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Ambion RNAlater Tissue Collection: RNA Stabilization Solution is an aqueous, nontoxic tissue storage reagent that rapidly permeates tissues to stabilize and protect cellular RNA
Expression of Orseolia oryzae nucleoside diphosphate kinase (OoNDPK) is enhanced in rice gall midge feeding on susceptible rice hosts and its over-expression leads to salt tolerance in Escherichia coli
Authors: DK Sinha, I Atray, JS Bentur, S Nair
The Asian rice gall midge, Orseolia oryzae, is a major dipteran pest of rice, with many known biotypes. The present investigation was initiated to understand the molecular mechanisms of infestation for developing novel integrated pest management strategies. We isolated and characterized a gene, nucleoside diphosphate kinase (OoNDPK), from the rice gall midge, encoding a protein with 169 amino acid residues and with a secretory signal sequence – an observation that assumes significance as salivary gland secretions have been implicated to play a major role in insect−plant interactions.
Authors: I Riedmaier, V Benes, J Blake, N Bretschneider, C Zinser, C Becker, HHD Meyer, MW Pfaffl
The abuse of anabolic substances in animal husbandry is forbidden within the EU and well controlled by detecting substance residues in different matrices. The application of newly designed drugs or substance cocktails represents big problems. Therefore developing sensitive test methods is important. The analysis of physiological changes caused by the use of anabolic agents on the molecular level, for example, by quantifying gene expression response, is a new approach to develop such screening methods. A novel technology for holistic gene expression analysis is RNA sequencing. In this study, the potential of this high-throughput method for the identification of biomarkers was evaluated. The effect of trenbolone acetate plus estradiol on gene expression in liver from Nguni heifers was analyzed with RNA sequencing.
Authors: G Giannoukos, DM Ciulla, K Huang, BJ Haas, J Izard, JZ Levin, J Livny, AM Earl, D Gevers, DV Ward, C Nusbaum, BW Birren, A Gnirke
We have developed a process for transcriptome analysis of bacterial communities that accommodates both intact and fragmented starting RNA and combines efficient rRNA removal with strand-specific RNA-seq. We applied this approach to an RNA mixture derived from three diverse cultured bacterial species and to RNA isolated from clinical stool samples. The resulting expression profiles were highly reproducible, enriched up to 40-fold for non-rRNA transcripts, and correlated well with profiles representing undepleted total RNA.
Authors: M-S Shiao, A Y-F Chang, B-Y Liao, Y-H Ching, M-Y J Lu, SM Chen, W-H Li
To sense numerous odorants and chemicals, animals have evolved a large number of olfactory receptor genes (Olfrs) in their genome. In particular, the house mouse has ∼1,100 genes in the Olfr gene family. This makes the mouse a good model organism to study Olfr genes and olfaction-related genes.
Authors: Y Li, T Taketo, YF Lau
Gonadal sex differentiation is an important developmental process, in which a bipotential primordial gonad undergoes two distinct pathways, i.e., testicular and ovarian differentiation, dependent on its genetic sex. Techniques of isolating fetal gonads at various developmental stages are valuable for studies on the molecular events involved in cell-fate determination, sex-speciﬁc somatic and germ-cell differentiation and structural organization. Here we describe various procedures for isolation of embryonic gonads at different developmental stages from embryos of timed-pregnant mice. The isolated fetal gonads can be used for a variety of studies, such as organ culture, gene and protein expression.
Authors: AD Bries, DS Weiner, R Jacquet, MJ Adamczyk, MA Morscher, E Lowder, MJ Askew, RP Steiner, WI Horne, WJ Landis
The effect of compression on the physis is generally defined by the Hueter-Volkmann principle, in which decreased linear growth of the physis results from increased compression. This investigation examined whether mechanically induced compression of rabbit physes causes changes in gene expression, cells, and extracellular components that promote physeal resilience and strength (type-II collagen and aggrecan) and cartilage hypertrophy (type-X collagen and matrix metalloprotease-13).
Authors: J Winkler, KC Kao
The presence of anti-microbial phenolic compounds, such as the model compound ferulic acid, in biomass hydrolysates pose significant challenges to the widespread use of biomass in conjunction with whole cell biocatalysis or fermentation. Currently, these inhibitory compounds must be removed through additional downstream processing or sufficiently diluted to create environments suitable for most industrially important microbial strains. Simultaneously, product toxicity must also be overcome to allow for efficient production of next generation biofuels such as n-butanol, isopropanol, and others from these low cost feedstocks.
