Short torch design for direct liquid sample introduction using conventional and micro-nebulizers for plasma spectrometry

DOEpatents

Montaser, Akbar [Potomac, MD; Westphal, Craig S [Landenberg, PA; Kahen, Kaveh [Montgomery Village, MD; Rutkowski, William F [Arlington, VA

2008-01-08

An apparatus and method for providing direct liquid sample introduction using a nebulizer are provided. The apparatus and method include a short torch having an inner tube and an outer tube, and an elongated adapter having a cavity for receiving the nebulizer and positioning a nozzle tip of the nebulizer a predetermined distance from a tip of the outer tube of the short torch. The predetermined distance is preferably about 2-5 mm.

Direct interplay between two candidate genes in FSHD muscular dystrophy

PubMed Central

Ferri, Giulia; Huichalaf, Claudia H.; Caccia, Roberta; Gabellini, Davide

2015-01-01

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common neuromuscular disorders. The major form of the disease (FSHD1) is linked to decrease in copy number of a 3.3-kb tandem repeated macrosatellite (D4Z4), located on chromosome 4q35. D4Z4 deletion alters chromatin structure of the locus leading to aberrant expression of nearby 4q35 genes. Given the high variability in disease onset and progression, multiple factors could contribute to the pathogenesis of FSHD. Among the FSHD candidate genes are double homeobox 4 (DUX4), encoded by the most telomeric D4Z4 unit, and FSHD region gene 1 (FRG1). DUX4 is a sequence-specific transcription factor. Here, we located putative DUX4 binding sites in the human FRG1 genomic area and we show specific DUX4 association to these regions. We found also that ectopically expressed DUX4 up-regulates the endogenous human FRG1 gene in healthy muscle cells, while DUX4 knockdown leads to a decrease in FRG1 expression in FSHD muscle cells. Moreover, DUX4 binds directly and specifically to its binding site located in the human FRG1 gene and transactivates constructs containing FRG1 genomic regions. Intriguingly, the mouse Frg1 genomic area lacks DUX4 binding sites and DUX4 is unable to activate the endogenous mouse Frg1 gene providing a possible explanation for the lack of muscle phenotype in DUX4 transgenic mice. Altogether, our results demonstrate that FRG1 is a direct DUX4 transcriptional target uncovering a novel regulatory circuit contributing to FSHD. PMID:25326393

Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

PubMed

Subburaj, Saminathan; Chung, Sung Jin; Lee, Choongil; Ryu, Seuk-Min; Kim, Duk Hyoung; Kim, Jin-Soo; Bae, Sangsu; Lee, Geung-Joo

2016-07-01

Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

CRISPR-mediated direct mutation of cancer genes in the mouse liver

PubMed Central

Xue, Wen; Chen, Sidi; Yin, Hao; Tammela, Tuomas; Papagiannakopoulos, Thales; Joshi, Nikhil S.; Cai, Wenxin; Yang, Gillian; Bronson, Roderick; Crowley, Denise G.; Zhang, Feng; Anderson, Daniel G.; Sharp, Phillip A.; Jacks, Tyler

2014-01-01

The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem (ES) cells1. Here we describe a new method of cancer model generation using the CRISPR/Cas system in vivo in wild-type mice. We have used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs)2–4 to the liver and directly target the tumor suppressor genes Pten5 and p536, alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology7, 8. Simultaneous targeting of Pten and p53 induced liver tumors that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumor tissue revealed insertion or deletion (indel) mutations of the tumor suppressor genes, including bi-allelic mutations of both Pten and p53 in tumors. Furthermore, co-injection of Cas9 plasmids harboring sgRNAs targeting the β-Catenin gene (Ctnnb1) and a single-stranded DNA (ssDNA) oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-Catenin. This study demonstrates the feasibility of direct mutation of tumor suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics. PMID:25119044

CRISPR-mediated direct mutation of cancer genes in the mouse liver.

PubMed

Xue, Wen; Chen, Sidi; Yin, Hao; Tammela, Tuomas; Papagiannakopoulos, Thales; Joshi, Nikhil S; Cai, Wenxin; Yang, Gillian; Bronson, Roderick; Crowley, Denise G; Zhang, Feng; Anderson, Daniel G; Sharp, Phillip A; Jacks, Tyler

2014-10-16

The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.

Development of in vivo gene therapy for hearing disorders: introduction of adeno-associated virus into the cochlea of the guinea pig.

PubMed

Lalwani, A K; Walsh, B J; Reilly, P G; Muzyczka, N; Mhatre, A N

1996-07-01

Gene therapy is currently being used to treat many disorders including cancer, viral infection and the degenerative and fatal diseases of the cardiovascular and the central nervous systems. However, the potential use of gene therapy for alleviation of hearing impairment has not been investigated despite the absence of effective therapy for most forms of inherited hearing disorders. The purpose of this study was to assess the feasibility of introducing genetic material directly into the peripheral auditory system using adeno-associated virus (AAV) as the transfection vector and Hartley guinea pigs as the animal model. Approximately 10(5) particles of AAV containing the bacterial beta-galactosidase (beta-gal) sequence with Ad 2 major late promoter were infused into the cochlea of the animal with the aid of an osmotic minipump. Animals were killed after 2 weeks. Two Hartley guinea pigs with intracochlear saline infusion and four unoperated (nonperfused) animals served as negative controls. Both, the infused and the contralateral, non-infused cochleae were harvested from each animal, decalcified, and embedded in paraffin. Sections, 8 microns in width, were cut from the embedded cochleae and assayed for beta-gal expression via immunohistochemistry. Animals perfused with AAV showed intense immunohistochemical reactivity in the spiral limbus, spiral ligament, spiral ganglion cells and the organ of Corti in the perfused cochlea and a much weaker staining but with similar pattern in the contralateral ear. Cochleae from saline-infused and unoperated animals were devoid of the DAB stain. This study demonstrates for the first time in vivo expression of a foreign gene within the mammalian inner ear resulting from its localized, AAV-mediated introduction. The ability to introduce and stably express exogenous genetic material in the peripheral auditory system will have both experimental and therapeutic benefits. These results lay the groundwork for future studies assessing the

Directed mutagenesis of the Rickettsia prowazekii pld gene encoding phospholipase D.

PubMed

Driskell, Lonnie O; Yu, Xue-jie; Zhang, Lihong; Liu, Yan; Popov, Vsevolod L; Walker, David H; Tucker, Aimee M; Wood, David O

2009-08-01

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The R. prowazekii pld gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a pld mutant of the virulent R. prowazekii strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted pld gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the pld mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.

Introduction of the second-generation direct-acting antivirals (DAAs) in chronic hepatitis C: a register-based study in Sweden.

PubMed

Frisk, P; Aggefors, K; Cars, T; Feltelius, N; Loov, S A; Wettermark, B; Weiland, O

2018-07-01

Introduction of the direct-acting antivirals (DAAs) for treatment of chronic hepatitis C (CHC) infection has been challenging in all health systems. In Sweden, a national protocol for managed introduction was developed. It was optional, but all county councils agreed to implement and follow it. The purpose of this study was to study (a) cure rates among all patients initiated on treatment in 2014-2015, (b) prescribers' adherence to the drug recommendations and treatment eligibility criteria in the protocol, and (c) introduction rate in the six Swedish healthcare regions. A cross-sectional study where national data from the Prescribed Drug Register and the quality register InfCare Hepatitis defined the study population, and clinical data from the Patient Register and InfCare Hepatitis were used to monitor outcomes. Descriptive statistics were used. A total of 3447 patients were initiated on treatment during 2014-2015. The overall cure rate, based on data from 85% of the cohort, was 96%, with variation between genotypes. Adherence to drug recommendations increased over time and varied between 43.2 and 94.2%. Adherence to the treatment eligibility criteria was initially 80% and increased to 87% when treatment restrictions were widened. The introduction rate differed initially between the regions and reached stable levels 15-18 months after the launch of the first DAA. The estimated overall cure rate was 96%, with some variations between genotypes. A high level of adherence to the introduction protocol as well as similar introduction rates in the health care regions indicate that the introduction protocol, alongside with other measures taken, contributed considerably to a rapid uptake and equal distribution of DAAs in Sweden.

Dual gene activation and knockout screen reveals directional dependencies in genetic networks. | Office of Cancer Genomics

Cancer.gov

Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair.

7 CFR 766.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

...) Have loan security being liquidated voluntarily or involuntarily. (b) The Agency services direct FLP... SPECIAL PROGRAMS DIRECT LOAN SERVICING-SPECIAL Overview § 766.1 Introduction. (a) This part describes the Agency's servicing policies for direct loan borrowers who: (1) Are financially distressed; (2) Are...

Direct adenovirus-mediated gene delivery to the temporomandibular joint in guinea-pigs.

PubMed

Kuboki, T; Nakanishi, T; Kanyama, M; Sonoyama, W; Fujisawa, T; Kobayashi, K; Ikeda, T; Kubo, T; Yamashita, A; Takigawa, M

1999-09-01

Adenovirus vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, recombinant adenovirus vectors harbouring beta-galactosidase gene (LacZ) was injected directly into the temporomandibular joints of Hartley guinea-pigs to clarify the in vivo transfer availability of the adenovirus vectors. Specifically, recombinant adenovirus harbouring LacZ gene (AxlCALacZ) was injected into the upper joint cavities of both mandibular joints of four male 6-week-old Hartley guinea-pigs. Either the same amount of recombinant adenovirus without LacZ gene (Axlw) suspension (placebo) or the same amount of phosphate-buffered saline solution (control) were injected into the upper joint cavities of both joints of another four male guinea-pigs. At 1, 2, 3 and 4 weeks after injection, the joints were dissected and the expression of delivered LacZ was examined by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining and reverse transcriptase-polymerase chain reaction (RT-PCR). To investigate the expression of transferred gene in other organs, total RNA was extracted from liver, kidney, heart and brain and the expression of LacZ mRNA and 18 S ribosomal RNA were analysed by RT-PCR. Clear expression of LacZ was observed in the articular surfaces of the temporal tubercle, articular disc and synovium of the temporomandibular joints even 4 weeks after injection in the AxlCALacZ-injected group, while no expression was detected in placebo and control groups. Histological examination confirmed that LacZ activity was clearly detected in a few cell layers of the articular surface tissues, which is much more efficient than in a previously study of the knee joint. In the other organs, expression of the delivered transgene was not observed. Based on these findings, direct gene delivery into the articular surface of the temporomandibular joint

Phage-Mediated Gene Therapy.

PubMed

Hosseinidoust, Zeinab

2017-01-01

Bacteriophages (bacterial viruses) have long been under investigation as vectors for gene therapy. Similar to other viral vectors, the phage coat proteins have evolved over millions of years to protect the viral genome from degradation post injection, offering protection for the valuable therapeutic sequence. However, what sets phage apart from other viral gene delivery vectors is their safety for human use and the relative ease by which foreign molecules can be expressed on the phage outer surface, enabling highly targeted gene delivery. The latter property also makes phage a popular choice for gene therapy target discovery through directed evolution. Although promising, phage-mediated gene therapy faces several outstanding challenges, the most notable being lower gene delivery efficiency compared to animal viruses, vector stability, and nondesirable immune stimulation. This review presents a critical review of promises and challenges of employing phage as gene delivery vehicles as well as an introduction to the concept of phage-based microbiome therapy as the new frontier and perhaps the most promising application of phage-based gene therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Direct multiplexed measurement of gene expression with color-coded probe pairs.

PubMed

Geiss, Gary K; Bumgarner, Roger E; Birditt, Brian; Dahl, Timothy; Dowidar, Naeem; Dunaway, Dwayne L; Fell, H Perry; Ferree, Sean; George, Renee D; Grogan, Tammy; James, Jeffrey J; Maysuria, Malini; Mitton, Jeffrey D; Oliveri, Paola; Osborn, Jennifer L; Peng, Tao; Ratcliffe, Amber L; Webster, Philippa J; Davidson, Eric H; Hood, Leroy; Dimitrov, Krassen

2008-03-01

We describe a technology, the NanoString nCounter gene expression system, which captures and counts individual mRNA transcripts. Advantages over existing platforms include direct measurement of mRNA expression levels without enzymatic reactions or bias, sensitivity coupled with high multiplex capability, and digital readout. Experiments performed on 509 human genes yielded a replicate correlation coefficient of 0.999, a detection limit between 0.1 fM and 0.5 fM, and a linear dynamic range of over 500-fold. Comparison of the NanoString nCounter gene expression system with microarrays and TaqMan PCR demonstrated that the nCounter system is more sensitive than microarrays and similar in sensitivity to real-time PCR. Finally, a comparison of transcript levels for 21 genes across seven samples measured by the nCounter system and SYBR Green real-time PCR demonstrated similar patterns of gene expression at all transcript levels.

Effects of FVIII immunity on hepatocyte and hematopoietic stem cell–directed gene therapy of murine hemophilia A

PubMed Central

Lytle, Allison M; Brown, Harrison C; Paik, Na Yoon; Knight, Kristopher A; Wright, J Fraser; Spencer, H Trent; Doering, Christopher B

2016-01-01

Immune responses to coagulation factors VIII (FVIII) and IX (FIX) represent primary obstacles to hemophilia treatment. Previously, we showed that hematopoietic stem cell (HSC) retroviral gene therapy induces immune nonresponsiveness to FVIII in both naive and preimmunized murine hemophilia A settings. Liver-directed adeno-associated viral (AAV)-FIX vector gene transfer achieved similar results in preclinical hemophilia B models. However, as clinical immune responses to FVIII and FIX differ, we investigated the ability of liver-directed AAV-FVIII gene therapy to affect FVIII immunity in hemophilia A mice. Both FVIII naive and preimmunized mice were administered recombinant AAV8 encoding a liver-directed bioengineered FVIII expression cassette. Naive animals receiving high or mid-doses subsequently achieved near normal FVIII activity levels. However, challenge with adjuvant-free recombinant FVIII induced loss of FVIII activity and anti-FVIII antibodies in mid-dose, but not high-dose AAV or HSC lentiviral (LV) vector gene therapy cohorts. Furthermore, unlike what was shown previously for FIX gene transfer, AAV-FVIII administration to hemophilia A inhibitor mice conferred no effect on anti-FVIII antibody or inhibitory titers. These data suggest that functional differences exist in the immune modulation achieved to FVIII or FIX in hemophilia mice by gene therapy approaches incorporating liver-directed AAV vectors or HSC-directed LV. PMID:26909355

Direct sample introduction-gas chromatography-mass spectrometry for the determination of haloanisole compounds in cork stoppers.

PubMed

Cacho, J I; Nicolás, J; Viñas, P; Campillo, N; Hernández-Córdoba, M

2016-12-02

A solventless analytical method is proposed for analyzing the compounds responsible for cork taint in cork stoppers. Direct sample introduction (DSI) is evaluated as a sample introduction system for the gas chromatography-mass spectrometry (GC-MS) determination of four haloanisoles (HAs) in cork samples. Several parameters affecting the DSI step, including desorption temperature and time, gas flow rate and other focusing parameters, were optimized using univariate and multivariate approaches. The proposed method shows high sensitivity and minimises sample handling, with detection limits of 1.6-2.6ngg -1 , depending on the compound. The suitability of the optimized procedure as a screening method was evaluated by obtaining decision limits (CCα) and detection capabilities (CCβ) for each analyte, which were found to be in 6.9-11.8 and 8.7-14.8ngg -1 , respectively, depending on the compound. Twenty-four cork samples were analysed, and 2,4,6-trichloroanisole was found in four of them at levels between 12.6 and 53ngg -1 . Copyright © 2016 Elsevier B.V. All rights reserved.

Gene doping detection: evaluation of approach for direct detection of gene transfer using erythropoietin as a model system.

PubMed

Baoutina, A; Coldham, T; Bains, G S; Emslie, K R

2010-08-01

As clinical gene therapy has progressed toward realizing its potential, concern over misuse of the technology to enhance performance in athletes is growing. Although 'gene doping' is banned by the World Anti-Doping Agency, its detection remains a major challenge. In this study, we developed a methodology for direct detection of the transferred genetic material and evaluated its feasibility for gene doping detection in blood samples from athletes. Using erythropoietin (EPO) as a model gene and a simple in vitro system, we developed real-time PCR assays that target sequences within the transgene complementary DNA corresponding to exon/exon junctions. As these junctions are absent in the endogenous gene due to their interruption by introns, the approach allows detection of trace amounts of a transgene in a large background of the endogenous gene. Two developed assays and one commercial gene expression assay for EPO were validated. On the basis of ability of these assays to selectively amplify transgenic DNA and analysis of literature on testing of gene transfer in preclinical and clinical gene therapy, it is concluded that the developed approach would potentially be suitable to detect gene doping through gene transfer by analysis of small volumes of blood using regular out-of-competition testing.

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems

PubMed Central

Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B.

2015-01-01

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing. PMID:26053390

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems.

PubMed

Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B

2015-01-01

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing.

Quantitative real-time monitoring of multi-elements in airborne particulates by direct introduction into an inductively coupled plasma mass spectrometer

NASA Astrophysics Data System (ADS)

Suzuki, Yoshinari; Sato, Hikaru; Hiyoshi, Katsuhiro; Furuta, Naoki

2012-10-01

A new calibration system for real-time determination of trace elements in airborne particulates was developed. Airborne particulates were directly introduced into an inductively coupled plasma mass spectrometer, and the concentrations of 15 trace elements were determined by means of an external calibration method. External standard solutions were nebulized by an ultrasonic nebulizer (USN) coupled with a desolvation system, and the resulting aerosol was introduced into the plasma. The efficiency of sample introduction via the USN was calculated by two methods: (1) the introduction of a Cr standard solution via the USN was compared with introduction of a Cr(CO)6 standard gas via a standard gas generator and (2) the aerosol generated by the USN was trapped on filters and then analyzed. The Cr introduction efficiencies obtained by the two methods were the same, and the introduction efficiencies of the other elements were equal to the introduction efficiency of Cr. Our results indicated that our calibration method for introduction efficiency worked well for the 15 elements (Ti, V, Cr, Mn, Co, Ni, Cu, Zn, As, Mo, Sn, Sb, Ba, Tl and Pb). The real-time data and the filter-collection data agreed well for elements with low-melting oxides (V, Co, As, Mo, Sb, Tl, and Pb). In contrast, the real-time data were smaller than the filter-collection data for elements with high-melting oxides (Ti, Cr, Mn, Ni, Cu, Zn, Sn, and Ba). This result implies that the oxides of these 8 elements were not completely fused, vaporized, atomized, and ionized in the initial radiation zone of the inductively coupled plasma. However, quantitative real-time monitoring can be realized after correction for the element recoveries which can be calculated from the ratio of real-time data/filter-collection data.

Introduction to bioinformatics.

PubMed

Can, Tolga

2014-01-01

Bioinformatics is an interdisciplinary field mainly involving molecular biology and genetics, computer science, mathematics, and statistics. Data intensive, large-scale biological problems are addressed from a computational point of view. The most common problems are modeling biological processes at the molecular level and making inferences from collected data. A bioinformatics solution usually involves the following steps: Collect statistics from biological data. Build a computational model. Solve a computational modeling problem. Test and evaluate a computational algorithm. This chapter gives a brief introduction to bioinformatics by first providing an introduction to biological terminology and then discussing some classical bioinformatics problems organized by the types of data sources. Sequence analysis is the analysis of DNA and protein sequences for clues regarding function and includes subproblems such as identification of homologs, multiple sequence alignment, searching sequence patterns, and evolutionary analyses. Protein structures are three-dimensional data and the associated problems are structure prediction (secondary and tertiary), analysis of protein structures for clues regarding function, and structural alignment. Gene expression data is usually represented as matrices and analysis of microarray data mostly involves statistics analysis, classification, and clustering approaches. Biological networks such as gene regulatory networks, metabolic pathways, and protein-protein interaction networks are usually modeled as graphs and graph theoretic approaches are used to solve associated problems such as construction and analysis of large-scale networks.

Effect of introduction of chondroitin sulfate into polymer-peptide conjugate responding to intracellular signals

NASA Astrophysics Data System (ADS)

Tomiyama, Tetsuro; Toita, Riki; Kang, Jeong-Hun; Koga, Haruka; Shiosaki, Shujiro; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki

2011-09-01

We recently developed a novel tumor-targeted gene delivery system responding to hyperactivated intracellular signals. Polymeric carrier for gene delivery consists of hydrophilic neutral polymer as main chains and cationic peptide substrate for target enzyme as side chains, and was named polymer-peptide conjugate (PPC). Introduction of chondroitin sulfate (CS), which induces receptor-medicated endocytosis, into polymers mainly with a high cationic charge density such as polyethylenimine can increase tumor-targeted gene delivery. In the present study, we examined whether introduction of CS into PPC containing five cationic amino acids can increase gene expression in tumor cells. Size and zeta potential of plasmid DNA (pDNA)/PPC/CS complex were <200 nm and between -10 and -15 mV, respectively. In tumor cell experiments, pDNA/PPC/CS complex showed lower stability and gene regulation, compared with that of pDNA/PPC. Moreover, no difference in gene expression was identified between positive and negative polymer. These results were caused by fast disintegration of pDNA/PPC/CS complexes in the presence of serum. Thus, we suggest that introduction of negatively charged CS into polymers with a low charge density may lead to low stability and gene regulation of complexes.

Variable directionality of gene expression changes across generations does not constitute negative evidence of epigenetic inheritance.

PubMed

Sharma, Abhay

2015-01-01

Transgenerational epigenetic inheritance in mammals has been controversial due to inherent difficulties in its experimental demonstration. A recent report has, however, opened a new front in the ongoing debate by claiming that endocrine disrupting chemicals, contrary to previous findings, do not cause effects across generations. This claim is based on the observation that gene expression changes induced by these chemicals in the exposed and unexposed generations are mainly in the opposite direction. This analysis shows that the pattern of gene expression reported in the two generations is not expected by chance and is suggestive of transmission across generations. A meta-analysis of diverse data sets related to endocrine disruptor-induced transgenerational gene expression alterations, including the data provided in the said report, further suggests that effects of endocrine disrupting chemicals persist in unexposed generations. Based on the prior evidence of phenotypic variability and gene expression alterations in opposite direction between generations, it is argued here that calling evidence of mismatched directionality in gene expression in experiments testing potential of environmental agents in inducing epigenetic inheritance of phenotypic traits as negative is untenable. This is expected to settle the newly raised doubts over epigenetic inheritance in mammals.

Forced expression of Hnf4a induces hepatic gene activation through directed differentiation.

PubMed

Yahoo, Neda; Pournasr, Behshad; Rostamzadeh, Jalal; Fathi, Fardin

2016-08-05

Embryonic stem (ES) cells are capable of unlimited self-renewal and have a diverse differentiation potential. These unique features make ES cells as an attractive source for developmental biology studies. Having the mature hepatocyte in the lab with functional activities is valuable in drug discovery studies. Overexpression of hepatocyte lineage-specific transcription factors (TFs) becomes a promising approach in pluripotent cell differentiation toward liver cells. Many studies generate transgenic ES cell lines to examine the effects of specific TFs overexpression in cell differentiation. In the present report, we have addressed whether a suspension or adherent model of differentiation is an appropriate way to study the role of Hnf4a overexpression. We generated ES cells that carried a doxycycline (Dox)-inducible Hnf4a using lentiviral vectors. The transduced cells were subjected to induced Hnf4a overexpression through both spontaneous and directed differentiation methods. Gene expression analysis showed substantially increased expression of hepatic gene markers, particularly Ttr and endogenous Hnf4a, in transduced cells differentiated by the directed approach. These results demonstrated that forced expression of TFs during directed differentiation would be an appropriate way to study relevant gene activation and the effects of overexpression in the context of hepatic differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

Drop-on-demand sample introduction system coupled with the flowing atmospheric-pressure afterglow for direct molecular analysis of complex liquid microvolume samples.

PubMed

Schaper, J Niklas; Pfeuffer, Kevin P; Shelley, Jacob T; Bings, Nicolas H; Hieftje, Gary M

2012-11-06

One of the fastest developing fields in analytical spectrochemistry in recent years is ambient desorption/ionization mass spectrometry (ADI-MS). This burgeoning interest has been due to the demonstrated advantages of the method: simple mass spectra, little or no sample preparation, and applicability to samples in the solid, liquid, or gaseous state. One such ADI-MS source, the flowing atmospheric-pressure afterglow (FAPA), is capable of direct analysis of solids just by aiming the source at the solid surface and sampling the produced ions into a mass spectrometer. However, direct introduction of significant volumes of liquid samples into this source has not been possible, as solvent loads can quench the afterglow and, thus, the formation of reagent ions. As a result, the analysis of liquid samples is preferably carried out by analyzing dried residues or by desorbing small amounts of liquid samples directly from the liquid surface. In the former case, reproducibility of sample introduction is crucial if quantitative results are desired. In the present study, introduction of liquid samples as very small droplets helps overcome the issues of sample positioning and reduced levels of solvent intake. A recently developed "drop-on-demand" (DOD) aerosol generator is capable of reproducibly producing very small volumes of liquid (∼17 pL). In this paper, the coupling of FAPA-MS and DOD is reported and applications are suggested. Analytes representing different classes of substances were tested and limits of detections were determined. Matrix tolerance was investigated for drugs of abuse and their metabolites by analyzing raw urine samples and quantification without the use of internal standards. Limits of detection below 2 μg/mL, without sample pretreatment, were obtained.

The Oral Introduction to the Instructional Film: A Closer Look.

ERIC Educational Resources Information Center

Arwady, Joseph W.

1980-01-01

Discusses reasons for an oral introduction to instructional films, types of oral introductions--i.e., substantive, attention-directing, anxiety-inducing, and advance organizers--and research related to these four areas. Recommendations for the use of the oral introduction are also offered. (RAO)

Gene Function Analysis in the Ubiquitous Human Commensal and Pathogen Malassezia Genus.

PubMed

Ianiri, Giuseppe; Averette, Anna F; Kingsbury, Joanne M; Heitman, Joseph; Idnurm, Alexander

2016-11-29

The genus Malassezia includes 14 species that are found on the skin of humans and animals and are associated with a number of diseases. Recent genome sequencing projects have defined the gene content of all 14 species; however, to date, genetic manipulation has not been possible for any species within this genus. Here, we develop and then optimize molecular tools for the transformation of Malassezia furfur and Malassezia sympodialis using Agrobacterium tumefaciens delivery of transfer DNA (T-DNA) molecules. These T-DNAs can insert randomly into the genome. In the case of M. furfur, targeted gene replacements were also achieved via homologous recombination, enabling deletion of the ADE2 gene for purine biosynthesis and of the LAC2 gene predicted to be involved in melanin biosynthesis. Hence, the introduction of exogenous DNA and direct gene manipulation are feasible in Malassezia species. Species in the genus Malassezia are a defining component of the microbiome of the surface of mammals. They are also associated with a wide range of skin disease symptoms. Many species are difficult to culture in vitro, and although genome sequences are available for the species in this genus, it has not been possible to assess gene function to date. In this study, we pursued a series of possible transformation methods and identified one that allows the introduction of DNA into two species of Malassezia, including the ability to make targeted integrations into the genome such that genes can be deleted. This research opens a new direction in terms of now being able to analyze gene functions in this little understood genus. These tools will contribute to define the mechanisms that lead to the commensalism and pathogenicity in this group of obligate fungi that are predominant on the skin of mammals. Copyright © 2016 Ianiri et al.

Liver-Directed Lentiviral Gene Therapy in a Dog Model of Hemophilia B

PubMed Central

Bartholomae, Cynthia C.; Volpin, Monica; Della Valle, Patrizia; Sanvito, Francesca; Sergi Sergi, Lucia; Gallina, Pierangela; Benedicenti, Fabrizio; Bellinger, Dwight; Raymer, Robin; Merricks, Elizabeth; Bellintani, Francesca; Martin, Samia; Doglioni, Claudio; D’Angelo, Armando; VandenDriessche, Thierry; Chuah, Marinee K.; Schmidt, Manfred; Nichols, Timothy; Montini, Eugenio; Naldini, Luigi

2017-01-01

We investigated the safety and efficacy of liver-directed gene therapy using lentiviral vectors in a large animal model of hemophilia B, and evaluated the risk of insertional mutagenesis in tumor-prone mouse models. We show that gene therapy using lentiviral vectors targeting expression of a canine factor IX transgene to hepatocytes was well-tolerated and provided stable long-term production of coagulation factor IX in dogs with hemophilia B. By exploiting three different mouse models designed to amplify the consequences of insertional mutagenesis, we show that no genotoxicity was detected with these lentiviral vectors. Our findings suggest that lentiviral vectors may be an attractive candidate for gene therapy targeted to the liver and may be useful for the treatment of hemophilia. PMID:25739762

Widespread and highly persistent gene transfer to the CNS by retrovirus vector in utero: implication for gene therapy to Krabbe disease.

PubMed

Shen, Jin-Song; Meng, Xing-Li; Yokoo, Takashi; Sakurai, Ken; Watabe, Kazuhiko; Ohashi, Toya; Eto, Yoshikatsu

2005-05-01

Brain-directed prenatal gene therapy may benefit some lysosomal storage diseases that affect the central nervous system (CNS) before birth. Our previous study showed that intrauterine introduction of recombinant adenoviruses into cerebral ventricles results in efficient gene transfer to the CNS in the mouse. However, transgene expression decreased with time due to the non-integrative property of adenoviral vectors. In this study, in order to obtain permanent gene transduction, we investigated the feasibility of retrovirus-mediated in utero gene transduction. Concentrated retrovirus encoding the LacZ gene was injected into the cerebral ventricles of the embryos of normal and twitcher mice (a murine model of Krabbe disease) at embryonic day 12. The distribution and maintenance of the transgene expression in the recipient brain were analyzed histochemically, biochemically and by the quantitative polymerase chain reaction method pre- and postnatally. Efficient and highly persistent gene transduction to the brain was achieved both in normal and the twitcher mouse. Transduced neurons, astrocytes and oligodendrocytes were distributed throughout the brain. The transduced LacZ gene, its transcript and protein expression in the brain were maintained for 14 months without decrement. In addition, gene transduction to multiple tissues other than the brain was also detected at low levels. This study suggests that brain-directed in utero gene transfer using retrovirus vector may be beneficial to the treatment of lysosomal storage diseases with severe brain damage early in life, such as Krabbe disease. Copyright (c) 2005 John Wiley & Sons, Ltd.

Drop-on-demand sample introduction system coupled with the flowing atmospheric-pressure afterglow for direct molecular analysis of complex liquid micro-volume samples

PubMed Central

Schaper, J. Niklas; Pfeuffer, Kevin P.; Shelley, Jacob T.; Bings, Nicolas H.

2012-01-01

One of the fastest developing fields in analytical spectrochemistry in recent years is ambient desorption/ionization mass spectrometry (ADI-MS). This burgeoning interest has been due to the demonstrated advantages of the method: simple mass spectra, little or no sample preparation, and applicability to samples in the solid, liquid, or gaseous state. One such ADI-MS source, the flowing atmospheric-pressure afterglow (FAPA), is capable of direct analysis of solids just by aiming the source at the solid surface and sampling the produced ions into a mass spectrometer. However, direct introduction of significant volumes of liquid samples into this source has not been possible, as solvent loads can quench the afterglow and, thus, the formation of reagent ions. As a result, the analysis of liquid samples is preferably carried out by analyzing dried residues or by desorbing small amounts of liquid samples directly from the liquid surface. In the former case, reproducibility of sample introduction is crucial if quantitative results are desired. In the present study, introduction of liquid samples as very small droplets helps overcome the issues of sample positioning and reduced levels of solvent intake. A recently developed “drop-on-demand” (DOD) aerosol generator is capable of reproducibly producing very small volumes of liquid (~17 pL). In this paper, the coupling of FAPA-MS and DOD is reported and applications are suggested. Analytes representing different classes of substances were tested and limits of detections were determined. Matrix tolerance was investigated for drugs of abuse and their metabolites by analyzing raw urine samples and quantification without the use of internal standards. Limits of detection below 2 µg/mL, without sample pretreatment, were obtained. PMID:23025277

Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.

