WorldWideScience

Sample records for rna sensitizes human

  1. MicroRNA-203 Modulates the Radiation Sensitivity of Human Malignant Glioma Cells

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Ji Hyun [Department of Radiation Oncology, Graduate School of Medicine, Seoul National University, Seoul (Korea, Republic of); Hwang, Yeo Hyun; Lee, David J.; Kim, Dan Hyo; Park, Ji Min [Medical Science Research Institute, Seoul National University Bundang Hospital, Kyeonggido (Korea, Republic of); Wu, Hong-Gyun [Department of Radiation Oncology, Graduate School of Medicine, Seoul National University, Seoul (Korea, Republic of); Kim, In Ah, E-mail: inah228@snu.ac.kr [Department of Radiation Oncology, Graduate School of Medicine, Seoul National University, Seoul (Korea, Republic of); Medical Science Research Institute, Seoul National University Bundang Hospital, Kyeonggido (Korea, Republic of); Cancer Research Institute, Seoul National University, Seoul (Korea, Republic of)

    2016-02-01

    Purpose: We investigated whether miR-203 could modulate the radiation sensitivity of glioblastoma (GBM) cells and which target gene(s) could be involved. Methods and Materials: Three human malignant glioma (MG) cell lines and normal human astrocytes were transfected with control microRNA, pre-miR-203, or antisense miR-203. Real-time PCR (RT-PCR), clonogenic assays, immunofluorescence, and invasion/migration assays were performed. To predict the target(s), bioinformatics analyses using microRNA target databases were performed. Results: Overexpression of miR-203 increased the radiation sensitivity of all 3 human MG cell lines and prolonged radiation-induced γ-H2AX foci formation. Bioinformatics analyses suggested that miR-203 could be involved in post-transcriptional control of DNA repair, PI3K/AKT, SRC, and JAK/STAT3 and the vascular signaling pathway. Western blot analysis validated the fact that miR-203 downregulated ATM, RAD51, SRC, PLD2, PI3K-AKT, JAK-STAT3, VEGF, HIF-1α, and MMP2. Overexpression of miR-203 inhibited invasion and migration potentials, downregulated SLUG and Vimentin, and upregulated Claudin-1 and ZO1. Conclusions: These data demonstrate that miR-203 potentially controls DNA damage repair via the PI3K/AKT and JAK/STAT3 pathways and may collectively contribute to the modulation of radiation sensitivity in MG cells by inhibiting DNA damage repair, prosurvival signaling, and epithelium-mesenchyme transition. Taken together, these findings demonstrate that miR-203 could be a target for overcoming the radiation resistance of GBM.

  2. MicroRNA-203 Modulates the Radiation Sensitivity of Human Malignant Glioma Cells

    International Nuclear Information System (INIS)

    Chang, Ji Hyun; Hwang, Yeo Hyun; Lee, David J.; Kim, Dan Hyo; Park, Ji Min; Wu, Hong-Gyun; Kim, In Ah

    2016-01-01

    Purpose: We investigated whether miR-203 could modulate the radiation sensitivity of glioblastoma (GBM) cells and which target gene(s) could be involved. Methods and Materials: Three human malignant glioma (MG) cell lines and normal human astrocytes were transfected with control microRNA, pre-miR-203, or antisense miR-203. Real-time PCR (RT-PCR), clonogenic assays, immunofluorescence, and invasion/migration assays were performed. To predict the target(s), bioinformatics analyses using microRNA target databases were performed. Results: Overexpression of miR-203 increased the radiation sensitivity of all 3 human MG cell lines and prolonged radiation-induced γ-H2AX foci formation. Bioinformatics analyses suggested that miR-203 could be involved in post-transcriptional control of DNA repair, PI3K/AKT, SRC, and JAK/STAT3 and the vascular signaling pathway. Western blot analysis validated the fact that miR-203 downregulated ATM, RAD51, SRC, PLD2, PI3K-AKT, JAK-STAT3, VEGF, HIF-1α, and MMP2. Overexpression of miR-203 inhibited invasion and migration potentials, downregulated SLUG and Vimentin, and upregulated Claudin-1 and ZO1. Conclusions: These data demonstrate that miR-203 potentially controls DNA damage repair via the PI3K/AKT and JAK/STAT3 pathways and may collectively contribute to the modulation of radiation sensitivity in MG cells by inhibiting DNA damage repair, prosurvival signaling, and epithelium-mesenchyme transition. Taken together, these findings demonstrate that miR-203 could be a target for overcoming the radiation resistance of GBM.

  3. SiRNA-mediated IGF-1R inhibition sensitizes human colon cancer SW480 cells to radiation

    International Nuclear Information System (INIS)

    Yavari, Kamal; Taghikhani, Mohammad; Mesbah-Namin, Seyed A.; Maragheh, Mohammad Ghannadi; Babaei, Mohammad Hosein; Arfaee, Ali Jabbary; Madani, Hossein; Mirzaei, Hamid Reza

    2010-01-01

    Purpose. Insulin like growth factor receptor 1 (IGF-1R) is well-documented to play a key role in radiation response and tumor radiosensitivity, thus offering an attractive clinic drug target to enhance tumor sensitivity to anti-cancer radiotherapy. Material and methods. Human colon carcinoma SW480 cells were transfected with the specific small interference RNA (siRNA) expression vector (pkD-shRNA-IGF-1R-V2) designed to target IGF-1R mRNA. The expression of IGF-1R mRNA and its protein among the transfected and untransfected cells were detected by semi-quantitative RT-PCR and ELISA assay. The changes in cell radiosensitivity were examined by MTT assay. Results. Transfection of mammalian expression vector pkD containing IGF-1R siRNA was shown to reduce IGF-1R mRNA levels by up to 95%. ELISA assay detected a similar inhibition of IGF-1R protein levels in cells transfected with IGF-1R siRNA. SW480 cells transfected with the expression vector for siRNA significantly rendered cells more sensitive to radiation and the highest radiation enhancement ratio was 2.02 ± 0.08. Conclusion. These data provide the first evidence that specific siRNA fragment (pkD-shRNA-IGF-1R-V2) targeting human IGF-1R mRNA is able to enhance colon cancer radiosensitivity. Also results indicated that, combining IGF-1R siRNA and radiation significantly enhances antitumor efficacy compared with either modality alone

  4. Radiation sensitivity of messenger RNA

    International Nuclear Information System (INIS)

    Ponta, H.; Pfennig-Yeh, M.L.; Herrlich, P.; Karlsruhe Univ.; Wagner, E.F.; Schweiger, M.

    1979-01-01

    Messenger RNA function is inactivated by irradiation with ultraviolet light. A unit length mRNA (in bases) is 2-3 times more sensitive than a unit length of DNA (in base pairs) with respect to the inactivation of template function. These data stem from four experimental systems all of which do not repair DNA: the translation of E. coli mRNA in rifampicin-treated cells, of T7 mRNA in infected E.coli, of f2 phage RNA in vivo, and of stable mRNA in chromosomeless minicells. The comparison of relative sensitivities to UV is relevant to the technique of UV mapping of transcription units which enjoys increasing popularity in pro- and eukaryotic genetic research. (orig.) [de

  5. Radiation sensitivity of messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

    Ponta, H; Pfennig-Yeh, M L; Herrlich, P [Kernforschungszentrum Karlsruhe G.m.b.H. (Germany, F.R.). Inst. fuer Genetik und Toxikologie von Spaltstoffen; Karlsruhe Univ. (TH) (Germany, F.R.). Inst. fuer Genetik); Wagner, E F; Schweiger, M [Innsbruck Univ. (Austria). Inst. fuer Biochemie

    1979-08-01

    Messenger RNA function is inactivated by irradiation with ultraviolet light. A unit length mRNA (in bases) is 2-3 times more sensitive than a unit length of DNA (in base pairs) with respect to the inactivation of template function. These data stem from four experimental systems all of which do not repair DNA: the translation of E. coli mRNA in rifampicin-treated cells, of T7 mRNA in infected E.coli, of f2 phage RNA in vivo, and of stable mRNA in chromosomeless minicells. The comparison of relative sensitivities to UV is relevant to the technique of UV mapping of transcription units which enjoys increasing popularity in pro- and eukaryotic genetic research.

  6. Sensitization of human carcinoma cells to alkylating agents by small interfering RNA suppression of 3-alkyladenine-DNA glycosylase.

    Science.gov (United States)

    Paik, Johanna; Duncan, Tod; Lindahl, Tomas; Sedgwick, Barbara

    2005-11-15

    One of the major cytotoxic lesions generated by alkylating agents is DNA 3-alkyladenine, which can be excised by 3-alkyladenine DNA glycosylase (AAG). Inhibition of AAG may therefore result in increased cellular sensitivity to chemotherapeutic alkylating agents. To investigate this possibility, we have examined the role of AAG in protecting human tumor cells against such agents. Plasmids that express small interfering RNAs targeted to two different regions of AAG mRNA were transfected into HeLa cervical carcinoma cells and A2780-SCA ovarian carcinoma cells. Stable derivatives of both cell types with low AAG protein levels were sensitized to alkylating agents. Two HeLa cell lines with AAG protein levels reduced by at least 80% to 90% displayed a 5- to 10-fold increase in sensitivity to methyl methanesulfonate, N-methyl-N-nitrosourea, and the chemotherapeutic drugs temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea. These cells showed no increase in sensitivity to UV light or ionizing radiation. After treatment with methyl methanesulfonate, AAG knockdown HeLa cells were delayed in S phase but accumulated in G2-M. Our data support the hypothesis that ablation of AAG activity in human tumor cells may provide a useful strategy to enhance the efficacy of current chemotherapeutic regimens that include alkylating agents.

  7. Development of Highly Sensitive and Specific mRNA Multiplex System (XCYR1) for Forensic Human Body Fluids and Tissues Identification

    Science.gov (United States)

    Xu, Yan; Xie, Jianhui; Cao, Yu; Zhou, Huaigu; Ping, Yuan; Chen, Liankang; Gu, Lihua; Hu, Wei; Bi, Gang; Ge, Jianye; Chen, Xin; Zhao, Ziqin

    2014-01-01

    The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples. PMID:24991806

  8. Transfer RNA and human disease

    Directory of Open Access Journals (Sweden)

    Jamie A Abbott

    2014-06-01

    Full Text Available Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA genes are hotspots for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase, mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers, and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing. Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

  9. Transfer RNA and human disease.

    Science.gov (United States)

    Abbott, Jamie A; Francklyn, Christopher S; Robey-Bond, Susan M

    2014-01-01

    Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are "hotspots" for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

  10. Transfecting Human Monocytes with RNA.

    Science.gov (United States)

    Dannull, Jens; Nair, Smita K

    2016-01-01

    Targeting monocytes as a delivery system for drugs or nucleic acids, and thereby harnessing their natural tissue-infiltrating capacity, has become an area of intense investigation in both basic and clinical research. Herein we describe an efficient method to deliver mRNA (messenger RNA) or siRNA (small interfering RNA) into human monocytes by electroporation. This method can be applied in the laboratory to monocytes isolated via magnetic bead-based techniques, or in a clinical setting using monocytes that were collected via counterflow centrifugation elutriation using the Elutra(®) Cell Separation System. We further demonstrate that electroporation of monocytes with RNA represents a robust and highly relevant approach to modify monocytes for cell-based therapies. Last, the procedure described can readily be adapted to monocytes from different species, hence facilitating research in animal models.

  11. Sensitivity, Specificity, and Clinical Value of Human Papillomavirus (HPV) E6/E7 mRNA Assay as a Triage Test for Cervical Cytology and HPV DNA Test ▿

    Science.gov (United States)

    Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Rossi, Paolo Giorgi

    2011-01-01

    There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of

  12. MicroRNA-139-5p affects cisplatin sensitivity in human nasopharyngeal carcinoma cells by regulating the epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Shao, Qianqian; Zhang, Pei; Ma, Yingye; Lu, Zhaoyi; Meng, Jie; Li, Hui; Wang, Xiaoming; Chen, Deshang; Zhang, Mingjie; Han, Yaofeng; Liu, Hao; Ma, Shiyin

    2018-04-30

    Nasopharyngeal carcinoma (NPC) is a head and neck cancer associated with poor prognosis. Many studies have shown that the epithelial-to-mesenchymal transition (EMT) is important in cancer progression, metastasis, and chemotherapy resistance and that microRNAs (miRNAs) play a key role in chemotherapy resistance associated with EMT. The miRNA miR-139-5p is downregulated in many human cancers and is closely related to tumor progression. The aim of this study was to investigate the ability of miR-139-5p to influence the cisplatin resistance, apoptosis, invasion and migration in NPC cells through the regulation of the EMT. We investigated these processes in parental HNE1 and cisplatin-resistant HNE1/DDP cells transfected with miR-139-5p inhibitors and mimics, respectively. Our results suggest that the upregulation of miR-139-5p expression inhibits proliferation, invasion, migration and EMT in human NPC cells. In addition, we found that miR-139-5p expression levels and DDP-induced apoptosis positively correlate in NPC cells. In conclusion, our results demonstrate that miR-139-5p can regulate the migration, invasion and DDP resistance in human NPC by modulating the EMT. The regulation of miR-139-5p levels might be a new approach to reverse EMT and DDP resistance and counteract metastasis and chemotherapy resistance in human NPC. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Double Stranded RNA in Human Seminal Plasma

    Directory of Open Access Journals (Sweden)

    Maxim V. Zagoskin

    2017-10-01

    Full Text Available Recently, human semen was shown to contain cell-free nucleic acids, such as DNA, long single stranded RNA, and small RNAs–miRNA and piRNA. The RNAs have been suggested to have potential biological roles as communication molecules between cells and in the temporal and spatial regulation of gene expression in the male reproductive system. Here we demonstrate that human seminal plasma contains a variety of cell-free dsRNAs, describe a robust method to isolate this type of nucleic acid in preparative amounts, and discuss the potential biological roles of these molecules in inheritance. dsRNA plays a role in a variety of biological processes, including gene regulation, is extremely stable and can gain access to cells from the extracellular medium. We suggest that one of the possible functions of dsRNA in human seminal plasma may be to influence human oocytes and therefore, influence the offspring. It also remains possible that these dsRNAs might have potential use as biomarkers for the study of human physiopathological conditions and genetic variation.

  14. Establishment of a sensitive system for analysis of human vaginal microbiota on the basis of rRNA-targeted reverse transcription-quantitative PCR.

    Science.gov (United States)

    Kurakawa, Takashi; Ogata, Kiyohito; Tsuji, Hirokazu; Kado, Yukiko; Takahashi, Takuya; Kida, Yumi; Ito, Masahiro; Okada, Nobuhiko; Nomoto, Koji

    2015-04-01

    Ten specific primer sets, for Lactobacillus gasseri, Lactobacillus crispatus, Atopobium vaginae, Gardnerella vaginalis, Mobiluncus curtisii, Chlamydia trachomatis/muridarum, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, and Bifidobacterium angulatum, were developed for quantitative analysis of vaginal microbiota. rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) analysis of the vaginal samples from 12 healthy Japanese volunteers using the new primer sets together with 25 existing primer sets revealed the diversity of their vaginal microbiota: Lactobacilli such as L. crispatus, L. gasseri, Lactobacillus jensenii, Lactobacillus iners, and Lactobacillus vaginalis, as the major populations at 10(7) cells/ml vaginal fluid, were followed by facultative anaerobes such as Streptococcus and strict anaerobes at lower population levels of 10(4) cells/ml or less. Certain bacterial vaginosis (BV)-related bacteria, such as G. vaginalis, A. vaginae, M. curtisii, and Prevotella, were also detected in some subjects. Especially in one subject, both G. vaginalis and A. vaginae were detected at high population levels of 10(8.8) and 10(8.9) cells/ml vaginal fluid, suggesting that she is an asymptomatic BV patient. These results suggest that the RT-qPCR system is effective for accurate analysis of major vaginal commensals and diagnosis of several vaginal infections. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. The Regulatory and Kinase Domains but Not the Interdomain Linker Determine Human Double-stranded RNA-activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs.

    Science.gov (United States)

    Sunita, S; Schwartz, Samantha L; Conn, Graeme L

    2015-11-20

    Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an important component of the innate immune system that presents a crucial first line of defense against viral infection. PKR has a modular architecture comprising a regulatory N-terminal dsRNA binding domain and a C-terminal kinase domain interposed by an unstructured ∼80-residue interdomain linker (IDL). Guided by sequence alignment, we created IDL deletions in human PKR (hPKR) and regulatory/kinase domain swap human-rat chimeric PKRs to assess the contributions of each domain and the IDL to regulation of the kinase activity by RNA. Using circular dichroism spectroscopy, limited proteolysis, kinase assays, and isothermal titration calorimetry, we show that each PKR protein is properly folded with similar domain boundaries and that each exhibits comparable polyinosinic-cytidylic (poly(rI:rC)) dsRNA activation profiles and binding affinities for adenoviral virus-associated RNA I (VA RNAI) and HIV-1 trans-activation response (TAR) RNA. From these results we conclude that the IDL of PKR is not required for RNA binding or mediating changes in protein conformation or domain interactions necessary for PKR regulation by RNA. In contrast, inhibition of rat PKR by VA RNAI and TAR RNA was found to be weaker than for hPKR by 7- and >300-fold, respectively, and each human-rat chimeric domain-swapped protein showed intermediate levels of inhibition. These findings indicate that PKR sequence or structural elements in the kinase domain, present in hPKR but absent in rat PKR, are exploited by viral non-coding RNAs to accomplish efficient inhibition of PKR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2017-01-01

    MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation. © 2017 Goldfarb and Cech; Published by Cold Spring Harbor Laboratory Press.

  17. Downregulation of MDM2 expression by RNAi inhibits LoVo human colorectal adenocarcinoma cells growth and the treatment of LoVo cells with mdm2siRNA3 enhances the sensitivity to cisplatin

    International Nuclear Information System (INIS)

    Yu Yan; Sun Ping; Sun Lichun; Liu Guoyi; Chen Guohua; Shang Lihua; Wu Hongbo; Hu Jing; Li Yue; Mao Yinling; Sui Guangjie; Sun Xiwen

    2006-01-01

    To investigate the biological effect of mdm2 in human colorectal adenocarcinoma LoVo cells, three mdm2siRNA constructions were recombinated and transient transfected into human colorectal adenocarcinoma LoVo cells with low differentiation character in vitro. The results showed that mdm2siRNA3 reduced mRNA level of mdm2 and protein level of mdm2, leading to proliferation inhibition on LoVo cells, and reduced tumor growth in nude mice. It was found that depletion of MDM2 in this pattern promoted apoptosis of LoVo cells and Cisplatin (DDP) treated in the mdm2siRNA3 transfected cell population would result in a substantial decrease by MTT colorimetry. Decreasing the MDM2 protein level in LoVo cells by RNAi could significantly inhibit tumor growth both in vitro and in vivo, which indicated that mdm2 gene played a definite role in the development and aggressiveness of human colon carcinoma. It also could be a therapeutic target in colorectal carcinoma. The synergistic activation of RNAi and cell toxicity agents indicated that the combination of chemotherapy and gene therapy will be a promising approach in the future

  18. A genome-wide siRNA screen to identify modulators of insulin sensitivity and gluconeogenesis.

    Directory of Open Access Journals (Sweden)

    Ruojing Yang

    Full Text Available BACKGROUND: Hepatic insulin resistance impairs insulin's ability to suppress hepatic glucose production (HGP and contributes to the development of type 2 diabetes (T2D. Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. METHODOLOGY/PRINCIPAL FINDINGS: To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC promoter (AH-G6PC cells. Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4 mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. CONCLUSIONS/SIGNIFICANCE: These results support the proposition that the proteins encoded by the genes identified in

  19. RNA.

    Science.gov (United States)

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  20. Structural Chemistry of Human RNA Methyltransferases.

    Science.gov (United States)

    Schapira, Matthieu

    2016-03-18

    RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

  1. Reproducible pattern of microRNA in normal human skin

    DEFF Research Database (Denmark)

    Holst, Line; Kaczkowski, Bogumil; Gniadecki, Robert

    2010-01-01

    RNA expression pattern in normal human skin. Here we investigated miRNA expression profiles from skin biopsies of 8 healthy volunteers taken from sun protected and mildly photo damaged skin using the modified protocol for miRNA extraction. We were able to show a constant pattern of miRNA expression between......MicroRNAs (miRNAs) regulate cell growth, differentiation and apoptosis via specific targeting of messenger RNA (mRNA). Aberrant mRNA expression contributes to pathological processes such as carcinogenesis. To take advantage of miRNA profiling in skin disease it is essential to investigate mi...... different individuals. We did not find any significant differences in miRNA expression between sun protected and mildly photodamaged skin. These results may be valuable for future design of studies on miRNA expression in skin disease....

  2. Reproducible pattern of microRNA in normal human skin

    DEFF Research Database (Denmark)

    Holst, Line; Kaczkowski, Bogumil; Gniadecki, Robert

    2010-01-01

    RNA expression pattern in normal human skin. Here we investigated miRNA expression profiles from skin biopsies of 8 healthy volunteers taken from sun protected and mildly photo damaged skin using the modified protocol for miRNA extraction. We were able to show a constant pattern of miRNA expression between...... different individuals. We did not find any significant differences in miRNA expression between sun protected and mildly photodamaged skin. These results may be valuable for future design of studies on miRNA expression in skin disease.......MicroRNAs (miRNAs) regulate cell growth, differentiation and apoptosis via specific targeting of messenger RNA (mRNA). Aberrant mRNA expression contributes to pathological processes such as carcinogenesis. To take advantage of miRNA profiling in skin disease it is essential to investigate mi...

  3. RAID: a comprehensive resource for human RNA-associated (RNA–RNA/RNA–protein) interaction

    Science.gov (United States)

    Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

    2014-01-01

    Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA–RNA/RNA–protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA–RNA interactions and 1619 RNA–protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA–RNA/RNA–protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA–RNA/RNA–protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. PMID:24803509

  4. Sets of RNA repeated tags and hybridization-sensitive fluorescent probes for distinct images of RNA in a living cell.

    Directory of Open Access Journals (Sweden)

    Takeshi Kubota

    Full Text Available BACKGROUND: Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. METHODOLOGY/PRINCIPAL FINDINGS: Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3'-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag-probe pairs. CONCLUSIONS/SIGNIFICANCE: A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging.

  5. Mechanisms of human immunodeficiency virus type 2 RNA packaging

    DEFF Research Database (Denmark)

    Ni, Na; Nikolaitchik, Olga A; Dilley, Kari A

    2011-01-01

    do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins......Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2...... proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results...

  6. Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation.

    Science.gov (United States)

    Alsaweed, Mohammed; Hepworth, Anna R; Lefèvre, Christophe; Hartmann, Peter E; Geddes, Donna T; Hassiotou, Foteini

    2015-10-01

    MicroRNA have been recently discovered in human milk signifying potentially important functions for both the lactating breast and the infant. Whilst human milk microRNA have started to be explored, little data exist on the evaluation of sample processing, and analysis to ensure that a full spectrum of microRNA can be obtained. Human milk comprises three main fractions: cells, skim milk, and lipids. Typically, the skim milk fraction has been measured in isolation despite evidence that the lipid fraction may contain more microRNA. This study aimed to standardize isolation of microRNA and total RNA from all three fractions of human milk to determine the most appropriate sampling and analysis procedure for future studies. Three different methods from eight commercially available kits were tested for their efficacy in extracting total RNA and microRNA from the lipid, skim, and cell fractions of human milk. Each fraction yielded different concentrations of RNA and microRNA, with the highest quantities found in the cell and lipid fractions, and the lowest in skim milk. The column-based phenol-free method was the most efficient extraction method for all three milk fractions. Two microRNAs were expressed and validated in the three milk fractions by qPCR using the three recommended extraction kits for each fraction. High expression levels were identified in the skim and lipid milk factions for these microRNAs. These results suggest that careful consideration of both the human milk sample preparation and extraction protocols should be made prior to embarking upon research in this area. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

  7. Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

    OpenAIRE

    Dibrov, Sergey; McLean, Jaime; Hermann, Thomas

    2011-01-01

    An oligonucleotide representing a regulatory element of human thymidylate synthase mRNA has been crystallized as a dimer. The structure of the asymmetric dimer has been determined at 1.97 Å resolution.

  8. tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Mayumi Okamoto

    2014-09-01

    Full Text Available Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD. The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.

  9. MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells.

    Directory of Open Access Journals (Sweden)

    Vanita Vanas

    Full Text Available Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin.

  10. Aptamer-miRNA-212 Conjugate Sensitizes NSCLC Cells to TRAIL

    Directory of Open Access Journals (Sweden)

    Margherita Iaboni

    2016-01-01

    Full Text Available TNF-related apoptosis-inducing ligand (TRAIL is a promising antitumor agent for its remarkable ability to selectively induce apoptosis in cancer cells, without affecting the viability of healthy bystander cells. The TRAIL tumor suppressor pathway is deregulated in many human malignancies including lung cancer. In human non-small cell lung cancer (NSCLC cells, sensitization to TRAIL therapy can be restored by increasing the expression levels of the tumor suppressor microRNA-212 (miR-212 leading to inhibition of the anti-apoptotic protein PED/PEA-15 implicated in treatment resistance. In this study, we exploited a previously described RNA aptamer inhibitor of the tyrosine kinase receptor Axl (GL21.T expressed on lung cancer cells, as a means to deliver miR-212 into human NSCLC cells expressing Axl. We demonstrate efficient delivery of miR-212 following conjugation of the miR to GL21.T (GL21.T-miR212 chimera. We show that the chimera downregulates PED and restores TRAIL-mediate cytotoxicity in cancer cells. Importantly, treatment of Axl+ lung cancer cells with the chimera resulted in (i an increase in caspase activation and (ii a reduction of cell viability in combination with TRAIL therapy. In conclusion, we demonstrate that the GL21.T-miR212 chimera can be employed as an adjuvant to TRAIL therapy for the treatment of lung cancer.

  11. 3'-5' RNA degradation pathways in human cells

    DEFF Research Database (Denmark)

    Lubas, Michal Szymon

    RNA synthesis and degradation are key steps in the regulation of gene expression in all living organisms. During the course of his PhD studies, Michal Lubas centred his research on the nuclear and cytoplasmic RNA turnover of both noncoding and coding RNAs in human cells. His proteomic studies...... revealed the interaction network of the main 3'-5' RNA degradation machinery – the RNA exosome complex. One of the key findings was the identification and characterisation of the Nuclear Exosome Targeting (NEXT) complex, important for nuclear functions of the exosome. Michal Lubas also studied the role...

  12. Nucleotide sequence of a human tRNA gene heterocluster

    International Nuclear Information System (INIS)

    Chang, Y.N.; Pirtle, I.L.; Pirtle, R.M.

    1986-01-01

    Leucine tRNA from bovine liver was used as a hybridization probe to screen a human gene library harbored in Charon-4A of bacteriophage lambda. The human DNA inserts from plaque-pure clones were characterized by restriction endonuclease mapping and Southern hybridization techniques, using both [3'- 32 P]-labeled bovine liver leucine tRNA and total tRNA as hybridization probes. An 8-kb Hind III fragment of one of these γ-clones was subcloned into the Hind III site of pBR322. Subsequent fine restriction mapping and DNA sequence analysis of this plasmid DNA indicated the presence of four tRNA genes within the 8-kb DNA fragment. A leucine tRNA gene with an anticodon of AAG and a proline tRNA gene with an anticodon of AGG are in a 1.6-kb subfragment. A threonine tRNA gene with an anticodon of UGU and an as yet unidentified tRNA gene are located in a 1.1-kb subfragment. These two different subfragments are separated by 2.8 kb. The coding regions of the three sequenced genes contain characteristic internal split promoter sequences and do not have intervening sequences. The 3'-flanking region of these three genes have typical RNA polymerase III termination sites of at least four consecutive T residues

  13. Performance comparison of digital microRNA profiling technologies applied on human breast cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Erik Knutsen

    Full Text Available MicroRNA profiling represents an important first-step in deducting individual RNA-based regulatory function in a cell, tissue, or at a specific developmental stage. Currently there are several different platforms to choose from in order to make the initial miRNA profiles. In this study we investigate recently developed digital microRNA high-throughput technologies. Four different platforms were compared including next generation SOLiD ligation sequencing and Illumina HiSeq sequencing, hybridization-based NanoString nCounter, and miRCURY locked nucleic acid RT-qPCR. For all four technologies, full microRNA profiles were generated from human cell lines that represent noninvasive and invasive tumorigenic breast cancer. This study reports the correlation between platforms, as well as a more extensive analysis of the accuracy and sensitivity of data generated when using different platforms and important consideration when verifying results by the use of additional technologies. We found all the platforms to be highly capable for microRNA analysis. Furthermore, the two NGS platforms and RT-qPCR all have equally high sensitivity, and the fold change accuracy is independent of individual miRNA concentration for NGS and RT-qPCR. Based on these findings we propose new guidelines and considerations when performing microRNA profiling.

  14. Detection of melatonin receptor mRNA in human muscle

    International Nuclear Information System (INIS)

    Li Lei

    2004-01-01

    To verify the expression of melatonin receptor mRNA in human, muscle, muscle beside vertebrae was collected to obtain total RNA and the mRNA of melatonin receptor was detected by RT-PCR method. The electrophoretic results of RT-PCR products by mt 1 and MT 2 primer were all positive and the sequence is corresponding with human melatonin receptor cDNA. It suggests that melatonin may act on the muscle beside vertebrae directly and regulate its growth and development. (authors)

  15. Mutation of miRNA target sequences during human evolution

    DEFF Research Database (Denmark)

    Gardner, Paul P; Vinther, Jeppe

    2008-01-01

    It has long-been hypothesized that changes in non-protein-coding genes and the regulatory sequences controlling expression could undergo positive selection. Here we identify 402 putative microRNA (miRNA) target sequences that have been mutated specifically in the human lineage and show that genes...... containing such deletions are more highly expressed than their mouse orthologs. Our findings indicate that some miRNA target mutations are fixed by positive selection and might have been involved in the evolution of human-specific traits....

  16. Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli

    DEFF Research Database (Denmark)

    Triman, K; Becker, E; Dammel, C

    1989-01-01

    Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance....... The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature......-sensitive spectinomycin resistance and two that produce unconditional loss of resistance. In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA. For the temperature-sensitive mutants, there is a lag period of about two generations between...

  17. microRNA Biomarkers to Generate Sensitivity to Abiraterone-Resistant Prostate Cancer

    Science.gov (United States)

    2017-09-01

    CYP17A1 inhibition with abiraterone in castration- resistant prostate cancer : induction of steroidogenesis and androgen receptor splice variants...AWARD NUMBER: W81XWH-15-1-0353 TITLE: microRNA Biomarkers to Generate Sensitivity to Abiraterone-Resistant Prostate Cancer PRINCIPAL...TITLE AND SUBTITLE microRNA Biomarkers to Generate Sensitivity to Abiraterone- Resistant Prostate Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER

  18. Novel pH-sensitive multifunctional envelope-type nanodevice for siRNA-based treatments for chronic HBV infection.

    Science.gov (United States)

    Yamamoto, Naoki; Sato, Yusuke; Munakata, Tsubasa; Kakuni, Masakazu; Tateno, Chise; Sanada, Takahiro; Hirata, Yuichi; Murakami, Shuko; Tanaka, Yasuhito; Chayama, Kazuaki; Hatakeyama, Hiroto; Hyodo, Mamoru; Harashima, Hideyoshi; Kohara, Michinori

    2016-03-01

    Antiviral agents including entecavir (ETV) suppress the replication of the hepatitis B virus (HBV) genome in human hepatocytes, but they do not reduce the abundance of viral proteins. The present study focused on effectively reducing viral protein levels. We designed siRNAs (HBV-siRNA) that target consensus sequences in HBV genomes. To prevent the emergence of escaped mutant virus, we mixed three HBV-siRNAs (HBV-siRNAmix); the mixture was encapsulated in a novel pH-sensitive multifunctional envelope-type nanodevice (MEND), a hepatocyte-specific drug delivery system. Coagulation factor 7 siRNA was used to assess delivery and knockdown efficiencies of MEND/siRNA treatments in mice. The potency of MEND/HBV-siRNAmix was evaluated in primary human hepatocytes and in chimeric mice with humanized liver persistently infected with HBV. Effective knockdown of targets, efficient delivery of siRNA, and liver-specific delivery were each observed with MEND. MEND/HBV-siRNA caused efficient reduction of HBsAg and HBeAg in vitro and in vivo. However, ETV treatment did not efficiently reduce HBsAg or HBeAg when compared with a single MEND/HBV-siRNAmix treatment. Furthermore, the suppressive effects of a single dose of MEND/HBV-siRNAmix persisted for 14days in vitro and in vivo. We demonstrated that MEND/HBV-siRNA controlled HBV more efficiently than did ETV. Furthermore, the effect of a single dose of MEND/HBV-siRNA persisted for a long time. These results indicated that MEND/HBV-siRNA may be a promising novel HBV treatment that is more effective than reverse transcriptase inhibitors. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  19. Cytochrome c oxidase subunit 1-based human RNA quantification to enhance mRNA profiling in forensic biology

    Directory of Open Access Journals (Sweden)

    Dong Zhao

    2017-01-01

    Full Text Available RNA analysis offers many potential applications in forensic science, and molecular identification of body fluids by analysis of cell-specific RNA markers represents a new technique for use in forensic cases. However, due to the nature of forensic materials that often admixed with nonhuman cellular components, human-specific RNA quantification is required for the forensic RNA assays. Quantification assay for human RNA has been developed in the present study with respect to body fluid samples in forensic biology. The quantitative assay is based on real-time reverse transcription-polymerase chain reaction of mitochondrial RNA cytochrome c oxidase subunit I and capable of RNA quantification with high reproducibility and a wide dynamic range. The human RNA quantification improves the quality of mRNA profiling in the identification of body fluids of saliva and semen because the quantification assay can exclude the influence of nonhuman components and reduce the adverse affection from degraded RNA fragments.

  20. A compromised yeast RNA polymerase II enhances UV sensitivity in the absence of global genome nucleotide excision repair.

    Science.gov (United States)

    Wong, J M; Ingles, C J

    2001-02-01

    Nucleotide excision repair is the major pathway responsible for removing UV-induced DNA damage, and is therefore essential for cell survival following exposure to UV radiation. In this report, we have assessed the contributions of some components of the RNA polymerase II (Pol II) transcription machinery to UV resistance in Saccharomyces cerevisiae. Deletion of the gene encoding the Pol II elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in the absence of global genome repair dependent on the RAD7 and RAD16 genes, a result seen previously with deletions of RAD26 and RAD28, yeast homologs of the human Cockayne syndrome genes CSB and CSA, respectively. A RAD7/16-dependent reduction in survival after UV irradiation was also seen in the presence of mutations in RNA Pol II that confer a defect in its response to SII, as well as with other mutations which reside in regions of the largest subunit of Pol II not involved in SII interactions. Indeed, an increase in UV sensitivity was achieved by simply decreasing the steadystate level of RNA Pol II. Truncation of the C-terminal domain and other RNA Pol II mutations conferred sensitivity to the ribonucleotide reductase inhibitor hydroxyurea and induction of RNR1 and RNR2 mRNAs after UV irradiation was attenuated in these mutant cells. That UV sensitivity can be a consequence of mutations in the RNA Pol II machinery in yeast cells suggests that alterations in transcriptional programs could underlie some of the pathophysiological defects seen in the human disease Cockayne syndrome.

  1. Radiation sensitivity of human malignant lymphocytes

    International Nuclear Information System (INIS)

    Seshadri, R.; Matthews, C.; Morley, A.A.

    1985-01-01

    A simple and rapid in vitro technique to assess the sensitivity of human malignant lymphocytes to roentgen irradiation is described. A variety of established malignant lymphocyte cell lines were cloned in microwells and clone survival was used as the end-point. The survival of the clonogenic malignant lymphocyte down to a fraction of approximately 0.001 could be measured accurately. Except for a T-cell line, the radiation sensitivities of the cell lines were similar to that of normal T-lymphocytes. (orig.)

  2. Re-inspection of small RNA sequence datasets reveals several novel human miRNA genes.

    Directory of Open Access Journals (Sweden)

    Thomas Birkballe Hansen

    Full Text Available BACKGROUND: miRNAs are key players in gene expression regulation. To fully understand the complex nature of cellular differentiation or initiation and progression of disease, it is important to assess the expression patterns of as many miRNAs as possible. Thereby, identifying novel miRNAs is an essential prerequisite to make possible a comprehensive and coherent understanding of cellular biology. METHODOLOGY/PRINCIPAL FINDINGS: Based on two extensive, but previously published, small RNA sequence datasets from human embryonic stem cells and human embroid bodies, respectively [1], we identified 112 novel miRNA-like structures and were able to validate miRNA processing in 12 out of 17 investigated cases. Several miRNA candidates were furthermore substantiated by including additional available small RNA datasets, thereby demonstrating the power of combining datasets to identify miRNAs that otherwise may be assigned as experimental noise. CONCLUSIONS/SIGNIFICANCE: Our analysis highlights that existing datasets are not yet exhaustedly studied and continuous re-analysis of the available data is important to uncover all features of small RNA sequencing.

  3. Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

    Science.gov (United States)

    2016-09-07

    sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

  4. Sensitive and long-term monitoring of intracellular microRNAs using a non-integrating cytoplasmic RNA vector.

    Science.gov (United States)

    Sano, Masayuki; Ohtaka, Manami; Iijima, Minoru; Nakasu, Asako; Kato, Yoshio; Nakanishi, Mahito

    2017-10-04

    MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression at the post-transcriptional level. Different types of cells express unique sets of miRNAs that can be exploited as potential molecular markers to identify specific cell types. Among the variety of miRNA detection methods, a fluorescence-based imaging system that utilises a fluorescent-reporter gene regulated by a target miRNA offers a major advantage for long-term tracking of the miRNA in living cells. In this study, we developed a novel fluorescence-based miRNA-monitoring system using a non-integrating cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because SeVdp vectors robustly and stably express transgenes, this system enabled sensitive monitoring of miRNAs by fluorescence microscopy. By applying this system for cellular reprogramming, we found that miR-124, but not miR-9, was significantly upregulated during direct neuronal conversion. Additionally, we were able to isolate integration-free human induced pluripotent stem cells by long-term tracking of let-7 expression. Notably, this system was easily expandable to allow detection of multiple miRNAs separately and simultaneously. Our findings provide insight into a powerful tool for evaluating miRNA expression during the cellular reprogramming process and for isolating reprogrammed cells potentially useful for medical applications.

  5. Thermo-sensitive nanoparticles for triggered release of siRNA.

    Science.gov (United States)

    Yang, Zheng; Cheng, Qiang; Jiang, Qian; Deng, Liandong; Liang, Zicai; Dong, Anjie

    2015-01-01

    Efficient delivery of small interfering RNA (siRNA) is crucially required for cancer gene therapy. Herein, a thermo-sensitive copolymer with a simple structure, poly (ethylene glycol) methyl ether acrylate-b-poly (N-isopropylacrylamide) (mPEG-b-PNIPAM) was developed. A novel kind of thermo-sensitive nanoparticles (DENPs) was constructed for the cold-shock triggered release of siRNA by double emulsion-solvent evaporation method using mPEG-b-PNIPAM and a cationic lipid, 3β [N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol [DC-Chol]. DENPs were observed by transmission electron microscopy and dynamical light scattering before and after 'cold shock' treatment. The encapsulation efficiency (EE) of siRNA in DENPs, which was measured by fluorescence spectrophotometer was 96.8% while it was significantly reduced to be 23.2% when DC-Chol was absent. DENPs/siRNA NPs exhibited a thermo-sensitive siRNA release character that the cumulatively released amount of siRNA from cold shock was approximately 2.2 folds higher after 7 days. In vitro luciferase silencing experiments indicated that DENPs showed potent gene silencing efficacy in HeLa-Luc cells (HeLa cells steadily expressed luciferase), which was further enhanced by a cold shock. Furthermore, MTT assay showed that cell viability with DENPs/siRNA up to 200 nM remained above 80%. We also observed that most of siRNA was accumulated in kidney mediated by DENPs instead of liver and spleen in vivo experiments. Thus, DENPs as a cold shock responsive quick release model for siRNA or hydrophilic macromolecules delivery provide a new way to nanocarrier design and clinic therapy.

  6. MicroRNA in Human Glioma

    Energy Technology Data Exchange (ETDEWEB)

    Li, Mengfeng, E-mail: limf@mail.sysu.edu.cn [Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Chinese Ministry of Education, Guangzhou 510080 (China); Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Li, Jun [Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Chinese Ministry of Education, Guangzhou 510080 (China); Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Liu, Lei; Li, Wei [Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Chinese Ministry of Education, Guangzhou 510080 (China); Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Yang, Yi [Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Chinese Ministry of Education, Guangzhou 510080 (China); Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Yuan, Jie [Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Chinese Ministry of Education, Guangzhou 510080 (China); Key Laboratory of Functional Molecules from Oceanic Microorganisms (Sun Yat-sen University), Department of Education of Guangdong Province, Guangzhou 510080 (China)

    2013-10-23

    Glioma represents a serious health problem worldwide. Despite advances in surgery, radiotherapy, chemotherapy, and targeting therapy, the disease remains one of the most lethal malignancies in humans, and new approaches to improvement of the efficacy of anti-glioma treatments are urgently needed. Thus, new therapeutic targets and tools should be developed based on a better understanding of the molecular pathogenesis of glioma. In this context, microRNAs (miRNAs), a class of small, non-coding RNAs, play a pivotal role in the development of the malignant phenotype of glioma cells, including cell survival, proliferation, differentiation, tumor angiogenesis, and stem cell generation. This review will discuss the biological functions of miRNAs in human glioma and their implications in improving clinical diagnosis, prediction of prognosis, and anti-glioma therapy.

  7. MicroRNA in Human Glioma

    International Nuclear Information System (INIS)

    Li, Mengfeng; Li, Jun; Liu, Lei; Li, Wei; Yang, Yi; Yuan, Jie

    2013-01-01

    Glioma represents a serious health problem worldwide. Despite advances in surgery, radiotherapy, chemotherapy, and targeting therapy, the disease remains one of the most lethal malignancies in humans, and new approaches to improvement of the efficacy of anti-glioma treatments are urgently needed. Thus, new therapeutic targets and tools should be developed based on a better understanding of the molecular pathogenesis of glioma. In this context, microRNAs (miRNAs), a class of small, non-coding RNAs, play a pivotal role in the development of the malignant phenotype of glioma cells, including cell survival, proliferation, differentiation, tumor angiogenesis, and stem cell generation. This review will discuss the biological functions of miRNAs in human glioma and their implications in improving clinical diagnosis, prediction of prognosis, and anti-glioma therapy

  8. Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

    Jbilo, O.; Barteles, C.F.; Chatonnet, A.; Toutant, J.P.; Lockridge, O.

    1994-12-31

    Tissue distribution of human acetyicholinesterase and butyryicholinesterase messenger RNA. 1 Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetyicholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.

  9. Human sensitivity to vertical self-motion.

    Science.gov (United States)

    Nesti, Alessandro; Barnett-Cowan, Michael; Macneilage, Paul R; Bülthoff, Heinrich H

    2014-01-01

    Perceiving vertical self-motion is crucial for maintaining balance as well as for controlling an aircraft. Whereas heave absolute thresholds have been exhaustively studied, little work has been done in investigating how vertical sensitivity depends on motion intensity (i.e., differential thresholds). Here we measure human sensitivity for 1-Hz sinusoidal accelerations for 10 participants in darkness. Absolute and differential thresholds are measured for upward and downward translations independently at 5 different peak amplitudes ranging from 0 to 2 m/s(2). Overall vertical differential thresholds are higher than horizontal differential thresholds found in the literature. Psychometric functions are fit in linear and logarithmic space, with goodness of fit being similar in both cases. Differential thresholds are higher for upward as compared to downward motion and increase with stimulus intensity following a trend best described by two power laws. The power laws' exponents of 0.60 and 0.42 for upward and downward motion, respectively, deviate from Weber's Law in that thresholds increase less than expected at high stimulus intensity. We speculate that increased sensitivity at high accelerations and greater sensitivity to downward than upward self-motion may reflect adaptations to avoid falling.

  10. Coordinated action of histone modification and microRNA regulations in human genome.

    Science.gov (United States)

    Wang, Xuan; Zheng, Guantao; Dong, Dong

    2015-10-10

    Both histone modifications and microRNAs (miRNAs) play pivotal role in gene expression regulation. Although numerous studies have been devoted to explore the gene regulation by miRNA and epigenetic regulations, their coordinated actions have not been comprehensively examined. In this work, we systematically investigated the combinatorial relationship between miRNA and epigenetic regulation by taking advantage of recently published whole genome-wide histone modification data and high quality miRNA targeting data. The results showed that miRNA targets have distinct histone modification patterns compared with non-targets in their promoter regions. Based on this finding, we proposed a machine learning approach to fit predictive models on the task to discern whether a gene is targeted by a specific miRNA. We found a considerable advantage in both sensitivity and specificity in diverse human cell lines. Finally, we found that our predicted miRNA targets are consistently annotated with Gene Ontology terms. Our work is the first genome-wide investigation of the coordinated action of miRNA and histone modification regulations, which provide a guide to deeply understand the complexity of transcriptional regulation. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. The determinants of alternative RNA splicing in human cells.

    Science.gov (United States)

    Ramanouskaya, Tatsiana V; Grinev, Vasily V

    2017-12-01

    Alternative splicing represents an important level of the regulation of gene function in eukaryotic organisms. It plays a critical role in virtually every biological process within an organism, including regulation of cell division and cell death, differentiation of tissues in the embryo and the adult organism, as well as in cellular response to diverse environmental factors. In turn, studies of the last decade have shown that alternative splicing itself is controlled by different mechanisms. Unfortunately, there is no clear understanding of how these diverse mechanisms, or determinants, regulate and constrain the set of alternative RNA species produced from any particular gene in every cell of the human body. Here, we provide a consolidated overview of alternative splicing determinants including RNA-protein interactions, epigenetic regulation via chromatin remodeling, coupling of transcription-to-alternative splicing, effect of secondary structures in pre-RNA, and function of the RNA quality control systems. We also extensively and critically discuss some mechanistic insights on coordinated inclusion/exclusion of exons during the formation of mature RNA molecules. We conclude that the final structure of RNA is pre-determined by a complex interplay between cis- and trans-acting factors. Altogether, currently available empirical data significantly expand our understanding of the functioning of the alternative splicing machinery of cells in normal and pathological conditions. On the other hand, there are still many blind spots that require further deep investigations.

  12. Novel targets for sensitizing breast cancer cells to TRAIL-induced apoptosis with siRNA delivery.

    Science.gov (United States)

    Thapa, Bindu; Bahadur Kc, Remant; Uludağ, Hasan

    2018-02-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in variety of cancer cells without affecting most normal cells, which makes it a promising agent for cancer therapy. However, TRAIL therapy is clinically not effective due to resistance induction. To identify novel regulators of TRAIL that can aid in therapy, protein targets whose silencing sensitized breast cancer cells against TRAIL were screened with an siRNA library against 446 human apoptosis-related proteins in MDA-231 cells. Using a cationic lipopolymer (PEI-αLA) for delivery of library members, 16 siRNAs were identified that sensitized the TRAIL-induced death in MDA-231 cells. The siRNAs targeting BCL2L12 and SOD1 were further evaluated based on the novelty and their ability to sensitize TRAIL induced cell death. Silencing both targets sensitized TRAIL-mediated cell death in MDA-231 cells as well as TRAIL resistant breast cancer cells, MCF-7. Combination of TRAIL and siRNA silencing BCL2L12 had no effect in normal human umbilical vein cells and human bone marrow stromal cell. The silencing of BCL2L12 and SOD1 enhanced TRAIL-mediated apoptosis in MDA-231 cells via synergistically activating capsase-3 activity. Hence, here we report siRNAs targeting BCL2L12 and SOD1 as a novel regulator of TRAIL-induced cell death in breast cancer cells, providing a new approach for enhancing TRAIL therapy for breast cancer. The combination of siRNA targeting BCL2L12 and TRAIL can be a highly effective synergistic pair in breast cancer cells with minimal effect on the non-transformed cells. © 2017 UICC.

  13. Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

    Science.gov (United States)

    Xia, Hongjie; Wang, Peipei; Wang, Guang-Chuan; Yang, Jie; Sun, Xianlin; Wu, Wenzhe; Qiu, Yang; Shu, Ting; Zhao, Xiaolu; Yin, Lei; Qin, Cheng-Feng; Hu, Yuanyang; Zhou, Xi

    2015-01-01

    RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our

  14. The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity.

    Science.gov (United States)

    Abbott, Jamie A; Guth, Ethan; Kim, Cindy; Regan, Cathy; Siu, Victoria M; Rupar, C Anthony; Demeler, Borries; Francklyn, Christopher S; Robey-Bond, Susan M

    2017-07-18

    Histidyl-tRNA synthetase (HARS) is a highly conserved translation factor that plays an essential role in protein synthesis. HARS has been implicated in the human syndromes Charcot-Marie-Tooth (CMT) Type 2W and Type IIIB Usher (USH3B). The USH3B mutation, which encodes a Y454S substitution in HARS, is inherited in an autosomal recessive fashion and associated with childhood deafness, blindness, and episodic hallucinations during acute illness. The biochemical basis of the pathophysiologies linked to USH3B is currently unknown. Here, we present a detailed functional comparison of wild-type (WT) and Y454S HARS enzymes. Kinetic parameters for enzymes and canonical substrates were determined using both steady state and rapid kinetics. Enzyme stability was examined using differential scanning fluorimetry. Finally, enzyme functionality in a primary cell culture was assessed. Our results demonstrate that the Y454S substitution leaves HARS amino acid activation, aminoacylation, and tRNA His binding functions largely intact compared with those of WT HARS, and the mutant enzyme dimerizes like the wild type does. Interestingly, during our investigation, it was revealed that the kinetics of amino acid activation differs from that of the previously characterized bacterial HisRS. Despite the similar kinetics, differential scanning fluorimetry revealed that Y454S is less thermally stable than WT HARS, and cells from Y454S patients grown at elevated temperatures demonstrate diminished levels of protein synthesis compared to those of WT cells. The thermal sensitivity associated with the Y454S mutation represents a biochemical basis for understanding USH3B.

  15. RNA-binding properties and RNA chaperone activity of human peroxiredoxin 1

    International Nuclear Information System (INIS)

    Kim, Ji-Hee; Lee, Jeong-Mi; Lee, Hae Na; Kim, Eun-Kyung; Ha, Bin; Ahn, Sung-Min; Jang, Ho Hee; Lee, Sang Yeol

    2012-01-01

    Highlights: ► hPrx1 has RNA-binding properties. ► hPrx1 exhibits helix-destabilizing activity. ► Cold stress increases hPrx1 level in the nuclear fraction. ► hPrx1 enhances the viability of cells exposed to cold stress. -- Abstract: Human peroxiredoxin 1 (hPrx1), a member of the peroxiredoxin family, detoxifies peroxide substrates and has been implicated in numerous biological processes, including cell growth, proliferation, differentiation, apoptosis, and redox signaling. To date, Prx1 has not been implicated in RNA metabolism. Here, we investigated the ability of hPrx1 to bind RNA and act as an RNA chaperone. In vitro, hPrx1 bound to RNA and DNA, and unwound nucleic acid duplexes. hPrx1 also acted as a transcription anti-terminator in an assay using an Escherichia coli strain containing a stem–loop structure upstream of the chloramphenicol resistance gene. The overall cellular level of hPrx1 expression was not increased at low temperatures, but the nuclear level of hPrx1 was increased. In addition, hPrx1 overexpression enhanced the survival of cells exposed to cold stress, whereas hPrx1 knockdown significantly reduced cell survival under the same conditions. These findings suggest that hPrx1 may perform biological functions as a RNA-binding protein, which are distinctive from known functions of hPrx1 as a reactive oxygen species scavenger.

  16. MicroRNA expression variability in human cervical tissues.

    Directory of Open Access Journals (Sweden)

    Patrícia M Pereira

    Full Text Available MicroRNAs (miRNAs are short (approximately 22 nt non-coding regulatory RNAs that control gene expression at the post-transcriptional level. Deregulation of miRNA expression has been discovered in a wide variety of tumours and it is now clear that they contribute to cancer development and progression. Cervical cancer is one of the most common cancers in women worldwide and there is a strong need for a non-invasive, fast and efficient method to diagnose the disease. We investigated miRNA expression profiles in cervical cancer using a microarray platform containing probes for mature miRNAs. We have evaluated miRNA expression profiles of a heterogeneous set of cervical tissues from 25 different patients. This set included 19 normal cervical tissues, 4 squamous cell carcinoma, 5 high-grade squamous intraepithelial lesion (HSIL and 9 low-grade squamous intraepithelial lesion (LSIL samples. We observed high variability in miRNA expression especially among normal cervical samples, which prevented us from obtaining a unique miRNA expression signature for this tumour type. However, deregulated miRNAs were identified in malignant and pre-malignant cervical tissues after tackling the high expression variability observed. We were also able to identify putative target genes of relevant candidate miRNAs. Our results show that miRNA expression shows natural variability among human samples, which complicates miRNA data profiling analysis. However, such expression noise can be filtered and does not prevent the identification of deregulated miRNAs that play a role in the malignant transformation of cervical squamous cells. Deregulated miRNAs highlight new candidate gene targets allowing for a better understanding of the molecular mechanism underlying the development of this tumour type.

  17. Poliovirus RNA polymerase: in vitro enzymatic activities, fidelity of replication, and characterization of a temperature-sensitive RNA-negative mutant

    International Nuclear Information System (INIS)

    Stokes, M.A.M.

    1985-01-01

    The in vitro activities of the purified poliovirus RNA polymerase were investigated in this study. The polymerase was shown to be a strict RNA dependent RNA polymerase. It only copied RNA templates but used either a DNA or RNA primer to initiate RNA synthesis. Partially purified polymerase has some DNA polymerase activities. Additional purification of the enzyme and studies with a mutant poliovirus RNA polymerase indicated that the DNA polymerase activities were due to a cellular polymerase. The fidelity of RNA replication in vitro by the purified poliovirus RNA polymerase was studied by measuring the rate of misincorporation of noncomplementary ribonucleotide monophosphates on synthetic homopolymeric RNA templates. The results showed that the ratio of noncomplementary to complementary ribonucleotides incorporated was 1-5 x 10 -3 . The viral polymerase of a poliovirus temperature sensitive RNA-negative mutant, Ts 10, was isolated. This study confirmed that the mutant was viable 33 0 , but was RNA negative at 39 0 . Characterization of the Ts 10 polymerase showed it was significantly more sensitive to heat inactivation than was the old-type polymerase. Highly purified poliovirions were found to contain several noncapsid proteins. At least two of these proteins were labeled by [ 35 S]methionine infected cells and appeared to be virally encoded proteins. One of these proteins was immunoprecipitated by anti-3B/sup vpg/ antiserum. This protein had the approximate Mr = 50,000 and appeared to be one of the previously identified 3B/sup vpg/ precursor proteins

  18. Clinical Impact of a Novel MicroRNA Chemo-Sensitivity Predictor in Gastrooesophageal Cancer

    DEFF Research Database (Denmark)

    Winther, Mette; Knudsen, Steen; Dahlgaard, Jesper

    2016-01-01

    to be independently associated with both OS (HR: 0.37 (95% CI: 0.16-0.87)) and DSS (HR: 0.32 (0.12-0.87)). CONCLUSION: The miRNA profile predictive for sensitivity to cisplatin, epirubicine and capecitabine was shown to be independently associated with OS and DSS in patients with gastrooesophageal cancer....

  19. MicroRNA-221 and -222 Regulate Radiation Sensitivity by Targeting the PTEN Pathway

    International Nuclear Information System (INIS)

    Zhang Chunzhi; Kang Chunsheng; Wang Ping; Cao Yongzhen; Lv Zhonghong; Yu Shizhu; Wang Guangxiu; Zhang Anling; Jia Zhifan; Han Lei; Yang Chunying; Ishiyama, Hiromichi; Teh, Bin S.; Xu Bo; Pu Peiyu

    2011-01-01

    Purpose: MicroRNAs (miRNAs) are noncoding RNAs inhibiting expression of numerous target genes by posttranscriptional regulation. miRNA-221 and miRNA-222 (miRNA-221/-222) expression is elevated in radioresistant tumor cell lines; however, it is not known whether and how miRNAs control cellular responses to ionizing irradiation. Methods and Materials: We used bioinformatic analyses, luciferase reporter assay, and genetic knockdown and biochemical assays to characterize the regulation pathways of miRNA-221/-222 in response to radiation treatment. Results: We identified the PTEN gene as a target of miRNA-221/-222. Furthermore, we found that knocking down miRNA-221/-222 by antisense oligonucleotides upregulated PTEN expression. Upregulated PTEN expression suppressed AKT activity and increased radiation-induced apoptosis, resulting in enhancement of radiosensitivity in tumor cells. Conclusions: miRNA-221/-222 control radiation sensitivity by regulating the PTEN/AKT pathway and can be explored as novel targets for radiosensitization.

  20. Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development.

    Science.gov (United States)

    Szabo, Linda; Morey, Robert; Palpant, Nathan J; Wang, Peter L; Afari, Nastaran; Jiang, Chuan; Parast, Mana M; Murry, Charles E; Laurent, Louise C; Salzman, Julia

    2015-06-16

    The pervasive expression of circular RNA is a recently discovered feature of gene expression in highly diverged eukaryotes, but the functions of most circular RNAs are still unknown. Computational methods to discover and quantify circular RNA are essential. Moreover, discovering biological contexts where circular RNAs are regulated will shed light on potential functional roles they may play. We present a new algorithm that increases the sensitivity and specificity of circular RNA detection by discovering and quantifying circular and linear RNA splicing events at both annotated and un-annotated exon boundaries, including intergenic regions of the genome, with high statistical confidence. Unlike approaches that rely on read count and exon homology to determine confidence in prediction of circular RNA expression, our algorithm uses a statistical approach. Using our algorithm, we unveiled striking induction of general and tissue-specific circular RNAs, including in the heart and lung, during human fetal development. We discover regions of the human fetal brain, such as the frontal cortex, with marked enrichment for genes where circular RNA isoforms are dominant. The vast majority of circular RNA production occurs at major spliceosome splice sites; however, we find the first examples of developmentally induced circular RNAs processed by the minor spliceosome, and an enriched propensity of minor spliceosome donors to splice into circular RNA at un-annotated, rather than annotated, exons. Together, these results suggest a potentially significant role for circular RNA in human development.