Authors: B Kasztelewicz, I Jankowska, J Pawłowska, J Teisseyre, K Dzierżanowska-Fangrat
Immunosuppressed pediatric transplant recipients are at risk of developing Epstein–Barr virus (EBV)-associated complications (such as post-transplant lymphoproliferative disorders). Monitoring of the EBV DNA level in blood alone has a low predictive value for the post-transplant course of EBV infection and its complications. Therefore, additional prognostic markers are widely sought. The study aim was to analyze EBV gene expression patterns and LMP1 polymorphism in relation to EBV DNA levels in pediatric liver transplant recipients. EBV load measurement, LMP1 variant, and gene expression analysis were performed in collected prospectively multiple blood samples from 30 patients.
Authors: CK Lee, SC Cary, AE Murray, RM Daniel
The equilibrium model, which describes the influence of temperature on enzyme activity, has been established as a valid and useful tool for characterizing enzyme eurythermalism and thermophily.
RNaseZap RNase Decontamination Solution is a surface decontamination solution that destroys RNases on contact. You simply spray RNaseZap® Solution onto the surface to be decontaminated and rinse it off with RNase-free water.
Authors: B Jurczyk, M Rapacz, K Budzisz, W Barcik, M Sasal
Strictly controlled and coordinated induction of CBF regulon (a set of genes regulated by CBF proteins) promotes plant freezing tolerance. CBFs regulate the expression of COR genes that confer freezing tolerance. COR14b in barley is one of the effector genes which seems to be important in resistance to combined freezing and photoinhibition of photosynthesis.
Ambion RNase T1 (E.C. 126.96.36.199) is isolated from an over-expressing E. coli strain containing the cloned Aspergillus oryzae gene and is an endonuclease that specifically cleaves 3' of G residues. Supplied in one tube containing 100,000 U (1,000 U/ µL).
Authors: GM Arachchilage, MJ Morris, S Basu
An RNA G-quadruplex library was synthesised and screened against kanamycin A as the ligand. Naturally occurring G-quadruplex forming sequences that differentially bind to kanamycin A were identified and characterized. This provides a simple and effective strategy for identification of potential intracellular G-quadruplex targets for a ligand.
Ambion RNAsecure Resuspension Solution (patents pending) is a unique non-enzymatic reagent that will irreversibly inactivate RNases in solution. It is supplied at working concentration for direct resuspension of RNA pellets. Ten tubes containing 1 mL each are provided.
Authors: JL Blum, BA Nyagode, MO James, ND Denslow
The organochlorine pesticide methoxychlor (MXC) is an environmental estrogen known to stimulate the expression of the egg-yolk protein, vitellogenin (Vtg) in fish species.
Ambion RNAlater Tissue Collection: RNA Stabilization Solution is an aqueous, nontoxic tissue storage reagent that rapidly permeates tissues to stabilize and protect cellular RNA.
Authors: CDS Duncan, J Mata
Most cellular processes are conducted by multi-protein complexes. However, little is known about how these complexes are assembled. In particular, it is not known if they are formed while one or more members of the complexes are being translated (cotranslational assembly). We took a genomic approach to address this question, by systematically identifying mRNAs associated with specific proteins. In a sample of 31 proteins from Schizosaccharomyces pombe that did not contain RNA–binding domains, we found that ~38% copurify with mRNAs that encode interacting proteins. For example, the cyclin-dependent kinase Cdc2p associates with the rum1 and cdc18 mRNAs, which encode, respectively, an inhibitor of Cdc2p kinase activity and an essential regulator of DNA replication.
This Ambion lipid-based formulation can be used to efficiently transfect adherent cells with siRNA as they are subcultured—without increased cytotoxicity. It is provided in one tube containing 1 mL.
Authors: Y-F Ren, G Li, J Wu, Y-F Xue, Y-J Song, L Lv, X-J Zhang, K-F Tang
It has been reported that decreased Dicer expression leads to Alu RNAs accumulation in human retinal pigmented epithelium cells, and Dicer may process the endogenous SINE/B1 RNAs (the rodent equivalent of the primate Alu RNAs) into small interfering RNAs (siRNAs). In this study, we aimed to address whether Dicer can process Alu RNAs and their common ancestor, 7SL RNA. Using Solexa sequencing technology, we showed that Alu-derived small RNAs accounted for 0.6% of the total cellular small RNAs in HepG2.2.15 cells, and the abundance decreased when Dicer was knocked down.
