

    2014-01-01

    Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK). The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth. PMID:25285524

  3. Stathmin mediates hepatocyte resistance to death from oxidative stress by down regulating JNK.

    Directory of Open Access Journals (Sweden)

    Enpeng Zhao

    Full Text Available Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK. The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth.

  4. Resistance to Powdery Mildews

    DEFF Research Database (Denmark)

    Siwoszek, Agnieszka Izabela

    in majority of them. Resistance to barley powdery mildew in the field is controlled by use of resistant varieties in a combination with fungicides. Early disease management is crucial for effective control. Yet, the pathogen commonly develops fungicide resistance due to simple point mutations. Several studies...... protection. In the present study, I provide an overview of the current knowledge about plant pathogens and plant disease resistance. I use Arabidopsis as a model to investigate the mechanism of non-host resistance, presumed to be the most durable and broad-spectrum form of resistance. I attempt to determine...

  5. Axl receptor tyrosine kinase is up-regulated in metformin resistant prostate cancer cells

    Science.gov (United States)

    Bansal, Nitu; Mishra, Prasun J.; Stein, Mark; DiPaola, Robert S.; Bertino, Joseph R.

    2015-01-01

    Recent epidemiological studies showed that metformin, a widely used anti-diabetic drug might prevent certain cancers. Metformin also has an anti-proliferative effect in preclinical studies of both hematologic malignancies as well as solid cancers and clinical studies testing metformin as an anti-cancer drug are in progress. However, all cancer types do not respond to metformin with the same effectiveness or acquire resistance. To understand the mechanism of acquired resistance and possibly its mechanism of action as an anti-proliferative agent, we developed metformin resistant LNCaP prostate cancer cells. Metformin resistant LNCaP cells had an increased proliferation rate, increased migration and invasion ability as compared to the parental cells, and expressed markers of epithelial-mesenchymal transition (EMT). A detailed gene expression microarray comparing the resistant cells to the wild type cells revealed that Edil2, Ereg, Axl, Anax2, CD44 and Anax3 were the top up-regulated genes and calbindin 2 and TPTE (transmembrane phosphatase with tensin homology) and IGF1R were down regulated. We focused on Axl, a receptor tyrosine kinase that has been shown to be up regulated in several drug resistance cancers. Here, we show that the metformin resistant cell line as well as castrate resistant cell lines that over express Axl were more resistant to metformin, as well as to taxotere compared to androgen sensitive LNCaP and CWR22 cells that do not overexpress Axl. Forced overexpression of Axl in LNCaP cells decreased metformin and taxotere sensitivity and knockdown of Axl in resistant cells increased sensitivity to these drugs. Inhibition of Axl activity by R428, a small molecule Axl kinase inhibitor, sensitized metformin resistant cells that overexpressed Axl to metformin. Inhibitors of Axl may enhance tumor responses to metformin and other chemotherapy in cancers that over express Axl. PMID:26036314

  6. Insecticide resistance in the dengue vector Aedes aegypti from Martinique: distribution, mechanisms and relations with environmental factors.

    Science.gov (United States)

    Marcombe, Sébastien; Mathieu, Romain Blanc; Pocquet, Nicolas; Riaz, Muhammad-Asam; Poupardin, Rodolphe; Sélior, Serge; Darriet, Frédéric; Reynaud, Stéphane; Yébakima, André; Corbel, Vincent; David, Jean-Philippe; Chandre, Fabrice

    2012-01-01

    Dengue is an important mosquito borne viral disease in Martinique Island (French West Indies). The viruses responsible for dengue are transmitted by Aedes aegypti, an indoor day-biting mosquito. The most effective proven method for disease prevention has been by vector control by various chemical or biological means. Unfortunately insecticide resistance has already been observed on the Island and recently showed to significantly reduce the efficacy of vector control interventions. In this study, we investigated the distribution of resistance and the underlying mechanisms in nine Ae. aegypti populations. Statistical multifactorial approach was used to investigate the correlations between insecticide resistance levels, associated mechanisms and environmental factors characterizing the mosquito populations. Bioassays revealed high levels of resistance to temephos and deltamethrin and susceptibility to Bti in the 9 populations tested. Biochemical assays showed elevated detoxification enzyme activities of monooxygenases, carboxylesterases and glutathione S-tranferases in most of the populations. Molecular screening for common insecticide target-site mutations, revealed the presence of the "knock-down resistance" V1016I Kdr mutation at high frequency (>87%). Real time quantitative RT-PCR showed the potential involvement of several candidate detoxification genes in insecticide resistance. Principal Component Analysis (PCA) performed with variables characterizing Ae. aegypti from Martinique permitted to underline potential links existing between resistance distribution and other variables such as agriculture practices, vector control interventions and urbanization. Insecticide resistance is widespread but not homogeneously distributed across Martinique. The influence of environmental and operational factors on the evolution of the resistance and mechanisms are discussed.

  7. Oxidation-resistant cermet

    Science.gov (United States)

    Phillips, W. M.

    1977-01-01

    Chromium metal alloys and chromium oxide ceramic are combined to produce cermets with oxidation-resistant properties. Application of cermets includes use in hot corrosive environments requiring strong resistive materials.

  8. Resistance seam welding

    International Nuclear Information System (INIS)

    Schueler, A.W.

    1977-01-01

    The advantages and disadvantages of the resistance seam welding process are presented. Types of seam welds, types of seam welding machines, seam welding power supplies, resistance seam welding parameters and seam welding characteristics of various metals

  9. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... how antimicrobial resistance both emerges and proliferates among bacteria. Over time, the use of antimicrobial drugs will result in the development of resistant strains of bacteria, complicating clinician's efforts to select the appropriate antimicrobial ...

  10. Electrical Methods: Resistivity Methods

    Science.gov (United States)

    Surface electrical resistivity surveying is based on the principle that the distribution of electrical potential in the ground around a current-carrying electrode depends on the electrical resistivities and distribution of the surrounding soils and rocks.

  11. Insulin Resistance and Prediabetes

    Science.gov (United States)

    ... Your Baby is Born Monogenic Diabetes Insulin Resistance & Prediabetes Insulin resistance and prediabetes occur when your body ... will stay in the healthy range. What is prediabetes? Prediabetes means your blood glucose levels are higher ...

  12. Antimicrobial (Drug) Resistance

    Science.gov (United States)

    ... with facebook share with twitter share with linkedin Antimicrobial (Drug) Resistance Go to Information for Researchers ► Credit: ... and infectious diseases. Why Is the Study of Antimicrobial (Drug) Resistance a Priority for NIAID? Over time, ...

  13. CONFERENCE REPORT ANTIRETROVIRAL RESISTANCE

    African Journals Online (AJOL)

    2004-08-02

    Aug 2, 2004 ... development of new agents with potential clinical utility for treating resistant ... data on the emergence of resistance among Thai women given zidovudine ... had achieved full virological suppression (viral load. < 50 copies/ml).