PubMed

Paquet, Dominik; Kwart, Dylan; Chen, Antonia; Sproul, Andrew; Jacob, Samson; Teo, Shaun; Olsen, Kimberly Moore; Gregg, Andrew; Noggle, Scott; Tessier-Lavigne, Marc

2016-05-05

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in

Liver-directed lentiviral gene therapy in a dog model of hemophilia B.

PubMed

Cantore, Alessio; Ranzani, Marco; Bartholomae, Cynthia C; Volpin, Monica; Valle, Patrizia Della; Sanvito, Francesca; Sergi, Lucia Sergi; Gallina, Pierangela; Benedicenti, Fabrizio; Bellinger, Dwight; Raymer, Robin; Merricks, Elizabeth; Bellintani, Francesca; Martin, Samia; Doglioni, Claudio; D'Angelo, Armando; VandenDriessche, Thierry; Chuah, Marinee K; Schmidt, Manfred; Nichols, Timothy; Montini, Eugenio; Naldini, Luigi

2015-03-04

We investigated the efficacy of liver-directed gene therapy using lentiviral vectors in a large animal model of hemophilia B and evaluated the risk of insertional mutagenesis in tumor-prone mouse models. We showed that gene therapy using lentiviral vectors targeting the expression of a canine factor IX transgene in hepatocytes was well tolerated and provided a stable long-term production of coagulation factor IX in dogs with hemophilia B. By exploiting three different mouse models designed to amplify the consequences of insertional mutagenesis, we showed that no genotoxicity was detected with these lentiviral vectors. Our findings suggest that lentiviral vectors may be an attractive candidate for gene therapy targeted to the liver and may be potentially useful for the treatment of hemophilia. Copyright © 2015, American Association for the Advancement of Science.

Direct and long-term detection of gene doping in conventional blood samples.

PubMed

Beiter, T; Zimmermann, M; Fragasso, A; Hudemann, J; Niess, A M; Bitzer, M; Lauer, U M; Simon, P

2011-03-01

The misuse of somatic gene therapy for the purpose of enhancing athletic performance is perceived as a coming threat to the world of sports and categorized as 'gene doping'. This article describes a direct detection approach for gene doping that gives a clear yes-or-no answer based on the presence or absence of transgenic DNA in peripheral blood samples. By exploiting a priming strategy to specifically amplify intronless DNA sequences, we developed PCR protocols allowing the detection of very small amounts of transgenic DNA in genomic DNA samples to screen for six prime candidate genes. Our detection strategy was verified in a mouse model, giving positive signals from minute amounts (20 μl) of blood samples for up to 56 days following intramuscular adeno-associated virus-mediated gene transfer, one of the most likely candidate vector systems to be misused for gene doping. To make our detection strategy amenable for routine testing, we implemented a robust sample preparation and processing protocol that allows cost-efficient analysis of small human blood volumes (200 μl) with high specificity and reproducibility. The practicability and reliability of our detection strategy was validated by a screening approach including 327 blood samples taken from professional and recreational athletes under field conditions.

CINCINNATA in Antirrhinum majus directly modulates genes involved in cytokinin and auxin signaling.

PubMed

Das Gupta, Mainak; Aggarwal, Pooja; Nath, Utpal

2014-12-01

Mutations in the CINCINNATA (CIN) gene in Antirrhinum majus and its orthologs in Arabidopsis result in crinkly leaves as a result of excess growth towards the leaf margin. CIN homologs code for TCP (TEOSINTE-BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTOR 1 AND 2) transcription factors and are expressed in a broad zone in a growing leaf distal to the proliferation zone where they accelerate cell maturation. Although a few TCP targets are known, the functional basis of CIN-mediated leaf morphogenesis remains unclear. We compared the global transcription profiles of wild-type and the cin mutant of A. majus to identify the targets of CIN. We cloned and studied the direct targets using RNA in situ hybridization, DNA-protein interaction, chromatin immunoprecipitation and reporter gene analysis. Many of the genes involved in the auxin and cytokinin signaling pathways showed altered expression in the cin mutant. Further, we showed that CIN binds to genomic regions and directly promotes the transcription of a cytokinin receptor homolog HISTIDINE KINASE 4 (AmHK4) and an IAA3/SHY2 (INDOLE-3-ACETIC ACID INDUCIBLE 3/SHORT HYPOCOTYL 2) homolog in A. majus. Our results suggest that CIN limits excess cell proliferation and maintains the flatness of the leaf surface by directly modulating the hormone pathways involved in patterning cell proliferation and differentiation during leaf growth. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Direct bisulfite sequencing for examination of DNA methylation with gene and nucleotide resolution from brain tissues.

PubMed

Parrish, R Ryley; Day, Jeremy J; Lubin, Farah D

2012-07-01

DNA methylation is an epigenetic modification that is essential for the development and mature function of the central nervous system. Due to the relevance of this modification to the transcriptional control of gene expression, it is often necessary to examine changes in DNA methylation patterns with both gene and single-nucleotide resolution. Here, we describe an in-depth basic protocol for direct bisulfite sequencing of DNA isolated from brain tissue, which will permit direct assessment of methylation status at individual genes as well as individual cytosine molecules/nucleotides within a genomic region. This method yields analysis of DNA methylation patterns that is robust, accurate, and reproducible, thereby allowing insights into the role of alterations in DNA methylation in brain tissue.

Identification of Direct Target Genes Using Joint Sequence and Expression Likelihood with Application to DAF-16

PubMed Central

Yu, Ron X.; Liu, Jie; True, Nick; Wang, Wei

2008-01-01

A major challenge in the post-genome era is to reconstruct regulatory networks from the biological knowledge accumulated up to date. The development of tools for identifying direct target genes of transcription factors (TFs) is critical to this endeavor. Given a set of microarray experiments, a probabilistic model called TRANSMODIS has been developed which can infer the direct targets of a TF by integrating sequence motif, gene expression and ChIP-chip data. The performance of TRANSMODIS was first validated on a set of transcription factor perturbation experiments (TFPEs) involving Pho4p, a well studied TF in Saccharomyces cerevisiae. TRANSMODIS removed elements of arbitrariness in manual target gene selection process and produced results that concur with one's intuition. TRANSMODIS was further validated on a genome-wide scale by comparing it with two other methods in Saccharomyces cerevisiae. The usefulness of TRANSMODIS was then demonstrated by applying it to the identification of direct targets of DAF-16, a critical TF regulating ageing in Caenorhabditis elegans. We found that 189 genes were tightly regulated by DAF-16. In addition, DAF-16 has differential preference for motifs when acting as an activator or repressor, which awaits experimental verification. TRANSMODIS is computationally efficient and robust, making it a useful probabilistic framework for finding immediate targets. PMID:18350157

5 CFR 2500.1 - Introduction.

Code of Federal Regulations, 2011 CFR

2011-01-01

... Administrative Personnel OFFICE OF ADMINISTRATION, EXECUTIVE OFFICE OF THE PRESIDENT INFORMATION SECURITY REGULATION § 2500.1 Introduction. (a) References. (1) Executive Order 12065, “National Security Information”, dated June 28, 1978. (2) Information Security Oversight Office Directive No. 1, “National Security...

5 CFR 2500.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Administrative Personnel OFFICE OF ADMINISTRATION, EXECUTIVE OFFICE OF THE PRESIDENT INFORMATION SECURITY REGULATION § 2500.1 Introduction. (a) References. (1) Executive Order 12065, “National Security Information”, dated June 28, 1978. (2) Information Security Oversight Office Directive No. 1, “National Security...

Assessing co-regulation of directly linked genes in biological networks using microarray time series analysis.

PubMed

Del Sorbo, Maria Rosaria; Balzano, Walter; Donato, Michele; Draghici, Sorin

2013-11-01

Differential expression of genes detected with the analysis of high throughput genomic experiments is a commonly used intermediate step for the identification of signaling pathways involved in the response to different biological conditions. The impact analysis was the first approach for the analysis of signaling pathways involved in a certain biological process that was able to take into account not only the magnitude of the expression change of the genes but also the topology of signaling pathways including the type of each interactions between the genes. In the impact analysis, signaling pathways are represented as weighted directed graphs with genes as nodes and the interactions between genes as edges. Edges weights are represented by a β factor, the regulatory efficiency, which is assumed to be equal to 1 in inductive interactions between genes and equal to -1 in repressive interactions. This study presents a similarity analysis between gene expression time series aimed to find correspondences with the regulatory efficiency, i.e. the β factor as found in a widely used pathway database. Here, we focused on correlations among genes directly connected in signaling pathways, assuming that the expression variations of upstream genes impact immediately downstream genes in a short time interval and without significant influences by the interactions with other genes. Time series were processed using three different similarity metrics. The first metric is based on the bit string matching; the second one is a specific application of the Dynamic Time Warping to detect similarities even in presence of stretching and delays; the third one is a quantitative comparative analysis resulting by an evaluation of frequency domain representation of time series: the similarity metric is the correlation between dominant spectral components. These three approaches are tested on real data and pathways, and a comparison is performed using Information Retrieval benchmark tools

Introduction to Pharmaceutical Biotechnology, Volume 1; Basic techniques and concepts

NASA Astrophysics Data System (ADS)

Bhatia, Saurabh; Goli, Divakar

2018-05-01

Animal biotechnology is a broad field including polarities of fundamental and applied research, as well as DNA science, covering key topics of DNA studies and its recent applications. In Introduction to Pharmaceutical Biotechnology, DNA isolation procedures followed by molecular markers and screening methods of the genomic library are explained. Interesting areas like isolation, sequencing and synthesis of genes, with the broader coverage on synthesis of genes, are also described. The book begins with an introduction to biotechnology and its main branches, explaining both the basic science and the applications of biotechnology-derived pharmaceuticals, with special emphasis on their clinical use. It then moves on to historical development and scope of biotechnology with an overall review of early applications that scientists employed long before the field was defined.

In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus.

PubMed

Nakamura, Akira; Takakura, Yasuaki; Kobayashi, Hideo; Hoshino, Takayuki

2005-08-01

An in vivo-directed evolutionary strategy was used to obtain a thermostabilized Escherichia coli hygromycin B phosphotransferase, using a host-vector system of Thermus thermophilus. Introduction of the mutant gene containing two amino acid substitutions, S52T and W238C, which was previously reported by Cannio et al. [J. Bacteriol., 180, 3237-3240 (1998)], did not confer hygromycin resistance on T. thermophilus cells at 55 degrees C; however, five spontaneously-generated independent mutants were obtained by selection of the transformants at this temperature. Each mutant gene contained one amino acid substitution of either A118V or T246A. Further selection with increasing temperature, at 58 degrees C and then 61 degrees C, led to acquisition of three more substitutions: D20G, S225P and Q226L. These mutations cumulatively influenced the maximum growth temperature of the T. thermophilus transformants in the presence of hygromycin; T. thermophilus carrying a mutant gene containing all the five substitutions was able to grow at up to 67 degrees C. This mutant gene, hph5, proved useful as a selection marker in the T. thermophilus host-vector system, either on the plasmid or by genome integration, at temperatures up to 65 degrees C.

Introduction of gold nanoparticles into myoglobin-Nafion film for direct electrochemistry application.

PubMed

Xie, Wenting; Kong, Linlin; Kan, Meixiu; Han, Dongmei; Wang, Xueji; Zhang, Hui-Min

2010-10-01

An effective myoglobin-Nafion film is prepared by introducing gold nanoparticles in through a simple procedure by ion-exchange combined with electrochemical reduction. Gold nanoparticles are highly dispersed in myoglobin-Nafion film with an average size of 2.3 +/- 0.2 nm. The electrochemical behavior of myoglobin entrapped in the film has been carefully investigated with cyclic voltammetry. The results show that the introduction of gold nanoparticles into myoglobin-Nafion film makes the direct electron transfer of myoglobin efficient. A pair of well-defined redox peaks for myoglobin heme Fe(II)/Fe(III) is observed with a formal potential of -0.150 V in 0.1 M phosphate buffer (pH 7.0). The electrochemical parameters of myoglobin in the composite film are further calculated with the results of the electron-transfer rate constant (k(s)) as 0.93 s(-1) and the charge transfer coefficient (alpha) as 0.69. The experimental results also demonstrate that the immobilized myoglobin retains its electrocatalytic activity for the reduction of hydrogen peroxide and the catalytic reduction peak of myoglobin appear in a linear relationship with H2O2 concentration in the range of 10.0-235.0 microM with correlation coefficient of 0.9970. Thus fabricated Au/Mb/Nafion electrode should give a new approach for developing redox protein or enzyme-based biosensors.

Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: Involvement of the human ERCC2 DNA repair gene

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

Flejter, W.L.; McDaniel, L.D.; Johns, D.

1992-01-01

Cultured cells from individuals afflicted with the genetically heterogeneous autosomal recessive disorder xeroderma pigmentosum (XP) exhibit sensitivity to UV radiation and defective nucleotide excision repair. Complementation of these mutant phenotypes after the introduction of single human chromosomes from repair-proficient cells into XP cells has provided a means of mapping the genes involved in this disease. The authors now report the phenotypic correction of XP cells from genetic complementation group D (XP-D) by a single human chromosome designated Tneo. Detailed molecular characterization of Tneo revealed a rearranged structure involving human chromosomes 16 and 19, including the excision repair cross-complementing 2 (ERCC2)more » gene from the previously described human DNA repair gene cluster at 19q13.2-q13.3. Direct transfer of a cosmid bearing the ERCC2 gene conferred UV resistance to XP-D cells.« less

Condensed Phase Membrane Introduction Mass Spectrometry with Direct Electron Ionization: On-line Measurement of PAHs in Complex Aqueous Samples

NASA Astrophysics Data System (ADS)

Termopoli, Veronica; Famiglini, Giorgio; Palma, Pierangela; Cappiello, Achille; Vandergrift, Gregory W.; Krogh, Erik T.; Gill, Chris G.

2016-02-01

Polycyclic aromatic hydrocarbons (PAHs) are USEPA regulated priority pollutants. Their low aqueous solubility requires very sensitive analytical methods for their detection, typically involving preconcentration steps. Presented is the first demonstrated `proof of concept' use of condensed phase membrane introduction mass spectrometry (CP-MIMS) coupled with direct liquid electron ionization (DEI) for the direct, on-line measurement of PAHs in aqueous samples. DEI is very well suited for the ionization of PAHs and other nonpolar compounds, and is not significantly influenced by the co-elution of matrix components. Linear calibration data for low ppb levels of aqueous naphthalene, anthracene, and pyrene is demonstrated, with measured detection limits of 4 ppb. Analytical response times (t10%-90% signal rise) ranged from 2.8 min for naphthalene to 4.7 min for pyrene. Both intra- and interday reproducibility has been assessed (<3% and 5% RSD, respectively). Direct measurements of ppb level PAHs spiked in a variety of real, complex environmental sample matrices is examined, including natural waters, sea waters, and a hydrocarbon extraction production waste water sample. For these spiked, complex samples, direct PAH measurement by CP-MIMS-DEI yielded minimal signal suppression from sample matrix effects (81%-104%). We demonstrate the use of this analytical approach to directly monitor real-time changes in aqueous PAH concentrations with potential applications for continuous on-line monitoring strategies and binding/adsorption studies in heterogeneous samples.

Characterization of a Vibrio alginolyticus Strain, Isolated from Alaskan Oysters, Carrying a Hemolysin Gene Similar to the Thermostable Direct Hemolysin-Related Hemolysin Gene (trh) of Vibrio parahaemolyticus▿

PubMed Central

González-Escalona, Narjol; Blackstone, George M.; DePaola, Angelo

2006-01-01

A Vibrio strain isolated from Alaskan oysters and classified by its biochemical characteristics as Vibrio alginolyticus possessed a thermostable direct hemolysin-related hemolysin (trh) gene previously reported only in Vibrio parahaemolyticus. This trh-like gene was cloned and sequenced and was 98% identical to the trh2 gene of V. parahaemolyticus. This gene seems to be functional since it was transcriptionally active in early-stationary-phase growing cells. To our knowledge, this is the first report of V. alginolyticus possessing a trh gene. PMID:17056701

Organization of the qa Gene Cluster in NEUROSPORA CRASSA: Direction of Transcription of the qa-3 Gene

PubMed Central

Strøman, Per; Reinert, William; Case, Mary E.; Giles, Norman H.

1979-01-01

In Neurospora crassa, the enzyme quinate (shikimate) dehydrogenase catalyzes the first reaction in the inducible quinic acid catabolic pathway and is encoded in the qa-3 gene of the qa cluster. In this cluster, the order of genes has been established as qa-1 qa-3 qa-4 qa-2. Amino-terminal sequences have been determined for purified quinate dehydrogenase from wild type and from UV-induced revertants in two different qa-3 mutants. These two mutants (M16 and M45) map at opposite ends of the qa-3 locus. In addition, mapping data (Case et al. 1978) indicate that the end of the qa-3 gene specified by M45 is closer to the adjacent qa-1 gene than is the end specified by the M16 mutant site. In one of the revertants (R45 from qa-3 mutant M45), the aminoterminal sequence for the first ten amino acids is identical to that of wild type. The other revertant (R1 from qa-3 mutant M16) differs from wild type at the amino-terminal end by a single altered residue at position three in the sequence. The observed change involves the substitution of an isoleucine in M16-R1 for a proline in wild type. This substitution requires a two-nucleotide change in the corresponding wild-type codon.——The combined genetic and biochemical data indicate that the qa-3 mutants M16 and M45 carry amino acid substitutions near the amino-terminal and carboxyl-terminal ends of the quinate dehydrogenase enzyme, respectively. On this basis we conclude that transcription of the qa-3 gene proceeds from the end specified by the M16 mutant site in the direction of the qa-1 gene. It appears probable that transcription is initiated from a promoter site within the qa cluster, possibly immediately adjacent to the qa-3 gene. PMID:159203

Rapid direct identification of Cryptococcus neoformans from pigeon droppings by nested PCR using CNLAC1 gene.

PubMed

Chae, H S; Park, G N; Kim, S H; Jo, H J; Kim, J T; Jeoung, H Y; An, D J; Kim, N H; Shin, B W; Kang, Y I; Chang, K S

2012-08-01

Isolation and identification of Cryptococcus neoformans and pathogenic yeast-like fungi from pigeon droppings has been taken for a long time and requires various nutrients for its growth. In this study, we attempted to establish a rapid direct identification method of Cr. neoformans from pigeon dropping samples by nested-PCR using internal transcribed spacer (ITS) CAP64 and CNLAC1 genes, polysaccharide capsule gene and laccase-associated gene to produce melanin pigment, respectively, which are common genes of yeasts. The ITS and CAP64 genes were amplified in all pathogenic yeasts, but CNLAC1 was amplified only in Cr. neoformans. The ITS gene was useful for yeast genotyping depending on nucleotide sequence. Homology of CAP64 genes among the yeasts were very high. The specificity of PCR using CNLAC1 was demonstrated in Cr. neoformans environmental strains but not in other yeast-like fungi. The CNLAC1 gene was detected in 5 serotypes of Cr. neoformans. The nested-PCR amplified up to 10(-11) μg of the genomic DNA and showed high sensitivity. All pigeon droppings among 31 Cr. neoformans-positive samples were positive and all pigeon droppings among 348 Cr. neoformans-negative samples were negative by the direct nested-PCR. In addition, after primary enrichment of pigeon droppings in Sabouraud dextrose broth, all Cr. neoformans-negative samples were negative by the nested-PCR, which showed high specificity. The nested-PCR showed high sensitivity without culture of pigeon droppings. Nested-PCR using CNLAC1 provides a rapid and reliable molecular diagnostic method to overcome weak points such as long culture time of many conventional methods.

Introduction of Complementary Foods to Infants.

PubMed

West, Christina

2017-01-01

While earlier food allergy prevention strategies implemented avoidance of allergenic foods in infancy, the current paradigm is shifting from avoidance to controlled exposure. This review focuses on the outcome of recent randomized controlled trials, which have examined the early introduction of allergenic foods for allergy prevention, and discusses the implementation of results in clinical practice. In infants at high risk of allergic disease, there is now direct evidence that regular early peanut consumption will reduce the prevalence of peanut allergy, compared to avoidance. Many international infant feeding guidelines already recommend complementary foods, including allergenic foods, to be introduced from 4 to 6 months of age irrespective of family history risk. Interim guidelines from 10 International Pediatric Allergy Associations state that healthcare providers should recommend the introduction of peanut-containing products into the diets of infants at high risk of allergic disease in countries where peanut allergy is prevalent. Direct translation of the results obtained from a cohort of high-risk infants to the general population has proved difficult, and issues regarding feasibility, safety, and cost-effectiveness have been raised. Five randomized placebo-controlled trials have assessed the effects of early egg exposure in infancy with varying results. In a recent comprehensive meta-analysis, there was moderate-certainty evidence that early versus late introduction of egg was associated with a reduced egg allergy risk. Although promising, optimal timing, doses, and if the feeding regimen should be stratified according to infant allergy risk remain to be determined. The single study that assessed introduction of multiple foods from 3 months whilst breastfeeding compared with exclusive breastfeeding until 6 months of age showed no reduction in food allergy prevalence. Future research should aim at optimizing infant feeding regimens and support a tolerogenic

Gene expression of human lung cancer cell line CL1-5 in response to a direct current electric field.

PubMed

Huang, Ching-Wen; Chen, Huai-Yi; Yen, Meng-Hua; Chen, Jeremy J W; Young, Tai-Horng; Cheng, Ji-Yen

2011-01-01

Electrotaxis is the movement of adherent living cells in response to a direct current (dc) electric field (EF) of physiological strength. Highly metastatic human lung cancer cells, CL1-5, exhibit directional migration and orientation under dcEFs. To understand the transcriptional response of CL1-5 cells to a dcEF, microarray analysis was performed in this study. A large electric-field chip (LEFC) was designed, fabricated, and used in this study. CL1-5 cells were treated with the EF strength of 0 mV/mm (the control group) and 300 mV/mm (the EF-treated group) for two hours. Signaling pathways involving the genes that expressed differently between the two groups were revealed. It was shown that the EF-regulated genes highly correlated to adherens junction, telomerase RNA component gene regulation, and tight junction. Some up-regulated genes such as ACVR1B and CTTN, and some down-regulated genes such as PTEN, are known to be positively and negatively correlated to cell migration, respectively. The protein-protein interactions of adherens junction-associated EF-regulated genes suggested that platelet-derived growth factor (PDGF) receptors and ephrin receptors may participate in sensing extracellular electrical stimuli. We further observed a high percentage of significantly regulated genes which encode cell membrane proteins, suggesting that dcEF may directly influence the activity of cell membrane proteins in signal transduction. In this study, some of the EF-regulated genes have been reported to be essential whereas others are novel for electrotaxis. Our result confirms that the regulation of gene expression is involved in the mechanism of electrotactic response.

Gene Function Analysis in the Ubiquitous Human Commensal and Pathogen Malassezia Genus

PubMed Central

Ianiri, Giuseppe; Averette, Anna F.; Kingsbury, Joanne M.; Heitman, Joseph

2016-01-01

ABSTRACT The genus Malassezia includes 14 species that are found on the skin of humans and animals and are associated with a number of diseases. Recent genome sequencing projects have defined the gene content of all 14 species; however, to date, genetic manipulation has not been possible for any species within this genus. Here, we develop and then optimize molecular tools for the transformation of Malassezia furfur and Malassezia sympodialis using Agrobacterium tumefaciens delivery of transfer DNA (T-DNA) molecules. These T-DNAs can insert randomly into the genome. In the case of M. furfur, targeted gene replacements were also achieved via homologous recombination, enabling deletion of the ADE2 gene for purine biosynthesis and of the LAC2 gene predicted to be involved in melanin biosynthesis. Hence, the introduction of exogenous DNA and direct gene manipulation are feasible in Malassezia species. PMID:27899504

Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

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

Mizoshiri, N.; Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto; Kishida, T.

Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genesmore » and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.« less

Application of the CRISPR/Cas9 gene editing technique to research on functional genomes of parasites.

PubMed

Cui, Yubao; Yu, Lili

2016-12-01

The clustered regularly-interspaced short palindromic repeats (CRISPR) structural family functions as an acquired immune system in prokaryotes. Gene editing techniques have co-opted CRISPR and the associated Cas nucleases to allow for the precise genetic modification of human cells, zebrafish, mice, and other eukaryotes. Indeed, this approach has been used to induce a variety of modifications including directed insertion/deletion (InDel) of bases, gene knock-in, introduction of mutations in both alleles of a target gene, and deletion of small DNA fragments. Thus, CRISPR technology offers a precise molecular tool for directed genome modification with a range of potential applications; further, its high mutation efficiency, simple process, and low cost provide additional advantages over prior editing techniques. This paper will provide an overview of the basic structure and function of the CRISPR gene editing system as well as current and potential applications to research on parasites. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

RNA interference can target pre-mRNA: consequences for gene expression in a Caenorhabditis elegans operon.

PubMed Central

Bosher, J M; Dufourcq, P; Sookhareea, S; Labouesse, M

1999-01-01

In nematodes, flies, trypanosomes, and planarians, introduction of double-stranded RNA results in sequence-specific inactivation of gene function, a process termed RNA interference (RNAi). We demonstrate that RNAi against the Caenorhabditis elegans gene lir-1, which is part of the lir-1/lin-26 operon, induced phenotypes very different from a newly isolated lir-1 null mutation. Specifically, lir-1(RNAi) induced embryonic lethality reminiscent of moderately strong lin-26 alleles, whereas the lir-1 null mutant was viable. We show that the lir-1(RNAi) phenotypes resulted from a severe loss of lin-26 gene expression. In addition, we found that RNAi directed against lir-1 or lin-26 introns induced similar phenotypes, so we conclude that lir-1(RNAi) targets the lir-1/lin-26 pre-mRNA. This provides direct evidence that RNA interference can prevent gene expression by targeting nuclear transcripts. Our results highlight that caution may be necessary when interpreting RNA interference without the benefit of mutant alleles. PMID:10545456

Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants

USDA-ARS?s Scientific Manuscript database

Direct cellobiose production from cellulose by a genetically modified fungus—Neurospora crassa, was explored in this study. A library of N. crassa sextuple beta-glucosidase (bgl) gene deletion strains was constructed. Various concentrations of cellobiose were detected in the culture broth of the N. ...

Gene-Environment Interactions in Schizophrenia: Review of Epidemiological Findings and Future Directions

PubMed Central

van Os, Jim; Rutten, Bart PF; Poulton, Richie

2008-01-01

Concern is building about high rates of schizophrenia in large cities, and among immigrants, cannabis users, and traumatized individuals, some of which likely reflects the causal influence of environmental exposures. This, in combination with very slow progress in the area of molecular genetics, has generated interest in more complicated models of schizophrenia etiology that explicitly posit gene-environment interactions (EU-GEI. European Network of Schizophrenia Networks for the Study of Gene Environment Interactions. Schizophrenia aetiology: do gene-environment interactions hold the key? [published online ahead of print April 25, 2008] Schizophr Res; S0920-9964(08) 00170–9). Although findings of epidemiological gene-environment interaction (G × E) studies are suggestive of widespread gene-environment interactions in the etiology of schizophrenia, numerous challenges remain. For example, attempts to identify gene-environment interactions cannot be equated with molecular genetic studies with a few putative environmental variables “thrown in”: G × E is a multidisciplinary exercise involving epidemiology, psychology, psychiatry, neuroscience, neuroimaging, pharmacology, biostatistics, and genetics. Epidemiological G × E studies using indirect measures of genetic risk in genetically sensitive designs have the advantage that they are able to model the net, albeit nonspecific, genetic load. In studies using direct molecular measures of genetic variation, a hypothesis-driven approach postulating synergistic effects between genes and environment impacting on a final common pathway, such as “sensitization” of mesolimbic dopamine neurotransmission, while simplistic, may provide initial focus and protection against the numerous false-positive and false-negative results that these investigations engender. Experimental ecogenetic approaches with randomized assignment may help to overcome some of the limitations of observational studies and allow for the additional

Cloning and expression in Escherichia coli of Vibrio parahaemolyticus thermostable direct hemolysin and thermolabile hemolysin genes.

PubMed

Taniguchi, H; Ohta, H; Ogawa, M; Mizuguchi, Y

1985-05-01

Two hemolysin genes of Vibrio parahaemolyticus WP1, a thermostable direct (TSD) hemolysin gene and a thermolabile hemolysin gene, were cloned into the pBR322 vector in Escherichia coli K-12 C600. A large amount of the TSD hemolysin produced in E. coli K-12 accumulated in the periplasmic space. The TSD hemolysin gene was localized on a 0.9-kilobase HindIII-BamHI fragment by identifying qualitatively the production of the TSD hemolysin by a reverse passive hemagglutination assay in the osmotic shock fluid. The thermolabile hemolysin gene was isolated on a 1.3-kilobase HindIII-PstI fragment by selection with the hemolysin on blood agar. Southern blot hybridization and colony hybridization experiments indicated that the TSD hemolysin gene was present in the chromosomal DNA of 15 Kanagawa phenomenon-positive strains but not in 14 negative strains, whereas the thermolabile hemolysin gene was detected in all strains. No homologous DNA sequences to TSD and thermolabile hemolysin genes were detected in the chromosomes of Vibrio cholerae, Vibrio vulnificus, non-O1 V. cholerae, and Vibrio anguillarum.

Cytoplasmic-genetic male sterility gene provides direct evidence for some hybrid rice recently evolving into weedy rice

PubMed Central

Zhang, Jingxu; Lu, Zuomei; Dai, Weimin; Song, Xiaoling; Peng, Yufa; Valverde, Bernal E.; Qiang, Sheng

2015-01-01

Weedy rice infests paddy fields worldwide at an alarmingly increasing rate. There is substantial evidence indicating that many weedy rice forms originated from or are closely related to cultivated rice. There is suspicion that the outbreak of weedy rice in China may be related to widely grown hybrid rice due to its heterosis and the diversity of its progeny, but this notion remains unsupported by direct evidence. We screened weedy rice accessions by both genetic and molecular marker tests for the cytoplasmic male sterility (CMS) genes (Wild abortive, WA, and Boro type, BT) most widely used in the production of indica and japonica three-line hybrid rice as a diagnostic trait of direct parenthood. Sixteen weedy rice accessions of the 358 tested (4.5%) contained the CMS-WA gene; none contained the CMS-BT gene. These 16 accessions represent weedy rices recently evolved from maternal hybrid rice derivatives, given the primarily maternal inheritance of this trait. Our results provide key direct evidence that hybrid rice can be involved in the evolution of some weedy rice accessions, but is not a primary factor in the recent outbreak of weedy rice in China. PMID:26012494

Cytoplasmic-genetic male sterility gene provides direct evidence for some hybrid rice recently evolving into weedy rice.