Authors: Q Yao, A-m Zhang, H Ma, S Lin, X-x Wang, J-g Sun, Z-t Chen
Lung cancer is the leading cause of cancer death worldwide. There is no effective early diagnostic technology for lung cancer. microRNAs (miRNAs) are noncoding RNA molecules which regulate the process of cell growth and differentiation in human cancers. Hsa-miR-155 (miR-155), highly expressed in non-small-cell lung cancer (NSCLC), can be used as a diagnostic marker for NSCLC.
Authors: N Zhu, D Zhang, S Chen, X Liu, L Lin, X Huang, Z Guo, J Liu, Y Wang, W Yuan, Y Qin
Inflammation is observed at all stages of atherosclerosis. The initial stage of atherosclerosis is characterized by recruitment of leukocytes to activated endothelial cells (ECs). MicroRNAs (miRNAs) are a class of 19–25 nucleotides, non-protein-coding RNAs that repress target gene expression by translational inhibition or mRNA degradation.
Authors: Y Wang, ATC Lee, JZI Ma, J Wang, J Ren, Y Yang, E Tantoso, K-B Li, LLPJ Ooi, P Tan, CGL Lee
Like other cancers, aberrant gene regulation features significantly in hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) were recently found to regulate gene expression at the post-transcriptional/translational levels. The expression profiles of 157 miRNAs were examined in 19 HCC patients, and 19 up-regulated and 3 down-regulated miRNAs were found to be associated with HCC. Putative gene targets of these 22 miRNAs were predicted in silico and were significantly enriched in 34 biological pathways, most of which are frequently dysregulated during carcinogenesis.
Ambion Anti-miR miRNA Inhibitors are chemically modified, single-stranded nucleic acids designed to specifically bind to and inhibit endogenous microRNA (miRNA) molecules.
Authors: N Nadiminty, R Tummala, W Lou, Y Zhu, J Zhang, X Chen, RW eVere White, HJ Kung, CP Evans, AC Gao
Castration-resistant prostate cancer continues to rely on androgen receptor (AR) expression. AR plays a central role in the development of prostate cancer and progression to castration resistance during and after androgen deprivation therapy. Here, we identified miR-let-7c as a key regulator of expression of AR. miR-let-7c suppresses AR expression and activity in human prostate cancer cells by targeting its transcription via c-Myc. Suppression of AR by let-7c leads to decreased cell proliferation of human prostate cancer cells. Down-regulation of Let-7c in prostate cancer specimens is inversely correlated with AR expression, whereas the expression of Lin28 (a repressor of let-7) is correlated positively with AR expression. Our study demonstrates that the miRNA let-7c plays an important role in the regulation of androgen signaling in prostate cancer by down-regulating AR expression. These results suggest that reconstitution of miR-let-7c may aid in targeting enhanced and hypersensitive AR in advanced prostate cancer.
Ambion Anti-miR miRNA Inhibitors are chemically modified, single-stranded nucleic acids designed to specifically bind to and inhibit endogenous microRNA (miRNA) molecules.
Authors: Castro RE, Ferreira DM, Afonso MB, Borralho PM, Machado MV, Cortez-Pinto H, Rodrigues CM.
BACKGROUND & AIMS:
Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of stages from simple steatosis to non-alcoholic steatohepatitis (NASH). However, disease pathogenesis remains largely unknown. microRNA (miRNA or miR) expression has recently been reported to be altered in human NASH, and modulated by ursodeoxycholic acid (UDCA) in the rat liver. Here, we aimed at evaluating the miR-34a/Sirtuin 1(SIRT1)/p53 pro-apoptotic pathway in human NAFLD, and to elucidate its function and modulation by UDCA in the rat liver and primary rat hepatocytes.
Ambion Pre-miR miRNA Precursor Molecules are small, chemically modified, double-stranded RNA molecules designed to mimic endogenous mature miRNAs. This ready-to-use miRNA mimic can be introduced into cells using transfection or electroporation parameters similar to those used for siRNAs.
Authors: T-C Huang, H-Y Chang, C-Y Chen, P-Y Wu, H Lee, Y-F Liao, W-M Hsu, H-C Huang, H-F Juan
Neuroblastoma is the most common extracranial solid tumor in children. We investigate whether miR-124, the abundant neuronal miRNA, plays a pivotal role in neuroblastoma. Knockdown of miR-124 promotes neuroblastoma SK-N-SH cell differentiation, cell cycle arrest and apoptosis. Further miR-124 is predicted to target aryl hydrocarbon receptor (AHR) which may promote neuroblastoma cell differentiation. We validate that miR-124 may suppress the expression of AHR by targeting its 3′-UTR. These results suggest that miR-124 could serve as a potential therapeutic target of neuroblastoma.