  14. Resistance to Linezolid

    DEFF Research Database (Denmark)

    Vester, Birte; Ntokou, Eleni

    2017-01-01

    Linezolid is an antimicrobial agent that binds to the bacterial ribosome and thereby inhibits protein synthesis. Soon after its release as a clinical drug, it became clear that bacteria could become resistant to linezolid. The resistance mechanisms are mainly causing alteration of the drug target...... site, but probably efflux might also play a role. The resistance is still rare in surveillance studies, but outbreaks of resistant clones from hospitals have been observed. So far the main mechanisms of resistance are occurrence of mutations in ribosomal genes or obtaining plasmids with a gene coding...... for a methyltransferase providing resistance. The most obvious way to avoid resistance may be development of derivatives of linezolid overcoming the known resistance mechanisms....

  15. Knockdown of ventral tegmental area mu-opioid receptors in rats prevents effects of social defeat stress: Implications for amphetamine cross-sensitization, social avoidance, weight regulation and expression of brain-derived neurotrophic factor

    Science.gov (United States)

    Johnston, Caitlin E.; Herschel, Daniel; Lasek, Amy W.; Hammer, Ronald P.; Nikulina, Ella M.

    2014-01-01

    Social defeat stress causes social avoidance and long-lasting cross-sensitization to psychostimulants, both of which are associated with increased brain-derived neurotrophic factor (BDNF) expression in the ventral tegmental area (VTA). Moreover, social stress upregulates VTA mu-opioid receptor (MOR) mRNA. In the VTA, MOR activation inhibits GABA neurons to disinhibit VTA dopamine neurons, thus providing a role for VTA MORs in the regulation of psychostimulant sensitization. The present study determined the effect of lentivirus-mediated MOR knockdown in the VTA on the consequences of intermittent social defeat stress, a salient and profound stressor in humans and rodents. Social stress exposure induced social avoidance and attenuated weight gain in animals with non-manipulated VTA MORs, but both these effects were prevented by VTA MOR knockdown. Rats with non-manipulated VTA MOR expression exhibited cross-sensitization to amphetamine challenge (1.0 mg/kg, i.p.), evidenced by a significant augmentation of locomotion. By contrast, knockdown of VTA MORs prevented stress-induced cross-sensitization without blunting the locomotor-activating effects of amphetamine. At the time point corresponding to amphetamine challenge, immunohistochemical analysis was performed to examine the effect of stress on VTA BDNF expression. Prior stress exposure increased VTA BDNF expression in rats with non-manipulated VTA MOR expression, while VTA MOR knockdown prevented stress-induced expression of VTA BDNF. Taken together, these results suggest that upregulation of VTA MOR is necessary for the behavioral and biochemical changes induced by social defeat stress. Elucidating VTA MOR regulation of stress effects on the mesolimbic system may provide new therapeutic targets for treating stress-induced vulnerability to substance abuse. PMID:25446676

  16. Resisting Organizational Change

    OpenAIRE

    Andersson, Gunnar

    2015-01-01

    We are continuously reminded of how change induces controversy and resistance, regardless of support. We repeatedly experience resistance in difficulties of implementation, little progress, and poor results, rather than increased productivity as anticipated. In a detailed account of how change plays out, a mosaic of what resistance looks like emerges. The picture is both familiar and absolutely concrete, and challenges the structural assumptions and dichotomies on support and resistance in an...

  17. Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda.

    Directory of Open Access Journals (Sweden)

    Renato A Carvalho

    Full Text Available The fall armyworm Spodoptera frugiperda is an economically important pest of small grain crops that occurs in all maize growing regions of the Americas. The intensive use of chemical pesticides for its control has led to the selection of resistant populations, however, to date, the molecular mechanisms underlying resistance have not been characterised. In this study the mechanisms involved in the resistance of two S. frugiperda strains collected in Brazil to chlorpyrifos (OP strain or lambda-cyhalothrin (PYR strain were investigated using molecular and genomic approaches. To examine the possible role of target-site insensitivity the genes encoding the organophosphate (acetylcholinesterase, AChE and pyrethroid (voltage-gated sodium channel, VGSC target-site proteins were PCR amplified. Sequencing of the S. frugiperda ace-1 gene identified several nucleotide changes in the OP strain when compared to a susceptible reference strain (SUS. These result in three amino acid substitutions, A201S, G227A and F290V, that have all been shown previously to confer organophosphate resistance in several other insect species. Sequencing of the gene encoding the VGSC in the PYR strain, identified mutations that result in three amino acid substitutions, T929I, L932F and L1014F, all of which have been shown previously to confer knockdown/super knockdown-type resistance in several arthropod species. To investigate the possible role of metabolic detoxification in the resistant phenotype of the OP and PYR stains all EST sequences available for S. frugiperda were used to design a gene-expression microarray. This was then used to compare gene expression in the resistant strains with the susceptible reference strain. Members of several gene families, previously implicated in metabolic resistance in other insects were found to be overexpressed in the resistant strains including glutathione S-transferases, cytochrome P450s and carboxylesterases. Taken together these results

  18. Investigating the molecular mechanisms of organophosphate and pyrethroid resistance in the fall armyworm Spodoptera frugiperda.

    Science.gov (United States)

    Carvalho, Renato A; Omoto, Celso; Field, Linda M; Williamson, Martin S; Bass, Chris

    2013-01-01

    The fall armyworm Spodoptera frugiperda is an economically important pest of small grain crops that occurs in all maize growing regions of the Americas. The intensive use of chemical pesticides for its control has led to the selection of resistant populations, however, to date, the molecular mechanisms underlying resistance have not been characterised. In this study the mechanisms involved in the resistance of two S. frugiperda strains collected in Brazil to chlorpyrifos (OP strain) or lambda-cyhalothrin (PYR strain) were investigated using molecular and genomic approaches. To examine the possible role of target-site insensitivity the genes encoding the organophosphate (acetylcholinesterase, AChE) and pyrethroid (voltage-gated sodium channel, VGSC) target-site proteins were PCR amplified. Sequencing of the S. frugiperda ace-1 gene identified several nucleotide changes in the OP strain when compared to a susceptible reference strain (SUS). These result in three amino acid substitutions, A201S, G227A and F290V, that have all been shown previously to confer organophosphate resistance in several other insect species. Sequencing of the gene encoding the VGSC in the PYR strain, identified mutations that result in three amino acid substitutions, T929I, L932F and L1014F, all of which have been shown previously to confer knockdown/super knockdown-type resistance in several arthropod species. To investigate the possible role of metabolic detoxification in the resistant phenotype of the OP and PYR stains all EST sequences available for S. frugiperda were used to design a gene-expression microarray. This was then used to compare gene expression in the resistant strains with the susceptible reference strain. Members of several gene families, previously implicated in metabolic resistance in other insects were found to be overexpressed in the resistant strains including glutathione S-transferases, cytochrome P450s and carboxylesterases. Taken together these results provide

  19. The knockdown of each component of the cysteine proteinase-adhesin complex of Entamoeba histolytica (EhCPADH) affects the expression of the other complex element as well as the in vitro and in vivo virulence.