  1. Comparative structural analysis of human DEAD-box RNA helicases.

    Directory of Open Access Journals (Sweden)

    Patrick Schütz

    2010-09-01

    Full Text Available DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

  2. Comparative structural analysis of human DEAD-box RNA helicases.

    Science.gov (United States)

    Schütz, Patrick; Karlberg, Tobias; van den Berg, Susanne; Collins, Ruairi; Lehtiö, Lari; Högbom, Martin; Holmberg-Schiavone, Lovisa; Tempel, Wolfram; Park, Hee-Won; Hammarström, Martin; Moche, Martin; Thorsell, Ann-Gerd; Schüler, Herwig

    2010-09-30

    DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

  3. mRNA related to insulin family in human placenta

    International Nuclear Information System (INIS)

    Younes, M.A.; D'Agostino, J.B.; Frazier, M.L.; Besch, P.K.

    1986-01-01

    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A + ) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A + ) RNA templates. Five hundred transformants were initially screened by colony hybridization using a 32 P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II

  4. mRNA related to insulin family in human placenta

    Energy Technology Data Exchange (ETDEWEB)

    Younes, M.A.; D' Agostino, J.B.; Frazier, M.L.; Besch, P.K.

    1986-03-01

    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A/sup +/) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A/sup +/) RNA templates. Five hundred transformants were initially screened by colony hybridization using a /sup 32/P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II.

  5. Specific down-regulation of XIAP with RNA interference enhances the sensitivity of canine tumor cell-lines to TRAIL and doxorubicin

    Directory of Open Access Journals (Sweden)

    Rothuizen Jan

    2006-09-01

    Full Text Available Abstract Background Apoptosis resistance occurs in various tumors. The anti-apoptotic XIAP protein is responsible for inhibiting apoptosis by reducing caspase-3 activation. Our aim is to evaluate whether RNA inhibition against XIAP increases the sensitivity of canine cell-lines for chemotherapeutics such as TRAIL and doxorubicin. We used small interfering RNA's (siRNA directed against XIAP in three cell-lines derived from bile-duct epithelia (BDE, mammary carcinoma (P114, and osteosarcoma (D17. These cell-lines represent frequently occurring canine cancers and are highly comparable to their human counterparts. XIAP down-regulation was measured by means of quantitative PCR (Q-PCR and Western blotting. The XIAP depleted cells were treated with a serial dilution of TRAIL or doxorubicin and compared to mock- and nonsense-treated controls. Viability was measured with a MTT assay. Results All XIAP siRNA treated cell-lines showed a mRNA down-regulation over 80 percent. Western blot analysis confirmed mRNA measurements. No compensatory effect of IAP family members was seen in XIAP depleted cells. The sensitivity of XIAP depleted cells for TRAIL was highest in BDE cells with an increase in the ED50 of 14-fold, compared to mock- and nonsense-treated controls. The sensitivity of P114 and D17 cell-lines increased six- and five-fold, respectively. Doxorubicin treatment in XIAP depleted cells increased sensitivity in BDE cells more than eight-fold, whereas P114 and D17 cell-lines showed an increase in sensitivity of three- and five-fold, respectively. Conclusion XIAP directed siRNA's have a strong sensitizing effect on TRAIL-reduced cell-viability and a smaller but significant effect with the DNA damaging drug doxorubicin. The increase in efficacy of chemotherapeutics with XIAP depletion provides the rationale for the use of XIAP siRNA's in insensitive canine tumors.

  6. Diagnostic Utility of Broad Range Bacterial 16S rRNA Gene PCR with Degradation of Human and Free Bacterial DNA in Bloodstream Infection Is More Sensitive Than an In-House Developed PCR without Degradation of Human and Free Bacterial DNA

    Directory of Open Access Journals (Sweden)

    Petra Rogina

    2014-01-01

    Full Text Available We compared a commercial broad range 16S rRNA gene PCR assay (SepsiTest to an in-house developed assay (IHP. We assessed whether CD64 index, a biomarker of bacterial infection, can be used to exclude patients with a low probability of systemic bacterial infection. From January to March 2010, 23 patients with suspected sepsis were enrolled. CD64 index, procalcitonin, and C-reactive protein were measured on admission. Broad range 16S rRNA gene PCR was performed from whole blood (SepsiTest or blood plasma (IHP and compared to blood culture results. Blood samples spiked with Staphylococcus aureus were used to assess sensitivity of the molecular assays in vitro. CD64 index was lower in patients where possible sepsis was excluded than in patients with microbiologically confirmed sepsis (P=0.004. SepsiTest identified more relevant pathogens than blood cultures (P=0.008; in three patients (13% results from blood culture and SepsiTest were congruent, whereas in four cases (17.4% relevant pathogens were detected by SepsiTest only. In vitro spiking experiments suggested equal sensitivity of SepsiTest and IHP. A diagnostic algorithm using CD64 index as a decision maker to perform SepsiTest shows improved detection of pathogens in patients with suspected blood stream infection and may enable earlier targeted antibiotic therapy.

  7. Inhibition of Src by microRNA-23b increases the cisplatin sensitivity of chondrosarcoma cells.

    Science.gov (United States)

    Huang, Kai; Chen, Jun; Yang, Mo-Song; Tang, Yu-Jun; Pan, Feng

    2017-01-01

    Chondrosarcomas are malignant cartilage-forming tumors from low-grade to high-grade aggressive tumors characterized by metastasis. Cisplatin is an effective DNA-damaging anti-tumor agent for the treatment against a wide variety of solid tumors. However, chondrosarcomas are notorious for their resistance to conventional chemo- and radio- therapies. In this study, we report miR-23b acts as a tumor suppressor in chondrosarcoma. The expressions of miR-23b are down-regulated in chondrosarcoma patient samples and cell lines compared with adjacent normal tissues and human primary chondrocytes. In addition, overexpression of miR-23b suppresses chondrosarcoma cell proliferation. By comparison of the cisplatin resistant chondrosarcoma cells and parental cells, we observed miR-23b was significantly down regulated in cisplatin resistant cells. Moreover, we demonstrate here Src kinase is a direct target of miR-23b in chondrosarcoma cells. Overexpression of miR-23b suppresses Src-Akt pathway, leading to the sensitization of cisplatin resistant chondrosarcoma cells to cisplatin. This chemo-sensitivity effect by the miR-23b-mediated inhibition of Src-Akt pathway is verified with the restoration of Src kinase in miR-23b-overespressing chondrosarcoma cells, resulting in the acquirement of resistance to cisplatin. In summary, our study reveals a novel role of miR-23b in cisplatin resistance in chondrosarcoma and will contribute to the development of the microRNA-targeted anti-cancer therapeutics.

  8. Genetics of human sensitivity to ultraviolet radiation

    Science.gov (United States)

    Cleaver, James E.

    1994-07-01

    the major human health effects of solar and artificial UV light occur from the UVB and UVC wavelength ranges and involve a variety of short-term and long-term deleterious changes to the skin and eyes. the more important initial damage to cellular macromolecules involves dimerization of adjacent pyrimidines in DNA to produce cyclobutane pyrimidine dimes, (6-4) pyrimidine- pyrimidone, and (6-4) dewar photoproducts. these photoproducts can be repaired by a genetically regulated enzyme system (nucleotide excision repair) which removes oligonucleotides 29-30 nucleotides long that contain the photoproducts, and synthesizes replacement patches. At least a dozen gene products are involved in the process of recognizing photoproducts in DNA, altering local DNA helicity and cleaving the polynucleotide chain at defined positions either side of a photoproduct. Hereditary mutations in many of these genes are recognized in the human genetic disorders xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). Several of the gene products have other functions involving the regulation of gene transcription which accounts for the complex clinical presentation of repair deficient diseases that involve sensitivity of the skin and eyes to UV light, increased solar carcinogenesis (in XP), demyelination, and ganglial calcification (in CS), hair abnormalities (in TTD), and developmental and neurological abnormalities

  9. Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases

    International Nuclear Information System (INIS)

    Cirullo, R.E.; Dana, S.; Wasmuth, J.J.

    1983-01-01

    A simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients has been developed that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, Chinese hamster cell lines have been constructed that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure

  10. MicroRNA signature of the human developing pancreas

    Directory of Open Access Journals (Sweden)

    Correa-Medina Mayrin

    2010-09-01

    Full Text Available Abstract Background MicroRNAs are non-coding RNAs that regulate gene expression including differentiation and development by either inhibiting translation or inducing target degradation. The aim of this study is to determine the microRNA expression signature during human pancreatic development and to identify potential microRNA gene targets calculating correlations between the signature microRNAs and their corresponding mRNA targets, predicted by bioinformatics, in genome-wide RNA microarray study. Results The microRNA signature of human fetal pancreatic samples 10-22 weeks of gestational age (wga, was obtained by PCR-based high throughput screening with Taqman Low Density Arrays. This method led to identification of 212 microRNAs. The microRNAs were classified in 3 groups: Group number I contains 4 microRNAs with the increasing profile; II, 35 microRNAs with decreasing profile and III with 173 microRNAs, which remain unchanged. We calculated Pearson correlations between the expression profile of microRNAs and target mRNAs, predicted by TargetScan 5.1 and miRBase altgorithms, using genome-wide mRNA expression data. Group I correlated with the decreasing expression of 142 target mRNAs and Group II with the increasing expression of 876 target mRNAs. Most microRNAs correlate with multiple targets, just as mRNAs are targeted by multiple microRNAs. Among the identified targets are the genes and transcription factors known to play an essential role in pancreatic development. Conclusions We have determined specific groups of microRNAs in human fetal pancreas that change the degree of their expression throughout the development. A negative correlative analysis suggests an intertwined network of microRNAs and mRNAs collaborating with each other. This study provides information leading to potential two-way level of combinatorial control regulating gene expression through microRNAs targeting multiple mRNAs and, conversely, target mRNAs regulated in

  11. RNA FISH for detecting expanded repeats in human diseases.

    Science.gov (United States)

    Urbanek, Martyna O; Krzyzosiak, Wlodzimierz J

    2016-04-01

    RNA fluorescence in situ hybridization (FISH) is a widely used technique for detecting transcripts in fixed cells and tissues. Many variants of RNA FISH have been proposed to increase signal strength, resolution and target specificity. The current variants of this technique facilitate the detection of the subcellular localization of transcripts at a single molecule level. Among the applications of RNA FISH are studies on nuclear RNA foci in diseases resulting from the expansion of tri-, tetra-, penta- and hexanucleotide repeats present in different single genes. The partial or complete retention of mutant transcripts forming RNA aggregates within the nucleoplasm has been shown in multiple cellular disease models and in the tissues of patients affected with these atypical mutations. Relevant diseases include, among others, myotonic dystrophy type 1 (DM1) with CUG repeats, Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3) with CAG repeats, fragile X-associated tremor/ataxia syndrome (FXTAS) with CGG repeats, myotonic dystrophy type 2 (DM2) with CCUG repeats, amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) with GGGGCC repeats and spinocerebellar ataxia type 32 (SCA32) with GGCCUG. In this article, we summarize the results obtained with FISH to examine RNA nuclear inclusions. We provide a detailed protocol for detecting RNAs containing expanded CAG and CUG repeats in different cellular models, including fibroblasts, lymphoblasts, induced pluripotent stem cells and murine and human neuronal progenitors. We also present the results of the first single-molecule FISH application in a cellular model of polyglutamine disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. ExpEdit: a webserver to explore human RNA editing in RNA-Seq experiments.

    Science.gov (United States)

    Picardi, Ernesto; D'Antonio, Mattia; Carrabino, Danilo; Castrignanò, Tiziana; Pesole, Graziano

    2011-05-01

    ExpEdit is a web application for assessing RNA editing in human at known or user-specified sites supported by transcript data obtained by RNA-Seq experiments. Mapping data (in SAM/BAM format) or directly sequence reads [in FASTQ/short read archive (SRA) format] can be provided as input to carry out a comparative analysis against a large collection of known editing sites collected in DARNED database as well as other user-provided potentially edited positions. Results are shown as dynamic tables containing University of California, Santa Cruz (UCSC) links for a quick examination of the genomic context. ExpEdit is freely available on the web at http://www.caspur.it/ExpEdit/.

  13. Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein–staphylococcal nuclease hybrid

    International Nuclear Information System (INIS)

    Mori, Tomoaki; Nakamura, Kento; Masaoka, Keisuke; Fujita, Yusuke; Morisada, Ryosuke; Mori, Koichi; Tobimatsu, Takamasa; Sera, Takashi

    2016-01-01

    Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms. - Highlights: • A novel RNA restriction enzyme using SNase was developed tor cleave viral RNA. • Our enzyme cleaved influenza RNA with rates >120-fold higher rates a PIN-fusion one. • Our artificial enzyme with the L5 linker showed the highest RNA cleavage rate. • Our artificial enzyme site-selectively cleaved influenza RNA in vitro.

  14. Androgen receptor signaling is required for androgen-sensitive human prostate cancer cell proliferation and survival

    Directory of Open Access Journals (Sweden)

    Day Wanda V

    2005-04-01

    Full Text Available Abstract Background Androgens and androgen receptors (AR regulate normal prostate development and growth. They also are involved in pathological development of prostatic diseases, including benign prostatic hyperplasia (BPH and prostate cancer (PCa. Antiandrogen therapy for PCa, in conjunction with chemical or surgical castration, offers initial positive responses and leads to massive prostate cell death. However, cancer cells later appear as androgen-independent PCa. To investigate the role of AR in prostate cell proliferation and survival, we introduced a vector-based small interfering RNA (siRNA. This siRNA targeted 5'-untranslated region of AR mRNA for extended suppression of AR expression in androgen-sensitive human prostate LNCaP cells. Results The siRNA design successfully suppressed endogenous AR expression, as revealed by western blotting and immunofluorescence staining in LNCaP cells. LNCaP cells did not proliferate in the absence of AR and underwent apoptosis, based on elevated phospho-Histone H2B expression and higher number of apoptotic body as compared to control cells. Conclusion We demonstrated that AR is vital for prostate cell proliferation and survival in this androgen-sensitive prostate cell line. These results further strengthen the hypothesis that AR can be a therapeutic target for treating androgen-sensitive stages of PCa. Unlike antiandorgens, however, siRNA targeting AR provides a direct inactivation of AR function through the suppression of AR protein expression.

  15. The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

    Science.gov (United States)

    Moser, Joanna J; Fritzler, Marvin J

    2010-10-18

    GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

  16. The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

    Directory of Open Access Journals (Sweden)

    Joanna J Moser

    2010-10-01

    Full Text Available GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA-mediated messenger RNA (mRNA silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC. To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells.RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1 miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2 astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3 miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4 the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells.The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

  17. Molecular structure and thermodynamic predictions to create highly sensitive microRNA biosensors

    International Nuclear Information System (INIS)

    Larkey, Nicholas E.; Brucks, Corinne N.; Lansing, Shan S.; Le, Sophia D.; Smith, Natasha M.; Tran, Victoria; Zhang, Lulu; Burrows, Sean M.

    2016-01-01

    Many studies have established microRNAs (miRNAs) as post-transcriptional regulators in a variety of intracellular molecular processes. Abnormal changes in miRNA have been associated with several diseases. However, these changes are sometimes subtle and occur at nanomolar levels or lower. Several biosensing hurdles for in situ cellular/tissue analysis of miRNA limit detection of small amounts of miRNA. Of these limitations the most challenging are selectivity and sensor degradation creating high background signals and false signals. Recently we developed a reporter+probe biosensor for let-7a that showed potential to mitigate false signal from sensor degradation. Here we designed reporter+probe biosensors for miR-26a-2-3p and miR-27a-5p to better understand the effect of thermodynamics and molecular structures of the biosensor constituents on the analytical performance. Signal changes from interactions between Cy3 and Cy5 on the reporters were used to understand structural aspects of the reporter designs. Theoretical thermodynamic values, single stranded conformations, hetero- and homodimerization structures, and equilibrium concentrations of the reporters and probes were used to interpret the experimental observations. Studies of the sensitivity and selectivity revealed 5–9 nM detection limits in the presence and absence of interfering off-analyte miRNAs. These studies will aid in determining how to rationally design reporter+probe biosensors to overcome hurdles associated with highly sensitive miRNA biosensing. - Highlights: • Challenges facing highly sensitive miRNA biosensor designs are addressed. • Thermodynamic and molecular structure design metrics for reporter+probe biosensors are proposed. • The influence of ideal and non-ideal reporter hairpin structures on reporter+probe formation and signal change are discussed. • 5–9 nM limits of detection were observed with no interference from off-analytes.

  18. Molecular structure and thermodynamic predictions to create highly sensitive microRNA biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Larkey, Nicholas E.; Brucks, Corinne N.; Lansing, Shan S.; Le, Sophia D.; Smith, Natasha M.; Tran, Victoria; Zhang, Lulu; Burrows, Sean M., E-mail: sean.burrows@oregonstate.edu

    2016-02-25

    Many studies have established microRNAs (miRNAs) as post-transcriptional regulators in a variety of intracellular molecular processes. Abnormal changes in miRNA have been associated with several diseases. However, these changes are sometimes subtle and occur at nanomolar levels or lower. Several biosensing hurdles for in situ cellular/tissue analysis of miRNA limit detection of small amounts of miRNA. Of these limitations the most challenging are selectivity and sensor degradation creating high background signals and false signals. Recently we developed a reporter+probe biosensor for let-7a that showed potential to mitigate false signal from sensor degradation. Here we designed reporter+probe biosensors for miR-26a-2-3p and miR-27a-5p to better understand the effect of thermodynamics and molecular structures of the biosensor constituents on the analytical performance. Signal changes from interactions between Cy3 and Cy5 on the reporters were used to understand structural aspects of the reporter designs. Theoretical thermodynamic values, single stranded conformations, hetero- and homodimerization structures, and equilibrium concentrations of the reporters and probes were used to interpret the experimental observations. Studies of the sensitivity and selectivity revealed 5–9 nM detection limits in the presence and absence of interfering off-analyte miRNAs. These studies will aid in determining how to rationally design reporter+probe biosensors to overcome hurdles associated with highly sensitive miRNA biosensing. - Highlights: • Challenges facing highly sensitive miRNA biosensor designs are addressed. • Thermodynamic and molecular structure design metrics for reporter+probe biosensors are proposed. • The influence of ideal and non-ideal reporter hairpin structures on reporter+probe formation and signal change are discussed. • 5–9 nM limits of detection were observed with no interference from off-analytes.

  19. Identification of nonviable genes affecting touch sensitivity in Caenorhabditis elegans using neuronally enhanced feeding RNA interference.

    Science.gov (United States)

    Chen, Xiaoyin; Cuadros, Margarete Diaz; Chalfie, Martin

    2015-01-09

    Caenorhabditis elegans senses gentle touch along the body via six touch receptor neurons. Although genetic screens and microarray analyses have identified several genes needed for touch sensitivity, these methods miss pleiotropic genes that are essential for the viability, movement, or fertility of the animals. We used neuronally enhanced feeding RNA interference to screen genes that cause lethality or paralysis when mutated, and we identified 61 such genes affecting touch sensitivity, including five positive controls. We confirmed 18 genes by using available alleles, and further studied one of them, tag-170, now renamed txdc-9. txdc-9 preferentially affects anterior touch response but is needed for tubulin acetylation and microtubule formation in both the anterior and posterior touch receptor neurons. Our results indicate that neuronally enhanced feeding RNA interference screens complement traditional mutageneses by identifying additional nonviable genes needed for specific neuronal functions. Copyright © 2015 Chen et al.

  20. Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9).

    Science.gov (United States)

    Fu, Qinqin; Yuan, Y Adam

    2013-03-01

    Intensive research interest has focused on small RNA-processing machinery and the RNA-induced silencing complex (RISC), key cellular machines in RNAi pathways. However, the structural mechanism regarding RISC assembly, the primary step linking small RNA processing and RNA-mediated gene silencing, is largely unknown. Human RNA helicase A (DHX9) was reported to function as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains (dsRBDs). Here, we report the crystal structures of human RNA helicase A (RHA) dsRBD1 and dsRBD2 domains in complex with dsRNAs, respectively. Structural analysis not only reveals higher siRNA duplex-binding affinity displayed by dsRBD1, but also identifies a crystallographic dsRBD1 pair of physiological significance in cooperatively recognizing dsRNAs. Structural observations are further validated by isothermal titration calorimetric (ITC) assay. Moreover, co-immunoprecipitation (co-IP) assay coupled with mutagenesis demonstrated that both dsRBDs are required for RISC association, and such association is mediated by dsRNA. Hence, our structural and functional efforts have revealed a potential working model for siRNA recognition by RHA tandem dsRBDs, and together they provide direct structural insights into RISC assembly facilitated by RHA.

  1. Identifying airway sensitizers: cytokine mRNA profiles induced by various anhydrides

    International Nuclear Information System (INIS)

    Plitnick, L.M.; Loveless, S.E.; Ladics, G.S.; Holsapple, M.P.; Smialowicz, R.J.; Woolhiser, M.R.; Anderson, P.K.; Smith, C.; Selgrade, M.J.K.

    2003-01-01

    Exposure to low molecular weight (LMW) chemicals in the workplace has been linked to a variety of respiratory effects. Within the LMW chemicals, one of the major classes involved in these effects are the acid anhydrides. The immunological basis of respiratory hypersensitivity involves CD4+ cells. By virtue of their induction of cytokines typical of CD4+ T-helper type 2 (Th2) cells--interleukin (IL)-4, 10, and 13--respiratory sensitizers may be identified and differentiated from contact sensitizers which induce Th1 cytokines (IL-2 and IFN-γ). Our previous work suggested that the ribonuclease protection assay (RPA) was useful in identifying the respiratory sensitizer, trimellitic anhydride (TMA), based on quantitative differences in Th2 cytokine mRNA as compared to the contact sensitizer dinitrochlorobenzene (DNCB). Therefore, the purpose of the studies described in this report was to expand the chemicals tested in the RPA. To this end, four acid anhydrides with known respiratory sensitization potential, TMA, maleic anhydride (MA), phthalic anhydride (PA) and hexahydrophthalic anhydride (HHPA), were tested. Although previously determined to induce immunologically equivalent responses in a local lymph node assay (LLNA), the initial dose chosen (2.5%) failed to induce Th2 cytokine mRNA expression. To determine if the lack of cytokine expression was related to dose, LLNAs were conducted at higher doses for each of the anhydrides. The highest doses evaluated (four- to six-fold higher than those used in the initial RPA) gave equivalent proliferative responses for the various anhydrides and were used for subsequent RPA testing. At these higher doses, significant increases in Th2 versus Th1 cytokine mRNA were observed for all anhydrides tested. These results suggest that the RPA has the potential to serve as a screen for the detection of LMW airway sensitizing chemicals. However, the basis for selecting immunologically equivalent doses may require some modification

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

    Directory of Open Access Journals (Sweden)

    Mallory E. Harden

    2017-01-01

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

  3. A Sensitive and Specific PCR Based Method for Identification of Cryptosporidium Sp. Using New Primers from 18S Ribosomal RNA

    Directory of Open Access Journals (Sweden)

    M Heydarnezhadi

    2011-09-01

    Full Text Available Background: The main goal of the present study was to develop a new sensitive and specific PCR based method for Identification of Cryptosporidium sp. using novel primers from 18S ribosomal RNA. Cryptosporidi­osis in high-risk host groups particularly in neonates and immuno-compromised individuals may result in death. To the best of our knowledge this is the first study regarding develop a new PCR based method to diagnose the cryptosporidiosis in Iran.Methods: A total of 850 human fecal samples from patients clinically suspected to cryptosporidiosis and 100 healthy and diarrheic cattle stool specimens were collected. The simplified formol-ether concentration method was carried out for all samples. They were then examined microscopically by modified Ziehl-Neel­sen staining method. Total DNA was extracted by QIA amp DNA stool mini kit was carried out by using designed prim­ers.Results: Twenty nine cases of cryptosporidiosis infection in human and 30 samples from cattle microscopi­cally were posi­tive. The described primary and nested PCR method could detect all Cryptospori­dium positive samples from human and cattle. Regards to suspected negative samples in pri­mary PCR examination, the Nested PCR could ap­prove two more positive results. Furthermore, Nested PCR analysis was able to detect one more case which was nega­tive in both microscopically examination and primary PCR. Specificity of the test was 100%. Sensitivity of Nested PCR in comparison to our gold standard; microscopy after Ridley concentration modified ziehl-Neelsen, was 100 %.Conclusion: Our developed PCR based method by using new primers devised from 18S ribosomal RNA revealed the ability for identification of the Cryptosporidium species such as C. parvum and C. huminis with high specificity and sensitivity.

  4. Correlation analyses revealed global microRNA-mRNA expression associations in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Wang, Lan; Zhu, Jiang; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Xiao-Wei; Xia, Wei; Xie, Fang-Fei; He, Pei; Bing, Peng-Fei; Qiu, Ying-Hua; Lin, Xiang; Lu, Xin; Zhang, Lei; Yi, Neng-Jun; Zhang, Yong-Hong; Lei, Shu-Feng

    2018-02-01

    MicroRNAs (miRNAs) can regulate gene expression through binding to complementary sites in the 3'-untranslated regions of target mRNAs, which will lead to existence of correlation in expression between miRNA and mRNA. However, the miRNA-mRNA correlation patterns are complex and remain largely unclear yet. To establish the global correlation patterns in human peripheral blood mononuclear cells (PBMCs), multiple miRNA-mRNA correlation analyses and expression quantitative trait locus (eQTL) analysis were conducted in this study. We predicted and achieved 861 miRNA-mRNA pairs (65 miRNAs, 412 mRNAs) using multiple bioinformatics programs, and found global negative miRNA-mRNA correlations in PBMC from all 46 study subjects. Among the 861 pairs of correlations, 19.5% were significant (P correlation network was complex and highlighted key miRNAs/genes in PBMC. Some miRNAs, such as hsa-miR-29a, hsa-miR-148a, regulate a cluster of target genes. Some genes, e.g., TNRC6A, are regulated by multiple miRNAs. The identified genes tend to be enriched in molecular functions of DNA and RNA binding, and biological processes such as protein transport, regulation of translation and chromatin modification. The results provided a global view of the miRNA-mRNA expression correlation profile in human PBMCs, which would facilitate in-depth investigation of biological functions of key miRNAs/mRNAs and better understanding of the pathogenesis underlying PBMC-related diseases.

  5. Altered expression of asparagine synthetase mRNA in human leukemic and carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Goodwin, L.O.; Guzowski, D.E.; Millan, C.A. [North Shore Univ. Hospital/Cornell Univ. Medical College, Manhasset, NY (United States)] [and others

    1994-09-01

    Asparagine synthetase (AS) is the enzyme responsible for the ATP-dependant conversion of aspartic acid to asparagine. The AS gene is expressed constitutively in most mammalian cells, including cells of the lymphoid lineage, as a 2 kb mRNA. In some leukemic phenotypes, AS expression is abrogated, resulting in no detectable enzyme activity. These cells are rendered sensitive to killing by L-asparaginase, which destroys extracellular asparagine. Prolonged treatment of leukemic cells with this agent can lead to resistance and the reappearance of AS activity, suggesting derepression of the AS gene, which has been shown to be regulated by intracellular levels of asparagine. Modulation of AS expression by asparagine employs cis and trans-acting elements involved in transcriptional and translational regulation. We have cloned and sequenced the human AS gene and surrounding sequence elements as well as the full-length cDNA. Using probes specific to the third and fourth exons of AS, we have identified an additional higher molecular weight mRNA (2.7 kb) in Northern blots derived from a chronic myelogenous leukemia and a colon carcinoma but not in normal lymphocytic or other human cell lines. We speculate that elements present in the cancer-derived mRNAs may be involved in the derepression of AS activity. This hypothesis is being evaluated by RNase protection assays using RNA isolated from a variety of human cell lines to characterize and elucidate the nature of this additional AS encoded message.

  6. Identification of Persistent RNA-DNA Hybrid Structures within the Origin of Replication of Human Cytomegalovirus

    OpenAIRE

    Prichard, Mark N.; Jairath, Sanju; Penfold, Mark E. T.; Jeor, Stephen St.; Bohlman, Marlene C.; Pari, Gregory S.

    1998-01-01

    Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates at the cis-acting origin of replication, oriLyt. oriLyt is a structurally complex region containing repeat elements and transcription factor binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA segment. These alkali-sensitive regions were the result of the degradation of two RNA species embedded within oriLyt and covalently linked to viral DNA. The virus-associa...

  7. The Complex Exogenous RNA Spectra in Human Plasma: An Interface with Human Gut Biota?

    Science.gov (United States)

    Wang, Kai; Li, Hong; Yuan, Yue; Etheridge, Alton; Zhou, Yong; Huang, David; Wilmes, Paul; Galas, David

    2012-01-01

    Human plasma has long been a rich source for biomarker discovery. It has recently become clear that plasma RNA molecules, such as microRNA, in addition to proteins are common and can serve as biomarkers. Surveying human plasma for microRNA biomarkers using next generation sequencing technology, we observed that a significant fraction of the circulating RNA appear to originate from exogenous species. With careful analysis of sequence error statistics and other controls, we demonstrated that there is a wide range of RNA from many different organisms, including bacteria and fungi as well as from other species. These RNAs may be associated with protein, lipid or other molecules protecting them from RNase activity in plasma. Some of these RNAs are detected in intracellular complexes and may be able to influence cellular activities under in vitro conditions. These findings raise the possibility that plasma RNAs of exogenous origin may serve as signaling molecules mediating for example the human-microbiome interaction and may affect and/or indicate the state of human health. PMID:23251414

  8. Landscape and variation of RNA secondary structure across the human transcriptome.

    OpenAIRE

    Wan, Y; Qu, K; Zhang, QC; Flynn, RA; Manor, O; Ouyang, Z; Zhang, J; Spitale, RC; Snyder, MP; Segal, E; Chang, HY

    2014-01-01

    In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comp...

  9. microRNA expression profiles in human peripheral blood lymphocytes cultured in modeled microgravity

    Data.gov (United States)

    National Aeronautics and Space Administration — In the present study we analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs) incubated in microgravity condition simulated by a...

  10. Co-LncRNA: investigating the lncRNA combinatorial effects in GO annotations and KEGG pathways based on human RNA-Seq data.

    Science.gov (United States)

    Zhao, Zheng; Bai, Jing; Wu, Aiwei; Wang, Yuan; Zhang, Jinwen; Wang, Zishan; Li, Yongsheng; Xu, Juan; Li, Xia

    2015-01-01

    Long non-coding RNAs (lncRNAs) are emerging as key regulators of diverse biological processes and diseases. However, the combinatorial effects of these molecules in a specific biological function are poorly understood. Identifying co-expressed protein-coding genes of lncRNAs would provide ample insight into lncRNA functions. To facilitate such an effort, we have developed Co-LncRNA, which is a web-based computational tool that allows users to identify GO annotations and KEGG pathways that may be affected by co-expressed protein-coding genes of a single or multiple lncRNAs. LncRNA co-expressed protein-coding genes were first identified in publicly available human RNA-Seq datasets, including 241 datasets across 6560 total individuals representing 28 tissue types/cell lines. Then, the lncRNA combinatorial effects in a given GO annotations or KEGG pathways are taken into account by the simultaneous analysis of multiple lncRNAs in user-selected individual or multiple datasets, which is realized by enrichment analysis. In addition, this software provides a graphical overview of pathways that are modulated by lncRNAs, as well as a specific tool to display the relevant networks between lncRNAs and their co-expressed protein-coding genes. Co-LncRNA also supports users in uploading their own lncRNA and protein-coding gene expression profiles to investigate the lncRNA combinatorial effects. It will be continuously updated with more human RNA-Seq datasets on an annual basis. Taken together, Co-LncRNA provides a web-based application for investigating lncRNA combinatorial effects, which could shed light on their biological roles and could be a valuable resource for this community. Database URL: http://www.bio-bigdata.com/Co-LncRNA/. © The Author(s) 2015. Published by Oxford University Press.

  11. High-throughput miRNA profiling of human melanoma blood samples

    Directory of Open Access Journals (Sweden)

    Rass Knuth

    2010-06-01

    Full Text Available Abstract Background MicroRNA (miRNA signatures are not only found in cancer tissue but also in blood of cancer patients. Specifically, miRNA detection in blood offers the prospect of a non-invasive analysis tool. Methods Using a microarray based approach we screened almost 900 human miRNAs to detect miRNAs that are deregulated in their expression in blood cells of melanoma patients. We analyzed 55 blood samples, including 20 samples of healthy individuals, 24 samples of melanoma patients as test set, and 11 samples of melanoma patients as independent validation set. Results A hypothesis test based approch detected 51 differentially regulated miRNAs, including 21 miRNAs that were downregulated in blood cells of melanoma patients and 30 miRNAs that were upregulated in blood cells of melanoma patients as compared to blood cells of healthy controls. The tets set and the independent validation set of the melanoma samples showed a high correlation of fold changes (0.81. Applying hierarchical clustering and principal component analysis we found that blood samples of melanoma patients and healthy individuals can be well differentiated from each other based on miRNA expression analysis. Using a subset of 16 significant deregulated miRNAs, we were able to reach a classification accuracy of 97.4%, a specificity of 95% and a sensitivity of 98.9% by supervised analysis. MiRNA microarray data were validated by qRT-PCR. Conclusions Our study provides strong evidence for miRNA expression signatures of blood cells as useful biomarkers for melanoma.

  12. RNA-DNA Differences Are Generated in Human Cells within Seconds after RNA Exits Polymerase II

    Directory of Open Access Journals (Sweden)

    Isabel X. Wang

    2014-03-01

    Full Text Available RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs in nascent RNA. Our results show that RDDs begin to occur in RNA chains ∼55 nt from the RNA polymerase II (Pol II active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA-editing mechanisms. Moreover, the 55 nt delay in appearance indicates that they do not arise during RNA synthesis by Pol II or as a direct consequence of modified base incorporation. Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in cotranscriptional RNA processing.

  13. Characterization of RNA isolated from eighteen different human tissues: results from a rapid human autopsy program.

    Science.gov (United States)

    Walker, Douglas G; Whetzel, Alexis M; Serrano, Geidy; Sue, Lucia I; Lue, Lih-Fen; Beach, Thomas G

    2016-09-01

    Many factors affect the integrity of messenger RNA from human autopsy tissues including postmortem interval (PMI) between death and tissue preservation and the pre-mortem agonal and disease states. In this communication, we describe RNA isolation and characterization of 389 samples from 18 different tissues from elderly donors who were participants in a rapid whole-body autopsy program located in Sun City, Arizona ( www.brainandbodydonationprogram.org ). Most tissues were collected within a PMI of 2-6 h (median 3.15 h; N = 455), but for this study, tissue from cases with longer PMIs (1.25-29.25 h) were included. RNA quality was assessed by RNA integrity number (RIN) and total yield (ng RNA/mg tissue). RIN correlated with PMI for heart (r = -0.531, p = 0.009) and liver (r = -558, p = 0.0017), while RNA yield correlated with PMI for colon (r = -485, p = 0.016) and skin (r = -0.460, p = 0.031). RNAs with the lowest integrity were from skin and cervix where 22.7 and 31.4 % of samples respectively failed to produce intact RNA; by contrast all samples from esophagus, lymph node, jejunum, lung, stomach, submandibular gland and kidney produced RNA with measurable RINs. Expression levels in heart RNA of 4 common housekeeping normalization genes showed significant correlations of Ct values with RIN, but only one gene, glyceraldehyde-3 phosphate dehydrogenase, showed a correlation of Ct with PMI. There were no correlations between RIN values obtained for liver, adrenal, cervix, esophagus and lymph node and those obtained from corresponding brain samples. We show that high quality RNA can be produced from most human autopsy tissues, though with significant differences between tissues and donors. The RNA stability and yield did not depend solely on PMI; other undetermined factors are involved, but these do not include the age of the donor.

  14. An in vitro human skin test for assessing sensitization potential.

    Science.gov (United States)

    Ahmed, S S; Wang, X N; Fielding, M; Kerry, A; Dickinson, I; Munuswamy, R; Kimber, I; Dickinson, A M

    2016-05-01

    Sensitization to chemicals resulting in an allergy is an important health issue. The current gold-standard method for identification and characterization of skin-sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in-vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro-haptens, respiratory sensitizers, non-sensitizing chemicals (including skin-irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non-sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in-vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process. Copyright © 2015 John Wiley & Sons, Ltd.

  15. DNA structure in human RNA polymerase II promoters

    DEFF Research Database (Denmark)

    Pedersen, Anders Gorm; Baldi, Pierre; Chauvin, Yves

    1998-01-01

    with a very low level of sequence similarity. The sequences, which include both TATA-containing and TATA-less promoters, are aligned by hidden Markov models. Using three different models of sequence-derived DNA bendability, the aligned promoters display a common structural profile with bendability being low...... protein in a manner reminiscent of DNA in a nucleosome. This notion is further supported by the finding that the periodic bendability is caused mainly by the complementary triplet pairs CAG/CTG and GGC/GCC, which previously have been found to correlate with nucleosome positioning. We present models where......The fact that DNA three-dimensional structure is important for transcriptional regulation begs the question of whether eukaryotic promoters contain general structural features independently of what genes they control. We present an analysis of a large set of human RNA polymerase II promoters...

  16. MicroRNA-target binding structures mimic microRNA duplex structures in humans.

    Directory of Open Access Journals (Sweden)

    Xi Chen

    Full Text Available Traditionally, researchers match a microRNA guide strand to mRNA sequences using sequence comparisons to predict its potential target genes. However, many of the predictions can be false positives due to limitations in sequence comparison alone. In this work, we consider the association of two related RNA structures that share a common guide strand: the microRNA duplex and the microRNA-target binding structure. We have analyzed thousands of such structure pairs and found many of them share high structural similarity. Therefore, we conclude that when predicting microRNA target genes, considering just the microRNA guide strand matches to gene sequences may not be sufficient--the microRNA duplex structure formed by the guide strand and its companion passenger strand must also be considered. We have developed software to translate RNA binding structure into encoded representations, and we have also created novel automatic comparison methods utilizing such encoded representations to determine RNA structure similarity. Our software and methods can be utilized in the other RNA secondary structure comparisons as well.

  17. Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

    Science.gov (United States)

    Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, Luísa

    2012-01-01

    Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its

  18. HumanViCe: Host ceRNA network in virus infected cells in human

    Directory of Open Access Journals (Sweden)

    Suman eGhosal

    2014-07-01

    Full Text Available Host-virus interaction via host cellular components has been an important field of research in recent times. RNA interference mediated by short interfering RNAs and microRNAs (miRNA, is a widespread anti-viral defence strategy. Importantly, viruses also encode their own miRNAs. In recent times miRNAs were identified as key players in host-virus interaction. Furthermore, viruses were shown to exploit the host miRNA networks to suite their own need. The complex cross-talk between host and viral miRNAs and their cellular and viral targets forms the environment for viral pathogenesis. Apart from protein-coding mRNAs, non-coding RNAs may also be targeted by host or viral miRNAs in virus infected cells, and viruses can exploit the host miRNA mediated gene regulatory network via the competing endogenous RNA effect. A recent report showed that viral U-rich non-coding RNAs called HSUR, expressed in primate virus herpesvirus saimiri (HVS infected T cells, were able to bind to three host miRNAs, causing significant alteration in cellular level for one of the miRNAs. We have predicted protein coding and non protein-coding targets for viral and human miRNAs in virus infected cells. We identified viral miRNA targets within host non-coding RNA loci from AGO interacting regions in three different virus infected cells. Gene ontology (GO and pathway enrichment analysis of the genes comprising the ceRNA networks in the virus infected cells revealed enrichment of key cellular signalling pathways related to cell fate decisions and gene transcription, like Notch and Wnt signalling pathways, as well as pathways related to viral entry, replication and virulence. We identified a vast number of non-coding transcripts playing as potential ceRNAs to the immune response associated genes; e.g. APOBEC family genes, in some virus infected cells. All these information are compiled in HumanViCe, a comprehensive database that provides the potential ceRNA networks in virus

  19. Integrated Analysis of Dysregulated ncRNA and mRNA Expression Profiles in Humans Exposed to Carbon Nanotubes.

    Directory of Open Access Journals (Sweden)

    Anna A Shvedova

    Full Text Available As the application of carbon nanotubes (CNT in consumer products continues to rise, studies have expanded to determine the associated risks of exposure on human and environmental health. In particular, several lines of evidence indicate that exposure to multi-walled carbon nanotubes (MWCNT could pose a carcinogenic risk similar to asbestos fibers. However, to date the potential markers of MWCNT exposure are not yet explored in humans.In the present study, global mRNA and ncRNA expression profiles in the blood of exposed workers, having direct contact with MWCNT aerosol for at least 6 months (n = 8, were compared with expression profiles of non-exposed (n = 7 workers (e.g., professional and/or technical staff from the same manufacturing facility.Significant changes in the ncRNA and mRNA expression profiles were observed between exposed and non-exposed worker groups. An integrative analysis of ncRNA-mRNA correlations was performed to identify target genes, functional relationships, and regulatory networks in MWCNT-exposed workers. The coordinated changes in ncRNA and mRNA expression profiles revealed a set of miRNAs and their target genes with roles in cell cycle regulation/progression/control, apoptosis and proliferation. Further, the identified pathways and signaling networks also revealed MWCNT potential to trigger pulmonary and cardiovascular effects as well as carcinogenic outcomes in humans, similar to those previously described in rodents exposed to MWCNTs.This study is the first to investigate aberrant changes in mRNA and ncRNA expression profiles in the blood of humans exposed to MWCNT. The significant changes in several miRNAs and mRNAs expression as well as their regulatory networks are important for getting molecular insights into the MWCNT-induced toxicity and pathogenesis in humans. Further large-scale prospective studies are necessary to validate the potential applicability of such changes in mRNAs and miRNAs as prognostic markers

  20. Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome

    DEFF Research Database (Denmark)

    Peng, Zhiyu; Cheng, Yanbing; Tan, Bertrand Chin-Ming

    2012-01-01

    a computational pipeline that carefully controls for false positives while calling RNA editing events from genome and whole-transcriptome data of the same individual. We identified 22,688 RNA editing events in noncoding genes and introns, untranslated regions and coding sequences of protein-coding genes. Most......RNA editing is a post-transcriptional event that recodes hereditary information. Here we describe a comprehensive profile of the RNA editome of a male Han Chinese individual based on analysis of ∼767 million sequencing reads from poly(A)(+), poly(A)(-) and small RNA samples. We developed...... changes (∼93%) converted A to I(G), consistent with known editing mechanisms based on adenosine deaminase acting on RNA (ADAR). We also found evidence of other types of nucleotide changes; however, these were validated at lower rates. We found 44 editing sites in microRNAs (miRNAs), suggesting a potential...

  1. Direct RNA-based detection of CTX-M β-lactamases in human blood samples.

    Science.gov (United States)

    Stein, Claudia; Makarewicz, Oliwia; Pfeifer, Yvonne; Brandt, Christian; Pletz, Mathias W

    2015-05-01

    Bloodstream infections with ESBL-producers are associated with increased mortality, which is due to delayed appropriate treatment resulting in clinical failure. Current routine diagnostics for detection of bloodstream infections consists of blood culture followed by species identification and susceptibility testing. In attempts to improve and accelerate diagnostic procedures, PCR-based methods have been developed. These methods focus on species identification covering only a limited number of ESBL coding genes. Therefore, they fail to cover the steadily further evolving genetic diversity of clinically relevant β-lactamases. We have recently designed a fast and novel RNA targeting method to detect and specify CTX-M alleles from bacterial cultures, based on an amplification-pyrosequencing approach. We further developed this assay towards a diagnostic tool for clinical use and evaluated its sensitivity and specificity when applied directly to human blood samples. An optimized protocol for mRNA isolation allows detection of specific CTX-M groups from as little as 100 CFU/mL blood via reverse transcription, amplification, and pyrosequencing directly from human EDTA blood samples as well as from pre-incubated human blood cultures with a turnaround time for test results of <7 h. Copyright © 2015 Elsevier GmbH. All rights reserved.

  2. Inhibition of Notch1 increases paclitaxel sensitivity to human breast cancer

    Institute of Scientific and Technical Information of China (English)

    Zhao Li; Ma Yongjie; Gu Feng; Fu Li

    2014-01-01

    Background Paclitaxel (PAC) is the first-line chemotherapy drug for most breast cancer patients,but clinical studies showed that some breast cancer patients were insensitive to PAC,which led to chemotherapy failure.It was reported that Notch1 signaling participated in drug resistance of breast cancer.Here,we show whether Notch1 expression is related to PAC sensitivity of breast cancer.Methods We employed Notch1 siRNA and Notch1 inhibitor,N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester (DAPT),to down regulate Notch1 expression in human breast cancer cells MDA-MB-231,and detected the inhibition effect by Western blotting and reverse trans cription-polymerase chain reaction,respectively.After 24 hours exposure to different concentration of PAC (0,1,5,10,15,20,and 25 μg/ml),the viability of the control group and experimental group cells was tested by MTT.We also examined the expression of Notch1 in PAC sensitive and nonsensitive breast cancer patients,respectively by immunohistochemistry (IHC).The PAC sensitivity of breast cancer patients were identified by collagen gel droplet embedded culture-drug sensitivity test (CD-DST).Results Down regulation of Notch1 expression by Notch1siRNA interference or Notch1 inhibitor increased the PAC sensitivity in MDA-MB-231 cells (P <0.05).Also,the expression of Notch1 in PAC sensitive patients was much lower than that of PAC non-sensitive patients (P <0.01).Conclusion Notch1 expression has an effect on PAC sensitivity in breast cancer patients,and the inhibition of Notch1 increases paclitaxel sensitivity to human breast cancer.

  3. Real-time dynamics of RNA Polymerase II clustering in live human cells

    Science.gov (United States)

    Cisse, Ibrahim

    2014-03-01

    Transcription is the first step in the central dogma of molecular biology, when genetic information encoded on DNA is made into messenger RNA. How this fundamental process occurs within living cells (in vivo) is poorly understood,[1] despite extensive biochemical characterizations with isolated biomolecules (in vitro). For high-order organisms, like humans, transcription is reported to be spatially compartmentalized in nuclear foci consisting of clusters of RNA Polymerase II, the enzyme responsible for synthesizing all messenger RNAs. However, little is known of when these foci assemble or their relative stability. We developed an approach based on photo-activation localization microscopy (PALM) combined with a temporal correlation analysis, which we refer to as tcPALM. The tcPALM method enables the real-time characterization of biomolecular spatiotemporal organization, with single-molecule sensitivity, directly in living cells.[2] Using tcPALM, we observed that RNA Polymerase II clusters form transiently, with an average lifetime of 5.1 (+/- 0.4) seconds. Stimuli affecting transcription regulation yielded orders of magnitude changes in the dynamics of the polymerase clusters, implying that clustering is regulated and plays a role in the cells ability to effect rapid response to external signals. Our results suggest that the transient crowding of enzymes may aid in rate-limiting steps of genome regulation.

  4. CID-miRNA: A web server for prediction of novel miRNA precursors in human genome

    International Nuclear Information System (INIS)

    Tyagi, Sonika; Vaz, Candida; Gupta, Vipin; Bhatia, Rohit; Maheshwari, Sachin; Srinivasan, Ashwin; Bhattacharya, Alok

    2008-01-01

    microRNAs (miRNA) are a class of non-protein coding functional RNAs that are thought to regulate expression of target genes by direct interaction with mRNAs. miRNAs have been identified through both experimental and computational methods in a variety of eukaryotic organisms. Though these approaches have been partially successful, there is a need to develop more tools for detection of these RNAs as they are also thought to be present in abundance in many genomes. In this report we describe a tool and a web server, named CID-miRNA, for identification of miRNA precursors in a given DNA sequence, utilising secondary structure-based filtering systems and an algorithm based on stochastic context free grammar trained on human miRNAs. CID-miRNA analyses a given sequence using a web interface, for presence of putative miRNA precursors and the generated output lists all the potential regions that can form miRNA-like structures. It can also scan large genomic sequences for the presence of potential miRNA precursors in its stand-alone form. The web server can be accessed at (http://mirna.jnu.ac.in/cidmirna/)

  5. Landscape and variation of RNA secondary structure across the human transcriptome.

    Science.gov (United States)

    Wan, Yue; Qu, Kun; Zhang, Qiangfeng Cliff; Flynn, Ryan A; Manor, Ohad; Ouyang, Zhengqing; Zhang, Jiajing; Spitale, Robert C; Snyder, Michael P; Segal, Eran; Chang, Howard Y

    2014-01-30

    In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comprehensive RSS map of human coding and non-coding RNAs. We identify unique RSS signatures that demarcate open reading frames and splicing junctions, and define authentic microRNA-binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1,900 transcribed single nucleotide variants (approximately 15% of all transcribed single nucleotide variants) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSSs. Selective depletion of 'riboSNitches' versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3' untranslated regions, binding sites of microRNAs and RNA-binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation.

  6. Next Generation Sequencing Analysis of Human Platelet PolyA+ mRNAs and rRNA-Depleted Total RNA

    Science.gov (United States)

    Kissopoulou, Antheia; Jonasson, Jon; Lindahl, Tomas L.; Osman, Abdimajid

    2013-01-01

    Background Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA. Materials and Methods We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT) isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts. Results Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq. Conclusion The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components compared with other cell and

  7. Phorbol esters induce interleukin 2 mRNA in sensitive but not in resistant EL4 cells

    International Nuclear Information System (INIS)

    Harrison, J.R.; Lynch, K.R.; Sando, J.J.

    1986-01-01

    Phorbol ester (PE) sensitive EL4 cells are growth-inhibited and produce interleukin 2 (IL2) when treated with PE. Resistant EL4 cells lack both responses. To determine whether the defect in resistant cells occurs pre or post-transcriptionally, an assay for IL2 mRNA was developed using a synthetic oligonucleotide to mouse IL2 as a probe. Total RNA (15 μg) from cells +/- PE was electrophoresed, blotted onto a cationic nylon membrane, and probed with radiolabeled oligomer. This probe hybridized to a 1.1 kb band in RNA from PE-treated sensitive cells. This RNA was detectable within 3h of PE administration, was clearly visible by 6h, and peaked by 9 to 12h. No bands hybridizing with the IL2 probe were detected in RNA isolated from unstimulated cells or from resistant EL4 cells at any time following PE stimulation. Since levels of the protooncogene c-myc have been shown to decrease in a number of cell lines during differentiation and growth inhibition, total RNA from EL4 cells was probed with a nick-translated plasmid containing the protein coding region of the c-myc gene. In PE sensitive cells, levels of c-myc RNA are markedly reduced by 3h. In a pilot experiment with resistant cells, c-myc levels appeared to remain constant. These results demonstrate that PE induced IL2 mRNA in PE sensitive but not resistant EL4 cells. Sensitive and resistant EL4 cell lines provide a useful model for the investigation of the regulation of gene expression by PE

  8. Human proteins that specifically bind to 8-oxoguanine-containing RNA and their responses to oxidative stress

    International Nuclear Information System (INIS)

    Hayakawa, Hiroshi; Fujikane, Aya; Ito, Riyoko; Matsumoto, Masaki; Nakayama, Keiichi I.; Sekiguchi, Mutsuo

    2010-01-01

    Research highlights: → We performed comprehensive survey for proteins that bind to oxidized RNA. → HNRNPD and HNRNPC proteins were identified as oxidized RNA binding proteins. → Knockdown of HNRNPD/C expression caused increased sensitivity to H 2 O 2 . → Amounts of HNRNPD protein rapidly decreased when cells were exposed to H 2 O 2 . -- Abstract: Exposure of cells to oxygen radicals damage various biologically important molecules. Among the oxidized bases produced in nucleic acids, 8-oxo-7,8-dihydroguanine (8-oxoguanine) is particularly important since it causes base mispairing. To ensure accurate gene expression, organisms must have a mechanism to discriminate 8-oxoguanine-containing RNA from normal transcripts. We searched for proteins that specifically bind to 8-oxoguanine-containing RNA from human HeLa cell extracts, and the candidate proteins were identified using mass spectrometry. Among the identified candidates, splicing isoform 1 of heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) and splicing isoform C1 of heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) exhibited strong abilities to bind to oxidized RNA. The amount of HNRNPD protein rapidly decreased when cells were exposed to hydrogen peroxide, an agent that enhances oxidative stress. Moreover, the suppression of HNRNPD expression by siRNA caused cells to exhibit an increased sensitivity to hydrogen peroxide. The application of siRNA against HNRNPC also caused an increase in sensitivity to hydrogen peroxide. Since no additive effect was observed with a combined addition of siRNAs for HNRNPD and HNRNPC, we concluded that the two proteins may function in the same mechanism for the accurate gene expression.

  9. Effect of aberrations in human eye on contrast sensitivity function

    Science.gov (United States)

    Quan, Wei; Wang, Feng-lin; Wang, Zhao-qi

    2011-06-01

    The quantitative analysis of the effect of aberrations in human eye on vision has important clinical value in the correction of aberrations. The wave-front aberrations of human eyes were measured with the Hartmann-Shack wave-front sensor and modulation transfer function (MTF) was computed from the wave-front aberrations. Contrast sensitivity function (CSF) was obtained from MTF and the retinal aerial image modulation (AIM). It is shown that the 2nd, 3rd, 4th, 5th, 6th Zernike aberrations deteriorate contrast sensitivity function. When the 2nd, 3rd, 4th, 5th, 6th Zernike aberrations are corrected high contrast sensitivity function can be obtained.

  10. Multivariate Models for Prediction of Human Skin Sensitization ...

    Science.gov (United States)

    One of the lnteragency Coordinating Committee on the Validation of Alternative Method's (ICCVAM) top priorities is the development and evaluation of non-animal approaches to identify potential skin sensitizers. The complexity of biological events necessary to produce skin sensitization suggests that no single alternative method will replace the currently accepted animal tests. ICCVAM is evaluating an integrated approach to testing and assessment based on the adverse outcome pathway for skin sensitization that uses machine learning approaches to predict human skin sensitization hazard. We combined data from three in chemico or in vitro assays - the direct peptide reactivity assay (DPRA), human cell line activation test (h-CLAT) and KeratinoSens TM assay - six physicochemical properties and an in silico read-across prediction of skin sensitization hazard into 12 variable groups. The variable groups were evaluated using two machine learning approaches , logistic regression and support vector machine, to predict human skin sensitization hazard. Models were trained on 72 substances and tested on an external set of 24 substances. The six models (three logistic regression and three support vector machine) with the highest accuracy (92%) used: (1) DPRA, h-CLAT and read-across; (2) DPRA, h-CLAT, read-across and KeratinoSens; or (3) DPRA, h-CLAT, read-across, KeratinoSens and log P. The models performed better at predicting human skin sensitization hazard than the murine

  11. Cockayne's syndrome: correlation of clinical features with cellular sensitivity of RNA synthesis to UV irradiation

    International Nuclear Information System (INIS)

    Lehmann, A.R.; Thompson, A.F.; Harcourt, S.A.

    1993-01-01

    Cockayne's syndrome (CS) is a rare autosomal recessive disorder with dwarfism, mental retardation, and otherwise clinically heterogeneous features. In cultured CS fibroblasts, the failure of RNA synthesis to recover to normal rates after UV-C irradiation provides a useful and relatively simple diagnostic test. We have measured post-UV-C RNA synthesis in 52 patients for whom a clinical diagnosis of CS was considered a possibility. Twenty-nine patients showed the defect characteristic of CS cells, and 23 had a normal response. We have attempted to correlate the cellular diagnosis with the different clinical features of the disorder. Clinical details of the patients were obtained from referring clinicians in the form of a questionnaire. Our results show that, apart from the cardinal features of dwarfism and mental retardation, sun sensitivity correlated best with a positive cellular diagnosis. Pigmentary retinopathy, gait defects, and dental caries were also good positive indicators, although several patients with a positive cellular diagnosis did not have these features. (Author)

  12. Sensitive chain specific radioimmunoassay for human immunoglobulins using monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Sikora, K; Alderson, T St.J.; Ellis, J [Ludwig Institute for Cancer Research, Cambridge (UK)

    1983-02-25

    A sensitive radioimmunoassay is described for human immunoglobulins. This solid-phase assay uses commercially available monoclonal antibodies and is specific for different Ig chain types. Levels of less than 20 ng/ml Ig are detectable. The assay is suitable for the analysis of human hybridoma supernatants.

  13. Sensitivity of risk parameters to human errors for a PWR

    International Nuclear Information System (INIS)

    Samanta, P.; Hall, R.E.; Kerr, W.

    1980-01-01

    Sensitivities of the risk parameters, emergency safety system unavailabilities, accident sequence probabilities, release category probabilities and core melt probability were investigated for changes in the human error rates within the general methodological framework of the Reactor Safety Study for a Pressurized Water Reactor (PWR). Impact of individual human errors were assessed both in terms of their structural importance to core melt and reliability importance on core melt probability. The Human Error Sensitivity Assessment of a PWR (HESAP) computer code was written for the purpose of this study

  14. Circular RNA expression profiling of human granulosa cells during maternal aging reveals novel transcripts associated with assisted reproductive technology outcomes.

    Directory of Open Access Journals (Sweden)

    Jing Cheng

    Full Text Available Circular RNAs (circRNAs are a unique class of endogenous RNAs which could be used as potential diagnostic and prognostic biomarkers of many diseases. Our study aimed to investigate circRNA profiles in human granulosa cells (GCs during maternal aging and to uncover age-related circRNA variations that potentially reflect decreased oocyte competence. CircRNAs in GCs from in vitro fertilization (IVF patients with young age (YA, ≤ 30 years and advanced age (AA, ≥ 38 years were profiled by microarray, and validated in 20 paired samples. The correlation between circRNAs expression and clinical characteristics was analyzed in additional 80 samples. Chip-based analysis revealed 46 up-regulated and 11 down-regulated circRNAs in AA samples (fold change > 2.0. Specifically, circRNA_103829, circRNA_103827 and circRNA_104816 were validated to be up-regulated, while circRNA_101889 was down-regulated in AA samples. After adjustment for gonadotropin treatment, only circRNA_103827 and circRNA_104816 levels were positively associated with maternal age (partial r = 0.332, P = 0.045; partial r = 0.473, P = 0.003; respectively. Moreover, circRNA_103827 and circRNA_104816 expressions in GCs were negatively correlated with the number of top quality embryos (r = -0.235, P = 0.036; r = -0.221, P = 0.049; respectively. Receiver operating characteristic (ROC curve analysis indicated that the performance of circRNA_103827 for live birth prediction reached 0.698 [0.570-0.825], with 77.2% sensitivity and 60.9% specificity (P = 0.006, and that of circRNA_104816 was 0.645 [0.507-0.783] (P = 0.043. Bioinformatics analysis revealed that both circRNAs were potentially involved in glucose metabolism, mitotic cell cycle, and ovarian steroidogenesis. Therefore, age-related up-regulation of circRNA_103827 and circRNA_104816 might be potential indicators of compromised follicular micro-environment which could be used to predict IVF prognosis, and improve female infertility

  15. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    International Nuclear Information System (INIS)

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M.

    1990-01-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures

  16. Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses.