Authors: M Liu, Z Wang, S Yang, W Zhang, S He, C Hu, H Zhu, L Quan, J Bai1, N Xu
Many studies have demonstrated the overexpression and amplification of the miR-17-92 cluster in malignant human cancers, including B-cell lymphomas and lung cancers. The purpose of this study was to investigate for the first time, the expression of the miR-17-92 cluster in esophageal squamous cell carcinoma (ESCC). The miR-17-92 cluster was found to be overexpressed in 21 out of 28 (75%) esophageal cancer samples.
Authors: MR Epis, A Barker, KM Giles, DJ Beveridge, PJ Leedman
ERBB-2 overexpression is associated with the development and progression of cancer and mediates its resistance to therapy. It has been suggested that post-transcriptional mechanisms control the overexpression of ERBB-2 in prostate cancer (PCa).
Authors: J Kazenwadel, MZ Michael, NL Harvey
The specification of arterial, venous, and lymphatic endothelial cell fate is critical during vascular development. Although the homeobox transcription factor, Prox1, is crucial for the specification and maintenance of lymphatic endothelial cell identity, little is known regarding the mechanisms that regulate Prox1 expression.
miRNA mimics are small, chemically modified double-stranded RNAs that mimic endogenous miRNAs and enable miRNA functional analysis by up-regulation of miRNA activity.
Authors: X Ye, MG Hemida, Y Qiu, PJ Hanson, HM Zhang, D Yang
Coxsackievirus B3 (CVB3) is one of the most prevalent causes of viral myocarditis and is associated with many other pathological conditions. CVB3 replication relies on host cellular machineries and causes direct damage to host cells. MicroRNAs have been found to regulate viral infections but their roles in CVB3 infection are still poorly understood.
Authors: H-H Kwok, P Y-K Yue, N-K Mak, R N-S Wong
Wrinkle formation is one of the primary characteristics of skin aging, the major cause of wrinkle is the loss of structural protein type I collagen in dermal layer of skin. Topical application of natural substances to reduce wrinkle is gaining attention in recent years. Although a number of polyphenoic compounds are suggested to prevent ultraviolet-induced wrinkle, very few of them are able to increase type I collagen synthesis directly. Ginseng has been known in folk medicine of its beneficial effect to skin.
The Ambion pMIR-REPORT miRNA Expression Reporter Vector System is for the measurement of miRNA expression in cells. It contains two mammalian expression vectors—one for cloning miRNA targets and evaluating miRNA regulation, and the other for the normalization of transfection efficiency.
MicroRNA-205 promotes keratinocyte migration via the lipid phosphatase SHIP2
Authors: J Yu, H Peng, Q Ruan, As Fatima, S Getsios, RM Lavker
microRNA-205 (miR-205) and miR-184 coordinately regulate the lipid phosphatase SHIP2 for Akt survival signaling in keratinocytes. As the PI3K-Akt pathway has also been implicated in regulating the actin cytoskeleton and cell motility, we investigated the role that these 2 miRNAs play in keratinocyte migration.
Ambion In Vivo siRNAs are designed using the proven Silencer® Select algorithm and incorporate chemical modifications for superior serum stability with in vivo delivery. The added serum stability does not compromise the performance of the siRNAs. Ambion In Vivo siRNAs have been shown to be non-toxic in vivo (mouse) and non-immunogenic (cell-based assays). In cell-based assays, Ambion® In Vivo siRNAs exhibit equivalent or better potency than Silencer
Single-Nucleotide Polymorphisms Inside MicroRNA Target Sites Influence Tumor Susceptibility
Authors: MS Nicoloso, H Sun, R Spizzo, H Kim, P Wickramasinghe, M Shimizu, SE Wojcik, J Ferdin, T Kunej, L Xiao, S Manoukian, G Secreto, F Ravagnani, X Wang, P Radice, CM Croce, RV Davuluri, GA Calin
Single-nucleotide polymorphisms (SNP) associated with polygenetic disorders, such as breast cancer (BC), can create, destroy, or modify microRNA (miRNA) binding sites; however, the extent to which SNPs interfere with miRNA gene regulation and affect cancer susceptibility remains largely unknown. We hypothesize that disruption of miRNA target binding by SNPs is a widespread mechanism relevant to cancer susceptibility.
Ambion TURBO DNA-free contains reagents for the efficient, complete digestion of DNA along with the removal of the enzyme and divalent cations post-digestion.
Authors: KA Miller, RS Phillips, J Mrázek, TR Hoover
Salmonella enterica is a globally significant bacterial food-borne pathogen that utilizes a variety of carbon sources. We report here that Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) uses d-glucosaminate (2-amino-2-deoxy-d-gluconic acid) as a carbon and nitrogen source via a previously uncharacterized mannose family phosphotransferase system (PTS) permease, and we designate the genes encoding the permease dgaABCD (d-glucosaminate PTS permease components EIIA, EIIB, EIIC, and EIID).