    Science.gov (United States)

    Ocádiz-Ruiz, Ramón; Fonseca, Wendy; Linford, Alicia S; Yoshino, Timothy P; Orozco, Esther; Rodríguez, Mario A

    2016-01-01

    Entamoeba histolytica is the protozoan parasite causative of human amoebiasis, disease responsible for 40 000-100 000 deaths annually. The cysteine proteinase-adhesin complex of this parasite (EhCPADH) is a heterodimeric protein formed by a cysteine protease (EhCP112) and an adhesin (EhADH) that plays an important role in the cytopathic mechanism of this parasite. The coding genes for EhCP112 and EhADH are adjacent in the E. histolytica genome, suggesting that their expression may be co-regulated, but this hypothesis has not yet been confirmed. Here, we performed the knockdown of EhCP112 and EhADH using gene-specific short-hairpin RNAs (shRNA), and the effect of these knockdowns on the expression of both complex components as well as on the in vitro and in vivo virulence was analysed. Results showed that the knockdown of one of the EhCPADH components produced a simultaneous downregulation of the other protein. Accordingly, a concomitant reduction in the overall expression of the complex was observed. The downregulation of each component also produced a significant decrease in the in vitro and in vivo virulence of trophozoites. These results demonstrated that the expression of EhCP112 and EhADH is co-regulated and confirmed that the EhCPADH complex plays an important role in E. histolytica virulence.

  20. Antibiotic resistance reservoirs

    NARCIS (Netherlands)

    Versluis, Dennis

    2016-01-01

    One of the major threats to human health in the 21st century is the emergence of pathogenic bacteria that are resistant to multiple antibiotics, thereby limiting treatment options. An important route through which pathogens become resistant is via acquisition of resistance genes from

  1. Resisting Mind Control.

    Science.gov (United States)

    Anderson, Susan M.; Zimbardo, Philip G.

    1980-01-01

    Provides conceptual analyses of mind control techniques along with practical advice on how to resist these techniques. The authors stress that effective mind control stems more from everyday social relations than from exotic technological gimmicks. Suggestions are given for resisting persuasion, resisting systems, and challenging the system.…

  2. Lentivirus-Mediated Knockdown of Astrocyte Elevated Gene-1 Inhibits Growth and Induces Apoptosis through MAPK Pathways in Human Retinoblastoma Cells.

    Directory of Open Access Journals (Sweden)

    Ying Chang

    Full Text Available To explore expression and function of astrocyte elevated gene-1 (AEG-1 in human retinoblastoma (RB.The expression of AEG-1 in histological sections of human RBs and in RB cell lines was examined using immunohistochemical staining and RT-PCR and Western blotting respectively. We knocked down AEG-1 gene levels by AEG-1-siRNA lentivirus transfection of human RB cell lines SO-RB50 and Y79, and using an MTT assay, we assessed the role of AEG-1 on RB cell proliferation. The biological significance of lentivirus transfection induced AEG-1 down-regulation was examined by assessing the apoptosis rate in the transfected RB cells by Annexin V-APC staining and flow cytometry. We additionally measured the expression of Bcl-2, Bax, cleaved-caspase-3 and caspase-3, and the phosphorylation and non-phosphorylation alternation of MAPKs.AEG-1 expression was detected to be strongly positive in the histological slides of 35 out of 54 (65% patients with RB. AEG-1 expression increased significantly (P<0.05 with tumor stage. In the RB cell lines SO-RB50, Y79 and WERI-RB1 as compared with retinal pigment epithelium cells, expression of AEG-1 mRNA and AEG-1 protein was significantly higher. In AEG-1-siRNA lentivirus transfected cell cultures as compared with negative control lentivirus transfected cell cultures, levels of AEG-1 mRNA and of AEG-1 protein (P<0.05 and cell growth rates (P<0.01 were significantly lower, and apoptosis rate (P<0.001, Bax/Bcl-2 ratio and cleaved-caspase-3 protein level were significantly increased. The P-ERK/ERK ratio was significantly decreased in the AEG-1-siRNA lentivirus transfected cell lines.Expression of AEG-1 was associated with RB, in histological slides of patients and in cell culture experiments. Lentivirus transfection induced knockdown of AEG-1 had a tumor suppressive effect, potentially by tumor cell apoptosis induction through inhibition of ERK.

  3. Knockdown of SCF(Skp2 function causes double-parked accumulation in the nucleus and DNA re-replication in Drosophila plasmatocytes.

    Directory of Open Access Journals (Sweden)

    Paul T Kroeger

    Full Text Available In Drosophila, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, we considered data from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. eater-Gal4;UAS-GFP flies were crossed to UAS-RNAi lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like, SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCF(Skp2 complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division

  4. Apoptosis induced by knockdown of uPAR and MMP-9 is mediated by inactivation of EGFR/STAT3 signaling in medulloblastoma.

    Directory of Open Access Journals (Sweden)

    Ramaprasada Rao Kotipatruni

    Full Text Available Medulloblastoma is a highly invasive cancer of central nervous system diagnosed mainly in children. Matrix metalloproteinase-9 (MMP-9 and urokinase plasminogen activator receptor (uPAR are over expressed in several cancers and well established for their roles in tumor progression. The present study is aimed to determine the consequences of targeting these molecules on medulloblastoma progression.Radiation is one of the foremost methods applied for treating cancer and considerable evidence showed that radiation elevated uPAR and MMP-9 expression in medulloblastoma cell. Therefore efforts are made to target these molecules in non-irradiated and irradiated medulloblastoma cells. Our results showed that siRNA-mediated knockdown of uPAR and MMP-9, either alone or in combination with radiation modulated a series of events leading to apoptosis. Down regulation of uPAR and MMP-9 inhibited the expression of anti-apoptotic molecules like Bcl-2, Bcl-xL, survivin, XIAP and cIAPI; activated BID cleavage, enhanced the expression of Bak and translocated cyctochrome C to cytosol. Capsase-3 and -9 activities were also increased in uPAR- and MMP-9-downregulated cells. The apoptosis induced by targeting MMP-9 and uPAR was initiated by inhibiting epidermal growth factor receptor (EGFR mediated activation of STAT3 and NF-κB related signaling molecules. Silencing uPAR and MMP-9 inhibited DNA binding activity of STAT3 and also reduced the recruitment of STAT3 protein at the promoter region of Bcl-2 and survivin genes. Our results suggest that inhibiting uPAR and MMP-9 reduced the expression of anti-apoptotic molecules by inactivating the transcriptional activity of STAT3. In addition, treating pre-established medulloblastoma with siRNAs against uPAR and MMP-9 both alone or in combination with radiation suppressed uPAR, MMP-9, EGFR, STAT3 expression and induced Bak activation leading to apoptosis.Taken together, our results illustrated that RNAi mediated targeting of

  5. DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells.

    Science.gov (United States)