    Directory of Open Access Journals (Sweden)

    Markus Hoffmann

    Full Text Available Bats (Chiroptera host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat or Yangochiroptera (genera Carollia and Tadarida for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV, a porcine coronavirus, or to infection mediated by the Spike (S protein of SARS-coronavirus (SARS-CoV incorporated into pseudotypes based on vesicular stomatitis virus (VSV. The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3 were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed.

  17. Uncovering layers of human RNA polymerase II transcription

    DEFF Research Database (Denmark)

    Jensen, Torben Heick

    In recent years DNA microarray and high-throughput sequencing technologies have challenged the “gene-centric” view that pre-mRNA is the only RNA species transcribed off protein-coding genes. Instead unorthodox transcription from within genic- and intergenic regions has been demonstrated to occur...

  18. Shift in genomic RNA patterns of human rotaviruses isolated from ...

    African Journals Online (AJOL)

    Rotalvirus-positive specimens from 322 infants and young children submitted to private patl1ology laboratories were analysed by polyacrylamide-gel electrophoresis of the viral RNA. A predominance of long RNA profiles occurred and a temporal shift in the genomic patterns was identified. An epidemic of the classic shorter ...

  19. Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide.

    Science.gov (United States)

    Li, Wei; Hou, Ting; Wu, Min; Li, Feng

    2016-01-01

    MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Slicer-independent mechanism drives small-RNA strand separation during human RISC assembly.

    Science.gov (United States)

    Park, June Hyun; Shin, Chanseok

    2015-10-30

    Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (AGOs 1-4 in humans). A fundamental step during RISC assembly involves the separation of two strands of a small RNA duplex, whereby only the guide strand is retained to form the mature RISC, a process not well understood. Despite the widely accepted view that 'slicer-dependent unwinding' via passenger-strand cleavage is a prerequisite for the assembly of a highly complementary siRNA into the AGO2-RISC, here we show by careful re-examination that 'slicer-independent unwinding' plays a more significant role in human RISC maturation than previously appreciated, not only for a miRNA duplex, but, unexpectedly, for a highly complementary siRNA as well. We discovered that 'slicer-dependency' for the unwinding was affected primarily by certain parameters such as temperature and Mg(2+). We further validate these observations in non-slicer AGOs (1, 3 and 4) that can be programmed with siRNAs at the physiological temperature of humans, suggesting that slicer-independent mechanism is likely a common feature of human AGOs. Our results now clearly explain why both miRNA and siRNA are found in all four human AGOs, which is in striking contrast to the strict small-RNA sorting system in Drosophila. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Multivariate Models for Prediction of Human Skin Sensitization Hazard

    Science.gov (United States)

    Strickland, Judy; Zang, Qingda; Paris, Michael; Lehmann, David M.; Allen, David; Choksi, Neepa; Matheson, Joanna; Jacobs, Abigail; Casey, Warren; Kleinstreuer, Nicole

    2016-01-01

    One of ICCVAM’s top priorities is the development and evaluation of non-animal approaches to identify potential skin sensitizers. The complexity of biological events necessary to produce skin sensitization suggests that no single alternative method will replace the currently accepted animal tests. ICCVAM is evaluating an integrated approach to testing and assessment based on the adverse outcome pathway for skin sensitization that uses machine learning approaches to predict human skin sensitization hazard. We combined data from three in chemico or in vitro assays—the direct peptide reactivity assay (DPRA), human cell line activation test (h-CLAT), and KeratinoSens™ assay—six physicochemical properties, and an in silico read-across prediction of skin sensitization hazard into 12 variable groups. The variable groups were evaluated using two machine learning approaches, logistic regression (LR) and support vector machine (SVM), to predict human skin sensitization hazard. Models were trained on 72 substances and tested on an external set of 24 substances. The six models (three LR and three SVM) with the highest accuracy (92%) used: (1) DPRA, h-CLAT, and read-across; (2) DPRA, h-CLAT, read-across, and KeratinoSens; or (3) DPRA, h-CLAT, read-across, KeratinoSens, and log P. The models performed better at predicting human skin sensitization hazard than the murine local lymph node assay (accuracy = 88%), any of the alternative methods alone (accuracy = 63–79%), or test batteries combining data from the individual methods (accuracy = 75%). These results suggest that computational methods are promising tools to effectively identify potential human skin sensitizers without animal testing. PMID:27480324

  2. Establishment of Lipofection for Studying miRNA Function in Human Adipocytes

    OpenAIRE

    Enlund, Eveliina; Fischer, Simon; Handrick, René; Otte, Kerstin; Debatin, Klaus-Michael; Wabitsch, Martin; Fischer-Posovszky, Pamela

    2014-01-01

    miRNA dysregulation has recently been linked to human obesity and its related complications such as type 2 diabetes. In order to study miRNA function in human adipocytes, we aimed for the modulation of mature miRNA concentration in these cells. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. Our objective therefore was to identify an efficient, non-viral transfection reagent capable of delivering small RNA...

  3. Temperature-sensitive mutations for live-attenuated Rift Valley fever vaccines: Implications from other RNA viruses

    Directory of Open Access Journals (Sweden)

    Shoko eNishiyama

    2015-08-01

    Full Text Available Rift Valley fever (RVF is a mosquito-borne zoonotic disease endemic to the African continent. RVF is characterized by high rate of abortions in ruminants and hemorrhagic fever, encephalitis or blindness in humans. RVF is caused by the Rift Valley fever virus (RVFV: genus Phlebovirus, family Bunyaviridae. Vaccination is the only known effective strategy to prevent the disease, but there are no licensed RVF vaccines available for humans. A live-attenuated vaccine candidate derived from the wild-type pathogenic Egyptian ZH548 strain, MP-12, has been conditionally licensed for veterinary use in the United States. MP-12 displays a temperature-sensitive (ts phenotype and does not replicate at 41oC. The ts mutation limits viral replication at a specific body temperature and may lead to an attenuation of the virus. Here we will review well-characterized ts mutations for RNA viruses, and further discuss the potential in designing novel live-attenuated vaccines for RVF.

  4. [Correlation between RNA Expression Level and Early PMI in Human Brain Tissue].

    Science.gov (United States)

    Lü, Y H; Ma, K J; Li, Z H; Gu, J; Bao, J Y; Yang, Z F; Gao, J; Zeng, Y; Tao, L; Chen, L

    2016-08-01

    To explore the correlation between the expression levels of several RNA markers in human brain tissue and early postmortem interval (PMI). Twelve individuals with known PMI (range from 4.3 to 22.5 h) were selected and total RNA was extracted from brain tissue. Eight commonly used RNA markers were chosen including β -actin, GAPDH, RPS29, 18S rRNA, 5S rRNA, U6 snRNA, miRNA-9 and miRNA-125b, and the expression levels were detected in brain tissue by real-time fluorescent quantitative PCR. The internal reference markers with stable expression in early PMI were screened using geNorm software and the relationship between its expression level and some relevant factors such as age, gender and cause of death were analyzed. RNA markers normalized by internal reference were inserted into the mathematic model established by previous research for PMI estimation using R software. Model quality was judged by the error rate calculated with estimated PMI. 5S rRNA, miRNA-9 and miRNA-125b showed quite stable expression and their expression levels had no relation with age, gender and cause of death. The error rate of estimated PMI using β -actin was 24.6%, while GAPDH was 41.0%. 5S rRNA, miRNA-9 and miRNA-125b are suitable as internal reference markers of human brain tissue owing to their stable expression in early PMI. The expression level of β -actin correlates well with PMI, which can be used as an additional index for early PMI estimation. Copyright© by the Editorial Department of Journal of Forensic Medicine

  5. DeepMirTar: a deep-learning approach for predicting human miRNA targets.

    Science.gov (United States)

    Wen, Ming; Cong, Peisheng; Zhang, Zhimin; Lu, Hongmei; Li, Tonghua

    2018-06-01

    MicroRNAs (miRNAs) are small noncoding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression by targeting messenger RNAs (mRNAs). Because the underlying mechanisms associated with miRNA binding to mRNA are not fully understood, a major challenge of miRNA studies involves the identification of miRNA-target sites on mRNA. In silico prediction of miRNA-target sites can expedite costly and time-consuming experimental work by providing the most promising miRNA-target-site candidates. In this study, we reported the design and implementation of DeepMirTar, a deep-learning-based approach for accurately predicting human miRNA targets at the site level. The predicted miRNA-target sites are those having canonical or non-canonical seed, and features, including high-level expert-designed, low-level expert-designed, and raw-data-level, were used to represent the miRNA-target site. Comparison with other state-of-the-art machine-learning methods and existing miRNA-target-prediction tools indicated that DeepMirTar improved overall predictive performance. DeepMirTar is freely available at https://github.com/Bjoux2/DeepMirTar_SdA. lith@tongji.edu.cn, hongmeilu@csu.edu.cn. Supplementary data are available at Bioinformatics online.

  6. High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

    Science.gov (United States)

    Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

    2016-01-01

    Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from RNA in RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

  7. Application of RNA interference in treating human diseases

    Indian Academy of Sciences (India)

    ference than either strand individually. After injection into ... antisense strand to messenger RNAs (mRNAs) that bear ... processing of longer dsRNA and stem loop precursors (Nov- ... RNAi has several applications in biomedical research,.

  8. Evidence of pestivirus RNA in human virus vaccines.

    Science.gov (United States)

    Harasawa, R; Tomiyama, T

    1994-01-01

    We examined live virus vaccines against measles, mumps, and rubella for the presence of pestivirus RNA or of pestiviruses by reverse transcription PCR. Pestivirus RNA was detected in two measles-mumps-rubella combined vaccines and in two monovalent vaccines against mumps and rubella. Nucleotide sequence analysis of the PCR products indicated that a modified live vaccine strain used for immunization of cattle against bovine viral diarrhea is not responsible for the contamination of the vaccines. Images PMID:8077414

  9. RNA isolation for transcriptomics of human and mouse small skin biopsies

    Directory of Open Access Journals (Sweden)

    Breit Timo M

    2011-10-01

    Full Text Available Abstract Background Isolation of RNA from skin biopsies presents a challenge, due to the tough nature of skin tissue and a high presence of RNases. As we lacked the dedicated equipment, i.e. homogenizer or bead-beater, needed for the available RNA from skin isolation methods, we adapted and tested our zebrafish single-embryo RNA-isolation protocol for RNA isolation from skin punch biopsies. Findings We tested our new RNA-isolation protocol in two experiments: a large-scale study with 97 human skin samples, and a small study with 16 mouse skin samples. Human skin was sampled with 4.0 mm biopsy punches and for the mouse skin different punch diameter sizes were tested; 1.0, 1.5, 2.0, and 2.5 mm. The average RNA yield in human samples was 1.5 μg with an average RNA quality RIN value of 8.1. For the mouse biopsies, the average RNA yield was 2.4 μg with an average RIN value of 7.5. For 96% of the human biopsies and 100% of the mouse biopsies we obtained enough high-quality RNA. The RNA samples were successfully tested in a transcriptomics analysis using the Affymetrix and Roche NimbleGen platforms. Conclusions Using our new RNA-isolation protocol, we were able to consistently isolate high-quality RNA, which is apt for further transcriptomics analysis. Furthermore, this method is already useable on biopsy material obtained with a punch diameter as small as 1.5 mm.

  10. Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells.

    Science.gov (United States)

    Dong, Fengping; Xie, Kabin; Chen, Yueying; Yang, Yinong; Mao, Yingwei

    2017-01-22

    CRISPR/Cas9 has been widely used for genomic editing in many organisms. Many human diseases are caused by multiple mutations. The CRISPR/Cas9 system provides a potential tool to introduce multiple mutations in a genome. To mimic complicated genomic variants in human diseases, such as multiple gene deletions or mutations, two or more small guide RNAs (sgRNAs) need to be introduced all together. This can be achieved by separate Pol III promoters in a construct. However, limited enzyme sites and increased insertion size lower the efficiency to make a construct. Here, we report a strategy to quickly assembly multiple sgRNAs in one construct using a polycistronic-tRNA-gRNA (PTG) strategy. Taking advantage of the endogenous tRNA processing system in mammalian cells, we efficiently express multiple sgRNAs driven using only one Pol III promoter. Using an all-in-one construct carrying PTG, we disrupt the deacetylase domain in multiple histone deacetylases (HDACs) in human cells simultaneously. We demonstrate that multiple HDAC deletions significantly affect the activation of the Wnt-signaling pathway. Thus, this method enables to efficiently target multiple genes and provide a useful tool to establish mutated cells mimicking human diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Differences in correlation of mRNA gene expression in mice sensitive and resistant to radiation-induced pulmonary fibrosis

    International Nuclear Information System (INIS)

    Johnston, C.J.; Piedboeuf, B.; Finkelstein, J.N.; Baggs, R.; Rubin, P.

    1995-01-01

    Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The purpose of this study was to determine if extracellular matrix protein and transforming growth factor β mRNA expression are altered late in the course of pulmonary fibrosis after irradiation, and then to determine if these changes differ between two strains of mice which vary in their sensitivity to radiation. Radiation-sensitive (C57BL/6) and radiation-resistant (C3H/HeJ) mice were irradiated with a single dose of 5 or 12.5 Gy to the thorax. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabeled cDNA probes for collagens I, III and IV, fibronectin, and transforming growth factor β 1 and β 3 . Autoradiographic data were quantified by video densitometry and results normalized to a control probe encoding for glyceralde-hyde-3-phosphate dehydrogenase. Alterations in mRNA abundance were observed in the sensitive mice at all times, while levels in the resistant mice were unaffected until 26 weeks after irradiation. The relationship between extracellular matrix protein per se and increased mRNA abundance suggests that late matrix protein accumulation may be a function of gene expression. Differences in levels of transforming growth factor βmRNA may lead to strain-dependent variation in fibrotic response and may also contribute to the radiation-induced component of pulmonary fibrosis. 32 refs., 5 figs

  12. Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.

    Science.gov (United States)

    Li, Wei; Jiang, Wei; Ding, Yongshun; Wang, Lei

    2015-09-15

    MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Structural insights into RNA processing by the human RISC-loading complex.

    Science.gov (United States)

    Wang, Hong-Wei; Noland, Cameron; Siridechadilok, Bunpote; Taylor, David W; Ma, Enbo; Felderer, Karin; Doudna, Jennifer A; Nogales, Eva

    2009-11-01

    Targeted gene silencing by RNA interference (RNAi) requires loading of a short guide RNA (small interfering RNA (siRNA) or microRNA (miRNA)) onto an Argonaute protein to form the functional center of an RNA-induced silencing complex (RISC). In humans, Argonaute2 (AGO2) assembles with the guide RNA-generating enzyme Dicer and the RNA-binding protein TRBP to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs and miRNAs from Dicer to AGO2. Here, using single-particle EM analysis, we show that human Dicer has an L-shaped structure. The RLC Dicer's N-terminal DExH/D domain, located in a short 'base branch', interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2.

  14. A riboswitch regulates RNA dimerization and packaging in human immunodeficiency virus type 1 virions

    NARCIS (Netherlands)

    Ooms, Marcel; Huthoff, Hendrik; Russell, Rodney; Liang, Chen; Berkhout, Ben

    2004-01-01

    The genome of retroviruses, including human immunodeficiency virus type I (HIV-1), consists of two identical RNA strands that are packaged as noncovalently linked dimers. The core packaging and dimerization signals are located in the downstream part of the untranslated leader of HIV-1 RNA-the Psi

  15. The RNA splicing factor ASF/SF2 inhibits human topoisomerase I mediated DNA relaxation

    DEFF Research Database (Denmark)

    Andersen, Félicie Faucon; Tange, Thomas Ø.; Sinnathamby, Thayaline

    2002-01-01

    Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around...

  16. RNase L controls terminal adipocyte differentiation, lipids storage and insulin sensitivity via CHOP10 mRNA regulation

    DEFF Research Database (Denmark)

    Fabre, Odile Martine Julie; Salehzada, T; Lambert, K

    2012-01-01

    Adipose tissue structure is altered during obesity, leading to deregulation of whole-body metabolism. Its function depends on its structure, in particular adipocytes number and differentiation stage. To better understand the mechanisms regulating adipogenesis, we have investigated the role...... is associated with CHOP10 mRNA and regulates its stability. CHOP10 expression is conserved in RNase L(-/-)-MEFs, maintaining preadipocyte state while impairing their terminal differentiation. RNase L(-/-)-MEFs have decreased lipids storage capacity, insulin sensitivity and glucose uptake. Expression of ectopic...... RNase L in RNase L(-/-)-MEFs triggers CHOP10 mRNA instability, allowing increased lipids storage, insulin response and glucose uptake. Similarly, downregulation of CHOP10 mRNA with CHOP10 siRNA in RNase L(-/-)-MEFs improves their differentiation in adipocyte. In vivo, aged RNase L(-)/(-) mice present...

  17. Next generation sequencing analysis of human platelet PolyA+ mRNAs and rRNA-depleted total RNA.

    Directory of Open Access Journals (Sweden)

    Antheia Kissopoulou

    Full Text Available BACKGROUND: Platelets are small anucleate cells circulating in the blood vessels where they play a key role in hemostasis and thrombosis. Here, we compared platelet RNA-Seq results obtained from polyA+ mRNA and rRNA-depleted total RNA. MATERIALS AND METHODS: We used purified, CD45 depleted, human blood platelets collected by apheresis from three male and one female healthy blood donors. The Illumina HiSeq 2000 platform was employed to sequence cDNA converted either from oligo(dT isolated polyA+ RNA or from rRNA-depleted total RNA. The reads were aligned to the GRCh37 reference assembly with the TopHat/Cufflinks alignment package using Ensembl annotations. A de novo assembly of the platelet transcriptome using the Trinity software package and RSEM was also performed. The bioinformatic tools HTSeq and DESeq from Bioconductor were employed for further statistical analyses of read counts. RESULTS: Consistent with previous findings our data suggests that mitochondrially expressed genes comprise a substantial fraction of the platelet transcriptome. We also identified high transcript levels for protein coding genes related to the cytoskeleton function, chemokine signaling, cell adhesion, aggregation, as well as receptor interaction between cells. Certain transcripts were particularly abundant in platelets compared with other cell and tissue types represented by RNA-Seq data from the Illumina Human Body Map 2.0 project. Irrespective of the different library preparation and sequencing protocols, there was good agreement between samples from the 4 individuals. Eighteen differentially expressed genes were identified in the two sexes at 10% false discovery rate using DESeq. CONCLUSION: The present data suggests that platelets may have a unique transcriptome profile characterized by a relative over-expression of mitochondrially encoded genes and also of genomic transcripts related to the cytoskeleton function, chemokine signaling and surface components

  18. A non-human primate model for gluten sensitivity.

    Directory of Open Access Journals (Sweden)

    Michael T Bethune

    2008-02-01

    Full Text Available Gluten sensitivity is widespread among humans. For example, in celiac disease patients, an inflammatory response to dietary gluten leads to enteropathy, malabsorption, circulating antibodies against gluten and transglutaminase 2, and clinical symptoms such as diarrhea. There is a growing need in fundamental and translational research for animal models that exhibit aspects of human gluten sensitivity.Using ELISA-based antibody assays, we screened a population of captive rhesus macaques with chronic diarrhea of non-infectious origin to estimate the incidence of gluten sensitivity. A selected animal with elevated anti-gliadin antibodies and a matched control were extensively studied through alternating periods of gluten-free diet and gluten challenge. Blinded clinical and histological evaluations were conducted to seek evidence for gluten sensitivity.When fed with a gluten-containing diet, gluten-sensitive macaques showed signs and symptoms of celiac disease including chronic diarrhea, malabsorptive steatorrhea, intestinal lesions and anti-gliadin antibodies. A gluten-free diet reversed these clinical, histological and serological features, while reintroduction of dietary gluten caused rapid relapse.Gluten-sensitive rhesus macaques may be an attractive resource for investigating both the pathogenesis and the treatment of celiac disease.

  19. MicroRNA-triggered, cascaded and catalytic self-assembly of functional ``DNAzyme ferris wheel'' nanostructures for highly sensitive colorimetric detection of cancer cells

    Science.gov (United States)

    Zhou, Wenjiao; Liang, Wenbin; Li, Xin; Chai, Yaqin; Yuan, Ruo; Xiang, Yun

    2015-05-01

    The construction of DNA nanostructures with various sizes and shapes has significantly advanced during the past three decades, yet the application of these DNA nanostructures for solving real problems is still in the early stage. On the basis of microRNA-triggered, catalytic self-assembly formation of the functional ``DNAzyme ferris wheel'' nanostructures, we show here a new signal amplification platform for highly sensitive, label-free and non-enzyme colorimetric detection of a small number of human prostate cancer cells. The microRNA (miR-141), which is catalytically recycled and reused, triggers isothermal self-assembly of a pre-designed, G-quadruplex sequence containing hairpin DNAs into ``DNAzyme ferris wheel''-like nanostructures (in association with hemin) with horseradish peroxidase mimicking activity. These DNAzyme nanostructures catalyze an intensified color transition of the probe solution for highly sensitive detection of miR-141 down to 0.5 pM with the naked eye, and the monitoring of as low as 283 human prostate cancer cells can also, theoretically, be achieved in a colorimetric approach. The work demonstrated here thus offers new opportunities for the construction of functional DNA nanostructures and for the application of these DNA nanostructures as an effective signal amplification means in the sensitive detection of nucleic acid biomarkers.

  20. Nonsense-Mediated RNA Decay Influences Human Embryonic Stem Cell Fate

    Directory of Open Access Journals (Sweden)

    Chih-Hong Lou

    2016-06-01

    Full Text Available Nonsense-mediated RNA decay (NMD is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.

  1. QSAR models of human data can enrich or replace LLNA testing for human skin sensitization

    OpenAIRE

    Alves, Vinicius M.; Capuzzi, Stephen J.; Muratov, Eugene; Braga, Rodolpho C.; Thornton, Thomas; Fourches, Denis; Strickland, Judy; Kleinstreuer, Nicole; Andrade, Carolina H.; Tropsha, Alexander

    2016-01-01

    Skin sensitization is a major environmental and occupational health hazard. Although many chemicals have been evaluated in humans, there have been no efforts to model these data to date. We have compiled, curated, analyzed, and compared the available human and LLNA data. Using these data, we have developed reliable computational models and applied them for virtual screening of chemical libraries to identify putative skin sensitizers. The overall concordance between murine LLNA and human skin ...

  2. Radiation sensitivity of human lung cancer cell lines

    International Nuclear Information System (INIS)

    Carmichael, J.; Degraff, W.G.; Gamson, J.; Russo, G.; Mitchell, J.B.; Gazdar, A.F.; Minna, J.D.; Levitt, M.L.

    1989-01-01

    X-Ray survival curves were determined using a panel of 17 human lung cancer cell lines, with emphasis on non-small cell lung cancer (NSCLC). In contrast to classic small cell lung cancer (SCLC) cell lines, NSCLC cell lines were generally less sensitive to radiation as evidenced by higher radiation survival curve extrapolation numbers, surviving fraction values following a 2Gy dose (SF2) and the mean inactivation dose values (D) values. The spectrum of in vitro radiation responses observed was similar to that expected in clinical practice, although mesothelioma was unexpectedly sensitive in vitro. Differences in radiosensitivity were best distinguished by comparison of SF2 values. Some NSCLC lines were relatively sensitive, and in view of this demonstrable variability in radiation sensitivity, the SF2 value may be useful for in vitro predictive assay testing of clinical specimens. (author)

  3. Alternative splicing of cyclooxygenase-1 mRNA in the human iris

    NARCIS (Netherlands)

    Dröge, M.J; van Sorge, A.A; van Haeringen, N.J; Quax, Wim; Zaagsma, Hans; Droge, MJ

    2003-01-01

    dIn homogenates of the human iris, the nonsteroidal antiinflammatory drug (NSAID) S(+)flurbiprofen has been reported to inhibit cyclooxygenase-1 (COX-1) 70-fold more potently than in human whole blood. We hypothesized that this difference may be due to alternative splicing of COX-1 mRNA in the human

  4. 1st International Symposium on Stress-Associated RNA Granules in Human Disease and Viral Infection

    Directory of Open Access Journals (Sweden)

    Bruce W. Banfield

    2014-09-01

    Full Text Available In recent years, important linkages have been made between RNA granules and human disease processes. On June 8-10 of this year, we hosted a new symposium, dubbed the 1st International Symposium on Stress-Associated RNA Granules in Human Disease and Viral Infection. This symposium brought together experts from diverse research disciplines ranging from cancer and neuroscience to infectious disease. This report summarizes speaker presentations and highlights current challenges in the field.

  5. Evaluation of Human Adipose Tissue Stromal Heterogeneity in Metabolic Disease Using Single Cell RNA-Seq

    Science.gov (United States)

    2017-09-01

    AWARD NUMBER: W81XWH-15-1-0251 TITLE: “Evaluation of Human Adipose Tissue Stromal Heterogeneity in Metabolic Disease Using Single Cell RNA...Heterogeneity in Metabolic Disease Using Single- Cell RNA-Seq 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Linus Tzu-Yen...ABSTRACT We have developed a robust protocol to generate single cell transcriptional profiles from subcutaneous adipose tissue samples of both human

  6. Differential sensitivity of Chironomus and human hemoglobin to gamma radiation

    International Nuclear Information System (INIS)

    Gaikwad, Pallavi S.; Panicker, Lata; Mohole, Madhura; Sawant, Sangeeta; Mukhopadhyaya, Rita; Nath, Bimalendu B.

    2016-01-01

    Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study. -- Highlights: •Comparison of radiation tolerant Chironomus Hb and radiation sensitive Human Hb. •Amino acid composition of midge and human heme confer differential hydrophobicity. •Heme pocket of evolutionarily ancient midge Hb provide gamma radiation resistivity.

  7. Differential sensitivity of Chironomus and human hemoglobin to gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Gaikwad, Pallavi S. [Stress Biology Research Laboratory, Department of Zoology, Savitribai Phule University, Pune, 411007 (India); Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085 (India); Panicker, Lata [Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085 (India); Mohole, Madhura; Sawant, Sangeeta [Bioinformatics Center, Savitribai Phule Pune University, Pune, 411007 (India); Mukhopadhyaya, Rita [Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085 (India); Nath, Bimalendu B., E-mail: bbnath@gmail.com [Stress Biology Research Laboratory, Department of Zoology, Savitribai Phule University, Pune, 411007 (India)

    2016-08-05

    Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study. -- Highlights: •Comparison of radiation tolerant Chironomus Hb and radiation sensitive Human Hb. •Amino acid composition of midge and human heme confer differential hydrophobicity. •Heme pocket of evolutionarily ancient midge Hb provide gamma radiation resistivity.

  8. Correlations between RNA and protein expression profiles in 23 human cell lines

    Directory of Open Access Journals (Sweden)

    Pontén Fredrik

    2009-08-01

    Full Text Available Abstract Background The Central Dogma of biology holds, in famously simplified terms, that DNA makes RNA makes proteins, but there is considerable uncertainty regarding the general, genome-wide correlation between levels of RNA and corresponding proteins. Therefore, to assess degrees of this correlation we compared the RNA profiles (determined using both cDNA- and oligo-based microarrays and protein profiles (determined immunohistochemically in tissue microarrays of 1066 gene products in 23 human cell lines. Results A high mean correlation coefficient (0.52 was obtained from the pairwise comparison of RNA levels determined by the two platforms. Significant correlations, with correlation coefficients exceeding 0.445, between protein and RNA levels were also obtained for a third of the specific gene products. However, the correlation coefficients between levels of RNA and protein products of specific genes varied widely, and the mean correlations between the protein and corresponding RNA levels determined using the cDNA- and oligo-based microarrays were 0.25 and 0.20, respectively. Conclusion Significant correlations were found in one third of the examined RNA species and corresponding proteins. These results suggest that RNA profiling might provide indirect support to antibodies' specificity, since whenever a evident correlation between the RNA and protein profiles exists, this can sustain that the antibodies used in the immunoassay recognized their cognate antigens.

  9. The Human Nuclear Exosome Targeting Complex Is Loaded onto Newly Synthesized RNA to Direct Early Ribonucleolysis

    Directory of Open Access Journals (Sweden)

    Michal Lubas

    2015-01-01

    Full Text Available The RNA exosome complex constitutes the major nuclear eukaryotic 3′-5′ exonuclease. Outside of nucleoli, the human nucleoplasmic exosome is directed to some of its substrates by the nuclear exosome targeting (NEXT complex. How NEXT targets RNA has remained elusive. Using an in vivo crosslinking approach, we report global RNA binding sites of RBM7, a key component of NEXT. RBM7 associates broadly with RNA polymerase II-derived RNA, including pre-mRNA and short-lived exosome substrates such as promoter upstream transcripts (PROMPTs, enhancer RNAs (eRNAs, and 3′-extended products from snRNA and replication-dependent histone genes. Within pre-mRNA, RBM7 accumulates at the 3′ ends of introns, and pulse-labeling experiments demonstrate that RBM7/NEXT defines an early exosome-targeting pathway for 3′-extended snoRNAs derived from such introns. We propose that RBM7 is generally loaded onto newly synthesized RNA to accommodate exosome action in case of available unprotected RNA 3′ ends.

  10. Identification of CELF1 RNA targets by CLIP-seq in human HeLa cells

    Directory of Open Access Journals (Sweden)

    Olivier Le Tonquèze

    2016-06-01

    Full Text Available The specific interactions between RNA-binding proteins and their target RNAs are an essential level to control gene expression. By combining ultra-violet cross-linking and immunoprecipitation (CLIP and massive SoliD sequencing we identified the RNAs bound by the RNA-binding protein CELF1, in human HeLa cells. The CELF1 binding sites deduced from the sequence data allow characterizing specific features of CELF1-RNA association. We present therefore the first map of CELF1 binding sites in human cells.

  11. Involvement of DNA topoisomerase I in transcription of human ribosomal RNA genes

    International Nuclear Information System (INIS)

    Zhang, H.; Wang, J.C.; Liu, L.F.

    1988-01-01

    Treatment of HeLa cells with a DNA topoisomerase I-specific inhibitor, camptothecin, results in rapid cessation of the synthesis of the 45S rRNA precursor. The inhibition of rRNA synthesis is reversible following drug removal and correlates with the presence of camptothecin-trapped topoisomerase I-DNA abortive complexes, which can be detected as topoisomerase I-linked DNA breaks upon lysis with sodium dodecyl sulfate. These breaks were found to be concentrated within the transcribed region of human rRNA genes. No such sites can be detected in the inactive human rRNA genes in mouse-human hybrid cells, suggesting a preferential association of topoisomerase I with actively transcribed genes. The distribution of RNA polymerase molecules along the transcription unit of human rRNA genes in camptothecin-treated HeLa cells, as assayed by nuclear run-on transcription, shows a graded decrease of the RNA polymerase density toward the 3' end of the transcription unit; the density is minimally affected near the 5' start of the transcription unit. These results suggest that DNA topoisomerase I is normally involved in the elongation step of transcription, especially when the transcripts are long, and that camptothecin interferes with this role

  12. Inhibition of Poly(A)-binding protein with a synthetic RNA mimic reduces pain sensitization in mice.

    Science.gov (United States)

    Barragán-Iglesias, Paulino; Lou, Tzu-Fang; Bhat, Vandita D; Megat, Salim; Burton, Michael D; Price, Theodore J; Campbell, Zachary T

    2018-01-02

    Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Comparatively little is known regarding the role of the regulatory factors bound to the 3' end of mRNA in nociceptor sensitization. Poly(A)-binding protein (PABP) stimulates translation initiation by bridging the Poly(A) tail to the eukaryotic initiation factor 4F complex associated with the mRNA cap. Here, we use unbiased assessment of PABP binding specificity to generate a chemically modified RNA-based competitive inhibitor of PABP. The resulting RNA mimic, which we designated as the Poly(A) SPOT-ON, is more stable than unmodified RNA and binds PABP with high affinity and selectivity in vitro. We show that injection of the Poly(A) SPOT-ON at the site of an injury can attenuate behavioral response to pain. Collectively, these results suggest that PABP is integral for nociceptive plasticity. The general strategy described here provides a broad new source of mechanism-based inhibitors for RNA-binding proteins and is applicable for in vivo studies.

  13. MicroRNA-9 enhances sensitivity to cetuximab in epithelial phenotype hepatocellular carcinoma cells through regulation of the eukaryotic translation initiation factor 5A-2.

    Science.gov (United States)

    Xue, Fei; Liang, Yuntian; Li, Zhenrong; Liu, Yanhui; Zhang, Hongwei; Wen, Yu; Yan, Lei; Tang, Qiang; Xiao, Erhui; Zhang, Dongyi

    2018-01-01

    Hepatocellular carcinoma (HCC) is one of the most widespread malignant human tumors worldwide. Treatment options include radiotherapy, surgical intervention and chemotherapy; however, drug resistance is an ongoing treatment concern. In the present study, the effects of a microRNA (miR/miRNA), miR-9, on the sensitivity of HCC cell lines to the epidermal growth factor receptor inhibitor, cetuximab, were examined. miR-9 has been proposed to serve a role in tumorigenesis and tumor progression. In the present study, bioinformatics analyses identified the eukaryotic translation initiation factor 5A2 (eIF-5A-2) as a target of miR-9. The expression levels of miR-9 and eIF-5A-2 were examined by reverse transcription-quantitative polymerase chain reaction and HCC cell lines were transfected with miR-9 mimics and inhibitors to determine the effects of the miRNA on cell proliferation and viability. The miR-9 mimic was revealed to significantly increase the sensitivity of epithelial phenotype HCC cells (Hep3B and Huh7) to cetuximab, while the miR-9 inhibitor triggered the opposite effect. There were no significant differences in sensitivity to cetuximab observed in mesenchymal phenotype HCC cells (SNU387 and SNU449). Cells lines displaying high expression levels of eIF-5A-2 were more resistant to cetuximab. Transfection of cells with a miR-9 mimic resulted in downregulation of the expression of eIF-5A-2 mRNA, while an miR-9 inhibitor increased expression. When expression of eIF-5A-2 was knocked down with siRNA, the effects of miR-9 on cetuximab sensitivity were no longer observed. Taken together, these data support a role for miR-9 in enhancing the sensitivity of epithelial phenotype HCC cells to cetuximab through regulation of eIF-5A-2.

  14. Identification of failure sequences sensitive to human error

    International Nuclear Information System (INIS)

    1987-06-01

    This report prepared by the participants of the technical committee meeting on ''Identification of Failure Sequences Sensitive to Human Error'' addresses the subjects discussed during the meeting and the conclusions reached by the committee. Chapter 1 reviews the INSAG recommendations and the main elements of the IAEA Programme in the area of human element. In Chapter 2 the role of human actions in nuclear power plants safety from insights of operational experience is reviewed. Chapter 3 is concerned with the relationship between probabilistic safety assessment and human performance associated with severe accident sequences. Chapter 4 addresses the role of simulators in view of training for accident conditions. Chapter 5 presents the conclusions and future trends. The seven papers presented by members of this technical committee are also included in this technical document. A separate abstract was prepared for each of these papers

  15. Sensitive detection of viral transcripts in human tumor transcriptomes.

    Directory of Open Access Journals (Sweden)

    Sven-Eric Schelhorn

    Full Text Available In excess of 12% of human cancer incidents have a viral cofactor. Epidemiological studies of idiopathic human cancers indicate that additional tumor viruses remain to be discovered. Recent advances in sequencing technology have enabled systematic screenings of human tumor transcriptomes for viral transcripts. However, technical problems such as low abundances of viral transcripts in large volumes of sequencing data, viral sequence divergence, and homology between viral and human factors significantly confound identification of tumor viruses. We have developed a novel computational approach for detecting viral transcripts in human cancers that takes the aforementioned confounding factors into account and is applicable to a wide variety of viruses and tumors. We apply the approach to conducting the first systematic search for viruses in neuroblastoma, the most common cancer in infancy. The diverse clinical progression of this disease as well as related epidemiological and virological findings are highly suggestive of a pathogenic cofactor. However, a viral etiology of neuroblastoma is currently contested. We mapped 14 transcriptomes of neuroblastoma as well as positive and negative controls to the human and all known viral genomes in order to detect both known and unknown viruses. Analysis of controls, comparisons with related methods, and statistical estimates demonstrate the high sensitivity of our approach. Detailed investigation of putative viral transcripts within neuroblastoma samples did not provide evidence for the existence of any known human viruses. Likewise, de-novo assembly and analysis of chimeric transcripts did not result in expression signatures associated with novel human pathogens. While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data, in principle, this is rendered less likely by the high sensitivity of our approach and the number of biological replicates

  16. A human torque teno virus encodes a microRNA that inhibits interferon signaling.

    Directory of Open Access Journals (Sweden)

    Rodney P Kincaid

    Full Text Available Torque teno viruses (TTVs are a group of viruses with small, circular DNA genomes. Members of this family are thought to ubiquitously infect humans, although causal disease associations are currently lacking. At present, there is no understanding of how infection with this diverse group of viruses is so prevalent. Using a combined computational and synthetic approach, we predict and identify miRNA-coding regions in diverse human TTVs and provide evidence for TTV miRNA production in vivo. The TTV miRNAs are transcribed by RNA polymerase II, processed by Drosha and Dicer, and are active in RISC. A TTV mutant defective for miRNA production replicates as well as wild type virus genome; demonstrating that the TTV miRNA is dispensable for genome replication in a cell culture model. We demonstrate that a recombinant TTV genome is capable of expressing an exogenous miRNA, indicating the potential utility of TTV as a small RNA vector. Gene expression profiling of host cells identifies N-myc (and STAT interactor (NMI as a target of a TTV miRNA. NMI transcripts are directly regulated through a binding site in the 3'UTR. SiRNA knockdown of NMI contributes to a decreased response to interferon signaling. Consistent with this, we show that a TTV miRNA mediates a decreased response to IFN and increased cellular proliferation in the presence of IFN. Thus, we add Annelloviridae to the growing list of virus families that encode miRNAs, and suggest that miRNA-mediated immune evasion can contribute to the pervasiveness associated with some of these viruses.

  17. A new sensitive method for detecting human endogenous (leukocyte) pyrogen.

    Science.gov (United States)

    Bodel, P; Miller, H

    1978-03-01

    Endogenous, or leukocyte pyrogen (EP), the mediator of fever, is currently detected by injection of pyrogen-containing supernatants into rabbits. This assay has been of little value in the study of human fever because it required injection of relatively large amounts of pyrogen. We now report that injection of medium containing human EP produces fever in mice. Supernatant from 1 c 10(5) granulocytes, stimulated by phagocytosis of staphylococci and incubated overnight, or 1 x 10(4) monocytes similarly treated, produce clear pyrogenic responses. This method for detecting EP is about 100-fold more sensitive than the rabbit assay, and it appears to be specific for EP. Preliminary studies of EP released by small samples of needle liver biopsies from febrile and afebrile patients suggests that this sensitive assay may be useful for investigations into the mechanisms of clinical fever.

  18. Nonsense mutations in the human β-globin gene affect mRNA metabolism

    International Nuclear Information System (INIS)

    Baserga, S.J.; Benz, E.J. Jr.

    1988-01-01

    A number of premature translation termination mutations (nonsense mutations) have been described in the human α- and β-globin genes. Studies on mRNA isolated from patients with β 0 -thalassemia have shown that for both the β-17 and the β-39 mutations less than normal levels of β-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human β-globin mRNA). In vitro studies using the cloned β-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human β-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation

  19. Elucidating Mechanisms of Molecular Recognition Between Human Argonaute and miRNA Using Computational Approaches

    KAUST Repository

    Jiang, Hanlun

    2016-12-06

    MicroRNA (miRNA) and Argonaute (AGO) protein together form the RNA-induced silencing complex (RISC) that plays an essential role in the regulation of gene expression. Elucidating the underlying mechanism of AGO-miRNA recognition is thus of great importance not only for the in-depth understanding of miRNA function but also for inspiring new drugs targeting miRNAs. In this chapter we introduce a combined computational approach of molecular dynamics (MD) simulations, Markov state models (MSMs), and protein-RNA docking to investigate AGO-miRNA recognition. Constructed from MD simulations, MSMs can elucidate the conformational dynamics of AGO at biologically relevant timescales. Protein-RNA docking can then efficiently identify the AGO conformations that are geometrically accessible to miRNA. Using our recent work on human AGO2 as an example, we explain the rationale and the workflow of our method in details. This combined approach holds great promise to complement experiments in unraveling the mechanisms of molecular recognition between large, flexible, and complex biomolecules.

  20. Lack of close relationship between three strains of human rhinoviruses as determined by their RNA sequences.

    Science.gov (United States)

    Yin, F H; Lonberg-Holm, K; Chan, S P

    1973-07-01

    The possible genomic homologies between three serotypes of human rhinoviruses (HRV 1A, HRV 2, and HRV 14) were investigated. First we confirmed that these viruses were unrelated by the criterion of the absence of common antigenic determinants on the surfaces of the native virions, as detected by cross-neutralization of complementfixation. RNA-RNA hybridization was then examined with purified, highly radioactive, double-stranded, replicative-form RNA and excess single-stranded virion RNA. Single-stranded RNA showed 100% homology with the minus strand from the replicative-form RNA of the same type of virus. HRV 1A, HRV 2, and HRV 14 showed low intertypic homologies; these were not significantly greater than those found between the rhinoviruses and polivirus, which were used as a negative control. The immunological relationship and the RNA homology between HRV 1A and HRV 1B were also examined by the above techniques. It was confirmed that HRV 1A and HRV 1B share some surface determinants and it was also found that HRV 1B RNA shares 70% homology with HRV 1A RNA.

  1. Elucidating Mechanisms of Molecular Recognition Between Human Argonaute and miRNA Using Computational Approaches.

    Science.gov (United States)

    Jiang, Hanlun; Zhu, Lizhe; Héliou, Amélie; Gao, Xin; Bernauer, Julie; Huang, Xuhui

    2017-01-01

    MicroRNA (miRNA) and Argonaute (AGO) protein together form the RNA-induced silencing complex (RISC) that plays an essential role in the regulation of gene expression. Elucidating the underlying mechanism of AGO-miRNA recognition is thus of great importance not only for the in-depth understanding of miRNA function but also for inspiring new drugs targeting miRNAs. In this chapter we introduce a combined computational approach of molecular dynamics (MD) simulations, Markov state models (MSMs), and protein-RNA docking to investigate AGO-miRNA recognition. Constructed from MD simulations, MSMs can elucidate the conformational dynamics of AGO at biologically relevant timescales. Protein-RNA docking can then efficiently identify the AGO conformations that are geometrically accessible to miRNA. Using our recent work on human AGO2 as an example, we explain the rationale and the workflow of our method in details. This combined approach holds great promise to complement experiments in unraveling the mechanisms of molecular recognition between large, flexible, and complex biomolecules.

  2. Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

    Science.gov (United States)

    Canuto, K. S.; Teixeira, A. F.; Rodrigues, J. A.; Paoli, F.; Nogueira, E. M.; Mencalha, A. L.; Fonseca, A. S.

    2017-06-01

    MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

  3. RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells

    International Nuclear Information System (INIS)

    Bueren, André O von; Shalaby, Tarek; Oehler-Jänne, Christoph; Arnold, Lucia; Stearns, Duncan; Eberhart, Charles G; Arcaro, Alexandre; Pruschy, Martin; Grotzer, Michael A

    2009-01-01

    With current treatment strategies, nearly half of all medulloblastoma (MB) patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis. To study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA) and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR) and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425). siRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT) expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector. In human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly

  4. RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells

    Directory of Open Access Journals (Sweden)

    Arcaro Alexandre

    2009-01-01

    Full Text Available Abstract Background With current treatment strategies, nearly half of all medulloblastoma (MB patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis. Methods To study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425. Results siRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector. Conclusion In human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly.

  5. Zebra finches are sensitive to prosodic features of human speech.

    Science.gov (United States)

    Spierings, Michelle J; ten Cate, Carel

    2014-07-22

    Variation in pitch, amplitude and rhythm adds crucial paralinguistic information to human speech. Such prosodic cues can reveal information about the meaning or emphasis of a sentence or the emotional state of the speaker. To examine the hypothesis that sensitivity to prosodic cues is language independent and not human specific, we tested prosody perception in a controlled experiment with zebra finches. Using a go/no-go procedure, subjects were trained to discriminate between speech syllables arranged in XYXY patterns with prosodic stress on the first syllable and XXYY patterns with prosodic stress on the final syllable. To systematically determine the salience of the various prosodic cues (pitch, duration and amplitude) to the zebra finches, they were subjected to five tests with different combinations of these cues. The zebra finches generalized the prosodic pattern to sequences that consisted of new syllables and used prosodic features over structural ones to discriminate between stimuli. This strong sensitivity to the prosodic pattern was maintained when only a single prosodic cue was available. The change in pitch was treated as more salient than changes in the other prosodic features. These results show that zebra finches are sensitive to the same prosodic cues known to affect human speech perception. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  6. Genome-wide RNA-seq analysis of human and mouse platelet transcriptomes

    Science.gov (United States)

    Rowley, Jesse W.; Oler, Andrew J.; Tolley, Neal D.; Hunter, Benjamin N.; Low, Elizabeth N.; Nix, David A.; Yost, Christian C.; Zimmerman, Guy A.

    2011-01-01

    Inbred mice are a useful tool for studying the in vivo functions of platelets. Nonetheless, the mRNA signature of mouse platelets is not known. Here, we use paired-end next-generation RNA sequencing (RNA-seq) to characterize the polyadenylated transcriptomes of human and mouse platelets. We report that RNA-seq provides unprecedented resolution of mRNAs that are expressed across the entire human and mouse genomes. Transcript expression and abundance are often conserved between the 2 species. Several mRNAs, however, are differentially expressed in human and mouse platelets. Moreover, previously described functional disparities between mouse and human platelets are reflected in differences at the transcript level, including protease activated receptor-1, protease activated receptor-3, platelet activating factor receptor, and factor V. This suggests that RNA-seq is a useful tool for predicting differences in platelet function between mice and humans. Our next-generation sequencing analysis provides new insights into the human and murine platelet transcriptomes. The sequencing dataset will be useful in the design of mouse models of hemostasis and a catalyst for discovery of new functions of platelets. Access to the dataset is found in the “Introduction.” PMID:21596849

  7. QSAR models of human data can enrich or replace LLNA testing for human skin sensitization

    Science.gov (United States)

    Alves, Vinicius M.; Capuzzi, Stephen J.; Muratov, Eugene; Braga, Rodolpho C.; Thornton, Thomas; Fourches, Denis; Strickland, Judy; Kleinstreuer, Nicole; Andrade, Carolina H.; Tropsha, Alexander

    2016-01-01

    Skin sensitization is a major environmental and occupational health hazard. Although many chemicals have been evaluated in humans, there have been no efforts to model these data to date. We have compiled, curated, analyzed, and compared the available human and LLNA data. Using these data, we have developed reliable computational models and applied them for virtual screening of chemical libraries to identify putative skin sensitizers. The overall concordance between murine LLNA and human skin sensitization responses for a set of 135 unique chemicals was low (R = 28-43%), although several chemical classes had high concordance. We have succeeded to develop predictive QSAR models of all available human data with the external correct classification rate of 71%. A consensus model integrating concordant QSAR predictions and LLNA results afforded a higher CCR of 82% but at the expense of the reduced external dataset coverage (52%). We used the developed QSAR models for virtual screening of CosIng database and identified 1061 putative skin sensitizers; for seventeen of these compounds, we found published evidence of their skin sensitization effects. Models reported herein provide more accurate alternative to LLNA testing for human skin sensitization assessment across diverse chemical data. In addition, they can also be used to guide the structural optimization of toxic compounds to reduce their skin sensitization potential. PMID:28630595

  8. The mRNA expression profile of metabolic genes relative to MHC isoform pattern in human skeletal muscles

    DEFF Research Database (Denmark)

    Plomgaard, Peter; Penkowa, Milena; Leick, Lotte

    2006-01-01

    The metabolic profile of rodent muscle is generally reflected in the myosin heavy chain (MHC) fiber-type composition. The present study was conducted to test the hypothesis that metabolic gene expression is not tightly coupled with MHC fiber-type composition for all genes in human skeletal muscle....... Triceps brachii, vastus lateralis quadriceps, and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers, because these muscles are characterized by different fiber-type compositions. As expected, citrate synthase and 3-hydroxyacyl dehydrogenase activity...... of a broad range of metabolic genes. The triceps muscle had two- to fivefold higher MHC IIa, phosphofructokinase, and LDH A mRNA content and two- to fourfold lower MHC I, lipoprotein lipase, CD36, hormone-sensitive lipase, and LDH B and hexokinase II mRNA than vastus lateralis or soleus. Interestingly...

  9. The miRNA and mRNA Signatures of Peripheral Blood Cells in Humans Infected with Trypanosoma brucei gambiense.

    Directory of Open Access Journals (Sweden)

    Smiths Lueong

    Full Text Available Simple, reliable tools for diagnosis of human African Trypanosomiases could ease field surveillance and enhance patient care. In particular, current methods to distinguish patients with (stage II and without (stage I brain involvement require samples of cerebrospinal fluid. We describe here an exploratory study to find out whether miRNAs from peripheral blood leukocytes might be useful in diagnosis of human trypanosomiasis, or for determining the stage of the disease. Using microarrays, we measured miRNAs in samples from Trypanosoma brucei gambiense-infected patients (9 stage I, 10 stage II, 8 seronegative parasite-negative controls and 12 seropositive, but parasite-negative subjects. 8 miRNAs (out of 1205 tested showed significantly lower expression in patients than in seronegative, parasite-negative controls, and 1 showed increased expression. There were no clear differences in miRNAs between patients in different disease stages. The miRNA profiles could not distinguish seropositive, but parasitologically negative samples from controls and results within this group did not correlate with those from the trypanolysis test. Some of the regulated miRNAs, or their predicted mRNA targets, were previously reported changed during other infectious diseases or cancer. We conclude that the changes in miRNA profiles of peripheral blood lymphocytes in human African trypanosomiasis are related to immune activation or inflammation, are probably disease-non-specific, and cannot be used to determine the disease stage. The approach has little promise for diagnostics but might yield information about disease pathology.

  10. MicroRNA and piRNA profiles in normal human testis detected by next generation sequencing.

    Directory of Open Access Journals (Sweden)

    Qingling Yang

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are the class of small endogenous RNAs that play an important regulatory role in cells by negatively affecting gene expression at transcriptional and post-transcriptional levels. There have been extensive studies aiming to discover miRNAs and to analyze their functions in the cells from a variety of species. However, there are no published studies of miRNA profiles in human testis using next generation sequencing (NGS technology. RESULTS: We employed Solexa sequencing technology to profile miRNAs in normal human testis. Total 770 known and 5 novel human miRNAs, and 20121 piRNAs were detected, indicating that the human testis has a complex population of small RNAs. The expression of 15 known and 5 novel detected miRNAs was validated by qRT-PCR. We have also predicted the potential target genes of the abundant known and novel miRNAs, and subjected them to GO and pathway analysis, revealing the involvement of miRNAs in many important biological phenomenon including meiosis and p53-related pathways that are implicated in the regulation of spermatogenesis. CONCLUSIONS: This study reports the first genome-wide miRNA profiles in human testis using a NGS approach. The presence of large number of miRNAs and the nature of their target genes suggested that miRNAs play important roles in spermatogenesis. Here we provide a useful resource for further elucidation of the regulatory role of miRNAs and piRNAs in the spermatogenesis. It may also facilitate the development of prophylactic strategies for male infertility.

  11. Reduction of bilirubin by targeting human heme oxygenase-1 through siRNA.

    Science.gov (United States)

    Xia, Zhen-Wei; Li, Chun-E; Jin, You-Xin; Shi, Yi; Xu, Li-Qing; Zhong, Wen-Wei; Li, Yun-Zhu; Yu, Shan-Chang; Zhang, Zi-Li

    2007-04-01

    Neonatal hyperbilirubinemia is a common clinical condition caused mainly by the increased production and decreased excretion of bilirubin. Current treatment is aimed at reducing the serum levels of bilirubin. Heme oxygenase-1 (HO-1) is a rate-limiting enzyme that generates bilirubin. In this study we intended to suppress HO-1 using the RNA interference technique. Small interfering RNA (siRNA)-A, -B, and -C were designed based on human HO-1 (hHO-1) mRNA sequences. siRNA was transfected into a human hepatic cell line (HL-7702). hHO-1 transcription and protein levels were then determined. In addition, the inhibitory effect of siRNA on hHO-1 was assessed in cells treated with hemin or transfected with an hHO-1 plasmid. siRNA-C showed the most potent suppressive effect on hHO-1. This inhibition is dose and time dependent. Compared with control, both hemin and hHO-1 plasmids up-regulated hHO-1 expression in HL-7702 cells. However, the up-regulation was significantly attenuated by siRNA-C. Furthermore, the decrease in hHO-1 activity was coincident with the suppression of its transcription. Finally, siRNA-C was shown to reduce hHO-1 enzymatic activity and bilirubin levels. Thus, this study provides a novel therapeutic rationale by blocking bilirubin formation via siRNA for preventing and treating neonatal hyperbilirubinemia and bilirubin encephalopathy at an early clinical stage.

  12. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

    Science.gov (United States)

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W; Grubert, Fabian; Candille, Sophie I; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L; Tang, Hua; Ricci, Emiliano; Snyder, Michael P

    2015-11-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. © 2015 Cenik et al.; Published by Cold Spring Harbor Laboratory Press.

  13. Effect of silencing of ATM expression by siRNA on radiosensitivity of human lung adenocarcinoma A549 cells

    International Nuclear Information System (INIS)

    Liu Xiaoqun; Qiao Tiankui

    2014-01-01

    Objective: To investigate the effect of silencing of ataxia-telangiectasia mutated (ATM) expression by plasmid-mediated RNA interference on the radiosensitivity of human lung adenocarcinoma A 549 cells. Methods: Eukaryotic expression plasmid containing ATM small interfering RNA (siRNA) (pSilencer2.1-ATM), as well as pSilencer2.1-nonspecific, was constructed.Lung adenocarcinoma A 549 cells were divided into positive group, negative group,and control group to be transfected with pSilencer2.1-ATM, pSilencer2.1-nonspecific, and no plasmid, respectively. The mRNA and protein expression of ATM was measured by RT-PCR and Western blot, respectively. The change in cell radiosensitivity was observed by colony-forming assay. Cell cycle and cell apoptosis were analyzed by flow cytometry. Results: The eukaryotic expression plasmid containing ATM siRNA was successfully constructed. The RT-PCR and Western blot demonstrated that the expression of ATM was down-regulated in the positive group. The sensitization enhancement ratios (D 0 ratios) for the positive group and negative group were 1.50 and 1.01, respectively. The flow cytometry revealed that the proportions of A 549 cells in G 1 and G 2 /M phases were significantly lower in the positive group than in the control group (51.27% vs 61.85%, P = 0.012; 6.34% vs 10.91%, P = 0.008) and that the apoptosis rate was significantly higher in the positive group than in the control group and negative group (49.31% vs 13.58%, P = 0.000; 49.31% vs 13.17%, P = 0.000). Conclusions: Silencing of ATM expression may increase the radiosensitivity of human lung adenocarcinoma A 549 cells, probably by affecting the cell cycle and promoting cell apoptosis. (authors)

  14. Sensitive and label-free detection of miRNA-145 by triplex formation.

    Science.gov (United States)

    Aviñó, Anna; Huertas, César S; Lechuga, Laura M; Eritja, Ramon

    2016-01-01

    The development of new strategies for detecting microRNAs (miRNAs) has become a crucial step in the diagnostic field. miRNA profiles depend greatly on the sample and the analytical platform employed, leading sometimes to contradictory results. In this work, we study the use of modified parallel tail-clamps to detect a miRNA sequence involved in tumor suppression by triplex formation. Thermal denaturing curves and circular dichroism (CD) measurements have been performed to confirm that parallel clamps carrying 8-aminoguanine form the most stable triplex structures with their target miRNA. The modified tail-clamps have been tested as bioreceptors in a surface plasmon resonance (SPR) biosensor for the detection of miRNA-145. The detection limit was improved 2.4 times demonstrating that a stable triplex structure is formed between target miRNA and 8-aminoguanine tail-clamp bioreceptor. This new approach is an essential step toward the label-free and reliable detection of miRNA signatures for diagnostic purposes.

  15. Sensitive voltammetric detection of yeast RNA based on its interaction with Victoria Blue B

    Directory of Open Access Journals (Sweden)

    WEI SUN

    2009-12-01

    Full Text Available Voltammetric studies of the interaction of yeast RNA (y-RNA with Victoria Blue B (VBB are described in this paper. Furthermore, a linear sweep voltammetric method for the detection of y-RNA was established. The reaction conditions, such as acidity and amount of buffer solution, the concentration of VBB, the reaction time and temperature, etc., were carefully investigated by second order derivative linear sweep voltammetry. Under the optimal conditions, the reduction peak current of VBB at –0.75 V decreased greatly after the addition of y-RNA to the solution without any shift of the reduction peak potential. Based on the decrease of the peak current, a new quantitative method for the determination of y-RNA was developed. The effects of co-existing substances on the determination were carefully investigated and three synthetic samples were determined with satisfactory results. The stoichiometry of the VBB–y-RNA complex was calculated by linear sweep voltammetry and the interaction mechanism is discussed.

  16. Establishment of lipofection for studying miRNA function in human adipocytes.

    Science.gov (United States)

    Enlund, Eveliina; Fischer, Simon; Handrick, René; Otte, Kerstin; Debatin, Klaus-Michael; Wabitsch, Martin; Fischer-Posovszky, Pamela

    2014-01-01

    miRNA dysregulation has recently been linked to human obesity and its related complications such as type 2 diabetes. In order to study miRNA function in human adipocytes, we aimed for the modulation of mature miRNA concentration in these cells. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. Our objective therefore was to identify an efficient, non-viral transfection reagent capable of delivering small RNAs into these cells. To achieve this, we compared the efficiencies of three transfection agents, Lipofectamine 2000, ScreenFect A and BPEI 1.2 k in delivering fluorescent-labelled siRNA into human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes and adipocytes. Downregulation of a specific target gene in response to miRNA mimic overexpression was assayed in SGBS cells and also in ex vivo differentiated primary human adipocytes. Our results demonstrated that while all three transfection agents were able to internalize the oligos, only lipofection resulted in the efficient downregulation of a specific target gene both in SGBS cells and in primary human adipocytes. Lipofectamine 2000 outperformed ScreenFect A in preadipocytes, but in adipocytes the two reagents gave comparable results making ScreenFect A a notable new alternative for the gold standard Lipofectamine 2000.