PubMed

Zhang, Jingxu; Lu, Zuomei; Dai, Weimin; Song, Xiaoling; Peng, Yufa; Valverde, Bernal E; Qiang, Sheng

2015-05-27

Weedy rice infests paddy fields worldwide at an alarmingly increasing rate. There is substantial evidence indicating that many weedy rice forms originated from or are closely related to cultivated rice. There is suspicion that the outbreak of weedy rice in China may be related to widely grown hybrid rice due to its heterosis and the diversity of its progeny, but this notion remains unsupported by direct evidence. We screened weedy rice accessions by both genetic and molecular marker tests for the cytoplasmic male sterility (CMS) genes (Wild abortive, WA, and Boro type, BT) most widely used in the production of indica and japonica three-line hybrid rice as a diagnostic trait of direct parenthood. Sixteen weedy rice accessions of the 358 tested (4.5%) contained the CMS-WA gene; none contained the CMS-BT gene. These 16 accessions represent weedy rices recently evolved from maternal hybrid rice derivatives, given the primarily maternal inheritance of this trait. Our results provide key direct evidence that hybrid rice can be involved in the evolution of some weedy rice accessions, but is not a primary factor in the recent outbreak of weedy rice in China.

Direct induction of T lymphocyte-specific gene expression by the mammalian Notch signaling pathway

PubMed Central

Reizis, Boris; Leder, Philip

2002-01-01

The Notch signaling pathway regulates the commitment and early development of T lymphocytes. We studied Notch-mediated induction of the pre-T cell receptor α (pTa) gene, a T-cell-specific transcriptional target of Notch. The pTa enhancer was activated by Notch signaling and contained binding sites for its nuclear effector, CSL. Mutation of the CSL-binding sites abolished enhancer induction by Notch and delayed the up-regulation of pTa transgene expression during T cell lineage commitment. These results show a direct mechanism of stage- and tissue-specific gene induction by the mammalian Notch/CSL signaling pathway. PMID:11825871

Directing 101.

ERIC Educational Resources Information Center

Pintoff, Ernest

Providing an introduction to anyone considering directing as a field of study or career, this book takes a broad look at the process of directing and encourages students and professionals alike to look outside of the movie industry for inspiration. Chapters in the book discuss selecting and acquiring material; budgeting and financing; casting and…

A direct molecular link between the autism candidate gene RORa and the schizophrenia candidate MIR137

NASA Astrophysics Data System (ADS)

Devanna, Paolo; Vernes, Sonja C.

2014-02-01

Retinoic acid-related orphan receptor alpha gene (RORa) and the microRNA MIR137 have both recently been identified as novel candidate genes for neuropsychiatric disorders. RORa encodes a ligand-dependent orphan nuclear receptor that acts as a transcriptional regulator and miR-137 is a brain enriched small non-coding RNA that interacts with gene transcripts to control protein levels. Given the mounting evidence for RORa in autism spectrum disorders (ASD) and MIR137 in schizophrenia and ASD, we investigated if there was a functional biological relationship between these two genes. Herein, we demonstrate that miR-137 targets the 3'UTR of RORa in a site specific manner. We also provide further support for MIR137 as an autism candidate by showing that a large number of previously implicated autism genes are also putatively targeted by miR-137. This work supports the role of MIR137 as an ASD candidate and demonstrates a direct biological link between these previously unrelated autism candidate genes.

Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

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

Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong

2010-02-19

Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna wasmore » similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.« less

Non-adaptive plasticity potentiates rapid adaptive evolution of gene expression in nature.

PubMed

Ghalambor, Cameron K; Hoke, Kim L; Ruell, Emily W; Fischer, Eva K; Reznick, David N; Hughes, Kimberly A

2015-09-17

Phenotypic plasticity is the capacity for an individual genotype to produce different phenotypes in response to environmental variation. Most traits are plastic, but the degree to which plasticity is adaptive or non-adaptive depends on whether environmentally induced phenotypes are closer or further away from the local optimum. Existing theories make conflicting predictions about whether plasticity constrains or facilitates adaptive evolution. Debate persists because few empirical studies have tested the relationship between initial plasticity and subsequent adaptive evolution in natural populations. Here we show that the direction of plasticity in gene expression is generally opposite to the direction of adaptive evolution. We experimentally transplanted Trinidadian guppies (Poecilia reticulata) adapted to living with cichlid predators to cichlid-free streams, and tested for evolutionary divergence in brain gene expression patterns after three to four generations. We find 135 transcripts that evolved parallel changes in expression within the replicated introduction populations. These changes are in the same direction exhibited in a native cichlid-free population, suggesting rapid adaptive evolution. We find 89% of these transcripts exhibited non-adaptive plastic changes in expression when the source population was reared in the absence of predators, as they are in the opposite direction to the evolved changes. By contrast, the remaining transcripts exhibiting adaptive plasticity show reduced population divergence. Furthermore, the most plastic transcripts in the source population evolved reduced plasticity in the introduction populations, suggesting strong selection against non-adaptive plasticity. These results support models predicting that adaptive plasticity constrains evolution, whereas non-adaptive plasticity potentiates evolution by increasing the strength of directional selection. The role of non-adaptive plasticity in evolution has received relatively

Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery.

PubMed

Ueki, Shoko; Magori, Shimpei; Lacroix, Benoît; Citovsky, Vitaly

2013-01-01

Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure.

Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I.

PubMed

Huang, Y; Ohtani, K; Iwanaga, R; Matsumura, Y; Nakamura, M

2001-03-01

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19(INK4d), a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-kappaB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-kappaB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of -444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.

Invasion success in Cogongrass (Imperata cylindrica): A population genetic approach exploring genetic diversity and historical introductions

Treesearch

Rima D. Lucardi; Lisa E. Wallace; Gary N. Ervin

2014-01-01

Propagule pressure significantly contributes to and limits the potential success of a biological invasion, especially during transport, introduction, and establishment. Events such as multiple introductions of foreign parent material and gene flow among them can increase genetic diversity in founding populations, often leading to greater invasion success. We applied...

Direct health costs of environmental tobacco smoke exposure and indirect health benefits due to smoking ban introduction.

PubMed

Hauri, Dimitri D; Lieb, Christoph M; Rajkumar, Sarah; Kooijman, Cornelis; Sommer, Heini L; Röösli, Martin

2011-06-01

Introducing comprehensive smoke-free policies to public places is expected to reduce health costs. This includes prevented health damages by avoiding environmental tobacco smoke (ETS) exposure as well as indirect health benefits from reduced tobacco consumption. The aim of this study was to estimate direct health costs of ETS exposure in public places and indirect health benefits from reduced tobacco consumption. We calculated attributable hospital days and years of life lost (YLL), based on the observed passive smoking and disease rates in Switzerland. The exposure-response associations of all relevant health outcomes were derived by meta-analysis from prospective cohort studies in order to calculate the direct health costs. To assess the indirect health benefits, a meta-analysis of smoking ban studies on hospital admissions for acute myocardial infarction was conducted. ETS exposure in public places in Switzerland causes 32,000 preventable hospital days (95% CI: 10,000-61,000), 3000 YLL (95% CI: 1000-5000), corresponding to health costs of 330 Mio CHF. The number of hospital days for ischaemic heart disease attributable to passive smoking is much larger if derived from smoking ban studies (41,000) than from prospective cohort studies (3200), resulting in additional health costs of 89 Mio CHF, which are attributed to the indirect health benefits of a smoking ban introduction. The example of smoking ban studies on ischaemic heart disease hospitalization rates suggests that total health costs that can be prevented with smoking bans are considerably larger than the costs arising from the direct health impact of ETS exposure in public places.

PMS2 gene mutational analysis: direct cDNA sequencing to circumvent pseudogene interference.

PubMed

Wimmer, Katharina; Wernstedt, Annekatrin

2014-01-01

The presence of highly homologous pseudocopies can compromise the mutation analysis of a gene of interest. In particular, when using PCR-based strategies, pseudogene co-amplification has to be effectively prevented. This is often achieved by using primers designed to be parental gene specific according to the reference sequence and by applying stringent PCR conditions. However, there are cases in which this approach is of limited utility. For example, it has been shown that the PMS2 gene exchanges sequences with one of its pseudogenes, named PMS2CL. This results in functional PMS2 alleles containing pseudogene-derived sequences at their 3'-end and in nonfunctional PMS2CL pseudogene alleles that contain gene-derived sequences. Hence, the paralogues cannot be distinguished according to the reference sequence. This shortcoming can be effectively circumvented by using direct cDNA sequencing. This approach is based on the selective amplification of PMS2 transcripts in two overlapping 1.6-kb RT-PCR products. In addition to avoiding pseudogene co-amplification and allele dropout, this method has also the advantage that it allows to effectively identify deletions, splice mutations, and de novo retrotransposon insertions that escape the detection of most DNA-based mutation analysis protocols.

The conserved role of Krox-20 in directing Hox gene expression during vertebrate hindbrain segmentation.

PubMed

Nonchev, S; Maconochie, M; Vesque, C; Aparicio, S; Ariza-McNaughton, L; Manzanares, M; Maruthainar, K; Kuroiwa, A; Brenner, S; Charnay, P; Krumlauf, R

1996-09-03

Transient segmentation in the hindbrain is a fundamental morphogenetic phenomenon in the vertebrate embryo, and the restricted expression of subsets of Hox genes in the developing rhombomeric units and their derivatives is linked with regional specification. Here we show that patterning of the vertebrate hindbrain involves the direct upregulation of the chicken and pufferfish group 2 paralogous genes, Hoxb-2 and Hoxa-2, in rhombomeres 3 and 5 (r3 and r5) by the zinc finger gene Krox-20. We identified evolutionarily conserved r3/r5 enhancers that contain high affinity Krox-20. binding sites capable of mediating transactivation by Krox-20. In addition to conservation of binding sites critical for Krox-20 activity in the chicken Hoxa-2 and pufferfish Hoxb-2 genes, the r3/r5 enhancers are also characterized by the presence of a number of identical motifs likely to be involved in cooperative interactions with Krox-20 during the process of hindbrain patterning in vertebrates.

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.

PubMed

Zheng, Zhi; Luo, Yuling; McMaster, Gary K

2006-07-01

Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis

NASA Astrophysics Data System (ADS)

Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S.; Qian, Pei-Yuan

2015-03-01

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning ``plug-and-play'' approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis.

PubMed

Li, Yongxin; Li, Zhongrui; Yamanaka, Kazuya; Xu, Ying; Zhang, Weipeng; Vlamakis, Hera; Kolter, Roberto; Moore, Bradley S; Qian, Pei-Yuan

2015-03-24

Bacilli are ubiquitous low G+C environmental Gram-positive bacteria that produce a wide assortment of specialized small molecules. Although their natural product biosynthetic potential is high, robust molecular tools to support the heterologous expression of large biosynthetic gene clusters in Bacillus hosts are rare. Herein we adapt transformation-associated recombination (TAR) in yeast to design a single genomic capture and expression vector for antibiotic production in Bacillus subtilis. After validating this direct cloning "plug-and-play" approach with surfactin, we genetically interrogated amicoumacin biosynthetic gene cluster from the marine isolate Bacillus subtilis 1779. Its heterologous expression allowed us to explore an unusual maturation process involving the N-acyl-asparagine pro-drug intermediates preamicoumacins, which are hydrolyzed by the asparagine-specific peptidase into the active component amicoumacin A. This work represents the first direct cloning based heterologous expression of natural products in the model organism B. subtilis and paves the way to the development of future genome mining efforts in this genus.

Gene expression profiling of immunomagnetically separated cells directly from stabilized whole blood for multicenter clinical trials

PubMed Central

2014-01-01

Background Clinically useful biomarkers for patient stratification and monitoring of disease progression and drug response are in big demand in drug development and for addressing potential safety concerns. Many diseases influence the frequency and phenotype of cells found in the peripheral blood and the transcriptome of blood cells. Changes in cell type composition influence whole blood gene expression analysis results and thus the discovery of true transcript level changes remains a challenge. We propose a robust and reproducible procedure, which includes whole transcriptome gene expression profiling of major subsets of immune cell cells directly sorted from whole blood. Methods Target cells were enriched using magnetic microbeads and an autoMACS® Pro Separator (Miltenyi Biotec). Flow cytometric analysis for purity was performed before and after magnetic cell sorting. Total RNA was hybridized on HGU133 Plus 2.0 expression microarrays (Affymetrix, USA). CEL files signal intensity values were condensed using RMA and a custom CDF file (EntrezGene-based). Results Positive selection by use of MACS® Technology coupled to transcriptomics was assessed for eight different peripheral blood cell types, CD14+ monocytes, CD3+, CD4+, or CD8+ T cells, CD15+ granulocytes, CD19+ B cells, CD56+ NK cells, and CD45+ pan leukocytes. RNA quality from enriched cells was above a RIN of eight. GeneChip analysis confirmed cell type specific transcriptome profiles. Storing whole blood collected in an EDTA Vacutainer® tube at 4°C followed by MACS does not activate sorted cells. Gene expression analysis supports cell enrichment measurements by MACS. Conclusions The proposed workflow generates reproducible cell-type specific transcriptome data which can be translated to clinical settings and used to identify clinically relevant gene expression biomarkers from whole blood samples. This procedure enables the integration of transcriptomics of relevant immune cell subsets sorted directly from

[Current status and prospects of gene doping detection].

PubMed

Wang, Wenjun; Zhang, Sichun; Xu, Jingjuan; Xia, Xinghua; Tian, Yaping; Zhang, Xinrong; Chen, Hong-Yuan

2008-07-01

The fast development of biotechnology promotes the development of doping. From recombinant protein to gene doping, there is a great challenge to their detection. The improvement of gene therapy and potential to enhance athletic performance open the door for gene doping. After a brief introduction of the concept of gene doping, the current status and prospects of gene doping detection are reviewed.

Helper-dependent adenoviral vectors for liver-directed gene therapy of primary hyperoxaluria type 1

PubMed Central

Castello, Raffaele; Borzone, Roberta; D’Aria, Stefania; Annunziata, Patrizia; Piccolo, Pasquale; Brunetti-Pierri, Nicola

2015-01-01

Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT) which catalyzes conversion of glyoxylate into glycine. AGT deficiency results in overproduction of oxalate which ultimately leads to end-stage renal disease and death. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only available therapy to prevent disease progression. Gene therapy is an attractive option to provide an alternative treatment for PH1. Towards this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. Compared to saline controls, AGT-deficient mice injected with an HDAd encoding the AGT under the control of a liver-specific promoter showed a significant reduction of hyperoxaluria and less increase of urinary oxalate following challenge with Ethylene Glycol (EG), a precursor of glyoxylate. These studies may thus pave the way to clinical application of HDAd for PH1 gene therapy. PMID:26609667

Prodrugs for Gene-Directed Enzyme-Prodrug Therapy (Suicide Gene Therapy)

PubMed Central

2003-01-01

This review focuses on the prodrugs used in suicide gene therapy. These prodrugs need to satisfy a number of criteria. They must be efficient and selective substrates for the activating enzyme, and be metabolized to potent cytotoxins preferably able to kill cells at all stages of the cell cycle. Both prodrugs and their activated species should have good distributive properties, so that the resulting bystander effects can maximize the effectiveness of the therapy, since gene transduction efficiencies are generally low. A total of 42 prodrugs explored for use in suicide gene therapy with 12 different enzymes are discussed, particularly in terms of their physiocochemical properties. An important parameter in determining bystander effects generated by passive diffusion is the lipophilicity of the activated form, a property conveniently compared by diffusion coefficients (log P for nonionizable compounds and log D7 for compounds containing an ionizable centre). Many of the early antimetabolite-based prodrugs provide very polar activated forms that have limited abilities to diffuse across cell membranes, and rely on gap junctions between cells for their bystander effects. Several later studies have shown that more lipophilic, neutral compounds have superior diffusion-based bystander effects. Prodrugs of DNA alkylating agents, that are less cell cycle-specific than antimetabolites and more effective against noncycling tumor cells, appear in general to be more active prodrugs, requiring less prolonged dosing schedules to be effective. It is expected that continued studies to optimize the bystander effects and other properties of prodrugs and the activated species they generate will contribute to improvements in the effectiveness of suicide gene therapy. PMID:12686722

PHD domain-mediated E3 ligase activity directs intramolecular sumoylation of an adjacent bromodomain required for gene silencing.

PubMed

Ivanov, Alexey V; Peng, Hongzhuang; Yurchenko, Vyacheslav; Yap, Kyoko L; Negorev, Dmitri G; Schultz, David C; Psulkowski, Elyse; Fredericks, William J; White, David E; Maul, Gerd G; Sadofsky, Moshe J; Zhou, Ming-Ming; Rauscher, Frank J

2007-12-14

Tandem PHD and bromodomains are often found in chromatin-associated proteins and have been shown to cooperate in gene silencing. Each domain can bind specifically modified histones: the mechanisms of cooperation between these domains are unknown. We show that the PHD domain of the KAP1 corepressor functions as an intramolecular E3 ligase for sumoylation of the adjacent bromodomain. The RING finger-like structure of the PHD domain is required for both Ubc9 binding and sumoylation and directs modification to specific lysine residues in the bromodomain. Sumoylation is required for KAP1-mediated gene silencing and functions by directly recruiting the SETDB1 histone methyltransferase and the CHD3/Mi2 component of the NuRD complex via SUMO-interacting motifs. Sumoylated KAP1 stimulates the histone methyltransferase activity of SETDB1. These data provide a mechanistic explanation for the cooperation of PHD and bromodomains in gene regulation and describe a function of the PHD domain as an intramolecular E3 SUMO ligase.

Earth System Monitoring, Introduction

NASA Astrophysics Data System (ADS)

Orcutt, John

This section provides sensing and data collection methodologies, as well as an understanding of Earth's climate parameters and natural and man-made phenomena, to support a scientific assessment of the Earth system as a whole, and its response to natural and human-induced changes. The coverage ranges from climate change factors and extreme weather and fires to oil spill tracking and volcanic eruptions. This serves as a basis to enable improved prediction and response to climate change, weather, and natural hazards as well as dissemination of the data and conclusions. The data collection systems include satellite remote sensing, aerial surveys, and land- and ocean-based monitoring stations. Our objective in this treatise is to provide a significant portion of the scientific and engineering basis of Earth system monitoring and to provide this in 17 detailed articles or chapters written at a level for use by university students through practicing professionals. The reader is also directed to the closely related sections on Ecological Systems, Introduction and also Climate Change Modeling Methodology, Introduction as well as Climate Change Remediation, Introduction to. For ease of use by students, each article begins with a glossary of terms, while at an average length of 25 print pages each, sufficient detail is presented for use by professionals in government, universities, and industries. The chapters are individually summarized below.

Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans.

PubMed Central

Napoli, C; Lemieux, C; Jorgensen, R

1990-01-01

We attempted to overexpress chalcone synthase (CHS) in pigmented petunia petals by introducing a chimeric petunia CHS gene. Unexpectedly, the introduced gene created a block in anthocyanin biosynthesis. Forty-two percent of plants with the introduced CHS gene produced totally white flowers and/or patterned flowers with white or pale nonclonal sectors on a wild-type pigmented background; none of hundreds of transgenic control plants exhibited such phenotypes. Progeny testing of one plant demonstrated that the novel color phenotype co-segregated with the introduced CHS gene; progeny without this gene were phenotypically wild type. The somatic and germinal stability of the novel color patterns was variable. RNase protection analysis of petal RNAs isolated from white flowers showed that, although the developmental timing of mRNA expression of the endogenous CHS gene was not altered, the level of the mRNA produced by this gene was reduced 50-fold from wild-type levels. Somatic reversion of plants with white flowers to phenotypically parental violet flowers was associated with a coordinate rise in the steady-state levels of the mRNAs produced by both the endogenous and the introduced CHS genes. Thus, in the altered white flowers, the expression of both genes was coordinately suppressed, indicating that expression of the introduced CHS gene was not alone sufficient for suppression of endogenous CHS transcript levels. The mechanism responsible for the reversible co-suppression of homologous genes in trans is unclear, but the erratic and reversible nature of this phenomenon suggests the possible involvement of methylation. PMID:12354959

A Thermostable β-Glucuronidase Obtained by Directed Evolution as a Reporter Gene in Transgenic Plants

PubMed Central

Xiong, Ai-Sheng; Peng, Ri-He; Zhuang, Jing; Chen, Jian-Min; Zhang, Bin; Zhang, Jian; Yao, Quan-Hong

2011-01-01

A β-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable β-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo β-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic β-glucuronidase activity, which is a welcome improvement in its use as a reporter. PMID:22096498

Cardiac gene therapy: Recent advances and future directions.

PubMed

Mason, Daniel; Chen, Yu-Zhe; Krishnan, Harini Venkata; Sant, Shilpa

2015-10-10

Gene therapy has the potential to serve as an adaptable platform technology for treating various diseases. Cardiovascular disease is a major cause of mortality in the developed world and genetic modification is steadily becoming a more plausible method to repair and regenerate heart tissue. Recently, new gene targets to treat cardiovascular disease have been identified and developed into therapies that have shown promise in animal models. Some of these therapies have advanced to clinical testing. Despite these recent successes, several barriers must be overcome for gene therapy to become a widely used treatment of cardiovascular diseases. In this review, we evaluate specific genetic targets that can be exploited to treat cardiovascular diseases, list the important delivery barriers for the gene carriers, assess the most promising methods of delivering the genetic information, and discuss the current status of clinical trials involving gene therapies targeted to the heart. Copyright © 2015 Elsevier B.V. All rights reserved.

Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

NASA Astrophysics Data System (ADS)

Travella, Silvia; Keller, Beat

Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

GlpR is a direct transcriptional repressor of fructose metabolic genes in Haloferax volcanii.

PubMed

Martin, Jonathan H; Rawls, Katie Sherwood; Chan, Jou Chin; Hwang, Sungmin; Martinez-Pastor, Mar; McMillan, Lana J; Prunetti, Laurence; Schmid, Amy K; Maupin-Furlow, Julie A

2018-06-18

DeoR-type helix-turn-helix (HTH) domain proteins are transcriptional regulators of sugar and nucleoside metabolism in diverse bacteria and occur in select archaea. In the model archaeon Haloferax volcanii , previous work implicated GlpR, a DeoR-type transcriptional regulator, in transcriptional repression of glpR and the gene encoding the fructose-specific phosphofructokinase ( pfkB ) during growth on glycerol. However, the global regulon governed by GlpR remained unclear. Here we compared transcriptomes of wild type and Δ glpR mutant strains grown on glycerol and glucose to detect significant transcript level differences for nearly 50 new genes regulated by GlpR. By coupling computational prediction of GlpR binding sequences with in vivo and in vitro DNA binding experiments, we determined that GlpR directly controls genes encoding enzymes in fructose degradation, including fructose bisphosphate aldolase, a central control point in glycolysis. GlpR also directly controls other transcription factors. In contrast, other metabolic pathways appear to be under indirect influence of GlpR. In vitro experiments demonstrated that GlpR purifies as a tetramer that binds the effector molecule fructose-1-phosphate (F1P). These results suggest that Hfx. volcanii GlpR functions as a direct negative regulator of fructose degradation during growth on carbon sources other than fructose, such as glucose and glycerol, and that GlpR bears striking functional similarity to bacterial DeoR-type regulators. IMPORTANCE Many archaea are extremophiles, able to thrive in habitats of extreme salinity, pH and temperature. These biological properties are ideal for applications in biotechnology. However, limited knowledge of archaeal metabolism is a bottleneck that prevents broad use of archaea as microbial factories for industrial products. Here we characterize how sugar uptake and use is regulated in a species that lives in high salinity. We demonstrate that a key sugar regulatory protein in

Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

PubMed Central

2009-01-01

Background One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive. These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels. The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. Results An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay

Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes.

PubMed

Bennett, Richard R; Schneider, Hal E; Estrella, Elicia; Burgess, Stephanie; Cheng, Andrew S; Barrett, Caitlin; Lip, Va; Lai, Poh San; Shen, Yiping; Wu, Bai-Lin; Darras, Basil T; Beggs, Alan H; Kunkel, Louis M

2009-10-18

One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive.These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels.The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in

The GLABRA2 homeodomain protein directly regulates CESA5 and XTH17 gene expression in Arabidopsis roots.

PubMed

Tominaga-Wada, Rumi; Iwata, Mineko; Sugiyama, Junji; Kotake, Toshihisa; Ishida, Tetsuya; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Okada, Kiyotaka; Wada, Takuji

2009-11-01

Arabidopsis root hair formation is determined by the patterning genes CAPRICE (CPC), GLABRA3 (GL3), WEREWOLF (WER) and GLABRA2 (GL2), but little is known about the later changes in cell wall material during root hair formation. A combined Fourier-transform infrared microspectroscopy-principal components analysis (FTIR-PCA) method was used to detect subtle differences in the cell wall material between wild-type and root hair mutants in Arabidopsis. Among several root hair mutants, only the gl2 mutation affected root cell wall polysaccharides. Five of the 10 genes encoding cellulose synthase (CESA1-10) and 4 of 33 xyloglucan endotransglucosylase (XTH1-33) genes in Arabidopsis are expressed in the root, but only CESA5 and XTH17 were affected by the gl2 mutation. The L1-box sequence located in the promoter region of these genes was recognized by the GL2 protein. These results indicate that GL2 directly regulates cell wall-related gene expression during root development.

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis.

PubMed

Muhar, Matthias; Ebert, Anja; Neumann, Tobias; Umkehrer, Christian; Jude, Julian; Wieshofer, Corinna; Rescheneder, Philipp; Lipp, Jesse J; Herzog, Veronika A; Reichholf, Brian; Cisneros, David A; Hoffmann, Thomas; Schlapansky, Moritz F; Bhat, Pooja; von Haeseler, Arndt; Köcher, Thomas; Obenauf, Anna C; Popow, Johannes; Ameres, Stefan L; Zuber, Johannes

2018-05-18

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Stromal-derived factor 1 directly promotes genes expressed within the ovulatory cascade in feline cumulus oocyte complexes.

PubMed

Rojo, Julieta L; Linari, Martina; Young, Kelly A; Peluffo, Marina C

2018-05-01

We hypothesized that the chemokine SDF1/CXCR4 system was present in feline cumulus-oocyte complexes (COCs) and that COCs cultured with SDF1 would directly upregulate gene expression in the ovulatory cascade. Ovaries (n = 50) were obtained from adult domestic cats during the breeding season and COCs were recovered from antral follicles. Because IVM media triggers cumulus-oocyte expansion, culture conditions needed to be optimized to study periovulatory genes. After optimization, the effects of 25 ng/ml SDF1 and the CXCR4 inhibitor were examined in a COC culture for 3, 12, and 24 h. MEM-hepes with 1% of charcoal stripped-FBS was the optimized culture medium, assessed by the expansion of COCs at 24 h in the gonadotropin (GNT) group but not in the media with serum alone. The mRNA expression of HAS2, TNFAIP6, PTX3, and AREG peaked at 3 h in GNT group as compared to all other groups (p < 0.05). COCs cultured with SDF1 showed increased HAS2 and TNFAIP6 mRNA expression at 3 h compared to negative controls and to the CXCR4 inhibitor group. CXCR4 and SDF1 immunostaining was present in both cumulus cells and the oocyte. These results demonstrate that GNT stimulation upregulates key periovulatory genes and expansion in feline COCs from antral follicles, and support the use of this culture system to examine molecular processes within the COC. In addition, SDF1 directly promotes key periovulatory genes in feline COCs, suggesting that the SDF1-CXCR4 pathway may extend its function beyond a chemoattractant, and may play a direct role within the COC.

The synthetic gestagen levonorgestrel directly affects gene expression in thyroid and pituitary glands of Xenopus laevis tadpoles.

PubMed

Lorenz, Claudia; Opitz, Robert; Trubiroha, Achim; Lutz, Ilka; Zikova, Andrea; Kloas, Werner

2016-08-01

The synthetic gestagen levonorgestrel (LNG) was previously shown to perturb thyroid hormone-dependent metamorphosis in Xenopus laevis. However, so far the mechanisms underlying the anti-metamorphic effects of LNG remained unknown. Therefore, a series of in vivo and ex vivo experiments was performed to identify potential target sites of LNG action along the pituitary-thyroid axis of X. laevis tadpoles. Prometamorphic tadpoles were treated in vivo with LNG (0.01-10nM) for 72h and brain-pituitary and thyroid tissue was analyzed for marker gene expression. While no treatment-related changes were observed in brain-pituitary tissue, LNG treatment readily affected thyroidal gene expression in tadpoles including decreased slc5a5 and iyd mRNA expression and a strong induction of dio2 and dio3 expression. When using an ex vivo organ explant culture approach, direct effects of LNG on both pituitary and thyroid gland gene expression were detecTable Specifically, treatment of pituitary explants with 10nM LNG strongly stimulated dio2 expression and concurrently suppressed tshb expression. In thyroid glands, ex vivo LNG treatment induced dio2 and dio3 mRNA expression in a thyrotropin-independent manner. When thyroid explants were cultured in thyrotropin-containing media, LNG caused similar gene expression changes as seen after 72h in vivo treatment including a very strong repression of thyrotropin-induced slc5a5 expression. Concerning the anti-thyroidal activity of LNG as seen under in vivo conditions, our ex vivo data provide clear evidence that LNG directly affects expression of genes important for thyroidal iodide handling as well as genes involved in negative feedback regulation of pituitary tshb expression. Copyright © 2016 Elsevier B.V. All rights reserved.

Bi-directional gene set enrichment and canonical correlation analysis identify key diet-sensitive pathways and biomarkers of metabolic syndrome.

PubMed

Morine, Melissa J; McMonagle, Jolene; Toomey, Sinead; Reynolds, Clare M; Moloney, Aidan P; Gormley, Isobel C; Gaora, Peadar O; Roche, Helen M

2010-10-07

Currently, a number of bioinformatics methods are available to generate appropriate lists of genes from a microarray experiment. While these lists represent an accurate primary analysis of the data, fewer options exist to contextualise those lists. The development and validation of such methods is crucial to the wider application of microarray technology in the clinical setting. Two key challenges in clinical bioinformatics involve appropriate statistical modelling of dynamic transcriptomic changes, and extraction of clinically relevant meaning from very large datasets. Here, we apply an approach to gene set enrichment analysis that allows for detection of bi-directional enrichment within a gene set. Furthermore, we apply canonical correlation analysis and Fisher's exact test, using plasma marker data with known clinical relevance to aid identification of the most important gene and pathway changes in our transcriptomic dataset. After a 28-day dietary intervention with high-CLA beef, a range of plasma markers indicated a marked improvement in the metabolic health of genetically obese mice. Tissue transcriptomic profiles indicated that the effects were most dramatic in liver (1270 genes significantly changed; p < 0.05), followed by muscle (601 genes) and adipose (16 genes). Results from modified GSEA showed that the high-CLA beef diet affected diverse biological processes across the three tissues, and that the majority of pathway changes reached significance only with the bi-directional test. Combining the liver tissue microarray results with plasma marker data revealed 110 CLA-sensitive genes showing strong canonical correlation with one or more plasma markers of metabolic health, and 9 significantly overrepresented pathways among this set; each of these pathways was also significantly changed by the high-CLA diet. Closer inspection of two of these pathways--selenoamino acid metabolism and steroid biosynthesis--illustrated clear diet-sensitive changes in

Bi-directional gene set enrichment and canonical correlation analysis identify key diet-sensitive pathways and biomarkers of metabolic syndrome

PubMed Central

2010-01-01

Background Currently, a number of bioinformatics methods are available to generate appropriate lists of genes from a microarray experiment. While these lists represent an accurate primary analysis of the data, fewer options exist to contextualise those lists. The development and validation of such methods is crucial to the wider application of microarray technology in the clinical setting. Two key challenges in clinical bioinformatics involve appropriate statistical modelling of dynamic transcriptomic changes, and extraction of clinically relevant meaning from very large datasets. Results Here, we apply an approach to gene set enrichment analysis that allows for detection of bi-directional enrichment within a gene set. Furthermore, we apply canonical correlation analysis and Fisher's exact test, using plasma marker data with known clinical relevance to aid identification of the most important gene and pathway changes in our transcriptomic dataset. After a 28-day dietary intervention with high-CLA beef, a range of plasma markers indicated a marked improvement in the metabolic health of genetically obese mice. Tissue transcriptomic profiles indicated that the effects were most dramatic in liver (1270 genes significantly changed; p < 0.05), followed by muscle (601 genes) and adipose (16 genes). Results from modified GSEA showed that the high-CLA beef diet affected diverse biological processes across the three tissues, and that the majority of pathway changes reached significance only with the bi-directional test. Combining the liver tissue microarray results with plasma marker data revealed 110 CLA-sensitive genes showing strong canonical correlation with one or more plasma markers of metabolic health, and 9 significantly overrepresented pathways among this set; each of these pathways was also significantly changed by the high-CLA diet. Closer inspection of two of these pathways - selenoamino acid metabolism and steroid biosynthesis - illustrated clear diet

Novel Directions for Diabetes Mellitus Drug Discovery

PubMed Central

Maiese, Kenneth; Chong, Zhao Zhong; Shang, Yan Chen; Wang, Shaohui

2012-01-01

Introduction Diabetes mellitus impacts almost 200 million individuals worldwide and leads to debilitating complications. New avenues of drug discovery must target the underlying cellular processes of oxidative stress, apoptosis, autophagy, and inflammation that can mediate multi-system pathology during diabetes mellitus. Areas Covered We examine novel directions for drug discovery that involve the β-nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide, the cytokine erythropoietin, the NAD+-dependent protein histone deacetylase SIRT1, the serine/threonine-protein kinase mammalian target of rapamycin (mTOR), and the wingless pathway. Implications for the targeting of these pathways that oversee gluconeogenic genes, insulin signaling and resistance, fatty acid beta-oxidation, inflammation, and cellular survival are presented. Expert Opinion Nicotinamide, erythropoietin, and the downstram pathways of SIRT1, mTOR, forkhead transcription factors, and wingless signaling offer exciting prospects for novel directions of drug discovery for the treatment of metabolic disorders. Future investigations must dissect the complex relationship and fine modulation of these pathways for the successful translation of robust reparative and regenerative strategies against diabetes mellitus and the complications of this disorder. PMID:23092114

The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth

NASA Technical Reports Server (NTRS)

Sedbrook, John C.; Carroll, Kathleen L.; Hung, Kai F.; Masson, Patrick H.; Somerville, Chris R.