    Hu, Hao; Gu, Yuanlong; Qian, Yi; Hu, Benshun; Zhu, Congyuan; Wang, Gaohe; Li, Jianping

    2014-09-12

    Pancreatic cancer is one of the most aggressive human malignancies with extremely poor prognosis. The moderate activity of the current standard gemcitabine and gemcitabine-based regimens was due to pre-existing or acquired chemo-resistance of pancreatic cancer cells. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in gemcitabine resistance, and studied the underlying mechanisms. We found that NU-7026 and NU-7441, two DNA-PKcs inhibitors, enhanced gemcitabine-induced cytotoxicity and apoptosis in PANC-1 pancreatic cancer cells. Meanwhile, PANC-1 cells with siRNA-knockdown of DNA-PKcs were more sensitive to gemcitabine than control PANC-1 cells. Through the co-immunoprecipitation (Co-IP) assay, we found that DNA-PKcs formed a complex with SIN1, the latter is an indispensable component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). DNA-PKcs-SIN1 complexation was required for Akt activation in PANC-1 cells, while inhibition of this complex by siRNA knockdown of DNA-PKcs/SIN1, or by DNA-PKcs inhibitors, prevented Akt phosphorylation in PANC-1 cells. Further, SIN1 siRNA-knockdown also facilitated gemcitabine-induced apoptosis in PANC-1 cells. Finally, DNA-PKcs and p-Akt expression was significantly higher in human pancreatic cancer tissues than surrounding normal tissues. Together, these results show that DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Harnessing the p53-PUMA Axis to Overcome DNA Damage Resistance in Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Xiaoguang Zhou

    2014-12-01

    Full Text Available Resistance to DNA damage–induced apoptosis is a hallmark of cancer and a major cause of treatment failure and lethal disease outcome. A tumor entity that is largely resistant to DNA-damaging therapies including chemo- or radiotherapy is renal cell carcinoma (RCC. This study was designed to explore the underlying molecular mechanisms of DNA damage resistance in RCC to develop strategies to resensitize tumor cells to DNA damage–induced apoptosis. Here, we show that apoptosis-resistant RCC cells have a disconnect between activation of p53 and upregulation of the downstream proapoptotic protein p53 upregulated modulator of apoptosis (PUMA. We demonstrate that this disconnect is not caused by gene-specific repression through CCCTC-binding factor (CTCF but instead by aberrant chromatin compaction. Treatment with an HDAC inhibitor was found to effectively reactivate PUMA expression on the mRNA and protein level and to revert resistance to DNA damage–induced cell death. Ectopic expression of PUMA was found to resensitize a panel of RCC cell lines to four different DNA-damaging agents tested. Remarkably, all RCC cell lines analyzed were wild-type for p53, and a knockdown was likewise able to sensitize RCC cells to acute genotoxic stress. Taken together, our results indicate that DNA damage resistance in RCC is reversible, involves the p53-PUMA axis, and is potentially targetable to improve the oncological outcomes of RCC patients.

  7. Aberrant Long Noncoding RNAs Expression Profiles Affect Cisplatin Resistance in Lung Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Lijuan Hu

    2017-01-01

    Full Text Available Background. Long noncoding RNAs (lncRNAs have been shown to be involved in the mechanism of cisplatin resistance in lung adenocarcinoma (LAD. However, the roles of lncRNAs in cisplatin resistance in LAD are not well understood. Methods. We used a high-throughput microarray to compare the lncRNA and mRNA expression profiles in cisplatin resistance cell A549/DDP and cisplatin sensitive cell A549. Several candidate cisplatin resistance-associated lncRNAs were verified by real-time quantitative reverse transcription polymerase chain reaction (PCR analysis. Results. We found that 1,543 lncRNAs and 1,713 mRNAs were differentially expressed in A549/DDP cell and A549 cell, hinting that many lncRNAs were irregular from cisplatin resistance in LAD. We also obtain the fact that 12 lncRNAs were aberrantly expressed in A549/DDP cell compared with A549 cell by quantitative PCR. Among these, UCA1 was the aberrantly expressed lncRNA and can significantly reduce the IC50 of cisplatin in A549/DDP cell after knockdown, while it can increase the IC50 of cisplatin after UCA1 was overexpressed in NCI-H1299. Conclusions. We obtained patterns of irregular lncRNAs and they may play a key role in cisplatin resistance of LAD.

  8. Nuclear respiratory factor-1 and bioenergetics in tamoxifen-resistant breast cancer cells

    International Nuclear Information System (INIS)

    Radde, Brandie N.; Ivanova, Margarita M.; Mai, Huy Xuan; Alizadeh-Rad, Negin; Piell, Kellianne; Van Hoose, Patrick; Cole, Marsha P.; Muluhngwi, Penn; Kalbfleisch, Ted S.; Rouchka, Eric C.; Hill, Bradford G.; Klinge, Carolyn M.

    2016-01-01

    Acquired tamoxifen (TAM) resistance is a significant clinical problem in treating patients with estrogen receptor α (ERα)+ breast cancer. We reported that ERα increases nuclear respiratory factor-1 (NRF-1), which regulates nuclear-encoded mitochondrial gene transcription, in MCF-7 breast cancer cells and NRF-1 knockdown stimulates apoptosis. Whether NRF-1 and target gene expression is altered in endocrine resistant breast cancer cells is unknown. We measured NRF-1and metabolic features in a cell model of progressive TAM-resistance. NRF-1 and its target mitochondrial transcription factor A (TFAM) were higher in TAM-resistant LCC2 and LCC9 cells than TAM-sensitive MCF-7 cells. Using extracellular flux assays we observed that LCC1, LCC2, and LCC9 cells showed similar oxygen consumption rate (OCR), but lower mitochondrial reserve capacity which was correlated with lower Succinate Dehydrogenase Complex, Subunit B in LCC1 and LCC2 cells. Complex III activity was lower in LCC9 than MCF-7 cells. LCC1, LCC2, and LCC9 cells had higher basal extracellular acidification (ECAR), indicating higher aerobic glycolysis, relative to MCF-7 cells. Mitochondrial bioenergetic responses to estradiol and 4-hydroxytamoxifen were reduced in the endocrine-resistant cells compared to MCF-7 cells. These results suggest the acquisition of altered metabolic phenotypes in response to long term antiestrogen treatment may increase vulnerability to metabolic stress. - Highlights: • NRF-1 and TFAM expression are higher in endocrine-resistant breast cancer cells. • Oxygen consumption rate is similar in endocrine-sensitive and resistant cells. • Mitochondrial reserve capacity is lower in endocrine-resistant cells. • Endocrine-resistant breast cancer cells have increased glycolysis. • Bioenergetic responses to E2 and tamoxifen are lower in endocrine-resistant cells.