Authors: H Singh, Y Li, PJ Fuller, C Harrison, J Rao, AN Stephens, G Nie
Helicoverpa armigera (Hübner) is one of the most polyphagous and cosmopolitan pest species, the larvae of which feed on numerous important crops. The gustatory system is critical in guiding insect feeding behavior.
Authors: G Ferrari, F Bignami, C Giacomini, S Franchini, P Rama
Purpose. To evaluate the safety/efficacy of topical infliximab, an anti-TNF-α monoclonal antibody, in a mouse model of ocular surface scarring.
Authors: PE Griffin, LF Roddam, YC Belessis, R Strachan, S Beggs, A Jaffe, MA Cooley
The Pseudomonas aeruginosa quorum sensing signal molecule N-3-oxododecanoyl-L-homoserine lactone (3OC12HSL) can inhibit function of the mammalian anti-inflammatory transcription factor peroxisome proliferator activated receptor (PPAR)γ, and can be degraded by human paraoxonase (PON)2. Because 3OC12HSL is detected in lungs of cystic fibrosis (CF) patients infected with P. aeruginosa, we investigated the relationship between P. aeruginosainfection and gene expression of PPARγ and PON2 in bronchoalveolar lavage fluid (BALF) of children with CF
Authors: Harmeet Singh, Ying Li, Peter J Fuller, Craig Harrison, Jyothsna Rao, Andrew N Stephens, and Guiying Nie
The high temperature requirement factor A3 (HtrA3) is a serine protease homologous to bacterial HtrA. Four human HtrAs have been identified. HtrA1 and HtrA3 share a high degree of domain organization and are downregulated in a number of cancers, suggesting a widespread loss of these proteases in cancer. This study examined how extensively the HtrA (HtrA1-3) proteins are downregulated in commonly used cancer cell lines and primary ovarian tumors.
Authors: Phoebe E. Griffin,Louise F. Roddam,Yvonne C. Belessis,Roxanne Strachan,Sean Beggs,Adam Jaffe,Margaret A. Cooley
The Pseudomonas aeruginosa quorum sensing signal molecule N-3-oxododecanoyl-l-homoserine lactone (3OC12HSL) can inhibit function of the mammalian anti-inflammatory transcription factor peroxisome proliferator activated receptor (PPAR)γ, and can be degraded by human paraoxonase (PON)2.
Authors: Q Zeng, MD Laiosa, DA Steeber, EM Biddle, Q Peng, C-H Yang
Dickeya dadantii 3937 is a gram-negative phytopathogenic bacterium that expresses genes encoding a type III secretion system (T3SS) in a bistable pattern when cultured in a homogeneous minimal media. In this work, we further characterized the bistable gene expression of T3SS at the single-cell level.
Authors: TJ Stark, JD Arnold, DH Spector, GW Yeo
Human cytomegalovirus (HCMV) contributes its own set of microRNAs (miRNAs) during lytic infection of cells, likely fine-tuning conditions important for viral replication. To enhance our understanding of this component of the HCMV-host transcriptome, we have conducted deep-sequencing analysis of small RNAs (smRNA-seq) from infected human fibroblast cells. We found that HCMV-encoded miRNAs accumulate to ∼20% of the total smRNA population at late stages of infection, and our analysis led to improvements in viral miRNA annotations and identification of two novel HCMV miRNAs, miR-US22 and miR-US33as.
Authors: HJ Mills, BK Reese, C St. Peter
The objective of this study was to determine shifts in the microbial community structure and potential function based on standard Integrated Ocean Drilling Program (IODP) storage procedures for sediment cores. Standard long-term storage protocols maintain sediment temperature at 4°C for mineralogy, geochemical, and/or geotechnical analysis whereas standard microbiological sampling immediately preserves sediments at −80°C. Storage at 4°C does not take into account populations may remain active over geologic time scales at temperatures similar to storage conditions.
Authors: L Yin, V Dragnea, CE Bauer
Heme-mediated regulation, presented in many biological processes, is achieved in part with proteins containing heme regulatory motif. In this study, we demonstrate that FLAG-tagged PpsR isolated from Rhodobacter sphaeroides cells contains bound heme.