2002-01-01

To investigate how roots respond to directional cues, we characterized a T-DNA-tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.

Home care packages: insights into the experiences of older people leading up to the introduction of consumer directed care in Australia.

PubMed

Day, Jenny; Taylor, Ann Clare Thorington; Summons, Peter; Van Der Riet, Pamela; Hunter, Sharyn; Maguire, Jane; Dilworth, Sophie; Bellchambers, Helen; Jeong, Sarah; Haydon, Gunilla; Harris, Margaret; Higgins, Isabel

2017-04-01

This paper reports phase one, conducted from March to June 2015, of a two-phase, qualitative descriptive study designed to explore the perceptions and experiences of older people before and after the introduction of consumer directed care (CDC) to home care packages (HCP) in Australia. Eligible consumers with a local HCP provider were mailed information about the study. Data collection occurred before the introduction of CDC and included face-to-face, in-depth interviews, summaries of interviews, field notes and reflective journaling. Semi-structured questions and 'emotional touchpoints' relating to home care were used to guide the interview conversation. Line-by-line data analysis, where significant statements were highlighted and clustered to reveal emergent themes, was used. Five older people, aged 81 to 91 years, participated in the study. The four emergent themes were: seeking quality and reciprocity in carer relationships; patchworking services; the waiting game; and technology with utility. Continuity of carers was central to the development of a trusting relationship and perceptions of care quality among older consumers. Care coordinators and workers should play a key role in ensuring older people receive timely information about CDC and their rights and responsibilities. Participants' use of contemporary technologies suggests opportunities to improve engagement of HCP clients in CDC.

Differences in selection drive olfactory receptor genes in different directions in dogs and wolf.

PubMed

Chen, Rui; Irwin, David M; Zhang, Ya-Ping

2012-11-01

The olfactory receptor (OR) gene family is the largest gene family found in mammalian genomes. It is known to evolve through a birth-and-death process. Here, we characterized the sequences of 16 segregating OR pseudogenes in the samples of the wolf and the Chinese village dog (CVD) and compared them with the sequences from dogs of different breeds. Our results show that the segregating OR pseudogenes in breed dogs are under strong purifying selection, while evolving neutrally in the CVD, and show a more complicated pattern in the wolf. In the wolf, we found a trend to remove deleterious polymorphisms and accumulate nondeleterious polymorphisms. On the basis of protein structure of the ORs, we found that the distribution of different types of polymorphisms (synonymous, nonsynonymous, tolerated, and untolerated) varied greatly between the wolf and the breed dogs. In summary, our results suggest that different forms of selection have acted on the segregating OR pseudogenes in the CVD since domestication, breed dogs after breed formation, and ancestral wolf population, which has driven the evolution of these genes in different directions.

A direct repeat of E-box-like elements is required for cell-autonomous circadian rhythm of clock genes

PubMed Central

Nakahata, Yasukazu; Yoshida, Mayumi; Takano, Atsuko; Soma, Haruhiko; Yamamoto, Takuro; Yasuda, Akio; Nakatsu, Toru; Takumi, Toru

2008-01-01

Background The circadian expression of the mammalian clock genes is based on transcriptional feedback loops. Two basic helix-loop-helix (bHLH) PAS (for Period-Arnt-Sim) domain-containing transcriptional activators, CLOCK and BMAL1, are known to regulate gene expression by interacting with a promoter element termed the E-box (CACGTG). The non-canonical E-boxes or E-box-like sequences have also been reported to be necessary for circadian oscillation. Results We report a new cis-element required for cell-autonomous circadian transcription of clock genes. This new element consists of a canonical E-box or a non-canonical E-box and an E-box-like sequence in tandem with the latter with a short interval, 6 base pairs, between them. We demonstrate that both E-box or E-box-like sequences are needed to generate cell-autonomous oscillation. We also verify that the spacing nucleotides with constant length between these 2 E-elements are crucial for robust oscillation. Furthermore, by in silico analysis we conclude that several clock and clock-controlled genes possess a direct repeat of the E-box-like elements in their promoter region. Conclusion We propose a novel possible mechanism regulated by double E-box-like elements, not to a single E-box, for circadian transcriptional oscillation. The direct repeat of the E-box-like elements identified in this study is the minimal required element for the generation of cell-autonomous transcriptional oscillation of clock and clock-controlled genes. PMID:18177499

Establishing a novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure for the direct detection of gene doping.

PubMed

Beiter, Thomas; Zimmermann, Martina; Fragasso, Annunziata; Armeanu, Sorin; Lauer, Ulrich M; Bitzer, Michael; Su, Hua; Young, William L; Niess, Andreas M; Simon, Perikles

2008-01-01

So far, the abuse of gene transfer technology in sport, so-called gene doping, is undetectable. However, recent studies in somatic gene therapy indicate that long-term presence of transgenic DNA (tDNA) following various gene transfer protocols can be found in DNA isolated from whole blood using conventional PCR protocols. Application of these protocols for the direct detection of gene doping would require almost complete knowledge about the sequence of the genetic information that has been transferred. Here, we develop and describe the novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure that overcomes this difficulty. Apart from the interesting perspectives that this spiPCR procedure offers in the fight against gene doping, this technology could also be of interest in biodistribution and biosafety studies for gene therapeutic applications.

Macrophage mediated PCI enhanced gene-directed enzyme prodrug therapy

NASA Astrophysics Data System (ADS)

Christie, Catherine E.; Zamora, Genesis; Kwon, Young J.; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

2015-03-01

Photochemical internalization (PCI) is a photodynamic therapy-based approach for improving the delivery of macromolecules and genes into the cell cytosol. Prodrug activating gene therapy (suicide gene therapy) employing the transduction of the E. coli cytosine deaminase (CD) gene into tumor cells, is a promising method. Expression of this gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-FC, to the toxic metabolite, 5-fluorouracil (5-FU). 5-FC may be particularly suitable for brain tumors, because it can readily cross the bloodbrain barrier (BBB). In addition the bystander effect, where activated drug is exported from the transfected cancer cells into the tumor microenvironment, plays an important role by inhibiting growth of adjacent tumor cells. Tumor-associated macrophages (TAMs) are frequently found in and around glioblastomas. Monocytes or macrophages (Ma) loaded with drugs, nanoparticles or photosensitizers could therefore be used to target tumors by local synthesis of chemo attractive factors. The basic concept is to combine PCI, to enhance the ex vivo transfection of a suicide gene into Ma, employing specially designed core/shell NP as gene carrier.

Modulation of Estrogen Response Element-Driven Gene Expressions and Cellular Proliferation with Polar Directions by Designer Transcription Regulators

PubMed Central

Muyan, Mesut; Güpür, Gizem; Yaşar, Pelin; Ayaz, Gamze; User, Sırma Damla; Kazan, Hasan Hüseyin; Huang, Yanfang

2015-01-01

Estrogen receptor α (ERα), as a ligand-dependent transcription factor, mediates 17β-estradiol (E2) effects. ERα is a modular protein containing a DNA binding domain (DBD) and transcription activation domains (AD) located at the amino- and carboxyl-termini. The interaction of the E2-activated ERα dimer with estrogen response elements (EREs) of genes constitutes the initial step in the ERE-dependent signaling pathway necessary for alterations of cellular features. We previously constructed monomeric transcription activators, or monotransactivators, assembled from an engineered ERE-binding module (EBM) using the ERα-DBD and constitutively active ADs from other transcription factors. Monotransactivators modulated cell proliferation by activating and repressing ERE-driven gene expressions that simulate responses observed with E2-ERα. We reasoned here that integration of potent heterologous repression domains (RDs) into EBM could generate monotransrepressors that alter ERE-bearing gene expressions and cellular proliferation in directions opposite to those observed with E2-ERα or monotransactivators. Consistent with this, monotransrepressors suppressed reporter gene expressions that emulate the ERE-dependent signaling pathway. Moreover, a model monotransrepressor regulated DNA synthesis, cell cycle progression and proliferation of recombinant adenovirus infected ER-negative cells through decreasing as well as increasing gene expressions with polar directions compared with E2-ERα or monotransactivator. Our results indicate that an ‘activator’ or a ‘repressor’ possesses both transcription activating/enhancing and repressing/decreasing abilities within a chromatin context. Offering a protein engineering platform to alter signal pathway-specific gene expressions and cell growth, our approach could also be used for the development of tools for epigenetic modifications and for clinical interventions wherein multigenic de-regulations are an issue. PMID:26295471

The RPG gene of Medicago truncatula controls Rhizobium-directed polar growth during infection

PubMed Central

Arrighi, Jean-François; Godfroy, Olivier; de Billy, Françoise; Saurat, Olivier; Jauneau, Alain; Gough, Clare

2008-01-01

Rhizobia can infect roots of host legume plants and induce new organs called nodules, in which they fix atmospheric nitrogen. Infection generally starts with root hair curling, then proceeds inside newly formed, intracellular tubular structures called infection threads. A successful symbiotic interaction relies on infection threads advancing rapidly at their tips by polar growth through successive cell layers of the root toward developing nodule primordia. To identify a plant component that controls this tip growth process, we characterized a symbiotic mutant of Medicago truncatula, called rpg for rhizobium-directed polar growth. In this mutant, nitrogen-fixing nodules were rarely formed due to abnormally thick and slowly progressing infection threads. Root hair curling was also abnormal, indicating that the RPG gene fulfils an essential function in the process whereby rhizobia manage to dominate the process of induced tip growth for root hair infection. Map-based cloning of RPG revealed a member of a previously unknown plant-specific gene family encoding putative long coiled-coil proteins we have called RRPs (RPG-related proteins) and characterized by an “RRP domain” specific to this family. RPG expression was strongly associated with rhizobial infection, and the RPG protein showed a nuclear localization, indicating that this symbiotic gene constitutes an important component of symbiotic signaling. PMID:18621693

The RPG gene of Medicago truncatula controls Rhizobium-directed polar growth during infection.

PubMed

Arrighi, Jean-François; Godfroy, Olivier; de Billy, Françoise; Saurat, Olivier; Jauneau, Alain; Gough, Clare

2008-07-15

Rhizobia can infect roots of host legume plants and induce new organs called nodules, in which they fix atmospheric nitrogen. Infection generally starts with root hair curling, then proceeds inside newly formed, intracellular tubular structures called infection threads. A successful symbiotic interaction relies on infection threads advancing rapidly at their tips by polar growth through successive cell layers of the root toward developing nodule primordia. To identify a plant component that controls this tip growth process, we characterized a symbiotic mutant of Medicago truncatula, called rpg for rhizobium-directed polar growth. In this mutant, nitrogen-fixing nodules were rarely formed due to abnormally thick and slowly progressing infection threads. Root hair curling was also abnormal, indicating that the RPG gene fulfils an essential function in the process whereby rhizobia manage to dominate the process of induced tip growth for root hair infection. Map-based cloning of RPG revealed a member of a previously unknown plant-specific gene family encoding putative long coiled-coil proteins we have called RRPs (RPG-related proteins) and characterized by an "RRP domain" specific to this family. RPG expression was strongly associated with rhizobial infection, and the RPG protein showed a nuclear localization, indicating that this symbiotic gene constitutes an important component of symbiotic signaling.

The Choice between MapMan and Gene Ontology for Automated Gene Function Prediction in Plant Science

PubMed Central

Klie, Sebastian; Nikoloski, Zoran

2012-01-01

Since the introduction of the Gene Ontology (GO), the analysis of high-throughput data has become tightly coupled with the use of ontologies to establish associations between knowledge and data in an automated fashion. Ontologies provide a systematic description of knowledge by a controlled vocabulary of defined structure in which ontological concepts are connected by pre-defined relationships. In plant science, MapMan and GO offer two alternatives for ontology-driven analyses. Unlike GO, initially developed to characterize microbial systems, MapMan was specifically designed to cover plant-specific pathways and processes. While the dependencies between concepts in MapMan are modeled as a tree, in GO these are captured in a directed acyclic graph. Therefore, the difference in ontologies may cause discrepancies in data reduction, visualization, and hypothesis generation. Here provide the first systematic comparative analysis of GO and MapMan for the case of the model plant species Arabidopsis thaliana (Arabidopsis) with respect to their structural properties and difference in distributions of information content. In addition, we investigate the effect of the two ontologies on the specificity and sensitivity of automated gene function prediction via the coupling of co-expression networks and the guilt-by-association principle. Automated gene function prediction is particularly needed for the model plant Arabidopsis in which only half of genes have been functionally annotated based on sequence similarity to known genes. The results highlight the need for structured representation of species-specific biological knowledge, and warrants caution in the design principles employed in future ontologies. PMID:22754563

Muscle-targeted hydrodynamic gene introduction of insulin-like growth factor-1 using polyplex nanomicelle to treat peripheral nerve injury.

PubMed

Nagata, Kazuya; Itaka, Keiji; Baba, Miyuki; Uchida, Satoshi; Ishii, Takehiko; Kataoka, Kazunori

2014-06-10

The recovery of neurologic function after peripheral nerve injury often remains incomplete because of the prolonged reinnervation process, which leads to skeletal muscle atrophy and articular contracture from disuse over time. To rescue the skeletal muscle and promote functional recovery, insulin-like growth factor-1 (IGF-1), a potent myogenic factor, was introduced into the muscle by hydrodynamic injection of IGF-1-expressing plasmid DNA using a biocompatible nonviral gene carrier, a polyplex nanomicelle. In a mouse model of sciatic nerve injury, the introduction of IGF-1 into the skeletal muscle of the paralyzed limb effectively alleviated a decrease in muscle weight compared with that in untreated control mice. Histologic analysis of the muscle revealed the IGF-1-expressing plasmid DNA (pDNA) to have a myogenic effect, inducing muscle hypertrophy with the upregulation of the myogenic regulatory factors, myogenin and MyoD. The evaluation of motor function by walking track analysis revealed that the group that received the hydrodynamic injection of IGF-1-expressing pDNA using the polyplex nanomicelle had significantly early recovery of motor function compared with groups receiving negative control pDNA and untreated controls. Early recovery of sensation in the distal area of sciatic nerve injury was also induced by the introduction of IGF-1-expressing pDNA, presumably because of the effect of secreted IGF-1 protein in the vicinity of the injured sciatic nerve exerting a synergistic effect with muscle hypertrophy, inducing a more favorable prognosis. This approach of introducing IGF-1 into skeletal muscle is promising for the treatment of peripheral nerve injury by promoting early motor function recovery. Copyright © 2014 Elsevier B.V. All rights reserved.

Exploration of new perspectives and limitations in Agrobacterium-mediated gene transfer technology. Final report, June 1, 1992--May 31, 1995

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

Marton, L.

1996-02-01

Genetic manipulation of plants often involves the introduction of homologous or partly homologous genes. Ectropic introduction of homologous sequences into plant genomes may trigger epigenetic changes, making expression of the genes unpredictable. The main project objective was to examine the feasibility of using Agrobacterium-mediated gene transfer for homologous gene targeting in plants.

Genotoxicity of retroviral hematopoietic stem cell gene therapy

PubMed Central

Trobridge, Grant D

2012-01-01

Introduction Retroviral vectors have been developed for hematopoietic stem cell (HSC) gene therapy and have successfully cured X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), adrenoleukodystrophy, and Wiskott-Aldrich syndrome. However, in HSC gene therapy clinical trials, genotoxicity mediated by integrated vector proviruses has led to clonal expansion, and in some cases frank leukemia. Numerous studies have been performed to understand the molecular basis of vector-mediated genotoxicity with the aim of developing safer vectors and safer gene therapy protocols. These genotoxicity studies are critical to advancing HSC gene therapy. Areas covered This review provides an introduction to the mechanisms of retroviral vector genotoxicity. It also covers advances over the last 20 years in designing safer gene therapy vectors, and in integration site analysis in clinical trials and large animal models. Mechanisms of retroviral-mediated genotoxicity, and the risk factors that contribute to clonal expansion and leukemia in HSC gene therapy are introduced. Expert opinion Continued research on virus–host interactions and next-generation vectors should further improve the safety of future HSC gene therapy vectors and protocols. PMID:21375467

System and method for introduction and stabilization of genes in Thermus sp.

DOEpatents

Kayser, Kevin J.; Park, Ho-Shin; Kilbane, II, John J.

2005-03-01

A method for introducing and stabilizing heterologous and recombinant genes in a thermophilic host in which a characteristic gene defining a detectable host characteristic is inactivated or deleted from the thermophilic host, resulting in a modified thermophilic host expressing an absence of the detectable host characteristic. A DNA fragment of interest is inserted into the modified thermophilic host together with an intact characteristic gene, whereby the detectable host characteristic is restored to the thermophilic host, thereby enabling detection and confirmation of successful transformation using plasmid vectors and integration of the DNA fragment into the chromosome of the thermophilic host.

A survey of disease connections for CD4+ T cell master genes and their directly linked genes.

PubMed

Li, Wentian; Espinal-Enríquez, Jesús; Simpfendorfer, Kim R; Hernández-Lemus, Enrique

2015-12-01

Genome-wide association studies and other genetic analyses have identified a large number of genes and variants implicating a variety of disease etiological mechanisms. It is imperative for the study of human diseases to put these genetic findings into a coherent functional context. Here we use system biology tools to examine disease connections of five master genes for CD4+ T cell subtypes (TBX21, GATA3, RORC, BCL6, and FOXP3). We compiled a list of genes functionally interacting (protein-protein interaction, or by acting in the same pathway) with the master genes, then we surveyed the disease connections, either by experimental evidence or by genetic association. Embryonic lethal genes (also known as essential genes) are over-represented in master genes and their interacting genes (55% versus 40% in other genes). Transcription factors are significantly enriched among genes interacting with the master genes (63% versus 10% in other genes). Predicted haploinsufficiency is a feature of most these genes. Disease-connected genes are enriched in this list of genes: 42% of these genes have a disease connection according to Online Mendelian Inheritance in Man (OMIM) (versus 23% in other genes), and 74% are associated with some diseases or phenotype in a Genome Wide Association Study (GWAS) (versus 43% in other genes). Seemingly, not all of the diseases connected to genes surveyed were immune related, which may indicate pleiotropic functions of the master regulator genes and associated genes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Research Issues and Language Program Direction. Issues in Language Program Direction: A Series of Annual Volumes.

ERIC Educational Resources Information Center

Heilenman, L. Kathy, Ed.

This collection of papers is divided into two parts. After "Introduction" (L. Kathy Heilenman), Part 1, "Research and Language Program Directors: The Relationship," includes "Research Domains and Language Program Direction" (Bill VanPatten); "Language Program Direction and the Modernist Agenda" (Celeste…

Experiences of older people following the introduction of consumer-directed care to home care packages: A qualitative descriptive study.

PubMed

Day, Jenny; Thorington Taylor, Ann C; Hunter, Sharyn; Summons, Peter; van der Riet, Pamela; Harris, Margaret; Maguire, Jane; Dilworth, Sophie; Jeong, Sarah; Bellchambers, Helen; Haydon, Gunilla; Higgins, Isabel

2018-06-12

To explore the experiences of older people receiving home care package (HCP) support following the introduction of consumer-directed care (CDC) by the Australian government on 1 July 2015. Thirty-one older people with existing HCP support from two service providers in regional New South Wales, Australia, participated in a face-to-face interview and/or a qualitative survey. Analysis revealed the theme of Choices: Preferences, constraints, balancing and choosing. Participants described choosing to live at home with HCP support; however, they were constrained by poor communication and information about service changes and options, personal budgets and access to future care. HCP services remained largely unchanged during transition to CDC. Many aspects of the initial implementation of CDC were challenging for older people. Clear, relevant and timely communication and information about CDC and its consequences for consumers appear to be needed to enhance CDC. © 2018 AJA Inc.

A brief introduction to web-based genome browsers.

PubMed

Wang, Jun; Kong, Lei; Gao, Ge; Luo, Jingchu

2013-03-01

Genome browser provides a graphical interface for users to browse, search, retrieve and analyze genomic sequence and annotation data. Web-based genome browsers can be classified into general genome browsers with multiple species and species-specific genome browsers. In this review, we attempt to give an overview for the main functions and features of web-based genome browsers, covering data visualization, retrieval, analysis and customization. To give a brief introduction to the multiple-species genome browser, we describe the user interface and main functions of the Ensembl and UCSC genome browsers using the human alpha-globin gene cluster as an example. We further use the MSU and the Rice-Map genome browsers to show some special features of species-specific genome browser, taking a rice transcription factor gene OsSPL14 as an example.

Transcription factor-based modulation of neural stem cell differentiation using direct protein transduction

PubMed Central

Stock, Kristin; Nolden, Lars; Edenhofer, Frank; Quandel, Tamara

2010-01-01

In contrast to conventional gene transfer strategies, the direct introduction of recombinant proteins into cells bypasses the risk of insertional mutagenesis and offers an alternative to genetic intervention. Here, we explore whether protein transduction of the gliogenic transcription factor Nkx2.2 can be used to promote oligodendroglial differentiation of mouse embryonic stem cell (ESC)-derived neural stem cells (NSC). To that end, a recombinant cell-permeant form of Nkx2.2 protein was generated. Exposure of ESC-derived NSC to the recombinant protein and initiation of differentiation resulted in a two-fold increase in the number of oligodendrocytes. Furthermore, Nkx2.2-transduced cells exhibited a more mature oligodendroglial phenotype. Comparative viral gene transfer studies showed that the biological effect of Nkx2.2 protein transduction is comparable to that obtained by lentiviral transduction. The results of this proof-of-concept study depict direct intracellular delivery of transcription factors as alternative modality to control lineage differentiation in NSC cultures without genetic modification. Electronic supplementary material The online version of this article (doi:10.1007/s00018-010-0347-1) contains supplementary material, which is available to authorized users. PMID:20352468

A potential target gene for the host-directed therapy of mycobacterial infection in murine macrophages

PubMed Central

Bao, Zhang; Chen, Ran; Zhang, Pei; Lu, Shan; Chen, Xing; Yao, Yake; Jin, Xiaozheng; Sun, Yilan; Zhou, Jianying

2016-01-01

Mycobacterium tuberculosis (MTB), one of the major bacterial pathogens for lethal infectious diseases, is capable of surviving within the phagosomes of host alveolar macrophages; therefore, host genetic variations may alter the susceptibility to MTB. In this study, to identify host genes exploited by MTB during infection, genes were non-selectively inactivated using lentivirus-based antisense RNA methods in RAW264.7 macrophages, and the cells that survived virulent MTB infection were then screened. Following DNA sequencing of the surviving cell clones, 26 host genes affecting susceptibility to MTB were identified and their pathways were analyzed by bioinformatics analysis. In total, 9 of these genes were confirmed as positive regulators of collagen α-5(IV) chain (Col4a5) expression, a gene encoding a type IV collagen subunit present on the cell surface. The knockdown of Col4a5 consistently suppressed intracellular mycobacterial viability, promoting the survival of RAW264.7 macrophages following mycobacterial infection. Furthermore, Col4a5 deficiency lowered the pH levels of intracellular vesicles, including endosomes, lysosomes and phagosomes in the RAW264.7 cells. Finally, the knockdown of Col4a5 post-translationally increased microsomal vacuolar-type H+-ATPase activity in macrophages, leading to the acidification of intracellular vesicles. Our findings reveal a novel role for Col4a5 in the regulation of macrophage responses to mycobacterial infection and identify Col4a5 as a potential target for the host-directed anti-mycobacterial therapy. PMID:27432120

Circadian clock gene LATE ELONGATED HYPOCOTYL directly regulates the timing of floral scent emission in Petunia

PubMed Central

Fenske, Myles P.; Hewett Hazelton, Kristen D.; Hempton, Andrew K.; Shim, Jae Sung; Yamamoto, Breanne M.; Riffell, Jeffrey A.; Imaizumi, Takato

2015-01-01

Flowers present a complex display of signals to attract pollinators, including the emission of floral volatiles. Volatile emission is highly regulated, and many species restrict emissions to specific times of the day. This rhythmic emission of scent is regulated by the circadian clock; however, the mechanisms have remained unknown. In Petunia hybrida, volatile emissions are dominated by products of the floral volatile benzenoid/phenylpropanoid (FVBP) metabolic pathway. Here we demonstrate that the circadian clock gene P. hybrida LATE ELONGATED HYPOCOTYL (LHY; PhLHY) regulates the daily expression patterns of the FVBP pathway genes and floral volatile production. PhLHY expression peaks in the morning, antiphasic to the expression of P. hybrida GIGANTEA (PhGI), the master scent regulator ODORANT1 (ODO1), and many other evening-expressed FVBP genes. Overexpression phenotypes of PhLHY in Arabidopsis caused an arrhythmic clock phenotype, which resembles those of LHY overexpressors. In Petunia, constitutive expression of PhLHY depressed the expression levels of PhGI, ODO1, evening-expressed FVBP pathway genes, and FVBP emission in flowers. Additionally, in the Petunia lines in which PhLHY expression was reduced, the timing of peak expression of PhGI, ODO1, and the FVBP pathway genes advanced to the morning. Moreover, PhLHY protein binds to cis-regulatory elements called evening elements that exist in promoters of ODO1 and other FVBP genes. Thus, our results imply that PhLHY directly sets the timing of floral volatile emission by restricting the expression of ODO1 and other FVBP genes to the evening in Petunia. PMID:26124104

Circadian clock gene LATE ELONGATED HYPOCOTYL directly regulates the timing of floral scent emission in Petunia.

PubMed

Fenske, Myles P; Hewett Hazelton, Kristen D; Hempton, Andrew K; Shim, Jae Sung; Yamamoto, Breanne M; Riffell, Jeffrey A; Imaizumi, Takato

2015-08-04

Flowers present a complex display of signals to attract pollinators, including the emission of floral volatiles. Volatile emission is highly regulated, and many species restrict emissions to specific times of the day. This rhythmic emission of scent is regulated by the circadian clock; however, the mechanisms have remained unknown. In Petunia hybrida, volatile emissions are dominated by products of the floral volatile benzenoid/phenylpropanoid (FVBP) metabolic pathway. Here we demonstrate that the circadian clock gene P. hybrida LATE ELONGATED HYPOCOTYL (LHY; PhLHY) regulates the daily expression patterns of the FVBP pathway genes and floral volatile production. PhLHY expression peaks in the morning, antiphasic to the expression of P. hybrida GIGANTEA (PhGI), the master scent regulator ODORANT1 (ODO1), and many other evening-expressed FVBP genes. Overexpression phenotypes of PhLHY in Arabidopsis caused an arrhythmic clock phenotype, which resembles those of LHY overexpressors. In Petunia, constitutive expression of PhLHY depressed the expression levels of PhGI, ODO1, evening-expressed FVBP pathway genes, and FVBP emission in flowers. Additionally, in the Petunia lines in which PhLHY expression was reduced, the timing of peak expression of PhGI, ODO1, and the FVBP pathway genes advanced to the morning. Moreover, PhLHY protein binds to cis-regulatory elements called evening elements that exist in promoters of ODO1 and other FVBP genes. Thus, our results imply that PhLHY directly sets the timing of floral volatile emission by restricting the expression of ODO1 and other FVBP genes to the evening in Petunia.

Gene-specific mechanisms direct glucocorticoid-receptor-driven repression of inflammatory response genes in macrophages

PubMed Central

Sacta, Maria A; Tharmalingam, Bowranigan; Coppo, Maddalena; Rollins, David A; Deochand, Dinesh K; Benjamin, Bradley; Yu, Li; Zhang, Bin; Hu, Xiaoyu; Li, Rong; Chinenov, Yurii

2018-01-01

The glucocorticoid receptor (GR) potently represses macrophage-elicited inflammation, however, the underlying mechanisms remain obscure. Our genome-wide analysis in mouse macrophages reveals that pro-inflammatory paused genes, activated via global negative elongation factor (NELF) dissociation and RNA Polymerase (Pol)2 release from early elongation arrest, and non-paused genes, induced by de novo Pol2 recruitment, are equally susceptible to acute glucocorticoid repression. Moreover, in both cases the dominant mechanism involves rapid GR tethering to p65 at NF-kB-binding sites. Yet, specifically at paused genes, GR activation triggers widespread promoter accumulation of NELF, with myeloid cell-specific NELF deletion conferring glucocorticoid resistance. Conversely, at non-paused genes, GR attenuates the recruitment of p300 and histone acetylation, leading to a failure to assemble BRD4 and Mediator at promoters and enhancers, ultimately blocking Pol2 initiation. Thus, GR displays no preference for a specific pro-inflammatory gene class; however, it effects repression by targeting distinct temporal events and components of transcriptional machinery. PMID:29424686

Direct-to-consumer marketing of evidence-based psychological interventions: introduction.

PubMed

Santucci, Lauren C; McHugh, R Kathryn; Barlow, David H

2012-06-01

The dissemination and implementation of evidence-based psychological interventions (EBPIs) to service provision settings has been a major challenge. Most efforts to disseminate and implement EBPIs have focused on clinicians and clinical systems as the consumers of these treatments and thus have targeted efforts to these groups. An alternative, complementary approach to achieve more widespread utilization of EBPIs is to disseminate directly to patients themselves. The aim of this special section is to explore several direct-to-consumer (i.e., patient) dissemination and education efforts currently underway. This manuscript highlights the rationale for direct-to-patient dissemination strategies as well as the application of marketing science to dissemination efforts. Achieving greater access to EBPIs will require the use of multiple approaches to overcome the many and varied barriers to successful dissemination and implementation. Copyright © 2011. Published by Elsevier Ltd.