  9. Nuclear respiratory factor-1 and bioenergetics in tamoxifen-resistant breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Radde, Brandie N.; Ivanova, Margarita M.; Mai, Huy Xuan; Alizadeh-Rad, Negin; Piell, Kellianne; Van Hoose, Patrick; Cole, Marsha P.; Muluhngwi, Penn; Kalbfleisch, Ted S. [Department of Biochemistry & Molecular Genetics, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, KY 40292 (United States); Rouchka, Eric C. [Bioinformatics and Biomedical Computing Laboratory, Department of Computer Engineering and Computer Science, University of Louisville, Louisville, KY 40292 (United States); Hill, Bradford G. [Department of Medicine, University of Louisville School of Medicine, Louisville, KY 40292 (United States); Klinge, Carolyn M., E-mail: carolyn.klinge@louisville.edu [Department of Biochemistry & Molecular Genetics, Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, KY 40292 (United States)

    2016-09-10

    Acquired tamoxifen (TAM) resistance is a significant clinical problem in treating patients with estrogen receptor α (ERα)+ breast cancer. We reported that ERα increases nuclear respiratory factor-1 (NRF-1), which regulates nuclear-encoded mitochondrial gene transcription, in MCF-7 breast cancer cells and NRF-1 knockdown stimulates apoptosis. Whether NRF-1 and target gene expression is altered in endocrine resistant breast cancer cells is unknown. We measured NRF-1and metabolic features in a cell model of progressive TAM-resistance. NRF-1 and its target mitochondrial transcription factor A (TFAM) were higher in TAM-resistant LCC2 and LCC9 cells than TAM-sensitive MCF-7 cells. Using extracellular flux assays we observed that LCC1, LCC2, and LCC9 cells showed similar oxygen consumption rate (OCR), but lower mitochondrial reserve capacity which was correlated with lower Succinate Dehydrogenase Complex, Subunit B in LCC1 and LCC2 cells. Complex III activity was lower in LCC9 than MCF-7 cells. LCC1, LCC2, and LCC9 cells had higher basal extracellular acidification (ECAR), indicating higher aerobic glycolysis, relative to MCF-7 cells. Mitochondrial bioenergetic responses to estradiol and 4-hydroxytamoxifen were reduced in the endocrine-resistant cells compared to MCF-7 cells. These results suggest the acquisition of altered metabolic phenotypes in response to long term antiestrogen treatment may increase vulnerability to metabolic stress. - Highlights: • NRF-1 and TFAM expression are higher in endocrine-resistant breast cancer cells. • Oxygen consumption rate is similar in endocrine-sensitive and resistant cells. • Mitochondrial reserve capacity is lower in endocrine-resistant cells. • Endocrine-resistant breast cancer cells have increased glycolysis. • Bioenergetic responses to E2 and tamoxifen are lower in endocrine-resistant cells.

  10. BAG3-mediated Mcl-1 stabilization contributes to drug resistance via interaction with USP9X in ovarian cancer.

    Science.gov (United States)

    Habata, Shutaro; Iwasaki, Masahiro; Sugio, Asuka; Suzuki, Miwa; Tamate, Masato; Satohisa, Seiro; Tanaka, Ryoichi; Saito, Tsuyoshi

    2016-07-01

    Paclitaxel in combination with carboplatin improves survival among patients with susceptible ovarian cancers, but no strategy has been established against resistant ovarian cancers. BAG3 (Bcl-2-associated athanogene 3) is one of six BAG family proteins, which are involved in such cellular processes as proliferation, migration and apoptosis. In addition, expression of BAG3 with Mcl-1, a Bcl-2 family protein, reportedly associates with resistance to chemotherapy. Our aim in this study was to evaluate the functional role of BAG3 and Mcl-1 in ovarian cancer chemoresistance and explore possible new targets for treatment. We found that combined expression of BAG3 and Mcl-1 was significantly associated with a poor prognosis in ovarian cancer patients. In vitro, BAG3 knockdown in ES2 clear ovarian cancer cells significantly increased the efficacy of paclitaxel in combination with the Mcl-1 antagonist MIM1, with or without the Bcl-2 family antagonist ABT737. Moreover, BAG3 was found to positively regulate Mcl-1 levels by binding to and inhibiting USP9X. Our data show that BAG3 and Mcl-1 are key mediators of resistance to chemotherapy in ovarian cancer. In BAG3 knockdown ES2 clear ovarian cancer cells, combination with ABT737 and MIM1 enhanced the efficacy of paclitaxel. These results suggest that inhibiting BAG3 in addition to anti-apoptotic Bcl-2 family proteins may be a useful therapeutic strategy for the treatment of chemoresistant ovarian cancers.

  11. Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

    International Nuclear Information System (INIS)

    Williamson, K.; Dickey, B.F.; Pyun, H.Y.; Navarro, J.

    1988-01-01

    The authors describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [ 3 H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [ 3 H]fMET-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, they also demonstrated fMET-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils

  12. Activation and regulation of arachidonic acid release in rabbit peritoneal neutrophils

    International Nuclear Information System (INIS)

    Tao, W.

    1988-01-01

    Arachidonic acid release in rabbit neutrophils can be enhanced by the addition of chemotactic fMet-Leu-Phe, platelet-activating factor, PAF, or the calcium ionophore A23187. Over 80% of the release [ 3 H]arachidonic acid comes from phosphatidylcholine and phosphatidylinositol. The release is dose-dependent and increases with increasing concentration of the stimulus. The A23187-induced release increases with increasing time of the stimulation. [ 3 H]arachidonic acid release, but not the rise in the concentration of intracellular calcium, is inhibited in pertussis toxin-treated neutrophils stimulated with PAF. The [ 3 H]arachidonic acid released by A23187 is potentiated while that release by fMET-Leu-Phe or PAF is inhibited in phorbol 12-myristate 13-acetate, PMA, treated rabbit neutrophils. The protein kinase C inhibitor 1-(5-isoquinoline sulfonyl)-2-methylpiperazine, H-7, has no effect on the potentiation by PMA of the A23187-induced release, it prevents the inhibition by PMA of the release produced by PAF or fMet-Leu-Phe. In addition, PMA increases arachidonic acid release in H-7-treated cells stimulated with fMet-Leu-Phe. The diacylglycerol kinase inhibitor R59022 increases the level of diacylglycerol in neutrophils stimulated with fMet-Leu-Phe. Furthermore, R59022 potentiates [ 3 H] arachidonic acid release produced by fMet-Leu-Phe. This potentiation is not inhibited by H-7, in fact, it is increased in H-7-treated neutrophils

  13. Overexpression of cytochrome P450 CYP6BG1 may contribute to chlorantraniliprole resistance in Plutella xylostella (L.).