  17. Establishment of lipofection for studying miRNA function in human adipocytes.

    Directory of Open Access Journals (Sweden)

    Eveliina Enlund

    Full Text Available miRNA dysregulation has recently been linked to human obesity and its related complications such as type 2 diabetes. In order to study miRNA function in human adipocytes, we aimed for the modulation of mature miRNA concentration in these cells. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. Our objective therefore was to identify an efficient, non-viral transfection reagent capable of delivering small RNAs into these cells. To achieve this, we compared the efficiencies of three transfection agents, Lipofectamine 2000, ScreenFect A and BPEI 1.2 k in delivering fluorescent-labelled siRNA into human Simpson-Golabi-Behmel syndrome (SGBS preadipocytes and adipocytes. Downregulation of a specific target gene in response to miRNA mimic overexpression was assayed in SGBS cells and also in ex vivo differentiated primary human adipocytes. Our results demonstrated that while all three transfection agents were able to internalize the oligos, only lipofection resulted in the efficient downregulation of a specific target gene both in SGBS cells and in primary human adipocytes. Lipofectamine 2000 outperformed ScreenFect A in preadipocytes, but in adipocytes the two reagents gave comparable results making ScreenFect A a notable new alternative for the gold standard Lipofectamine 2000.

  18. Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing.

    Science.gov (United States)

    Jones, Christopher P; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin

    2013-02-01

    The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA(Lys3). Host cell tRNA(Lys) is selectively packaged into HIV-1 through a specific interaction between the major tRNA(Lys)-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA(Lys3) is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA(Lys) and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA(Lys3) in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA(Lys) to increase the efficiency of tRNA(Lys3) annealing to viral RNA.

  19. Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA.

    Science.gov (United States)

    Lu, Song; Chow, Christie C; Zhou, Junwei; Leung, Po Sing; Tsui, Stephen K; Lui, Kathy O

    2016-01-01

    In this chapter, we describe a highly efficient genetic modification strategy for human pancreatic progenitor cells using modified mRNA-encoding GFP and Neurogenin-3. The properties of modified mRNA offer an invaluable platform to drive protein expression, which has broad applicability in pathway regulation, directed differentiation, and lineage specification. This approach can also be used to regulate expression of other pivotal transcription factors during pancreas development and might have potential therapeutic values in regenerative medicine.

  20. The Functional Characterization of Long Noncoding RNA SPRY4-IT1 in Human Melanoma Cells

    OpenAIRE

    Mazar, Joseph; Zhao, Wei; Khalil, Ahmad M.; Lee, Bongyong; Shelley, John; Govindarajan, Subramaniam S.; Yamamoto, Fumiko; Ratnam, Maya; Aftab, Muhammad Nauman; Collins, Sheila; Finck, Brian N.; Han, Xianlin; Mattick, John S.; Dinger, Marcel E.; Perera, Ranjan J.

    2014-01-01

    Expression of the long noncoding RNA (lncRNA) SPRY4-IT1 is low in normal human melanocytes but high in melanoma cells. siRNA knockdown of SPRY4-IT1 blocks melanoma cell invasion and proliferation, and increases apoptosis. To investigate its function further, we affinity purified SPRY4-IT1 from melanoma cells and used mass spectrometry to identify the protein lipin 2, an enzyme that converts phosphatidate to diacylglycerol (DAG), as a major binding partner. SPRY4-IT1 knockdown increases the ac...

  1. The human cap-binding complex is functionally connected to the nuclear RNA exosome

    DEFF Research Database (Denmark)

    Andersen, Peter Refsing; Domanski, Michal; Kristiansen, Maiken Søndergaard

    2013-01-01

    Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome...... and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA) and then further connects, together with the ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis...

  2. Keratin-18 and microRNA-122 complement alanine aminotransferase as novel safety biomarkers for drug-induced liver injury in two human cohorts.

    Science.gov (United States)

    Thulin, Petra; Nordahl, Gunnar; Gry, Marcus; Yimer, Getnet; Aklillu, Eleni; Makonnen, Eyasu; Aderaye, Getachew; Lindquist, Lars; Mattsson, C Mikael; Ekblom, Björn; Antoine, Daniel J; Park, B Kevin; Linder, Stig; Harrill, Alison H; Watkins, Paul B; Glinghammar, Björn; Schuppe-Koistinen, Ina

    2014-03-01

    There is a demand for more sensitive, specific and predictive biomarkers for drug-induced liver injury (DILI) than the gold standard used today, alanine aminotransferase (ALT). The aim of this study was to qualify novel DILI biomarkers (keratin-18 markers M65/M30, microRNA-122, glutamate dehydrogenase and alpha-foetoprotein) in human DILI. Levels of the novel biomarkers were measured by enzyme-linked immunosorbent assay or real-time quantitative reverse-transcription PCR (qRT-PCR) in two human DILI cohorts: a human volunteer study with acetaminophen and a human immunodeficiency virus (HIV)/tuberculosis (TB) study. In the acetaminophen study, serum M65 and microRNA-122 levels were significantly increased at an earlier time point than ALT. Furthermore, the maximal elevation of M65 and microRNA-122 exceeded the increase in ALT. In the HIV/TB study, all the analysed novel biomarkers increased after 1 week of treatment. In contrast to ALT, the novel biomarkers remained stable in a human cohort with exercise-induced muscular injury. M65 and microRNA-122 are potential biomarkers of DILI superior to ALT with respect to sensitivity and specificity. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Biobanking of human pancreas cancer tissue: impact of ex-vivo procurement times on RNA quality.

    Science.gov (United States)

    Rudloff, Udo; Bhanot, Umesh; Gerald, William; Klimstra, David S; Jarnagin, William R; Brennan, Murray F; Allen, Peter J

    2010-08-01

    Tissue banking has become a major initiative at many oncology centers. The influence of warm ex-vivo ischemia times, storage times, and biobanking protocols on RNA integrity and subsequent microarray data is not well documented. A prospective institutional review board-approved protocol for the banking of abdominal neoplasms was initiated at Memorial Sloan-Kettering Cancer Center in 2001. Sixty-four representative pancreas cancer specimens snap-frozen at various ex-vivo procurement times (1 h) and banked during three time periods (2001-2004, 2004-2006, 2006-2008) were processed. RNA integrity was determined by microcapillary electrophoresis using the RNA integrity number (RIN) algorithm and by results of laser-capture microdissection (LCM). Overall, 42% of human pancreas cancer specimens banked under a dedicated protocol yielded RNA with a RIN of > or =7. Limited warm ex-vivo ischemia times did not negatively impact RNA quality (percentage of tissue with total RNA with RIN of > or =7 for 60 min, 42%), and long-term storage of banked pancreas cancer biospecimens did not negatively influence RNA quality (total RNA with RIN of > or =7 banked 2001-2004, 44%; 2004-2006, 38%; 2006-2008, 50%). RNA retrieved from pancreatic cancer samples with RIN of > or =7 subject to LCM yielded RNA suitable for further downstream applications. Fresh-frozen pancreas tissue banked within a standardized research protocol yields high-quality RNA in approximately 50% of specimens and can be used for enrichment by LCM. Quality of tissues of the biobank were not adversely impacted by limited variations of warm ischemia times or different storage periods. This study shows the challenges and investments required to initiate and maintain high-quality tissue repositories.

  4. 3' terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2013-09-21

    Post-transcriptional 3' end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3' RACE coupled with high-throughput sequencing to characterize the 3' terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes. The 3' terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3' terminus of an in vitro transcribed MRP RNA control and the differing 3' terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR). 3' RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3' terminal sequences of noncoding RNAs.

  5. 3′ terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing

    Science.gov (United States)

    2013-01-01

    Background Post-transcriptional 3′ end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3′ RACE coupled with high-throughput sequencing to characterize the 3′ terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes. Results The 3′ terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3′ terminus of an in vitro transcribed MRP RNA control and the differing 3′ terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR). Conclusions 3′ RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3′ terminal sequences of noncoding RNAs. PMID:24053768

  6. Expression of a novel non-coding mitochondrial RNA in human proliferating cells.

    Science.gov (United States)

    Villegas, Jaime; Burzio, Veronica; Villota, Claudio; Landerer, Eduardo; Martinez, Ronny; Santander, Marcela; Martinez, Rodrigo; Pinto, Rodrigo; Vera, María I; Boccardo, Enrique; Villa, Luisa L; Burzio, Luis O

    2007-01-01

    Previously, we reported the presence in mouse cells of a mitochondrial RNA which contains an inverted repeat (IR) of 121 nucleotides (nt) covalently linked to the 5' end of the mitochondrial 16S RNA (16S mtrRNA). Here, we report the structure of an equivalent transcript of 2374 nt which is over-expressed in human proliferating cells but not in resting cells. The transcript contains a hairpin structure comprising an IR of 815 nt linked to the 5' end of the 16S mtrRNA and forming a long double-stranded structure or stem and a loop of 40 nt. The stem is resistant to RNase A and can be detected and isolated after digestion with the enzyme. This novel transcript is a non-coding RNA (ncRNA) and several evidences suggest that the transcript is synthesized in mitochondria. The expression of this transcript can be induced in resting lymphocytes stimulated with phytohaemagglutinin (PHA). Moreover, aphidicolin treatment of DU145 cells reversibly blocks proliferation and expression of the transcript. If the drug is removed, the cells re-assume proliferation and over-express the ncmtRNA. These results suggest that the expression of the ncmtRNA correlates with the replicative state of the cell and it may play a role in cell proliferation.

  7. Salivary Proteome Patterns Affecting Human Salt Taste Sensitivity.

    Science.gov (United States)

    Stolle, Theresa; Grondinger, Freya; Dunkel, Andreas; Meng, Chen; Médard, Guillaume; Kuster, Bernhard; Hofmann, Thomas

    2017-10-25

    To investigate the role of perireceptor events in inter-individual variability in salt taste sensitivity, 31 volunteers were monitored in their detection functions for sodium chloride (NaCl) and classified into sensitive (0.6-1.7 mmol/L), medium-sensitive (1.8-6.9 mmol/L), and nonsensitive (7.0-11.2 mmol/L) subjects. Chemosensory intervention of NaCl-sensitive (S + ) and nonsensitive (S - ) panellists with potassium chloride, ammonium chloride, and sodium gluconate showed the salt taste sensitivity to be specific for NaCl. As no significant differences were found between S + and S - subjects in salivary sodium and protein content, salivary proteome differences and their stimulus-induced dynamic changes were analyzed by tryptic digestion, iTRAQ labeling, and liquid chromatography-tandem mass spectrometry analysis. Differences in the salivary proteome between S + and S - subjects were found primarily in resting saliva and were largely independent of the dynamic alterations observed upon salt stimulation. Gene ontology enrichment analysis of key proteins, i.e., immunoglobulin heavy constant y1, myeloblastin, cathepsin G, and kallikrein, revealed significantly increased serine-type endopeptidase activity for the S + group, while the S - group exhibited augmented cysteine-type endopeptidase inhibitor activity by increased abundances in lipocalin-1 and cystatin-D, -S, and -SN, respectively. As proteases have been suggested to facilitate transepithelial sodium transport by cleaving the y-subunit of the epithelial sodium channel (ENaC) and protease inhibitors have been shown to reduce ENaC-mediated sodium transport, the differentially modulated proteolytic activity patterns observed in vivo for S + and S - subjects show evidence of them playing a crucial role in affecting human NaCl sensitivity.

  8. Sensitive periods in human development: evidence from musical training.

    Science.gov (United States)

    Penhune, Virginia B

    2011-10-01

    One of the primary goals of cognitive neuroscience is to understand the interaction between genes, development and specific experience. A particularly fascinating example of this interaction is a sensitive period - a time during development when experience has a differential effect on behavior and the brain. Behavioral and brain imaging studies in musicians have provided suggestive evidence for a possible sensitive period for musical training; showing that musicians who began training early show better task performance and greater changes in auditory and motor regions of the brain. However, these studies have not controlled for likely differences between early- (ET) and late-trained (LT) musicians in the number of years of musical experience. This review presents behavioral work from our laboratory comparing the performance of ET (before age seven) and LT musicians who were matched for years of experience on the ability to tap in synchrony with auditory and visual rhythms. The results demonstrate the existence of a possible sensitive period for musical training that has its greatest impact on measures of sensorimotor integration. Work on motor learning in children and how this might relate to the observed sensitive period effect is also reviewed. These studies are described in the context of what is currently known about sensitive periods in animals and humans; drawing on evidence from anatomy and physiology, studies of deafness, as well as structural and functional neuroimaging studies in trained musicians. The possible mechanisms underlying sensitive periods for musical training are discussed based on current theories describing the influence of both low-level features of sensory experience and higher-level cognitive processing. Copyright © 2011 Elsevier Srl. All rights reserved.

  9. Scavenger receptors in human airway epithelial cells: role in response to double-stranded RNA.

    Directory of Open Access Journals (Sweden)

    Audrey Dieudonné

    Full Text Available Scavenger receptors and Toll-like receptors (TLRs cooperate in response to danger signals to adjust the host immune response. The TLR3 agonist double stranded (dsRNA is an efficient activator of innate signalling in bronchial epithelial cells. In this study, we aimed at defining the role played by scavenger receptors expressed by bronchial epithelial cells in the control of the innate response to dsRNA both in vitro and in vivo. Expression of several scavenger receptor involved in pathogen recognition was first evaluated in human bronchial epithelial cells in steady-state and inflammatory conditions. Their implication in the uptake of dsRNA and the subsequent cell activation was evaluated in vitro by competition with ligand of scavenger receptors including maleylated ovalbumin and by RNA silencing. The capacity of maleylated ovalbumin to modulate lung inflammation induced by dsRNA was also investigated in mice. Exposure to tumor necrosis factor-α increased expression of the scavenger receptors LOX-1 and CXCL16 and the capacity to internalize maleylated ovalbumin, whereas activation by TLR ligands did not. In contrast, the expression of SR-B1 was not modulated in these conditions. Interestingly, supplementation with maleylated ovalbumin limited dsRNA uptake and inhibited subsequent activation of bronchial epithelial cells. RNA silencing of LOX-1 and SR-B1 strongly blocked the dsRNA-induced cytokine production. Finally, administration of maleylated ovalbumin in mice inhibited the dsRNA-induced infiltration and activation of inflammatory cells in bronchoalveolar spaces and lung draining lymph nodes. Together, our data characterize the function of SR-B1 and LOX-1 in bronchial epithelial cells and their implication in dsRNA-induced responses, a finding that might be relevant during respiratory viral infections.

  10. Discovery and Development of Therapeutic Drugs against Lethal Human RNA Viruses: a Multidisciplinary Assault.

    Science.gov (United States)

    1991-07-16

    AD-A239 742 AD GRANT NO: DAMD17-89-Z-9021 TITLE: DISCOVERY AND DEVELOPMENT OF THERAPEUTIC DRUGS AGAINST LETHAL HUMAN RNA VIRUSES: A MULTIDISCIPLINARY...62787A871 AB WrJDA317987 11. TITLE (Include Securty Classification) DISCOVERY AND DEVELOPMENT OF THERAPEUTIC DRUGS AGAINST LETHAL HUMAN RNA VIRUSES: A...G. R. Pettit, III, D.-S. Huang, and G. R. Pettit, 23rd Int’l. Horticulture Congress, Italy, 8/27 - 9/1/90. "Bryostatins Define the Role of Protein

  11. Gene-specific correlation of RNA and protein levels in human cells and tissues

    DEFF Research Database (Denmark)

    Edfors, Fredrik; Danielsson, Frida; Hallström, Björn M.

    2016-01-01

    An important issue for molecular biology is to establish whether transcript levels of a given gene can be used as proxies for the corresponding protein levels. Here, we have developed a targeted proteomics approach for a set of human non-secreted proteins based on parallel reaction monitoring...... to measure, at steady-state conditions, absolute protein copy numbers across human tissues and cell lines and compared these levels with the corresponding mRNA levels using transcriptomics. The study shows that the transcript and protein levels do not correlate well unless a gene-specific RNA-to-protein (RTP...

  12. 13C spin relaxation measurements in RNA: Sensitivity and resolution improvement using spin-state selective correlation experiments

    International Nuclear Information System (INIS)

    Boisbouvier, Jerome; Brutscher, Bernhard; Simorre, Jean-Pierre; Marion, Dominique

    1999-01-01

    A set of new NMR pulse sequences has been designed for the measurement of 13 C relaxation rate constants in RNA and DNA bases: the spin-lattice relaxation rate constant R(C z ), the spin-spin relaxation rate constant R(C + ), and the CSA-dipolar cross-correlated relaxation rate constant Γ C,CH xy . The use of spin-state selective correlation techniques provides increased sensitivity and spectral resolution. Sensitivity optimised C-C filters are included in the pulse schemes for the suppression of signals originating from undesired carbon isotopomers. The experiments are applied to a 15% 13 C-labelled 33-mer RNA-theophylline complex. The measured R(C + )/Γ C,CH xy ratios indicate that 13 C CSA tensors do not vary significantly for the same type of carbon (C 2 , C 6 , C 8 ), but that they differ from one type to another. In addition, conformational exchange effects in the RNA bases are detected as a change in the relaxation decay of the narrow 13 C doublet component when varying the spacing of a CPMG pulse train. This new approach allows the detection of small exchange effects with a higher precision compared to conventional techniques

  13. mRNA transfection of mouse and human neural stem cell cultures.

    Directory of Open Access Journals (Sweden)

    Samuel McLenachan

    Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

  14. mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

    Science.gov (United States)

    McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J.; Chen, Fred K.

    2013-01-01

    The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. PMID:24386231

  15. mRNA transfection of mouse and human neural stem cell cultures.

    Science.gov (United States)

    McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J; Chen, Fred K

    2013-01-01

    The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

  16. RNA-Guided Activation of Pluripotency Genes in Human Fibroblasts

    DEFF Research Database (Denmark)

    Xiong, Kai; Zhou, Yan; Blichfeld, Kristian Aabo

    2017-01-01

    -associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts...... could be obtained from these SAM fibroblasts. In conclusion, our study showed that CRISPR/Cas9-based ATFs are potent to activate and maintain transcription of endogenous human pluripotent genes. However, future improvements of the system are still required to improve activation efficiency and cellular...

  17. The human nuclear poly(a-binding protein promotes RNA hyperadenylation and decay.

    Directory of Open Access Journals (Sweden)

    Stefan M Bresson

    Full Text Available Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A-binding protein (PABPN1, the poly(A polymerases (PAPs, PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. The sensitivity of human mesenchymal stem cells to ionizing radiation

    International Nuclear Information System (INIS)

    Chen, M.-F.; Lin, C.-T.; Chen, W.-C.; Yang, C.-T.; Chen, C.-C.; Liao, S.-K.; Liu, J.M.; Lu, C.-H.; Lee, K.-D.

    2006-01-01

    Purpose: Recent studies have shown that mesenchymal stem cells (MSCs) obtained from bone marrow transplantation patients originate from the host. This clinical observation suggests that MSCs in their niches could be resistant to irradiation. However, the biologic responses of bone marrow MSCs to irradiation have rarely been described in the literature. Methods and Materials: In this study, human bone marrow-derived, clonally expanded MSCs were used to investigate their sensitivity to irradiation in vitro, and the cellular mechanisms that may facilitate resistance to irradiation. The human lung cancer cell line A549 and the breast cancer cell line HCC1937 were used as controls for radiosensitivity; the former line has been shown to be radioresistant and the latter radiosensitive. We then examined their in vitro biologic changes and sensitivities to radiation therapy. Results: Our results suggest that MSCs are characterized as resistant to irradiation. Several cellular mechanisms were demonstrated that may facilitate resistance to irradiation: ATM protein phosphorylation, activation of cell-cycle checkpoints, double-strand break repair by homologous recombination and nonhomologous end joining (NHEJ), and the antioxidant capacity for scavenging reactive oxygen species. Conclusions: As demonstrated, MSCs possess a better antioxidant reactive oxygen species-scavenging capacity and active double-strand break repair to facilitate their radioresistance. These findings provide a better understanding of radiation-induced biologic responses in MSCs and may lead to the development of better strategies for stem cell treatment and cancer therapy

  20. How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing.

    Science.gov (United States)

    Koralewska, Natalia; Hoffmann, Weronika; Pokornowska, Maria; Milewski, Marek; Lipinska, Andrea; Bienkowska-Szewczyk, Krystyna; Figlerowicz, Marek; Kurzynska-Kokorniak, Anna

    2016-01-01

    Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

  1. Generation of human induced pluripotent stem cells using non-synthetic mRNA.

    Science.gov (United States)

    Rohani, L; Fabian, C; Holland, H; Naaldijk, Y; Dressel, R; Löffler-Wirth, H; Binder, H; Arnold, A; Stolzing, A

    2016-05-01

    Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  2. MicroRNA-211 Regulates Oxidative Phosphorylation and Energy Metabolism in Human Vitiligo.

    Science.gov (United States)

    Sahoo, Anupama; Lee, Bongyong; Boniface, Katia; Seneschal, Julien; Sahoo, Sanjaya K; Seki, Tatsuya; Wang, Chunyan; Das, Soumen; Han, Xianlin; Steppie, Michael; Seal, Sudipta; Taieb, Alain; Perera, Ranjan J

    2017-09-01

    Vitiligo is a common chronic skin disorder characterized by loss of epidermal melanocytes and progressive depigmentation. Vitiligo has complex immune, genetic, environmental, and biochemical causes, but the exact molecular mechanisms of vitiligo development and progression, particularly those related to metabolic control, are poorly understood. In this study we characterized the human vitiligo cell line PIG3V and the normal human melanocyte line HEM-l by RNA sequencing, targeted metabolomics, and shotgun lipidomics. Melanocyte-enriched microRNA-211, a known metabolic switch in nonpigmented melanoma cells, was severely down-regulated in vitiligo cell line PIG3V and skin biopsy samples from vitiligo patients, whereas its predicted targets PPARGC1A, RRM2, and TAOK1 were reciprocally up-regulated. microRNA-211 binds to PGC1-α 3' untranslated region locus and represses it. Although mitochondrial numbers were constant, mitochondrial complexes I, II, and IV and respiratory responses were defective in vitiligo cells. Nanoparticle-coated microRNA-211 partially augmented the oxygen consumption rate in PIG3V cells. The lower oxygen consumption rate, changes in lipid and metabolite profiles, and increased reactive oxygen species production observed in vitiligo cells appear to be partly due to abnormal regulation of microRNA-211 and its target genes. These genes represent potential biomarkers and therapeutic targets in human vitiligo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Cigarette smoking decreases global microRNA expression in human alveolar macrophages.

    Directory of Open Access Journals (Sweden)

    Joel W Graff

    Full Text Available Human alveolar macrophages are critical components of the innate immune system. Cigarette smoking-induced changes in alveolar macrophage gene expression are linked to reduced resistance to pulmonary infections and to the development of emphysema/COPD. We hypothesized that microRNAs (miRNAs could control, in part, the unique messenger RNA (mRNA expression profiles found in alveolar macrophages of cigarette smokers. Activation of macrophages with different stimuli in vitro leads to a diverse range of M1 (inflammatory and M2 (anti-inflammatory polarized phenotypes that are thought to mimic activated macrophages in distinct tissue environments. Microarray mRNA data indicated that smoking promoted an "inverse" M1 mRNA expression program, defined by decreased expression of M1-induced transcripts and increased expression of M1-repressed transcripts with few changes in M2-regulated transcripts. RT-PCR arrays identified altered expression of many miRNAs in alveolar macrophages of smokers and a decrease in global miRNA abundance. Stratification of human subjects suggested that the magnitude of the global decrease in miRNA abundance was associated with smoking history. We found that many of the miRNAs with reduced expression in alveolar macrophages of smokers were predicted to target mRNAs upregulated in alveolar macrophages of smokers. For example, miR-452 is predicted to target the transcript encoding MMP12, an important effector of smoking-related diseases. Experimental antagonism of miR-452 in differentiated monocytic cells resulted in increased expression of MMP12. The comprehensive mRNA and miRNA expression profiles described here provide insight into gene expression regulation that may underlie the adverse effects cigarette smoking has on alveolar macrophages.

  4. DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome.

    Science.gov (United States)

    Aktaş, Tuğçe; Avşar Ilık, İbrahim; Maticzka, Daniel; Bhardwaj, Vivek; Pessoa Rodrigues, Cecilia; Mittler, Gerhard; Manke, Thomas; Backofen, Rolf; Akhtar, Asifa

    2017-04-06

    Transposable elements are viewed as 'selfish genetic elements', yet they contribute to gene regulation and genome evolution in diverse ways. More than half of the human genome consists of transposable elements. Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome. Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability. Alu elements are the main targets of the RNA-editing enzyme ADAR and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC, but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant nuclear RNA helicase, binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post

  5. Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells Toward a Retinal Lineage.

    Science.gov (United States)

    Sridhar, Akshayalakshmi; Ohlemacher, Sarah K; Langer, Kirstin B; Meyer, Jason S

    2016-04-01

    The derivation of human induced pluripotent stem cells (hiPSCs) from patient-specific sources has allowed for the development of novel approaches to studies of human development and disease. However, traditional methods of generating hiPSCs involve the risks of genomic integration and potential constitutive expression of pluripotency factors and often exhibit low reprogramming efficiencies. The recent description of cellular reprogramming using synthetic mRNA molecules might eliminate these shortcomings; however, the ability of mRNA-reprogrammed hiPSCs to effectively give rise to retinal cell lineages has yet to be demonstrated. Thus, efforts were undertaken to test the ability and efficiency of mRNA-reprogrammed hiPSCs to yield retinal cell types in a directed, stepwise manner. hiPSCs were generated from human fibroblasts via mRNA reprogramming, with parallel cultures of isogenic human fibroblasts reprogrammed via retroviral delivery of reprogramming factors. New lines of mRNA-reprogrammed hiPSCs were established and were subsequently differentiated into a retinal fate using established protocols in a directed, stepwise fashion. The efficiency of retinal differentiation from these lines was compared with retroviral-derived cell lines at various stages of development. On differentiation, mRNA-reprogrammed hiPSCs were capable of robust differentiation to a retinal fate, including the derivation of photoreceptors and retinal ganglion cells, at efficiencies often equal to or greater than their retroviral-derived hiPSC counterparts. Thus, given that hiPSCs derived through mRNA-based reprogramming strategies offer numerous advantages owing to the lack of genomic integration or constitutive expression of pluripotency genes, such methods likely represent a promising new approach for retinal stem cell research, in particular, those for translational applications. In the current report, the ability to derive mRNA-reprogrammed human induced pluripotent stem cells (hi

  6. A SELEX-screened aptamer of human hepatitis B virus RNA encapsidation signal suppresses viral replication.

    Directory of Open Access Journals (Sweden)

    Hui Feng

    Full Text Available BACKGROUND: The specific interaction between hepatitis B virus (HBV polymerase (P protein and the ε RNA stem-loop on pregenomic (pg RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking ε in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a SELEX approach, based on a new in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP, to identify potential ε decoys in two large ε RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an ε decoy that competitively inhibits P protein binding to the authentic ε signal on pgRNA. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the first successful identification of human HBV ε aptamers by an in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ε decoy RNAs to interfere with viral P-ε complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B.

  7. Genomic binding profiles of functionally distinct RNA polymerase III transcription complexes in human cells.

    Science.gov (United States)

    Moqtaderi, Zarmik; Wang, Jie; Raha, Debasish; White, Robert J; Snyder, Michael; Weng, Zhiping; Struhl, Kevin

    2010-05-01

    Genome-wide occupancy profiles of five components of the RNA polymerase III (Pol III) machinery in human cells identified the expected tRNA and noncoding RNA targets and revealed many additional Pol III-associated loci, mostly near short interspersed elements (SINEs). Several genes are targets of an alternative transcription factor IIIB (TFIIIB) containing Brf2 instead of Brf1 and have extremely low levels of TFIIIC. Strikingly, expressed Pol III genes, unlike nonexpressed Pol III genes, are situated in regions with a pattern of histone modifications associated with functional Pol II promoters. TFIIIC alone associates with numerous ETC loci, via the B box or a novel motif. ETCs are often near CTCF binding sites, suggesting a potential role in chromosome organization. Our results suggest that human Pol III complexes associate preferentially with regions near functional Pol II promoters and that TFIIIC-mediated recruitment of TFIIIB is regulated in a locus-specific manner.

  8. Genotypes of Pestivirus RNA detected n anti influenza virus vaccines for human use

    Directory of Open Access Journals (Sweden)

    M. Giangaspero

    2004-02-01

    Full Text Available Nine polyvalent human influenza virus vaccines were tested by reverse transcriptase-polymerase chain reaction (RT-PCR for the presence of pestivirus RNA. Samples were selected from manufacturers in Europe and the USA. Three samples of the nine vaccines tested (33.3% gave positive results for pestivirus RNA. The 5´-untranslated genomic region sequence of the contaminant pestivirus RNA was analysed based on primary nucleotide sequence homology and on secondary sequence structures characteristic to genotypes. Two sequences belonged to Pestivirus type-1 (bovine viral diarrhoea virus [BVDV] species, genotypes BVDV-1b and BVDV-1e. These findings confirm previous reports, suggesting an improvement in preventive measures against contamination of biological products for human use.

  9. A path-based measurement for human miRNA functional similarities using miRNA-disease associations

    Science.gov (United States)

    Ding, Pingjian; Luo, Jiawei; Xiao, Qiu; Chen, Xiangtao

    2016-09-01

    Compared with the sequence and expression similarity, miRNA functional similarity is so important for biology researches and many applications such as miRNA clustering, miRNA function prediction, miRNA synergism identification and disease miRNA prioritization. However, the existing methods always utilized the predicted miRNA target which has high false positive and false negative to calculate the miRNA functional similarity. Meanwhile, it is difficult to achieve high reliability of miRNA functional similarity with miRNA-disease associations. Therefore, it is increasingly needed to improve the measurement of miRNA functional similarity. In this study, we develop a novel path-based calculation method of miRNA functional similarity based on miRNA-disease associations, called MFSP. Compared with other methods, our method obtains higher average functional similarity of intra-family and intra-cluster selected groups. Meanwhile, the lower average functional similarity of inter-family and inter-cluster miRNA pair is obtained. In addition, the smaller p-value is achieved, while applying Wilcoxon rank-sum test and Kruskal-Wallis test to different miRNA groups. The relationship between miRNA functional similarity and other information sources is exhibited. Furthermore, the constructed miRNA functional network based on MFSP is a scale-free and small-world network. Moreover, the higher AUC for miRNA-disease prediction indicates the ability of MFSP uncovering miRNA functional similarity.

  10. Insulin sensitivity is independent of lipid binding protein trafficking at the plasma membrane in human skeletal muscle

    DEFF Research Database (Denmark)

    Jordy, Andreas Børsting; Serup, Annette Karen; Karstoft, Kristian

    2014-01-01

    The aim of the present study was to investigate lipid-induced regulation of lipid binding proteins in human skeletal muscle and the impact hereof on insulin sensitivity. Eleven healthy male subjects underwent a 3-day hyper-caloric and high-fat diet regime. Muscle biopsies were taken before......-regulated by increased fatty acid availability. This suggests a time dependency in the up-regulation of FAT/CD36 and FABPpm protein during high availability of plasma fatty acids. Furthermore, we did not detect FATP1 and FATP4 protein in giant sarcolemmal vesicles obtained from human skeletal muscle. In conclusion......, this study shows that a short-term lipid-load increases mRNA content of key lipid handling proteins in human muscle. However, decreased insulin sensitivity after high-fat diet is not accompanied with relocation of FAT/CD36 or FABPpm protein to the sarcolemma. Finally, FATP1 and FATP4 protein could...

  11. Downregulation of eIF4G by microRNA-503 enhances drug sensitivity of MCF-7/ADR cells through suppressing the expression of ABC transport proteins.

    Science.gov (United States)

    Pan, Xia; Yang, Xiaoyan; Zang, Jinglei; Zhang, Si; Huang, Nan; Guan, Xinxin; Zhang, Jianhua; Wang, Zhihui; Li, Xi; Lei, Xiaoyong

    2017-06-01

    Overexpression of adenosine triphosphate-binding cassette (ABC) transport protein is emerging as a critical contributor to anticancer drug resistance. The eukaryotic translation initiation factor (eIF) 4F complex, the key modulator of mRNA translation, is regulated by the phosphoinositide 3-kinase-AKT-mammalian target of rapamycin pathway in anticancer drug-resistant tumors. The present study demonstrated the roles of ABC translation protein alterations in the acquisition of the Adriamycin (ADM)-resistant phenotype of MCF-7 human breast cells. Quantitative polymerase chain reaction and western blot analysis were applied to examine the differences in mRNA and protein levels, respectively. It was found that the expression of the ABC sub-family B member 1, ABC sub-family C member 1 and ABC sub-family G member 2 transport proteins were upregulated in MCF-7/ADR cells. An MTT assay was used to detect the cell viability, from the results MCF-7/ADR cells were less sensitive to ADM, tamoxifen (TAM) and taxol (TAX) treatment compared with MCF-7 cells. We predicted that the 3'-untranslated region of eukaryotic translation initiation factor 4-γ 1 (eIF4G) contains a potential miRNA binding site for microRNA (miR)-503 through using computational programs. These binding sites were confirmed by luciferase reporter assays. eIF4G mRNA degradation was accelerated in cells transfected with miR-503 mimics. Furthermore, it was demonstrated that eIF4G and ABC translation proteins were significantly downregulated in MCF-7/ADR cells after transfection with miR-503. It was found that miR-503 mimics could sensitize the cells to treatment with ADM, TAM and TAX. These findings demonstrated for the first time that eIF4G acted as a key factor in MCF-7/ADR cells, and may be an efficient agent for preventing and reversing multi-drug resistance in breast cancer.

  12. Elucidating Mechanisms of Molecular Recognition Between Human Argonaute and miRNA Using Computational Approaches

    KAUST Repository

    Jiang, Hanlun; Zhu, Lizhe; Hé liou, Amé lie; Gao, Xin; Bernauer, Julie; Huang, Xuhui

    2016-01-01

    that are geometrically accessible to miRNA. Using our recent work on human AGO2 as an example, we explain the rationale and the workflow of our method in details. This combined approach holds great promise to complement experiments in unraveling the mechanisms

  13. Generation of human induced pluripotent stem cells using non-synthetic mRNA

    Directory of Open Access Journals (Sweden)

    L. Rohani

    2016-05-01

    Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.

  14. New families of human regulatory RNA structures identified by comparative analysis of vertebrate genomes

    DEFF Research Database (Denmark)

    Parker, Brian John; Moltke, Ida; Roth, Adam

    2011-01-01

    a comparative method, EvoFam, for genome-wide identification of families of regulatory RNA structures, based on primary sequence and secondary structure similarity. We apply EvoFam to a 41-way genomic vertebrate alignment. Genome-wide, we identify 220 human, high-confidence families outside protein...

  15. On the biased nucleotide composition of the human coronavirus RNA genome

    NARCIS (Netherlands)

    Berkhout, Ben; van Hemert, Formijn

    2015-01-01

    We investigated the nucleotide composition of the RNA genome of the six human coronaviruses. Some general coronavirus characteristics were apparent (e.g. high U, low C count), but we also detected species-specific signatures. Most strikingly, the high U and low C proportions are quite variable and

  16. Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

    Directory of Open Access Journals (Sweden)

    Barredo Julio C

    2002-03-01

    Full Text Available Abstract Background We describe an alternative method to determine mRNA half-life (t1/2 based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp. To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h. Conclusions We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

  17. Visfatin mRNA expression in human subcutaneous adipose tissue is regulated by exercise

    DEFF Research Database (Denmark)

    Frydelund-Larsen, Lone; Åkerström, Thorbjörn; Nielsen, Søren

    2006-01-01

    in abdominal subcutaneous adipose tissue and skeletal muscle biopsies obtained from healthy young men at time points 0, 3, 4.5, 6, 9, and 24 h in relation to either 3 h of ergometer cycle exercise at 60% of Vo(2 max) or rest. Adipose tissue visfatin mRNA expression increased threefold at the time points 3, 4......Visfatin [pre-beta-cell colony-enhancing factor (PBEF)] is a novel adipokine that is produced by adipose tissue, skeletal muscle, and liver and has insulin-mimetic actions. Regular exercise enhances insulin sensitivity. In the present study, we therefore examined visfatin mRNA expression.......5, and 6 h in response to exercise (n = 8) compared with preexercise samples and compared with the resting control group (n = 7, P = 0.001). Visfatin mRNA expression in skeletal muscle was not influenced by exercise. The exercise-induced increase in adipose tissue visfatin was, however, not accompanied...

  18. Enhanced expression of IL-8 in normal human keratinocytes and human keratinocyte cell line HaCaT in vitro after stimulation with contact sensitizers, tolerogens and irritants.

    Science.gov (United States)

    Mohamadzadeh, M; Müller, M; Hultsch, T; Enk, A; Saloga, J; Knop, J

    1994-12-01

    To investigate the interleukin-8 production of keratinocytes after stimulation in vitro we have used various agents: (i) contact sensitizer (2,4-dinitrofluorobenzene, 3-n-pentadecylcatechol); (ii) tolerogen (5-methyl-3-n-pentadecylcatechol); (iii) irritant (sodium lauryl sulfate). Interleukin-8 gene expression was assessed by northern blot hybridization of the total cytoplasmic RNA extracted from subconfluent normal human keratinocyte cultures and the keratinocyte cell line HaCaT using a radiolabeled DNA probe specific for human interleukin-8. Interleukin-8 gene expression was markedly increased upon in vitro stimulation after 1-6 h with contact sensitizers, tolerogen and the irritant. In contrast, interleukin-8 production was not detectable in unstimulated normal human keratinocytes or the HaCaT keratinocyte cell line. These results suggest that the induction and production of interleukin-8 is a response to nonspecific stimuli and may play a critical role in the early response to immunogenic or inflammatory signals in man.

  19. Quality Improvement to Demonstrate the Lack of Reliability of the Human Papillomavirus mRNA Assay to Identify Women With Latent Human Papillomavirus Infections.

    Science.gov (United States)

    Cotton, Sarah; Brown, Robert E; Nugent, Elizabeth K; Robazetti, Sonia C; Berens, Pamela D; Smith, Judith A

    2018-04-01

    To assess the consistency between human papillomavirus (HPV) mRNA testing in women with a history of previous HPV infections diagnosed by HPV DNA assay and the potential effects on follow-up HPV screening. This was a quality improvement study that used data from a pathology laboratory software database reviewed from November 2014 to June 2016 to identify female patients aged 30 years or older with greater than one HPV-positive result, including one or more HPV mRNA assay results and one or more documented HPV DNA assay results for comparison. Previous correlative cytology and colposcopic histopathology were also documented. American College of Obstetricians and Gynecologists' cervical cancer screening guidelines were used to compare potential differences in follow-up recommendations. Four hundred twenty-five charts for female patients 30 years of age or older were identified with one or more prior high-risk HPV infections by DNA assay. There was a 69.3% difference in HPV mRNA results compared with previous HPV DNA-positive results. There was a potential change in follow-up for 71.7% of patients with one prior high-risk-HPV-positive result and 60.0% of patients with two or more prior high-risk HPV-positive results. There were 231 colposcopy reports evaluated in this study. Of these, 62 (26.8%) were abnormal colposcopy reports, including 45 low-grade squamous intraepithelial lesions, 15 high-grade squamous intraepithelial lesions, and two cancers. Twenty-five (40.3%) abnormal colposcopy findings were in patients with a history of at least than two prior HPV DNA-positive results and a report of currently being HPV-negative with the mRNA assay. The HPV mRNA assays are less sensitive for detection of latent HPV infections compared with HPV DNA assays. Based on these data and the potential change in follow-up care, the HPV mRNA assay should not be used for a primary screening tool for cervical cancer. Many pathology laboratories have shifted to using the HPV mRNA assay

  20. CRISPR/Cas9-mediated noncoding RNA editing in human cancers.

    Science.gov (United States)

    Yang, Jie; Meng, Xiaodan; Pan, Jinchang; Jiang, Nan; Zhou, Chengwei; Wu, Zhenhua; Gong, Zhaohui

    2018-01-02

    Cancer is characterized by multiple genetic and epigenetic alterations, including a higher prevalence of mutations of oncogenes and/or tumor suppressors. Mounting evidences have shown that noncoding RNAs (ncRNAs) are involved in the epigenetic regulation of cancer genes and their associated pathways. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) system, a revolutionary genome-editing technology, has shed light on ncRNA-based cancer therapy. Here, we briefly introduce the classifications and mechanisms of CRISPR/Cas9 system. Importantly, we mainly focused on the applications of CRISPR/Cas9 system as a molecular tool for ncRNA (microRNA, long noncoding RNA and circular RNA, etc.) editing in human cancers, and the novel techniques that are based on CRISPR/Cas9 system. Additionally, the off-target effects and the corresponding solutions as well as the challenges toward CRISPR/Cas9 were also evaluated and discussed. Long- and short-ncRNAs have been employed as targets in precision oncology, and CRISPR/Cas9-mediated ncRNA editing may provide an excellent way to cure cancer.

  1. Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription.

    Science.gov (United States)

    Hicks, Jessica A; Li, Liande; Matsui, Masayuki; Chu, Yongjun; Volkov, Oleg; Johnson, Krystal C; Corey, David R

    2017-08-15

    In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Formation of tRNA granules in the nucleus of heat-induced human cells

    International Nuclear Information System (INIS)

    Miyagawa, Ryu; Mizuno, Rie; Watanabe, Kazunori; Ijiri, Kenichi

    2012-01-01

    Highlights: ► tRNAs are tranlocated into the nucleus in heat-induced HeLa cells. ► tRNAs form the unique granules in the nucleus. ► tRNA ganules overlap with nuclear stress granules. -- Abstract: The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA Met (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA Met was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

  3. Phenylbutyrate Sensitizes Human Glioblastoma Cells Lacking Wild-Type P53 Function to Ionizing Radiation

    International Nuclear Information System (INIS)

    Lopez, Carlos A.; Feng, Felix Y.; Herman, Joseph M.; Nyati, Mukesh K.; Lawrence, Theodore S.; Ljungman, Mats

    2007-01-01

    Purpose: Histone deacetylase (HDAC) inhibitors induce growth arrest, differentiation, and apoptosis in cancer cells. Phenylbutyrate (PB) is a HDAC inhibitor used clinically for treatment of urea cycle disorders. Because of its low cytotoxicity, cerebrospinal fluid penetration, and high oral bioavailability, we investigated PB as a potential radiation sensitizer in human glioblastoma cell lines. Methods and Materials: Four glioblastoma cell lines were selected for this study. Phenylbutyrate was used at a concentration of 2 mM, which is achievable in humans. Western blots were used to assess levels of acetylated histone H3 in tumor cells after treatment with PB. Flow cytometry was used for cell cycle analysis. Clonogenic assays were performed to assess the effect of PB on radiation sensitivity. We used shRNA against p53 to study the role of p53 in radiosensitization. Results: Treatment with PB alone resulted in hyperacetylation of histones, confirmed by Western blot analysis. The PB alone resulted in cytostatic effects in three cell lines. There was no evidence of G 1 arrest, increase in sub-G 1 fraction or p21 protein induction. Clonogenic assays showed radiosensitization in two lines harboring p53 mutations, with enhancement ratios (± SE) of 1.5 (± 0.2) and 1.3 (± 0.1), respectively. There was no radiopotentiating effect in two cell lines with wild-type p53, but knockdown of wild-type p53 resulted in radiosensitization by PB. Conclusions: Phenylbutyrate can produce p21-independent cytostasis, and enhances radiation sensitivity in p53 mutant human glioblastoma cells in vitro. This suggests the potential application of combined PB and radiotherapy in glioblastoma harboring mutant p53

  4. Nuclear modifier MTO2 modulates the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Xiangyu He

    Full Text Available The phenotypic manifestations of mitochondrial DNA (mtDNA mutations are modulated by mitochondrial DNA haplotypes, nuclear modifier genes and environmental factors. The yeast mitochondrial 15S rRNA C1477G (P(R or P(R 454 mutation corresponds to the human 12S rRNA C1494T and A1555G mutations, which are well known as primary factors for aminoglycoside-induced nonsyndromic deafness. Here we report that the deletion of the nuclear modifier gene MTO2 suppressed the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae. First, the strain with a single mtDNA C1477G mutation exhibited hypersensitivity to neomycin. Functional assays indicated that the steady-state transcription level of mitochondrial DNA, the mitochondrial respiratory rate, and the membrane potential decreased significantly after neomycin treatment. The impaired mitochondria could not produce sufficient energy to maintain cell viability. Second, when the mto2 null and the mitochondrial C1477G mutations co-existed (mto2(P(R, the oxygen consumption rate in the double mutant decreased markedly compared to that of the control strains (MTO2(P(S, mto2(P(S and MTO2(P(R. The expression levels of the key glycolytic genes HXK2, PFK1 and PYK1 in the mto2(P(R strain were stimulated by neomycin and up-regulated by 89%, 112% and 55%, respectively. The enhanced glycolysis compensated for the respiratory energy deficits, and could be inhibited by the glycolytic enzyme inhibitor. Our findings in yeast will provide a new insight into the pathogenesis of human deafness.

  5. MicroRNA Expression Profiles in Cultured Human Fibroblasts in Space

    Science.gov (United States)

    Wu, Honglu; Lu, Tao; Jeevarajan, John; Rohde, Larry; Zhang, Ye

    2014-01-01

    Microgravity, or an altered gravity environment from the static 1g, has been shown to influence global gene expression patterns and protein levels in living organisms. However, it is unclear how these changes in gene and protein expressions are related to each other or are related to other factors regulating such changes. A different class of RNA, the small non-coding microRNA (miRNA), can have a broad effect on gene expression networks by mainly inhibiting the translation process. Previously, we investigated changes in the expression of miRNA and related genes under simulated microgravity conditions on the ground using the NASA invented bioreactor. In comparison to static 1 g, simulated microgravity altered a number of miRNAs in human lymphoblastoid cells. Pathway analysis with the altered miRNAs and RNA expressions revealed differential involvement of cell communication and catalytic activity, as well as immune response signaling and NGF activation of NF-kB pathways under simulated microgravity condition. The network analysis also identified several projected networks with c- Rel, ETS1 and Ubiquitin C as key factors. In a flight experiment on the International Space Station (ISS), we will investigate the effects of actual spaceflight on miRNA expressions in nondividing human fibroblast cells in mostly G1 phase of the cell cycle. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. In addition to miRNA expressions, we will investigate the effects of spaceflight on the cellular response to DNA damages from bleomycin treatment.

  6. Application of a Novel and Automated Branched DNA in Situ Hybridization Method for the Rapid and Sensitive Localization of mRNA Molecules in Plant Tissues

    Directory of Open Access Journals (Sweden)

    Andrew J. Bowling

    2014-04-01

    Full Text Available Premise of the study: A novel branched DNA detection technology, RNAscope in situ hybridization (ISH, originally developed for use on human clinical and animal tissues, was adapted for use in plant tissue in an attempt to overcome some of the limitations associated with traditional ISH assays. Methods and Results: Zea mays leaf tissue was formaldehyde fixed and paraffin embedded (FFPE and then probed with the RNAscope ISH assay for two endogenous genes, phosphoenolpyruvate carboxylase (PEPC and phosphoenolpyruvate carboxykinase (PEPCK. Results from both manual and automated methods showed tissue- and cell-specific mRNA localization patterns expected from these well-studied genes. Conclusions: RNAscope ISH is a sensitive method that generates high-quality, easily interpretable results from FFPE plant tissues. Automation of the RNAscope method on the Ventana Discovery Ultra platform allows significant advantages for repeatability, reduction in variability, and flexibility of workflow processes.

  7. MicroRNA-138 is a potential regulator of memory performance in humans

    Directory of Open Access Journals (Sweden)

    Julia eSchröder

    2014-07-01

    Full Text Available Genetic factors underlie a substantial proportion of individual differences in cognitive functions in humans, including processes related to episodic and working memory. While genetic association studies have proposed several candidate memory genes, these currently explain only a minor fraction of the phenotypic variance. Here, we performed genome-wide screening on 13 episodic and working memory phenotypes in 1,318 participants of the Berlin Aging Study II aged 60 years or older. The analyses highlight a number of novel single nucleotide polymorphisms (SNPs associated with memory performance, including one located in a putative regulatory region of microRNA (miRNA hsa-mir-138-5p (rs9882688, P-value = 7.8x10-9. Expression quantitative trait locus analyses on next-generation RNA-sequencing data revealed that rs9882688 genotypes show a significant correlation with the expression levels of this miRNA in 309 human lymphoblastoid cell lines (P-value = 5x10-4. In silico modeling of other top-ranking GWAS signals identified an additional memory-associated SNP in the 3' untranslated region (3'UTR of DCP1B, a gene encoding a core component of the mRNA decapping complex in humans, predicted to interfere with hsa-mir-138-5p binding. This prediction was confirmed in vitro by luciferase assays showing differential binding of hsa-mir-138-5p to 3'UTR reporter constructs in two human cell lines (HEK293: P-value = 0.0470; SH-SY5Y: P-value = 0.0866. Finally, expression profiling of hsa-mir-138-5p and DCP1B mRNA in human post-mortem brain tissue revealed that both molecules are expressed simultaneously in frontal cortex and hippocampus, suggesting that the proposed interaction between hsa-mir-138-5p and DCP1B may also take place in vivo. In summary, by combining unbiased genome-wide screening with extensive in silico modeling, in vitro functional assays, and gene expression profiling, our study identified miRNA-138 as a potential molecular regulator of human memory

  8. MicroRNA-330-5p as a Putative Modulator of Neoadjuvant Chemoradiotherapy Sensitivity in Oesophageal Adenocarcinoma.

    LENUS (Irish Health Repository)

    Bibby, Becky A S

    2015-01-01

    Oesophageal adenocarcinoma (OAC) is the sixth most common cause of cancer deaths worldwide, and the 5-year survival rate for patients diagnosed with the disease is approximately 17%. The standard of care for locally advanced disease is neoadjuvant chemotherapy or, more commonly, combined neoadjuvant chemoradiation therapy (neo-CRT) prior to surgery. Unfortunately, ~60-70% of patients will fail to respond to neo-CRT. Therefore, the identification of biomarkers indicative of patient response to treatment has significant clinical implications in the stratification of patient treatment. Furthermore, understanding the molecular mechanisms underpinning tumour response and resistance to neo-CRT will contribute towards the identification of novel therapeutic targets for enhancing OAC sensitivity to CRT. MicroRNAs (miRNA\\/miR) function to regulate gene and protein expression and play a causal role in cancer development and progression. MiRNAs have also been identified as modulators of key cellular pathways associated with resistance to CRT. Here, to identify miRNAs associated with resistance to CRT, pre-treatment diagnostic biopsy specimens from patients with OAC were analysed using miRNA-profiling arrays. In pre-treatment biopsies miR-330-5p was the most downregulated miRNA in patients who subsequently failed to respond to neo-CRT. The role of miR-330 as a potential modulator of tumour response and sensitivity to CRT in OAC was further investigated in vitro. Through vector-based overexpression the E2F1\\/p-AKT survival pathway, as previously described, was confirmed as a target of miR-330 regulation. However, miR-330-mediated alterations to the E2F1\\/p-AKT pathway were insufficient to significantly alter cellular sensitivity to chemotherapy (cisplatin and 5-flurouracil). In contrast, silencing of miR-330-5p enhanced, albeit subtly, cellular resistance to clinically relevant doses of radiation. This study highlights the need for further investigation into the potential of

  9. microRNA-320/RUNX2 axis regulates adipocytic differentiation of human mesenchymal (skeletal) stem cells

    DEFF Research Database (Denmark)

    Hamam, D; Ali, D; Vishnubalaji, R

    2014-01-01

    The molecular mechanisms promoting lineage-specific commitment of human mesenchymal (skeletal or stromal) stem cells (hMSCs) into adipocytes (ADs) are not fully understood. Thus, we performed global microRNA (miRNA) and gene expression profiling during adipocytic differentiation of h...... differentiation and accelerated formation of mature ADs in ex vivo cultures. Integrated analysis of bioinformatics and global gene expression profiling in miR-320c overexpressing cells and during adipocytic differentiation of hMSC identified several biologically relevant gene targets for miR-320c including RUNX2...

  10. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    International Nuclear Information System (INIS)

    Rumi, Mohammad; Ishihara, Shunji; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-01

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor α-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use

  11. A Multifunctional Envelope-Type Nano Device Containing a pH-Sensitive Cationic Lipid for Efficient Delivery of Short Interfering RNA to Hepatocytes In Vivo.

    Science.gov (United States)

    Sato, Yusuke; Harashima, Hideyoshi; Kohara, Michinori

    2016-01-01

    Various types of nanoparticles have been developed with the intent of efficiently delivering short interfering RNA (siRNA) to hepatocytes to date. To achieve efficient SiRNA delivery, various aspects of the delivery processes and physical properties need to be considered. We recently developed an original lipid nanoparticle, a multifunctional envelope-type nano device (MEND) containing YSK05, a pH-sensitive cationic lipid (YSK05-MEND). The YSK05-MEND with SiRNA in its formulation showed hepatocyte-specific uptake and robust gene silencing in hepatocytes after intravenous administration. Here, we describe the procedure used in the preparation and characterization method of the YSK05-MEND.

  12. Downregulation of telomerase activity in human promyelocytic cell line using RNA interference.

    Science.gov (United States)

    Miri-Moghaddam, E; Deezagi, A; Soheili, Z S

    2009-12-01

    Telomerase is a ribonucleoprotein complex. It consists of two main components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA. High telomerase activity is present in most malignant cells, but it is barely detectable in majority of somatic cells. The direct correlation between telomerase reactivation and carcinogens has made hTERT a key target for anticancer therapeutic studies. In this study, for the first time, we evaluated the ability of the new generation of short interfering RNA (siRNA) to regulate telomerase activity in the human promyelocytic leukemia cell line (HL-60). Transient transfection cell line by hTERT siRNAs resulted in statistically significant suppression of hTERT messenger RNAs which were detected by quantitative real-time polymerase chain reaction, while the expressed hTERT protein levels were measured by flow cytometry. The results of telomeric repeat amplification protocol showed that telomerase activity was significantly reduced upon transfection of the HL-60 cell line with hTERT siRNAs. The results of this study showed that telomerase activity and cell proliferation were efficiently inhibited in the hTERT siRNA-treated leukemic cell line.

  13. Intergenic and repeat transcription in human, chimpanzee and macaque brains measured by RNA-Seq.

    Directory of Open Access Journals (Sweden)

    Augix Guohua Xu

    Full Text Available Transcription is the first step connecting genetic information with an organism's phenotype. While expression of annotated genes in the human brain has been characterized extensively, our knowledge about the scope and the conservation of transcripts located outside of the known genes' boundaries is limited. Here, we use high-throughput transcriptome sequencing (RNA-Seq to characterize the total non-ribosomal transcriptome of human, chimpanzee, and rhesus macaque brain. In all species, only 20-28% of non-ribosomal transcripts correspond to annotated exons and 20-23% to introns. By contrast, transcripts originating within intronic and intergenic repetitive sequences constitute 40-48% of the total brain transcriptome. Notably, some repeat families show elevated transcription. In non-repetitive intergenic regions, we identify and characterize 1,093 distinct regions highly expressed in the human brain. These regions are conserved at the RNA expression level across primates studied and at the DNA sequence level across mammals. A large proportion of these transcripts (20% represents 3'UTR extensions of known genes and may play roles in alternative microRNA-directed regulation. Finally, we show that while transcriptome divergence between species increases with evolutionary time, intergenic transcripts show more expression differences among species and exons show less. Our results show that many yet uncharacterized evolutionary conserved transcripts exist in the human brain. Some of these transcripts may play roles in transcriptional regulation and contribute to evolution of human-specific phenotypic traits.

  14. In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

    DEFF Research Database (Denmark)

    Nielsen, M. E.; Rasmussen, I. A.; Kristensen, S. G.

    2011-01-01

    significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the follicular......Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24...... and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated...

  15. Discrimination of skin sensitizers from non-sensitizers by interleukin-1α and interleukin-6 production on cultured human keratinocytes.

    Science.gov (United States)

    Jung, Daun; Che, Jeong-Hwan; Lim, Kyung-Min; Chun, Young-Jin; Heo, Yong; Seok, Seung Hyeok

    2016-09-01

    In vitro testing methods for classifying sensitizers could be valuable alternatives to in vivo sensitization testing using animal models, such as the murine local lymph node assay (LLNA) and the guinea pig maximization test (GMT), but there remains a need for in vitro methods that are more accurate and simpler to distinguish skin sensitizers from non-sensitizers. Thus, the aim of our study was to establish an in vitro assay as a screening tool for detecting skin sensitizers using the human keratinocyte cell line, HaCaT. HaCaT cells were exposed to 16 relevant skin sensitizers and 6 skin non-sensitizers. The highest dose used was the dose causing 75% cell viability (CV75) that we determined by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The levels of extracellular production of interleukin-1α (IL-1α) and IL-6 were measured. The sensitivity of IL-1α was 63%, specificity was 83% and accuracy was 68%. In the case of IL-6, sensitivity: 69%, specificity: 83% and accuracy: 73%. Thus, this study suggests that measuring extracellular production of pro-inflammatory cytokines IL-1α and IL-6 by human HaCaT cells may potentially classify skin sensitizers from non-sensitizers. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  16. A-to-I RNA editing: the "ADAR" side of human cancer.

    Science.gov (United States)

    Galeano, Federica; Tomaselli, Sara; Locatelli, Franco; Gallo, Angela

    2012-05-01

    Carcinogenesis is a complex, multi-stage process depending on both endogenous and exogenous factors. In the past years, DNA mutations provided important clues to the comprehension of the molecular pathways involved in numerous cancers. Recently, post-transcriptional modification events, such as RNA editing, are emerging as new players in several human diseases, including tumours. A-to-I RNA editing changes the nucleotide sequence of target RNAs, introducing A-to-I/G "mutations". Since ADAR enzymes catalyse this nucleotide conversion, their expression/activity is essential and finely regulated in normal cells. This review summarizes the available knowledge on A-to-I RNA editing in the cancer field, giving a new view on how ADARs may play a role in carcinogenesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral

    Directory of Open Access Journals (Sweden)

    Claire M. Smith

    2016-08-01

    Full Text Available Defective interfering (DI viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8 was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1; it comprises 395 nucleotides and is packaged in the DI virion in place of a full-length genome segment 1. Given intranasally, 244/PR8 DI virus protects mice and ferrets from clinical influenza caused by a number of different influenza A subtypes and interferes with production of infectious influenza A virus in cells in culture. However, evidence that DI influenza viruses are active in cells of the human respiratory tract is lacking. Here we show that 244/PR8 DI RNA is replicated by an influenza A challenge virus in human lung diploid fibroblasts, bronchial epithelial cells, and primary nasal basal cells, and that the yield of challenge virus is significantly reduced in a dose-dependent manner indicating that DI influenza virus has potential as a human antiviral.