Challenges to develop nitrogen-fixing cereals by direct nif-gene transfer.

PubMed

Curatti, Leonardo; Rubio, Luis M

2014-08-01

Some regions of the developing world suffer low cereal production yields due to low fertilizer inputs, among other factors. Biological N2 fixation, catalyzed by the prokaryotic enzyme nitrogenase, is an alternative to the use of synthetic N fertilizers. The molybdenum nitrogenase is an O2-labile metalloenzyme composed of the NifDK and NifH proteins, which biosyntheses require a number of nif gene products. A challenging strategy to increase cereal crop productivity in a scenario of low N fertilization is the direct transfer of nif genes into cereals. The sensitivity of nitrogenase to O2 and the apparent complexity of nitrogenase biosynthesis are the main barriers identified so far. Expression of active NifH requires the products of nifM, nifH, and possibly nifU and nifS, whereas active NifDK requires the products of nifH, nifD, nifK, nifB, nifE, nifN, and possibly nifU, nifS, nifQ, nifV, nafY, nifW and nifZ. Plastids and mitochondria are potential subcellular locations for nitrogenase. Both could provide the ATP and electrons required for nitrogenase to function but they differ in their internal O2 levels and their ability to incorporate ammonium into amino acids. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Stratification of clear cell renal cell carcinoma (ccRCC) genomes by gene-directed copy number alteration (CNA) analysis

PubMed Central

Thiesen, H.-J.; Steinbeck, F.; Maruschke, M.; Koczan, D.; Ziems, B.; Hakenberg, O. W.

2017-01-01

Tumorigenic processes are understood to be driven by epi-/genetic and genomic alterations from single point mutations to chromosomal alterations such as insertions and deletions of nucleotides up to gains and losses of large chromosomal fragments including products of chromosomal rearrangements e.g. fusion genes and proteins. Overall comparisons of copy number alterations (CNAs) presented in 48 clear cell renal cell carcinoma (ccRCC) genomes resulted in ratios of gene losses versus gene gains between 26 ccRCC Fuhrman malignancy grades G1 (ratio 1.25) and 20 G3 (ratio 0.58). Gene losses and gains of 15762 CNA genes were mapped to 795 chromosomal cytoband loci including 280 KEGG pathways. CNAs were classified according to their contribution to Fuhrman tumour gradings G1 and G3. Gene gains and losses turned out to be highly structured processes in ccRCC genomes enabling the subclassification and stratification of ccRCC tumours in a genome-wide manner. CNAs of ccRCC seem to start with common tumour related gene losses flanked by CNAs specifying Fuhrman grade G1 losses and CNA gains favouring grade G3 tumours. The appearance of recurrent CNA signatures implies the presence of causal mechanisms most likely implicated in the pathogenesis and disease-outcome of ccRCC tumours distinguishing lower from higher malignant tumours. The diagnostic quality of initial 201 genes (108 genes supporting G1 and 93 genes G3 phenotypes) has been successfully validated on published Swiss data (GSE19949) leading to a restricted CNA gene set of 171 CNA genes of which 85 genes favour Fuhrman grade G1 and 86 genes Fuhrman grade G3. Regarding these gene sets overall survival decreased with the number of G3 related gene losses plus G3 related gene gains. CNA gene sets presented define an entry to a gene-directed and pathway-related functional understanding of ongoing copy number alterations within and between individual ccRCC tumours leading to CNA genes of prognostic and predictive value. PMID

Stratification of clear cell renal cell carcinoma (ccRCC) genomes by gene-directed copy number alteration (CNA) analysis.

PubMed

Thiesen, H-J; Steinbeck, F; Maruschke, M; Koczan, D; Ziems, B; Hakenberg, O W

2017-01-01

Tumorigenic processes are understood to be driven by epi-/genetic and genomic alterations from single point mutations to chromosomal alterations such as insertions and deletions of nucleotides up to gains and losses of large chromosomal fragments including products of chromosomal rearrangements e.g. fusion genes and proteins. Overall comparisons of copy number alterations (CNAs) presented in 48 clear cell renal cell carcinoma (ccRCC) genomes resulted in ratios of gene losses versus gene gains between 26 ccRCC Fuhrman malignancy grades G1 (ratio 1.25) and 20 G3 (ratio 0.58). Gene losses and gains of 15762 CNA genes were mapped to 795 chromosomal cytoband loci including 280 KEGG pathways. CNAs were classified according to their contribution to Fuhrman tumour gradings G1 and G3. Gene gains and losses turned out to be highly structured processes in ccRCC genomes enabling the subclassification and stratification of ccRCC tumours in a genome-wide manner. CNAs of ccRCC seem to start with common tumour related gene losses flanked by CNAs specifying Fuhrman grade G1 losses and CNA gains favouring grade G3 tumours. The appearance of recurrent CNA signatures implies the presence of causal mechanisms most likely implicated in the pathogenesis and disease-outcome of ccRCC tumours distinguishing lower from higher malignant tumours. The diagnostic quality of initial 201 genes (108 genes supporting G1 and 93 genes G3 phenotypes) has been successfully validated on published Swiss data (GSE19949) leading to a restricted CNA gene set of 171 CNA genes of which 85 genes favour Fuhrman grade G1 and 86 genes Fuhrman grade G3. Regarding these gene sets overall survival decreased with the number of G3 related gene losses plus G3 related gene gains. CNA gene sets presented define an entry to a gene-directed and pathway-related functional understanding of ongoing copy number alterations within and between individual ccRCC tumours leading to CNA genes of prognostic and predictive value.

Regulatory analysis of the mouse Hoxb3 gene: multiple elements work in concert to direct temporal and spatial patterns of expression.

PubMed

Kwan, C T; Tsang, S L; Krumlauf, R; Sham, M H

2001-04-01

The expression pattern of the mouse Hoxb3 gene is exceptionally complex and dynamic compared with that of other members of the Hoxb cluster. There are multiple types of transcripts for Hoxb3 gene, and the anterior boundaries of its expression vary at different stages of development. Two enhancers flanking Hoxb3 on the 3' and 5' sides regulate Hoxb2 and Hoxb4, respectively, and these control regions define the two ends of a 28-kb interval in and around the Hoxb3 locus. To assay the regulatory potential of DNA fragments in this interval we have used transgenic analysis with a lacZ reporter gene to locate cis-elements for directing the dynamic patterns of Hoxb3 expression. Our detailed analysis has identified four new and widely spaced cis-acting regulatory regions that can together account for major aspects of the Hoxb3 expression pattern. Elements Ib, IIIa, and IVb control gene expression in neural and mesodermal tissues; element Va controls mesoderm-specific gene expression. The most anterior neural expression domain of Hoxb3 is controlled by an r5 enhancer (element IVa); element IIIa directs reporter expression in the anterior spinal cord and hindbrain up to r6, and the region A enhancer (in element I) mediates posterior neural expression. Hence, the regulation of segmental expression of Hoxb3 in the hindbrain is different from that of Hoxa3, as two separate enhancer elements contribute to expression in r5 and r6. The mesoderm-specific element (Va) directs reporter expression to prevertebra C1 at 12.5 dpc, which is the anterior limit of paraxial mesoderm expression for Hoxb3. When tested in combinations, these cis-elements appear to work as modules in an additive manner to recapitulate the major endogenous expression patterns of Hoxb3 during embryogenesis. Together our study shows that multiple control elements direct reporter gene expression in diverse tissue-, temporal-, and spatially restricted subset of the endogenous Hoxb3 expression domains and work in

The position of DNA cleavage by TALENs and cell synchronization influences the frequency of gene editing directed by single-stranded oligonucleotides.

PubMed

Rivera-Torres, Natalia; Strouse, Bryan; Bialk, Pawel; Niamat, Rohina A; Kmiec, Eric B

2014-01-01

With recent technological advances that enable DNA cleavage at specific sites in the human genome, it may now be possible to reverse inborn errors, thereby correcting a mutation, at levels that could have an impact in a clinical setting. We have been developing gene editing, using single-stranded DNA oligonucleotides (ssODNs), as a tool to direct site specific single base changes. Successful application of this technique has been demonstrated in many systems ranging from bacteria to human (ES and somatic) cells. While the frequency of gene editing can vary widely, it is often at a level that does not enable clinical application. As such, a number of stimulatory factors such as double-stranded breaks are known to elevate the frequency significantly. The majority of these results have been discovered using a validated HCT116 mammalian cell model system where credible genetic and biochemical readouts are available. Here, we couple TAL-Effector Nucleases (TALENs) that execute specific ds DNA breaks with ssODNs, designed specifically to repair a missense mutation, in an integrated single copy eGFP gene. We find that proximal cleavage, relative to the mutant base, is key for enabling high frequencies of editing. A directionality of correction is also observed with TALEN activity upstream from the target base being more effective in promoting gene editing than activity downstream. We also find that cells progressing through S phase are more amenable to combinatorial gene editing activity. Thus, we identify novel aspects of gene editing that will help in the design of more effective protocols for genome modification and gene therapy in natural genes.

Direct Imaging of Gene-Carrier Complexes in Animal Cells

NASA Astrophysics Data System (ADS)

Lin, Alison J.; Slack, Nelle L.; Ahmad, Ayesha; Matsumoto, Brian; Safinya, Cyrus R.

1998-03-01

Cationic lipids are promising gene carriers for DNA transfection. Establishing the correlations between structures of cationic lipid/DNA complexes (CL-DNA) and pathways of transfection will greatly aid us in achieving the optimal CL-DNA transfections. Our first step is to determine the uptake mechanism of DNA by studying the interactions and structures of DNA and cationic lipids. X-ray diffraction shows that the CL-DNA undergoes structural phase transitions from lamellar( J. Raedler, I. Koltover, T. Salditt, C. R. Safinya, Science 275, 810 (1997).) to inverted hexagonal self-assemblies as we change the lipid composition. X-ray diffraction and optical microscopy techniques are used to directly image the progress of the CL-DNA in mouse L-cells and unravel the complex structure in-situ. Fluorescence and confocal optical microscopy techniques allow us to monitor the interactions between the complexes and different organelles in the cell cytoplasm. Current results indicate that once inside cells, complexes containing DOPE follow a different pathway from those containing DOPC. This research is funded by NSF-DMR-9624091, PRF-31352-AC7, and Los Alamos-STB/UC:96-108.

In the Beginning-There Is the Introduction-and Your Study Hypothesis.

PubMed

Vetter, Thomas R; Mascha, Edward J

2017-05-01

Writing a manuscript for a medical journal is very akin to writing a newspaper article-albeit a scholarly one. Like any journalist, you have a story to tell. You need to tell your story in a way that is easy to follow and makes a compelling case to the reader. Although recommended since the beginning of the 20th century, the conventional Introduction-Methods-Results-And-Discussion (IMRAD) scientific reporting structure has only been the standard since the 1980s. The Introduction should be focused and succinct in communicating the significance, background, rationale, study aims or objectives, and the primary (and secondary, if appropriate) study hypotheses. Hypothesis testing involves posing both a null and an alternative hypothesis. The null hypothesis proposes that no difference or association exists on the outcome variable of interest between the interventions or groups being compared. The alternative hypothesis is the opposite of the null hypothesis and thus typically proposes that a difference in the population does exist between the groups being compared on the parameter of interest. Most investigators seek to reject the null hypothesis because of their expectation that the studied intervention does result in a difference between the study groups or that the association of interest does exist. Therefore, in most clinical and basic science studies and manuscripts, the alternative hypothesis is stated, not the null hypothesis. Also, in the Introduction, the alternative hypothesis is typically stated in the direction of interest, or the expected direction. However, when assessing the association of interest, researchers typically look in both directions (ie, favoring 1 group or the other) by conducting a 2-tailed statistical test because the true direction of the effect is typically not known, and either direction would be important to report.

A Simple Model to Rank Shellfish Farming Areas Based on the Risk of Disease Introduction and Spread.

PubMed

Thrush, M A; Pearce, F M; Gubbins, M J; Oidtmann, B C; Peeler, E J

2017-08-01

The European Union Council Directive 2006/88/EC requires that risk-based surveillance (RBS) for listed aquatic animal diseases is applied to all aquaculture production businesses. The principle behind this is the efficient use of resources directed towards high-risk farm categories, animal types and geographic areas. To achieve this requirement, fish and shellfish farms must be ranked according to their risk of disease introduction and spread. We present a method to risk rank shellfish farming areas based on the risk of disease introduction and spread and demonstrate how the approach was applied in 45 shellfish farming areas in England and Wales. Ten parameters were used to inform the risk model, which were grouped into four risk themes based on related pathways for transmission of pathogens: (i) live animal movement, (ii) transmission via water, (iii) short distance mechanical spread (birds) and (iv) long distance mechanical spread (vessels). Weights (informed by expert knowledge) were applied both to individual parameters and to risk themes for introduction and spread to reflect their relative importance. A spreadsheet model was developed to determine quantitative scores for the risk of pathogen introduction and risk of pathogen spread for each shellfish farming area. These scores were used to independently rank areas for risk of introduction and for risk of spread. Thresholds were set to establish risk categories (low, medium and high) for introduction and spread based on risk scores. Risk categories for introduction and spread for each area were combined to provide overall risk categories to inform a risk-based surveillance programme directed at the area level. Applying the combined risk category designation framework for risk of introduction and spread suggested by European Commission guidance for risk-based surveillance, 4, 10 and 31 areas were classified as high, medium and low risk, respectively. © 2016 Crown copyright.

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease.

PubMed

Doetschman, Thomas; Georgieva, Teodora

2017-03-03

Genetic engineering of model organisms and cultured cells has for decades provided important insights into the mechanisms underlying cardiovascular development and disease. In the past few years the development of several nuclease systems has broadened the range of model/cell systems that can be engineered. Of these, the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has become the favorite for its ease of application. Here we will review this RNA-guided nuclease system for gene editing with respect to its usefulness for cardiovascular studies and with an eye toward potential therapy. Studies on its off-target activity, along with approaches to minimize this activity will be given. The advantages of gene editing versus gene targeting in embryonic stem cells, including the breadth of species and cell types to which it is applicable, will be discussed. We will also cover its use in iPSC for research and possible therapeutic purposes; and we will review its use in muscular dystrophy studies where considerable progress has been made toward dystrophin correction in mice. The CRISPR/Ca9s system is also being used for high-throughput screening of genes, gene regulatory regions, and long noncoding RNAs. In addition, the CRISPR system is being used for nongene-editing purposes such as activation and inhibition of gene expression, as well as for fluorescence tagging of chromosomal regions and individual mRNAs to track their cellular location. Finally, an approach to circumvent the inability of post-mitotic cells to support homologous recombination-based gene editing will be presented. In conclusion, applications of the CRISPR/Cas system are expanding at a breath-taking pace and are revolutionizing approaches to gain a better understanding of human diseases. © 2017 American Heart Association, Inc.

Direct stimulation of immediate-early genes by intranuclear insulin in trypsin-treated H35 hepatoma cells.

PubMed Central

Lin, Y J; Harada, S; Loten, E G; Smith, R M; Jarett, L

1992-01-01

H35 hepatoma cells were treated with trypsin to abolish insulin binding and insulin-stimulated receptor kinase activity. Insulin was, however, internalized by fluid-phase endocytosis in trypsin-treated cells. Furthermore, nuclear accumulation of insulin was similar in control and trypsin-treated hepatoma cells. Northern blot analysis revealed insulin increased g33 and c-fos mRNA concentrations identically in control and trypsin-treated cells but had no effect on beta 2-microglobulin mRNA. Actinomycin D treatment prior to or after insulin addition demonstrated that insulin increased gene transcription and had no effect on mRNA degradation. These studies suggest that the accumulation of intact insulin in cell nuclei may be directly involved in the increased transcription of immediate-early genes. Images PMID:1409684

PHD Domain-Mediated E3 Ligase Activity Directs Intramolecular Sumoylation of an Adjacent Bromodomain which is Required for Gene Silencing

PubMed Central

Ivanov, Alexey V.; Peng, Hongzhuang; Yurchenko, Vyacheslav; Yap, Kyoko L.; Negorev, Dmitri G.; Schultz, David C.; Psulkowski, Elyse; Fredericks, William J.; White, David E.; Maul, Gerd G.; Sadofsky, Moshe J.; Zhou, Ming-Ming; Rauscher, Frank J.

2015-01-01

SUMMARY Tandem PHD and bromodomains are often found in chromatin-associated proteins and have been shown to cooperate in gene silencing. Each domain can bind specifically modified histones: the mechanisms of cooperation between these domains are unknown. We show that the PHD domain of the KAP1 corepressor functions as an intramolecular E3 ligase for sumoylation of the adjacent bromodomain. The RING finger-like structure of the PHD domain is required for both Ubc9 binding and sumoylation and directs modification to specific lysine residues in the bromodomain. Sumoylation is required for KAP1-mediated gene silencing and functions by directly recruiting the SETDB1 histone methyltransferase and the CHD3/Mi2 component of the NuRD complex via SUMO interacting motifs. Sumoylated KAP1 stimulates the histone methyltransferase activity of SETDB1. These data provide a mechanistic explanation for the cooperation of PHD and bromodomains in gene regulation and describe a new function of the PHD domain as an intramolecular E3 SUMO ligase. PMID:18082607

Gene delivery to skin using biolistics.

PubMed

Heiser, William C

2006-06-01

INTRODUCTION Biolistics ("biological ballistics") or particle bombardment provides a rapid and simple physical procedure for delivering genes into cells (Klein et al. 1987; Yang et al. 1990). The technique has many advantages-plasmids may be used for delivery, DNA theoretically can be delivered to any cell type, and genes may be delivered to cells in vitro, ex vivo, or in vivo. DNA-coated gold particles are distributed evenly along the length of the tubing, which is subsequently cut into short sections of cartridges to be used in a gene gun. The Helios Gene Gun uses a pulse of helium to launch the DNA-coated particles, spreading them onto the target cells.

Hypoxia-response element (HRE)-directed transcriptional regulation of the rat lysyl oxidase gene in response to cobalt and cadmium.

PubMed

Gao, Song; Zhou, Jing; Zhao, Yinzhi; Toselli, Paul; Li, Wande

2013-04-01

Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5'-ACGTG-3') exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10-100 μM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)-treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at -387/-383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation.

Direct sample introduction gas chromatography and mass spectrometry for the determination of phthalate esters in cleaning products.

PubMed

Cacho, J I; Campillo, N; Viñas, P; Hernández-Córdoba, M

2015-02-06

A method using direct sample introduction (DSI) coupled to gas chromatography-mass spectrometry (GC-MS) is developed for the determination of six phthalate esters (dimethyl, diethyl, dibutyl, butylbenzyl, diethylhexyl and dioctyl phthalate) in cleaning products. The different variables involved in the DSI step, including venting time and temperature, vaporisation time and temperature, injector heating temperature and gas flow rate and pressure, were evaluated and optimised using Taguchi orthogonal arrays. The proposed method, using calibration against methanolic standards, showed good linearity in the 0.05-15 μg g(-1) range and good repeatability, with RSD values ranging from 3.5% to 5.7%. Quantification limits between 0.010 and 0.041 μg g(-1), depending on the compound, were attained, while recovery assays provided values from 83% to 115%. Twenty-seven cleaning products were analysed using the DSI-GC-MS method, being four phthalates (dimethyl, diethyl, dibutyl and diethylhexyl phthalate) found in fourteen of them at concentration levels in the 0.1-21 μg g(-1) range. Compared with the most common GC injection technique, which uses the split/splitless injector, the proposed DSI procedure provided larger peak areas and lower detection limits, as result of the greater injected volume and reduction in noise. Copyright © 2014 Elsevier B.V. All rights reserved.

Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.

PubMed

Christiaens, H; Leer, R J; Pouwels, P H; Verstraete, W

1992-12-01

The conjugated bile acid hydrolase gene from the silage isolate Lactobacillus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For the screening of this hydrolase gene within the gene bank, a direct plate assay developed by Dashkevicz and Feighner (M. P. Dashkevicz and S. D. Feighner, Appl. Environ. Microbiol. 53:331-336, 1989) was adapted to the growth requirements of E. coli. Because of hydrolysis and medium acidification, hydrolase-active colonies were surrounded with big halos of precipitated, free bile acids. This phenomenon was also obtained when the gene was cloned into a multicopy shuttle vector and subsequently reintroduced into the parental Lactobacillus strain. The cbh gene and surrounding regions were characterized by nucleotide sequence analysis. The deduced amino acid sequence was shown to have 52% similarity with a penicillin V amidase from Bacillus sphaericus. Preliminary characterization of the gene product showed that it is a cholylglycine hydrolase (EC 3.5.1.24) with only slight activity against taurine conjugates. The optimum pH was between 4.7 and 5.5. Optimum temperature ranged from 30 to 45 degrees C. Southern blot analysis indicated that the cloned gene has similarity with genomic DNA of bile acid hydrolase-active Lactobacillus spp. of intestinal origin.

Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity

PubMed Central

Yonehara, Keisuke; Fiscella, Michele; Drinnenberg, Antonia; Esposti, Federico; Trenholm, Stuart; Krol, Jacek; Franke, Felix; Scherf, Brigitte Gross; Kusnyerik, Akos; Müller, Jan; Szabo, Arnold; Jüttner, Josephine; Cordoba, Francisco; Reddy, Ashrithpal Police; Németh, János; Nagy, Zoltán Zsolt; Munier, Francis; Hierlemann, Andreas; Roska, Botond

2016-01-01

Summary Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease. Video Abstract PMID:26711119

Potential Direct Regulators of the Drosophila yellow Gene Identified by Yeast One-Hybrid and RNAi Screens

PubMed Central

Kalay, Gizem; Lusk, Richard; Dome, Mackenzie; Hens, Korneel; Deplancke, Bart; Wittkopp, Patricia J.

2016-01-01

The regulation of gene expression controls development, and changes in this regulation often contribute to phenotypic evolution. Drosophila pigmentation is a model system for studying evolutionary changes in gene regulation, with differences in expression of pigmentation genes such as yellow that correlate with divergent pigment patterns among species shown to be caused by changes in cis- and trans-regulation. Currently, much more is known about the cis-regulatory component of divergent yellow expression than the trans-regulatory component, in part because very few trans-acting regulators of yellow expression have been identified. This study aims to improve our understanding of the trans-acting control of yellow expression by combining yeast-one-hybrid and RNAi screens for transcription factors binding to yellow cis-regulatory sequences and affecting abdominal pigmentation in adults, respectively. Of the 670 transcription factors included in the yeast-one-hybrid screen, 45 showed evidence of binding to one or more sequence fragments tested from the 5′ intergenic and intronic yellow sequences from D. melanogaster, D. pseudoobscura, and D. willistoni, suggesting that they might be direct regulators of yellow expression. Of the 670 transcription factors included in the yeast-one-hybrid screen, plus another TF previously shown to be genetically upstream of yellow, 125 were also tested using RNAi, and 32 showed altered abdominal pigmentation. Nine transcription factors were identified in both screens, including four nuclear receptors related to ecdysone signaling (Hr78, Hr38, Hr46, and Eip78C). This finding suggests that yellow expression might be directly controlled by nuclear receptors influenced by ecdysone during early pupal development when adult pigmentation is forming. PMID:27527791

Life cycle replacement by gene introduction under an allee effect in periodical cicadas.

PubMed

Nariai, Yukiko; Hayashi, Saki; Morita, Satoru; Umemura, Yoshitaka; Tainaka, Kei-ichi; Sota, Teiji; Cooley, John R; Yoshimura, Jin

2011-04-06

Periodical cicadas (Magicicada spp.) in the USA are divided into three species groups (-decim, -cassini, -decula) of similar but distinct morphology and behavior. Each group contains at least one species with a 17-year life cycle and one with a 13-year cycle; each species is most closely related to one with the other cycle. One explanation for the apparent polyphyly of 13- and 17-year life cycles is that populations switch between the two cycles. Using a numerical model, we test the general feasibility of life cycle switching by the introduction of alleles for one cycle into populations of the other cycle. Our results suggest that fitness reductions at low population densities of mating individuals (the Allee effect) could play a role in life cycle switching. In our model, if the 13-year cycle is genetically dominant, a 17-year cycle population will switch to a 13-year cycle given the introduction of a few 13-year cycle alleles under a moderate Allee effect. We also show that under a weak Allee effect, different year-classes ("broods") with 17-year life cycles can be generated. Remarkably, the outcomes of our models depend only on the dominance relationships of the cycle alleles, irrespective of any fitness advantages.

Novel Random Mutagenesis Method for Directed Evolution.

PubMed

Feng, Hong; Wang, Hai-Yan; Zhao, Hong-Yan

2017-01-01

Directed evolution is a powerful strategy for gene mutagenesis, and has been used for protein engineering both in scientific research and in the biotechnology industry. The routine method for directed evolution was developed by Stemmer in 1994 (Stemmer, Proc Natl Acad Sci USA 91, 10747-10751, 1994; Stemmer, Nature 370, 389-391, 1994). Since then, various methods have been introduced, each of which has advantages and limitations depending upon the targeted genes and procedure. In this chapter, a novel alternative directed evolution method which combines mutagenesis PCR with dITP and fragmentation by endonuclease V is described. The kanamycin resistance gene is used as a reporter gene to verify the novel method for directed evolution. This method for directed evolution has been demonstrated to be efficient, reproducible, and easy to manipulate in practice.

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

PubMed Central

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

2016-01-01

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

Functional genetics for all: engineered nucleases, CRISPR and the gene editing revolution.

PubMed

Gilles, Anna F; Averof, Michalis

2014-01-01

Developmental biology, as all experimental science, is empowered by technological advances. The availability of genetic tools in some species - designated as model organisms - has driven their use as major platforms for understanding development, physiology and behavior. Extending these tools to a wider range of species determines whether (and how) we can experimentally approach developmental diversity and evolution. During the last two decades, comparative developmental biology (evo-devo) was marked by the introduction of gene knockdown and deep sequencing technologies that are applicable to a wide range of species. These approaches allowed us to test the developmental role of specific genes in diverse species, to study biological processes that are not accessible in established models and, in some cases, to conduct genome-wide screens that overcome the limitations of the candidate gene approach. The recent discovery of CRISPR/Cas as a means of precise alterations into the genome promises to revolutionize developmental genetics. In this review we describe the development of gene editing tools, from zinc-finger nucleases to TALENs and CRISPR, and examine their application in gene targeting, their limitations and the opportunities they present for evo-devo. We outline their use in gene knock-out and knock-in approaches, and in manipulating gene functions by directing molecular effectors to specific sites in the genome. The ease-of-use and efficiency of CRISPR in diverse species provide an opportunity to close the technology gap that exists between established model organisms and emerging genetically-tractable species.

Gene Expression Profile Analysis is Directly Affected by the Selected Reference Gene: The Case of Leaf-Cutting Atta Sexdens

PubMed Central

Máximo, Wesley P. F.; Zanetti, Ronald; Paiva, Luciano V.

2018-01-01

Although several ant species are important targets for the development of molecular control strategies, only a few studies focus on identifying and validating reference genes for quantitative reverse transcription polymerase chain reaction (RT-qPCR) data normalization. We provide here an extensive study to identify and validate suitable reference genes for gene expression analysis in the ant Atta sexdens, a threatening agricultural pest in South America. The optimal number of reference genes varies according to each sample and the result generated by RefFinder differed about which is the most suitable reference gene. Results suggest that the RPS16, NADH and SDHB genes were the best reference genes in the sample pool according to stability values. The SNF7 gene expression pattern was stable in all evaluated sample set. In contrast, when using less stable reference genes for normalization a large variability in SNF7 gene expression was recorded. There is no universal reference gene suitable for all conditions under analysis, since these genes can also participate in different cellular functions, thus requiring a systematic validation of possible reference genes for each specific condition. The choice of reference genes on SNF7 gene normalization confirmed that unstable reference genes might drastically change the expression profile analysis of target candidate genes. PMID:29419794

Identification of the interleukin 4 receptor alpha gene as a direct target for p73.

PubMed

Sasaki, Yasushi; Mita, Hiroaki; Toyota, Minoru; Ishida, Setsuko; Morimoto, Ichiro; Yamashita, Toshiharu; Tanaka, Toshihiro; Imai, Kohzoh; Nakamura, Yusuke; Tokino, Takashi

2003-12-01

p73 has a high degree of structural homology to p53 and can activate transcription of p53-responsive genes. However, analysis of p73-deficient mice revealed a marked divergence in the physiological activities of p53 family genes and distinguishes p73 from p53. Mice deficient for p73 exhibit profound defects, including hippocampal dysgenesis, chronic infection, and inflammation, as well as abnormalities in pheromone sensory pathways. p73 plays important roles in neurogenesis, sensory pathways, and homeostatic regulation. Here, we found that the interleukin 4 receptor alpha (IL-4Ralpha) gene is up-regulated by p73 but not significantly by p53 in several human cancer cell lines. IL-4Ralphatranscription is also activated in response to cisplatin, a DNA-damaging agent known to induce p73. By using small interference RNA designed to target p73, we demonstrated that silencing endogenous p73 abrogates the induction of the IL-4Ralpha gene after cisplatin treatment. Furthermore, we identified a p73-binding site in the first intron of the IL-4Ralpha gene that can directly interact with the p73 protein in vivo. This p73-binding site consists of eight copies of a 10-bp consensus p53-binding motif and is a functional response element that is relatively specific for p73 among the p53 family. p73beta promoted localized nucleosomal acetylation through recruitment of coactivator p300, indicating that p73 regulates transcription of IL-4Ralpha through the unique p73-binding site. We also found that p73beta-transfected tumor cells are sensitive to IL-4-mediated apoptosis. Our data suggest that IL-4Ralpha could mediate, in part, certain immune responses and p73-dependent cell death.

Comparative Evaluation of Two Serial Gene Expression Experiments | Division of Cancer Prevention

Cancer.gov

Stuart G. Baker, 2014 Introduction This program fits biologically relevant response curves in comparative analysis of the two gene expression experiments involving same genes but under different scenarios and at least 12 responses. The program outputs gene pairs with biologically relevant response curve shapes including flat, linear, sigmoid, hockey stick, impulse and step

An Introduction to Fund Raising: The Newcomers' Guide to Development.

ERIC Educational Resources Information Center

Faust, Paula J., Ed.

A comprehensive introduction to all phases of college fund-raising is presented in 14 chapters. Attention is directed to the philosophy behind development, development's role in institutional advancement, and the personal qualities that lead to success in fund-raising. Basic principles also are covered for the following: annual giving, the capital…

Oxytocin receptor gene polymorphisms are associated with human directed social behavior in dogs (Canis familiaris).

PubMed

Kis, Anna; Bence, Melinda; Lakatos, Gabriella; Pergel, Enikő; Turcsán, Borbála; Pluijmakers, Jolanda; Vas, Judit; Elek, Zsuzsanna; Brúder, Ildikó; Földi, Levente; Sasvári-Székely, Mária; Miklósi, Adám; Rónai, Zsolt; Kubinyi, Enikő

2014-01-01

The oxytocin system has a crucial role in human sociality; several results prove that polymorphisms of the oxytocin receptor gene are related to complex social behaviors in humans. Dogs' parallel evolution with humans and their adaptation to the human environment has made them a useful species to model human social interactions. Previous research indicates that dogs are eligible models for behavioral genetic research, as well. Based on these previous findings, our research investigated associations between human directed social behaviors and two newly described (-212AG, 19131AG) and one known (rs8679684) single nucleotide polymorphisms (SNPs) in the regulatory regions (5' and 3' UTR) of the oxytocin receptor gene in German Shepherd (N = 104) and Border Collie (N = 103) dogs. Dogs' behavior traits have been estimated in a newly developed test series consisting of five episodes: Greeting by a stranger, Separation from the owner, Problem solving, Threatening approach, Hiding of the owner. Buccal samples were collected and DNA was isolated using standard protocols. SNPs in the 3' and 5' UTR regions were analyzed by polymerase chain reaction based techniques followed by subsequent electrophoresis analysis. The gene-behavior association analysis suggests that oxytocin receptor gene polymorphisms have an impact in both breeds on (i) proximity seeking towards an unfamiliar person, as well as their owner, and on (ii) how friendly dogs behave towards strangers, although the mediating molecular regulatory mechanisms are yet unknown. Based on these results, we conclude that similarly to humans, the social behavior of dogs towards humans is influenced by the oxytocin system.