    Science.gov (United States)

    Li, Xiuxia; Li, Ran; Zhu, Bin; Gao, Xiwu; Liang, Pei

    2018-06-01

    The diamondback moth Plutella xylostella (L.) is the most widely distributed pest of cruciferous crops and has developed resistance to most commonly used insecticides, including chlorantraniliprole. Resistance to chlorantraniliprole is likely caused by mutations of the target, the ryanodine receptor, and/or mediated by an increase in detoxification enzyme activities. Although target-site resistance is documented in detail, resistance mediated by increased metabolism has rarely been reported. The activity of cytochrome P450 was significantly higher in two resistant P. xylostella populations than in a susceptible one. Among ten detected cytochrome P450 genes, CYP6BG1 was significantly overexpressed (over 80-fold) in a field-resistant population compared with expression in a susceptible one. Knockdown of CYP6BG1 by RNA interference dramatically reduced the 7-ethoxycoumarin-O-deethylase (7-ECOD) activity of P450 by 45.5% and increased the toxicity of chlorantraniliprole toward P. xylostella by 26.8% at 48 h postinjection of double-stranded RNA. By contrast, overexpression of CYP6BG1 in a transgenic Drosophila melanogaster line significantly decreased the toxicity of the insecticide to the transgenic flies. Overexpression of CYP6BG1 may contribute to chlorantraniliprole resistance in P. xylostella. Our findings will provide new insights into the mechanisms of resistance to diamide insecticides in other insect pests. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  14. Methylation of WTH3, a possible drug resistant gene, inhibits p53 regulated expression

    International Nuclear Information System (INIS)

    Tian, Kegui; Wang, Yuezeng; Huang, Yu; Sun, Boqiao; Li, Yuxin; Xu, Haopeng

    2008-01-01

    Previous results showed that over-expression of the WTH3 gene in MDR cells reduced MDR1 gene expression and converted their resistance to sensitivity to various anticancer drugs. In addition, the WTH3 gene promoter was hypermethylated in the MCF7/AdrR cell line and primary drug resistant breast cancer epithelial cells. WTH3 was also found to be directly targeted and up regulated by the p53 gene. Furthermore, over expression of the WTH3 gene promoted the apoptotic phenotype in various host cells. To further confirm WTH3's drug resistant related characteristics, we recently employed the small hairpin RNA (shRNA) strategy to knockdown its expression in HEK293 cells. In addition, since the WTH3 promoter's p53-binding site was located in a CpG island that was targeted by methylation, we were interested in testing the possible effect this epigenetic modification had on the p53 transcription factor relative to WTH3 expression. To do so, the in vitro methylation method was utilized to examine the p53 transgene's influence on either the methylated or non-methylated WTH3 promoter. The results generated from the gene knockdown strategy showed that reduction of WTH3 expression increased MDR1 expression and elevated resistance to Doxorubicin as compared to the original control cells. Data produced from the methylation studies demonstrated that DNA methylation adversely affected the positive impact of p53 on WTH3 promoter activity. Taken together, our studies provided further evidence that WTH3 played an important role in MDR development and revealed one of its transcription regulatory mechanisms, DNA methylation, which antagonized p53's positive impact on WTH3 expression

  15. Challenges to Resistance Welding

    DEFF Research Database (Denmark)

    Song, Quanfeng

    This report originates from the compulsory defense during my Ph.D. study at the Technical University of Denmark. Resistance welding is an old and well-proven technology. Yet the emergence of more and more new materials, new designs, invention off new joining techniques, and more stringent...... requirement in quality have imposed challenges to the resistance welding. More some research and development have to be done to adapt the old technology to the manufacturing industry of the 21st century. In the 1st part of the report, the challenging factors to the resistance welding are reviewed. Numerical...... simulation of resistance welding has been under development for many years. Yet it is no easy to make simulation results reliable and accurate because of the complexity of resistance welding process. In the 2nd part of the report numerical modeling of resistance welding is reviewed, some critical factors...

  16. Multidrug-Resistant Candida

    DEFF Research Database (Denmark)

    Arendrup, Maiken Cavling; Patterson, Thomas F

    2017-01-01

    Invasive Candida infections remain an important cause of morbidity and mortality, especially in hospitalized and immunocompromised or critically ill patients. A limited number of antifungal agents from only a few drug classes are available to treat patients with these serious infections. Resistance...... can be either intrinsic or acquired. Resistance mechanisms are not exchanged between Candida; thus, acquired resistance either emerges in response to an antifungal selection pressure in the individual patient or, more rarely, occur due to horizontal transmission of resistant strains between patients....... Although multidrug resistance is uncommon, increasing reports of multidrug resistance to the azoles, echinocandins, and polyenes have occurred in several Candida species, most notably Candida glabrata and more recently Candida auris. Drivers are overall antifungal use, subtherapeutic drug levels at sites...

  17. WNT4 mediates estrogen receptor signaling and endocrine resistance in invasive lobular carcinoma cell lines.

    Science.gov (United States)

    Sikora, Matthew J; Jacobsen, Britta M; Levine, Kevin; Chen, Jian; Davidson, Nancy E; Lee, Adrian V; Alexander, Caroline M; Oesterreich, Steffi

    2016-09-20

    Invasive lobular carcinoma (ILC) of the breast typically presents with clinical biomarkers consistent with a favorable response to endocrine therapies, and over 90 % of ILC cases express the estrogen receptor (ER). However, a subset of ILC cases may be resistant to endocrine therapies, suggesting that ER biology is unique in ILC. Using ILC cell lines, we previously demonstrated that ER regulates a distinct gene expression program in ILC cells, and we hypothesized that these ER-driven pathways modulate the endocrine response in ILC. One potential novel pathway is via the Wnt ligand WNT4, a critical signaling molecule in mammary gland development regulated by the progesterone receptor. The ILC cell lines MDA-MB-134-VI, SUM44PE, and BCK4 were used to assess WNT4 gene expression and regulation, as well as the role of WNT4 in estrogen-regulated proliferation. To assess these mechanisms in the context of endocrine resistance, we developed novel ILC endocrine-resistant long-term estrogen-deprived (ILC-LTED) models. ILC and ILC-LTED cell lines were used to identify upstream regulators and downstream signaling effectors of WNT4 signaling. ILC cells co-opted WNT4 signaling by placing it under direct ER control. We observed that ER regulation of WNT4 correlated with use of an ER binding site at the WNT4 locus, specifically in ILC cells. Further, WNT4 was required for endocrine response in ILC cells, as WNT4 knockdown blocked estrogen-induced proliferation. ILC-LTED cells remained dependent on WNT4 for proliferation, by either maintaining ER function and WNT4 regulation or uncoupling WNT4 from ER and upregulating WNT4 expression. In the latter case, WNT4 expression was driven by activated nuclear factor kappa-B signaling in ILC-LTED cells. In ILC and ILC-LTED cells, WNT4 led to suppression of CDKN1A/p21, which is critical for ILC cell proliferation. CDKN1A knockdown partially reversed the effects of WNT4 knockdown. WNT4 drives a novel signaling pathway in ILC cells, with a

  18. Resistance in Everyday Life

    DEFF Research Database (Denmark)

    This book is about resistance in everyday life, illustrated through empirical contexts from different parts of the world. Resistance is a widespread phenomenon in biological, social and psychological domains of human cultural development. Yet, it is not well articulated in the academic literature....... The contributors deal with strategies for handling dissent by individuals or groups, specifically dissent through resistance. Resistance can be a location of intense personal, interpersonal and cultural negotiation, and that is the primary reason for interest in this phenomenon. Ordinary life events contain...