  18. Understanding alcoholism through microRNA signatures in brains of human alcoholics

    Directory of Open Access Journals (Sweden)

    R. Dayne eMayfield

    2012-04-01

    Full Text Available Advances in the fields of genomics and genetics in the last decade have identified a large number of genes that can potentially influence alcohol-drinking behavior in humans as well as animal models. Consequently, the task of identifying efficient molecular targets that could be used to develop effective therapeutics against the disease has become increasingly daunting. One of the reasons for this is the fact that each of the many alcohol-responsive genes only contributes a small effect to the overall mechanism and disease phenotype, as is characteristic of complex traits. Current research trends are hence shifting towards the analysis of gene networks rather than emphasizing individual genes. The discovery of microRNAs and their mechanisms of action on regulation of transcript level and protein translation have made evident the utility of these small non-coding RNA molecules that act as central coordinators of multiple cross-communicating cellular pathways. Cells exploit the fact that a single microRNA can target hundreds of mRNA transcripts and that a single mRNA transcript can be simultaneously targeted by distinct microRNAs, to ensure fine-tuned and/or redundant control over a large number of cellular functions. By the same token, we can use these properties of microRNAs to develop novel, targeted strategies to combat complex disorders. In this review, we will focus on recent discoveries of microRNA signatures in brain of human alcoholics supporting the hypothesis that changes in gene expression and regulation by microRNAs are responsible for long-term neuroadaptations occurring during development of alcoholism. We also discuss insights into the potential modulation of epigenetic regulators by a subset of microRNAs. Taken together, microRNA activity may be controlling many of the cellular mechanisms already known to be involved in the development of alcoholism, and suggests potential targets for the development of novel therapeutic

  19. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K, E-mail: Jamboor.vishwanatha@unthsc.edu [Department of Molecular Biology and Immunology and Institute for Cancer Research, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-11-04

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high ({approx}97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  20. ADAR RNA editing in human disease; more to it than meets the I.

    Science.gov (United States)

    Gallo, Angela; Vukic, Dragana; Michalík, David; O'Connell, Mary A; Keegan, Liam P

    2017-09-01

    We review the structures and functions of ADARs and their involvements in human diseases. ADAR1 is widely expressed, particularly in the myeloid component of the blood system, and plays a prominent role in promiscuous editing of long dsRNA. Missense mutations that change ADAR1 residues and reduce RNA editing activity cause Aicardi-Goutières Syndrome, a childhood encephalitis and interferonopathy that mimics viral infection and resembles an extreme form of Systemic Lupus Erythmatosus (SLE). In Adar1 mouse mutant models aberrant interferon expression is prevented by eliminating interferon activation signaling from cytoplasmic dsRNA sensors, indicating that unedited cytoplasmic dsRNA drives the immune induction. On the other hand, upregulation of ADAR1 with widespread promiscuous RNA editing is a prominent feature of many cancers and particular site-specific RNA editing events are also affected. ADAR2 is most highly expressed in brain and is primarily required for site-specific editing of CNS transcripts; recent findings indicate that ADAR2 editing is regulated by neuronal excitation for synaptic scaling of glutamate receptors. ADAR2 is also linked to the circadian clock and to sleep. Mutations in ADAR2 could contribute to excitability syndromes such as epilepsy, to seizures, to diseases involving neuronal plasticity defects, such as autism and Fragile-X Syndrome, to neurodegenerations such as ALS, or to astrocytomas or glioblastomas in which reduced ADAR2 activity is required for oncogenic cell behavior. The range of human disease associated with ADAR1 mutations may extend further to include other inflammatory conditions while ADAR2 mutations may affect psychiatric conditions.

  1. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    Science.gov (United States)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.

    2011-11-01

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  2. Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

    International Nuclear Information System (INIS)

    Mukerjee, Anindita; Shankardas, Jwalitha; Ranjan, Amalendu P; Vishwanatha, Jamboor K

    2011-01-01

    Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (∼97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

  3. New families of human regulatory RNA structures identified by comparative analysis of vertebrate genomes.

    Science.gov (United States)

    Parker, Brian J; Moltke, Ida; Roth, Adam; Washietl, Stefan; Wen, Jiayu; Kellis, Manolis; Breaker, Ronald; Pedersen, Jakob Skou

    2011-11-01

    Regulatory RNA structures are often members of families with multiple paralogous instances across the genome. Family members share functional and structural properties, which allow them to be studied as a whole, facilitating both bioinformatic and experimental characterization. We have developed a comparative method, EvoFam, for genome-wide identification of families of regulatory RNA structures, based on primary sequence and secondary structure similarity. We apply EvoFam to a 41-way genomic vertebrate alignment. Genome-wide, we identify 220 human, high-confidence families outside protein-coding regions comprising 725 individual structures, including 48 families with known structural RNA elements. Known families identified include both noncoding RNAs, e.g., miRNAs and the recently identified MALAT1/MEN β lincRNA family; and cis-regulatory structures, e.g., iron-responsive elements. We also identify tens of new families supported by strong evolutionary evidence and other statistical evidence, such as GO term enrichments. For some of these, detailed analysis has led to the formulation of specific functional hypotheses. Examples include two hypothesized auto-regulatory feedback mechanisms: one involving six long hairpins in the 3'-UTR of MAT2A, a key metabolic gene that produces the primary human methyl donor S-adenosylmethionine; the other involving a tRNA-like structure in the intron of the tRNA maturation gene POP1. We experimentally validate the predicted MAT2A structures. Finally, we identify potential new regulatory networks, including large families of short hairpins enriched in immunity-related genes, e.g., TNF, FOS, and CTLA4, which include known transcript destabilizing elements. Our findings exemplify the diversity of post-transcriptional regulation and provide a resource for further characterization of new regulatory mechanisms and families of noncoding RNAs.

  4. Demonstration of Hepatitis C Virus RNA with In Situ Hybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver

    Directory of Open Access Journals (Sweden)

    Kazuya Shiogama

    2013-01-01

    Full Text Available Background. In situ hybridization (ISH with high sensitivity has been requested to demonstrate hepatitis C virus (HCV RNA in formalin-fixed, paraffin-embedded (FFPE sections of the liver. Methods. ISH employing a locked-nucleic-acid- (LNA-modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected and of needle-biopsied livers from HCV-infected patients. Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82% of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73% of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions. HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma. Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.

  5. Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung.

    Science.gov (United States)

    Tagalakis, Aristides D; Munye, Mustafa M; Ivanova, Rositsa; Chen, Hanpeng; Smith, Claire M; Aldossary, Ahmad M; Rosa, Luca Z; Moulding, Dale; Barnes, Josephine L; Kafetzis, Konstantinos N; Jones, Stuart A; Baines, Deborah L; Moss, Guy W J; O'Callaghan, Christopher; McAnulty, Robin J; Hart, Stephen L

    2018-05-10

    Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air-liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (V t ), short circuit current (I sc ), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced V t , the amiloride-sensitive I sc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  6. Mechanistic study on the nuclear modifier gene MSS1 mutation suppressing neomycin sensitivity of the mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhou, Qiyin; Wang, Wei; He, Xiangyu; Zhu, Xiaoyu; Shen, Yaoyao; Yu, Zhe; Wang, Xuexiang; Qi, Xuchen; Zhang, Xuan; Fan, Mingjie; Dai, Yu; Yang, Shuxu; Yan, Qingfeng

    2014-01-01

    The phenotypic manifestation of mitochondrial DNA (mtDNA) mutations can be modulated by nuclear genes and environmental factors. However, neither the interaction among these factors nor their underlying mechanisms are well understood. The yeast Saccharomyces cerevisiae mtDNA 15S rRNA C1477G mutation (PR) corresponds to the human 12S rRNA A1555G mutation. Here we report that a nuclear modifier gene mss1 mutation suppresses the neomycin-sensitivity phenotype of a yeast C1477G mutant in fermentable YPD medium. Functional assays show that the mitochondrial function of the yeast C1477G mutant was impaired severely in YPD medium with neomycin. Moreover, the mss1 mutation led to a significant increase in the steady-state level of HAP5 (heme activated protein), which greatly up-regulated the expression of glycolytic transcription factors RAP1, GCR1, and GCR2 and thus stimulated glycolysis. Furthermore, the high expression of the key glycolytic enzyme genes HXK2, PFK1 and PYK1 indicated that enhanced glycolysis not only compensated for the ATP reduction from oxidative phosphorylation (OXPHOS) in mitochondria, but also ensured the growth of the mss1(PR) mutant in YPD medium with neomycin. This study advances our understanding of the phenotypic manifestation of mtDNA mutations.

  7. Random small interfering RNA library screen identifies siRNAs that induce human erythroleukemia cell differentiation.

    Science.gov (United States)

    Fan, Cuiqing; Xiong, Yuan; Zhu, Ning; Lu, Yabin; Zhang, Jiewen; Wang, Song; Liang, Zicai; Shen, Yan; Chen, Meihong

    2011-03-01

    Cancers are characterized by poor differentiation. Differentiation therapy is a strategy to alleviate malignant phenotypes by inducing cancer cell differentiation. Here we carried out a combinatorial high-throughput screen with a random siRNA library on human erythroleukemia K-562 cell differentiation. Two siRNAs screened from the library were validated to be able to induce erythroid differentiation to varying degrees, determined by CD235 and globin up-regulation, GATA-2 down-regulation, and cell growth inhibition. The screen we performed here is the first trial of screening cancer differentiation-inducing agents from a random siRNA library, demonstrating that a random siRNA library can be considered as a new resource in efforts to seek new therapeutic agents for cancers. As a random siRNA library has a broad coverage for the entire genome, including known/unknown genes and protein coding/non-coding sequences, screening using a random siRNA library can be expected to greatly augment the repertoire of therapeutic siRNAs for cancers.

  8. BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells.

    Science.gov (United States)

    Awe, Jason P; Crespo, Agustin Vega; Li, You; Kiledjian, Megerditch; Byrne, James A

    2013-02-06

    The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics.

  9. MicroRNA Signature of Human Microvascular Endothelium Infected with Rickettsia rickettsii

    Directory of Open Access Journals (Sweden)

    Abha Sahni

    2017-07-01

    Full Text Available MicroRNAs (miRNAs mediate gene silencing by destabilization and/or translational repression of target mRNA. Infection of human microvascular endothelial cells as primary targets of Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, triggers host responses appertaining to alterations in cellular gene expression. Microarray-based profiling of endothelial cells infected with R. rickettsii for 3 or 24 h revealed differential expression of 33 miRNAs, of which miRNAs129-5p, 200a-3p, 297, 200b-3p, and 595 were identified as the top five up-regulated miRNAs (5 to 20-fold, p ≤ 0.01 and miRNAs 301b-3p, 548a-3p, and 377-3p were down-regulated (2 to 3-fold, p ≤ 0.01. Changes in the expression of selected miRNAs were confirmed by q-RT-PCR in both in vitro and in vivo models of infection. As potential targets, expression of genes encoding NOTCH1, SMAD2, SMAD3, RIN2, SOD1, and SOD2 was either positively or negatively regulated. Using a miRNA-specific mimic or inhibitor, NOTCH1 was determined to be a target of miRNA 200a-3p in R. rickettsii-infected human dermal microvascular endothelial cells (HMECs. Predictive interactome mapping suggested the potential for miRNA-mediated modulation of regulatory gene networks underlying important host cell signaling pathways. This first demonstration of altered endothelial miRNA expression provides new insights into regulatory elements governing mechanisms of host responses and pathogenesis during human rickettsial infections.

  10. UVA and UVB irradiation differentially regulate microRNA expression in human primary keratinocytes.

    Directory of Open Access Journals (Sweden)

    Anne Kraemer

    Full Text Available MicroRNA (miRNA-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2, which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis.

  11. UVA and UVB Irradiation Differentially Regulate microRNA Expression in Human Primary Keratinocytes

    Science.gov (United States)

    Kraemer, Anne; Chen, I-Peng; Henning, Stefan; Faust, Alexandra; Volkmer, Beate; Atkinson, Michael J.; Moertl, Simone; Greinert, Ruediger

    2013-01-01

    MicroRNA (miRNA)-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2), which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis. PMID:24391759

  12. MicroRNA-224 promotes the sensitivity of osteosarcoma cells to cisplatin by targeting Rac1.

    Science.gov (United States)

    Geng, Shuo; Gu, Lina; Ju, Fang; Zhang, Hepeng; Wang, Yiwen; Tang, Han; Bi, ZhengGang; Yang, Chenglin

    2016-09-01

    Osteosarcoma is the most common primary bone tumour in children and adolescents. Accumulating evidence has shown that microRNAs (miRNAs) participate in the development of almost all types of cancer. Here, we investigated the role of miR-224 in the development and progression of osteosarcoma. We demonstrated that miR-224 was down-regulated in osteosarcoma cell lines and tissues. Lower miR-224 levels were correlated with shorter survivalin osteosarcoma patients. Furthermore, overexpression of miR-224 suppressed osteosarcoma cell proliferation, migration and invasion and contributed to the increased sensitivity of MG-63 cells to cisplatin. We identified Rac1 as a direct target gene of miR-224 in osteosarcoma. Rac1 expression was up-regulated in the osteosarcoma cell lines and tissues, and there was an inverse correlation between Rac1 and miR-224 expression in osteosarcoma tissues. Furthermore, rescuing Rac1 expression decreased the sensitivity of miR-224-overexpressing MG-63 cells to cisplatin. We also demonstrated that ectopic expression of Rac1 promoted the proliferation, migration and invasion of miR-224-overexpressing MG-63 cells. These data suggest that miR-224 plays a tumour suppressor role in the development of osteosarcoma and is related to the sensitivity of osteosarcoma to cisplatin. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  13. MicroRNA-451 sensitizes lung cancer cells to cisplatin through regulation of Mcl-1.

    Science.gov (United States)

    Cheng, Dezhi; Xu, Yi; Sun, Changzheng; He, Zhifeng

    2016-12-01

    As one of the most widely used chemotherapy drugs for lung cancer, chemoresistance of cisplatin (DPP) is one of the major hindrances in treatment of this malignancy. The microRNAs (miRNAs) have been identified to mediate chemotherapy drug resistance. MiR-451 as a tumor suppressor has been evaluated its potential effect on the sensitivity of cancer cells to DDP. However, the role of miR-451 in regulatory mechanism of chemosensitivity in lung cancer cells is still largely unknown. In this study, we first constructed a cisplatin-resistant A549 cell line (A549/DPP) accompanied with a decreased expression of miR-451 and an increased expression of Mcl-1in the drug resistant cells compared with the parental cells. Exogenous expression of miR-451 level in A549/DPP was found to sensitize their reaction to the treatment of cisplatin, which coincides with reduced expression of Mcl-1. Interestingly, Mcl-1 knockdown in A549/DPP cells increased the chemosensitivity to DPP, suggesting the dependence of Mcl-1 regulation in miR-451 activity. Moreover, miR-451 can restore cisplatin treatment response in cisplatin-resistant xenografts in vivo, while Mcl-1 protein levels were decreased. Thus, these findings provided that in lung cancer cells, tumor suppressor miR-451 enhanced DPP sensitivity via regulation of Mcl-1 expression, which could be served as a novel therapeutic target for the treatment of chemotherapy resistant in lung cancer.

  14. Knockdown of Ki-67 by dicer-substrate small interfering RNA sensitizes bladder cancer cells to curcumin-induced tumor inhibition.

    Directory of Open Access Journals (Sweden)

    Sivakamasundari Pichu

    Full Text Available Transitional cell carcinoma (TCC of the urinary bladder is the most common cancer of the urinary tract. Most of the TCC cases are of the superficial type and are treated with transurethral resection (TUR. However, the recurrence rate is high and the current treatments have the drawback of inducing strong systemic toxicity or cause painful cystitis. Therefore, it would be of therapeutic value to develop novel concepts and identify novel drugs for the treatment of bladder cancer. Ki-67 is a large nucleolar phosphoprotein whose expression is tightly linked to cell proliferation, and curcumin, a phytochemical derived from the rhizome Curcuma longa, has been shown to possess powerful anticancer properties. In this study, we evaluated the combined efficacy of curcumin and a siRNA against Ki-67 mRNA (Ki-67-7 in rat (AY-27 and human (T-24 bladder cancer cells. The anticancer effects were assessed by the determination of cell viability, apoptosis and cell cycle analysis. Ki-67-7 (10 nM and curcumin (10 µM, when treated independently, were moderately effective. However, in their combined presence, proliferation of bladder cancer cells was profoundly (>85% inhibited; the rate of apoptosis in the combined presence of curcumin and Ki-67-7 (36% was greater than that due to Ki-67-7 (14% or curcumin (13% alone. A similar synergy between curcumin and Ki-67-7 in inducing cell cycle arrest was also observed. Western blot analysis suggested that pretreatment with Ki-67-7 sensitized bladder cancer cells to curcumin-mediated apoptosis and cell cycle arrest by p53- and p21-independent mechanisms. These data suggest that a combination of anti-Ki-67 siRNA and curcumin could be a viable treatment against the proliferation of bladder cancer cells.

  15. Widespread molecular patterns associated with drug sensitivity in breast cancer cell lines, with implications for human tumors.

    Directory of Open Access Journals (Sweden)

    Chad J Creighton

    Full Text Available BACKGROUND: Recent landmark studies have profiled cancer cell lines for molecular features, along with measuring the corresponding growth inhibitory effects for specific drug compounds. These data present a tool for determining which subsets of human cancer might be more responsive to particular drugs. To this end, the NCI-DREAM-sponsored DREAM7: Drug Sensitivity Prediction Challenge (sub-challenge 1 set out to predict the sensitivities of 18 breast cancer cell lines to 31 previously untested compounds, on the basis of molecular profiling data and a training subset of cell lines. METHODS AND RESULTS: With 47 teams submitting blinded predictions, team Creighton scored third in terms of overall accuracy. Team Creighton's method was simple and straightforward, incorporated multiple expression data types (RNA-seq, gene array, RPPA, and incorporated all profiled features (not only the "best" predictive ones. As an extension of the approach, cell line data, from public datasets of expression profiling coupled with drug sensitivities (Barretina, Garnett, Heiser were used to "predict" the drug sensitivities in human breast tumors (using data from The Cancer Genome Atlas. Drug sensitivity correlations within human breast tumors showed differences by expression-based subtype, with many associations in line with the expected (e.g. Lapatinib sensitivity in HER2-enriched cancers and others inviting further study (e.g. relative resistance to PI3K inhibitors in basal-like cancers. CONCLUSIONS: Molecular patterns associated with drug sensitivity are widespread, with potentially hundreds of genes that could be incorporated into making predictions, as well as offering biological clues as to the mechanisms involved. Applying the cell line patterns to human tumor data may help generate hypotheses on what tumor subsets might be more responsive to therapies, where multiple cell line datasets representing various drugs may be used, in order to assess consistency of

  16. MicroRNA signature of cis-platin resistant vs. cis-platin sensitive ovarian cancer cell lines

    Directory of Open Access Journals (Sweden)

    Kumar Smriti

    2011-09-01

    Full Text Available Abstract Background Ovarian cancer is the leading cause of death from gynecologic cancer in women worldwide. According to the National Cancer Institute, ovarian cancer has the highest mortality rate among all the reproductive cancers in women. Advanced stage diagnosis and chemo/radio-resistance is a major obstacle in treating advanced ovarian cancer. The most commonly employed chemotherapeutic drug for ovarian cancer treatment is cis-platin. As with most chemotherapeutic drugs, many patients eventually become resistant to cis-platin and therefore, diminishing its effect. The efficacy of current treatments may be improved by increasing the sensitivity of cancer cells to chemo/radiation therapies. Methods The present study is focused on identifying the differential expression of regulatory microRNAs (miRNAs between cis-platin sensitive (A2780, and cis-platin resistant (A2780/CP70 cell lines. Cell proliferation assays were conducted to test the sensitivity of the two cell lines to cis-platin. Differential expression patterns of miRNA between cis-platin sensitive and cis-platin resistant cell lines were analyzed using novel LNA technology. Results Our results revealed changes in expression of 11 miRNAs out of 1,500 miRNAs analyzed. Out of the 11 miRNAs identified, 5 were up-regulated in the A2780/CP70 cell line and 6 were down regulated as compared to cis-platin sensitive A2780 cells. Our microRNA data was further validated by quantitative real-time PCR for these selected miRNAs. Ingenuity Pathway Analysis (IPA and Kyoto Encyclopedia of Genes and Genomes (KEGG analysis was performed for the selected miRNAs and their putative targets to identify the potential pathways and networks involved in cis-platin resistance. Conclusions Our data clearly showed the differential expression of 11 miRNAs in cis-platin resistant cells, which could potentially target many important pathways including MAPK, TGF-β signaling, actin cytoskeleton, ubiquitin mediated

  17. Patterns of dioxin-altered mRNA expression in livers of dioxin-sensitive versus dioxin-resistant rats

    Energy Technology Data Exchange (ETDEWEB)

    Franc, Monique A. [University of Toronto, Department of Pharmacology and Toxicology, Medical Sciences Building, Toronto, ON (Canada); Johnson and Johnson Pharmaceutical Research and Development, Department of Pharmacogenomics, 1000 Route 202 South, P.O. Box 300, Raritan, NJ (United States); Moffat, Ivy D.; Boutros, Paul C.; Okey, Allan B. [University of Toronto, Department of Pharmacology and Toxicology, Medical Sciences Building, Toronto, ON (Canada); Tuomisto, Jouni T.; Tuomisto, Jouko [National Public Health Institute, Department of Environmental Health, Centre for Environmental Health Risk Analysis, Kuopio (Finland); Pohjanvirta, Raimo [University of Helsinki, Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, Helsinki (Finland)

    2008-11-15

    Dioxins exert their major toxicologic effects by binding to the aryl hydrocarbon receptor (AHR) and altering gene transcription. Numerous dioxin-responsive genes previously were identified both by conventional biochemical and molecular techniques and by recent mRNA expression microarray studies. However, of the large set of dioxin-responsive genes the specific genes whose dysregulation leads to death remain unknown. To identify specific genes that may be involved in dioxin lethality we compared changes in liver mRNA levels following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in three strains/lines of dioxin-sensitive rats with changes in three dioxin-resistant rat strains/lines. The three dioxin-resistant strains/lines all harbor a large deletion in the transactivation domain of the aryl hydrocarbon receptor (AHR). Despite this deletion, many genes exhibited a ''Type-I'' response - that is, their responses were similar in dioxin-sensitive and dioxin-resistant rats. Several genes that previously were well established as being dioxin-responsive or under AHR regulation emerged as Type-I responses (e.g. CYP1A1, CYP1A2, CYP1B1 and Gsta3). In contrast, a relatively small number of genes exhibited a Type-II response - defined as a difference in responsiveness between dioxin-sensitive and dioxin-resistant rat strains. Type-II genes include: malic enzyme 1, ubiquitin C, cathepsin L, S-adenosylhomocysteine hydrolase and ferritin light chain 1. In silico searches revealed that AH response elements are conserved in the 5'-flanking regions of several genes that respond to TCDD in both the Type-I and Type-II categories. The vast majority of changes in mRNA levels in response to 100 {mu}g/kg TCDD were strain-specific; over 75% of the dioxin-responsive clones were affected in only one of the six strains/lines. Selected genes were assessed by quantitative RT-PCR in dose-response and time-course experiments and responses of some genes were

  18. Comparison of viral RNA electrophoresis and indirect ELISA methods in the diagnosis of human rotavirus infection

    Energy Technology Data Exchange (ETDEWEB)

    Avendano, L F; Dubinovsky, S; James, Jr, H D

    1984-01-01

    A total of 177 stool samples from Chilean diarrhea patients under two years of age were tested for rotavirus by two methods - the indirect enzyme-linked immunosorbent assay (indirect ELISA) and viral RNA electrophoresis in agarose gels (v RNA EPH). Fifty of the specimens came from patients with acute diarrhea and 127 came from patients with protracted diarrhea. The indirect ELISA testing was performed at the National Institutes of Health in the United States: the electrophoretic testing was carried out in Santiago, Chile by the authors. The electrophoretic method detected rotavirus in 36% of the acute samples and 25% of the samples from protracted cases, while the indirect ELISA method detected rotavirus in higher percentages of samples - 46% and 38%, respectively. These results support the conclusion that v RNA EPH is a less sensitive method for detecting rotavirus than the indirect ELISA. Nevertheless, the former method's high specificity, ease of application, and low cost make it a worthwhile alternative to indirect ELISA. Thus, considering the important role played by rotavirus in infant diarrhea and the need for a diagnostic technique that can be incorporated into the routines of medical center laboratories in developing countries, there is good reason to conclude that v RNA EPH is a useful tool for studying rotavirus diarrhea. 18 refs, 3 tabs. Also published in the Bol. Oficina Sanit. Panam. (1984) v. 97(1), p. 1-7 (In Spanish).

  19. Comparison of viral RNA electrophoresis and indirect ELISA methods in the diagnosis of human rotavirus infection

    International Nuclear Information System (INIS)

    Avendano, L.F.; Dubinovsky, S.

    1984-01-01

    A total of 177 stool samples from Chilean diarrhea patients under two years of age were tested for rotavirus by two methods - the indirect enzyme-linked immunosorbent assay (indirect ELISA) and viral RNA electrophoresis in agarose gels (v RNA EPH). Fifty of the specimens came from patients with acute diarrhea and 127 came from patients with protracted diarrhea. The indirect ELISA testing was performed at the National Institutes of Health in the United States: the electrophoretic testing was carried out in Santiago, Chile by the authors. The electrophoretic method detected rotavirus in 36% of the acute samples and 25% of the samples from protracted cases, while the indirect ELISA method detected rotavirus in higher percentages of samples - 46% and 38%, respectively. These results support the conclusion that v RNA EPH is a less sensitive method for detecting rotavirus than the indirect ELISA. Nevertheless, the former method's high specificity, ease of application, and low cost make it a worthwhile alternative to indirect ELISA. Thus, considering the important role played by rotavirus in infant diarrhea and the need for a diagnostic technique that can be incorporated into the routines of medical center laboratories in developing countries, there is good reason to conclude that v RNA EPH is a useful tool for studying rotavirus diarrhea. (author)

  20. Combined sequencing of mRNA and DNA from human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Florian Mertes

    2016-06-01

    Full Text Available Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO database under accession number GSE69471.

  1. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    International Nuclear Information System (INIS)

    Li Li; Li Xincang; Li Lu; Wang Jinxing; Jin Wenrui

    2011-01-01

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10 -14 mol L -1 . Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  2. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

    Li Li [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Li Xincang [School of Life Sciences, Shandong University, Jinan 250100 (China); Li Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2011-01-24

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10{sup -14} mol L{sup -1}. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  3. MicroRNA Regulation of Human Genes Essential for Influenza A (H7N9 Replication.

    Directory of Open Access Journals (Sweden)

    Stefan Wolf

    Full Text Available Influenza A viruses are important pathogens of humans and animals. While seasonal influenza viruses infect humans every year, occasionally animal-origin viruses emerge to cause pandemics with significantly higher morbidity and mortality rates. In March 2013, the public health authorities of China reported three cases of laboratory confirmed human infection with avian influenza A (H7N9 virus, and subsequently there have been many cases reported across South East Asia and recently in North America. Most patients experience severe respiratory illness, and morbidity with mortality rates near 40%. No vaccine is currently available and the use of antivirals is complicated due the frequent emergence of drug resistant strains. Thus, there is an imminent need to identify new drug targets for therapeutic intervention. In the current study, a high-throughput screening (HTS assay was performed using microRNA (miRNA inhibitors to identify new host miRNA targets that reduce influenza H7N9 replication in human respiratory (A549 cells. Validation studies lead to a top hit, hsa-miR-664a-3p, that had potent antiviral effects in reducing H7N9 replication (TCID50 titers by two logs. In silico pathway analysis revealed that this microRNA targeted the LIF and NEK7 genes with effects on pro-inflammatory factors. In follow up studies using siRNAs, anti-viral properties were shown for LIF. Furthermore, inhibition of hsa-miR-664a-3p also reduced virus replication of pandemic influenza A strains H1N1 and H3N2.

  4. Involvement of hGLD-2 in cytoplasmic polyadenylation of human p53 mRNA

    DEFF Research Database (Denmark)

    Glahder, Jacob-Andreas Harald; Norrild, Bodil

    2011-01-01

    Cytoplasmic polyadenylation is a post-transcriptional mechanism regulating mRNA stability and translation. The human p53 3'-untranslated region (3'-UTR) contains two regions similar to cytoplasmic polyadenylation elements (CPEs) just upstream of the poly(A) hexanucleotide. Evaluation of the p53 CPE......-like elements was performed by luciferase reporter assays, qPCR, and poly(A) assays. Herein, we report the down regulation of a luciferase reporter fused to the p53 3'-UTR, when human CPE-binding protein 1 (hCPEB1) is overexpressed. This inhibition is partially rescued when hCPEB1fused to hGLD-2 [a human...... cytoplasmic poly(A) polymerase] is overexpressed instead. The stability of a luciferase mRNA containing the p53 3'-UTR downstream, is decreased when hCPEB1 is overexpressed as seen by qPCR. Expression of hGLD-2 restores the mRNA stability. This is due to elongation of the poly(A) tail as seen by a PCR...

  5. Role of MicroRNA-1 in Human Cancer and Its Therapeutic Potentials

    Directory of Open Access Journals (Sweden)

    Chao Han

    2014-01-01

    Full Text Available While the mechanisms of human cancer development are not fully understood, evidence of microRNA (miRNA, miR dysregulation has been reported in many human diseases, including cancer. miRs are small noncoding RNA molecules that regulate posttranscriptional gene expression by binding to complementary sequences in the specific region of gene mRNAs, resulting in downregulation of gene expression. Not only are certain miRs consistently dysregulated across many cancers, but they also play critical roles in many aspects of cell growth, proliferation, metastasis, apoptosis, and drug resistance. Recent studies from our group and others revealed that miR-1 is frequently downregulated in various types of cancer. Through targeting multiple oncogenes and oncogenic pathways, miR-1 has been demonstrated to be a tumor suppressor gene that represses cancer cell proliferation and metastasis and promotes apoptosis by ectopic expression. In this review, we highlight recent findings on the aberrant expression and functional significance of miR-1 in human cancers and emphasize its significant values for therapeutic potentials.

  6. Hypoxia regulates microRNA expression in the human carotid body

    International Nuclear Information System (INIS)

    Mkrtchian, Souren; Lee, Kian Leong; Kåhlin, Jessica; Ebberyd, Anette; Poellinger, Lorenz; Fagerlund, Malin Jonsson; Eriksson, Lars I.

    2017-01-01

    The carotid body (CB) is the key sensing organ for physiological oxygen levels in the body. Under conditions of low oxygen (hypoxia), the CB plays crucial roles in signaling to the cardiorespiratory center in the medulla oblongata for the restoration of oxygen homeostasis. How hypoxia regulates gene expression in the human CB remains poorly understood. While limited information on transcriptional regulation in animal CBs is available, the identity and impact of important post-transcriptional regulators such as non-coding RNAs, and in particular miRNAs are not known. Here we show using ex vivo experiments that indeed a number of miRNAs are differentially regulated in surgically removed human CB slices when acute hypoxic conditions were applied. Analysis of the hypoxia-regulated miRNAs shows that they target biological pathways with upregulation of functions related to cell proliferation and immune response and downregulation of cell differentiation and cell death functions. Comparative analysis of the human CB miRNAome with the global miRNA expression patterns of a large number of different human tissues showed that the CB miRNAome had a unique profile which reflects its highly specialized functional status. Nevertheless, the human CB miRNAome is most closely related to the miRNA expression pattern of brain tissues indicating that they may have the most similar developmental origins. - Highlights: • Hypoxia triggers differential expression of many miRNAs in the human carotid body. • This can lead to the upregulation of proliferation and immune response functions. • CB expression profile in the carotid body resembles the miRNA expression pattern in the brain. • miRNAs are involved in the regulation of carotid body functions including oxygen sensing.

  7. Hypoxia regulates microRNA expression in the human carotid body

    Energy Technology Data Exchange (ETDEWEB)

    Mkrtchian, Souren, E-mail: souren.mkrtchian@ki.se [Section for Anesthesiology and Intensive Care Medicine, Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Lee, Kian Leong, E-mail: csilkl@nus.edu.sg [Cancer Science Institute of Singapore, National University of Singapore, 117599 Singapore (Singapore); Kåhlin, Jessica [Section for Anesthesiology and Intensive Care Medicine, Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Function Perioperative Medicine and Intensive Care, Karolinska University Hospital, SE-171 76 Stockholm (Sweden); Ebberyd, Anette [Section for Anesthesiology and Intensive Care Medicine, Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Poellinger, Lorenz [Cancer Science Institute of Singapore, National University of Singapore, 117599 Singapore (Singapore); Department of Cell and Molecular Biology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Fagerlund, Malin Jonsson; Eriksson, Lars I. [Section for Anesthesiology and Intensive Care Medicine, Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm (Sweden); Function Perioperative Medicine and Intensive Care, Karolinska University Hospital, SE-171 76 Stockholm (Sweden)

    2017-03-15

    The carotid body (CB) is the key sensing organ for physiological oxygen levels in the body. Under conditions of low oxygen (hypoxia), the CB plays crucial roles in signaling to the cardiorespiratory center in the medulla oblongata for the restoration of oxygen homeostasis. How hypoxia regulates gene expression in the human CB remains poorly understood. While limited information on transcriptional regulation in animal CBs is available, the identity and impact of important post-transcriptional regulators such as non-coding RNAs, and in particular miRNAs are not known. Here we show using ex vivo experiments that indeed a number of miRNAs are differentially regulated in surgically removed human CB slices when acute hypoxic conditions were applied. Analysis of the hypoxia-regulated miRNAs shows that they target biological pathways with upregulation of functions related to cell proliferation and immune response and downregulation of cell differentiation and cell death functions. Comparative analysis of the human CB miRNAome with the global miRNA expression patterns of a large number of different human tissues showed that the CB miRNAome had a unique profile which reflects its highly specialized functional status. Nevertheless, the human CB miRNAome is most closely related to the miRNA expression pattern of brain tissues indicating that they may have the most similar developmental origins. - Highlights: • Hypoxia triggers differential expression of many miRNAs in the human carotid body. • This can lead to the upregulation of proliferation and immune response functions. • CB expression profile in the carotid body resembles the miRNA expression pattern in the brain. • miRNAs are involved in the regulation of carotid body functions including oxygen sensing.

  8. Sensitivity to ionizing radiation and chemotherapeutic agents in gemcitabine-resistant human tumor cell lines

    International Nuclear Information System (INIS)

    Bree, Chris van; Kreder, Natasja Castro; Loves, Willem J.P.; Franken, Nicolaas A.P.; Peters, Godefridus J.; Haveman, Jaap

    2002-01-01

    Purpose: To determine cross-resistance to anti-tumor treatments in 2',2'difluorodeoxycytidine (dFdC, gemcitabine)-resistant human tumor cells. Methods and Materials: Human lung carcinoma cells SW-1573 (SWp) were made resistant to dFdC (SWg). Sensitivity to cisplatin (cDDP), paclitaxel, 5-fluorouracil (5-FU), methotrexate (MTX), cytarabine (ara-C), and dFdC was measured by a proliferation assay. Radiosensitivity and radioenhancement by dFdC of this cell panel and the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000 were determined by clonogenic assay. Bivariate flowcytometry was performed to study cell cycle changes. Results: In the SWg, a complete deoxycytidine kinase (dCK) deficiency was found on mRNA and protein level. This was accompanied by a 10-fold decrease in dCK activity which resulted in the >1000-fold resistance to dFdC. Sensitivity to other anti-tumor drugs was not altered, except for ara-C (>100-fold resistance). Radiosensitivity was not altered in the dFdC-resistant cell lines SWg and AG6000. High concentrations (50-100 μM dFdC) induced radioenhancement in the dFdC-resistant cell lines similar to the radioenhancement obtained at lower concentrations (10 nM dFdC) in the parental lines. An early S-phase arrest was found in all cell lines after dFdC treatment where radioenhancement was achieved. Conclusions: In the dFdC-resistant lung tumor cell line SWg, the deficiency in dCK is related to the resistance to dFdC and ara-C. No cross-resistance was observed to other anti-tumor drugs used for the treatment in lung cancer. Sensitivity to ionizing radiation was not altered in two different dFdC-resistant cell lines. Resistance to dFdC does not eliminate the ability of dFdC to sensitize cells to radiation

  9. Formation of tRNA granules in the nucleus of heat-induced human cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyagawa, Ryu [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan); Mizuno, Rie [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Watanabe, Kazunori, E-mail: watanabe@ric.u-tokyo.ac.jp [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Ijiri, Kenichi [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer tRNAs are tranlocated into the nucleus in heat-induced HeLa cells. Black-Right-Pointing-Pointer tRNAs form the unique granules in the nucleus. Black-Right-Pointing-Pointer tRNA ganules overlap with nuclear stress granules. -- Abstract: The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA{sup Met} (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA{sup Met} was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

  10. Cockayne's syndrome: correlation of clinical features with cellular sensitivity of RNA synthesis to UV irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Lehmann, A.R.; Thompson, A.F.; Harcourt, S.A. (Medical Research Council, Brighton (United Kingdom). Cell Mutation Unit); Stefanini, Miria (Consiglio Nazionale delle Ricerche, Pavia (Italy). Ist. di Genetica Biochimica ed Evoluzionistica); Norris, P.G. (Addenbrooke' s Hospital, Cambridge (United Kingdom))

    1993-08-01

    Cockayne's syndrome (CS) is a rare autosomal recessive disorder with dwarfism, mental retardation, and otherwise clinically heterogeneous features. In cultured CS fibroblasts, the failure of RNA synthesis to recover to normal rates after UV-C irradiation provides a useful and relatively simple diagnostic test. We have measured post-UV-C RNA synthesis in 52 patients for whom a clinical diagnosis of CS was considered a possibility. Twenty-nine patients showed the defect characteristic of CS cells, and 23 had a normal response. We have attempted to correlate the cellular diagnosis with the different clinical features of the disorder. Clinical details of the patients were obtained from referring clinicians in the form of a questionnaire. Our results show that, apart from the cardinal features of dwarfism and mental retardation, sun sensitivity correlated best with a positive cellular diagnosis. Pigmentary retinopathy, gait defects, and dental caries were also good positive indicators, although several patients with a positive cellular diagnosis did not have these features. (Author).

  11. MicroRNA Profiling in Aqueous Humor of Individual Human Eyes by Next-Generation Sequencing.

    Science.gov (United States)

    Wecker, Thomas; Hoffmeier, Klaus; Plötner, Anne; Grüning, Björn Andreas; Horres, Ralf; Backofen, Rolf; Reinhard, Thomas; Schlunck, Günther

    2016-04-01

    Extracellular microRNAs (miRNAs) in aqueous humor were suggested to have a role in transcellular signaling and may serve as disease biomarkers. The authors adopted next-generation sequencing (NGS) techniques to further characterize the miRNA profile in single samples of 60 to 80 μL human aqueous humor. Samples were obtained at the outset of cataract surgery in nine independent, otherwise healthy eyes. Four samples were used to extract RNA and generate sequencing libraries, followed by an adapter-driven amplification step, electrophoretic size selection, sequencing, and data analysis. Five samples were used for quantitative PCR (qPCR) validation of NGS results. Published NGS data on circulating miRNAs in blood were analyzed in comparison. One hundred fifty-eight miRNAs were consistently detected by NGS in all four samples; an additional 59 miRNAs were present in at least three samples. The aqueous humor miRNA profile shows some overlap with published NGS-derived inventories of circulating miRNAs in blood plasma with high prevalence of human miR-451a, -21, and -16. In contrast to blood, miR-184, -4448, -30a, -29a, -29c, -19a, -30d, -205, -24, -22, and -3074 were detected among the 20 most prevalent miRNAs in aqueous humor. Relative expression patterns of miR-451a, -202, and -144 suggested by NGS were confirmed by qPCR. Our data illustrate the feasibility of miRNA analysis by NGS in small individual aqueous humor samples. Intraocular cells as well as blood plasma contribute to the extracellular aqueous humor miRNome. The data suggest possible roles of miRNA in intraocular cell adhesion and signaling by TGF-β and Wnt, which are important in intraocular pressure regulation and glaucoma.

  12. The prognostic potential and carcinogenesis of long non-coding RNA TUG1 in human cholangiocarcinoma

    OpenAIRE

    Xu, Yi; Leng, Kaiming; Li, Zhenglong; Zhang, Fumin; Zhong, Xiangyu; Kang, Pengcheng; Jiang, Xingming; Cui, Yunfu

    2017-01-01

    Cholangiocarcinoma (CCA) is a fatal disease with increasing worldwide incidence and is characterized by poor prognosis due to its poor response to conventional chemotherapy or radiotherapy. Long non-coding RNAs (lncRNAs) play key roles in multiple human cancers, including CCA. Cancer progression related lncRNA taurine-up-regulated gene 1 (TUG1) was reported to be involved in human carcinomas. However, the impact of TUG1 in CCA is unclear. The aim of this study was to explore the expression pa...

  13. Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells

    International Nuclear Information System (INIS)

    Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha; Beisiegel, Ulrike; Heeren, Joerg

    2008-01-01

    There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged withdrawal of the peroxisome proliferator activated receptor γ (PPARγ) agonist rosiglitazone. Differentiated hMSC-Tert developed the characteristic monolocular phenotype of mature adipocytes. The expression of adipocyte specific markers was highly increased during differentiation. Most importantly, the presence of the PPARγ agonist rosiglitazone was not required for the stable expression of lipoprotein lipase, adipocyte fatty acid binding protein and perilipin on mRNA and protein levels. Adiponectin expression was post-transcriptionally down-regulated in the absence of rosiglitazone. Insulin sensitivity as measured by insulin-induced phosphorylation of Akt and S6 ribosomal protein was also independent of rosiglitazone. In addition to commonly used adipogenic markers, we investigated further PPARγ-stimulated proteins with a role in lipid metabolism. We observed an increase of lipoprotein receptor (VLDLR, LRP1) and apolipoprotein E expression during differentiation. Despite this increased expression, the receptor-mediated endocytosis of lipoproteins was decreased in differentiated adipocytes, suggesting that these proteins may have an additional function in adipose tissue beyond lipoprotein uptake

  14. RNA of Enterococcus faecalis Strain EC-12 Is a Major Component Inducing Interleukin-12 Production from Human Monocytic Cells.

    Directory of Open Access Journals (Sweden)

    Ryoichiro Nishibayashi

    Full Text Available Interleukin-12 (IL-12 is an important cytokine for the immunomodulatory effects of lactic acid bacteria (LAB. Using murine immune cells, we previously reported that the RNA of Enterococcus faecalis EC-12, a LAB strain exerting probiotic-like beneficial effects, is the major IL-12-inducing immunogenic component. However, it was recently revealed that bacterial RNA can be a ligand for Toll-like receptor (TLR 13, which is only expressed in mice. Because TLR13 is not expressed in humans, the immuno-stimulatory and -modulatory effects of LAB RNA in human cells should be augmented excluding TLR13 contribution. In experiment 1 of this study, the role of LAB RNA in IL-12 induction in human immune cells was studied using three LAB strains, E.faecalis EC-12, Lactobacillus gasseri JCM5344, and Bifidobacterium breve JCM1192. RNase A treatment of heat-killed LAB significantly decreased the IL-12 production of human peripheral blood mononuclear cells on stimulation, while RNase III treatment revealed virtually no effects. Further, IL-12 production against heat-killed E. faecalis EC-12 was abolished by depleting monocytes. These results demonstrated that single stranded RNA (ssRNA of LAB is a strong inducer of IL-12 production from human monocytes. In experiment 2, major receptor for ssRNA of E. faecalis EC-12 was identified using THP-1 cells, a human monocytic cell line. The type of RNA molecules of E. faecalis EC-12 responsible for IL-12 induction was also identified. IL-12 production induced by the total RNA of E. faecalis EC-12 was significantly reduced by the treatment of siRNA for TLR8 but not for TLR7. Furthermore, both 23S and 16S rRNA, but not mRNA, of E. faecalis EC-12 markedly induced IL-12 production from THP-1 cells. These results suggested that the recognition of ssRNA of E. faecalis EC-12 was mediated by TLR8 and that rRNA was the RNA molecule that exhibited IL-12-inducing ability in human cells.

  15. MicroRNA (miRNA Signaling in the Human CNS in Sporadic Alzheimer’s Disease (AD-Novel and Unique Pathological Features

    Directory of Open Access Journals (Sweden)

    Yuhai Zhao

    2015-12-01

    Full Text Available Of the approximately ~2.65 × 103 mature microRNAs (miRNAs so far identified in Homo sapiens, only a surprisingly small but select subset—about 35–40—are highly abundant in the human central nervous system (CNS. This fact alone underscores the extremely high selection pressure for the human CNS to utilize only specific ribonucleotide sequences contained within these single-stranded non-coding RNAs (ncRNAs for productive miRNA–mRNA interactions and the down-regulation of gene expression. In this article we will: (i consolidate some of our still evolving ideas concerning the role of miRNAs in the CNS in normal aging and in health, and in sporadic Alzheimer’s disease (AD and related forms of chronic neurodegeneration; and (ii highlight certain aspects of the most current work in this research field, with particular emphasis on the findings from our lab of a small pathogenic family of six inducible, pro-inflammatory, NF-κB-regulated miRNAs including miRNA-7, miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a and miRNA-155. This group of six CNS-abundant miRNAs significantly up-regulated in sporadic AD are emerging as what appear to be key mechanistic contributors to the sporadic AD process and can explain much of the neuropathology of this common, age-related inflammatory neurodegeneration of the human CNS.

  16. MicroRNA (miRNA) Signaling in the Human CNS in Sporadic Alzheimer’s Disease (AD)-Novel and Unique Pathological Features

    Science.gov (United States)

    Zhao, Yuhai; Pogue, Aileen I.; Lukiw, Walter J.

    2015-01-01

    Of the approximately ~2.65 × 103 mature microRNAs (miRNAs) so far identified in Homo sapiens, only a surprisingly small but select subset—about 35–40—are highly abundant in the human central nervous system (CNS). This fact alone underscores the extremely high selection pressure for the human CNS to utilize only specific ribonucleotide sequences contained within these single-stranded non-coding RNAs (ncRNAs) for productive miRNA–mRNA interactions and the down-regulation of gene expression. In this article we will: (i) consolidate some of our still evolving ideas concerning the role of miRNAs in the CNS in normal aging and in health, and in sporadic Alzheimer’s disease (AD) and related forms of chronic neurodegeneration; and (ii) highlight certain aspects of the most current work in this research field, with particular emphasis on the findings from our lab of a small pathogenic family of six inducible, pro-inflammatory, NF-κB-regulated miRNAs including miRNA-7, miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a and miRNA-155. This group of six CNS-abundant miRNAs significantly up-regulated in sporadic AD are emerging as what appear to be key mechanistic contributors to the sporadic AD process and can explain much of the neuropathology of this common, age-related inflammatory neurodegeneration of the human CNS. PMID:26694372

  17. Effect of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) on the growth and radiotherapeutic sensitivity of human lymphoma cell lines

    International Nuclear Information System (INIS)

    Yu Zeyang; Fan Wo; Li Dongqing; Zhu Ran; Wang Yongqing; Wu Jinchang

    2008-01-01

    Objective: To explore the inhibitory effect and radiation sensitization of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) on human lymphoma cell lines. Methods: Human lymphoma cell lines Raji and Daudi were treated with rAd-p53, radiation therapy and combined treatment, respectively. The cell growth inhibition was assessed by MTT. The p53 protein expression was detected by Western blotting, and p53 mRNA was detected by BT-PCB. Results: The MTT results showed that the inhibitory effect and radiosensitivity enhancement of rAd-p53 on human lymphoma cell lines were not obvious [Raji: (27.5±4.1)%; Daudi: (28.1±1.6)%]. The results of Western blotting and BT-PCB showed that extrinsic p53 protein and p53 mRNA were expressed to some degree, but not at high-level. In addition, the results didn't demonstrate obvious radiosensitivity enhancement. Conclusions: The role of inhibition and radiosensitivity enhancement of rAd-p53 was not significant on human lymphoma cell lines. (authors)

  18. SL1 RNA gene recovery from Enterobius vermicularis ancient DNA in pre-Columbian human coprolites.

    Science.gov (United States)

    Iñiguez, Alena Mayo; Reinhard, Karl; Carvalho Gonçalves, Marcelo Luiz; Ferreira, Luiz Fernando; Araújo, Adauto; Paulo Vicente, Ana Carolina

    2006-11-01

    Enterobius vermicularis, pinworm, is one of the most common helminths worldwide, infecting nearly a billion people at all socio-economic levels. In prehistoric populations the paleoparasitological findings show a pinworm homogeneous distribution among hunter-gatherers in North America, intensified with the advent of agriculture. This same increase also occurred in the transition from nomad hunter-gatherers to sedentary farmers in South America, although E. vermicularis infection encompasses only the ancient Andean peoples, with no record among the pre-Colombian populations in the South American lowlands. However, the outline of pinworm paleoepidemiology has been supported by microscopic finding of eggs recovered from coprolites. Since molecular techniques are precise and sensitive in detecting pathogen ancient DNA (aDNA), and also could provide insights into the parasite evolutionary history, in this work we have performed a molecular paleoparasitological study of E. vermicularis. aDNA was recovered and pinworm 5S rRNA spacer sequences were determined from pre-Columbian coprolites (4110 BC-AD 900) from four different North and South American archaeological sites. The sequence analysis confirmed E. vermicularis identity and revealed a similarity among ancient and modern sequences. Moreover, polymorphisms were identified at the relative positions 160, 173 and 180, in independent coprolite samples from Tulán, San Pedro de Atacama, Chile (1080-950 BC). We also verified the presence of peculiarities (Splicing leader (SL1) RNA sequence, spliced donor site, the Sm antigen biding site, and RNA secondary structure) which characterise the SL1 RNA gene. The analysis shows that the SL1 RNA gene of contemporary pinworms was present in pre-Columbian E. vermicularis by 6110 years ago. We were successful in detecting E. vermicularis aDNA even in coprolites without direct microscopic evidence of the eggs, improving the diagnosis of helminth infections in the past and further

  19. Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiaojie [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Taizhou Polytechnic College, Taizhou (China); Zhang, Ling; Shao, Yueting; Liang, Zuowen; Shao, Chen; Wang, Bo; Guo, Baofeng; Li, Na; Zhao, Xuejian [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Li, Yang, E-mail: lyang@jlu.edu.cn [Department of Pathophysiology, Prostate Diseases Prevention and Treatment Research Centre, Norman Bethune Medical School, Jilin University, Changchun (China); Xu, Deqi [Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Neu3 is as one of the sialidases and regulates cell surface functions. Black-Right-Pointing-Pointer A Neu3-specific siRNA inhibited prostrate cancer cell invasion and migration. Black-Right-Pointing-Pointer The Neu3-specific siRNA inhibited prostate cancer metastasis in mice. Black-Right-Pointing-Pointer Targeting Neu3 may have utility for gene-based therapy of human cancer metastasis. -- Abstract: Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed

  20. Transient Gene and miRNA Expression Profile Changes of Confluent Human Fibroblast Cells in Space

    Science.gov (United States)

    Zhang, Ye; Lu, Tao; Wong, Michael; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Wang, Xiaoyu; Wu, Honglu

    2015-01-01

    Microgravity or an altered gravity environment from the static 1 gravitational constant has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of the cells. Whether non-dividing cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted on the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days for investigations of gene and miRNA (microRNA) expression profile changes in these cells. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly even though they were confluent, as measured by the expression of the protein Ki-67 positive cells, and the cells in space grew slightly faster. Gene and miRNA expression data indicated activation of NF(sub kappa)B (nuclear factor kappa-light-chain-enhancer of activated B cells) and other growth related pathways involving HGF and VEGF in the flown cells. On Day 14 when the cells were mostly non-dividing, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples in respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeleton changes by immunohistochemistry staining of the cells with antibodies for alpha-tubulin showed no difference between the flight and ground samples. Results of our study suggest that in true non-dividing human fibroblast cells, microgravity in

  1. Comparison of risk sensitivity to human errors in the Oconee and LaSalle PRAs

    International Nuclear Information System (INIS)

    Wong, S.; Higgins, J.

    1991-01-01

    This paper describes the comparative analyses of plant risk sensitivity to human errors in the Oconee and La Salle Probabilistic Risk Assessment (PRAs). These analyses were performed to determine the reasons for the observed differences in the sensitivity of core melt frequency (CMF) to changes in human error probabilities (HEPs). Plant-specific design features, PRA methods, and the level of detail and assumptions in the human error modeling were evaluated to assess their influence risk estimates and sensitivities

  2. Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells.

    Science.gov (United States)

    Slanina, H; Schmutzler, M; Christodoulides, M; Kim, K S; Schubert-Unkmeir, A

    2012-01-01

    Expression of exogenous DNA or small interfering RNA (siRNA) in vitro is significantly affected by the particular delivery system utilized. In this study, we evaluated the transfection efficiency of plasmid DNA and siRNA into human brain microvascular endothelial cells (HBMEC) and meningioma cells, which constitute the blood-cerebrospinal fluid barrier, a target of meningitis-causing pathogens. Chemical transfection methods and various lipofection reagents including Lipofectamin™, FuGene™, or jetPRIME®, as well as physical transfection methods and electroporation techniques were applied. To monitor the transfection efficiencies, HBMEC and meningioma cells were transfected with the reporter plasmid pTagGFP2-actin vector, and efficiency of transfection was estimated by fluorescence microscopy and flow cytometry. We established protocols based on electroporation using Cell Line Nucleofector® Kit V with the Amaxa® Nucleofector® II system from Lonza and the Neon® Transfection system from Invitrogen resulting in up to 41 and 82% green fluorescent protein-positive HBMEC, respectively. Optimal transfection solutions, pulse programs and length were evaluated. We furthermore demonstrated that lipofection is an efficient method to transfect meningioma cells with a transfection efficiency of about 81%. Finally, we applied the successful electroporation protocols to deliver synthetic siRNA to HBMEC and analyzed the role of the actin-binding protein cortactin in Neisseria meningitidis pathogenesis. Copyright © 2012 S. Karger AG, Basel.

  3. The origins and evolutionary history of human non-coding RNA regulatory networks.

    Science.gov (United States)

    Sherafatian, Masih; Mowla, Seyed Javad

    2017-04-01

    The evolutionary history and origin of the regulatory function of animal non-coding RNAs are not well understood. Lack of conservation of long non-coding RNAs and small sizes of microRNAs has been major obstacles in their phylogenetic analysis. In this study, we tried to shed more light on the evolution of ncRNA regulatory networks by changing our phylogenetic strategy to focus on the evolutionary pattern of their protein coding targets. We used available target databases of miRNAs and lncRNAs to find their protein coding targets in human. We were able to recognize evolutionary hallmarks of ncRNA targets by phylostratigraphic analysis. We found the conventional 3'-UTR and lesser known 5'-UTR targets of miRNAs to be enriched at three consecutive phylostrata. Firstly, in eukaryata phylostratum corresponding to the emergence of miRNAs, our study revealed that miRNA targets function primarily in cell cycle processes. Moreover, the same overrepresentation of the targets observed in the next two consecutive phylostrata, opisthokonta and eumetazoa, corresponded to the expansion periods of miRNAs in animals evolution. Coding sequence targets of miRNAs showed a delayed rise at opisthokonta phylostratum, compared to the 3' and 5' UTR targets of miRNAs. LncRNA regulatory network was the latest to evolve at eumetazoa.

  4. Design of a Sensitive and Selective Electrochemical Aptasensor for the Determination of the Complementary cDNA of miRNA-145 Based on the Intercalation and Electrochemical Reduction of Doxorubicin.

    Science.gov (United States)

    Mohamadi, Maryam; Mostafavi, Ali; Torkzadeh-Mahani, Masoud

    2017-11-01

    The aim of this research was the determination of a microRNA (miRNA) using a DNA electrochemical aptasensor. In this biosensor, the complementary complementary DNA (cDNA) of miRNA-145 (a sense RNA transcript) was the target strand and the cDNA of miRNA-145 was the probe strand. Both cDNAs can be the product of the reverse transcriptase-polymerase chain reaction of miRNA. The proposed aptasensor's function was based on the hybridization of target strands with probes immobilized on the surface of a working electrode and the subsequent intercalation of doxorubicin (DOX) molecules functioning as the electroactive indicators of any double strands that formed. Electrochemical transduction was performed by measuring the cathodic current resulting from the electrochemical reduction of the intercalated molecules at the electrode surface. In the experiment, because many DOX molecules accumulated on each target strand on the electrode surface, amplification was inherently easy, without a need for enzymatic or complicated amplification strategies. The proposed aptasensor also had the excellent ability to regenerate as a result of the melting of the DNA duplex. Moreover, the use of DNA probe strands obviated the challenges of working with an RNA probe, such as sensitivity to RNase enzyme. In addition to the linear relationship between the electrochemical signal and the concentration of the target strands that ranged from 2.0 to 80.0 nM with an LOD of 0.27 nM, the proposed biosensor was clearly capable of distinguishing between complementary (target strand) and noncomplementary sequences. The presented biosensor was successfully applied for the quantification of DNA strands corresponding to miRNA-145 in human serum samples.

  5. Decreased expression of microRNA let-7i and its association with chemotherapeutic response in human gastric cancer

    Directory of Open Access Journals (Sweden)

    Liu Kun

    2012-10-01

    Full Text Available Abstract Background MicroRNA let-7i has been proven to be down-regulated in many human malignancies and correlated with tumor progression and anticancer drug resistance. Our study aims to characterize the contribution of miRNA let-7i to the initiation and malignant progression of locally advanced gastric cancer (LAGC, and evaluate its possible value in neoadjuvant chemotherapeutic efficacy prediction. Methods Eighty-six previously untreated LAGC patients who underwent preoperative chemotherapy and radical resection were included in our study. Let-7i expression was examined for pairs of cancer tissues and corresponding normal adjacent tissues (NATs, using quantitative RT-PCR. The relationship of let-7i level to clinicopathological characteristics, pathologic tumor regression grades after chemotherapy, and overall survival (OS was also investigated. Results Let-7i was significantly down-regulated in most tumor tissues (78/86: 91% compared with paired NATs (P P =0.024 independently of other clinicopathological factors, including tumor node metastasis (TNM stage (HR = 3.226, P = 0.013, depth of infiltration (HR = 4.167, P P = 0.037. Conclusions These findings indicate that let-7i may be a good candidate for use a therapeutic target and a potential tissue marker for the prediction of chemotherapeutic sensitivity and prognosis in LAGC patients.

  6. The RNA helicase DDX1 is involved in restricted HIV-1 Rev function in human astrocytes

    International Nuclear Information System (INIS)

    Fang Jianhua; Acheampong, Edward; Dave, Rajnish; Wang Fengxiang; Mukhtar, Muhammad; Pomerantz, Roger J.