Evaluation of introduction of the Haemophilus influenzae vaccine in Côte d’Ivoire

PubMed

Yohou, Kévin Sylvestre; Aka, Nicaise Lepri; Noufe, Soualihou; Douba, Alfred; Assi Assi, Bernard; Dagnan, Simplice N Cho

2016-11-25

Introduction: Côte d’Ivoire introduced the Haemophilus influenzae type b vaccine into the EPI in March 2009. Following this introduction, an evaluation was conducted in 2012 in order to evaluate the vaccine introduction process. Methods: Data collection methods consisted of document review, structured interviews and direct observation. This study collected information from six health region officials, 12 health districts and 36 healthcare institutions. Seventy-two mothers or child carers were also interviewed. Collected data were processed and analysed by Excel, Epi Info and SPSS. Results: A vaccine introduction plan was developed, but was not communicated at the operational level. The planned training for district health care providers was conducted eighteen months after introduction of the vaccine. None of the vaccinating centres had communication support about the new vaccine. Temperature recording was regularly performed in 92% of district deposits and 68% of vaccinating centres. Deteriorated vaccines were observed in 6% of vaccinating centres. Only 3.5% of parents had been informed about introduction of the vaccine. Increased immunization coverage for the third dose of pentavalent vaccine was observed in one half of health districts. Conclusion: Evaluation of the introduction of Haemophilus influenzae type b vaccine highlightsthe strengths and weaknesses of the health system and provides lessons for the introduction of other vaccines into the expanded programme on immunization.

CaSPIAN: A Causal Compressive Sensing Algorithm for Discovering Directed Interactions in Gene Networks

PubMed Central

Emad, Amin; Milenkovic, Olgica

2014-01-01

We introduce a novel algorithm for inference of causal gene interactions, termed CaSPIAN (Causal Subspace Pursuit for Inference and Analysis of Networks), which is based on coupling compressive sensing and Granger causality techniques. The core of the approach is to discover sparse linear dependencies between shifted time series of gene expressions using a sequential list-version of the subspace pursuit reconstruction algorithm and to estimate the direction of gene interactions via Granger-type elimination. The method is conceptually simple and computationally efficient, and it allows for dealing with noisy measurements. Its performance as a stand-alone platform without biological side-information was tested on simulated networks, on the synthetic IRMA network in Saccharomyces cerevisiae, and on data pertaining to the human HeLa cell network and the SOS network in E. coli. The results produced by CaSPIAN are compared to the results of several related algorithms, demonstrating significant improvements in inference accuracy of documented interactions. These findings highlight the importance of Granger causality techniques for reducing the number of false-positives, as well as the influence of noise and sampling period on the accuracy of the estimates. In addition, the performance of the method was tested in conjunction with biological side information of the form of sparse “scaffold networks”, to which new edges were added using available RNA-seq or microarray data. These biological priors aid in increasing the sensitivity and precision of the algorithm in the small sample regime. PMID:24622336

Gene flow among established Puerto Rican populations of the exotic tree species, Albizia lebbeck.

PubMed

Dunphy, B K; Hamrick, J L

2005-04-01

We estimate gene flow and patterns of genetic diversity in Albizia lebbeck, an invasive leguminous tree in the dry forest of southwestern Puerto Rico. Genetic diversity estimates calculated for 10 populations of 24 trees each indicated that these populations may have been formed from multiple introductions. The presence of unique genotypes in the northernmost populations suggests that novel genotypes are still immigrating into the area. This combination of individuals from disparate locations led to high estimates of genetic diversity (He = 0.266, P = 0.67). Indirect estimates of gene flow indicate that only 0.69 migrants per generation move between populations, suggesting that genetic diversity within populations should decrease due to genetic drift. Since migration-drift equilibrium was not found, however, this estimate needs to be viewed with caution. The regular production of pods in this outcrossing species (tm = 0.979) indicates that sufficient outcross pollen is received to insure successful reproduction. Direct estimates of gene flow indicate that between 44 and 100% of pollen received by trees in four small stands of trees (n < 11) was foreign. The role of gene flow in facilitating the spread of this invasive plant species is discussed.

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells.

PubMed

Nakamura, Mikiko; Suzuki, Ayako; Akada, Junko; Yarimizu, Tohru; Iwakiri, Ryo; Hoshida, Hisashi; Akada, Rinji

2015-08-01

Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained. For this purpose, we investigated two enhancer reagents, polyethylene glycol and tRNA, for a chemical transfection method. The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized. The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3'-terminus of any gene. The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures.

Introduction to Psychomotor Skills (NS 117): Competency-Based Course Syllabus.

ERIC Educational Resources Information Center

Brady, Marilyn H.; Wells, Tanya G.

"Introduction to Psychomotor Skills" (NS 117) is the first of seven core courses in the associate degree nursing program at Chattanooga State Technical Community College. The course was designed to help students develop competencies in psychomotor skills necessary to assume the role of provider of direct patient care. The course syllabus for NS…

Rapid detection of translation-terminating mutations at the adenomatous polyposis coli (APC) gene by direct protein truncation test

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

Van Der Luut, R.; Khan, P.M.; Van Leeuwen, C.

Familial adenomatous polyposis (FAP) is usually associated with protein truncating mutations in the adenomatous polyposis coli (APC) gene. The APC mutations are known to play a major role in colorectal carcinogensis. For the identification of protein truncating mutations of the APC gene, the authors developed a rapid, sensitive, and direct screening procedure. The technique is based on the in vitro transcription and translation of the genomic PCR products and is called the protein truncation test. Samples of DNA from individual FAP patients, members of a FAP family, colorectal tumors, and colorectal tumor-derived cell lines were used to show the effectivenessmore » of this method. 9 refs., 2 figs.« less

Liver-directed gene therapy corrects cardiovascular lesions in feline mucopolysaccharidosis type I.

PubMed

Hinderer, Christian; Bell, Peter; Gurda, Brittney L; Wang, Qiang; Louboutin, Jean-Pierre; Zhu, Yanqing; Bagel, Jessica; O'Donnell, Patricia; Sikora, Tracey; Ruane, Therese; Wang, Ping; Haskins, Mark E; Wilson, James M

2014-10-14

Patients with mucopolysaccharidosis type I (MPS I), a genetic deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), exhibit accumulation of glycosaminoglycans in tissues, with resulting diverse clinical manifestations including neurological, ocular, skeletal, and cardiac disease. MPS I is currently treated with hematopoietic stem cell transplantation or weekly enzyme infusions, but these therapies have significant drawbacks for patient safety and quality of life and do not effectively address some of the most critical clinical sequelae, such as life-threatening cardiac valve involvement. Using the naturally occurring feline model of MPS I, we tested liver-directed gene therapy as a means of achieving long-term systemic IDUA reconstitution. We treated four MPS I cats at 3-5 mo of age with an adeno-associated virus serotype 8 vector expressing feline IDUA from a liver-specific promoter. We observed sustained serum enzyme activity for 6 mo at ∼ 30% of normal levels in one animal, and in excess of normal levels in three animals. Remarkably, treated animals not only demonstrated reductions in glycosaminoglycan storage in most tissues, but most also exhibited complete resolution of aortic valve lesions, an effect that has not been previously observed in this animal model or in MPS I patients treated with current therapies. These data point to clinically meaningful benefits of the robust enzyme expression achieved with hepatic gene transfer that extend beyond the economic and quality of life advantages over lifelong enzyme infusions.

Non-coplanar polychlorinated biphenyls (PCBs) are direct agonists for the human pregnane-X receptor and constitutive androstane receptor, and activate target gene expression in a tissue-specific manner

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

Al-Salman, Fadheela; Plant, Nick, E-mail: N.Plant@Surrey.ac.uk

The polychlorinated biphenyl group possesses high environmental persistence, leading to bioaccumulation and a number of adverse effects in mammals. Whilst coplanar PCBs elicit their toxic effects through agonism of the aryl hydrocarbon receptor; however, non-coplanar PCBs are not ligands for AhR, but may be ligands for members of the nuclear receptor family of proteins. To better understand the biological actions of non-coplanar PCBs, we have undertaken a systematic analysis of their ability to activate PXR and CAR-mediated effects. Cells were exposed to a range of non-coplanar PCBs (99, 138, 153, 180 and 194), or the coplanar PCB77: Direct activation ofmore » PXR and CAR was measured using a mammalian receptor activation assay in human liver cells, with rifampicin and CITCO used as positive controls ligands for PXR and CAR, respectively; activation of target gene expression was examined using reporter gene plasmids for CYP3A4 and MDR1 transfected into liver, intestine and lung cell lines. Several of the non-coplanar PCBs directly activated PXR and CAR, whilst the coplanar PCB77 did not. Non-coplanar PCBs were also able to activate PXR/CAR target gene expression in a substitution- and tissue-specific manner. Non-coplanar PCBs act as direct activators for the nuclear receptors PXR and CAR, and are able to elicit transcriptional activation of target genes in a substitution- and tissue-dependent manner. Chronic activation of PXR/CAR is linked to adverse effects and must be included in any risk assessment of PCBs. -- Highlights: ► Several Non-coplanar PCBs are able to directly activate both PXR and CAR in vitro. ► PCB153 is the most potent direct activator of PXR and CAR nuclear receptors. ► Non-coplanar PCB activation of CYP3A4/MDR1 reporter genes is structure-dependent. ► Non-coplanar PCB activate CYP3A4/MDR1 reporter genes in a tissue-dependent. ► PCB153 is the most potent activator of PXR/CAR target gene in all tissues.« less

Direct cloning of the trxB gene that encodes thioredoxin reductase.

PubMed Central

Russel, M; Model, P

1985-01-01

A strain was constructed which contains mutations in the genes encoding thioredoxin (trxA) and thioredoxin reductase (trxB) such that filamentous phage f1 cannot grow. The complementation of either mutation with its wild-type allele permits phage growth. We used this strain to select f1 phage which contain a cloned trxB gene. The location of the gene on the cloned fragment was determined, and its protein product was identified. Plasmid subclones that contain this gene overproduce thioredoxin reductase. Images PMID:2989245

Cluster Analysis in Nursing Research: An Introduction, Historical Perspective, and Future Directions.

PubMed

Dunn, Heather; Quinn, Laurie; Corbridge, Susan J; Eldeirawi, Kamal; Kapella, Mary; Collins, Eileen G

2017-05-01

The use of cluster analysis in the nursing literature is limited to the creation of classifications of homogeneous groups and the discovery of new relationships. As such, it is important to provide clarity regarding its use and potential. The purpose of this article is to provide an introduction to distance-based, partitioning-based, and model-based cluster analysis methods commonly utilized in the nursing literature, provide a brief historical overview on the use of cluster analysis in nursing literature, and provide suggestions for future research. An electronic search included three bibliographic databases, PubMed, CINAHL and Web of Science. Key terms were cluster analysis and nursing. The use of cluster analysis in the nursing literature is increasing and expanding. The increased use of cluster analysis in the nursing literature is positioning this statistical method to result in insights that have the potential to change clinical practice.

Hypoxia-Response Element (HRE)–Directed Transcriptional Regulation of the Rat Lysyl Oxidase Gene in Response to Cobalt and Cadmium

PubMed Central

Li, Wande

2013-01-01

Lysyl oxidase (LO) catalyzes crosslink of collagen, elastin, and histone H1, stabilizing the extracellular matrix and cell nucleus. This enzyme displays dual functions for tumorigenesis, i.e., as a tumor suppressor inactivating the ras oncogene and as a tumor promoter enhancing malignant cell metastasis. To elucidate LO transcriptional regulation, we have cloned the 804 base pair region upstream of the translation start site (ATG) of the rat LO gene with the maximal promoter activity. Computer analysis indicated that at least four hypoxia-response element (HRE) consensuses (5′-ACGTG-3′) exist in the cloned LO promoter. Treatment of rat lung fibroblasts (RFL6) with CoCl2 (Co, 10–100 μM), a chemical hypoxia reagent, enhanced LO mRNA expression and promoter activities. Overexpression of LO was associated with upregulation of hypoxia-inducible factor (HIF)-1α at mRNA levels in cobalt (Co)–treated cells. Thus, LO is a hypoxia-responsive gene. Dominant negative-HIF-1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition, and in vitro translated HIF-1α binding assays indicated that only one HRE mapped at −387/−383 relative to ATG was functionally active among four consensuses. Site-directed mutation of this HRE significantly diminished the Co-induced and LO promoter-directed expression of the reporter gene. Cadmium (Cd), an inducer of reactive oxygen species, inhibited HIF-1α mRNA expression and HIF-1α binding to the LO gene in Co-treated cells as revealed by RT-PCR and ChIP assays, respectively. Thus, modulation of the HRE activity by Co and Cd plays a critical role in LO gene transactivation. PMID:23161664

Magnetic nanoparticles: Applications in gene delivery and gene therapy.

PubMed

Majidi, Sima; Zeinali Sehrig, Fatemeh; Samiei, Mohammad; Milani, Morteza; Abbasi, Elham; Dadashzadeh, Kianoosh; Akbarzadeh, Abolfazl

2016-06-01

Gene therapy is defined as the direct transfer of genetic material to tissues or cells for the treatment of inherited disorders and acquired diseases. For gene delivery, magnetic nanoparticles (MNPs) are typically combined with a delivery platform to encapsulate the gene, and promote cell uptake. Delivery technologies that have been used with MNPs contain polymeric, viral, as well as non-viral platforms. In this review, we focus on targeted gene delivery using MNPs.

Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

PubMed

Kaulich, Manuel; Lee, Yeon J; Lönn, Peter; Springer, Aaron D; Meade, Bryan R; Dowdy, Steven F

2015-04-20

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Trichostatin A specifically improves the aberrant expression of transcription factor genes in embryos produced by somatic cell nuclear transfer

PubMed Central

Inoue, Kimiko; Oikawa, Mami; Kamimura, Satoshi; Ogonuki, Narumi; Nakamura, Toshinobu; Nakano, Toru; Abe, Kuniya; Ogura, Atsuo

2015-01-01

Although mammalian cloning by somatic cell nuclear transfer (SCNT) has been established in various species, the low developmental efficiency has hampered its practical applications. Treatment of SCNT-derived embryos with histone deacetylase (HDAC) inhibitors can improve their development, but the underlying mechanism is still unclear. To address this question, we analysed gene expression profiles of SCNT-derived 2-cell mouse embryos treated with trichostatin A (TSA), a potent HDAC inhibitor that is best used for mouse cloning. Unexpectedly, TSA had no effect on the numbers of aberrantly expressed genes or the overall gene expression pattern in the embryos. However, in-depth investigation by gene ontology and functional analyses revealed that TSA treatment specifically improved the expression of a small subset of genes encoding transcription factors and their regulatory factors, suggesting their positive involvement in de novo RNA synthesis. Indeed, introduction of one of such transcription factors, Spi-C, into the embryos at least partially mimicked the TSA-induced improvement in embryonic development by activating gene networks associated with transcriptional regulation. Thus, the effects of TSA treatment on embryonic gene expression did not seem to be stochastic, but more specific than expected, targeting genes that direct development and trigger zygotic genome activation at the 2-cell stage. PMID:25974394

COX inhibitors directly alter gene expression: role in cancer prevention?

PubMed Central

Wang, Xingya; Baek, Seung Joon; Eling, Thomas

2016-01-01

Inflammation is an important contributor to the development and progression of human cancers. Inflammatory lipid metabolites, prostaglandins, formed from arachidonic acid by prostaglandin H synthases commonly called cyclooxygenases (COXs) bind to specific receptors that activate signaling pathways driving the development and progression of tumors. Inhibitors of prostaglandin formation, COX inhibitors, or nonsteroidal anti-inflammatory drugs (NSAIDs) are well documented as agents that inhibit tumor growth and with long-term use prevent tumor development. NSAIDs also alter gene expression independent of COX inhibition and these changes in gene expression also appear to contribute to the anti-tumorigenic activity of these drugs. Many NSAIDs, as illustrated by sulindac sulfide, alter gene expressions by altering the expression or phosphorylation status of the transcription factors specificity protein 1 and early growth response-1 with the balance between these two events resulting in increases or decreases in specific target genes. In this review, we have summarized and discussed the various genes altered by this mechanism after NSAID treatment and how these changes in expression relate to the anti-tumorigenic activity. A major focus of the review is on NSAID-activated gene (NAG-1) or growth differentiation factor 15. This unique member of the TGF-β superfamily is highly induced by NSAIDs and numerous drugs and chemicals with anti-tumorigenic activities. Investigations with a transgenic mouse expressing the human NAG-1 suggest it acts to suppress tumor development in several mouse models of cancer. The biochemistry and biology of NAG-1 were discussed as potential contributor to cancer prevention by COX inhibitors. PMID:22020924

Tightly-wound miniknot vectors for gene therapy: a potential improvement over supercoiled minicircle DNA.

PubMed

Tolmachov, Oleg E

2010-04-01

Minimized derivatives of bacterial plasmids with removed bacterial backbones are promising vectors for the efficient delivery and for the long-term expression of therapeutic genes. The absence of the bacterial plasmid backbone, a known inducer of innate immune response and a known silencer of transgene expression, provides a partial explanation for the high efficiency of gene transfer using minimized DNA vectors. Supercoiled minicircle DNA is a type of minimized DNA vector obtained via intra-plasmid recombination in bacteria. Minicircle vectors seem to get an additional advantage from their physical compactness, which reduces DNA damage due to the mechanical stress during gene delivery. An independent topological means for DNA compression is knotting, with some knotted DNA isoforms offering superior compactness. I propose that, firstly, knotted DNA can be a suitable compact DNA form for the efficient transfection of a range of human cells with therapeutic genes, and, secondly, that knotted minimized DNA vectors without bacterial backbones ("miniknot" vectors) can surpass supercoiled minicircle DNA vectors in the efficiency of therapeutic gene delivery. Crucially, while the introduction of a single nick to a supercoiled DNA molecule leads to the loss of the compact supercoiled status, the introduction of nicks to knotted DNA does not change knotting. Tight miniknot vectors can be readily produced by the direct action of highly concentrated type II DNA topoisomerase on minicircle DNA or, alternatively, by annealing of the 19-base cohesive ends of the minimized vectors confined within the capsids of Escherichia coli bacteriophage P2 or its satellite bacteriophage P4. After reaching the nucleoplasm of the target cell, the knotted DNA is expected to be unknotted through type II topoisomerase activity and thus to become available for transcription, chromosomal integration or episomal maintenance. The hypothesis can be tested by comparing the gene transfer efficiency

Local gene transfection in the cochlea (Review).

PubMed

Xia, Li; Yin, Shankai

2013-07-01

There is much interest in the potential application of vector-induced gene therapeutic approaches to several forms of hearing disorders due to the poor efficacy of existing treatments. The cochlea is an ideal site for local gene transfection due to its anatomical encapsulation and fluid flow within its ducts. However, this requires the development of novel technologies in materials science and microbial supply vectors for target gene delivery. This review focuses on the introduction of various viral and non-viral vectors as well as injection approaches to transfecting cochlear cells in vivo. Finally, the perspective of local gene therapy was discussed. Therapeutic approaches using local gene transfection may provide a means of cochlear cell and tissue protection and treatment in cases of exogenous hearing loss and endogenous disorders.

Efficient disruption of Zebrafish genes using a Gal4-containing gene trap

PubMed Central

2013-01-01

Background External development and optical transparency of embryos make zebrafish exceptionally suitable for in vivo insertional mutagenesis using fluorescent proteins to visualize expression patterns of mutated genes. Recently developed Gene Breaking Transposon (GBT) vectors greatly improve the fidelity and mutagenicity of transposon-based gene trap vectors. Results We constructed and tested a bipartite GBT vector with Gal4-VP16 as the primary gene trap reporter. Our vector also contains a UAS:eGFP cassette for direct detection of gene trap events by fluorescence. To confirm gene trap events, we generated a UAS:mRFP tester line. We screened 270 potential founders and established 41 gene trap lines. Three of our gene trap alleles display homozygous lethal phenotypes ranging from embryonic to late larval: nsf tpl6, atp1a3atpl10 and flrtpl19. Our gene trap cassette is flanked by direct loxP sites, which enabled us to successfully revert nsf tpl6, atp1a3atpl10 and flrtpl19 gene trap alleles by injection of Cre mRNA. The UAS:eGFP cassette is flanked by direct FRT sites. It can be readily removed by injection of Flp mRNA for use of our gene trap alleles with other tissue-specific GFP-marked lines. The Gal4-VP16 component of our vector provides two important advantages over other GBT vectors. The first is increased sensitivity, which enabled us to detect previously unnoticed expression of nsf in the pancreas. The second advantage is that all our gene trap lines, including integrations into non-essential genes, can be used as highly specific Gal4 drivers for expression of other transgenes under the control of Gal4 UAS. Conclusions The Gal4-containing bipartite Gene Breaking Transposon vector presented here retains high specificity for integrations into genes, high mutagenicity and revertibility by Cre. These features, together with utility as highly specific Gal4 drivers, make gene trap mutants presented here especially useful to the research community. PMID:24034702

Recent gyrokinetic turbulence insights with GENE and direct comparison with experimental measurements

NASA Astrophysics Data System (ADS)

Goerler, Tobias

2017-10-01

Throughout the last years direct comparisons between gyrokinetic turbulence simulations and experimental measurements have been intensified substantially. Such studies are largely motivated by the urgent need for reliable transport predictions for future burning plasma devices and the associated necessity for validating the numerical tools. On the other hand, they can be helpful to assess the way a particular diagnostic experiences turbulence and provide ideas for further optimization and the physics that may not yet be accessible. Here, synthetic diagnostics, i.e. models that mimic the spatial and sometimes temporal response of the experimental diagnostic, play an important role. In the contribution at hand, we focus on recent gyrokinetic GENE simulations dedicated to ASDEX Upgrade L-mode plasmas and comparison with various turbulence measurements. Particular emphasis will be given to density fluctuation spectra which are experimentally accessible via Doppler reflectometry. A sophisticated synthetic diagnostic involving a fullwave code has recently been established and solves the long-lasting question on different spectral roll-overs in gyrokinetic and measured spectra as well as the potentially different power laws in the O- and X-mode signals. The demonstrated agreement furthermore extends the validation data base deep into spectral space and confirms a proper coverage of the turbulence cascade physics. The flux-matched GENE simulations are then used to study the sensitivity of the latter to the main microinstability drive and investigate the energetics at the various scales. Additionally, electron scale turbulence based modifications of the high-k power law spectra in such plasmas will be presented and their visibility in measurable signals be discussed.

Direct comparison of administration routes for AAV8-mediated ocular gene therapy.

PubMed

Igarashi, Tsutomu; Miyake, Koichi; Asakawa, Nagisa; Miyake, Noriko; Shimada, Takashi; Takahashi, Hiroshi

2013-05-01

We recently demonstrated that direct subretinal (SR) injection of adeno-associated virus (AAV) type 8 (AAV8) into photoreceptor cells and retinal pigment epithelium (RPE) is a highly efficient model of gene delivery. The current study compared transduction efficiency and expression patterns associated with various routes of vector administration. The efficacy of intravitreal (VT), SR and subconjunctival (SC) injections for delivery of AAV8-derived vectors, i.e. those expressing luciferase (Luc) and enhanced green fluorescent protein (GFP) - AAV8/Luc and AAV8/GFP, respectively - were compared in an animal (mouse) model (n = 8 mice/group). Transduction efficiency and expression patterns were examined at post-injection weeks 1 and 2, and months 1, 3, 6 and 12 via in vivo imaging. One year after AAV injection, AAV8/Luc-treated mice exhibited stable and sustained high expression of vector in the VT and SR groups, but not in the SC group (VT:SR:SC = 3,218:2,923:115; 1 × 10(5 )photons/s). Histological analysis showed that GFP expression was observed in the inner retina of VT group mice, and in photoreceptor cells and RPE of SR group mice, whereas no GFP expression was noted in the SC group. Electroretinography (ERG) revealed adverse effects following SR delivery. Results suggest that both SR and VT injections of AAV8 vectors are useful routes for administering ocular gene therapy, and stress the importance of selecting an appropriate administration route, i.e. one that targets specific cells, for treating ocular disorders.

Adaptive population divergence and directional gene flow across steep elevational gradients in a climate-sensitive mammal.

PubMed

Waterhouse, Matthew D; Erb, Liesl P; Beever, Erik A; Russello, Michael A

2018-06-01

The ecological effects of climate change have been shown in most major taxonomic groups; however, the evolutionary consequences are less well-documented. Adaptation to new climatic conditions offers a potential long-term mechanism for species to maintain viability in rapidly changing environments, but mammalian examples remain scarce. The American pika (Ochotona princeps) has been impacted by recent climate-associated extirpations and range-wide reductions in population sizes, establishing it as a sentinel mammalian species for climate change. To investigate evidence for local adaptation and reconstruct patterns of genomic diversity and gene flow across rapidly changing environments, we used a space-for-time design and restriction site-associated DNA sequencing to genotype American pikas along two steep elevational gradients at 30,966 SNPs and employed independent outlier detection methods that scanned for genotype-environment associations. We identified 338 outlier SNPs detected by two separate analyses and/or replicated in both transects, several of which were annotated to genes involved in metabolic function and oxygen transport. Additionally, we found evidence of directional gene flow primarily downslope from high-elevation populations, along with reduced gene flow at outlier loci. If this trend continues, elevational range contractions in American pikas will likely be from local extirpation rather than upward movement of low-elevation individuals; this, in turn, could limit the potential for adaptation within this landscape. These findings are of particular relevance for future conservation and management of American pikas and other elevationally restricted, thermally sensitive species. © 2018 John Wiley & Sons Ltd.

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

PubMed Central

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

2013-01-01

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

Estimating the rate of biological introductions: Lessepsian fishes in the Mediterranean.

PubMed

Belmaker, Jonathan; Brokovich, Eran; China, Victor; Golani, Daniel; Kiflawi, Moshe

2009-04-01

Sampling issues preclude the direct use of the discovery rate of exotic species as a robust estimate of their rate of introduction. Recently, a method was advanced that allows maximum-likelihood estimation of both the observational probability and the introduction rate from the discovery record. Here, we propose an alternative approach that utilizes the discovery record of native species to control for sampling effort. Implemented in a Bayesian framework using Markov chain Monte Carlo simulations, the approach provides estimates of the rate of introduction of the exotic species, and of additional parameters such as the size of the species pool from which they are drawn. We illustrate the approach using Red Sea fishes recorded in the eastern Mediterranean, after crossing the Suez Canal, and show that the two approaches may lead to different conclusions. The analytical framework is highly flexible and could provide a basis for easy modification to other systems for which first-sighting data on native and introduced species are available.

Photochemical internalization-mediated nonviral gene transfection: polyamine core-shell nanoparticles as gene carrier

NASA Astrophysics Data System (ADS)

Zamora, Genesis; Wang, Frederick; Sun, Chung-Ho; Trinidad, Anthony; Kwon, Young Jik; Cho, Soo Kyung; Berg, Kristian; Madsen, Steen J.; Hirschberg, Henry

2014-10-01

The overall objective of the research was to investigate the utility of photochemical internalization (PCI) for the enhanced nonviral transfection of genes into glioma cells. The PCI-mediated introduction of the tumor suppressor gene phosphatase and tensin homolog (PTEN) or the cytosine deaminase (CD) pro-drug activating gene into U87 or U251 glioma cell monolayers and multicell tumor spheroids were evaluated. In the study reported here, polyamine-DNA gene polyplexes were encapsulated in a nanoparticle (NP) with an acid degradable polyketal outer shell. These NP synthetically mimic the roles of viral capsid and envelope, which transport and release the gene, respectively. The effects of PCI-mediated suppressor and suicide genes transfection efficiency employing either "naked" polyplex cores alone or as NP-shelled cores were compared. PCI was performed with the photosensitizer AlPcS2a and λ=670-nm laser irradiance. The results clearly demonstrated that the PCI can enhance the delivery of both the PTEN or CD genes in human glioma cell monolayers and multicell tumor spheroids. The transfection efficiency, as measured by cell survival and inhibition of spheroid growth, was found to be significantly greater at suboptimal light and DNA levels for shelled NPs compared with polyamine-DNA polyplexes alone.

Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.

PubMed

Ceccarelli, A; Zhukovskaya, N; Kawata, T; Bozzaro, S; Williams, J

2000-12-01

The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.

[Research progress on the cloning of Mendel's gene in pea (Pisum sativum L.) and its application in genetics teaching].

PubMed

He, Feng-Hua; Zhu, Bi-Yan; Gao, Feng; Li, Shao-Shan; Li, Niang-Hui

2013-07-01

One hundred and fifty years ago, Gregor Mendel investigated the segregation of seven traits in pea (Pisum sativum) and established the law of segregation and the law of independent assortment in genetics. After the two laws of genetics were rediscovered in 1900, the seven traits have been extensively investigated in the fields of plant physiology and biochemistry as well as in the cell and molecular levels. Recently, with the development of molecular technology in genetics, four genes for seed shape (R), stem length (Le), cotyledon colour (I), and flower colour (A) have been cloned and sequenced; and another three genes for immature pod colour (Gp), fasciation (Fa) and pod form (V) have been located in the linkage groups, respectively. The identification and cloning of the four Mendel's genes will help deeply understand the basic concept of gene in many respects: like the diversity of gene function, the different origins for gene mutation in molecular level, and the molecular nature of a dominant gene or a recessive gene. In teaching of genetics, the introduction of most recent research advancements of cloning of Mendel's genes to the students and the interpretation of the Mendel's laws in molecular level will help students promote their learning interests in genetics and help students grasp the whole content from classical genetics to molecular genetics and the developmental direction of this subject.

Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming.

PubMed

Rubio, Alicia; Luoni, Mirko; Giannelli, Serena G; Radice, Isabella; Iannielli, Angelo; Cancellieri, Cinzia; Di Berardino, Claudia; Regalia, Giulia; Lazzari, Giovanna; Menegon, Andrea; Taverna, Stefano; Broccoli, Vania

2016-11-18

The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.

Complete TCRα gene locus control region activity in T cells derived in vitro from embryonic stem cells

PubMed Central

Lahiji, Armin; Kučerová-Levisohn, Martina; Lovett, Jordana; Holmes, Roxanne; Zúñiga-Pflücker, Juan Carlos; Ortiz, Benjamin D.