  19. Resistent tuberkulose i Danmark

    DEFF Research Database (Denmark)

    Thomsen, V O; Johansen, I S; Bauer, J O

    2001-01-01

    INTRODUCTION: Increased rates of multidrug-resistant (MDR) tuberculosis (TB) has been reported from countries close to Denmark. We evaluated the incidence of drug resistance in Denmark in order to determine the magnitude of the problem. MATERIALS AND METHODS: Susceptibility testing was performed......-cluster. Among all patients with isoniazid- and streptomycin-resistance, 77.0% had clustered strains. DISCUSSION: In conclusion, although drug resistance among untreated Danes was close to the rate estimated in good national programmes, close monitoring is needed in future years, as active transmission...

  20. Antipastorialism : Resistant Georgic Mode

    National Research Council Canada - National Science Library

    Zimmerman, Donald

    2000-01-01

    .... Abolitionists, women, Afro-British slaves, and those who protested land enclosure developed a multivalent, resistant mode of writing, which I name 'antipastoralism', that countered orthodox, poetical...

  1. Galectin-1-Induced Autophagy Facilitates Cisplatin Resistance of Hepatocellular Carcinoma.

    Directory of Open Access Journals (Sweden)

    Yu-Chi Su

    Full Text Available Hepatocellular carcinoma (HCC is one of the most common cancers in Taiwan. Although chemotherapy is the primary treatment for HCC patients, drug resistance often leads to clinical failure. Galectin-1 is a beta-galactoside binding lectin which is up-regulated in HCC patients and promotes tumor growth by mediating cancer cell adhesion, migration and proliferation, but its role in chemoresistance of HCC is poorly understood. In this study we found that galectin-1 is able to lead to chemoresistance against cisplatin treatment, and subsequent inhibition has reversed the effect of cell death in HCC cells. Moreover, galectin-1 was found to induce autophagic flux in HCC cells. Inhibition of autophagy by inhibitors or knockdown of Atg5 cancels galectin-1-induced cisplatin resistance in HCC cells. Increase of mitophagy triggered by galectin-1 was found to reduce the mitochondrial potential loss and apoptosis induced by cisplatin treatment. Finally, using an in situ hepatoma mouse model, we clearly demonstrated that inhibition of galectin-1 by thiodigalactoside could significantly augment the anti-HCC effect of cisplatin. Taken together, our findings offer a new insight into the chemoresistance galectin-1 causes against cisplatin treatment, and points to a potential approach to improve the efficacy of cisplatin in the treatment of HCC patients.

  2. Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Keisuke Tabara

    Full Text Available Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs. However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2 or BIBW2992 (pan-TKI of EGFR family proteins. Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.

  3. Forkhead Box Protein C2 Promotes Epithelial-Mesenchymal Transition, Migration and Invasion in Cisplatin-Resistant Human Ovarian Cancer Cell Line (SKOV3/CDDP

    Directory of Open Access Journals (Sweden)

    Chanjuan Li

    2016-08-01

    Full Text Available Background/Aims: Forkhead Box Protein C2 (FOXC2 has been reported to be overexpressed in a variety of human cancers. However, it is unclear whether FOXC2 regulates epithelial-mesenchymal transition (EMT in CDDP-resistant ovarian cancer cells. The aim of this study is to investigate the effects of FOXC2 on EMT and invasive characteristics of CDDP-resistant ovarian cancer cells and the underlying molecular mechanism. Methods: MTT, Western blot, scratch wound healing, matrigel transwell invasion, attachment and detachment assays were performed to detect half maximal inhibitory concentration (IC50 of CDDP, expression of EMT-related proteins and invasive characteristics in CDDP-resistant ovarian cancer cell line (SKOV3/CDDP and its parental cell line (SKOV3. Small hairpin RNA (shRNA was used to knockdown FOXC2 and analyze the effect of FOXC2 knockdown on EMT and invasive characteristics of SKOV3/CDDP cells. Also, the effect of FOXC2 upregulation on EMT and invasive characteristics of SKOV3 cells was analyzed. Furthermore, the molecular mechanism underlying FOXC2-regulating EMT in ovarian cancer cells was determined. Results: Compared with parental SKOV3 cell line, SKOV3/CDDP showed higher IC50 of CDDP (43.26μM (PConclusions: Taken together, these data demonstrate that FOXC2 may be a promoter of EMT phenotype in CDDP-resistant ovarian cancer cells and a potential therapeutic target for the treatment of advanced ovarian cancer.

  4. Endoplasmic reticulum stress regulates inflammation and insulin resistance in skeletal muscle from pregnant women.

    Science.gov (United States)

    Liong, Stella; Lappas, Martha

    2016-04-15

    Sterile inflammation and infection are key mediators of inflammation and peripheral insulin resistance associated with gestational diabetes mellitus (GDM). Studies have shown endoplasmic reticulum (ER) stress to induce inflammation and insulin resistance associated with obesity and type 2 diabetes, however is paucity of studies investigating the effects of ER stress in skeletal muscle on inflammation and insulin resistance associated with GDM. ER stress proteins IRE1α, GRP78 and XBP-1s were upregulated in skeletal muscle of obese pregnant women, whereas IRE1α was increased in GDM women. Suppression of ER stress, using ER stress inhibitor tauroursodeoxycholic acid (TUDCA) or siRNA knockdown of IRE1α and GRP78, significantly downregulated LPS-, poly(I:C)- or IL-1β-induced production of IL-6, IL-8, IL-1β and MCP-1. Furthermore, LPS-, poly(I:C)- or TNF-α-induced insulin resistance was improved following suppression of ER stress, by increasing insulin-stimulated phosphorylation of IR-β, IRS-1, GLUT-4 expression and glucose uptake. In summary, our inducible obesity and GDM-like models suggests that the development of GDM may be involved in activating ER stress-induced inflammation and insulin resistance in human skeletal muscle. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. STAT1 pathway mediates amplification of metastatic potential and resistance to therapy.

    Directory of Open Access Journals (Sweden)

    Nikolai N Khodarev

    Full Text Available BACKGROUND: Traditionally IFN/STAT1 signaling is connected with an anti-viral response and pro-apoptotic tumor-suppressor functions. Emerging functions of a constitutively activated IFN/STAT1 pathway suggest an association with an aggressive tumor phenotype. We hypothesized that tumor clones that constitutively overexpress this pathway are preferentially selected by the host microenvironment due to a resistance to STAT1-dependent cytotoxicity and demonstrate increased metastatic ability combined with increased resistance to genotoxic stress. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that clones of B16F1 tumors grown in the lungs of syngeneic C57BL/6 mice demonstrate variable transcriptional levels of IFN/STAT1 pathway expression. Tumor cells that constitutively overexpress the IFN/STAT1 pathway (STAT1(H genotype are selected by the lung microenvironment. STAT1(H tumor cells also demonstrate resistance to IFN-gamma (IFNgamma, ionizing radiation (IR, and doxorubicin relative to parental B16F1 and low expressors of the IFN/STAT1 pathway (STAT1(L genotype. Stable knockdown of STAT1 reversed the aggressive phenotype and decreased both lung colonization and resistance to genotoxic stress. CONCLUSIONS: Our results identify a pathway activated by tumor-stromal interactions thereby selecting for pro-metastatic and therapy-resistant tumor clones. New therapies targeted against the IFN/STAT1 signaling pathway may provide an effective strategy to treat or sensitize aggressive tumor clones to conventional cancer therapies and potentially prevent distant organ colonization.