Authors: PE Griffin, LF Roddam, YC Belessis, R Strachan, S Beggs, A Jaffe, MA Cooley
The Pseudomonas aeruginosa quorum sensing signal molecule N-3-oxododecanoyl-L-homoserine lactone (3OC12HSL) can inhibit function of the mammalian anti-inflammatory transcription factor peroxisome proliferator activated receptor (PPAR)γ, and can be degraded by human paraoxonase (PON)2. Because 3OC12HSL is detected in lungs of cystic fibrosis (CF) patients infected with P. aeruginosa, we investigated the relationship between P. aeruginosa infection and gene expression of PPARγ and PON2 in bronchoalveolar lavage fluid (BALF) of children with CF.
Authors: P Mamidala, AJ Wijeratne, S Wijeratne, K Kornacker, B Sudhamalla, LJ Rivera-Vega, A Hoelmer, T Meulia, SC Jones, O Mittapalli
Bed bugs (Cimex lectularius) are hematophagous nocturnal parasites of humans that have attained high impact status due to their worldwide resurgence. The sudden and rampant resurgence of C. lectularius has been attributed to numerous factors including frequent international travel, narrower pest management practices, and insecticide resistance.
Authors: MS Box, V Coustham, C Dean, JS Mylne
Many experiments in modern plant molecular biology require the processing of large numbers of samples for a variety of applications from mutant screens to the analysis of natural variants. A severe bottleneck to many such analyses is the acquisition of good yields of high quality RNA suitable for use in sensitive downstream applications such as real time quantitative reverse-transcription-polymerase chain reaction (real time qRT-PCR). Although several commercial kits are available for high-throughput RNA extraction in 96-well format, only one non-kit method has been described in the literature using the commercial reagent TRIZOL.
Authors: A-B Hachmann, E Sevim, A Gaballa, DL Popham, H Antelmann, JD Helmann
Daptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca2+-dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, DapR1, from the model bacterium Bacillus subtilis 168. DapR1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. DapR1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels.
Authors: T Shan, D Lan, L Li, C Wang, L Cui, W Zhang, X Hua, C Zhu, W Zhao, E Delwart
Using random PCR amplification followed by plasmid subcloning and DNA sequencing, we detected bocavirus related sequences in 9 out of 17 porcine stool samples. Using primer walking, we sequenced the nearly complete genomes of two highly divergent bocaviruses we provisionally named porcine bocavirus 1 isolate H18 (PBoV1-H18) and porcine bocavirus 2 isolate A6 (PBoV2-A6) which differed by 51.8% in their NS1 protein.
Authors: S Shuxian, W Sheng, S Schachtele, J Lokensgard
Production of reactive oxygen species (ROS) and proinflammatory cytokines by microglial cells in response to viral brain infection contributes to both pathogen clearance and neuronal damage. In the present study, we examined the effect of herpes simplex virus (HSV)-1-induced, NADPH oxidase-derived ROS in activating mitogen-activated protein kinases (MAPKs) as well as driving cytokine and chemokine expression in primary murine microglia.
Authors: M Panigaj, H Glier, M Wildova, K Holada
Prion diseases have been observed to deregulate the transcription of erythroid genes, and prion protein knockout mice have demonstrated a diminished response to experimental anemia. To investigate the role of the cellular prion protein (PrPC) in erythropoiesis, we studied the protein’s expression on mouse erythroid precursors in vivo and utilized an in vitro model of the erythroid differentiation of murine erythroleukemia cells (MEL) to evaluate the effect of silencing PrPC through RNA interference. The expression of PrPC and selected differentiation markers was analyzed by quantitative multicolor flow cytometry, western blot analysis and quantitative RT-PCR. The silencing of PrPC expression in MEL cells was achieved by expression of shRNAmir from an integrated retroviral vector genome.
Krüppel-like factor 4 regulates adaptive expression of the zinc transporter Zip4 in mouse small intestine
Authors: JP Liuzzi, L Guo, S-M Chang, RJ Cousins
Epithelial cells of the small intestine are the site of zinc absorption. Intestinal uptake of zinc is inversely proportional to the dietary supply of this essential micronutrient. The mechanism responsible for this adaptive differential in apical zinc transport is not known. The zinc transporter Zip4 (Slc39a4) is essential for adequate enteric zinc uptake.
Ambion's RNase-free DNase I is of the highest purity available and is recommended to degrade DNA in the presence of RNA when the absence of RNase is critical to maintain the integrity of the RNA.
Authors: TI Jones, JCJ Chen, F Rahimov, S Homma, P Arashiro, ML Beermann, OD King, JB Miller, LM Kunkel, CP Emerson Jr, KR Wagner, PL Jones
Facioscapulohumeral muscular dystrophy (FSHD), the most prevalent myopathy afflicting both children and adults, is predominantly associated with contractions in the 4q35-localized macrosatellite D4Z4 repeat array.