2013-01-01

Locus Control Regions (LCR) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin’s gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. Herein we report manifestation of all key features of mouse T cell receptor (TCR)-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells (ESC). High level, copy number-related TCRα LCR-linked reporter gene expression levels are cell type-restricted in this system, and upregulated during the expected stage transition of T cell development. We further report that de novo introduction of TCRα LCR linked transgenes into existing T cell lines yields incomplete LCR activity. Together, these data indicate that establishing full TCRα LCR activity requires critical molecular events occurring prior to final T-lineage determination. This study additionally validates a novel, tractable and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering. PMID:23720809

Directed Neural Differentiation of Mouse Embryonic Stem Cells Is a Sensitive System for the Identification of Novel Hox Gene Effectors

PubMed Central

Bami, Myrto; Episkopou, Vasso; Gavalas, Anthony; Gouti, Mina

2011-01-01

The evolutionarily conserved Hox family of homeodomain transcription factors plays fundamental roles in regulating cell specification along the anterior posterior axis during development of all bilaterian animals by controlling cell fate choices in a highly localized, extracellular signal and cell context dependent manner. Some studies have established downstream target genes in specific systems but their identification is insufficient to explain either the ability of Hox genes to direct homeotic transformations or the breadth of their patterning potential. To begin delineating Hox gene function in neural development we used a mouse ES cell based system that combines efficient neural differentiation with inducible Hoxb1 expression. Gene expression profiling suggested that Hoxb1 acted as both activator and repressor in the short term but predominantly as a repressor in the long run. Activated and repressed genes segregated in distinct processes suggesting that, in the context examined, Hoxb1 blocked differentiation while activating genes related to early developmental processes, wnt and cell surface receptor linked signal transduction and cell-to-cell communication. To further elucidate aspects of Hoxb1 function we used loss and gain of function approaches in the mouse and chick embryos. We show that Hoxb1 acts as an activator to establish the full expression domain of CRABPI and II in rhombomere 4 and as a repressor to restrict expression of Lhx5 and Lhx9. Thus the Hoxb1 patterning activity includes the regulation of the cellular response to retinoic acid and the delay of the expression of genes that commit cells to neural differentiation. The results of this study show that ES neural differentiation and inducible Hox gene expression can be used as a sensitive model system to systematically identify Hox novel target genes, delineate their interactions with signaling pathways in dictating cell fate and define the extent of functional overlap among different Hox

Comparative analysis of the Rotarix™ vaccine strain and G1P[8] rotaviruses detected before and after vaccine introduction in Belgium.

PubMed

Zeller, Mark; Heylen, Elisabeth; Tamim, Sana; McAllen, John K; Kirkness, Ewen F; Akopov, Asmik; De Coster, Sarah; Van Ranst, Marc; Matthijnssens, Jelle

2017-01-01

G1P[8] rotaviruses are responsible for the majority of human rotavirus infections worldwide. The effect of universal mass vaccination with rotavirus vaccines on circulating G1P[8] rotaviruses is still poorly understood. Therefore we analyzed the complete genomes of the Rotarix™ vaccine strain, and 70 G1P[8] rotaviruses, detected between 1999 and 2010 in Belgium (36 before and 34 after vaccine introduction) to investigate the impact of rotavirus vaccine introduction on circulating G1P[8] strains. All rotaviruses possessed a complete Wa-like genotype constellation, but frequent intra-genogroup reassortments were observed as well as multiple different cluster constellations circulating in a single season. In addition, identical cluster constellations were found to circulate persistently over multiple seasons. The Rotarix™ vaccine strain possessed a unique cluster constellation that was not present in currently circulating G1P[8] strains. At the nucleotide level, the VP6, VP2 and NSP2 gene segments of Rotarix™ were relatively distantly related to any Belgian G1P[8] strain, but other gene segments of Rotarix™ were found in clusters also containing circulating Belgian strains. At the amino acid level, the genetic distance between Rotarix™ and circulating Belgian strains was considerably lower, except for NSP1. When we compared the Belgian G1P[8] strains collected before and after vaccine introduction a reduction in the proportion of strains that were found in the same cluster as the Rotarix™ vaccine strain was observed for most gene segments. The reduction in the proportion of strains belonging to the same cluster may be the result of the vaccine introduction, although natural fluctuations cannot be ruled out.

27 CFR 53.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 2 2010-04-01 2010-04-01 false Introduction. 53.1 Section... THE TREASURY (CONTINUED) FIREARMS MANUFACTURERS EXCISE TAXES-FIREARMS AND AMMUNITION Introduction § 53.1 Introduction. The regulations in this part (part 53, subchapter C, chapter I, title 27, Code of...

Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice.

PubMed

Wert, S E; Glasser, S W; Korfhagen, T R; Whitsett, J A

1993-04-01

Transgenic animals bearing a chimeric gene containing 5'-flanking regions of the human surfactant protein C (SP-C) gene ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene were analyzed by in situ hybridization histochemistry to determine the temporal and spatial distribution of transgene expression during organogenesis of the murine lung. Ontogenic expression of the SP-C-CAT gene was compared to that of the endogenous SP-C gene and to the Clara cell CC10 gene. High levels of SP-C-CAT expression were observed as early as Day 10 of gestation in epithelial cells of the primordial lung buds. Low levels of endogenous SP-C mRNA were detected a day later, but only in the more distal epithelial cells of the newly formed, primitive, lobar bronchi. On Gestational Days 13 through 16, transcripts for both the endogenous and chimeric gene were restricted to distal epithelial elements of the branching bronchial tubules and were no longer detected in the more proximal regions of the bronchial tree. Although high levels of SP-C-CAT expression were maintained throughout organogenesis, endogenous SP-C expression increased dramatically on Gestational Day 15, coincident with acinar tubule differentiation at the lung periphery. Low levels of endogenous CC10 expression were detected by Gestational Day 16 in both lobar and segmental bronchi. By the time of birth, CC10 transcripts were expressed at high levels in the trachea and at all levels of the bronchial tree; endogenous SP-C mRNA was restricted to epithelial cells of the terminal alveolar saccules; and SP-C-CAT expression was now detected in both alveolar and bronchiolar epithelial cells. These results indicate that (1) cis-acting regulatory elements of the human SP-C gene can direct high levels of foreign gene expression to epithelial cells of the embryonic mouse lung; (2) expression of the human SP-C-CAT chimeric gene is developmentally regulated, exhibiting a morphogenic expression pattern similar, but not

Introduction

USDA-ARS?s Scientific Manuscript database

This publication represents an introduction to a special issue of the journal General and Comparative Endocrinology dedicated to Insect Endocrinology. The issue addresses a number of aspects of invertebrate neuropeptide research including identification of novel invertebrate neuropeptide sequences ...

Application of GIS in foreign direct investment decision support system

NASA Astrophysics Data System (ADS)

Zhou, Jianlan; Sun, Koumei

2007-06-01

It is important to make decisions on how to attract foreign direct investment (FDI) to China and know how the inequality of FDI introduction by locational different provinces. Following background descriptions on China's FDI economic environments and FDI-related policies, this paper demonstrates the uses of geographical information system (GIS) and multi-criterion decision-making (MCDM) framework in solving a spatial multi-objective problem of evaluating and ranking China's provinces for FDI introduction. It implements a foreign direct investment decision support system, which reveals the main determinants of FDI in China and gives some results of regional geographical analysis over spatial data.

Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL

PubMed Central

Habu, Yuichiro; Miyano-Kurosaki, Naoko; Kitano, Michiko; Endo, Yumihiko; Yukita, Masakazu; Ohira, Shigeru; Takaku, Hiroaki; Nashimoto, Masayuki; Takaku, Hiroshi

2005-01-01

The tRNA 3′-processing endoribonuclease (tRNase Z or 3′ tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3′ trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression. PMID:15647506

Definition of Cis-Acting Elements Regulating Expression of the Drosophila Melanogaster Ninae Opsin Gene by Oligonucleotide-Directed Mutagenesis

PubMed Central

Mismer, D.; Rubin, G. M.

1989-01-01

We have analyzed the cis-acting regulatory sequences of the Rh1 (ninaE) gene in Drosophila melanogaster by P-element-mediated germline transformation of indicator genes transcribed from mutant ninaE promoter sequences. We have previously shown that a 200-bp region extending from -120 to +67 relative to the transcription start site is sufficient to obtain eye-specific expression from the ninaE promoter. In the present study, 22 different 4-13-bp sequences in the -120/+67 promoter region were altered by oligonucleotide-directed mutagenesis. Several of these sequences were found to be required for proper promoter function; two of these are conserved in the promoter of the homologous gene isolated from the related species Drosophila virilis. Alteration of a conserved 9-bp sequence results in aberrant, low level expression in the body. Alteration of a separate 11-bp sequence, found in the promoter regions of several photoreceptor-specific genes of Drosophila, results in an approximately 15-fold reduction in promoter efficiency but without apparent alteration of tissue-specificity. A protein factor capable of interacting with this 11-bp sequence has been detected by DNaseI footprinting in embryonic nuclear extracts. Finally, we have further characterized two separable enhancer sequences previously shown to be required for normal levels of expression from this promoter. PMID:2521839

Population differentiation and behavioural association of the two 'personality' genes DRD4 and SERT in dunnocks (Prunella modularis).

PubMed

Holtmann, B; Grosser, S; Lagisz, M; Johnson, S L; Santos, E S A; Lara, C E; Robertson, B C; Nakagawa, S

2016-02-01

Quantifying the variation in behaviour-related genes within and between populations provides insight into how evolutionary processes shape consistent behavioural traits (i.e. personality). Deliberate introductions of non-native species offer opportunities to investigate how such genes differ between native and introduced populations and how polymorphisms in the genes are related to variation in behaviour. Here, we compared the genetic variation of the two 'personality' genes, DRD4 and SERT, between a native (United Kingdom, UK) and an introduced (New Zealand, NZ) population of dunnocks, Prunella modularis. The NZ population showed a significantly lower number of single nucleotide polymorphisms (SNPs) compared to the UK population. Standardized F'st estimates of the personality genes and neutral microsatellites indicate that selection (anthropogenic and natural) probably occurred during and post the introduction event. Notably, the largest genetic differentiation was found in the intronic regions of the genes. In the NZ population, we also examined the association between polymorphisms in DRD4 and SERT and two highly repeatable behavioural traits: flight-initiation distance and mating status (promiscuous females and cobreeding males). We found 38 significant associations (for different allele effect models) between the two behavioural traits and the studied genes. Further, 22 of the tested associations showed antagonistic allele effects for males and females. Our findings illustrate how introduction events and accompanying ecological changes could influence the genetic diversity of behaviour-related genes. © 2015 John Wiley & Sons Ltd.

Environmental history impacts gene expression during diapause development in the alfalfa leafcutting bee, Megachile rotundata

USDA-ARS?s Scientific Manuscript database

Our understanding of the mechanisms controlling insect diapause has increased dramatically with the introduction of global gene expression techniques, such as RNAseq. However, little attention has been given to how ecologically relevant field conditions may affect gene expression during diapause dev...

The Direct Loan Reconciliation Game.

ERIC Educational Resources Information Center

Department of Education, Washington, DC.

This training guide for a one-day workshop provides an introduction to the William D. Ford Federal Direct Loan Program for administrative personnel at higher education institutions. The six sections of the guide, each corresponding to a workshop session, include activity sheets, questions for participants to answer, and space for notes. Following…

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm.

PubMed

Elwell, Jennifer A; Lovato, TyAnna L; Adams, Melanie M; Baca, Erica M; Lee, Thai; Cripps, Richard M

2015-04-15

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arises through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. Copyright © 2015 Elsevier Inc. All rights reserved.

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm

PubMed Central

Elwell, Jennifer A.; Lovato, TyAnna L.; Adams, Melanie M.; Baca, Erica M.; Lee, Thai; Cripps, Richard M.

2015-01-01

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arise through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist expression in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. PMID:25704510

Gene therapy for carcinoma of the breast

PubMed Central

Stoff-Khalili, MA; Dall, P; Curiel, DT

2007-01-01

In view of the limited success of available treatment modalities for breast cancer, alternative and complementary strategies need to be developed. The delineation of the molecular basis of breast cancer provides the possibility of specific intervention by gene therapy through the introduction of genetic material for therapeutic purposes. In this regard, several gene therapy approaches for carcinoma of the breast have been developed. These approaches can be divided into six broad categories: (1) mutation compensation, (2) molecular chemotherapy, (3) proapoptotic gene therapy, (4) antiangiogenic gene therapy, (5) genetic immunopotentiation, and (6) genetic modulation of resistance/sensitivity. Clinical trials for breast cancer have been initiated to evaluate safety, toxicity, and efficacy. Combined modality therapy with gene therapy and chemotherapy or radiation therapy has shown promising results. It is expected that as new therapeutic targets and approaches are identified and advances in vector design are realized, gene therapy will play an increasing role in clinical breast cancer treatment. PMID:16410823

Long-term reversal of diabetes in non-obese diabetic mice by liver-directed gene therapy.

PubMed

Ren, Binhai; O'Brien, Bronwyn A; Byrne, Michelle R; Ch'ng, Edwin; Gatt, Prudence N; Swan, M Anne; Nassif, Najah T; Wei, Ming Q; Gijsbers, Rik; Debyser, Zeger; Simpson, Ann M

2013-01-01

Type 1 diabetes (T1D) results from an autoimmune attack against the insulin-producing β-cells of the pancreas. The present study aimed to reverse T1D by gene therapy. We used a novel surgical technique, which involves isolating the liver from the circulation before the delivery of a lentiviral vector carrying furin-cleavable human insulin (INS-FUR) or empty vector to the livers of diabetic non-obese diabetic mice (NOD). This was compared with the direct injection of the vector into the portal circulation. Mice were monitored for body weight and blood glucose. Intravenous glucose tolerance tests were performed. Expression of insulin and pancreatic transcription factors was determined by the reverse transcriptase-polymerase chain reaction and immunohistochemistry and immunoelectron microscopy was used to localise insulin. Using the novel surgical technique, we achieved long-term transduction (42% efficiency) of hepatocytes, restored normoglycaemia for 150 days (experimental endpoint) and re-established normal glucose tolerance. We showed the expression of β-cell transcription factors, murine insulin, glucagon and somatostatin, and hepatic storage of insulin in granules. The expression of hepatic markers, C/EBP-β, G6PC, AAT and GLUI was down-regulated in INS-FUR-treated livers. Liver function tests remained normal, with no evidence of intrahepatic inflammation or autoimmune destruction of the insulin-secreting liver tissue. By comparison, direct injection of INS-FUR reduced blood glucose levels, and no pancreatic transdifferentiation or normal glucose tolerance was observed. This gene therapy protocol has, for the first time, permanently reversed T1D with normal glucose tolerance in NOD mice and, as such, represents a novel therapeutic strategy for the treatment of T1D. Copyright © 2013 John Wiley & Sons, Ltd.

Soils: An Introduction, Fifth Edition

NASA Astrophysics Data System (ADS)

Baisden, W. Troy

Understanding the links among global biogeochemical cycles, ecology, hydrology and climate demands a knowledge base that has traditionally been considered soil science. However, for soil science to play a role in this understanding, geologists, hydrologists, ecologists, climatologists, and many others must have a fundamental understanding of soil science. Do introductory soil science texts speak to this audience?To address this question, I reviewed the fifth edition of a textbook that set out in its original edition to accomplish just this goal—to be the introductory soil science text for students outside the discipline of soil science. As such, Singer and Munns' Soils:An Introduction must be compared to The Nature and Properties of Soils by N.C. Brady and R.R. Weil, a standard text directly descended from a first edition published in 1922.

Introduction to Geostatistics

NASA Astrophysics Data System (ADS)

Kitanidis, P. K.

1997-05-01

Introduction to Geostatistics presents practical techniques for engineers and earth scientists who routinely encounter interpolation and estimation problems when analyzing data from field observations. Requiring no background in statistics, and with a unique approach that synthesizes classic and geostatistical methods, this book offers linear estimation methods for practitioners and advanced students. Well illustrated with exercises and worked examples, Introduction to Geostatistics is designed for graduate-level courses in earth sciences and environmental engineering.

Investigations on the direct introduction of cigarette smoke for trace elements analysis by inductively coupled plasma mass spectrometry

NASA Astrophysics Data System (ADS)

Chang, Michael J.; Naworal, John D.; Walker, Kathleen; Connell, Chris T.

2003-11-01

Direct introduction of mainstream cigarette smoke into an inductively coupled plasma mass spectrometry (ICP-MS) has been investigated with respect to its feasibility for on-line analysis of trace elements. An automated apparatus was designed and built interfacing a smoking machine with an ICP-MS for smoke generation, collection, injection and analysis. Major and minor elements present in the particulate phase and the gas phase of mainstream cigarette smoke of 2R4F reference cigarettes have been qualitatively identified by examination of their full mass spectra. This method provides a rapid-screening analysis of the transfer of trace elements into mainstream smoke during cigarette combustion. A full suite of elements present in the whole cigarette smoke has been identified, including As, B, Ba, Br, Cd, Cl, Cs, Cu, Hg, I, K, Li, Mn, Na, Pb, Rb, Sb, Sn, Tl and Zn. Of these elements, the major portions of B, Ba, Cs, Cu, K, Li, Mn, Na, Pb, Rb, Sn, Tl and Zn are present in the particulate phase, whereas the major portion of Hg is present in the gas phase. As, Br, Cd, Cl, I and Sb exist in a distribution between the gas phase and the particulate phase. Depending on the element, the precision of measurement ranges from 5 to 25% in terms of relative standard deviation of peak height and peak area, based on the fourth puff of 2R4F mainstream cigarette smoke analyzed in five smoking replicates.

Adaptive population divergence and directional gene flow across steep elevational gradients in a climate‐sensitive mammal

USGS Publications Warehouse

Waterhouse, Matthew D.; Erb, Liesl P.; Beever, Erik; Russello, Michael A.

2018-01-01

The American pika is a thermally sensitive, alpine lagomorph species. Recent climate-associated population extirpations and genetic signatures of reduced population sizes range-wide indicate the viability of this species is sensitive to climate change. To test for potential adaptive responses to climate stress, we sampled pikas along two elevational gradients (each ~470 to 1640 m) and employed three outlier detection methods, BAYESCAN, LFMM, and BAYPASS, to scan for genotype-environment associations in samples genotyped at 30,763 SNP loci. We resolved 173 loci with robust evidence of natural selection detected by either two independent analyses or replicated in both transects. A BLASTN search of these outlier loci revealed several genes associated with metabolic function and oxygen transport, indicating natural selection from thermal stress and hypoxia. We also found evidence of directional gene flow primarily downslope from large high-elevation populations and reduced gene flow at outlier loci, a pattern suggesting potential impediments to the upward elevational movement of adaptive alleles in response to contemporary climate change. Finally, we documented evidence of reduced genetic diversity associated the south-facing transect and an increase in corticosterone stress levels associated with inbreeding. This study suggests the American pika is already undergoing climate-associated natural selection at multiple genomic regions. Further analysis is needed to determine if the rate of climate adaptation in the American pika and other thermally sensitive species will be able to keep pace with rapidly changing climate conditions.

The Introduction of Middle Schools in the Northern Territory: Processes and Reality

ERIC Educational Resources Information Center

Palmer, Bill

2006-01-01

The year 2006 has been a year in which a decision on the introduction of middle schools has been made by the Labor government of the Northern Territory. The initial impetus for the change came from the 2003 Secondary Education Review "Future Directions for Secondary Education in the Northern Territory," chaired by Gregor Ramsay. There…

Introduction of Peptidase Genes from Lactobacillus delbrueckii subsp. lactis into Lactococcus lactis and Controlled Expression

PubMed Central

Wegmann, U.; Klein, J. R.; Drumm, I.; Kuipers, O. P.; Henrich, B.

1999-01-01

Peptidases PepI, PepL, PepW, and PepG from Lactobacillus delbrueckii subsp. lactis, which have no counterparts in Lactococcus lactis, and peptidase PepQ were examined to determine their potential to confer new peptidolytic properties to lactococci. Controllable expression of the corresponding genes (pep genes) was achieved by constructing translational fusions with the promoter of the nisA gene (PnisA). A suitable host strain, UKLc10, was constructed by chromosomal integration of the genes encoding the NisRK two-component system into the fivefold peptidase-deficient mutant IM16 of L. lactis. Recombinants of this strain were used to analyze growth, peptidase activities, peptide utilization, and intracellular protein cleavage products. After nisin induction of PnisA::pep fusions, all of the peptidases were visible as distinct bands in protein gels. Despite the fact that identical transcription and translation signals were used to express the pep genes, the relative amounts of individual peptidases varied considerably. All of the peptidases exhibited activities in extracts of recombinant UKLc10 clones, but only PepL and PepG allowed the clones to utilize specific peptide substrates as sources of essential amino acids. In milk medium, induction of pepG and induction of pepW resulted in growth acceleration. The activities of all five peptidases during growth in milk medium were revealed by high-performance liquid chromatography analyses of intracellular amino acid and peptide pools. PMID:10543778

Characterization and Expression Analysis of Genes Directing Galactomannan Synthesis in Coffee

PubMed Central

Pré, Martial; Caillet, Victoria; Sobilo, Julien; McCarthy, James

2008-01-01

Background and Aims Galactomannans act as storage reserves for the seeds in some plants, such as guar (Cyamopsis tetragonoloba) and coffee (Coffea arabica and Coffea canephora). In coffee, the galactomannans can represent up to 25 % of the mass of the mature green coffee grain, and they exert a significant influence on the production of different types of coffee products. The objective of the current work was to isolate and characterize cDNA encoding proteins responsible for galactomannan synthesis in coffee and to study the expression of the corresponding transcripts in the developing coffee grain from C. arabica and C. canephora, which potentially exhibit slight galactomannan variations. Comparative gene expression analysis was also carried out for several other tissues of C. arabica and C. canephora. Methods cDNA banks, RACE-PCR and genome walking were used to generate full-length cDNA for two putative coffee mannan synthases (ManS) and two galactomannan galactosyl transferases (GMGT). Gene-specific probe-primer sets were then generated and used to carry out comparative expression analysis of the corresponding genes in different coffee tissues using quantitative RT-PCR Key Results Two of the putative galactomannan biosynthetic genes, ManS1 and GMGT1, were demonstrated to have very high expression in the developing coffee grain of both Coffea species during endosperm development, consistent with our proposal that these two genes are responsible for the production of the majority of the galactomannans found in the grain. In contrast, the expression data presented indicates that the ManS2 gene product is probably involved in the synthesis of the galactomannans found in green tissue. Conclusions The identification of genes implicated in galactomannan synthesis in coffee are presented. The data obtained will enable more detailed studies on the biosynthesis of this important component of coffee grain and contribute to a better understanding of some functional

Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice.

PubMed

Sato, Masahiro; Inada, Emi; Saitoh, Issei; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi

2013-11-01

The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ. © 2013 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptions are made.

Advances in gene therapy for heart failure.

PubMed

Fish, Kenneth M; Ishikawa, Kiyotake

2015-04-01

Chronic heart failure is expected to increase its social and economic burden as a consequence of improved survival in patients with acute cardiac events. Cardiac gene therapy holds significant promise in heart failure treatment for patients with currently very limited or no treatment options. The introduction of adeno-associated virus (AAV) gene vector changed the paradigm of cardiac gene therapy, and now it is the primary vector of choice for chronic heart failure gene therapy in clinical and preclinical studies. Recently, there has been significant progress towards clinical translation in this field spearheaded by AAV-1 mediated sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) gene therapy targeting chronic advanced heart failure patients. Meanwhile, several independent laboratories are reporting successful gene therapy approaches in clinically relevant large animal models of heart failure and some of these approaches are expected to enter clinical trials in the near future. This review will focus on gene therapy approaches targeting heart failure that is in clinical trials and those close to its initial clinical trial application.

Introduction

USDA-ARS?s Scientific Manuscript database

The introduction to the second edition of the Compendium of Apple and Pear Diseases contains a general description of genus and species of commercial importance, some general information about growth and fruiting habits as well as recent production statistics. A general description of major scion c...

Project T.E.A.M. (Technical Education Advancement Modules). Introduction to Industrial Physics.

ERIC Educational Resources Information Center

Whisenhunt, James E.

This instructional guide, one of a series developed by the Technical Education Advancement Modules (TEAM) project, is a 20-hour introduction to industrial physics that explains and demonstrates to industrial maintenance mechanics the direct relationship of physics to machinery. Project TEAM is intended to upgrade basic technical competencies of…

A novel needleless liquid jet injection methodology for improving direct cardiac gene delivery: An optimization of parameters, AAV mediated therapy and investigation of host responses in ischemic heart failure

NASA Astrophysics Data System (ADS)

Fargnoli, Anthony Samuel

Heart disease remains the leading cause of mortality and morbidity worldwide, with 22 million new patients diagnosed annually. Essentially, all present therapies have significant cost burden to the healthcare system, yet fail to increase survival rates. One key employed strategy is the genetic reprogramming of cells to increase contractility via gene therapy, which has advanced to Phase IIb Clinical Trials for advanced heart failure patients. It has been argued that the most significant barrier preventing FDA approval are resolving problems with safe, efficient myocardial delivery, whereby direct injection in the infarct and remote tissue areas is not clinically feasible. Here, we aim to: (1) Improve direct cardiac gene delivery through the development of a novel liquid jet device approach (2) Compare the new method against traditional IM injection with two different vector constructions and evaluate outcome (3) Evaluate the host response resulting from both modes of direct cardiac injection, then advance a drug/gene combination with controlled release nanoparticle formulations.

NTL8 Regulates Trichome Formation in Arabidopsis by Directly Activating R3 MYB Genes TRY and TCL11[OPEN

PubMed Central

Tian, Hainan; Wang, Xianling; Guo, Hongyan; Cheng, Yuxin; Hou, Chunjiang

2017-01-01

The NAM, ATAF1/2, and CUC (NAC) are plant-specific transcription factors that regulate multiple aspects of plant growth and development and plant response to environmental stimuli. We report here the identification of NTM1-LIKE8 (NTL8), a membrane-associated NAC transcription factor, as a novel regulator of trichome formation in Arabidopsis (Arabidopsis thaliana). From an activation-tagged Arabidopsis population, we identified a dominant, gain-of-function mutant with glabrous inflorescence stem. By using plasmid rescue and RT-PCR analyses, we found that NTL8 was tagged; thus, the mutant was named ntl8-1 Dominant (ntl8-1D). Recapitulation experiment further confirmed that the phenotype observed in the ntl8-1D mutant was caused by elevated expression of NTL8. Quantitative RT-PCR results showed that the expression level of the single-repeat R3 MYB genes TRIPTYCHON (TRY) and TRICHOMELESS1 (TCL1) was elevated in the ntl8-1D mutant. Genetic analyses demonstrated that NTL8 acts upstream of TRY and TCL1 in the regulation of trichome formation. When recruited to the promoter region of the reporter gene Gal4:GUS by a fused GAL4 DNA-binding domain, NTL8 activated the expression of the reporter gene. Chromatin immunoprecipitation results indicated that TRY and TCL1 are direct targets of NTL8. However, NTL8 did not interact with SQUAMOSA PROMOTER BINDING PROTEIN LIKE9, another transcription factor that regulates the expression of TRY and TCL1, in yeast and plant cells. Taken together, our results suggest that NTL8 negatively regulates trichome formation in Arabidopsis by directly activating the expression of TRY and TCL1. PMID:28649093

Chemical modification of chitosan for efficient gene therapy.

PubMed

Jiang, Hu-Lin; Cui, Peng-Fei; Xie, Rong-Lin; Cho, Chong-Su

2014-01-01

Gene therapy involves the introduction of foreign genetic material into cells in order to exert a therapeutic effect. Successful gene therapy relies on effective vector system. Viral vectors are highly efficient in transfecting cells, but the undesirable complications limit their therapeutic applications. As a natural biopolymer, chitosan has been considered to be a good gene carrier candidate due to its ideal character which combines biocompatibility, low toxicity with high cationic density together. However, the low cell specificity and low transfection efficiency of chitosan as a gene carrier need to be overcome before undertaking clinical trials. This chapter is principally on those endeavors such as chemical modifications using cell-specific ligands and stimuli-response groups as well as penetrating modifications that have been done to increase the performances of chitosan in gene therapy. © 2014 Elsevier Inc. All rights reserved.

Staph ID/R: a Rapid Method for Determining Staphylococcus Species Identity and Detecting the mecA Gene Directly from Positive Blood Culture

PubMed Central

Pasko, Chris; Dunn, John; Jaeckel, Heidi; Nieuwlandt, Dan; Weed, Diane; Woodruff, Evelyn; Zheng, Xiaotian

2012-01-01

Rapid diagnosis of staphylococcal bacteremia directs appropriate antimicrobial therapy, leading to improved patient outcome. We describe herein a rapid test (<75 min) that can identify the major pathogenic strains of Staphylococcus to the species level as well as the presence or absence of the methicillin resistance determinant gene, mecA. The test, Staph ID/R, combines a rapid isothermal nucleic acid amplification method, helicase-dependent amplification (HDA), with a chip-based array that produces unambiguous visible results. The analytic sensitivity was 1 CFU per reaction for the mecA gene and was 1 to 250 CFU per reaction depending on the staphylococcal species present in the positive blood culture. Staph ID/R has excellent specificity as well, with no cross-reactivity observed. We validated the performance of Staph ID/R by testing 104 frozen clinical positive blood cultures and comparing the results with rpoB gene or 16S rRNA gene sequencing for species identity determinations and mecA gene PCR to confirm mecA gene results. Staph ID/R agreed with mecA gene PCR for all samples and agreed with rpoB/16S rRNA gene sequencing in all cases except for one sample that contained a mixture of two staphylococcal species, one of which Staph ID/R correctly identified, for an overall agreement of 99.0% (P < 0.01). Staph ID/R could potentially be used to positively affect patient management for Staphylococcus-mediated bacteremia. PMID:22170912

Urban landscape genomics identifies fine-scale gene flow patterns in an avian invasive.

PubMed

Low, G W; Chattopadhyay, B; Garg, K M; Irestedt, M; Ericson, Pgp; Yap, G; Tang, Q; Wu, S; Rheindt, F E

2018-01-01

Invasive species exert a serious impact on native fauna and flora and have been the target of many eradication and management efforts worldwide. However, a lack of data on population structure and history, exacerbated by the recency of many species introductions, limits the efficiency with which such species can be kept at bay. In this study we generated a novel genome of high assembly quality and genotyped 4735 genome-wide single nucleotide polymorphic (SNP) markers from 78 individuals of an invasive population of the Javan Myna Acridotheres javanicus across the island of Singapore. We inferred limited population subdivision at a micro-geographic level, a genetic patch size (~13-14 km) indicative of a pronounced dispersal ability, and barely an increase in effective population size since introduction despite an increase of four to five orders of magnitude in actual population size, suggesting that low population-genetic diversity following a bottleneck has not impeded establishment success. Landscape genomic analyses identified urban features, such as low-rise neighborhoods, that constitute pronounced barriers to gene flow. Based on our data, we consider an approach targeting the complete eradication of Javan Mynas across Singapore to be unfeasible. Instead, a mixed approach of localized mitigation measures taking into account urban geographic features and planning policy may be the most promising avenue to reducing the adverse impacts of this urban pest. Our study demonstrates how genomic methods can directly inform the management and control of invasive species, even in geographically limited datasets with high gene flow rates.

Oxytocin Receptor Gene Polymorphisms Are Associated with Human Directed Social Behavior in Dogs (Canis familiaris)

PubMed Central

Lakatos, Gabriella; Pergel, Enikő; Turcsán, Borbála; Pluijmakers, Jolanda; Vas, Judit; Elek, Zsuzsanna; Brúder, Ildikó; Földi, Levente; Sasvári-Székely, Mária; Miklósi, Ádám; Rónai, Zsolt; Kubinyi, Enikő

2014-01-01

The oxytocin system has a crucial role in human sociality; several results prove that polymorphisms of the oxytocin receptor gene are related to complex social behaviors in humans. Dogs' parallel evolution with humans and their adaptation to the human environment has made them a useful species to model human social interactions. Previous research indicates that dogs are eligible models for behavioral genetic research, as well. Based on these previous findings, our research investigated associations between human directed social behaviors and two newly described (−212AG, 19131AG) and one known (rs8679684) single nucleotide polymorphisms (SNPs) in the regulatory regions (5′ and 3′ UTR) of the oxytocin receptor gene in German Shepherd (N = 104) and Border Collie (N = 103) dogs. Dogs' behavior traits have been estimated in a newly developed test series consisting of five episodes: Greeting by a stranger, Separation from the owner, Problem solving, Threatening approach, Hiding of the owner. Buccal samples were collected and DNA was isolated using standard protocols. SNPs in the 3′ and 5′ UTR regions were analyzed by polymerase chain reaction based techniques followed by subsequent electrophoresis analysis. The gene–behavior association analysis suggests that oxytocin receptor gene polymorphisms have an impact in both breeds on (i) proximity seeking towards an unfamiliar person, as well as their owner, and on (ii) how friendly dogs behave towards strangers, although the mediating molecular regulatory mechanisms are yet unknown. Based on these results, we conclude that similarly to humans, the social behavior of dogs towards humans is influenced by the oxytocin system. PMID:24454713

Direct-to-consumer advertising of prescription medicines: a counter argument.