    2005-01-01

    Productive infection by human immunodeficiency virus type I (HIV-1) in the central nervous system (CNS) involves mainly macrophages and microglial cells. A frequency of less than 10% of human astrocytes is estimated to be infectable with HIV-1. Nonetheless, this relatively low percentage of infected astrocytes, but associated with a large total number of astrocytic cells in the CNS, makes human astrocytes a critical part in the analyses of potential HIV-1 reservoirs in vivo. Investigations in astrocytic cell lines and primary human fetal astrocytes revealed that limited HIV-1 replication in these cells resulted from low-level viral entry, transcription, viral protein processing, and virion maturation. Of note, a low ratio of unspliced versus spliced HIV-1-specific RNA was also investigated, as Rev appeared to act aberrantly in astrocytes, via loss of nuclear and/or nucleolar localization and diminished Rev-mediated function. Host cellular machinery enabling Rev function has become critical for elucidation of diminished Rev activity, especially for those factors leading to RNA metabolism. We have recently identified a DEAD-box protein, DDX1, as a Rev cellular co-factor and now have explored its potential importance in astrocytes. Cells were infected with HIV-1 pseudotyped with envelope glycoproteins of amphotropic murine leukemia viruses (MLV). Semi-quantitative reverse transcriptase-polymerase chain reactions (RT-PCR) for unspliced, singly-spliced, and multiply-spliced RNA clearly showed a lower ratio of unspliced/singly-spliced over multiply-spliced HIV-1-specific RNA in human astrocytes as compared to Rev-permissive, non-glial control cells. As well, the cellular localization of Rev in astrocytes was cytoplasmically dominant as compared to that of Rev-permissive, non-glial controls. This endogenous level of DDX1 expression in astrocytes was demonstrated directly to lead to a shift of Rev sub-cellular distribution dominance from nuclear and/or nucleolar to

  7. Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras

    Science.gov (United States)

    Wheeler, Lee Adam; Trifonova, Radiana; Vrbanac, Vladimir; Basar, Emre; McKernan, Shannon; Xu, Zhan; Seung, Edward; Deruaz, Maud; Dudek, Tim; Einarsson, Jon Ivar; Yang, Linda; Allen, Todd M.; Luster, Andrew D.; Tager, Andrew M.; Dykxhoorn, Derek M.; Lieberman, Judy

    2011-01-01

    The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission. PMID:21576818

  8. Intracellular human papillomavirus E6, E7 mRNA quantification predicts CIN 2+ in cervical biopsies better than Papanicolaou screening for women regardless of age.

    Science.gov (United States)

    Pierry, Deirdre; Weiss, Gerald; Lack, Benjamin; Chen, Victor; Fusco, Judy

    2012-08-01

    Cervical cancer screening in women younger than 30 years relies on cervical cytology because of the poor performance of human papillomavirus (HPV) DNA testing in this age group. To determine the performance of in-cell HPV E6, E7 mRNA quantification (HPV OncoTect) for the detection of high-grade cervical intraepithelial neoplasia in women younger than 30 years. We analyzed 3133 cytology specimens from a screening population of women aged 19-75 years investigate HPV OncoTect as a triage/secondary screening test for atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) cytology in women younger than 30 years. Test results were compared to histology in 246 cases. The sensitivity of E6, E7 mRNA was 89% for CIN 2+ and 100% for CIN 3+ lesions in women 30 years and older. In women younger than 30 years, the sensitivity of E6, E7 mRNA for CIN 2+ lesions was 88% for CIN 2+ and 92% for CIN 3+ lesions. Abnormal cytology (≥ASCUS) exhibited a sensitivity of 89% for CIN 2+ and 100% for CIN 3+ in women 30 years and older and 96% sensitivity for CIN 2+ and 93% sensitivity for CIN 3+ in women younger than 30. The specificity of E6, E7 mRNA was >80% for CIN 2+ and CIN 3+ in both groups of women compared to a specificity of abnormal cytology of ASCUS/LSIL triage in women including those younger than 30 years.

  9. Analysis of miRNA and mRNA Expression Profiles Highlights Alterations in Ionizing Radiation Response of Human Lymphocytes under Modeled Microgravity

    Science.gov (United States)

    Casara, Silvia; Sales, Gabriele; Lanfranchi, Gerolamo; Celotti, Lucia; Mognato, Maddalena

    2012-01-01

    Background Ionizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity, a condition of weightlessness experienced by astronauts during space missions, which could have a synergistic action on cells, increasing the risk of radiation exposure. Methodology/Principal Findings We analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of γ-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover, let-7i*, miR-7, miR-7-1*, miR-27a, miR-144, miR-200a, miR-598, miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles, carried out on PBL of the same donors, identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of “Response to DNA damage” is enriched when PBL are incubated in 1 g but not in MMG. Moreover, some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses. Conclusions/Significance On the whole, by integrating the transcriptome and microRNome, we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL. PMID:22347458

  10. Experimental study of APE1 RNA interference enhancing the sensitivity of neutron radiation in osteosarcoma

    International Nuclear Information System (INIS)

    Wang Dong; Qing Yi; Zhong Zhaoyang; Li Zengpeng; Zhang Xinhong; Yang Yuxin

    2007-01-01

    Objective: To knock down APE1 gene expression in HOS cells, and explore its antitumor effects in combination with 252 Cf neutron radiotherapy. Methods: pSilence APE1 siBNA plasmid was transfected into HOS cells by SuperFect Transfection liposome. The transfected HOS cells were irradiated by 252 Cf neutron, then MTY assay, clone formation assay and alkaline comet assay were used to detect the radiobiological reaction, and cell apoptosis was detected with flow cytometry. Results: The D 37 value was 3.02 vs. 2.42 in the control and transfected HOS cells respectively after irradiation with 252 Cf neutron, the DMF value is 1.43. The tail moments and cell apoptosis rate at 200, 500 and 1000 cGy showed significant difference between the two groups (P 252 Cf neutron radiotherapy may be a promising approach to therapy of human osteosarcoma in the future. (authors)

  11. siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts.

    Science.gov (United States)

    Lokossou, Adjimon Gatien; Toufaily, Chirine; Vargas, Amandine; Barbeau, Benoit

    2016-09-20

    Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

  12. Proanthocyanidins modulate microRNA expression in human HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Anna Arola-Arnal

    Full Text Available Mi(croRNAs are small non-coding RNAs of 18-25 nucleotides in length that modulate gene expression at the post-transcriptional level. These RNAs have been shown to be involved in a several biological processes, human diseases and metabolic disorders. Proanthocyanidins, which are the most abundant polyphenol class in the human diet, have positive health effects on a variety of metabolic disorders such as inflammation, obesity, diabetes and insulin resistance. The present study aimed to evaluate whether proanthocyanidin-rich natural extracts modulate miRNA expression. Using microarray analysis and Q-PCR, we investigated miRNA expression in HepG2 cells treated with proanthocyanidins. Our results showed that when HepG2 cells were treated with grape seed proanthocyanidin extract (GSPE, cocoa proanthocyanidin extract (CPE or pure epigallocatechin gallate isolated from green tea (EGCG, fifteen, six and five differentially expressed miRNAs, respectively, were identified out of 904 mRNAs. Specifically, miR-30b* was downregulated by the three treatments, and treatment with GSPE or CPE upregulated miR-1224-3p, miR-197 and miR-532-3p. Therefore, these results provide evidence of the capacity of dietary proanthocyanidins to influence microRNA expression, suggesting a new mechanism of action of proanthocyanidins.

  13. Deep RNA-Seq analysis reveals unexpected features of human prostate basal epithelial cells

    Directory of Open Access Journals (Sweden)

    Dingxiao Zhang

    2016-03-01

    Full Text Available Prostate cancer is the second leading cause of cancer-related deaths among American men [1]. The prostate gland mainly contains basal and luminal cells, which are constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here, for the first time, we describe a whole-genome transcriptome analysis of human benign prostatic basal and luminal populations by using deep RNA sequencing (GSE67070 [2]. Combined with comprehensive molecular and biological characterizations, we show that the differential gene expression profiles account for their distinct functional phenotypes. Strikingly, in contrast to luminal cells, basal cells preferentially express gene categories associated with stem cells, neural and neuronal development, and RNA processing. Of clinical relevance, the treatment failed castration-resistant and anaplastic prostate cancers molecularly resemble a basal-like phenotype. We also identified genes associated with patient clinical outcome. Therefore, we provide a gene expression resource for understanding human prostate epithelial lineages, and link the cell-type specific gene signatures to subtypes of prostate cancer development. Keywords: Prostate epithelial cells, Basal cells, Luminal cells, RNA-seq

  14. Orthogonal use of a human tRNA synthetase active site to achieve multifunctionality.

    Science.gov (United States)

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A; Schimmel, Paul; Yang, Xiang-Lei

    2010-01-01

    Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.

  15. MicroRNA-223 Enhances Radiation Sensitivity of U87MG Cells In Vitro and In Vivo by Targeting Ataxia Telangiectasia Mutated

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Liping; Zhu, Ji [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Zaorsky, Nicholas G. [Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania (United States); Deng, Yun [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Wu, Xingzhong [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Liu, Yong [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Liu, Fangqi; Cai, Guoxiang; Gu, Weilie [Department of Colorectal Cancer, Fudan University, Shanghai Cancer Center, Shanghai (China); Shen, Lijun [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Zhang, Zhen, E-mail: zhenzhang6@hotmail.com [Departments of Radiation Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China)

    2014-03-15

    Purpose: Ataxia telangiectasia mutated (ATM) protein is important in the DNA damage response because it repairs radiation-induced damage in cancers. We examined the effect of microRNA-223 (miR-223), a regulator of ATM expression, on radiation sensitivity of cancer cells. Methods and Materials: Human embryonic kidney 293 T (293T) cells were infected with pLL3.7-miR-223 plasmid to generate the pLL3.7-miR-223 and -empty virus (EV) lentivirus (miR-223 and EV). A dual luciferase assay in which the reporter contained wild-type 3′ untranslated region (UTR) of ATM was performed. U87MG cells were infected with miR-223 or EV to establish the overexpressed stable cell lines (U87-223 or U87-EV, respectively). Cells were irradiated in vitro, and dose enhancement ratios at 2 Gy (DER{sub 2}) were calculated. Hind legs of BALB/c athymic mice were injected with U87-223 or U87-EV cells; after 2 weeks, half of the tumors were irradiated. Tumor volumes were tracked for a total of 5 weeks. Results: The dual luciferase reporter assay showed a significant reduction in luciferase activity of 293T cells cotransfected with miR-223 and the ATM 3′UTR compared to that in EV control. Overexpression of miR-223 in U87MG cells showed that ATM expression was significantly downregulated in the U87-223 cells compared to that in U87-EV (ATM/β-actin mRNA 1.0 vs 1.5, P<.05). U87-223 cells were hypersensitive to radiation compared to U87-EV cells in vitro (DER{sub 2} = 1.32, P<.01). Mice injected with miR-223-expressing tumors had almost the same tumors after 3 weeks (1.5 cm{sup 3} vs 1.7 cm{sup 3}). However, irradiation significantly decreased tumor size in miR-223-expressing tumors compared to those in controls (0.033 cm{sup 3} vs 0.829 cm{sup 3}). Conclusions: miR-223 overexpression downregulates ATM expression and sensitizes U87 cells to radiation in vitro and in vivo. MicroRNA-223 may be a novel cancer-targeting therapy, although its cancer- and patient-specific roles are

  16. LncRNA Expression Profile of Human Thoracic Aortic Dissection by High-Throughput Sequencing.

    Science.gov (United States)

    Sun, Jie; Chen, Guojun; Jing, Yuanwen; He, Xiang; Dong, Jianting; Zheng, Junmeng; Zou, Meisheng; Li, Hairui; Wang, Shifei; Sun, Yili; Liao, Wangjun; Liao, Yulin; Feng, Li; Bin, Jianping

    2018-01-01

    In this study, the long non-coding RNA (lncRNA) expression profile in human thoracic aortic dissection (TAD), a highly lethal cardiovascular disease, was investigated. Human TAD (n=3) and normal aortic tissues (NA) (n=3) were examined by high-throughput sequencing. Bioinformatics analyses were performed to predict the roles of aberrantly expressed lncRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to validate the results. A total of 269 lncRNAs (159 up-regulated and 110 down-regulated) and 2, 255 mRNAs (1 294 up-regulated and 961 down-regulated) were aberrantly expressed in human TAD (fold-change> 1.5, PTAD than in NA. The predicted binding motifs of three up-regulated lncRNAs (ENSG00000248508, ENSG00000226530, and EG00000259719) were correlated with up-regulated RUNX1 (R=0.982, PTAD. These findings suggest that lncRNAs are novel potential therapeutic targets for human TAD. © 2018 The Author(s). Published by S. Karger AG, Basel.

  17. Machine Learning Approaches for Predicting Human Skin Sensitization Hazard

    Science.gov (United States)

    One of ICCVAM’s top priorities is the development and evaluation of non-animal approaches to identify potential skin sensitizers. The complexity of biological events necessary for a substance to elicit a skin sensitization reaction suggests that no single in chemico, in vit...

  18. Multivariate Models for Prediction of Skin Sensitization Hazard in Humans

    Science.gov (United States)

    One of ICCVAM’s highest priorities is the development and evaluation of non-animal approaches to identify potential skin sensitizers. The complexity of biological events necessary for a substance to elicit a skin sensitization reaction suggests that no single alternative me...

  19. Analytical Performances of Human Immunodeficiency Virus Type 1 RNA-Based Amplix® Real-Time PCR Platform for HIV-1 RNA Quantification

    Directory of Open Access Journals (Sweden)

    Christian Diamant Mossoro-Kpinde

    2016-01-01

    Full Text Available Objectives. We evaluated the performances of Amplix real-time PCR platform developed by Biosynex (Strasbourg, France, combining automated station extraction (Amplix station 16 Dx and real-time PCR (Amplix NG, for quantifying plasma HIV-1 RNA by lyophilized HIV-1 RNA-based Amplix reagents targeting gag and LTR, using samples from HIV-1-infected adults from Central African Republic. Results. Amplix real-time PCR assay showed low limit of detection (28 copies/mL, across wide dynamic range (1.4–10 log copies/mL, 100% sensitivity and 99% specificity, high reproducibility, and accuracy with mean bias < 5%. The assay showed excellent correlations and concordance of 95.3% with the reference HIV-1 RNA load assay (Roche, with mean absolute bias of +0.097 log copies/mL by Bland-Altman analysis. The assay was able to detect and quantify the most prevalent HIV-1 subtype strains and the majority of non-B subtypes, CRFs of HIV-1 group M, and HIV-1 groups N and O circulating in Central Africa. The Amplix assay showed 100% sensitivity and 99.6% specificity to diagnose virological failure in clinical samples from antiretroviral drug-experienced patients. Conclusions. The HIV-1 RNA-based Amplix real-time PCR platform constitutes sensitive and reliable system for clinical monitoring of HIV-1 RNA load in HIV-1-infected children and adults, particularly adapted to intermediate laboratory facilities in sub-Saharan Africa.

  20. Characterization of an Hfq dependent antisense sRNA in the Gram-positive human pathogen Listeria monocytogenes

    DEFF Research Database (Denmark)

    Nielsen, Jesper Sejrup; Lei Kristensen, Lisbeth; Hanghøj Chrisitansen, Mie

    between sRNA and target mRNA rely on the RNA chaperone Hfq. Hfq is a ubiquitous protein found in almost all genres of bacterial life. However, so far its role as an RNA chaperone has only been described in Gram-negative species such as Escherichia coli and Salmonella (Vogel, J. 2009). We previously...... identified several Hfq-binding sRNAs in the Gram-positive human pathogen L. monocytogenes (Christiansen et al 2006). Through bioinformatics, we have identified a number of candidate targets for one of these sRNAs (LhrA). Here, we present the characterization of one of these targets. Our results suggest...

  1. miRNA-720 controls stem cell phenotype, proliferation and differentiation of human dental pulp cells.

    Directory of Open Access Journals (Sweden)

    Emilio Satoshi Hara

    Full Text Available Dental pulp cells (DPCs are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP cells from human DPCs and periodontal ligament cells (PDLCs, and performed a locked nucleic acid (LNA-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs, which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.

  2. MicroRNA expression profiles in human cancer cells after ionizing radiation

    International Nuclear Information System (INIS)

    Niemoeller, Olivier M; Niyazi, Maximilian; Corradini, Stefanie; Zehentmayr, Franz; Li, Minglun; Lauber, Kirsten; Belka, Claus

    2011-01-01

    MicroRNAs are regulators of central cellular processes and are implicated in the pathogenesis and prognosis of human cancers. MicroRNAs also modulate responses to anti-cancer therapy. In the context of radiation oncology microRNAs were found to modulate cell death and proliferation after irradiation. However, changes in microRNA expression profiles in response to irradiation have not been comprehensively analyzed so far. The present study's intend is to present a broad screen of changes in microRNA expression following irradiation of different malignant cell lines. 1100 microRNAs (Sanger miRBase release version 14.0) were analyzed in six malignant cell lines following irradiation with clinically relevant doses of 2.0 Gy. MicroRNA levels 6 hours after irradiation were compared to microRNA levels in non-irradiated cells using the 'Geniom Biochip MPEA homo sapiens'. Hierarchical clustering analysis revealed a pattern, which significantly (p = 0.014) discerned irradiated from non-irradiated cells. The expression levels of a number of microRNAs known to be involved in the regulation of cellular processes like apoptosis, proliferation, invasion, local immune response and radioresistance (e. g. miR-1285, miR-24-1, miR-151-5p, let-7i) displayed 2 - 3-fold changes after irradiation. Moreover, several microRNAs previously not known to be radiation-responsive were discovered. Ionizing radiation induced significant changes in microRNA expression profiles in 3 glioma and 3 squamous cell carcinoma cell lines. The functional relevance of these changes is not addressed but should by analyzed by future work especially focusing on clinically relevant endpoints like radiation induced cell death, proliferation, migration and metastasis

  3. Diurnal Variations of Human Circulating Cell-Free Micro-RNA.

    Directory of Open Access Journals (Sweden)

    Niels H H Heegaard

    Full Text Available A 24-hour light and dark cycle-dependent rhythmicity pervades physiological processes in virtually all living organisms including humans. These regular oscillations are caused by external cues to endogenous, independent biological time-keeping systems (clocks. The rhythm is reflected by gene expression that varies in a circadian and specific fashion in different organs and tissues and is regulated largely by dynamic epigenetic and post-transcriptional mechanisms. This leads to well-documented oscillations of specific electrolytes, hormones, metabolites, and plasma proteins in blood samples. An emerging, important class of gene regulators is short single-stranded RNA (micro-RNA, miRNA that interferes post-transcriptionally with gene expression and thus may play a role in the circadian variation of gene expression. MiRNAs are promising biomarkers by virtue of their disease-specific tissue expression and because of their presence as stable entities in the circulation. However, no studies have addressed the putative circadian rhythmicity of circulating, cell-free miRNAs. This question is important both for using miRNAs as biological markers and for clues to miRNA function in the regulation of circadian gene expression. Here, we investigate 92 miRNAs in plasma samples from 24 young male, healthy volunteers repeatedly sampled 9 times during a 24-hour stay in a regulated environment. We demonstrate that a third (26/79 of the measurable plasma miRNAs (using RT-qPCR on a microfluidic system exhibit a rhythmic behavior and are distributed in two main phase patterns. Some of these miRNAs weakly target known clock genes and many have strong targets in intracellular MAPK signaling pathways. These novel findings highlight the importance of considering bio-oscillations in miRNA biomarker studies and suggest the further study of a set of specific circulating miRNAs in the regulation and functioning of biological clocks.

  4. High resolution autoradiographic studies of RNA, protein and DNA synthesis during human eosinophil granulocytopoiesis

    International Nuclear Information System (INIS)

    Wickramasinghe, S.N.; Hughes, M.

    1978-01-01

    Human bone marrow cells which had been incubated with [ 3 H] uridine or [ 3 H]leucine for 1 h were studied using the technique of electron microscope-autoradiography. The autoradiographs revealed the presence of newly-synthesized RNA and protein molecules within or on a proportion of (1) the primary and secondary granules in all classes of eosinophil precursors and (2) the secondary granules in eosinophil granulocytes. It is suggested that the granule-associated RNA molecules may be concerned with the synthesis of at least some of the new protein molecules which were incorporated into the limiting membrane or substance of eosinophil granules long after the immature primary granule stage. Studies of eosinophil precursors which had been incubated with [ 3 H]thymidine for 1 h showed that the eosinophil granules did not label with this DNA precursor. (author)

  5. RNA profiles of porcine embryos during genome activation reveal complex metabolic switch sensitive to in vitro conditions

    DEFF Research Database (Denmark)

    Østrup, Olga; Olbricht, Gayla; Østrup, Esben

    2013-01-01

    produced in vitro. Overall, our data are in good accordance with previously published, genome-wide profiling data in other species. Moreover, comparison with mouse and human embryos showed striking overlap in functional annotation of transcripts during the EGA, suggesting conserved basic mechanisms...... a handful of reports characterize changing transcriptome profiles and resulting metabolic changes in cleavage stage embryos. The aims of the current study were to investigate RNA profiles of in vivo developed (ivv) and in vitro produced (ivt) porcine embryos before (2-cell stage) and after (late 4-cell...... from oocyte and are imposed either before oocyte aspiration or during in vitro maturation. IVT embryos have altered content of apoptotic factors, cell cycle regulation factors and spindle components, and transcription factors, which all may contribute to reduced developmental competence of embryos...

  6. Yeast RNA viruses as indicators of exosome activity: human exosome hCsl4p participates in RNA degradation in Saccharomyces cerevisiae'.

    Science.gov (United States)

    Ramírez-Garrastacho, Manuel; Esteban, Rosa

    2011-12-01

    The exosome is an evolutionarily conserved 10-mer complex involved in RNA metabolism, located in both the nucleus and the cytoplasm. The cytoplasmic exosome plays an important role in mRNA turnover through its 3'→5' exonucleolytic activity. The superkiller (SKI) phenotype of yeast was originally identified as an increase of killer toxin production due to elevated levels of the L-A double-stranded RNA (dsRNA) Totivirus and its satellite toxin-encoding M dsRNA. Most SKI genes were later shown to be either components of the exosome or modulators of its activity. Variations in the amount of Totivirus are, thus, good indicators of yeast exosome activity, and can be used to analyse its components. Furthermore, if exosome proteins of higher eukaryotes were functional in S. cerevisiae, these viruses would provide a simple tool to analyse their function. In this work, we have found that hCSL4, the human orthologue of SKI4 in the yeast exosome, rescues the null phenotype of the deletion mutant. hCsl4p shares with Ski4p conserved S1 RNA-binding domains, but lacks the N-terminal third of Ski4p. Nevertheless, it interacts with the Dis3p exonuclease of yeast exosome, and partially complements the superkiller phenotype of ski4-1 mutation. The elimination of the N-terminal third of Ski4p does not affect its activity, indicating that it is dispensable for RNA degradation. We have also identified the point mutation G152E in hCSL4, equivalent to the ski4-1 mutation G253E, which impairs the activity of the protein, thus validating our approach of using yeast RNA virus to analyse the exosome of higher eukaryotes. Copyright © 2011 John Wiley & Sons, Ltd.

  7. Developmental changes in hypothalamic oxytocin and oxytocin receptor mRNA expression and their sensitivity to fasting in male and female rats.

    Science.gov (United States)

    Matsuzaki, Toshiya; Iwasa, Takeshi; Munkhzaya, Munkhsaikhan; Tungalagsuvd, Altankhuu; Kawami, Takako; Murakami, Masahiro; Yamasaki, Mikio; Yamamoto, Yuri; Kato, Takeshi; Kuwahara, Akira; Yasui, Toshiyuki; Irahara, Minoru

    2015-04-01

    Oxytocin (OT) affects the central nervous system and is involved in a variety of social and non-social behaviors. Recently, the role played by OT in energy metabolism and its organizational effects on estrogen receptor alpha (ER-α) during the neonatal period have gained attention. In this study, the developmental changes in the hypothalamic mRNA levels of OT, the OT receptor (OTR), and ER-α were evaluated in male and female rats. In addition, the fasting-induced changes in the hypothalamic mRNA levels of OT and the OTR were evaluated. Hypothalamic explants were taken from postnatal day (PND) 10, 20, and 30 rats, and the mRNA level of each molecule was measured. Hypothalamic OT mRNA expression increased throughout the developmental period in both sexes. The rats' hypothalamic OTR mRNA levels were highest on PND 10 and decreased throughout the developmental period. In the male rats, the hypothalamic mRNA levels of ER-α were higher on PND 30 than on PND 10. On the other hand, no significant differences in hypothalamic ER-α mRNA expression were detected among the examined time points in the female rats, although hypothalamic ER-α mRNA expression tended to be higher on PND 30 than on PND 10. Significant positive correlations were detected between hypothalamic OT and ER-α mRNA expression in both the male and female rats. Hypothalamic OT mRNA expression was not affected by fasting at any of the examined time points in either sex. These results indicate that hypothalamic OT expression is not sensitive to fasting during the developmental period. In addition, as a positive correlation was detected between hypothalamic OT and ER-α mRNA expression, these two molecules might interact with each other to induce appropriate neuronal development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Evidence for rRNA 2'-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes.

    Science.gov (United States)

    Erales, Jenny; Marchand, Virginie; Panthu, Baptiste; Gillot, Sandra; Belin, Stéphane; Ghayad, Sandra E; Garcia, Maxime; Laforêts, Florian; Marcel, Virginie; Baudin-Baillieu, Agnès; Bertin, Pierre; Couté, Yohann; Adrait, Annie; Meyer, Mélanie; Therizols, Gabriel; Yusupov, Marat; Namy, Olivier; Ohlmann, Théophile; Motorin, Yuri; Catez, Frédéric; Diaz, Jean-Jacques

    2017-12-05

    Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2'-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2'-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.

  9. An effective tumor-targeting strategy utilizing hypoxia-sensitive siRNA delivery system for improved anti-tumor outcome.

    Science.gov (United States)

    Kang, Lin; Fan, Bo; Sun, Ping; Huang, Wei; Jin, Mingji; Wang, Qiming; Gao, Zhonggao

    2016-10-15

    Hypoxia is a feature of most solid tumors, targeting hypoxia is considered as the best validated yet not extensively exploited strategy in cancer therapy. Here, we reported a novel tumor-targeting strategy using a hypoxia-sensitive siRNA delivery system. In the study, 2-nitroimidazole (NI), a hydrophobic component that can be converted to hydrophilic 2-aminoimidazole (AI) through bioreduction under hypoxic conditions, was conjugated to the alkylated polyethyleneimine (bPEI1.8k-C6) to form amphiphilic bPEI1.8k-C6-NI polycations. bPEI1.8k-C6-NI could self-assemble into micelle-like aggregations in aqueous, which contributed to the improved stability of the bPEI1.8k-C6-NI/siRNA polyplexes, resulted in increased cellular uptake. After being transported into the hypoxic tumor cells, the selective nitro-to-amino reduction would cause structural change and elicit a relatively loose structure to facilitate the siRNA dissociation in the cytoplasm, for enhanced gene silencing efficiency ultimately. Therefore, the conflict between the extracellular stability and the intracellular siRNA release ability of the polyplexes was solved by introducing the hypoxia-responsive unit. Consequently, the survivin-targeted siRNA loaded polyplexes shown remarkable anti-tumor effect not only in hypoxic cells, but also in tumor spheroids and tumor-bearing mice, indicating that the hypoxia-sensitive siRNA delivery system had great potential for tumor-targeted therapy. Hypoxia is one of the most remarkable features of most solid tumors, and targeting hypoxia is considered as the best validated strategy in cancer therapy. However, in the past decades, there were few reports about using this strategy in the drug delivery system, especially in siRNA delivery system. Therefore, we constructed a hypoxia-sensitive siRNA delivery system utilizing a hypoxia-responsive unit, 2-nitroimidazole, by which the unavoidable conflict between improved extracellular stability and promoted intracellular siRNA

  10. Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18

    Science.gov (United States)

    Smirnov, Alexandre; Entelis, Nina; Martin, Robert P.; Tarassov, Ivan

    2011-01-01

    5S rRNA is an essential component of ribosomes of all living organisms, the only known exceptions being mitochondrial ribosomes of fungi, animals, and some protists. An intriguing situation distinguishes mammalian cells: Although the mitochondrial genome contains no 5S rRNA genes, abundant import of the nuclear DNA-encoded 5S rRNA into mitochondria was reported. Neither the detailed mechanism of this pathway nor its rationale was clarified to date. In this study, we describe an elegant molecular conveyor composed of a previously identified human 5S rRNA import factor, rhodanese, and mitochondrial ribosomal protein L18, thanks to which 5S rRNA molecules can be specifically withdrawn from the cytosolic pool and redirected to mitochondria, bypassing the classic nucleolar reimport pathway. Inside mitochondria, the cytosolic 5S rRNA is shown to be associated with mitochondrial ribosomes. PMID:21685364

  11. Characterization of a "TRAMP-like" co-factor of the human RNA exosome

    DEFF Research Database (Denmark)

    Christensen, Marianne Skovgaard; Kristiansen, Maiken Søndergaard; Lubas, Michal Szymon

    Genome-wide studies in yeast, plants and humans have revealed numerous new transcripts in what was previously thought to be silent DNA or junk DNA. One class of non-coding transcript discovered recently is the PROMoter uPstream Transcripts (PROMPTs), which is only seen upon depletion of the RNA...... exosome, the major 3’-5’ exonuclease complex in human cells. PROMPTs have a lot in common with the yeast Cryptic Unstable Transcripts (CUTs), which are degraded by the concerted effort of the exosome, and its co-factor complex TRAMP (Trf4p/Air1p/Mtr4p). We have identified human proteins with functional...... similarities to components of the yeast TRAMP complex, and show that these are involved in the degradation of PROMPTs. While, these proteins form transient complexes with the exosome, our preliminary results also indicate that complex formation can occur directly with catalytic components of the exosome...

  12. Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Te, E-mail: liute79@yahoo.com [School of Environmental Science and Engineering, Donghua University, Shanghai 201620 (China); Shanghai Geriatric Institute of Chinese Medicine, Shanghai 200031 (China); Cheng, Weiwei [International Peace Maternity and Child Health Hospital, Shanghai Jiaotong University, Shanghai 200030 (China); Huang, Yongyi [Laboratoire PROTEE, Batiment R, Universite du Sud Toulon-Var, 83957 LA GARDE Cedex (France); Huang, Qin; Jiang, Lizhen [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Guo, Lihe, E-mail: liute79@yahoo.com [Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2012-02-15

    Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal. Highlights: Black-Right-Pointing-Pointer microRNA-145 inhibits Sox2 expression in human iPS cells. Black-Right-Pointing-Pointer microRNA-145 suppresses the self-renewal and pluripotency of human iPS cells. Black-Right-Pointing-Pointer HuAECs regulate expression of microRNA-145 and Sox2 in human iPS cells. Black-Right-Pointing-Pointer HuAECs feeder

  13. Human amniotic epithelial cell feeder layers maintain human iPS cell pluripotency via inhibited endogenous microRNA-145 and increased Sox2 expression

    International Nuclear Information System (INIS)

    Liu, Te; Cheng, Weiwei; Huang, Yongyi; Huang, Qin; Jiang, Lizhen; Guo, Lihe

    2012-01-01

    Currently, human induced pluripotent stem (iPS) cells were generated from patient or disease-specific sources and share the same key properties as embryonic stem cells. This makes them attractive for personalized medicine, drug screens or cellular therapy. Long-term cultivation and maintenance of normal iPS cells in an undifferentiated self-renewing state are a major challenge. Our previous studies have shown that human amniotic epithelial cells (HuAECs) could provide a good source of feeder cells for mouse and human embryonic stem cells, or spermatogonial stem cells, but the mechanism for this is unknown. Here, we examined the effect of endogenous microRNA-145 regulation on Sox2 expression in human iPS cells by HuAECs feeder cells regulation, and in turn on human iPS cells pluripotency. We found that human IPS cells transfected with a microRNA-145 mutant expressed Sox2 at high levels, allowing iPS to maintain a high level of AP activity in long-term culture and form teratomas in SCID mice. Expression of stem cell markers was increased in iPS transfected with the microRNA-145 mutant, compared with iPS was transfected with microRNA-145. Besides, the expression of Drosha proteins of the microRNA-processor complex, required for the generation of precursor pre-miRNA, was significantly increased in human iPS cells cultured on MEF but not on HuAECs. Taken together, these results suggest that endogenous Sox2 expression may be regulated by microRNA-145 in human iPS cells with HuAECs feeder cells, and Sox2 is a crucial component required for maintenance of them in an undifferentiated, proliferative state capable of self-renewal. Highlights: ► microRNA-145 inhibits Sox2 expression in human iPS cells. ► microRNA-145 suppresses the self-renewal and pluripotency of human iPS cells. ► HuAECs regulate expression of microRNA-145 and Sox2 in human iPS cells. ► HuAECs feeder layers maintain human iPS cells pluripotency. ► HuAECs negatively regulates the synthesis of

  14. Toehold-mediated nonenzymatic amplification circuit on graphene oxide fluorescence switching platform for sensitive and homogeneous microRNA detection

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Ru; Liao, Yuhui; Zhou, Xiaoming, E-mail: zhouxm@scnu.edu.cn; Xing, Da, E-mail: xingda@scnu.edu.cn

    2015-08-12

    A novel graphene oxide (GO) fluorescence switch-based homogenous system has been developed to solve two problems that are commonly encountered in conventional GO-based biosensors. First, with the assistance of toehold-mediated nonenzymatic amplification (TMNA), the sensitivity of this system greatly surpasses that of previously described GO-based biosensors, which are always limited to the nM range due to the lack of efficient signal amplification. Second, without enzymatic participation in amplification, the unreliability of detection resulting from nonspecific desorption of DNA probes on the GO surface by enzymatic protein can be avoided. Moreover, the interaction mechanism of the double-stranded TMNA products contains several single-stranded toeholds at two ends and GO has also been explored with combinations of atomic force microscopy imaging, zeta potential detection, and fluorescence assays. It has been shown that the hybrids can be anchored to the surface of GO through the end with more unpaired bases, and that the other end, which has weaker interaction with GO, can escape GO adsorption due to the robustness of the central dsDNA structures. We verified this GO fluorescence switch-based detection system by detecting microRNA 21, an overexpressed non-encoding gene in a variety of malignant cells. Rational design of the probes allowed the isothermal nonenzymatic reaction to achieve more than 100-fold amplification efficiency. The detection results showed that our strategy has a detection limit of 10 pM and a detection range of four orders of magnitude. - Highlights: • This paper explored the interaction mechanism of TMNA products with GO surface. • This homogeneous and isothermal system permits a detection limit of 10 pM for microRNA. • This nonenzymatic strategy can avoid nonspecific desorption caused by enzyme protein. • The interaction model can be used to explore the application ability of nonenzymatic circuit.

  15. Differential sensitivity to human communication in dogs, wolves, and human infants.

    Science.gov (United States)

    Topál, József; Gergely, György; Erdohegyi, Agnes; Csibra, Gergely; Miklósi, Adám

    2009-09-04

    Ten-month-old infants persistently search for a hidden object at its initial hiding place even after observing it being hidden at another location. Recent evidence suggests that communicative cues from the experimenter contribute to the emergence of this perseverative search error. We replicated these results with dogs (Canis familiaris), who also commit more search errors in ostensive-communicative (in 75% of the total trials) than in noncommunicative (39%) or nonsocial (17%) hiding contexts. However, comparative investigations suggest that communicative signals serve different functions for dogs and infants, whereas human-reared wolves (Canis lupus) do not show doglike context-dependent differences of search errors. We propose that shared sensitivity to human communicative signals stems from convergent social evolution of the Homo and the Canis genera.

  16. Large-scale benchmarking reveals false discoveries and count transformation sensitivity in 16S rRNA gene amplicon data analysis methods used in microbiome studies

    DEFF Research Database (Denmark)

    Thorsen, Jonathan; Brejnrod, Asker Daniel; Mortensen, Martin Steen

    2016-01-01

    BACKGROUND: There is an immense scientific interest in the human microbiome and its effects on human physiology, health, and disease. A common approach for examining bacterial communities is high-throughput sequencing of 16S rRNA gene hypervariable regions, aggregating sequence-similar amplicons...

  17. Multivariate Models for Prediction of Human Skin Sensitization Hazard.

    Science.gov (United States)

    One of the lnteragency Coordinating Committee on the Validation of Alternative Method's (ICCVAM) top priorities is the development and evaluation of non-animal approaches to identify potential skin sensitizers. The complexity of biological events necessary to produce skin sensiti...

  18. Structural requirements for the binding of tRNA Lys3 to reverse transcriptase of the human immunodeficiency virus type 1

    NARCIS (Netherlands)

    Oude Essink, B. B.; Das, A. T.; Berkhout, B.

    1995-01-01

    Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) RNA genome is primed by the cellular tRNA Lys3 molecule. Packaging of this tRNA primer during virion assembly is thought to be mediated by specific interactions with the reverse transcriptase (RT) protein. Portions of the tRNA

  19. Suppression of NRF2–ARE activity sensitizes chemotherapeutic agent-induced cytotoxicity in human acute monocytic leukemia cells

    International Nuclear Information System (INIS)

    Peng, Hui; Wang, Huihui; Xue, Peng; Hou, Yongyong; Dong, Jian; Zhou, Tong; Qu, Weidong; Peng, Shuangqing; Li, Jin; Carmichael, Paul L.; Nelson, Bud; Clewell, Rebecca; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

    2016-01-01

    Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the antioxidant response element (ARE)-dependent transcription, plays a pivotal role in chemical detoxification in normal and tumor cells. Consistent with previous findings that NRF2–ARE contributes to chemotherapeutic resistance of cancer cells, we found that stable knockdown of NRF2 by lentiviral shRNA in human acute monocytic leukemia (AML) THP-1 cells enhanced the cytotoxicity of several chemotherapeutic agents, including arsenic trioxide (As 2 O 3 ), etoposide and doxorubicin. Using an ARE-luciferase reporter expressed in several human and mouse cells, we identified a set of compounds, including isonicotinic acid amides, isoniazid and ethionamide, that inhibited NRF2–ARE activity. Treatment of THP-1 cells with ethionamide, for instance, significantly reduced mRNA expression of multiple ARE-driven genes under either basal or As 2 O 3 -challenged conditions. As determined by cell viability and cell cycle, suppression of NRF2–ARE by ethionamide also significantly enhanced susceptibility of THP-1 and U937 cells to As 2 O 3 -induced cytotoxicity. In THP-1 cells, the sensitizing effect of ethionamide on As 2 O 3 -induced cytotoxicity was highly dependent on NRF2. To our knowledge, the present study is the first to demonstrate that ethionamide suppresses NRF2–ARE signaling and disrupts the transcriptional network of the antioxidant response in AML cells, leading to sensitization to chemotherapeutic agents. - Highlights: • Identification of novel inhibitors of ARE-dependent transcription • Suppression of NRF2–ARE sensitizes THP-1 cells to chemotherapy. • Ethionamide suppresses ARE-dependent transcriptional activity. • Ethionamide and isoniazid increase the cytotoxicity of As 2 O 3 in AML cells. • Sensitization of THP-1 cells to As 2 O 3 toxicity by ethionamide is NRF2-dependent.

  20. Non-codingRNA sequence variations in human chronic lymphocytic leukemia and colorectal cancer.

    Science.gov (United States)

    Wojcik, Sylwia E; Rossi, Simona; Shimizu, Masayoshi; Nicoloso, Milena S; Cimmino, Amelia; Alder, Hansjuerg; Herlea, Vlad; Rassenti, Laura Z; Rai, Kanti R; Kipps, Thomas J; Keating, Michael J; Croce, Carlo M; Calin, George A

    2010-02-01

    Cancer is a genetic disease in which the interplay between alterations in protein-coding genes and non-coding RNAs (ncRNAs) plays a fundamental role. In recent years, the full coding component of the human genome was sequenced in various cancers, whereas such attempts related to ncRNAs are still fragmentary. We screened genomic DNAs for sequence variations in 148 microRNAs (miRNAs) and ultraconserved regions (UCRs) loci in patients with chronic lymphocytic leukemia (CLL) or colorectal cancer (CRC) by Sanger technique and further tried to elucidate the functional consequences of some of these variations. We found sequence variations in miRNAs in both sporadic and familial CLL cases, mutations of UCRs in CLLs and CRCs and, in certain instances, detected functional effects of these variations. Furthermore, by integrating our data with previously published data on miRNA sequence variations, we have created a catalog of DNA sequence variations in miRNAs/ultraconserved genes in human cancers. These findings argue that ncRNAs are targeted by both germ line and somatic mutations as well as by single-nucleotide polymorphisms with functional significance for human tumorigenesis. Sequence variations in ncRNA loci are frequent and some have functional and biological significance. Such information can be exploited to further investigate on a genome-wide scale the frequency of genetic variations in ncRNAs and their functional meaning, as well as for the development of new diagnostic and prognostic markers for leukemias and carcinomas.

  1. Comprehensive microRNA profiling in B-cells of human centenarians by massively parallel sequencing

    Directory of Open Access Journals (Sweden)

    Gombar Saurabh

    2012-07-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are small, non-coding RNAs that regulate gene expression and play a critical role in development, homeostasis, and disease. Despite their demonstrated roles in age-associated pathologies, little is known about the role of miRNAs in human aging and longevity. Results We employed massively parallel sequencing technology to identify miRNAs expressed in B-cells from Ashkenazi Jewish centenarians, i.e., those living to a hundred and a human model of exceptional longevity, and younger controls without a family history of longevity. With data from 26.7 million reads comprising 9.4 × 108 bp from 3 centenarian and 3 control individuals, we discovered a total of 276 known miRNAs and 8 unknown miRNAs ranging several orders of magnitude in expression levels, a typical characteristics of saturated miRNA-sequencing. A total of 22 miRNAs were found to be significantly upregulated, with only 2 miRNAs downregulated, in centenarians as compared to controls. Gene Ontology analysis of the predicted and validated targets of the 24 differentially expressed miRNAs indicated enrichment of functional pathways involved in cell metabolism, cell cycle, cell signaling, and cell differentiation. A cross sectional expression analysis of the differentially expressed miRNAs in B-cells from Ashkenazi Jewish individuals between the 50th and 100th years of age indicated that expression levels of miR-363* declined significantly with age. Centenarians, however, maintained the youthful expression level. This result suggests that miR-363* may be a candidate longevity-associated miRNA. Conclusion Our comprehensive miRNA data provide a resource for further studies to identify genetic pathways associated with aging and longevity in humans.

  2. Structural profiles of human miRNA families from pairwise clustering

    DEFF Research Database (Denmark)

    Kaczkowski, Bogumil; Þórarinsson, Elfar; Reiche, Kristin

    2009-01-01

    secondary structure already predicted, little is known about the patterns of structural conservation among pre-miRNAs. We address this issue by clustering the human pre-miRNA sequences based on pairwise, sequence and secondary structure alignment using FOLDALIGN, followed by global multiple alignment...... of obtained clusters by WAR. As a result, the common secondary structure was successfully determined for four FOLDALIGN clusters: the RF00027 structural family of the Rfam database and three clusters with previously undescribed consensus structures. Availability: http://genome.ku.dk/resources/mirclust...

  3. MicroRNA-223 Expression Is Upregulated in Insulin Resistant Human Adipose Tissue

    Directory of Open Access Journals (Sweden)

    Tung-Yueh Chuang

    2015-01-01

    Full Text Available MicroRNAs (miRNAs are short noncoding RNAs involved in posttranscriptional regulation of gene expression and influence many cellular functions including glucose and lipid metabolism. We previously reported that adipose tissue (AT from women with polycystic ovary syndrome (PCOS or controls with insulin resistance (IR revealed a differentially expressed microRNA (miRNA profile, including upregulated miR-93 in PCOS patients and in non-PCOS women with IR. Overexpressed miR-93 directly inhibited glucose transporter isoform 4 (GLUT4 expression, thereby influencing glucose metabolism. We have now studied the role of miR-223, which is also abnormally expressed in the AT of IR subjects. Our data indicates that miR-223 is significantly overexpressed in the AT of IR women, regardless of whether they had PCOS or not. miR-223 expression in AT was positively correlated with HOMA-IR. Unlike what is reported in cardiomyocytes, overexpression of miR-223 in human differentiated adipocytes was associated with a reduction in GLUT4 protein content and insulin-stimulated glucose uptake. In addition, our data suggests miR-223 regulates GLUT4 expression by direct binding to its 3′ untranslated region (3′UTR. In conclusion, in AT miR-223 is an IR-related miRNA that may serve as a potential therapeutic target for the treatment of IR-related disorders.

  4. Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors

    Energy Technology Data Exchange (ETDEWEB)

    Yakubov, Eduard [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel); Rechavi, Gidi [Cancer Research Center, Chaim Sheba Medical Center, Tel-Hashomer and Sackler School of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rozenblatt, Shmuel [Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Tel-Aviv (Israel); Givol, David, E-mail: david.givol@weizmann.ac.il [Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot (Israel)

    2010-03-26

    Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.

  5. The Human Microbiome and Understanding the 16S rRNA Gene in Translational Nursing Science.

    Science.gov (United States)

    Ames, Nancy J; Ranucci, Alexandra; Moriyama, Brad; Wallen, Gwenyth R

    As more is understood regarding the human microbiome, it is increasingly important for nurse scientists and healthcare practitioners to analyze these microbial communities and their role in health and disease. 16S rRNA sequencing is a key methodology in identifying these bacterial populations that has recently transitioned from use primarily in research to having increased utility in clinical settings. The objectives of this review are to (a) describe 16S rRNA sequencing and its role in answering research questions important to nursing science; (b) provide an overview of the oral, lung, and gut microbiomes and relevant research; and (c) identify future implications for microbiome research and 16S sequencing in translational nursing science. Sequencing using the 16S rRNA gene has revolutionized research and allowed scientists to easily and reliably characterize complex bacterial communities. This type of research has recently entered the clinical setting, one of the best examples involving the use of 16S sequencing to identify resistant pathogens, thereby improving the accuracy of bacterial identification in infection control. Clinical microbiota research and related requisite methods are of particular relevance to nurse scientists-individuals uniquely positioned to utilize these techniques in future studies in clinical settings.

  6. Mi-DISCOVERER: A bioinformatics tool for the detection of mi-RNA in human genome.

    Science.gov (United States)

    Arshad, Saadia; Mumtaz, Asia; Ahmad, Freed; Liaquat, Sadia; Nadeem, Shahid; Mehboob, Shahid; Afzal, Muhammad

    2010-11-27

    MicroRNAs (miRNAs) are 22 nucleotides non-coding RNAs that play pivotal regulatory roles in diverse organisms including the humans and are difficult to be identified due to lack of either sequence features or robust algorithms to efficiently identify. Therefore, we made a tool that is Mi-Discoverer for the detection of miRNAs in human genome. The tools used for the development of software are Microsoft Office Access 2003, the JDK version 1.6.0, BioJava version 1.0, and the NetBeans IDE version 6.0. All already made miRNAs softwares were web based; so the advantage of our project was to make a desktop facility to the user for sequence alignment search with already identified miRNAs of human genome present in the database. The user can also insert and update the newly discovered human miRNA in the database. Mi-Discoverer, a bioinformatics tool successfully identifies human miRNAs based on multiple sequence alignment searches. It's a non redundant database containing a large collection of publicly available human miRNAs.

  7. Deciphering the transcriptional circuitry of microRNA genes expressed during human monocytic differentiation

    KAUST Repository

    Schmeier, Sebastian; MacPherson, Cameron R; Essack, Magbubah; Kaur, Mandeep; Schaefer, Ulf; Suzuki, Harukazu; Hayashizaki, Yoshihide; Bajic, Vladimir B.

    2009-01-01

    Background: Macrophages are immune cells involved in various biological processes including host defence, homeostasis, differentiation, and organogenesis. Disruption of macrophage biology has been linked to increased pathogen infection, inflammation and malignant diseases. Differential gene expression observed in monocytic differentiation is primarily regulated by interacting transcription factors (TFs). Current research suggests that microRNAs (miRNAs) degrade and repress translation of mRNA, but also may target genes involved in differentiation. We focus on getting insights into the transcriptional circuitry regulating miRNA genes expressed during monocytic differentiation. Results: We computationally analysed the transcriptional circuitry of miRNA genes during monocytic differentiation using in vitro time-course expression data for TFs and miRNAs. A set of TF?miRNA associations was derived from predicted TF binding sites in promoter regions of miRNA genes. Time-lagged expression correlation analysis was utilised to evaluate the TF?miRNA associations. Our analysis identified 12 TFs that potentially play a central role in regulating miRNAs throughout the differentiation process. Six of these 12 TFs (ATF2, E2F3, HOXA4, NFE2L1, SP3, and YY1) have not previously been described to be important for monocytic differentiation. The remaining six TFs are CEBPB, CREB1, ELK1, NFE2L2, RUNX1, and USF2. For several miRNAs (miR-21, miR-155, miR-424, and miR-17-92), we show how their inferred transcriptional regulation impacts monocytic differentiation. Conclusions: The study demonstrates that miRNAs and their transcriptional regulatory control are integral molecular mechanisms during differentiation. Furthermore, it is the first study to decipher on a large-scale, how miRNAs are controlled by TFs during human monocytic differentiation. Subsequently, we have identified 12 candidate key controllers of miRNAs during this differentiation process. 2009 Schmeier et al; licensee Bio

  8. Deciphering the transcriptional circuitry of microRNA genes expressed during human monocytic differentiation

    KAUST Repository

    Schmeier, Sebastian

    2009-12-10

    Background: Macrophages are immune cells involved in various biological processes including host defence, homeostasis, differentiation, and organogenesis. Disruption of macrophage biology has been linked to increased pathogen infection, inflammation and malignant diseases. Differential gene expression observed in monocytic differentiation is primarily regulated by interacting transcription factors (TFs). Current research suggests that microRNAs (miRNAs) degrade and repress translation of mRNA, but also may target genes involved in differentiation. We focus on getting insights into the transcriptional circuitry regulating miRNA genes expressed during monocytic differentiation. Results: We computationally analysed the transcriptional circuitry of miRNA genes during monocytic differentiation using in vitro time-course expression data for TFs and miRNAs. A set of TF?miRNA associations was derived from predicted TF binding sites in promoter regions of miRNA genes. Time-lagged expression correlation analysis was utilised to evaluate the TF?miRNA associations. Our analysis identified 12 TFs that potentially play a central role in regulating miRNAs throughout the differentiation process. Six of these 12 TFs (ATF2, E2F3, HOXA4, NFE2L1, SP3, and YY1) have not previously been described to be important for monocytic differentiation. The remaining six TFs are CEBPB, CREB1, ELK1, NFE2L2, RUNX1, and USF2. For several miRNAs (miR-21, miR-155, miR-424, and miR-17-92), we show how their inferred transcriptional regulation impacts monocytic differentiation. Conclusions: The study demonstrates that miRNAs and their transcriptional regulatory control are integral molecular mechanisms during differentiation. Furthermore, it is the first study to decipher on a large-scale, how miRNAs are controlled by TFs during human monocytic differentiation. Subsequently, we have identified 12 candidate key controllers of miRNAs during this differentiation process. 2009 Schmeier et al; licensee Bio

  9. Forced selection of a human immunodeficiency virus type 1 variant that uses a non-self tRNA primer for reverse transcription: Involvement of viral RNA sequences and the reverse transcriptase enzyme

    NARCIS (Netherlands)

    Abbink, Truus E. M.; Beerens, Nancy; Berkhout, Ben

    2004-01-01

    Human immunodeficiency virus type 1 uses the tRNA(3)(Lys) molecule as a selective primer for reverse transcription. This primer specificity is imposed by sequence complementarity between the tRNA primer and two motifs in the viral RNA genome: the primer-binding site (PBS) and the primer activation

  10. Localization of calcium-binding proteins and GABA transporter (GAT-1) messenger RNA in the human subthalamic nucleus

    International Nuclear Information System (INIS)

    Augood, S.J.; Waldvogel, H.J.; Muenkle, M.C.; Faull, R.L.M.; Emson, P.C.

    1999-01-01

    The distribution of messenger RNA encoding the human GAT-1 (a high-affinity GABA transporter) was investigated in the subthalamic nucleus of 10 neurologically normal human post mortem cases. Further, the distribution of messenger RNA and protein encoding the three neuronally expressed calcium-binding proteins (calbindin D28k, parvalbumin and calretinin) was similarly investigated using in situ hybridization and immunohistochemical techniques. Cellular sites of calbindin D28k, parvalbumin, calretinin and GAT-1 messenger RNA expression were localized using human-specific oligonucleotide probes radiolabelled with [ 35 S]dATP. Sites of protein localization were visualized using specific anti-calbindin D28k, anti-parvalbumin and anti-calretinin antisera. Examination of emulsion-coated tissue sections processed for in situ hybridization revealed an intense signal for GAT-1 messenger RNA within the human subthalamic nucleus, indeed the majority of Methylene Blue-counterstained cells were enriched in this transcript. Further, a marked heterogeneity was noted with regard to the expression of the messenger RNA's encoding the three calcium-binding proteins; this elliptical nucleus was highly enriched in parvalbumin messenger RNA-positive neurons and calretinin mRNA-positive cells but not calbindin messenger RNA-positive cells. Indeed, only an occasional calbindin messenger RNA-positive cell was detected within the mediolateral extent of the nucleus. In marked contrast, numerous parvalbumin messenger RNA-positive cells and calretinin messenger RNA-positive cells were detected and they were topographically distributed; parvalbumin messenger RNA-positive cells were highly enriched in the dorsal subthalamic nucleus extending mediolaterally; calretinin messenger RNA-positive cells were more enriched ventrally although some degree of overlap was apparent. Computer-assisted analysis of the average cross-sectional somatic area of parvalbumin, calretinin and GAT-1 messenger RNA

  11. Quantitative relationship between the local lymph node assay and human skin sensitization assays.

    Science.gov (United States)

    Schneider, K; Akkan, Z

    2004-06-01

    The local lymph node assay (LLNA) is a new test method which allows for the quantitative assessment of sensitizing potency in the mouse. Here, we investigate the quantitative correlation between results from the LLNA and two human sensitization tests--specifically, human repeat insult patch tests (HRIPTs) and human maximization tests (HMTs). Data for 57 substances were evaluated, of which 46 showed skin sensitizing properties in human tests, whereas 11 yielded negative results in humans. For better comparability data from mouse and human tests were transformed to applied doses per skin area, which ranged over four orders of magnitude for the substances considered. Regression analysis for the 46 human sensitizing substances revealed a significant positive correlation between the LLNA and human tests. The correlation was better between LLNA and HRIPT data (n=23; r=0.77) than between LLNA and HMT data (n=38; r=0.65). The observed scattering of data points is related to various uncertainties, in part associated with insufficiencies of data from older HMT studies. Predominantly negative results in the LLNA for another 11 substances which showed no skin sensitizing activity in human maximization tests further corroborate the correspondence between LLNA and human tests. Based on this analysis, the LLNA can be considered a reliable basis for relative potency assessments for skin sensitizers. Proposals are made for the regulatory exploitation of the LLNA: four potency groups can be established, and assignment of substances to these groups according to the outcome of the LLNA can be used to characterize skin sensitizing potency in substance-specific assessments. Moreover, based on these potency groups, a more adequate consideration of sensitizing substances in preparations becomes possible. It is proposed to replace the current single concentration limit for skin sensitizers in preparations, which leads to an all or nothing classification of a preparation as sensitizing to

  12. Deep RNA sequencing reveals dynamic regulation of myocardial noncoding RNAs in failing human heart and remodeling with mechanical circulatory support.

    Science.gov (United States)

    Yang, Kai-Chien; Yamada, Kathryn A; Patel, Akshar Y; Topkara, Veli K; George, Isaac; Cheema, Faisal H; Ewald, Gregory A; Mann, Douglas L; Nerbonne, Jeanne M

    2014-03-04

    Microarrays have been used extensively to profile transcriptome remodeling in failing human heart, although the genomic coverage provided is limited and fails to provide a detailed picture of the myocardial transcriptome landscape. Here, we describe sequencing-based transcriptome profiling, providing comprehensive analysis of myocardial mRNA, microRNA (miRNA), and long noncoding RNA (lncRNA) expression in failing human heart before and after mechanical support with a left ventricular (LV) assist device (LVAD). Deep sequencing of RNA isolated from paired nonischemic (NICM; n=8) and ischemic (ICM; n=8) human failing LV samples collected before and after LVAD and from nonfailing human LV (n=8) was conducted. These analyses revealed high abundance of mRNA (37%) and lncRNA (71%) of mitochondrial origin. miRNASeq revealed 160 and 147 differentially expressed miRNAs in ICM and NICM, respectively, compared with nonfailing LV. Among these, only 2 (ICM) and 5 (NICM) miRNAs are normalized with LVAD. RNASeq detected 18 480, including 113 novel, lncRNAs in human LV. Among the 679 (ICM) and 570 (NICM) lncRNAs differentially expressed with heart failure, ≈10% are improved or normalized with LVAD. In addition, the expression signature of lncRNAs, but not miRNAs or mRNAs, distinguishes ICM from NICM. Further analysis suggests that cis-gene regulation represents a major mechanism of action of human cardiac lncRNAs. The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

  13. MicroRNA-101 regulates T-cell acute lymphoblastic leukemia progression and chemotherapeutic sensitivity by targeting Notch1.

    Science.gov (United States)

    Qian, Lu; Zhang, Wanggang; Lei, Bo; He, Aili; Ye, Lianhong; Li, Xingzhou; Dong, Xin

    2016-11-01

    The present study aimed to investigate the role of microRNA (miR)-101 in acute lymphoblastic leukemia progression and chemoresistance. Furthermore, a novel target gene of miR-101 was identified. Here, we confirmed that miR-101 was significantly downregulated in the blood samples of patients with T-cell acute lymphoblastic leukemia (T-ALL) compared with the healthy controls, as determined by reverse transcription quantitative polymerase chain reaction (RTqPCR) analysis. The in vitro experiments demonstrated that miR-101 significantly repressed the proliferation and invasion, and induced potent apoptosis in Jurkat cells, as determined by CCK-8, flow cytometer and cell invasion assays. Luciferase assay confirmed that Notch1 was a target gene of miR-101, and western blotting showed that miR-101 suppressed the expression of Notch1 at the protein level. Moreover, functional restoration assays revealed that Notch1 mediates the effects of miR-101 on Jurkat cell proliferation, apoptosis and invasion. miR-101 enhanced the sensitivity of Jurkat cells to the chemotherapeutic agent adriamycin. Taken together, our results show for the first time that miR-101 acts as a tumor suppressor in T-cell acute lymphoblastic leukaemia and it could enhance chemotherapeutic sensitivity. Furthermore, Notch1 was identified to be a novel target of miR-101. This study indicates that miR-101 may represent a potential therapeutic target for T-cell acute lymphoblastic leukemia intervention.

  14. Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients.

    Science.gov (United States)

    Lueders, Tillmann; Manefield, Mike; Friedrich, Michael W

    2004-01-01

    Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.