Authors: LS Reinert, L Harder, CK Holm, MB Iversen, KA Horan, F Dagnæs-Hansen, BP Ulhøi, TH Holm, TH Mogensen, T Owens, JR Nyengaard, AR Thomsen, SR Paludan
Herpes simplex viruses (HSVs) are highly prevalent neurotropic viruses. While they can replicate lytically in cells of the epithelial lineage, causing lesions on mucocutaneous surfaces, HSVs also establish latent infections in neurons, which act as reservoirs of virus for subsequent reactivation events. Immunological control of HSV involves activation of innate immune pattern-recognition receptors such as TLR3, which detects doublestranded RNA and induces type I IFN expression.
Authors: R Bansal, P Mamidala, MA Rouf Mian, O Mittapalli, AP Michel
Quantitative real-time polymerase chain reaction (qRT-PCR) is a common and robust tool for accurate quantification of mRNA transcripts. To normalize results, a housekeeping gene ([HKG], reference gene or endogenous control gene) is mandatory. Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a significant soybean, Glycine max (L.) Merr., pest, yet gene expression and functional genomics studies are hindered by a lack of stable HKGs.
Authors: C-C Yang, H Liu, SL Chen, T-H Wang, C-L Hsieh, Y Huang, S-J Chen, H-Cn Chen, B Yat-Ming Yung, B Chin-Ming Tan
Skeletal myogenesis involves highly coordinated steps that integrate developmental cues at the chromatin of muscle progenitors. Here, we identify Myb-binding protein 1a (Mybbp1a) as a novel negative regulator of muscle-specific gene expression and myoblast differentiation. The mode of action of Mybbp1a was linked to promoter regulation as illustrated by its interaction with MyoD at the genomic regions of silent muscle-specific genes as well as its negative effect on MyoD-mediated transcriptional activity. We propose that Mybbp1a exerts its repressive role by inducing a less permissible chromatin structure following recruitment of negative epigenetic modifiers such as HDAC1/2 and Suv39h1.
Authors: J Chen, A Stahl, NM Krah, MR Seaward, RJ Dennison, P Sapieha, J Hua, CJ Hatton, AM Juan, CM Aderman, KL Willett, KI Guerin, A Mammoto, M Campbell, LEH Smith
Ischemic proliferative retinopathy, characterized by pathological retinal neovascularization, is a major cause of blindness in working-age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathological vessels in retinopathy, we investigated the potential role of Wnt signaling in pathological retinal vascular growth in proliferative retinopathy.
Authors: AM Hashem, AS Flaman, A Farnsworth, EG Brown, G Van Domselaar, R He, X Li
Influenza viruses cause serious infections that can be prevented or treated using vaccines or antiviral agents, respectively. While vaccines are effective, they have a number of limitations, and influenza strains resistant to currently available anti-influenza drugs are increasingly isolated. This necessitates the exploration of novel anti-influenza therapies.
Authors: KL Adams, R Percifield, JF Wendel
Most eukaryotes have undergone genome doubling at least once during their evolutionary history. Hybridization followed by genome doubling (allopolyploidization) is a prominent mode of speciation in plants, leading to phenotypic novelty and changes in genome structure and gene expression. Molecular events that take place immediately after polyploid formation can be studied using newly synthesized allopolyploids. Here we studied the extent of gene silencing in a newly created and genomically stable allotetraploid cotton, of genotype AAGG, using an AFLP-cDNA display screen. Over 2000 transcripts were screened and ~ 5% of the duplicated genes in the allotetraploid were inferred to have been silenced or downregulated. Sequencing of 24 AFLP-cDNA fragments revealed genes with a variety of functions
TURBO DNase cleaves double-stranded DNA nonspecifically to leave 5' phosphorylated oligodeoxynucleotides. It has increased affinity for DNA-binding and remains active in the presence of salt.
Authors: KM Dorn, JD Fankhauser, DL Wyse, MD Marks
Field pennycress (Thlaspi arvense L.) has potential as an oilseed crop that may be grown during fall (autumn) and winter months in the Midwestern United States and harvested in the early spring as a biodiesel feedstock. There has been little agronomic improvement in pennycress through traditional breeding.
Used as a component in many hybridization protocols, Ambion Salmon Sperm DNA is rigorously tested for RNase and DNase contamination and is suspended in nuclease-free water at a concentration of 10 mg⁄mL.