  6. Sustained miRNA-mediated Knockdown of Mutant AAT With Simultaneous Augmentation of Wild-type AAT Has Minimal Effect on Global Liver miRNA Profiles

    OpenAIRE

    2013-01-01

    α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concom...

  7. Survivin knockdown increased anti-cancer effects of (−)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells

    International Nuclear Information System (INIS)

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

    2012-01-01

    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-κB), invasive (MMP-2 and MMP-9), and angiogenic (VEGF and b-FGF) factors. Also, in vitro

  8. Xeroderma Pigmentosum Group A Promotes Autophagy to Facilitate Cisplatin Resistance in Melanoma Cells through the Activation of PARP1.

    Science.gov (United States)

    Ge, Rui; Liu, Lin; Dai, Wei; Zhang, Weigang; Yang, Yuqi; Wang, Huina; Shi, Qiong; Guo, Sen; Yi, Xiuli; Wang, Gang; Gao, Tianwen; Luan, Qi; Li, Chunying

    2016-06-01

    Xeroderma pigmentosum group A (XPA), a key protein in the nucleotide excision repair pathway, has been shown to promote the resistance of tumor cells to chemotherapeutic drugs by facilitating the DNA repair process. However, the role of XPA in the resistance of melanoma to platinum-based drugs like cisplatin is largely unknown. In this study, we initially found that XPA was expressed at higher levels in cisplatin-resistant melanoma cells than in cisplatin-sensitive ones. Furthermore, the knockdown of XPA not only increased cellular apoptosis but also inhibited cisplatin-induced autophagy, which rendered the melanoma cells more sensitive to cisplatin. Moreover, we discovered that the increased XPA in resistant melanoma cells promoted poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) activation and that the inhibition of PARP1 could attenuate the cisplatin-induced autophagy. Finally, we proved that the inhibition of PARP1 and the autophagy process made resistant melanoma cells more susceptible to cisplatin treatment. Our study shows that XPA can promote cell-protective autophagy in a DNA repair-independent manner by enhancing the activation of PARP1 in melanoma cells resistant to cisplatin and that the XPA-PARP1-mediated autophagy process can be targeted to overcome cisplatin resistance in melanoma chemotherapy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Antibiotic resistance in animals.

    Science.gov (United States)

    Barton, Mary D; Pratt, Rachael; Hart, Wendy S

    2003-01-01

    There is currently no systematic surveillance or monitoring of antibiotic resistance in Australian animals. Registration of antibiotics for use in animals is tightly controlled and has been very conservative. Fluoroquinolones have not been registered for use in food producing animals and other products have been removed from the market because of human health concerns. In the late 1970s, the Animal Health Committee coordinated a survey of resistance in Salmonella and Escherichia coli isolates from cattle, pigs and poultry and in bovine Staphylococcus aureus. Some additional information is available from published case reports. In samples collected prior to the withdrawal of avoparcin from the market, no vancomycin resistant Enterococcus faecium or Enterococcus faecalis were detected in samples collected from pigs, whereas some vanA enterococci, including E. faecium and E. faecalis, were found in chickens. No vanB enterococci were detected in either species. Virginiamycin resistance was common in both pig and poultry isolates. Multiple resistance was common in E. coli and salmonellae isolates. No fluoroquinolone resistance was found in salmonellae, E. coli or Campylobacter. Beta-lactamase production is common in isolates from bovine mastitis, but no methicillin resistance has been detected. However, methicillin resistance has been reported in canine isolates of Staphylococcus intermedius and extended spectrum beta-lactamase producing E. coli has been found in dogs.

  10. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... Skip to common links HHS U.S. Department of Health and Human Services U.S. Food and Drug Administration A to Z Index Follow FDA En Español Search FDA Submit search ... & Health Antimicrobial Resistance Animation of Antimicrobial Resistance Share Tweet ...

  11. Resistance and conformity

    NARCIS (Netherlands)

    Sumter, S.R.; Bokhorst, C.L.; Westenberg, P.M.; Levesque, R.J.R.

    2011-01-01

    Resistance to peer influence, or the ability to resist making choices or adopting views under the implicit or explicit influence of your peers, is expected to undergo changes during adolescence. Two developmental trajectories have emerged from the field. On the one hand, adolescents show a temporary

  12. Mechanisms of Antibiotic Resistance

    Science.gov (United States)

    Munita, Jose M.; Arias, Cesar A.

    2015-01-01

    Emergence of resistance among the most important bacterial pathogens is recognized as a major public health threat affecting humans worldwide. Multidrug-resistant organisms have emerged not only in the hospital environment but are now often identified in community settings, suggesting that reservoirs of antibiotic-resistant bacteria are present outside the hospital. The bacterial response to the antibiotic “attack” is the prime example of bacterial adaptation and the pinnacle of evolution. “Survival of the fittest” is a consequence of an immense genetic plasticity of bacterial pathogens that trigger specific responses that result in mutational adaptations, acquisition of genetic material or alteration of gene expression producing resistance to virtually all antibiotics currently available in clinical practice. Therefore, understanding the biochemical and genetic basis of resistance is of paramount importance to design strategies to curtail the emergence and spread of resistance and devise innovative therapeutic approaches against multidrug-resistant organisms. In this chapter, we will describe in detail the major mechanisms of antibiotic resistance encountered in clinical practice providing specific examples in relevant bacterial pathogens. PMID:27227291

  13. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... Skip to common links HHS U.S. Department of Health and Human Services U.S. Food and Drug Administration ... Tobacco Products Animal & Veterinary Home Animal & Veterinary Safety & Health Antimicrobial Resistance Animation of Antimicrobial Resistance Share Tweet ...

  14. Animation of Antimicrobial Resistance

    Medline Plus

    Full Text Available ... menu Skip to common links HHS U.S. Department of Health and Human Services U.S. Food and Drug Administration ... Tobacco Products Animal & Veterinary Home Animal & Veterinary Safety & Health Antimicrobial Resistance Animation of Antimicrobial Resistance Share Tweet Linkedin Pin it More ...

  15. Phenotypic Resistance to Antibiotics

    Directory of Open Access Journals (Sweden)

    Jose L. Martinez

    2013-04-01

    Full Text Available The development of antibiotic resistance is usually associated with genetic changes, either to the acquisition of resistance genes, or to mutations in elements relevant for the activity of the antibiotic. However, in some situations resistance can be achieved without any genetic alteratio