  15. Strong inverse correlation between microRNA-125b and human papillomavirus DNA in productive infection.

    Science.gov (United States)

    Nuovo, Gerard J; Wu, Xin; Volinia, Stefano; Yan, Fengting; di Leva, Gianpiero; Chin, Nena; Nicol, Alcina F; Jiang, Jinmai; Otterson, Gregory; Schmittgen, Thomas D; Croce, Carlo

    2010-09-01

    Infection by the human papillomavirus (HPV) is a cause of cervical intraepithelial neoplasia (CIN) and cancer. microRNA (miRNA) in situ analysis of the transformation zone epithelia, the site of initial cervical HPV infection, showed that miRNAs let-7c, -99a, 26a, and 125b were the most abundantly expressed. In situ testing of CIN 1 showed a dramatic reduction in miR-125b expression in the koilocytes, the cytologic marker of productive HPV infection. A marked reduction in miR-125b was likewise observed in the HPV-infected cells of the condyloma acuminatum, verruca vulgaris, and epidermodysplasia verruciformis. Reverse transcriptase in situ polymerase chain reaction (PCR) showed that the pre-miRNA 125b was present in the koilocyte, suggesting direct inactivation of the mature miRNA. HEK cells transfected with only the antimiR-125b showed perinuclear halos equivalent to HPV-infected koilocytes. NIH 3T3 cells transfected with the HPV 16 full-length genome and mimetic miR-125b showed a marked reduction in viral DNA and protein synthesis by quantitative PCR and in situ-based analyses, respectively (P=0.002). Alternatively, cotransfection with anti-miR-125b and HPV 16 markedly increased HPV DNA (P=0.002). Sequence analyses showed strong homology between L2 of different HPV genotypes and miR-125b. Transfection with HPV 16 L2 resulted in a marked reduction in miR-125b levels in the NIH 3T3 cells. HPV L2-induced inactivation of miR-125b is associated with the classic cytologic changes of the koilocyte, and the exogenous application of mimetic miR-125b markedly inhibits HPV DNA synthesis.

  16. The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

    International Nuclear Information System (INIS)

    Ziehr, Ben; Lenarcic, Erik; Cecil, Chad; Moorman, Nathaniel J.

    2016-01-01

    Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.

  17. Alternative splicing and extensive RNA editing of human TPH2 transcripts.

    Directory of Open Access Journals (Sweden)

    Maik Grohmann

    Full Text Available Brain serotonin (5-HT neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2 in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1 is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre-mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour.

  18. The eIF4AIII RNA helicase is a critical determinant of human cytomegalovirus replication

    Energy Technology Data Exchange (ETDEWEB)

    Ziehr, Ben; Lenarcic, Erik; Cecil, Chad; Moorman, Nathaniel J., E-mail: nmoorman@med.unc.edu

    2016-02-15

    Human cytomegalovirus (HCMV) was recently shown to encode a large number of spliced mRNAs. While the nuclear export of unspliced viral transcripts has been extensively studied, the role of host mRNA export factors in HCMV mRNA trafficking remains poorly defined. We found that the eIF4AIII RNA helicase, a component of the exon junction complex, was necessary for efficient virus replication. Depletion of eIF4AIII limited viral DNA accumulation, export of viral mRNAs from the nucleus, and the production of progeny virus. However eIF4AIII was dispensable for the association of viral transcripts with ribosomes. We found that pateamine A, a natural compound that inhibits both eIF4AI/II and eIF4AIII, has potent antiviral activity and inhibits HCMV replication throughout the virus lytic cycle. Our results demonstrate that eIF4AIII is required for efficient HCMV replication, and suggest that eIF4A family helicases may be a new class of targets for the development of host-directed antiviral therapeutics. - Highlights: • The host eIF4AIII RNA helicase is required for efficient HCMV replication. • Depleting eIF4AIII inhibited the nuclear export of HCMV mRNAs. • HCMV mRNAs did not require eIF4AIII to associate with polyribosomes. • The eIF4A family helicases may be new targets for host-directed antiviral drugs.

  19. Comparison of preribosomal RNA processing pathways in yeast, plant and human cells - focus on coordinated action of endo- and exoribonucleases.

    Science.gov (United States)

    Tomecki, Rafal; Sikorski, Pawel J; Zakrzewska-Placzek, Monika

    2017-07-01

    Proper regulation of ribosome biosynthesis is mandatory for cellular adaptation, growth and proliferation. Ribosome biogenesis is the most energetically demanding cellular process, which requires tight control. Abnormalities in ribosome production have severe consequences, including developmental defects in plants and genetic diseases (ribosomopathies) in humans. One of the processes occurring during eukaryotic ribosome biogenesis is processing of the ribosomal RNA precursor molecule (pre-rRNA), synthesized by RNA polymerase I, into mature rRNAs. It must not only be accurate but must also be precisely coordinated with other phenomena leading to the synthesis of functional ribosomes: RNA modification, RNA folding, assembly with ribosomal proteins and nucleocytoplasmic RNP export. A multitude of ribosome biogenesis factors ensure that these events take place in a correct temporal order. Among them are endo- and exoribonucleases involved in pre-rRNA processing. Here, we thoroughly present a wide spectrum of ribonucleases participating in rRNA maturation, focusing on their biochemical properties, regulatory mechanisms and substrate specificity. We also discuss cooperation between various ribonucleolytic activities in particular stages of pre-rRNA processing, delineating major similarities and differences between three representative groups of eukaryotes: yeast, plants and humans. © 2017 Federation of European Biochemical Societies.

  20. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: Evidence for differential gene expression

    International Nuclear Information System (INIS)

    Kim, Sunyoung; Baltimore, D.; Byrn, R.; Groopman, J.

    1989-01-01

    The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. The authors have established a single-cycle growth condition for HIV in H9 cells, a human CD4 + lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes

  1. shRNA-mediated EMMPRIN silencing inhibits human leukemic monocyte lymphoma U937 cell proliferation and increases chemosensitivity to adriamycin.

    Science.gov (United States)

    Gao, Hui; Jiang, Qixiao; Han, Yantao; Peng, Jianjun; Wang, Chunbo

    2015-03-01

    EMMPRIN is a widely distributed cell surface glycoprotein, which plays an important role in tumor progression and confers resistance to some chemotherapeutic drugs. Recent studies have shown that EMMPRIN overexpression indicates poor prognosis in acute myeloid leukemia (AML). However, little was known on the role of EMMPRIN in leukemia. Human leukemia cell line U937 was stably transfected with a EMMPRIN-targeted shRNA-containing vector to investigate the effect of EMMPRIN on cellular functions. EMMPRIN expression was monitored by qRT-PCR and Western blotting. Cell viability and proliferation were determined by trypan blue exclusion and BrdU labeling, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. Cytotoxicity of chemotherapeutic agent adriamycin on cells was assessed by MTT assay. Knockdown of EMMPRIN gene significantly inhibited cell viability and decreased cell proliferation. Fluorescence-activated cell-sorting analysis revealed that the reduced EMMPRIN expression resulted in cell cycle arrest at G1 phase and induced apoptosis. Meanwhile, western blotting analysis showed that EMMPRIN knockdown was associated with downregulation of cell cycle- and apoptosis-related molecules including cyclin D1, cyclin E, as well as increase in cleavage of caspase-3 and PARP. This study also showed that silencing of EMMPRIN sensitized U937 cells to Adriamycin. EMMPRIN is involved in proliferation, growth, and chemosensitivity of human AML line U937, indicating that EMMPRIN may be a promising therapeutic target for AML.

  2. A microRNA activity map of human mesenchymal tumors: connections to oncogenic pathways; an integrative transcriptomic study

    Directory of Open Access Journals (Sweden)

    Fountzilas Elena

    2012-07-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are nucleic acid regulators of many human mRNAs, and are associated with many tumorigenic processes. miRNA expression levels have been used in profiling studies, but some evidence suggests that expression levels do not fully capture miRNA regulatory activity. In this study we integrate multiple gene expression datasets to determine miRNA activity patterns associated with cancer phenotypes and oncogenic pathways in mesenchymal tumors – a very heterogeneous class of malignancies. Results Using a computational method, we identified differentially activated miRNAs between 77 normal tissue specimens and 135 sarcomas and we validated many of these findings with microarray interrogation of an independent, paraffin-based cohort of 18 tumors. We also showed that miRNA activity is imperfectly correlated with miRNA expression levels. Using next-generation miRNA sequencing we identified potential base sequence alterations which may explain differential activity. We then analyzed miRNA activity changes related to the RAS-pathway and found 21 miRNAs that switch from silenced to activated status in parallel with RAS activation. Importantly, nearly half of these 21 miRNAs were predicted to regulate integral parts of the miRNA processing machinery, and our gene expression analysis revealed significant reductions of these transcripts in RAS-active tumors. These results suggest an association between RAS signaling and miRNA processing in which miRNAs may attenuate their own biogenesis. Conclusions Our study represents the first gene expression-based investigation of miRNA regulatory activity in human sarcomas, and our findings indicate that miRNA activity patterns derived from integrated transcriptomic data are reproducible and biologically informative in cancer. We identified an association between RAS signaling and miRNA processing, and demonstrated sequence alterations as plausible causes for differential miRNA activity

  3. MnO2 nanosheet mediated "DD-A" FRET binary probes for sensitive detection of intracellular mRNA.

    Science.gov (United States)

    Ou, Min; Huang, Jin; Yang, Xiaohai; Quan, Ke; Yang, Yanjing; Xie, Nuli; Wang, Kemin

    2017-01-01

    The donor donor-acceptor (DD-A) FRET model has proven to have a higher FRET efficiency than donor-acceptor acceptor (D-AA), donor-acceptor (D-A), and donor donor-acceptor acceptor (DD-AA) FRET models. The in-tube and in-cell experiments clearly demonstrate that the "DD-A" FRET binary probes can indeed increase the FRET efficiency and provide higher imaging contrast, which is about one order of magnitude higher than the ordinary "D-A" model. Furthermore, MnO 2 nanosheets were employed to deliver these probes into living cells for intracellular TK1 mRNA detection because they can adsorb ssDNA probes, penetrate across the cell membrane and be reduced to Mn 2+ ions by intracellular GSH. The results indicated that the MnO 2 nanosheet mediated "DD-A" FRET binary probes are capable of sensitive and selective sensing gene expression and chemical-stimuli changes in gene expression levels in cancer cells. We believe that the MnO 2 nanosheet mediated "DD-A" FRET binary probes have the potential as a simple but powerful tool for basic research and clinical diagnosis.

  4. Lack of a Close Relationship Between Three Strains of Human Rhinoviruses as Determined by Their RNA Sequences 1

    Science.gov (United States)

    Yin, Fay H.; Lonberg-Holm, K.; Chan, S. P.

    1973-01-01

    The possible genomic homologies between three serotypes of human rhinoviruses (HRV 1A, HRV 2, and HRV 14) were investigated. First we confirmed that these viruses were unrelated by the criterion of the absence of common antigenic determinants on the surfaces of the native virions, as detected by cross-neutralization of complementfixation. RNA-RNA hybridization was then examined with purified, highly radioactive, double-stranded, replicative-form RNA and excess single-stranded virion RNA. Single-stranded RNA showed 100% homology with the minus strand from the replicative-form RNA of the same type of virus. HRV 1A, HRV 2, and HRV 14 showed low intertypic homologies; these were not significantly greater than those found between the rhinoviruses and polivirus, which were used as a negative control. The immunological relationship and the RNA homology between HRV 1A and HRV 1B were also examined by the above techniques. It was confirmed that HRV 1A and HRV 1B share some surface determinants and it was also found that HRV 1B RNA shares 70% homology with HRV 1A RNA. PMID:4126194

  5. Human microRNA oncogenes and tumor suppressors show significantly different biological patterns: from functions to targets.

    Directory of Open Access Journals (Sweden)

    Dong Wang

    Full Text Available MicroRNAs (miRNAs are small noncoding RNAs which play essential roles in many important biological processes. Therefore, their dysfunction is associated with a variety of human diseases, including cancer. Increasing evidence shows that miRNAs can act as oncogenes or tumor suppressors, and although there is great interest in research into these cancer-associated miRNAs, little is known about them. In this study, we performed a comprehensive analysis of putative human miRNA oncogenes and tumor suppressors. We found that miRNA oncogenes and tumor suppressors clearly show different patterns in function, evolutionary rate, expression, chromosome distribution, molecule size, free energy, transcription factors, and targets. For example, miRNA oncogenes are located mainly in the amplified regions in human cancers, whereas miRNA tumor suppressors are located mainly in the deleted regions. miRNA oncogenes tend to cleave target mRNAs more frequently than miRNA tumor suppressors. These results indicate that these two types of cancer-associated miRNAs play different roles in cancer formation and development. Moreover, the patterns identified here can discriminate novel miRNA oncogenes and tumor suppressors with a high degree of accuracy. This study represents the first large-scale bioinformatic analysis of human miRNA oncogenes and tumor suppressors. Our findings provide help for not only understanding of miRNAs in cancer but also for the specific identification of novel miRNAs as miRNA oncogenes and tumor suppressors. In addition, the data presented in this study will be valuable for the study of both miRNAs and cancer.

  6. cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

    DEFF Research Database (Denmark)

    Young, M F; Kerr, J M; Termine, J D

    1990-01-01

    A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells...... osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb....

  7. Caffeine markedly sensitizes human mesothelioma cell lines to pemetrexed

    Science.gov (United States)

    Min, Sang Hee; Goldman, I. David; Zhao, Rongbao

    2013-01-01

    Pemetrexed is a new generation antifolate approved for the treatment of mesothelioma and non-small cell lung cancer. Caffeine is known to augment radiation or chemotherapeutic drug-induced cell killing. The current study addresses the impact of caffeine on the activity of pemetrexed in mesothelioma cell lines. Caffeine enhanced pemetrexed activity in all four mesothelioma cell lines tested (H2052, H2373, H28 and MSTO-211H). Caffeine sensitized H2052 cells in a dose- and schedule-dependent manner, and was associated with a markedly decreased clonogenic survival. Caffeine sensitization occurred only in cells subjected to pulse, but not continuous, exposure to pemetrexed. Similar pemetrexed sensitization was also observed with the clinically better tolerated caffeine analog, theobromine. Pemetrexed sensitization by caffeine was associated with an increase in pemetrexed-induced phosphorylation of ataxia-telangiectasia-mutated (ATM) and Chk1. These data indicate that caffeine and its analog, theobromine, may be a useful approach to enhance pemetrexed-based chemotherapy. PMID:17594092

  8. The prognostic potential and carcinogenesis of long non-coding RNA TUG1 in human cholangiocarcinoma.

    Science.gov (United States)

    Xu, Yi; Leng, Kaiming; Li, Zhenglong; Zhang, Fumin; Zhong, Xiangyu; Kang, Pengcheng; Jiang, Xingming; Cui, Yunfu

    2017-09-12

    Cholangiocarcinoma (CCA) is a fatal disease with increasing worldwide incidence and is characterized by poor prognosis due to its poor response to conventional chemotherapy or radiotherapy. Long non-coding RNAs (lncRNAs) play key roles in multiple human cancers, including CCA. Cancer progression related lncRNA taurine-up-regulated gene 1 (TUG1) was reported to be involved in human carcinomas. However, the impact of TUG1 in CCA is unclear. The aim of this study was to explore the expression pattern of TUG1 and evaluate its clinical significance as well as prognostic potential in CCA. In addition, the functional roles of TUG1 including cell proliferation, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT), were evaluated after TUG1 silencing. Our data demonstrated up-regulation of TUG1 in both CCA tissues and cell lines. Moreover, overexpression of TUG1 is linked to tumor size ( p =0.005), TNM stage ( p =0.013), postoperative recurrence ( p =0.036) and overall survival ( p =0.010) of CCA patients. Furthermore, down-regulation of TUG1 following RNA silencing reduced cell growth and increased apoptosis in CCA cells. Additionally, TUG1 suppression inhibited metastasis potential in vitro by reversing EMT. Overall, our results suggest that TUG1 may be a rational CCA-related prognostic factor and therapeutic target.

  9. Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

    Science.gov (United States)

    Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

    2011-01-01

    Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

  10. A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1.

    Science.gov (United States)

    Wang, Yuru; Havel, Jocelyn; Beal, Peter A

    2015-11-20

    Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. ADAR1 and ADAR2 are two members of the family that have been shown to be catalytically active. Earlier, we reported a phenotypic screen for the study of human ADAR2 using budding yeast S. cerevisiae as the host system. While this screen has been successfully applied to the study of ADAR2, it failed with ADAR1. Here, we report a new reporter that uses a novel editing substrate and is suitable for the study of ADAR1. We screened plasmid libraries with randomized codons for two important residues in human ADAR1 (G1007 and E1008). The screening results combined with in vitro deamination assays led to the identification of mutants that are more active than the wild type protein. Furthermore, a screen of the ADAR1 E1008X library with a reporter construct bearing an A•G mismatch at the editing site suggests one role for the residue at position 1008 is to sense the identity of the base pairing partner for the editing site adenosine. This work has provided a starting point for future in vitro evolution studies of ADAR1 and led to new insight into ADAR's editing site selectivity.

  11. Computational study of ‘HUB’ microRNA in human cardiac diseases

    Science.gov (United States)

    Krishnan, Remya; Nair, Achuthsankar S.; Dhar, Pawan K.

    2017-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs ~22 nucleotides long that do not encode for proteins but have been reported to influence gene expression in normal and abnormal health conditions. Though a large body of scientific literature on miRNAs exists, their network level profile linking molecules with their corresponding phenotypes, is less explored. Here, we studied a network of 191 human miRNAs reported to play a role in 30 human cardiac diseases. Our aim was to study miRNA network properties like hubness and preferred associations, using data mining, network graph theory and statistical analysis. A total of 16 miRNAs were found to have a disease node connectivity of >5 edges (i.e., they were linked to more than 5 diseases) and were considered hubs in the miRNAcardiac disease network. Alternatively, when diseases were considered as hubs, >10 of miRNAs showed up on each ‘disease hub node’. Of all the miRNAs associated with diseases, 19 miRNAs (19/24= 79.1% of upregulated events) were found to be upregulated in atherosclerosis. The data suggest micro RNAs as early stage biological markers in cardiac conditions with potential towards microRNA based therapeutics. PMID:28479745

  12. MicroRNA-143 Downregulates Interleukin-13 Receptor Alpha1 in Human Mast Cells

    Directory of Open Access Journals (Sweden)

    Jianqiu Cheng

    2013-08-01

    Full Text Available MicroRNA-143 (miR-143 was found to be downregulated in allergic rhinitis, and bioinformatics analysis predicted that IL-13Rα1 was a target gene of miR-143. To understand the molecular mechanisms of miR-143 involved in the pathogenesis of allergic inflammation, recombinant miR-143 plasmid vectors were constructed, and human mast cell-1(HMC-1 cells which play a central role in the allergic response were used for study. The plasmids were transfected into HMC-1 cells using a lentiviral vector. Expression of IL-13Rα1 mRNA was then detected by reverse transcriptase polymerase chain reaction (RT-PCR and Western Blotting. The miR-143 lentiviral vector was successfully stably transfected in HMC-1 cells for target gene expression. Compared to the control, the target gene IL-13Rα1 was less expressed in HMC-1 transfected with miR-143 as determined by RT-PCR and Western Blotting (p < 0.05; this difference in expression was statistically significant and the inhibition efficiency was 71%. It indicates that miR-143 directly targets IL-13Rα1 and suppresses IL-13Rα1 expression in HMC-1 cells. Therefore, miR-143 may be associated with allergic reaction in human mast cells.

  13. Chronological changes in microRNA expression in the developing human brain.

    Directory of Open Access Journals (Sweden)

    Michael P Moreau

    Full Text Available MicroRNAs (miRNAs are endogenously expressed noncoding RNA molecules that are believed to regulate multiple neurobiological processes. Expression studies have revealed distinct temporal expression patterns in the developing rodent and porcine brain, but comprehensive profiling in the developing human brain has not been previously reported.We performed microarray and TaqMan-based expression analysis of all annotated mature miRNAs (miRBase 10.0 as well as 373 novel, predicted miRNAs. Expression levels were measured in 48 post-mortem brain tissue samples, representing gestational ages 14-24 weeks, as well as early postnatal and adult time points.Expression levels of 312 miRNAs changed significantly between at least two of the broad age categories, defined as fetal, young, and adult.We have constructed a miRNA expression atlas of the developing human brain, and we propose a classification scheme to guide future studies of neurobiological function.

  14. RNA-Seq Analysis of Human Trigeminal and Dorsal Root Ganglia with a Focus on Chemoreceptors.

    Directory of Open Access Journals (Sweden)

    Caroline Flegel

    Full Text Available The chemosensory capacity of the somatosensory system relies on the appropriate expression of chemoreceptors, which detect chemical stimuli and transduce sensory information into cellular signals. Knowledge of the complete repertoire of the chemoreceptors expressed in human sensory ganglia is lacking. This study employed the next-generation sequencing technique (RNA-Seq to conduct the first expression analysis of human trigeminal ganglia (TG and dorsal root ganglia (DRG. We analyzed the data with a focus on G-protein coupled receptors (GPCRs and ion channels, which are (potentially involved in chemosensation by somatosensory neurons in the human TG and DRG. For years, transient receptor potential (TRP channels have been considered the main group of receptors for chemosensation in the trigeminal system. Interestingly, we could show that sensory ganglia also express a panel of different olfactory receptors (ORs with putative chemosensory function. To characterize OR expression in more detail, we performed microarray, semi-quantitative RT-PCR experiments, and immunohistochemical staining. Additionally, we analyzed the expression data to identify further known or putative classes of chemoreceptors in the human TG and DRG. Our results give an overview of the major classes of chemoreceptors expressed in the human TG and DRG and provide the basis for a broader understanding of the reception of chemical cues.

  15. Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1.

    Science.gov (United States)

    Darlix, J L; Gabus, C; Nugeyre, M T; Clavel, F; Barré-Sinoussi, F

    1990-12-05

    The retroviral genome consists of two identical RNA molecules joined at their 5' ends by the Dimer Linkage Structure (DLS). To study the mechanism of dimerization and the DLS of HIV-1 RNA, large amounts of bona fide HIV-1 RNA and of mutants have been synthesized in vitro. We report that HIV-1 RNA forms dimeric molecules and that viral nucleocapsid (NC) protein NCp15 greatly activates dimerization. Deletion mutagenesis in the RNA 5' 1333 nucleotides indicated that a small domain of 100 nucleotides, located between positions 311 to 415 from the 5' end, is necessary and sufficient to promote HIV-1 RNA dimerization. This dimerization domain encompasses an encapsidation element located between the 5' splice donor site and initiator AUG of gag and shows little sequence variations in different strains of HIV-1. Furthermore, cross-linking analysis of the interactions between NC and HIV-1 RNA (311 to 415) locates a major contact site in the encapsidation element of HIV-1 RNA. The genomic RNA dimer is tightly associated with nucleocapsid protein molecules in avian and murine retroviruses, and this ribonucleoprotein structure is believed to be the template for reverse transcription. Genomic RNA-protein interactions have been analyzed in human immunodeficiency virus (HIV) virions and results showed that NC protein molecules are tightly bound to the genomic RNA dimer. Since retroviral RNA dimerization and packaging appear to be under the control of the same cis element, the encapsidation sequences, and trans-acting factor, the NC protein, they are probably related events in the course of virion assembly.

  16. Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach.

    Directory of Open Access Journals (Sweden)

    Jordan R Plews

    2010-12-01

    Full Text Available Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine.In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3β, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5'-aza-2'-deoxycytidine and cultured in human embryonic stem cell (ES medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days.Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells.

  17. miRNA signature and Dicer requirement during human endometrial stromal decidualization in vitro.

    Directory of Open Access Journals (Sweden)

    Carlos Estella

    Full Text Available Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human

  18. Human glioblastoma-associated microglia/monocytes express a distinct RNA profile compared to human control and murine samples.

    Science.gov (United States)

    Szulzewsky, Frank; Arora, Sonali; de Witte, Lot; Ulas, Thomas; Markovic, Darko; Schultze, Joachim L; Holland, Eric C; Synowitz, Michael; Wolf, Susanne A; Kettenmann, Helmut

    2016-08-01

    Glioblastoma (GBM) is the most aggressive brain tumor in adults. It is strongly infiltrated by microglia and peripheral monocytes that support tumor growth. In the present study we used RNA sequencing to compare the expression profile of CD11b(+) human glioblastoma-associated microglia/monocytes (hGAMs) to CD11b(+) microglia isolated from non-tumor samples. Hierarchical clustering and principal component analysis showed a clear separation of the two sample groups and we identified 334 significantly regulated genes in hGAMs. In comparison to human control microglia hGAMs upregulated genes associated with mitotic cell cycle, cell migration, cell adhesion, and extracellular matrix organization. We validated the expression of several genes associated with extracellular matrix organization in samples of human control microglia, hGAMs, and the hGAMs-depleted fraction via qPCR. The comparison to murine GAMs (mGAMs) showed that both cell populations share a significant fraction of upregulated transcripts compared with their respective controls. These genes were mostly related to mitotic cell cycle. However, in contrast to murine cells, human GAMs did not upregulate genes associated to immune activation. Comparison of human and murine GAMs expression data to several data sets of in vitro-activated human macrophages and murine microglia showed that, in contrast to mGAMs, hGAMs share a smaller overlap to these data sets in general and in particular to cells activated by proinflammatory stimulation with LPS + INFγ or TNFα. Our findings provide new insights into the biology of human glioblastoma-associated microglia/monocytes and give detailed information about the validity of murine experimental models. GLIA 2016 GLIA 2016;64:1416-1436. © 2016 Wiley Periodicals, Inc.

  19. The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

    Science.gov (United States)

    Zuo, Ping; Manley, James L.

    1994-04-01

    ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

  20. Is There a Sensitive Period in Human Incest Avoidance?

    Directory of Open Access Journals (Sweden)

    Liqun Luo

    2011-04-01

    Full Text Available Many studies support the proposition that early cosocialization with opposite-sex children has the effect of inhibiting later mutual sexual attraction, but the existence of a period in the life cycle in which individuals are sensitive to the effect of early cosocialization has been a matter of controversy. Drawing on earlier traditional psychological research, and on more recent work guided by parental investment theory, we hypothesized that only for maternal perinatal association (MPA-absent males a less-than-around-three-years age difference with the sister can predict stronger aversion to sibling incest. The results corroborated the hypothesis. The results can be interpreted as support for the existence of a sensitive period as well as for the potent role of MPA. Cross-cultural comparative studies were called on to further test the hypothesis.

  1. Prevalence of Human Papillomavirus Infection in Unselected SurePath Samples Using the APTIMA HPV mRNA Assay

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte M

    2013-01-01

    The APTIMA Human Papillomavirus (HPV) Assay detects E6/E7 mRNA from 14 human papillomavirus genotypes. Horizon was a population-based split-sample study among well-screened women, with an aim to compare APTIMA, Hybrid Capture 2 (HC2), and liquid-based cytology (LBC) using SurePath samples. APTIMA...

  2. Evaluation of automated RNA-extraction technology and a qualitative HCV assay for sensitivity and detection of HCV RNA in pool-screening systems

    NARCIS (Netherlands)

    Beld, M.; Habibuw, M. R.; Rebers, S. P.; Boom, R.; Reesink, H. W.

    2000-01-01

    BACKGROUND: The objective of this study was the evaluation of NAT technology for the detection of HCV RNA in plasma pools according to the recommendations of the Paul Ehrlich Institute (5000 IU/mL/donation) and the Committee for Proprietary Medical Products (100 IU/mL/manufacturing pool). STUDY

  3. Human sensitization to Prosopis juliflora antigen in Saudi Arabia.

    Science.gov (United States)

    Al-Frayh, A; Hasnain, S M; Gad-El-Rab, M O; Al-Turki, T; Al-Mobeireek, K; Al-Sedairy, S T

    1999-01-01

    Allergenicity to Prosopis juliflora pollen antigen has been reported from only a few countries, including the US, South Africa, India and Kuwait. In some parts of Saudi Arabia, species of Prosopis have been introduced by the millions as roadside ornamentation. There appear to be four flowering seasons during which pollen grains float in all directions. However, the role of Prosopis pollen as the sensitizing and/or triggering agent of allergic asthma and/or rhinitis in the Kingdom has never been evaluated. A total of 473 allergic patients suffering from bronchial asthma in four different geographical regions (Abha, Qassim, Hofuf and Gizan), and attending allergy clinics and chest disease centers of university and Ministry of Health hospitals in the region were tested for immediate hypersensitivity reaction to Prosopis juliflora allergens. Airborne pollen grains at one center were also studied for one full year, using volumetric sampling techniques. A total of 76.1% patients in Qassim, 37.5% in Gizan, 29% in Abha and 11% in Hofuf reacted positively to Prosopis antigen. Multiple sensitivities to other pollen antigens were detected in all patients. The level of airborne Prosopis pollen detected in Gizan exceeded 90 grains m -3 of air. In view of the documented evidence of Prosopis-involved allergenicity, the role of Prosopis pollen as a sensitizing factor in Saudi Arabia has been confirmed. However, the cause of elicitation of symptoms in many multiple sensitive patients, together with the question of cross-reactivities, needs thorough and detailed investigation. In vitro confirmation of all positive results is also required to incriminate Prosopis as one of the major allergens in parts of Saudi Arabia.

  4. Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters

    Directory of Open Access Journals (Sweden)

    Mamoru Oshiki

    2018-04-01

    Full Text Available Detection and genotyping of pathogenic RNA viruses in human and environmental samples are useful for monitoring the circulation and prevalence of these pathogens, whereas a conventional PCR assay followed by Sanger sequencing is time-consuming and laborious. The present study aimed to develop a high-throughput detection-and-genotyping tool for 11 human RNA viruses [Aichi virus; astrovirus; enterovirus; norovirus genogroup I (GI, GII, and GIV; hepatitis A virus; hepatitis E virus; rotavirus; sapovirus; and human parechovirus] using a microfluidic device and next-generation sequencer. Microfluidic nested PCR was carried out on a 48.48 Access Array chip, and the amplicons were recovered and used for MiSeq sequencing (Illumina, Tokyo, Japan; genotyping was conducted by homology searching and phylogenetic analysis of the obtained sequence reads. The detection limit of the 11 tested viruses ranged from 100 to 103 copies/μL in cDNA sample, corresponding to 101–104 copies/mL-sewage, 105–108 copies/g-human feces, and 102–105 copies/g-digestive tissues of oyster. The developed assay was successfully applied for simultaneous detection and genotyping of RNA viruses to samples of human feces, sewage, and artificially contaminated oysters. Microfluidic nested PCR followed by MiSeq sequencing enables efficient tracking of the fate of multiple RNA viruses in various environments, which is essential for a better understanding of the circulation of human pathogenic RNA viruses in the human population.

  5. Optimizing human activity patterns using global sensitivity analysis.

    Science.gov (United States)

    Fairchild, Geoffrey; Hickmann, Kyle S; Mniszewski, Susan M; Del Valle, Sara Y; Hyman, James M

    2014-12-01

    Implementing realistic activity patterns for a population is crucial for modeling, for example, disease spread, supply and demand, and disaster response. Using the dynamic activity simulation engine, DASim, we generate schedules for a population that capture regular (e.g., working, eating, and sleeping) and irregular activities (e.g., shopping or going to the doctor). We use the sample entropy (SampEn) statistic to quantify a schedule's regularity for a population. We show how to tune an activity's regularity by adjusting SampEn, thereby making it possible to realistically design activities when creating a schedule. The tuning process sets up a computationally intractable high-dimensional optimization problem. To reduce the computational demand, we use Bayesian Gaussian process regression to compute global sensitivity indices and identify the parameters that have the greatest effect on the variance of SampEn. We use the harmony search (HS) global optimization algorithm to locate global optima. Our results show that HS combined with global sensitivity analysis can efficiently tune the SampEn statistic with few search iterations. We demonstrate how global sensitivity analysis can guide statistical emulation and global optimization algorithms to efficiently tune activities and generate realistic activity patterns. Though our tuning methods are applied to dynamic activity schedule generation, they are general and represent a significant step in the direction of automated tuning and optimization of high-dimensional computer simulations.

  6. Promoter Analysis Reveals Globally Differential Regulation of Human Long Non-Coding RNA and Protein-Coding Genes

    KAUST Repository

    Alam, Tanvir

    2014-10-02

    Transcriptional regulation of protein-coding genes is increasingly well-understood on a global scale, yet no comparable information exists for long non-coding RNA (lncRNA) genes, which were recently recognized to be as numerous as protein-coding genes in mammalian genomes. We performed a genome-wide comparative analysis of the promoters of human lncRNA and protein-coding genes, finding global differences in specific genetic and epigenetic features relevant to transcriptional regulation. These two groups of genes are hence subject to separate transcriptional regulatory programs, including distinct transcription factor (TF) proteins that significantly favor lncRNA, rather than coding-gene, promoters. We report a specific signature of promoter-proximal transcriptional regulation of lncRNA genes, including several distinct transcription factor binding sites (TFBS). Experimental DNase I hypersensitive site profiles are consistent with active configurations of these lncRNA TFBS sets in diverse human cell types. TFBS ChIP-seq datasets confirm the binding events that we predicted using computational approaches for a subset of factors. For several TFs known to be directly regulated by lncRNAs, we find that their putative TFBSs are enriched at lncRNA promoters, suggesting that the TFs and the lncRNAs may participate in a bidirectional feedback loop regulatory network. Accordingly, cells may be able to modulate lncRNA expression levels independently of mRNA levels via distinct regulatory pathways. Our results also raise the possibility that, given the historical reliance on protein-coding gene catalogs to define the chromatin states of active promoters, a revision of these chromatin signature profiles to incorporate expressed lncRNA genes is warranted in the future.

  7. Migration of human antigen-presenting cells in a human skin graft onto nude mice model after contact sensitization

    NARCIS (Netherlands)

    Hoefakker, S.; Balk, H.P.; Boersma, W.J.A.; Joost, T. van; Notten, W.R.F.; Claassen, E.

    1995-01-01

    Fluorescent contact chemical allergens provoke sensitization after application on both syngeneic and allogeneic skin grafts in mice. We attempted to determine whether the functional activity in a contact sensitization response of human skin graft was affected at the level of antigen uptake and

  8. What did domestication do to dogs? A new account of dogs' sensitivity to human actions.

    Science.gov (United States)

    Udell, Monique A R; Dorey, Nicole R; Wynne, Clive D L

    2010-05-01

    Over the last two decades increasing evidence for an acute sensitivity to human gestures and attentional states in domestic dogs has led to a burgeoning of research into the social cognition of this highly familiar yet previously under-studied animal. Dogs (Canis lupus familiaris) have been shown to be more successful than their closest relative (and wild progenitor) the wolf, and than man's closest relative, the chimpanzee, on tests of sensitivity to human social cues, such as following points to a container holding hidden food. The "Domestication Hypothesis" asserts that during domestication dogs evolved an inherent sensitivity to human gestures that their non-domesticated counterparts do not share. According to this view, sensitivity to human cues is present in dogs at an early age and shows little evidence of acquisition during ontogeny. A closer look at the findings of research on canine domestication, socialization, and conditioning, brings the assumptions of this hypothesis into question. We propose the Two Stage Hypothesis, according to which the sensitivity of an individual animal to human actions depends on acceptance of humans as social companions, and conditioning to follow human limbs. This offers a more parsimonious explanation for the domestic dog's sensitivity to human gestures, without requiring the use of additional mechanisms. We outline how tests of this new hypothesis open directions for future study that offer promise of a deeper understanding of mankind's oldest companion.

  9. X-ray sensitivity of human tumor cells in vitro

    International Nuclear Information System (INIS)

    Weichselbaum, R.R.; Nove, J.; Little, J.B.

    1980-01-01

    Clonally-derived cells from ten human malignant tumors considered radiocurable (breast, neuroblastoma, medulloblastoma) or non-radiocurable (osteosarcoma, hypernephroma, glioblastoma, melanoma) were studied in cell culture and their in vitro x-ray survival curve parameters determined (anti n, D 0 ). There were no significant differences among the tumor cell lines suggesting that survival parameters in vitro do not explain differences in clinical radiocurability. Preliminary investigation with density inhibited human tumor cells indicate that such an approach may yield information regarding inherent cellular differences in radiocurability

  10. Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease.

    Science.gov (United States)

    Mattijssen, Sandy; Hinson, Ella R; Onnekink, Carla; Hermanns, Pia; Zabel, Bernhard; Cresswell, Peter; Pruijn, Ger J M

    2011-07-01

    RNase MRP is a conserved endoribonuclease, in humans consisting of a 267-nucleotide RNA associated with 7-10 proteins. Mutations in its RNA component lead to several autosomal recessive skeletal dysplasias, including cartilage-hair hypoplasia (CHH). Because the known substrates of mammalian RNase MRP, pre-ribosomal RNA, and RNA involved in mitochondrial DNA replication are not likely involved in CHH, we analyzed the effects of RNase MRP (and the structurally related RNase P) depletion on mRNAs using DNA microarrays. We confirmed the upregulation of the interferon-inducible viperin mRNA by RNAi experiments and this appeared to be independent of the interferon response. We detected two cleavage sites for RNase MRP/RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of a mRNA by RNase MRP/RNase P in human cells. Implications for the involvement in the pathophysiology of CHH are discussed.

  11. Noncoding RNA in the Transcriptional Landscape of Human Neural Progenitor Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Patrick eHecht

    2015-10-01

    Full Text Available Increasing evidence suggests that noncoding RNAs play key roles in cellular processes, particularly in the brain. The present study used RNA sequencing to identify the transcriptional landscape of two human neural progenitor cell lines, SK-N-SH and ReNcell CX, as they differentiate into human cortical projection neurons. Protein coding genes were found to account for 54.8% and 57.0% of expressed genes, respectively, and alignment of RNA sequencing reads revealed that only 25.5-28.1% mapped to exonic regions of the genome. Differential expression analysis in the two cell lines identified altered gene expression in both protein coding and noncoding RNAs as they undergo neural differentiation with 222 differentially expressed genes observed in SK-N-SH cells and 19 differentially expressed genes in ReNcell CX. Interestingly, genes showing differential expression in SK-N-SH cells are enriched in genes implicated in autism spectrum disorder, but not in gene sets related to cancer or Alzheimer’s disease. Weighted gene co-expression network analysis (WGCNA was used to detect modules of co-expressed protein coding and noncoding RNAs in SK-N-SH cells and found four modules to be associated with neural differentiation. These modules contain varying levels of noncoding RNAs ranging from 10.7% to 49.7% with gene ontology suggesting roles in numerous cellular processes important for differentiation. These results indicate that noncoding RNAs are highly expressed in human neural progenitor cells and likely hold key regulatory roles in gene networks underlying neural differentiation and neurodevelopmental disorders.

  12. Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast

    Science.gov (United States)

    Díaz, Paula; Sibley, Colin P.; Greenwood, Susan L.

    2016-01-01

    Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO2), reactive oxygen species (ROS) and potassium (K+) channels. Here we test the hypothesis that K+ channels mediate the effects of pO2 and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO2. On days 3–5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO2-sensitive voltage-gated K+ (KV) channels, or ROS (10–1000μM H2O2). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and 86Rb (K+) efflux assessed to estimate syncytiotrophoblast K+ permeability. hCG secretion and 86Rb efflux were significantly greater in explants maintained in 21% pO2 than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO2, and reduced 86Rb efflux at 21% but not 6% pO2. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO2 and unaffected by 4-AP/TEA. H2O2 stimulated 86Rb efflux and hCG secretion at normoxia but decreased 86Rb efflux, without affecting hCG secretion, at 21% pO2. 4-AP/TEA-sensitive K+ channels participate in pO2-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and 86Rb efflux are pO2-dependent but causal links between the two remain to be established. PMID:26863525

  13. RNA interference-based therapeutics for human immunodeficiency virus HIV-1 treatment: synthetic siRNA or vector-based shRNA?

    Science.gov (United States)

    Subramanya, Sandesh; Kim, Sang-Soo; Manjunath, N; Shankar, Premlata

    2010-02-01

    Despite the clinical benefits of highly active antiretroviral therapy (HAART), the prospect of life-long antiretroviral treatment poses significant problems, which has spurred interest in developing new drugs and strategies to treat HIV infection and eliminate persistent viral reservoirs. RNAi has emerged as a therapeutic possibility for HIV. We discuss progress in overcoming hurdles to translating transient and stable RNAi enabling technologies to clinical application for HIV; covering the past 2 - 3 years. HIV inhibition can be achieved by transfection of chemically or enzymatically synthesized siRNAs or by DNA-based vector systems expressing short hairpin RNAs (shRNAs) that are processed intracellularly into siRNA. We compare these approaches, focusing on technical and safety issues that will guide the choice of strategy for clinical use. Introduction of synthetic siRNA into cells or its stable endogenous production using vector-driven shRNA have been shown to suppress HIV replication in vitro and, in some instances, in vivo. Each method has advantages and limitations in terms of ease of delivery, duration of silencing, emergence of escape mutants and potential toxicity. Both appear to have potential as future therapeutics for HIV, once the technical and safety issues of each approach are overcome.

  14. Human error in strabismus surgery: Quantification with a sensitivity analysis

    NARCIS (Netherlands)

    S. Schutte (Sander); J.R. Polling (Jan Roelof); F.C.T. van der Helm (Frans); H.J. Simonsz (Huib)

    2009-01-01

    textabstractBackground: Reoperations are frequently necessary in strabismus surgery. The goal of this study was to analyze human-error related factors that introduce variability in the results of strabismus surgery in a systematic fashion. Methods: We identified the primary factors that influence

  15. Human error in strabismus surgery : Quantification with a sensitivity analysis

    NARCIS (Netherlands)

    Schutte, S.; Polling, J.R.; Van der Helm, F.C.T.; Simonsz, H.J.

    2008-01-01

    Background- Reoperations are frequently necessary in strabismus surgery. The goal of this study was to analyze human-error related factors that introduce variability in the results of strabismus surgery in a systematic fashion. Methods- We identified the primary factors that influence the outcome of

  16. Variable high pressure processing sensitivities for GII human noroviruses

    Science.gov (United States)

    Human norovirus (HuNoV) is the leading cause of foodborne diseases worldwide. High pressure processing (HPP) is one of the most promising non-thermal technologies for decontamination of viral pathogens in foods. However, the survival of HuNoVs by HPP is poorly understood because these viruses cann...

  17. Structure of the central RNA recognition motif of human TIA-1 at 1.95 A resolution

    International Nuclear Information System (INIS)

    Kumar, Amit O.; Swenson, Matthew C.; Benning, Matthew M.; Kielkopf, Clara L.

    2008-01-01

    T-cell-restricted intracellular antigen-1 (TIA-1) regulates alternative pre-mRNA splicing in the nucleus, and mRNA translation in the cytoplasm, by recognizing uridine-rich sequences of RNAs. As a step towards understanding RNA recognition by this regulatory factor, the X-ray structure of the central RNA recognition motif (RRM2) of human TIA-1 is presented at 1.95 A resolution. Comparison with structurally homologous RRM-RNA complexes identifies residues at the RNA interfaces that are conserved in TIA-1-RRM2. The versatile capability of RNP motifs to interact with either proteins or RNA is reinforced by symmetry-related protein-protein interactions mediated by the RNP motifs of TIA-1-RRM2. Importantly, the TIA-1-RRM2 structure reveals the locations of mutations responsible for inhibiting nuclear import. In contrast with previous assumptions, the mutated residues are buried within the hydrophobic interior of the domain, where they would be likely to destabilize the RRM fold rather than directly inhibit RNA binding

  18. CLIP-seq analysis of multi-mapped reads discovers novel functional RNA regulatory sites in the human transcriptome.

    Science.gov (United States)

    Zhang, Zijun; Xing, Yi

    2017-09-19

    Crosslinking or RNA immunoprecipitation followed by sequencing (CLIP-seq or RIP-seq) allows transcriptome-wide discovery of RNA regulatory sites. As CLIP-seq/RIP-seq reads are short, existing computational tools focus on uniquely mapped reads, while reads mapped to multiple loci are discarded. We present CLAM (CLIP-seq Analysis of Multi-mapped reads). CLAM uses an expectation-maximization algorithm to assign multi-mapped reads and calls peaks combining uniquely and multi-mapped reads. To demonstrate the utility of CLAM, we applied it to a wide range of public CLIP-seq/RIP-seq datasets involving numerous splicing factors, microRNAs and m6A RNA methylation. CLAM recovered a large number of novel RNA regulatory sites inaccessible by uniquely mapped reads. The functional significance of these sites was demonstrated by consensus motif patterns and association with alternative splicing (splicing factors), transcript abundance (AGO2) and mRNA half-life (m6A). CLAM provides a useful tool to discover novel protein-RNA interactions and RNA modification sites from CLIP-seq and RIP-seq data, and reveals the significant contribution of repetitive elements to the RNA regulatory landscape of the human transcriptome. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Data in support of transcriptional regulation and function of Fas-antisense long noncoding RNA during human erythropoiesis

    Directory of Open Access Journals (Sweden)

    Olga Villamizar

    2016-06-01

    Full Text Available This paper describes data related to a research article titled, “Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death” [1]. Long noncoding RNAs (lncRNAs are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis. Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf described in the research article. Also included are 5′ untranslated sequences (UTR for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing erythroblasts derived from human CD34+ cells. End point and quantitative RT-PCR data for cDNA prepared using random hexamers versus oligo(dT18 revealed that lncRNA Saf is not effectively polyadenylated. Finally, we include flow cytometry histograms demonstrating Fas levels on maturing erythroblasts derived from human CD34+ cells transduced using mock conditions or with lentivirus particles encoding for Saf.

  20. Evidence supporting oral sensitivity to complex carbohydrates independent of sweet taste sensitivity in humans.

    Directory of Open Access Journals (Sweden)

    Julia Y Q Low

    Full Text Available Compared to simple sugars, complex carbohydrates have been assumed invisible to taste. However, two recent studies proposed that there may be a perceivable taste quality elicited by complex carbohydrates independent of sweet taste. There is precedent with behavioural studies demonstrating that rats are very attracted to complex carbohydrates, and that complex carbohydrates are preferred to simple sugars at low concentrations. This suggests that rats may have independent taste sensors for simple sugars and complex carbohydrates. The aim of this paper is to investigate oral sensitivities of two different classes of complex carbohydrates (a soluble digestible and a soluble non-digestible complex carbohydrate, and to compare these to other caloric and non-nutritive sweeteners in addition to the prototypical tastes using two commonly used psychophysical measures. There were strong correlations between the detection thresholds and mean intensity ratings for complex carbohydrates (maltodextrin, oligofructose (r = 0.94, P 0.05. However, moderate correlations were observed between perceived intensities of complex carbohydrates and sweeteners (r = 0.48-0.61, P < 0.05. These data provide evidence that complex carbohydrates can be sensed in the oral cavity over a range of concentrations independent of sweet taste sensitivity at low concentrations, but with partial overlap with sweet taste intensity at higher concentrations.

  1. Onconase responsive genes in human mesothelioma cells: implications for an RNA damaging therapeutic agent

    International Nuclear Information System (INIS)

    Altomare, Deborah A; Rybak, Susanna M; Pei, Jianming; Maizel, Jacob V; Cheung, Mitchell; Testa, Joseph R; Shogen, Kuslima

    2010-01-01

    Onconase represents a new class of RNA-damaging drugs. Mechanistically, Onconase is thought to internalize, where it degrades intracellular RNAs such as tRNA and double-stranded RNA, and thereby suppresses protein synthesis. However, there may be additional or alternative mechanism(s) of action. In this study, microarray analysis was used to compare gene expression profiles in untreated human malignant mesothelioma (MM) cell lines and cells exposed to 5 μg/ml Onconase for 24 h. A total of 155 genes were found to be regulated by Onconase that were common to both epithelial and biphasic MM cell lines. Some of these genes are known to significantly affect apoptosis (IL-24, TNFAIP3), transcription (ATF3, DDIT3, MAFF, HDAC9, SNAPC1) or inflammation and the immune response (IL-6, COX-2). RT-PCR analysis of selected up- or down-regulated genes treated with varying doses and times of Onconase generally confirmed the expression array findings in four MM cell lines. Onconase treatment consistently resulted in up-regulation of IL-24, previously shown to have tumor suppressive activity, as well as ATF3 and IL-6. Induction of ATF3 and the pro-apoptotic factor IL-24 by Onconase was highest in the two most responsive MM cell lines, as defined by DNA fragmentation analysis. In addition to apoptosis, gene ontology analysis indicated that pathways impacted by Onconase include MAPK signaling, cytokine-cytokine-receptor interactions, and Jak-STAT signaling. These results provide a broad picture of gene activity after treatment with a drug that targets small non-coding RNAs and contribute to our overall understanding of MM cell response to Onconase as a therapeutic strategy. The findings provide insights regarding mechanisms that may contribute to the efficacy of this novel drug in clinical trials of MM patients who have failed first line chemotherapy or radiation treatment

  2. Heritability in the efficiency of nonsense-mediated mRNA decay in humans.

    LENUS (Irish Health Repository)

    Seoighe, Cathal

    2010-01-01

    BACKGROUND: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. PRINCIPAL FINDINGS: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. CONCLUSIONS: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3.

  3. Heritability in the efficiency of nonsense-mediated mRNA decay in humans

    KAUST Repository

    Seoighe, Cathal

    2010-07-21

    Background: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. Principal Findings: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. Conclusions: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3. © 2010 Seoighe, Gehring.

  4. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

    Science.gov (United States)

    Eskildsen, Tilde; Taipaleenmäki, Hanna; Stenvang, Jan; Abdallah, Basem M.; Ditzel, Nicholas; Nossent, Anne Yael; Bak, Mads; Kauppinen, Sakari; Kassem, Moustapha

    2011-01-01

    Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3′ UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central role in promoting osteoblast differentiation, as a bona fide target of miR-138. We show that miR-138 attenuates bone formation in vivo, at least in part by inhibiting the focal adhesion kinase signaling pathway. Our findings suggest that pharmacological inhibition of miR-138 by antimiR-138 could represent a therapeutic strategy for enhancing bone formation in vivo. PMID:21444814

  5. Effect of ethanol of the radiation sensitivity of human hemoglobin

    International Nuclear Information System (INIS)

    Szweda-Lewandowska, Z.; Puchala, M.

    1981-01-01

    Radiation sensitivity of oxy-, deoxy-, and methemoglobin (HbOs, Hbbj, and MetHb) in water solutions containing 0.2 M ethanol and in ethanol-free solutions was compared. Radiation sensitivity was estimated on the basis of changes in absorbance at the Soret band (a = 430 nm for deoxyhemoglobin), changes in the absorbance ration Avqv/Avwt determined after conversion of irradiated preparations to methemoglobin, and changes in the value of parameters describing the reaction of hemoglobin oxygenation. The protection coefficient p of hemoglobin by ethanol (ratio of a change in the absence of ethanol to that in its presence) calculated from changes in absorbance at the Soret band equaled about 1.5 at a 4-Mrad dose in all bases except MetHb irradiated in air for which p was much higher (about 3.2). The protection coefficient p' calculated from Dtx values for changes in Avchemically bondv/Avwt equaled 2.2 for HbOs, and 2.8 for MetHb for preparations irradiated in air; p' = 1.7 for Hbbj and 1.8 for MetHb for preparations irradiated under argon. On the basis of these results, the role of /sup ./OH radicals and oxygen in the radiation damage of hemoglobin is discussed

  6. Human sensitization to Prosopis Juliflora antigen in Saudi Arabia

    International Nuclear Information System (INIS)

    Al-Frayh, A.; Gad-el-Rab, Mohammed O.; Al-Mobeireek, K.; Al-Turki, T.; Hasnain, Syed M.; Al-Sedairy, Sultan T.

    1999-01-01

    Allergenicity Prosopis juliflora pollen antigen has been reported fromonly a few countries, including the US, South Africa, India and Kuwait. Insome parts of Saudi Arabia, species of Prosopis have been introduced by themillions as roadside ornamentation. There appear to be four flowering seasonsduring which pollen grains float in all directions. However, the role ofProsopis pollen as the sensitizing and/or rhinitis in the Kingdom has neverbeen evaluated. A total of 473 allergic patients suffering from the bronchialasthma in four different geographical regions (Abha, Qassim, Hofuf, Gizan),and attending allergy clinics and chest disease centers of university andMinistry of Health hospitals in the region were tested for immediatehypersensitivity reaction to Prosopis Juliflora allergens. Airborne pollengrains at one center were also studied for one full year, using volumetricsampling techniques. A total of 76.1% patients in Qassim, 37.5% in Gizan, 29%in Abha and 11% in Hofuf reacted positively to Prosopis antigen. Multiplesensitivities to other pollen antigens were detected in all patients. Thelevel of airborne Prosopis pollen detected in Gizan exceeded 90 grains m ofair. In view of documented evidence of Prosopis pollen as a sensitizingfactor in Saudi Arabia has been confirmed. However the cause of elicitationof symptoms in many multiple sensitive patients, together with the questionof cross-reactivities, needs thorough and detailed investigation. In vitroconfirmation of all positive results is also required to incriminate Prosopisas one of the major allergens in parts of Saudi Arabia. (author)

  7. Human gut microbes impact host serum metabolome and insulin sensitivity

    DEFF Research Database (Denmark)

    Pedersen, Helle Krogh; Gudmundsdottir, Valborg; Nielsen, Henrik Bjørn

    2016-01-01

    Insulin resistance is a forerunner state of ischaemic cardiovascular disease and type 2 diabetes. Here we show how the human gut microbiome impacts the serum metabolome and associates with insulin resistance in 277 non-diabetic Danish individuals. The serum metabolome of insulin-resistant individ......Insulin resistance is a forerunner state of ischaemic cardiovascular disease and type 2 diabetes. Here we show how the human gut microbiome impacts the serum metabolome and associates with insulin resistance in 277 non-diabetic Danish individuals. The serum metabolome of insulin......-resistant individuals is characterized by increased levels of branched-chain amino acids (BCAAs), which correlate with a gut microbiome that has an enriched biosynthetic potential for BCAAs and is deprived of genes encoding bacterial inward transporters for these amino acids. Prevotella copri and Bacteroides vulgatus...

  8. Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a modified RNA system

    Directory of Open Access Journals (Sweden)

    Kyung-Ok Uhm

    2017-10-01

    Full Text Available We generated human induced pluripotent stem cells (KSCBi002-B and KSCBi002-B-1 from the dermal fibroblasts of a donor using a modified RNA-based gene delivery method. According to GTG-banding analysis, the generated KSCBi002-B line has a cytogenetic abnormality (46,XY, t(1;4(q21;q25 that is distinct from that of the donor, whereas KSCBi002-B-1 has a normal karyotype (46,XY. These cell lines can be useful as a model for characterizing the hiPSCs generated by a non-viral and non-integrative system, or as a chromosomal balanced translocation model. These two cell lines are registered and available from the National Stem Cell Bank, Korea National Institute of Health.

  9. Predicting human miRNA target genes using a novel evolutionary methodology

    KAUST Repository

    Aigli, Korfiati; Kleftogiannis, Dimitrios A.; Konstantinos, Theofilatos; Spiros, Likothanassis; Athanasios, Tsakalidis; Seferina, Mavroudi

    2012-01-01

    The discovery of miRNAs had great impacts on traditional biology. Typically, miRNAs have the potential to bind to the 3'untraslated region (UTR) of their mRNA target genes for cleavage or translational repression. The experimental identification of their targets has many drawbacks including cost, time and low specificity and these are the reasons why many computational approaches have been developed so far. However, existing computational approaches do not include any advanced feature selection technique and they are facing problems concerning their classification performance and their interpretability. In the present paper, we propose a novel hybrid methodology which combines genetic algorithms and support vector machines in order to locate the optimal feature subset while achieving high classification performance. The proposed methodology was compared with two of the most promising existing methodologies in the problem of predicting human miRNA targets. Our approach outperforms existing methodologies in terms of classification performances while selecting a much smaller feature subset. © 2012 Springer-Verlag.

  10. Predicting human miRNA target genes using a novel evolutionary methodology

    KAUST Repository

    Aigli, Korfiati

    2012-01-01

    The discovery of miRNAs had great impacts on traditional biology. Typically, miRNAs have the potential to bind to the 3\\'untraslated region (UTR) of their mRNA target genes for cleavage or translational repression. The experimental identification of their targets has many drawbacks including cost, time and low specificity and these are the reasons why many computational approaches have been developed so far. However, existing computational approaches do not include any advanced feature selection technique and they are facing problems concerning their classification performance and their interpretability. In the present paper, we propose a novel hybrid methodology which combines genetic algorithms and support vector machines in order to locate the optimal feature subset while achieving high classification performance. The proposed methodology was compared with two of the most promising existing methodologies in the problem of predicting human miRNA targets. Our approach outperforms existing methodologies in terms of classification performances while selecting a much smaller feature subset. © 2012 Springer-Verlag.

  11. Keratin14 mRNA expression in human pneumocytes during quiescence, repair and disease.

    Directory of Open Access Journals (Sweden)

    Marco Confalonieri

    Full Text Available The lung alveoli slowly self-renew pneumocytes, but their facultative regeneration capacity is rapidly efficient after an injury, so fibrosis infrequently occurs. We recently observed Keratin 14 (KRT14 expression during diffuse alveolar damage (DAD, but not in controls. We wonder if KRT14 may be a marker of pneumocyte transition from quiescence to regeneration. Quantitative PCR and Western blot analyses highlighted the presence of KRT14 (mRNA and protein only in human lung samples with DAD or interstitial lung disease (ILD. In the exponentially growing cell lines A549 and H441, the mRNA and protein levels of KRT14 peaked at day one after cell seeding and decreased at day two, opposite to what observed for the proliferation marker E2F1. The inverse relation of KRT14 versus E2F1 expression holds true also for other proliferative markers, such as cyclin E1 and cyclin D1. Of interest, we also found that E2F1 silencing caused cell cycle arrest and increased KRT14 expression, whilst E2F1 stimulation induced cell cycle progression and decreased KRT14. KRT14 also increased in proliferative pneumocytes (HPAEpiC just before transdifferentiation. Overall, our results suggest that KRT14 is a viable biomarker of pneumocyte activation, and repair/regeneration. The involvement of KRT14 in regenerative process may suggest a novel pharmaceutical target to accelerate lung repair.