Authors: Ravindra B. Chalamalasetty, William C. Dunty Jr, Kristin K. Biris,Rieko Ajima, Michelina Iacovino, Arica Beisaw, Lionel Feigenbaum, Deborah L. Chapman, Jeong Kyo Yoon, Michael Kyba & Terry P. Yamaguchi
Segmentation is an organizing principle of body plans. The segmentation clock, a molecular oscillator best illustrated by the cyclic expression of Notch signalling genes, controls the periodic cleavage of somites from unsegmented presomitic mesoderm during vertebrate segmentation. Wnt3a controls the spatiotemporal expression of cyclic Notch genes; however, the underlying mechanisms remain obscure. Here we show by transcriptional proﬁ ling of Wnt3a−/− embryos that the bHLH transcription factor, Mesogenin1 (Msgn1) , is a direct target gene of Wnt3a. To identify Msgn1 targets, we conducted genome-wide studies of Msgn1 activity in embryonic stem cells. We show that Msgn1 is a major transcriptional activator of a Notch signalling program and synergizes with Notch to trigger clock gene expression. Msgn1 also indirectly regulates cyclic genes in the Fgf and Wnt pathways. Thus, Msgn1 is a central component of a transcriptional cascade that translates a spatial Wnt3a gradient into a temporal pattern of clock gene expression.
The Ambion Decade Marker System produces a set of radiolabeled RNA molecules of 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, and 10 nucleotides. The molecular weight markers are ideally suited for experiments where RNA molecules less than 150 nucleotides in length are being studied. Sufficient reagents are provided for 10 marker preparation reactions. This marker set is particularly useful for research laboratories studying tRNA, snRNA, snoRNA, miRNA, siRNA and other small RNA molecules.
Authors: B Khraiwesh
Small RNAs (20–24 nucleotides long and nonprotein coding) have been increasingly investigated. They are responsible for phenomena described as RNA interference (RNAi), cosuppression, gene silencing, or quelling. Major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biosynthesis. MiRNAs control the expression of cognate target genes by binding to reverse complementary sequences, resulting in cleavage or translational inhibition of the target RNA. SiRNAs have similar structure, function, and biogenesis as miRNAs; siRNAs derive from long double-stranded RNA of transgenes, endogenous repeat sequences, or transposons. Understanding these fundamental processes requires the sensitive and specific detection of small RNA species. In this report, we present a simple Northern blot protocol for small RNAs in transgenic plants.
The Ambion pMIR-REPORT miRNA Expression Reporter Vector System is for the measurement of miRNA expression in cells. It contains two mammalian expression vectors-—one for cloning miRNA targets and evaluating miRNA regulation, and the other for the normalization of transfection efficiency.
Authors: N Patel, SM Tahara, P Malik, VK Kalra
Plasminogen activator inhibitor-1 (PAI-1) is a key physiological inhibitor of fibrinolysis. Previously, we reported placenta growth factor (PlGF) mediated transcriptional upregulation of PAI-1 mRNAexpression via activation of hypoxia-inducible factor-1α and activator protein-1 in human pulmonary microvascular endothelial cells (HPMVEC); which resulted in elevated PAI-1 in humans with sickle cell anemia (SCA).
We offer a wide variety of antibodies against key signaling proteins, cellular markers and organelles as well as cell junctions and CD markers.
Authors: D Zhou, Y Li, L Lin, L Zhou, P Igarashi, Y Liu
β-Catenin is a unique intracellular protein functioning as an integral component of the cell–cell adherens complex and a principal signaling protein mediating canonical Wnt signaling. Little is known about its function in adult kidneys in the normal physiologic state or after acute kidney injury (AKI). To study this, we generated conditional knockout mice in which the β-catenin gene was specifically disrupted in renal tubules (Ksp-β-cat−/−). These mice were phenotypically normal with no appreciable defects in kidney morphology and function. In the absence of β-catenin, γ-catenin functionally substituted for it in E-cadherin binding, thereby sustaining the integrity of epithelial adherens junctions in the kidneys. In AKI induced by ischemia reperfusion or folic acid, the loss of tubular β-catenin substantially aggravated renal lesions.
The last step in every RNA isolation protocol is to resuspend the purified RNA pellet. After painstakingly isolating the RNA, it is crucial that the pellet be suspended and stored in a safe, RNase-free solution.
Authors: D Duan, K-x Zheng, Y Shen, R Cao, L Jiang, Z Lu, X Yan, J Li
MicroRNAs (miRNAs) are key biological regulators and promising disease markers whose detection technologies hold great potentials in advancing fundamental research and medical diagnostics. Currently, miRNAs in biological samples have to be labeled before being applied to most high-throughput assays.