  12. Targeting Rad50 sensitizes human nasopharyngeal carcinoma cells to radiotherapy

    International Nuclear Information System (INIS)

    Chang, Lihong; Huang, Jiancong; Wang, Kai; Li, Jingjia; Yan, Ruicheng; Zhu, Ling; Ye, Jin; Wu, Xifu; Zhuang, Shimin; Li, Daqing; Zhang, Gehua

    2016-01-01

    The Mre11-Rad50-Nbs1 (MRN) complex is well known for its crucial role in initiating DNA double strand breaks (DSBs) repair pathways to resistant irradiation (IR) injury and thus facilitating radioresistance which severely reduces radiocurability of nasopharyngeal cancer (NPC). Targeting native cellular MRN function would sensitize NPC cells to IR. A recombinant adenovirus containing a mutant Rad50 gene (Ad-RAD50) expressing Rad50 zinc hook domain but lacking the ATPase domain and the Mre11 interaction domain was constructed to disrupt native cellular MRN functions. The effects of Ad-RAD50 on the MRN functions were assessed in NPC cells lines using western blot, co-immunoprecipitation and confocal microscopy analyses. The increased radiosensitivity of transient Ad-RAD50 to IR was examined in NPC cells, including MTT assay, colony formation. The molecular mechanisms of radiosensitization were confirmed by neutral comet assay and western bolts. Nude mice subcutaneous injection, tumor growth curve and TUNEL assay were used to evaluate tumor regression and apoptosis in vivo. Rad50 is remarkably upregulated in NPC cells after IR, implying the critical role of Rad50 in MRN functions. The transient expression of this mutant Rad50 decreased the levels of native cellular Rad50, Mre11 and Nbs1, weakened the interactions among these proteins, abrogated the G2/M arrest induced by DSBs and reduced the DNA repair ability in NPC cells. A combination of IR and mutant RAD50 therapy produced significant tumor cytotoxicity in vitro, with a corresponding increase in DNA damage, prevented proliferation and cell viability. Furthermore, Ad-RAD50 sensitized NPC cells to IR by causing dramatic tumor regression and inducing apoptosis in vivo. Our findings define a novel therapeutic approach to NPC radiosensitization via targeted native cellular Rad50 disruption. The online version of this article (doi:10.1186/s12885-016-2190-8) contains supplementary material, which is available to

  13. Analysis of clustered point mutations in the human ribosomal RNA gene promoter by transient expression in vivo

    International Nuclear Information System (INIS)

    Jones, M.H.; Learned, R.M.; Tjian, R.

    1988-01-01

    The authors have mapped the cis regulatory elements required in vivo for initiation at the human rRNA promoter by RNA polymerase I. Transient expression in COS-7 cells was used to evaluate the transcription phenotype of clustered base substitution mutations in the human rRNA promoter. The promoter consists of two major elements: a large upstream region, composed of several domains, that lies between nucleotides -234 and -107 relative to the transcription initiation site and affects transcription up to 100-fold and a core element that lies between nucleotides -45 and +20 and affects transcription up to 1000-fold. The upstream regions is able to retain partial function when positioned within 100-160 nucleotides of the transcription initiation site, but it cannot stimulate transcription from distances of ≥ 600 nucleotides. In addition, they demonstrate, using mouse-human hybrid rRNA promoters, that the sequences responsible for human species-specific transcription in vivo appear to reside in both the core and upstream elements, and sequences from the mouse rRNA promoter cannot be substituted for them

  14. Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers.

    Science.gov (United States)

    De Rocquigny, H; Gabus, C; Vincent, A; Fournié-Zaluski, M C; Roques, B; Darlix, J L

    1992-07-15

    The nucleocapsid (NC) of human immunodeficiency virus type 1 consists of a large number of NC protein molecules, probably wrapping the dimeric RNA genome within the virion inner core. NC protein is a gag-encoded product that contains two zinc fingers flanked by basic residues. In human immunodeficiency virus type 1 virions, NCp15 is ultimately processed into NCp7 and p6 proteins. During virion assembly the retroviral NC protein is necessary for core formation and genomic RNA encapsidation, which are essential for virus infectivity. In vitro NCp15 activates viral RNA dimerization, a process most probably linked in vivo to genomic RNA packaging, and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. To characterize the domains of human immunodeficiency virus type 1 NC protein necessary for its various functions, the 72-amino acid NCp7 and several derived peptides were synthesized in a pure form. We show here that synthetic NCp7 with or without the two zinc fingers has the RNA annealing activities of NCp15. Further deletions of the N-terminal 12 and C-terminal 8 amino acids, leading to a 27-residue peptide lacking the finger domains, have little or no effect on NC protein activity in vitro. However deletion of short sequences containing basic residues flanking the first finger leads to a complete loss of NC protein activity. It is proposed that the basic residues and the zinc fingers cooperate to select and package the genomic RNA in vivo. Inhibition of the viral RNA binding and annealing activities associated with the basic residues flanking the first zinc finger of NC protein could therefore be used as a model for the design of antiviral agents.

  15. Human Long Noncoding RNA Regulation of Stem Cell Potency and Differentiation

    Directory of Open Access Journals (Sweden)

    Seahyoung Lee

    2017-01-01

    Full Text Available Because of their capability of differentiation into lineage-specific cells, stem cells are an attractive therapeutic modality in regenerative medicine. To develop an effective stem cell-based therapeutic strategy with predictable results, deeper understanding of the underlying molecular mechanisms of stem cell differentiation and/or pluripotency maintenance is required. Thus, reviewing the key factors involved in the transcriptional and epigenetic regulation of stem cell differentiation and maintenance is important. Accumulating data indicate that long noncoding RNAs (lncRNAs mediate numerous biological processes, including stem cell differentiation and maintenance. Here, we review recent findings on the human lncRNA regulation of stem cell potency and differentiation. Although the clinical implication of these lncRNAs is only beginning to be elucidated, it is anticipated that lncRNAs will become important therapeutic targets in the near future.

  16. Silencing of cytosolic NADP(+)-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine.

    Science.gov (United States)

    Lee, Su-Min; Park, Sin Young; Shin, Seoung Woo; Kil, In Sup; Yang, Eun Sun; Park, Jeen-Woo

    2009-02-01

    Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.

  17. The small RNA content of human sperm reveals pseudogene-derived piRNAs complementary to protein-coding genes

    Science.gov (United States)

    Pantano, Lorena; Jodar, Meritxell; Bak, Mads; Ballescà, Josep Lluís; Tommerup, Niels; Oliva, Rafael; Vavouri, Tanya

    2015-01-01

    At the end of mammalian sperm development, sperm cells expel most of their cytoplasm and dispose of the majority of their RNA. Yet, hundreds of RNA molecules remain in mature sperm. The biological significance of the vast majority of these molecules is unclear. To better understand the processes that generate sperm small RNAs and what roles they may have, we sequenced and characterized the small RNA content of sperm samples from two human fertile individuals. We detected 182 microRNAs, some of which are highly abundant. The most abundant microRNA in sperm is miR-1246 with predicted targets among sperm-specific genes. The most abundant class of small noncoding RNAs in sperm are PIWI-interacting RNAs (piRNAs). Surprisingly, we found that human sperm cells contain piRNAs processed from pseudogenes. Clusters of piRNAs from human testes contain pseudogenes transcribed in the antisense strand and processed into small RNAs. Several human protein-coding genes contain antisense predicted targets of pseudogene-derived piRNAs in the male germline and these piRNAs are still found in mature sperm. Our study provides the most extensive data set and annotation of human sperm small RNAs to date and is a resource for further functional studies on the roles of sperm small RNAs. In addition, we propose that some of the pseudogene-derived human piRNAs may regulate expression of their parent gene in the male germline. PMID:25904136

  18. Sensitivity analysis of human brain structural network construction

    Directory of Open Access Journals (Sweden)

    Kuang Wei

    2017-12-01

    Full Text Available Network neuroscience leverages diffusion-weighted magnetic resonance imaging and tractography to quantify structural connectivity of the human brain. However, scientists and practitioners lack a clear understanding of the effects of varying tractography parameters on the constructed structural networks. With diffusion images from the Human Connectome Project (HCP, we characterize how structural networks are impacted by the spatial resolution of brain atlases, total number of tractography streamlines, and grey matter dilation with various graph metrics. We demonstrate how injudicious combinations of highly refined brain parcellations and low numbers of streamlines may inadvertently lead to disconnected network models with isolated nodes. Furthermore, we provide solutions to significantly reduce the likelihood of generating disconnected networks. In addition, for different tractography parameters, we investigate the distributions of values taken by various graph metrics across the population of HCP subjects. Analyzing the ranks of individual subjects within the graph metric distributions, we find that the ranks of individuals are affected differently by atlas scale changes. Our work serves as a guideline for researchers to optimize the selection of tractography parameters and illustrates how biological characteristics of the brain derived in network neuroscience studies can be affected by the choice of atlas parcellation schemes. Diffusion tractography has been proven to be a promising noninvasive technique to study the network properties of the human brain. However, how various tractography and network construction parameters affect network properties has not been studied using a large cohort of high-quality data. We utilize data provided by the Human Connectome Project to characterize the changes to network properties induced by varying the brain parcellation atlas scales, the number of reconstructed tractography tracks, and the degree of grey

  19. Evaluation of human skin tests for potential dermal irritant and contact sensitizing products: a position paper

    NARCIS (Netherlands)

    Loveren H van; Jong WH de; Garssen J; LPI

    1998-01-01

    Prediction of human cutaneous irritation and sensitization in view of hazard identification has primarily relied on the use of laboratory animals. Such studies in laboratory animals have been very instrumental in the detection of potential contact sensitizing agents. There are however many

  20. Sensitivity-enhanced 13C MR spectroscopy of the human brain at 3 Tesla.

    NARCIS (Netherlands)

    Klomp, D.W.J.; Renema, W.K.J.; Graaf, M. de; Galan, B.E. de; Kentgens, A.P.M.; Heerschap, A.

    2006-01-01

    A new coil design for sensitivity-enhanced 13C MR spectroscopy (MRS) of the human brain is presented. The design includes a quadrature transmit/receive head coil optimized for 13C MR sensitivity. Loss-less blocking circuits inside the coil conductors allow this coil to be used inside a homogeneous

  1. Sensitivity-enhanced C-13 MR spectroscopy of the human brain at 3 Tesla

    NARCIS (Netherlands)

    Klomp, D.W.J.; Renema, W.K.J.; Graaf, M. van der; Galan, B.E. de; Kentgens, A.P.M.; Heerschap, A.

    2006-01-01

    A new coil design for sensitivity-enhanced C-13 MR spectroscopy (MRS) of the human brain is presented. The design includes a quadrature transmit/receive head coil optimized for C-13 MR sensitivity. Loss-less blocking circuits inside the coil conductors allow this coil to be used inside a homogeneous

  2. MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

    Directory of Open Access Journals (Sweden)

    Erkko Ylösmäki

    Full Text Available MicroRNAs (miRNAs are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5 in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

  3. Curcumin promotes apoptosis in A549/DDP multidrug-resistant human lung adenocarcinoma cells through an miRNA signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jian, E-mail: zhangjian197011@yahoo.com [Department of Respiratory Medicine, Xijing Hospital, The Fourth Military Medical University, Xi' an 710032 (China); Zhang, Tao [Department of Thoracic Surgery, Tangdu Hospital, The Fourth Military Medical University, Xi' an 710038 (China); Ti, Xinyu; Shi, Jieran; Wu, Changgui; Ren, Xinling [Department of Respiratory Medicine, Xijing Hospital, The Fourth Military Medical University, Xi' an 710032 (China); Yin, Hong, E-mail: yinnhong@yahoo.com [The Medical Image Center, Xijing Hospital, The Fourth Military Medical University, Xi' an 710032 (China)

    2010-08-13

    Research highlights: {yields} Curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells {yields} Curcumin promotes apoptosis in A549/DDP cells through a miRNA signaling pathway {yields} Curcumin induces A549/DDP cell apoptosis by downregulating miR-186* {yields} miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin -- Abstract: Curcumin extracted from the rhizomes of Curcuma longa L. has been shown to have inhibitory effects on cancers through its anti-proliferative and pro-apoptotic activities. Emerging evidence demonstrates that curcumin can overcome drug resistance to classical chemotherapies. Thus, the mechanisms underlying the anti-tumor activities of curcumin require further study. In our study, we first demonstrated that curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells. Further studies showed that curcumin altered miRNA expression; in particular, significantly downregulated the expression of miR-186* in A549/DDP. In addition, transfection of cells with a miR-186* inhibitor promoted A549/DDP apoptosis, and overexpression of miR-186* significantly inhibited curcumin-induced apoptosis in A549/DDP cells. These observations suggest that miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin.

  4. Curcumin promotes apoptosis in A549/DDP multidrug-resistant human lung adenocarcinoma cells through an miRNA signaling pathway

    International Nuclear Information System (INIS)

    Zhang, Jian; Zhang, Tao; Ti, Xinyu; Shi, Jieran; Wu, Changgui; Ren, Xinling; Yin, Hong

    2010-01-01

    Research highlights: → Curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells → Curcumin promotes apoptosis in A549/DDP cells through a miRNA signaling pathway → Curcumin induces A549/DDP cell apoptosis by downregulating miR-186* → miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin -- Abstract: Curcumin extracted from the rhizomes of Curcuma longa L. has been shown to have inhibitory effects on cancers through its anti-proliferative and pro-apoptotic activities. Emerging evidence demonstrates that curcumin can overcome drug resistance to classical chemotherapies. Thus, the mechanisms underlying the anti-tumor activities of curcumin require further study. In our study, we first demonstrated that curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells. Further studies showed that curcumin altered miRNA expression; in particular, significantly downregulated the expression of miR-186* in A549/DDP. In addition, transfection of cells with a miR-186* inhibitor promoted A549/DDP apoptosis, and overexpression of miR-186* significantly inhibited curcumin-induced apoptosis in A549/DDP cells. These observations suggest that miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin.

  5. [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.

  6. Carbon nanotube-polyamidoamine dendrimer hybrid-modified electrodes for highly sensitive electrochemical detection of microRNA24.

    Science.gov (United States)

    Li, Fengye; Peng, Jing; Zheng, Qiong; Guo, Xiang; Tang, Hao; Yao, Shouzhuo

    2015-01-01

    A simple and ultrasensitive microRNA (miRNA) electrochemical biosensor employing multiwalled carbon nanotube (MWCNT)-polyamidoamine (PAMAM) dendrimer and methylene blue (MB) redox indicator is reported in this work. The assay utilizes a glass carbon (GC) electrode modified with MWCNT-PAMAM, on which the oligonucleotide capture probes are immobilized. The electrochemical detection of miRNAs is completed by measuring the reduction signal change of MB before and after the probe hybridization with target miRNA (miRNA24 is used as a model case). The MWCNT-PAMAM/GC electrode shows greatly enhanced signal to MB reduction in contrast to bare GC electrode. The functionalization of MWCNT with PAMAM maintains the electrochemical property of MWCNT to MB reduction but minimizes the undesired adsorption of MB on the MWCNT surface. The effect of experimental variables on the miRNA detection is investigated and optimized. A detection limit of 0.5 fM and a linear peak current density-concentration relationship up to 100 nM are obtained following 60 min hybridization. The proposed assay is successfully used to detect miRNA24 in total RNA sample extracted from HeLa cells.

  7. Hunted woolly monkeys (Lagothrix poeppigii show threat-sensitive responses to human presence.

    Directory of Open Access Journals (Sweden)

    Sarah Papworth

    Full Text Available Responding only to individuals of a predator species which display threatening behaviour allows prey species to minimise energy expenditure and other costs of predator avoidance, such as disruption of feeding. The threat sensitivity hypothesis predicts such behaviour in prey species. If hunted animals are unable to distinguish dangerous humans from non-dangerous humans, human hunting is likely to have a greater effect on prey populations as all human encounters should lead to predator avoidance, increasing stress and creating opportunity costs for exploited populations. We test the threat sensitivity hypothesis in wild Poeppigi's woolly monkeys (Lagothrix poeppigii in Yasuní National Park, Ecuador, by presenting human models engaging in one of three behaviours "hunting", "gathering" or "researching". These experiments were conducted at two sites with differing hunting pressures. Visibility, movement and vocalisations were recorded and results from two sites showed that groups changed their behaviours after being exposed to humans, and did so in different ways depending on the behaviour of the human model. Results at the site with higher hunting pressure were consistent with predictions based on the threat sensitivity hypothesis. Although results at the site with lower hunting pressure were not consistent with the results at the site with higher hunting pressure, groups at this site also showed differential responses to different human behaviours. These results provide evidence of threat-sensitive predator avoidance in hunted primates, which may allow them to conserve both time and energy when encountering humans which pose no threat.

  8. Human adipocytes are highly sensitive to intermittent hypoxia induced NF-kappaB activity and subsequent inflammatory gene expression

    International Nuclear Information System (INIS)

    Taylor, Cormac T.; Kent, Brian D.; Crinion, Sophie J.; McNicholas, Walter T.; Ryan, Silke

    2014-01-01

    Highlights: • Intermittent hypoxia (IH) leads to NF-κB activation in human primary adipocytes. • Adipocytes bear higher pro-inflammatory potential than other human primary cells. • IH leads to upregulation of multiple pro-inflammatory genes in human adipocytes. - Abstract: Introduction: Intermittent hypoxia (IH)-induced activation of pro-inflammatory pathways is a major contributing factor to the cardiovascular pathophysiology associated with obstructive sleep apnea (OSA). Obesity is commonly associated with OSA although it remains unknown whether adipose tissue is a major source of inflammatory mediators in response to IH. The aim of this study was to test the hypothesis that IH leads to augmented inflammatory responses in human adipocytes when compared to cells of non-adipocyte lineages. Methods and results: Human primary subcutaneous and visceral adipocytes, human primary microvascular pulmonary endothelial cells (HUMEC-L) and human primary small airway epithelial cells (SAEC) were exposed to 0, 6 or 12 cycles of IH or stimulated with tumor necrosis factor (TNF)-α. IH led to a robust increase in NF-κB DNA-binding activity in adipocytes compared with normoxic controls regardless of whether the source of adipocytes was visceral or subcutaneous. Notably, the NF-κB response of adipocytes to both IH and TNF-α was significantly greater than that in HUMEC-L and SAEC. Western blotting confirmed enhanced nuclear translocation of p65 in adipocytes in response to IH, accompanied by phosphorylation of I-κB. Parallel to p65 activation, we observed a significant increase in secretion of the adipokines interleukin (IL)-8, IL-6 and TNF-α with IH in adipocytes accompanied by significant upregulation of mRNA expression. PCR-array suggested profound influence of IH on pro-inflammatory gene expression in adipocytes. Conclusion: Human adipocytes demonstrate strong sensitivity to inflammatory gene expression in response to acute IH and hence, adipose tissue may be a key

  9. Human adipocytes are highly sensitive to intermittent hypoxia induced NF-kappaB activity and subsequent inflammatory gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, Cormac T. [School of Medicine and Medical Science, The Conway Institute, University College Dublin (Ireland); Kent, Brian D.; Crinion, Sophie J.; McNicholas, Walter T. [School of Medicine and Medical Science, The Conway Institute, University College Dublin (Ireland); Pulmonary and Sleep Disorders Unit, St. Vincent’s University Hospital, Dublin (Ireland); Ryan, Silke, E-mail: silke.ryan@ucd.ie [School of Medicine and Medical Science, The Conway Institute, University College Dublin (Ireland); Pulmonary and Sleep Disorders Unit, St. Vincent’s University Hospital, Dublin (Ireland)

    2014-05-16

    Highlights: • Intermittent hypoxia (IH) leads to NF-κB activation in human primary adipocytes. • Adipocytes bear higher pro-inflammatory potential than other human primary cells. • IH leads to upregulation of multiple pro-inflammatory genes in human adipocytes. - Abstract: Introduction: Intermittent hypoxia (IH)-induced activation of pro-inflammatory pathways is a major contributing factor to the cardiovascular pathophysiology associated with obstructive sleep apnea (OSA). Obesity is commonly associated with OSA although it remains unknown whether adipose tissue is a major source of inflammatory mediators in response to IH. The aim of this study was to test the hypothesis that IH leads to augmented inflammatory responses in human adipocytes when compared to cells of non-adipocyte lineages. Methods and results: Human primary subcutaneous and visceral adipocytes, human primary microvascular pulmonary endothelial cells (HUMEC-L) and human primary small airway epithelial cells (SAEC) were exposed to 0, 6 or 12 cycles of IH or stimulated with tumor necrosis factor (TNF)-α. IH led to a robust increase in NF-κB DNA-binding activity in adipocytes compared with normoxic controls regardless of whether the source of adipocytes was visceral or subcutaneous. Notably, the NF-κB response of adipocytes to both IH and TNF-α was significantly greater than that in HUMEC-L and SAEC. Western blotting confirmed enhanced nuclear translocation of p65 in adipocytes in response to IH, accompanied by phosphorylation of I-κB. Parallel to p65 activation, we observed a significant increase in secretion of the adipokines interleukin (IL)-8, IL-6 and TNF-α with IH in adipocytes accompanied by significant upregulation of mRNA expression. PCR-array suggested profound influence of IH on pro-inflammatory gene expression in adipocytes. Conclusion: Human adipocytes demonstrate strong sensitivity to inflammatory gene expression in response to acute IH and hence, adipose tissue may be a key

  10. Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

    Science.gov (United States)

    Almalki, Abdulraheem; Alston, Charlotte L; Parker, Alasdair; Simonic, Ingrid; Mehta, Sarju G; He, Langping; Reza, Mojgan; Oliveira, Jorge M A; Lightowlers, Robert N; McFarland, Robert; Taylor, Robert W; Chrzanowska-Lightowlers, Zofia M A

    2014-01-01

    Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe). © 2013. Published by Elsevier B.V. All rights reserved.

  11. RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise

    DEFF Research Database (Denmark)

    Haas, Claus; Hanson, E; Anjos, M J

    2013-01-01

    samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used......A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10-0.01 µl saliva, 5-0.01 µl semen) and, optionally, bona fide or mock casework...... different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin...

  12. Characterization and vectorization of siRNA targeting RET/PTC1 in human papillary thyroid carcinoma cells

    Directory of Open Access Journals (Sweden)

    Massade L.

    2011-10-01

    Full Text Available RET/PTC1 fusion oncogene is the most common genetic alteration identified to date in thyroid papillary carcinomas (PTC and represents a good target for small interfering RNA (siRNA. Our aim was: i to target the RET/PTC1 oncogene by siRNAs, ii to assess the knockdown effects on cell growth and cell cycle regulation and iii to vectorize it in order to protect it from degradation. Methods. Human cell lines expressing RET/PTC1 were transfected by siRNA RET/PTC1, inhibition of the oncogene expression was assessed by qRT-PCR and by Western blot. Conjugation of siRNA RET/PTC1 to squalene was performed by coupling it to squalene. In vivo studies are performed in nude mice. Conclusion. In this short communication, we report the main published results obtained during last years.

  13. Characterization of DNA polymerase. beta. mRNA: cell-cycle growth response in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Zmudzka, B Z; Fornace, A; Collins, J; Wilson, S H

    1988-10-25

    DNA polymerase ..beta.. (..beta..-polymerase) is a housekeeping enzyme involved in DNA repair in vertebrate cells. The authors used a cDNA probe to study abundance of ..beta..-polymerase mRNA in cultured human cells. The mRNA level in synchronized HeLa cells, representing different stages of the cell-cycle, varied only slightly. Contact inhibited fibroblasts AG-1522 contained the same level of mRNA as growing cells. The steady-state level of mRNA in fibroblasts is equivalent to 6 molecules per cell. The results indicate that the ..beta..-polymerase transcript is low abundance and is neither cell-cycles nor growth phase responsive.

  14. Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

    International Nuclear Information System (INIS)

    Zambetti, G.; Stein, J.; Stein, G.

    1987-01-01

    The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression

  15. Rifampicin sensitivity of residual RNA synthesis in Escherichia coli cells exposed to ultraviolet radiation and combined ultraviolet and γ radiations

    International Nuclear Information System (INIS)

    Prakash, R.K.; Netrawali, M.S.; Pradhan, D.S.

    1976-01-01

    UV-irradiation prevents rifampicin inhibition of the initiation of RNA synthesis by E.coli cells, but such rifampicin insensitivity is not exhibited by the residual RNA synthesis in γ-irradiated cells. Studies of the rate of [ 3 H]-uridine incorporation by E.coli cells at various times of incubation have been used to show that when γ-irradiation was given either before or after UV-irradiation of cells, the observed rifampicin insensitivity of residual RNA synthesis in the UV-irradiated cells was obliterated. RNA synthesis in cells subjected to combined exposures of UV- and γ-radiations was lowered to a lesser extent than that in the cells exposed to UV-irradiation alone. Possible mechanisms are discussed. (U.K.)

  16. Rifampicin sensitivity of residual RNA synthesis in Escherichia coli cells exposed to ultraviolet radiation and combined ultraviolet and. gamma. radiations

    Energy Technology Data Exchange (ETDEWEB)

    Prakash, R K; Netrawali, M S; Pradhan, D S [Bhabha Atomic Research Centre, Bombay (India). Biochemistry and Food Technology Div.

    1976-09-01

    UV-irradiation prevents rifampicin inhibition of the initiation of RNA synthesis by E.coli cells, but such rifampicin insensitivity is not exhibited by the residual RNA synthesis in ..gamma..-irradiated cells. Studies of the rate of (/sup 3/H)-uridine incorporation by E.coli cells at various times of incubation have been used to show that when ..gamma.. irradiation was given either before or after uv-irradiation of cells, the observed rifampicin insensitivity of residual RNA synthesis in the uv-irradiated cells was obliterated. RNA synthesis in cells subjected to combined exposures of uv- and ..gamma..-radiations was lowered to a lesser extent than that in the cells exposed to uv-irradiation alone. Possible mechanisms are discussed.

  17. Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans.

    Science.gov (United States)

    Josephson, Anna; Trifunovski, Alexandra; Widmer, Hans Ruedi; Widenfalk, Johan; Olson, Lars; Spenger, Christian

    2002-11-18

    Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain. Copyright 2002 Wiley-Liss, Inc.

  18. mRNA transfection of mouse and human neural stem cell cultures

    OpenAIRE

    McLenachan, Samuel; Zhang, D.; Palomo, A.B.; Edel, Michael John; Chen, F.K.

    2013-01-01

    The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has ...

  19. Mapping posttranscriptional regulation of the human glycome uncovers microRNA defining the glycocode

    OpenAIRE

    Agrawal, Praveen; Kurcon, Tomasz; Pilobello, Kanoelani T.; Rakus, John F.; Koppolu, Sujeethraj; Liu, Zhongyin; Batista, Bianca S.; Eng, William S.; Hsu, Ku-Lung; Liang, Yaxuan; Mahal, Lara K.

    2014-01-01

    Carbohydrates (glycans) are complex cell surface molecules that control multiple aspects of cell biology, including cell–cell communication, cancer metastasis, and inflammation. Glycan biosynthesis requires the coordination of many enzymes, but how this is regulated is not well understood. Herein we show that microRNA (miRNA), small noncoding RNA, are a major regulator of cell surface glycosylation. We map miRNA expression onto carbohydrate signatures obtained by using lectin microarrays, a g...

  20. Increased Levels of Cell-Free Human Placental Lactogen mRNA at 28-32 Gestational Weeks in Plasma of Pregnant Women With Placenta Previa and Invasive Placenta

    Science.gov (United States)

    Sekizawa, Akihiko; Ventura, Walter; Koide, Keiko; Hori, Kyouko; Okai, Takashi; Masashi, Yoshida; Furuya, Kenichi; Mizumoto, Yoshifumi

    2014-01-01

    We compared the levels of cell-free human placental lactogen (hPL) messenger RNA (mRNA) in maternal plasma at 28 to 32 weeks of gestation between women with diagnosis of placenta previa or invasive placenta and women with an uneventful pregnancy. Sensitivity and specificity of hPL mRNA for the prediction of invasive placenta were further explored. Plasma hPL mRNA were quantified by real-time reverse-transcriptase polymerase chain reaction in women with placenta previa (n = 13), invasive placenta (n = 5), and normal pregnancies (n = 92). Median (range) hPL mRNA was significantly higher in women with placenta previa, 782 (10-2301) copies/mL of plasma, and in those with invasive placenta, 615 (522-2102) copies/mL of plasma, when compared to normal pregnancies, 90 (4-4407) copies/mL of plasma, P < .01 and P < .05, respectively. We found a sensitivity of 100% and a specificity of 61.5% for the prediction of invasive placenta among women with placenta previa. In conclusion, expression of hPL mRNA is increased in plasma of women with placenta previa and invasive placenta at 28 to 32 weeks of gestation. PMID:23744883

  1. Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

    Science.gov (United States)

    Marquet, R; Baudin, F; Gabus, C; Darlix, J L; Mougel, M; Ehresmann, C; Ehresmann, B

    1991-05-11

    The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process.

  2. Vibration sensitivity of human muscle spindles and Golgi tendon organs.

    Science.gov (United States)

    Fallon, James B; Macefield, Vaughan G

    2007-07-01

    The responses of the various muscle receptors to vibration are more complicated than a naïve categorization into stretch (muscle spindle primary ending), length (muscle spindle secondary endings), and tension (Golgi tendon organs) receptors. To emphasize the similarity of responses to small length changes, we recorded from 58 individual muscle afferents subserving receptors in the ankle or toe dorsiflexors of awake human subjects (32 primary endings, 20 secondary endings, and six Golgi tendon organs). Transverse sinusoidal vibration was applied to the distal tendon of the receptor-bearing muscle, while subjects either remained completely relaxed or maintained a weak isometric contraction of the appropriate muscle. In relaxed muscle, few units responded in a 1:1 manner to vibration, and there was no evidence of a preferred frequency of activation. In active muscle the response profiles of all three receptor types overlapped, with no significant difference in threshold between receptor types. These results emphasize that when intramuscular tension increases during a voluntary contraction, Golgi tendon organs and muscle spindle secondary endings, not just muscle spindle primary endings, can effectively encode small imposed length changes.

  3. Sensitivity of risk parameters to human errors in reactor safety study for a PWR

    International Nuclear Information System (INIS)

    Samanta, P.K.; Hall, R.E.; Swoboda, A.L.

    1981-01-01

    Sensitivities of the risk parameters, emergency safety system unavailabilities, accident sequence probabilities, release category probabilities and core melt probability were investigated for changes in the human error rates within the general methodological framework of the Reactor Safety Study (RSS) for a Pressurized Water Reactor (PWR). Impact of individual human errors were assessed both in terms of their structural importance to core melt and reliability importance on core melt probability. The Human Error Sensitivity Assessment of a PWR (HESAP) computer code was written for the purpose of this study. The code employed point estimate approach and ignored the smoothing technique applied in RSS. It computed the point estimates for the system unavailabilities from the median values of the component failure rates and proceeded in terms of point values to obtain the point estimates for the accident sequence probabilities, core melt probability, and release category probabilities. The sensitivity measure used was the ratio of the top event probability before and after the perturbation of the constituent events. Core melt probability per reactor year showed significant increase with the increase in the human error rates, but did not show similar decrease with the decrease in the human error rates due to the dominance of the hardware failures. When the Minimum Human Error Rate (M.H.E.R.) used is increased to 10 -3 , the base case human error rates start sensitivity to human errors. This effort now allows the evaluation of new error rate data along with proposed changes in the man machine interface

  4. Human skeletal muscle perilipin 2 and 3 expression varies with insulin sensitivity

    DEFF Research Database (Denmark)

    Vigelsø Hansen, Andreas; Prats Gavalda, Clara; Ploug, Thorkil

    2013-01-01

    Background: Impaired insulin sensitivity may partly arise from a dysregulated lipid metabolism in human skeletal muscle. This study investigates the expression levels of perilipin 2, 3, and 5, and four key lipases in human skeletal muscle from the subjects that exhibit a range from normal to very...

  5. A sensitive chain specific radioimmunoassay for human immunoglobulins using monoclonal antibodies

    International Nuclear Information System (INIS)

    Sikora, K.; Alderson, T.St.J.; Ellis, J.

    1983-01-01

    A sensitive radioimmunoassay is described for human immunoglobulins. This solid-phase assay uses commercially available monoclonal antibodies and is specific for different Ig chain types. Levels of less than 20 ng/ml Ig are detectable. The assay is suitable for the analysis of human hybridoma supernatants. (Auth.)

  6. Sensitivity to ionizing radiation and chemotherapeutic agents in gemcitabine-resistant human tumor cell lines

    NARCIS (Netherlands)

    van Bree, Chris; Castro Kreder, Natasja; Loves, Willem J. P.; Franken, Nicolaas A. P.; Peters, Godefridus J.; Haveman, Jaap

    2002-01-01

    Purpose: To determine cross-resistance to anti-tumor treatments in 2',2'difluorodeoxycytidine (dFdC, gemcitabine)-resistant human tumor cells. Methods and Materials: Human lung carcinoma cells SW-1573 (SWp) were made resistant to dFdC (SWg). Sensitivity to cisplatin (cDDP), paclitaxel,

  7. TERRA Expression Levels Do Not Correlate With Telomere Length and Radiation Sensitivity in Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Alexandra eSmirnova

    2013-05-01

    Full Text Available Mammalian telomeres are transcribed into long non-coding telomeric RNA molecules (TERRA that seem to play a role in the maintenance of telomere stability. In human cells, CpG island promoters drive TERRA transcription and are regulated by methylation. It was suggested that the amount of TERRA may be related to telomere length. To test this hypothesis we measured telomere length and TERRA levels in single clones isolated from five human cell lines: HeLa (cervical carcinoma, BRC-230 (breast cancer, AKG and GK2 (gastric cancers and GM847 (SV40 immortalized skin fibroblasts. We observed great clonal heterogeneity both in TRF (Terminal Restriction Fragment length and in TERRA levels. However, these two parameters did not correlate with each other. Moreover, cell survival to γ-rays did not show a significant variation among the clones, suggesting that, in this cellular system, the intra-population variability in telomere length and TERRA levels does not influence sensitivity to ionizing radiation. This conclusion was supported by the observation that in a cell line in which telomeres were greatly elongated by the ectopic expression of telomerase, TERRA expression levels and radiation sensitivity were similar to the parental HeLa cell line.

  8. Dependencies of the radiation sensitivity of human tooth enamel in EPR dosimetry

    International Nuclear Information System (INIS)

    Wieser, A.; El-Faramawy, N.; Meckbach, R.

    2001-01-01

    The EPR dose response of tooth enamel was determined for human molars collected in Egypt. The influence of age, gender and residence of the tooth donors as well as tooth position and sample preparation on EPR sensitivity and its variability over the enamel samples was investigated. The EPR sensitivity and its variability were found to depend only on the sample preparation procedure. The variability in EPR sensitivity of enamel from Egyptian teeth was maximally 10% and the mean sensitivity was in good agreement with that of German teeth

  9. Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

    2018-01-01

    DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1 ) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P correlation (R T4  > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

  10. Molecular Architecture of the Human Mediator–RNA Polymerase II–TFIIF Assembly

    Science.gov (United States)

    Bernecky, Carrie; Grob, Patricia; Ebmeier, Christopher C.; Nogales, Eva; Taatjes, Dylan J.

    2011-01-01

    The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The structure of the human Mediator–pol II interface is not well-characterized, whereas attempts to structurally define the Mediator–pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator–pol II–TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator–pol II–TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft) are exposed within the Mediator–pol II–TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator–CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator–pol II complexes lacking TFIIF reveal that TFIIF plays a key role in

  11. Genome-wide mRNA and miRNA expression data analysis to screen for markers involved in sarcomagenesis in human chondrosarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Biju Issac

    2014-12-01

    Full Text Available Genes and miRNAs involved in sarcomagenesis related pathways are unknown and therefore signaling events leading to mesenchymal cell transformation to sarcoma are poorly elucidated. Exiqon and Illumina microarray study on human chondrosarcoma JJ012 and chondrocytes C28 cell lines to compare and analyze the differentially expressed miRNAs and their gene targets was recently published in the Journal Tumor Biology in 2014. Here we describe in details the contents and quality controls for the miRNA and gene expression data associated with the study that is relevant to this dataset.

  12. Identification of the Specific Interactors of the Human Lariat RNA Debranching Enzyme 1 Protein

    Directory of Open Access Journals (Sweden)

    So Masaki

    2015-02-01

    Full Text Available In eukaryotes, pre-mRNA splicing is an essential step for gene expression. We have been analyzing post-splicing intron turnover steps in higher eukaryotes. Here, we report protein interaction between human Debranching enzyme 1 (hDbr1 and several factors found in the Intron Large (IL complex, which is an intermediate complex of the intron degradation pathway. The hDbr1 protein specifically interacts with xeroderma pigmentosum, complementeation group A (XPA-binding protein 2 (Xab2. We also attempted to identify specific interactors of hDbr1. Co-immunoprecipitation experiments followed by mass spectrometry analysis identified a novel protein as one of the specific interactors of hDbr1. This protein is well conserved among many species and shows the highest similarity to yeast Drn1, so it is designated as human Dbr1 associated ribonuclease 1 (hDrn1. hDrn1 directly interacts with hDbr1 through protein–protein interaction. Furthermore, hDrn1 shuttles between the nucleus and the cytoplasm, as hDbr1 protein does. These findings suggest that hDrn1 has roles in both the nucleus and the cytoplasm, which are highly likely to involve hDbr1.

  13. MicroRNA-433 negatively regulates the expression of thymidylate synthase (TYMS) responsible for 5-fluorouracil sensitivity in HeLa cells

    International Nuclear Information System (INIS)

    Gotanda, Keisuke; Hirota, Takeshi; Matsumoto, Nozomi; Ieiri, Ichiro

    2013-01-01

    Thymidylate synthase (TYMS) is an important folate-dependent enzyme in DNA synthesis and an important target for cancer chemotherapy. High TYMS expression levels in tumors are generally associated with resistance to 5-fluorouracil (5-FU). The cause of the variability in TYMS expression is still not fully understood, however, only a small proportion of the TYMS expression can be explained by TYMS genetic polymorphisms. The purpose of this study is to identify novel microRNAs (miRNAs) which regulate the expression of TYMS and to determine whether miRNAs binding to the 3′-untranslated region (UTR) of TYMS mRNA affect the proliferation of HeLa cells treated with 5-FU. An in silico search was performed to find potential binding sites of miRNAs in TYMS mRNA. The efficacy of predicted miRNAs at the 3′-UTR of TYMS mRNA was evaluated using a dual-luciferase reporter assay. TYMS mRNA and protein expression in HeLa cells was quantified with real-time RT-PCR and Western blotting, respectively. The effects of miR-433 on cell proliferative activity were determined by WST-8 assay. The overexpression of miR-433 was associated with significantly decreased reporter activity in the plasmid containing the 3′-UTR of TYMS mRNA (P < 0.01). The levels of TYMS mRNA and protein in HeLa cells were significantly decreased by the overexpression of miR-433 (P < 0.05). Furthermore, miR-433 increased inhibition of cell proliferation in HeLa cells treated with 5-FU at over 2.0 μM. The results indicate that miR-433 post-transcriptionally regulates the expression of TYMS mRNA and protein, and increases sensitivity to 5-FU in HeLa cells. This is the first report showing that a miRNA regulating TYMS expression has a significant impact on sensitivity to 5-FU treatment

  14. Irradiation sensitivity of human and porcine mesenchymal stem cells

    International Nuclear Information System (INIS)

    Singh, S.

    2009-01-01

    Surgical resection, chemotherapy, radiotherapy, and combinations thereof are a plethora of possible treatment modalities of head and neck malignancies. Treatment regimens including radiotherapy however put jaws at risk of subsequent osteoradionecrosis. Besides cancer cells, irradiation impacts on all tissue-inherent cells, including mesenchymal stem cells (MSCs). Since it is the bone and bone marrow MSC, which contributes to bone regeneration through proliferation and osteogenic differentiation of its progeny, the influence of irradiation on MSC viability and the respective differentiation capacity appears to be critical. However to date, only a few reports picked MSCs role out as a pivotal topic. As a first attempt, we irradiated human bone derived MSC in vitro. With increasing doses the cells self-renewal capabilities were greatly reduced. Notably however, the mitotically stalled cells were still capable of differentiating into osteoblasts and preadipocytes. Next, the mandibles of Sus scrofa domestica were irradiated with a total dose of 18 Gy. At different time points post radiatio, MSCs were isolated from bone autopsies. In comparison between irradiated and non- irradiated samples, no significant differences regarding the proliferation and osteogenic differentiation potential of tissue specific MSC became apparent Therefore, pig mandibles were irradiated with doses of 9 and 18 Gy, and MSCs were isolated immediately afterwards. No significant differences between the untreated and bone irradiated with 9 Gy with respect of proliferation and osteogenic differentiation were observed. Cells isolated from 18 Gy irradiated specimens exhibited a greatly reduced osteogenic differentiation capacity, and during the first two weeks proliferation rates of explanted cells were greatly diminished. Thereafter, cells recovered and showed proliferation behaviour comparable to control samples. These results imply that MSCs can cope with irradiation up to relatively high doses

  15. ELFN1-AS1: A Novel Primate Gene with Possible MicroRNA Function Expressed Predominantly in Human Tumors

    Directory of Open Access Journals (Sweden)

    Dmitrii E. Polev

    2014-01-01

    Full Text Available Human gene LOC100505644 uncharacterized LOC100505644 [Homo sapiens] (Entrez Gene ID 100505644 is abundantly expressed in tumors but weakly expressed in few normal tissues. Till now the function of this gene remains unknown. Here we identified the chromosomal borders of the transcribed region and the major splice form of the LOC100505644-specific transcript. We characterised the major regulatory motifs of the gene and its splice sites. Analysis of the secondary structure of the major transcript variant revealed a hairpin-like structure characteristic for precursor microRNAs. Comparative genomic analysis of the locus showed that it originated in primates de novo. Taken together, our data indicate that human gene LOC100505644 encodes some non-protein coding RNA, likely a microRNA. It was assigned a gene symbol ELFN1-AS1 (ELFN1 antisense RNA 1 (non-protein coding. This gene combines features of evolutionary novelty and predominant expression in tumors.

  16. The Crystal Structure of the RNA-Dependent RNA Polymerase from Human Rhinovirus: A Dual Function Target for Common Cold Antiviral Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Love, Robert A.; Maegley, Karen A.; Yu, Xiu; Ferre, RoseAnn; Lingardo, Laura K.; Diehl, Wade; Parge, Hans E.; Dragovich, Peter S.; Fuhrman, Shella A. (Pfizer)

    2010-11-16

    Human rhinoviruses (HRV), the predominant members of the Picornaviridae family of positive-strand RNA viruses, are the major causative agents of the common cold. Given the lack of effective treatments for rhinoviral infections, virally encoded proteins have become attractive therapeutic targets. The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) denoted 3D{sup pol}, which is responsible for replicating the viral genome and for synthesizing a protein primer used in the replication. Here the crystal structures for three viral serotypes (1B, 14, and 16) of HRV 3D{sup pol} have been determined. The three structures are very similar to one another, and to the closely related poliovirus (PV) 3D{sup pol} enzyme. Because the reported PV crystal structure shows significant disorder, HRV 3D{sup pol} provides the first complete view of a picornaviral RdRp. The folding topology of HRV 3D{sup pol} also resembles that of RdRps from hepatitis C virus (HCV) and rabbit hemorrhagic disease virus (RHDV) despite very low sequence homology.

  17. Targeting human breast cancer cells by an oncolytic adenovirus using microRNA-targeting strategy.

    Science.gov (United States)

    Shayestehpour, Mohammad; Moghim, Sharareh; Salimi, Vahid; Jalilvand, Somayeh; Yavarian, Jila; Romani, Bizhan; Mokhtari-Azad, Talat

    2017-08-15

    MicroRNA-targeting strategy is a promising approach that enables oncolytic viruses to replicate in tumor cells but not in normal cells. In this study, we targeted adenoviral replication toward breast cancer cells by inserting ten complementary binding sites for miR-145-5p downstream of E1A gene. In addition, we evaluated the effect of increasing miR-145 binding sites on inhibition of virus replication. Ad5-control and adenoviruses carrying five or ten copies of miR145-5p target sites (Ad5-5miR145T, Ad5-10miR145T) were generated and inoculated into MDA-MB-453, BT-20, MCF-7 breast cancer cell lines and human mammary epithelial cells (HMEpC). Titer of Ad5-10miR145T in HMEpC was significantly lower than Ad5-control titer. Difference between the titer of these two viruses at 12, 24, 36, and 48h after infection was 1.25, 2.96, 3.06, and 3.77 log TCID 50 . No significant difference was observed between the titer of both adenoviruses in MDA-MB-453, BT-20 and MCF-7 cells. The infectious titer of adenovirus containing 10 miR-145 binding sites in HMEpC cells at 24, 36, and 48h post-infection was 1.7, 2.08, and 4-fold, respectively, lower than the titer of adenovirus carrying 5 miR-145 targets. Our results suggest that miR-145-targeting strategy provides selectivity for adenovirus replication in breast cancer cells. Increasing the number of miRNA binding sites within the adenoviral genome confers more selectivity for viral replication in cancer cells. Copyright © 2017. Published by Elsevier B.V.

  18. MicroRNA Mediated Chemokine Responses in Human Airway Smooth Muscle Cells.

    Directory of Open Access Journals (Sweden)

    Mythili Dileepan

    Full Text Available Airway smooth muscle (ASM cells play a critical role in the pathophysiology of asthma due to their hypercontractility and their ability to proliferate and secrete inflammatory mediators. microRNAs (miRNAs are gene regulators that control many signaling pathways and thus serve as potential therapeutic alternatives for many diseases. We have previously shown that miR-708 and miR-140-3p regulate the MAPK and PI3K signaling pathways in human ASM (HASM cells following TNF-α exposure. In this study, we investigated the regulatory effect of these miRNAs on other asthma-related genes. Microarray analysis using the Illumina platform was performed with total RNA extracted from miR-708 (or control miR-transfected HASM cells. Inhibition of candidate inflammation-associated gene expression was further validated by qPCR and ELISA. The most significant biologic functions for the differentially expressed gene set included decreased inflammatory response, cytokine expression and signaling. qPCR revealed inhibition of expression of CCL11, CXCL10, CCL2 and CXCL8, while the release of CCL11 was inhibited in miR-708-transfected cells. Transfection of cells with miR-140-3p resulted in inhibition of expression of CCL11, CXCL12, CXCL10, CCL5 and CXCL8 and of TNF-α-induced CXCL12 release. In addition, expression of RARRES2, CD44 and ADAM33, genes known to contribute to the pathophysiology of asthma, were found to be inhibited in miR-708-transfected cells. These results demonstrate that miR-708 and miR-140-3p exert distinct effects on inflammation-associated gene expression and biological function of ASM cells. Targeting these miRNA networks may provide a novel therapeutic mechanism to down-regulate airway inflammation and ASM proliferation in asthma.

  19. Genetic characterization of human-pathogenic Cyclospora cayetanensis parasites from three endemic regions at the 18S ribosomal RNA locus.

    Science.gov (United States)

    Sulaiman, Irshad M; Ortega, Ynes; Simpson, Steven; Kerdahi, Khalil

    2014-03-01

    Cyclospora cayetanensis is an apicocomplexan parasite that infects the gastrointestinal tract and causes acute diarrheal disease in humans. In recent years, this human-pathogenic parasite has led to several foodborne outbreaks in the United States and Canada, mostly associated with imported produce. Understanding the biology and epidemiology of C. cayetanensis is difficult because little is known about its origin, possible zoonotic reservoirs, and genetic relationships with other coccidian parasites. Recently, we developed a 70kDa heat shock protein (HSP70) gene based nested PCR protocol for detection of C. cayetanensis parasite and sequenced the PCR products of 16 human isolates from Nepal, Mexico, and Peru. In this study, we have characterized the regions of 18S ribosomal RNA (rRNA) gene of 17 human C. cayetanensis isolates for molecular detection, and also to ascertain the genetic diversity of this parasite. The 18S rRNA primer sets were further tested by PCR amplification followed by nucleotide sequencing of the PCR amplified products of previously characterized C. cayetanensis isolates from three endemic regions at HSP70 locus. Although no genetic polymorphism was observed at the regions of HSP70 locus characterized in our previous study, the data analysis of this study revealed a minor genetic diversity at the 18S rRNA locus among the C. cayetanensis isolates. The 18S rRNA gene-based nested PCR protocol provides a useful genetic marker for the detection of C. cayetanensis parasite and confirms it as a genetically distinct species in genus Cyclospora. The results also supported lack of geographic segregation and existence of genetically homogeneous population for the C. cayetanensis parasites both at the HSP70 as well as at the18S rRNA loci. Published by Elsevier B.V.

  20. The human nucleolar protein FTSJ3 associates with NIP7 and functions in pre-rRNA processing.

    Directory of Open Access Journals (Sweden)

    Luis G Morello

    Full Text Available NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A' to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells.

  1. Computational sequence analysis of predicted long dsRNA transcriptomes of major crops reveals sequence complementarity with human genes.

    Science.gov (United States)

    Jensen, Peter D; Zhang, Yuanji; Wiggins, B Elizabeth; Petrick, Jay S; Zhu, Jin; Kerstetter, Randall A; Heck, Gregory R; Ivashuta, Sergey I

    2013-01-01

    Long double-stranded RNAs (long dsRNAs) are precursors for the effector molecules of sequence-specific RNA-based gene silencing in eukaryotes. Plant cells can contain numerous endogenous long dsRNAs. This study demonstrates that such endogenous long dsRNAs in plants have sequence complementarity to human genes. Many of these complementary long dsRNAs have perfect sequence complementarity of at least 21 nucleotides to human genes; enough complementarity to potentially trigger gene silencing in targeted human cells if delivered in functional form. However, the number and diversity of long dsRNA molecules in plant tissue from crops such as lettuce, tomato, corn, soy and rice with complementarity to human genes that have a long history of safe consumption supports a conclusion that long dsRNAs do not present a significant dietary risk.

  2. An ultraviolet-sensitive maternal mRNA encoding a cytoskeletal protein may be involved in axis formation in the ascidian embryo

    International Nuclear Information System (INIS)

    Jeffery, W.R.

    1990-01-01

    Ultraviolet (uv) irradiation of the vegetal hemisphere of fertilized eggs during ooplasmic segregation inhibits subsequent gastrulation and axis formation in ascidian embryos. The molecular basis of this phenomenon was investigated in by comparing in vivo protein synthesis and in vitro mRNA translation in normal and uv-irradiated embryos of the ascidian Styela clava. Analysis of protein synthesis by [35S]methionine incorporation, two-dimensional (2D) gel electrophoresis, and autoradiography showed that only 21 of 433 labeled polypeptides were missing or decreased in labeling intensity in uv-irradiated embryos. The most prominent of these was a 30,000 molecular weight (pI 6.0) polypeptide (p30). Extraction of gastrulae with the nonionic detergent Triton X-100 showed that p30 is retained in the detergent insoluble residue, suggesting that it is associated with the cytoskeleton. Several lines of evidence suggest that p30 may be involved in axis formation. First, p30 labeling peaks during gastrulation, when the embryonic axis is being established. Second, axis formation and p30 labeling are abolished by the same threshold uv dose, which is distinct from that required to inactivate muscle cell development. Third, the uv sensitivity period for abolishing p30 labeling and axis formation are both restricted to ooplasmic segregation. In vitro translation of egg RNA followed by 2D gel electrophoresis and autoradiography of the protein products showed that p30 is encoded by a maternal mRNA. The translation of p30 mRNA was abolished by uv irradiation of fertilized eggs during ooplasmic segregation suggesting that this message is a uv-sensitive target. The results are consistent with the hypothesis that uv irradiation blocks gastrulation and axis formation by inhibiting the translation of maternal mRNA localized in the vegetal hemisphere of the fertilized egg

  3. Research resources: comparative microRNA profiles in human corona radiata cells and cumulus oophorus cells detected by next-generation small RNA sequencing.

    Directory of Open Access Journals (Sweden)

    Xian-Hong Tong

    Full Text Available During folliculogenesis, cumulus cells surrounding the oocyte differentiate into corona radiata cells (CRCs and cumulus oophorus cells (COCs, which are involved in gonadal steroidogenesis and the development of germ cells. Several studies suggested that microRNAs (miRNAs play an important regulatory role at the post-transcriptional level in cumulus cells. However, comparative miRNA profiles and associated processes in human CRCs and COCs have not been reported before. In this study, miRNA profiles were obtained from CRCs and COCs using next generation sequencing in women undergoing controlled ovarian stimulation for IVF. A total of 785 and 799 annotated miRNAs were identified in CRCs and COCs, while high expression levels of six novel miRNAs were detected both in CRCs and in COCs. In addition, different expression patterns in CRCs and COCs were detected in 72 annotated miRNAs. To confirm the miRNA profile in COCs and CRCs, quantitative real-time PCR was used to validate the expression of annotated miRNAs, differentially expressed miRNAs, and novel miRNAs. The miRNAs in the let-7 family were found to be involved in the regulation of a broad range of biological processes in both cumulus cell populations, which was accompanied by a large amount of miRNA editing. Bioinformatics analysis showed that amino acid and energy metabolism were targeted significantly by miRNAs that were differentially expressed between CRCs and COCs. Our work extends the current knowledge of the regulatory role of miRNAs and their targeted pathways in folliculogenesis, and provides novel candidates for molecular biomarkers in the research of female infertility.

  4. [Expression of Jagged1 mRNA in human epithelial ovarian carcinoma tissues and effect of RNA interference of Jagged1 on growth of xenograft in nude mice].

    Science.gov (United States)

    Liu, G Y; Gao, Z H; Li, L; Song, T T; Sheng, X G

    2016-06-25

    To investigate the expression of Jagged1 in human epithelial ovarian carcinoma tissues and the effect of Jagged1 on growth of xenograft in nude mice. (1) Forty-eight cases of ovarian cancer and 30 cases of patients with benign epithelial ovarian tumor in the Henan Province Xinxiang Central Hospital during Feb. 2011 to Mar. 2014 were enrolled in this study. The mRNA expression of Jagged1, Notch1 and the downstream target genes Hes1, Hey1 were analyzed by using realtime PCR method. (2) The ovarian cancer xenograft models in nude mice were constructed by injecting SKOV3 cells in axillary subcutaneouswere. The nude mice were randomly divided into Jagged1 interference group, blank plasmid group and control group. Each group had 10 mice. They were transfected with pcDNA3.1(+)-siRNA-Jagged1, blank plasmid pDC3.1 and phosphate buffer, respectively. The tumor volumes and tumor masses were measured 14 days after transfection and the inhibition rate was calculated. The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues after transfection in each group was detected by using realtime PCR technique and the relative protein expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues was detected by utilizing western blot method. (1) The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in ovarian cancer tissues were higher than benign ovarian tumor tissues, the differences were statistically significant (Ptissues of nude micein Jagged1 interference group were lower than that in the other two groups, the differences were statistically significant (Ptissues of nude mice among the three groups (P>0.05). Jagged1 is highly expressed in epithelial ovarian carcinoma. Jagged1 gene interference in xenograft tumor can inhibit ovarian cancer cell growth and improve tumor suppressor rate, which probably play roles by inhibiting Notch1 signaling pathway.

  5. CBFA1 and topoisomerase I mRNA levels decline during cellular aging of human trabecular osteoblasts

    DEFF Research Database (Denmark)

    Christiansen, Mette; Kveiborg, M.; Kassem, M.

    2000-01-01

    In order to understand the reasons for age-related impairment of the function of bone forming osteoblasts, we have examined the steady-state mRNA levels of the transcription factor CBFA1 and topoisomerase I during cellular aging of normal human trabecular osteoblasts, by the use of semiquantitati...

  6. UBL5 is essential for pre-mRNA splicing and sister chromatid cohesion in human cells

    DEFF Research Database (Denmark)

    Oka, Yasuyoshi; Varmark, Hanne; Vitting-Seerup, Kristoffer

    2014-01-01

    UBL5 is an atypical ubiquitin-like protein, whose function in metazoans remains largely unexplored. We show that UBL5 is required for sister chromatid cohesion maintenance in human cells. UBL5 primarily associates with spliceosomal proteins, and UBL5 depletion decreases pre-mRNA splicing efficien...

  7. Impaired RNA splicing of 5'-regulatory sequences of the astroglial glutamate transporter EAAT2 in human astrocytoma

    NARCIS (Netherlands)

    Münch, C.; Penndorf, A.; Schwalenstöcker, B.; Troost, D.; Ludolph, A. C.; Ince, P.; Meyer, T.

    2001-01-01

    A loss of the glutamate transporter EAAT2 has been reported in the neoplastic transformation of astrocytic cells and astrocytoma. The RNA expression of EAAT2 and five 5'-regulatory splice variants was investigated to identify alterations of the post-transcriptional EAAT2 gene regulation in human

  8. SIMULTANEOUS EXPRESSION AND REGULATION OF G-CSF AND IL-6 MESSENGER-RNA IN ADHERENT HUMAN MONOCYTES AND FIBROBLASTS

    NARCIS (Netherlands)

    VELLENGA, E; VANDERVINNE, B; DEWOLF, JTM; HALIE, MR

    The regulation of granulocyte-colony stimulating factor (G-CSF) and interleukin-6 (IL-6) mRNA was studied in human adherent monocytes in response to the protein kinase C activator, oleolyl-acetylglycerol (OAG), the calcium-ionophore A23187 and the cyclic AMP elevating agents, dibutyryl c-AMP

  9. Human blood RNA stabilization in samples collected and transported for a large biobank

    Science.gov (United States)

    2012-01-01

    Background The Norwegian Mother and Child Cohort Study (MoBa) is a nation-wide population-based pregnancy cohort initiated in 1999, comprising more than 108.000 pregnancies recruited between 1999 and 2008. In this study we evaluated the feasibility of integrating RNA analyses into existing MoBa protocols. We compared two different blood RNA collection tube systems – the PAXgene™ Blood RNA system and the Tempus™ Blood RNA system - and assessed the effects of suboptimal blood volumes in collection tubes and of transportation of blood samples by standard mail. Endpoints to characterize the samples were RNA quality and yield, and the RNA transcript stability of selected genes. Findings High-quality RNA could be extracted from blood samples stabilized with both PAXgene and Tempus tubes. The RNA yields obtained from the blood samples collected in Tempus tubes were consistently higher than from PAXgene tubes. Higher RNA yields were obtained from cord blood (3 – 4 times) compared to adult blood with both types of tubes. Transportation of samples by standard mail had moderate effects on RNA quality and RNA transcript stability; the overall RNA quality of the transported samples was high. Some unexplained changes in gene expression were noted, which seemed to correlate with suboptimal blood volumes collected in the tubes. Temperature variations during transportation may also be of some importance. Conclusions Our results strongly suggest that special collection tubes are necessary for RNA stabilization and they should be used for establishing new biobanks. We also show that the 50,000 samples collected in the MoBa biobank provide RNA of high quality and in sufficient amounts to allow gene expression analyses for studying the association of disease with altered patterns of gene expression. PMID:22988904

  10. Suppression of NRF2–ARE activity sensitizes chemotherapeutic agent-induced cytotoxicity in human acute monocytic leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Hui [The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC (United States); Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing (China); Wang, Huihui [School of Public Health, China Medical University, 77 Puhe Road, Shenyang No