PubMed

Mintzes, Barbara; Mangin, Dee

2009-12-01

Direct-to-consumer advertising of prescription-only medicines is currently only permitted in the USA and New Zealand. Barbara Mintzes and Dee Mangin provide their case against direct-to-consumer advertising of prescription medicines, arguing that its wider introduction would lead to a 'Pharma-knows-best' culture.

Introduction.

ERIC Educational Resources Information Center

Fortner, Rosanne

1994-01-01

Provides an introduction to Project WET (Water Education for Teachers). Examines approaches to water education, how the Project WET program is different from other water education projects, how the program is implemented in the classroom, what kinds of materials are available, and how to obtain Project WET. (LZ)

Histidine-rich stabilized polyplexes for cMet-directed tumor-targeted gene transfer

NASA Astrophysics Data System (ADS)

Kos, Petra; Lächelt, Ulrich; Herrmann, Annika; Mickler, Frauke Martina; Döblinger, Markus; He, Dongsheng; Krhač Levačić, Ana; Morys, Stephan; Bräuchle, Christoph; Wagner, Ernst

2015-03-01

Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing

Analysis of the volatile organic matter of engine piston deposits by direct sample introduction thermal desorption gas chromatography/mass spectrometry.

PubMed

Diaby, M; Kinani, S; Genty, C; Bouchonnet, S; Sablier, M; Le Negrate, A; El Fassi, M

2009-12-01

This article establishes an alternative method for the characterization of volatiles organic matter (VOM) contained in deposits of the piston first ring grooves of diesel engines using a ChromatoProbe direct sample introduction (DSI) device coupled to gas chromatography/mass spectrometry (GC/MS) analysis. The addition of an organic solvent during thermal desorption leads to an efficient extraction and a good chromatographic separation of extracted products. The method was optimized investigating the effects of several solvents, the volume added to the solid sample, and temperature programming of the ChromatoProbe DSI device. The best results for thermal desorption were found using toluene as an extraction solvent and heating the programmable temperature injector from room temperature to 300 degrees C with a temperature step of 105 degrees C. With the use of the optimized thermal desorption conditions, several components have been positively identified in the volatile fraction of the deposits: aromatics, antioxidants, and antioxidant degradation products. Moreover, this work highlighted the presence of diesel fuel in the VOM of the piston deposits and gave new facts on the absence of the role of diesel fuel in the deposit formation process. Most importantly, it opens the possibility of quickly performing the analysis of deposits with small amounts of samples while having a good separation of the volatiles.

From Genetics to Genomics: A Short Introduction for Pediatric Neurologists.

PubMed

Neubauer, Bernd A; Lemke, Johannes R

2016-01-01

It is estimated that in humans approximately 50% of all 22500 genes are needed for the development and maintenance of the nervous system. The introduction of high-throughput technology in genetic analysis has therefore major implications, not only for the investigation of specific disease entities but also for the diagnostic workup of single individuals with neurologic disorders of genetic origin. A short primer for clinicians is presented, addressing aspects of current developments in medical genomics. Significant findings of the last years are exemplified in an educational manner to provide a basic understanding of disease mechanisms that were unraveled by recent genomic analysis. Georg Thieme Verlag KG Stuttgart · New York.

Isotope dilution ICP-MS with laser-assisted sample introduction for direct determination of sulfur in petroleum products.

PubMed

Boulyga, Sergei F; Heilmann, Jens; Heumann, Klaus G

2005-08-01

Inductively coupled plasma isotope dilution mass spectrometry (ICP-IDMS) with direct laser-assisted introduction of isotope-diluted samples into the plasma, using a laser ablation system with high ablation rates, was developed for accurate sulfur determinations in different petroleum products such as 'sulfur-free' premium gasoline, diesel fuel, and heating oil. Two certified gas oil reference materials were analyzed for method validation. Two different 34S-enriched spike compounds, namely, elementary sulfur dissolved in xylene and dibenzothiophene in hexane, were synthesized and tested for their usefulness in this isotope dilution technique. The isotope-diluted sample was adsorbed on a filter-paper-like material, which was fixed in a special holder for irradiation by the laser beam. Under these conditions no time-dependent spike/analyte fractionation was only observed for the dibenzothiophene spike during the laser ablation process, which means that the measured 34S/32S isotope ratio of the isotope-diluted sample remained constant-a necessary precondition for accurate results with the isotope dilution technique. A comparison of LA-ICP-IDMS results with the certified values of the gas oil reference materials and with results obtained from ICP-IDMS analyses with wet sample digestion demonstrated the accuracy of the new LA-ICP-IDMS method in the concentration range of 9.2 microg g(-1) ('sulfur-free' premium gasoline) to 10.4 mg g(-1) (gas oil reference material BCR 107). The detection limit for sulfur by LA-ICP-IDMS is 0.04 microg g(-1) and the analysis time is only about 10 min, which therefore also qualifies this method for accurate determinations of low sulfur contents in petroleum products on a routine level.

Invasion trajectory of alien trees: the role of introduction pathway and planting history.

PubMed

Donaldson, Jason E; Hui, Cang; Richardson, David M; Robertson, Mark P; Webber, Bruce L; Wilson, John R U

2014-05-01

Global change is driving a massive rearrangement of the world's biota. Trajectories of distributional shifts are shaped by species traits, the recipient environment and driving forces with many of the driving forces directly due to human activities. The relative importance of each in determining the distributions of introduced species is poorly understood. We consider 11 Australian Acacia species introduced to South Africa for different reasons (commercial forestry, dune stabilization and ornamentation) to determine how features of the introduction pathway have shaped their invasion history. Projections from species distribution models (SDMs) were developed to assess how the reason for introduction influences the similarity between climatic envelopes in native and alien ranges. A lattice model for an idealized invasion was developed to assess the relative contribution of intrinsic traits and introduction dynamics on the abundance and extent over the course of simulated invasions. SDMs show that alien populations of ornamental species in South Africa occupy substantially different climate space from their native ranges, whereas species introduced for forestry occupy a similar climate space in native and introduced ranges. This may partly explain the slow spread rates observed for some alien ornamental plants. Such mismatches are likely to become less pronounced with the current drive towards 'eco gardens' resulting in more introductions of ornamental species with a close climate match between native and newly introduced regions. The results from the lattice model showed that the conditions associated with the introduction pathway (especially introduction pressure) dominate early invasion dynamics. The placement of introduction foci in urban areas limited the extent and abundance of invasive populations. Features of introduction events appear to initially mask the influence of intrinsic species traits on invasions and help to explain the relative success of species

A Look to Future Directions in Gene Therapy Research for Monogenic Diseases

PubMed Central

Porteus, Matthew H; Connelly, Jon P; Pruett, Shondra M

2006-01-01

The concept of gene therapy has long appealed to biomedical researchers and clinicians because it promised to treat certain diseases at their origins. In the last several years, there have been several trials in which patients have benefited from gene therapy protocols. This progress, however, has revealed important problems, including the problem of insertional oncogenesis. In this review, which focuses on monogenic diseases, we discuss the problem of insertional oncogenesis and identify areas for future research, such as developing more quantitative assays for risk and efficacy, and ways of minimizing the genotoxic effects of gene therapy protocols, which will be important if gene therapy is to fulfill its conceptual promise. PMID:17009872

EIN3 and ORE1 Accelerate Degreening during Ethylene-Mediated Leaf Senescence by Directly Activating Chlorophyll Catabolic Genes in Arabidopsis

PubMed Central

Qiu, Kai; Li, Zhongpeng; Yang, Zhen; Chen, Junyi; Wu, Shouxin; Zhu, Xiaoyu; Gao, Shan; Gao, Jiong; Ren, Guodong; Kuai, Benke; Zhou, Xin

2015-01-01

Degreening, caused by chlorophyll degradation, is the most obvious symptom of senescing leaves. Chlorophyll degradation can be triggered by endogenous and environmental cues, and ethylene is one of the major inducers. ETHYLENE INSENSITIVE3 (EIN3) is a key transcription factor in the ethylene signaling pathway. It was previously reported that EIN3, miR164, and a NAC (NAM, ATAF, and CUC) transcription factor ORE1/NAC2 constitute a regulatory network mediating leaf senescence. However, how this network regulates chlorophyll degradation at molecular level is not yet elucidated. Here we report a feed-forward regulation of chlorophyll degradation that involves EIN3, ORE1, and chlorophyll catabolic genes (CCGs). Gene expression analysis showed that the induction of three major CCGs, NYE1, NYC1 and PAO, by ethylene was largely repressed in ein3 eil1 double mutant. Dual-luciferase assay revealed that EIN3 significantly enhanced the promoter activity of NYE1, NYC1 and PAO in Arabidopsis protoplasts. Furthermore, Electrophoretic mobility shift assay (EMSA) indicated that EIN3 could directly bind to NYE1, NYC1 and PAO promoters. These results reveal that EIN3 functions as a positive regulator of CCG expression during ethylene-mediated chlorophyll degradation. Interestingly, ORE1, a senescence regulator which is a downstream target of EIN3, could also activate the expression of NYE1, NYC1 and PAO by directly binding to their promoters in EMSA and chromatin immunoprecipitation (ChIP) assays. In addition, EIN3 and ORE1 promoted NYE1 and NYC1 transcriptions in an additive manner. These results suggest that ORE1 is also involved in the direct regulation of CCG transcription. Moreover, ORE1 activated the expression of ACS2, a major ethylene biosynthesis gene, and subsequently promoted ethylene production. Collectively, our work reveals that EIN3, ORE1 and CCGs constitute a coherent feed-forward loop involving in the robust regulation of ethylene-mediated chlorophyll degradation

Degeneration and domestication of a selfish gene in yeast: molecular evolution versus site-directed mutagenesis.

PubMed

Koufopanou, Vassiliki; Burt, Austin

2005-07-01

VDE is a homing endonuclease gene in yeasts with an unusual evolutionary history including horizontal transmission, degeneration, and domestication into the mating-type switching locus HO. We investigate here the effects of these features on its molecular evolution. In addition, we correlate rates of evolution with results from site-directed mutagenesis studies. Functional elements have lower rates of evolution than degenerate ones and higher conservation at functionally important sites. However, functionally important and unimportant sites are equally likely to have been involved in the evolution of new function during the domestication of VDE into HO. The domestication event also indicates that VDE has been lost in some species and that VDE has been present in yeasts for more than 50 Myr.

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo

PubMed Central

Coles, Andrew H.; Osborn, Maire F.; Alterman, Julia F.; Turanov, Anton A.; Godinho, Bruno M.D.C.; Kennington, Lori; Chase, Kathryn; Aronin, Neil

2016-01-01

Preclinical development of RNA interference (RNAi)-based therapeutics requires a rapid, accurate, and robust method of simultaneously quantifying mRNA knockdown in hundreds of samples. The most well-established method to achieve this is quantitative real-time polymerase chain reaction (qRT-PCR), a labor-intensive methodology that requires sample purification, which increases the potential to introduce additional bias. Here, we describe that the QuantiGene® branched DNA (bDNA) assay linked to a 96-well Qiagen TissueLyser II is a quick and reproducible alternative to qRT-PCR for quantitative analysis of mRNA expression in vivo directly from tissue biopsies. The bDNA assay is a high-throughput, plate-based, luminescence technique, capable of directly measuring mRNA levels from tissue lysates derived from various biological samples. We have performed a systematic evaluation of this technique for in vivo detection of RNAi-based silencing. We show that similar quality data is obtained from purified RNA and tissue lysates. In general, we observe low intra- and inter-animal variability (around 10% for control samples), and high intermediate precision. This allows minimization of sample size for evaluation of oligonucleotide efficacy in vivo. PMID:26595721

Insights into the Introduction History and Population Genetic Dynamics of the Nile Monitor (Varanus niloticus) in Florida

PubMed Central

Dowell, Stephanie A.; Campbell, Todd S.

2016-01-01

Invasive species are widely recognized as important drivers of the ongoing biodiversity crisis. The US state of Florida is especially susceptible to the proliferation of invasive reptiles, and nonnative lizards currently outnumber native lizard species. At present, there are 3 documented breeding populations of the Nile monitor (Varanus niloticus) in different regions of Southern Florida, and these populations are considered potential dangers to threatened, fossorial endemics, such as burrowing owls, American crocodiles, and gopher tortoises. Nevertheless, at present, both the introduction histories of these populations and the degree to which they are connected by gene flow are not known. To address these issues, we genotyped V. niloticus from Cape Coral, Homestead Air Reserve Base, and West Palm Beach at 17 microsatellite loci and conducted a variety of analyses to assess both intrapopulation genetic diversity, the degree of gene flow between populations, and the most likely introduction scenario. The results of our analyses demonstrate that all 3 populations have limited genetic diversity (mean number of effective alleles across loci in all 3 populations ~ 2.00) and are highly differentiated from one another (G ST = 0.268; G″ST = 0.628). Our results also suggest that these populations resulted from independent introduction events that occurred within the past few decades. Consequently, we advise that wildlife managers focus management efforts on containment of existing populations and intensification of monitoring efforts on potential migration corridors. PMID:26971010

Search Engine for Antimicrobial Resistance: A Cloud Compatible Pipeline and Web Interface for Rapidly Detecting Antimicrobial Resistance Genes Directly from Sequence Data.

PubMed

Rowe, Will; Baker, Kate S; Verner-Jeffreys, David; Baker-Austin, Craig; Ryan, Jim J; Maskell, Duncan; Pearce, Gareth

2015-01-01

Antimicrobial resistance remains a growing and significant concern in human and veterinary medicine. Current laboratory methods for the detection and surveillance of antimicrobial resistant bacteria are limited in their effectiveness and scope. With the rapidly developing field of whole genome sequencing beginning to be utilised in clinical practice, the ability to interrogate sequencing data quickly and easily for the presence of antimicrobial resistance genes will become increasingly important and useful for informing clinical decisions. Additionally, use of such tools will provide insight into the dynamics of antimicrobial resistance genes in metagenomic samples such as those used in environmental monitoring. Here we present the Search Engine for Antimicrobial Resistance (SEAR), a pipeline and web interface for detection of horizontally acquired antimicrobial resistance genes in raw sequencing data. The pipeline provides gene information, abundance estimation and the reconstructed sequence of antimicrobial resistance genes; it also provides web links to additional information on each gene. The pipeline utilises clustering and read mapping to annotate full-length genes relative to a user-defined database. It also uses local alignment of annotated genes to a range of online databases to provide additional information. We demonstrate SEAR's application in the detection and abundance estimation of antimicrobial resistance genes in two novel environmental metagenomes, 32 human faecal microbiome datasets and 126 clinical isolates of Shigella sonnei. We have developed a pipeline that contributes to the improved capacity for antimicrobial resistance detection afforded by next generation sequencing technologies, allowing for rapid detection of antimicrobial resistance genes directly from sequencing data. SEAR uses raw sequencing data via an intuitive interface so can be run rapidly without requiring advanced bioinformatic skills or resources. Finally, we show that SEAR

Targeting of HPV-16+ Epithelial Cancer Cells by TCR Gene Engineered T Cells Directed against E6.

PubMed

Draper, Lindsey M; Kwong, Mei Li M; Gros, Alena; Stevanović, Sanja; Tran, Eric; Kerkar, Sid; Raffeld, Mark; Rosenberg, Steven A; Hinrichs, Christian S

2015-10-01

The E6 and E7 oncoproteins of HPV-associated epithelial cancers are in principle ideal immunotherapeutic targets, but evidence that T cells specific for these antigens can recognize and kill HPV(+) tumor cells is limited. We sought to determine whether TCR gene engineered T cells directed against an HPV oncoprotein can successfully target HPV(+) tumor cells. T-cell responses against the HPV-16 oncoproteins were investigated in a patient with an ongoing 22-month disease-free interval after her second resection of distant metastatic anal cancer. T cells genetically engineered to express an oncoprotein-specific TCR from this patient's tumor-infiltrating T cells were tested for specific reactivity against HPV(+) epithelial tumor cells. We identified, from an excised metastatic anal cancer tumor, T cells that recognized an HLA-A*02:01-restricted epitope of HPV-16 E6. The frequency of the dominant T-cell clonotype from these cells was approximately 400-fold greater in the patient's tumor than in her peripheral blood. T cells genetically engineered to express the TCR from this clonotype displayed high avidity for an HLA-A*02:01-restricted epitope of HPV-16, and they showed specific recognition and killing of HPV-16(+) cervical, and head and neck cancer cell lines. These findings demonstrate that HPV-16(+) tumors can be targeted by E6-specific TCR gene engineered T cells, and they provide the foundation for a novel cellular therapy directed against HPV-16(+) malignancies, including cervical, oropharyngeal, anal, vulvar, vaginal, and penile cancers. ©2015 American Association for Cancer Research.

Defined single-gene and multi-gene deletion mutant collections in Salmonella enterica sv Typhimurium.

PubMed

Porwollik, Steffen; Santiviago, Carlos A; Cheng, Pui; Long, Fred; Desai, Prerak; Fredlund, Jennifer; Srikumar, Shabarinath; Silva, Cecilia A; Chu, Weiping; Chen, Xin; Canals, Rocío; Reynolds, M Megan; Bogomolnaya, Lydia; Shields, Christine; Cui, Ping; Guo, Jinbai; Zheng, Yi; Endicott-Yazdani, Tiana; Yang, Hee-Jeong; Maple, Aimee; Ragoza, Yury; Blondel, Carlos J; Valenzuela, Camila; Andrews-Polymenis, Helene; McClelland, Michael

2014-01-01

We constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available.

Direct Broadcast Satellite: Radio Program

NASA Astrophysics Data System (ADS)

Hollansworth, James E.

1992-10-01

NASA is committed to providing technology development that leads to the introduction of new commercial applications for communications satellites. The Direct Broadcast Satellite-Radio (DBS-R) Program is a joint effort between The National Aeronautics and Space Administration (NASA) and The United States Information Agency/Voice of America (USIA/VOA) directed at this objective. The purpose of this program is to define the service and develop the technology for a direct-to-listener satellite sound broadcasting system. The DBS-R Program, as structured by NASA and VOA, is now a three-phase program designed to help the U.S. commercial communications satellite and receiver industry bring about this new communications service. Major efforts are being directed towards frequency planning hardware and service development, service demonstration, and experimentation with new satellite and receiver technology.

Reverse engineering of gene regulatory networks.

PubMed

Cho, K H; Choo, S M; Jung, S H; Kim, J R; Choi, H S; Kim, J

2007-05-01

Systems biology is a multi-disciplinary approach to the study of the interactions of various cellular mechanisms and cellular components. Owing to the development of new technologies that simultaneously measure the expression of genetic information, systems biological studies involving gene interactions are increasingly prominent. In this regard, reconstructing gene regulatory networks (GRNs) forms the basis for the dynamical analysis of gene interactions and related effects on cellular control pathways. Various approaches of inferring GRNs from gene expression profiles and biological information, including machine learning approaches, have been reviewed, with a brief introduction of DNA microarray experiments as typical tools for measuring levels of messenger ribonucleic acid (mRNA) expression. In particular, the inference methods are classified according to the required input information, and the main idea of each method is elucidated by comparing its advantages and disadvantages with respect to the other methods. In addition, recent developments in this field are introduced and discussions on the challenges and opportunities for future research are provided.

An Introduction to the Sources of Delivery Error for Direct-Fire Ballistic Projectiles

DTIC Science & Technology

2013-07-01

Ballistic mismatch has also been used to quantify the difference in target impacts using different gun tubes ...the angle between the local “upwards” direction of the gun tube and the vertical direction as defined by gravity. Cant results from the gun tube ...Determining Optimal Tube Shape for Reduction of Jump Error for Tank Fleets Using Fleet Zero. Presented at the 20th International Symposium on Ballistics

Gene Therapy in the Cornea: 2005-present

PubMed Central

Mohan, Rajiv R.; Tovey, Jonathan C.K.; Sharma, Ajay; Tandon, Ashish

2011-01-01

Successful restoration of vision in human patients with gene therapy affirmed its promise to cure ocular diseases and disorders. The efficacy of gene therapy is contingent upon vector and mode of therapeutic DNA introduction into targeted cells/tissues. The cornea is an ideal tissue for gene therapy due to its ease of access and relative immune-privilege. Considerable progress has been made in the field of corneal gene therapy in last 5 years. Several new gene transfer vectors, techniques and approaches have evolved. Although corneal gene therapy is still in its early stages of development, the potential of gene-based interventions to treat corneal abnormalities have begun to surface. Identification of next generation viral and nanoparticle vectors, characterization of delivered gene levels, localization, and duration in the cornea, and significant success in controlling corneal disorders, particularly fibrosis and angiogenesis, in experimental animal disease models, with no major side effects have propelled gene therapy a step closer towards establishing gene-based therapies for corneal blindness. Recently, researchers have assessed the delivery of therapeutic genes for corneal diseases and disorders due to trauma, infections, chemical, mechanical, and surgical injury, and/or abnormal wound healing. This review provides an update on the developments in gene therapy for corneal diseases and discusses the barriers that hinder its utilization for delivering genes in the cornea. PMID:21967960

Selfish Gene Algorithm Vs Genetic Algorithm: A Review

NASA Astrophysics Data System (ADS)

Ariff, Norharyati Md; Khalid, Noor Elaiza Abdul; Hashim, Rathiah; Noor, Noorhayati Mohamed

2016-11-01

Evolutionary algorithm is one of the algorithms inspired by the nature. Within little more than a decade hundreds of papers have reported successful applications of EAs. In this paper, the Selfish Gene Algorithms (SFGA), as one of the latest evolutionary algorithms (EAs) inspired from the Selfish Gene Theory which is an interpretation of Darwinian Theory ideas from the biologist Richards Dawkins on 1989. In this paper, following a brief introduction to the Selfish Gene Algorithm (SFGA), the chronology of its evolution is presented. It is the purpose of this paper is to present an overview of the concepts of Selfish Gene Algorithm (SFGA) as well as its opportunities and challenges. Accordingly, the history, step involves in the algorithm are discussed and its different applications together with an analysis of these applications are evaluated.

Mumps virus F gene and HN gene sequencing as a molecular tool to study mumps virus transmission.

PubMed

Gouma, Sigrid; Cremer, Jeroen; Parkkali, Saara; Veldhuijzen, Irene; van Binnendijk, Rob S; Koopmans, Marion P G

2016-11-01

Various mumps outbreaks have occurred in the Netherlands since 2004, particularly among persons who had received 2 doses of measles, mumps, and rubella (MMR) vaccination. Genomic typing of pathogens can be used to track outbreaks, but the established genotyping of mumps virus based on the small hydrophobic (SH) gene sequences did not provide sufficient resolution. Therefore, we expanded the sequencing to include fusion (F) gene and haemagglutinin-neuraminidase (HN) gene sequences in addition to the SH gene sequences from 109 mumps virus genotype G strains obtained between 2004 and mid 2015 in the Netherlands. When the molecular information from these 3 genes was combined, we were able to identify separate mumps virus clusters and track mumps virus transmission. The analyses suggested that multiple mumps virus introductions occurred in the Netherlands between 2004 and 2015 resulting in several mumps outbreaks throughout this period, whereas during some local outbreaks the molecular data pointed towards endemic circulation. Combined analysis of epidemiological data and sequence data collected in 2015 showed good support for the phylogenetic clustering. Copyright © 2016 Elsevier B.V. All rights reserved.

26 CFR 44.0-1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 16 2010-04-01 2010-04-01 true Introduction. 44.0-1 Section 44.0-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) MISCELLANEOUS EXCISE TAXES TAXES ON WAGERING; EFFECTIVE JANUARY 1, 1955 Introduction § 44.0-1 Introduction. (a) In general. The...

7 CFR 767.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... SPECIAL PROGRAMS INVENTORY PROPERTY MANAGEMENT Overview § 767.1 Introduction. (a) Purpose. This part... 7 Agriculture 7 2010-01-01 2010-01-01 false Introduction. 767.1 Section 767.1 Agriculture... financial interest. ...

Direct Loan Overview Workshop. Participant's Guide.

ERIC Educational Resources Information Center

Department of Education, Washington, DC.

This training guide for a one-day workshop provides an introduction to the William D. Ford Federal Direct Loan Program for administrative personnel at higher educations institutions. The seven sections of the guide, each corresponding to a single workshop session, include activity sheets, questions for participants to answer, and space for notes.…

26 CFR 41.0-1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 16 2010-04-01 2010-04-01 true Introduction. 41.0-1 Section 41.0-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) MISCELLANEOUS EXCISE TAXES EXCISE TAX ON USE OF CERTAIN HIGHWAY MOTOR VEHICLES Introduction § 41.0-1 Introduction. The regulations in...

26 CFR 49.0-1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 16 2010-04-01 2010-04-01 true Introduction. 49.0-1 Section 49.0-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) MISCELLANEOUS EXCISE TAXES FACILITIES AND SERVICES EXCISE TAXES Introduction § 49.0-1 Introduction. The regulations in this part 49 are...

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.

PubMed

Rivera-Torres, Natalia; Banas, Kelly; Bialk, Pawel; Bloh, Kevin M; Kmiec, Eric B

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

PubMed Central

Rivera-Torres, Natalia; Bialk, Pawel; Bloh, Kevin M.; Kmiec, Eric B.

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and

Direct Detection of OXA-48 Carbapenemase Gene in Lysate Samples through Changes in Mechanical Properties of DNA Monolayers upon Hybridization.

PubMed

Domínguez, Carmen M; Ramos, Daniel; Mingorance, Jesús; Fierro, José L G; Tamayo, Javier; Calleja, Montserrat

2018-01-02

Carbapenem-resistant Enterobacteriaceae have recently become an important cause of morbidity and mortality due to healthcare-associated infections. Most commonly used diagnostic methods are incompatible with fast and accurate directed therapy. We report here the direct identification of the bla OXA48 gene, which codes for the carbapenemase OXA-48, in lysate samples from Klebsiella pneumoniae. The method is PCR-free and label-free. It is based on the measurement of changes in the stiffness of DNA self-assembled monolayers anchored to microcantilevers that occur as a consequence of the hybridization. The stiffness of the DNA layer is measured through changes of the sensor resonance frequency upon hybridization and at varying relative humidity.

The Himalayas as a directional barrier to gene flow.

PubMed

Gayden, Tenzin; Cadenas, Alicia M; Regueiro, Maria; Singh, Nanda B; Zhivotovsky, Lev A; Underhill, Peter A; Cavalli-Sforza, Luigi L; Herrera, Rene J

2007-05-01

High-resolution Y-chromosome haplogroup analyses coupled with Y-short tandem repeat (STR) haplotypes were used to (1) investigate the genetic affinities of three populations from Nepal--including Newar, Tamang, and people from cosmopolitan Kathmandu (referred to as "Kathmandu" subsequently)--as well as a collection from Tibet and (2) evaluate whether the Himalayan mountain range represents a geographic barrier for gene flow between the Tibetan plateau and the South Asian subcontinent. The results suggest that the Tibetans and Nepalese are in part descendants of Tibeto-Burman-speaking groups originating from Northeast Asia. All four populations are represented predominantly by haplogroup O3a5-M134-derived chromosomes, whose Y-STR-based age (+/-SE) was estimated at 8.1+/-2.9 thousand years ago (KYA), more recent than its Southeast Asian counterpart. The most pronounced difference between the two regions is reflected in the opposing high-frequency distributions of haplogroups D in Tibet and R in Nepal. With the exception of Tamang, both Newar and Kathmandu exhibit considerable similarities to the Indian Y-haplogroup distribution, particularly in their haplogroup R and H composition. These results indicate gene flow from the Indian subcontinent and, in the case of haplogroup R, from Eurasia as well, a conclusion that is also supported by the admixture analysis. In contrast, whereas haplogroup D is completely absent in Nepal, it accounts for 50.6% of the Tibetan Y-chromosome gene pool. Coalescent analyses suggest that the expansion of haplogroup D derivatives--namely, D1-M15 and D3-P47 in Tibet--involved two different demographic events (5.1+/-1.8 and 11.3+/-3.7 KYA, respectively) that are more recent than those of D2-M55 representatives common in Japan. Low frequencies, relative to Nepal, of haplogroup J and R lineages in Tibet are also consistent with restricted gene flow from the subcontinent. Yet the presence of haplogroup O3a5-M134 representatives in Nepal

Multimedia presentations on the human genome: Implementation and assessment of a teaching program for the introduction to genome science using a poster and animations.

PubMed

Kano, Kei; Yahata, Saiko; Muroi, Kaori; Kawakami, Masahiro; Tomoda, Mari; Miyaki, Koichi; Nakayama, Takeo; Kosugi, Shinji; Kato, Kazuto

2008-11-01

Genome science, including topics such as gene recombination, cloning, genetic tests, and gene therapy, is now an established part of our daily lives; thus we need to learn genome science to better equip ourselves for the present day. Learning from topics directly related to the human has been suggested to be more effective than learning from Mendel's peas not only because many students do not understand that plants are organisms, but also because human biology contains important social and health issues. Therefore, we have developed a teaching program for the introduction to genome science, whose subjects are focused on the human genome. This program comprises mixed multimedia presentations: a large poster with illustrations and text on the human genome (a human genome map for every home), and animations on the basics of genome science. We implemented and assessed this program at four high schools. Our results indicate that students felt that they learned about the human genome from the program and some increases in students' understanding were observed with longer exposure to the mixed multimedia presentations. Copyright © 2008 International Union of Biochemistry and Molecular Biology, Inc.

Molecular Cloning of Human Gene(s) Directing the Synthesis of Nervous System Cholinesterases

DTIC Science & Technology

1987-09-01

and shed light on the unknown physiological function of these serine hydrolases in proliferating and differentiating cells. In pheochromocytoma cells...Reiness, C.G., Reichardt, L.F. and Hall, Z.W. (1981) Cellular localization of the molecular forms of acetylcholinesterase in rat pheochromocytoma PC12...melanogaster: Structural gene for acetylcholinesterase with an unusual 5’ leader. The EMBO J., 2949-2954. 99. Merken, L., Simons, M.J., Swillens, S

Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription*

PubMed Central

Layer, Justin H.; Weil, P. Anthony

2013-01-01

We have previously shown that yeast TFIID provides coactivator function on the promoters of ribosomal protein-encoding genes (RPGs) by making direct contact with the transactivator repressor activator protein 1 (Rap1). Further, our structural studies of assemblies generated with purified Rap1, TFIID, and TFIIA on RPG enhancer-promoter DNA indicate that Rap1-TFIID interaction induces dramatic conformational rearrangements of enhancer-promoter DNA and TFIID-bound TFIIA. These data indicate a previously unknown yet critical role for yeast TFIIA in the integration of activator-TFIID contacts with promoter conformation and downstream preinitiation complex formation and/or function. Here we describe the use of systematic mutagenesis to define how specific TFIIA contacts contribute to these processes. We have verified that TFIIA is required for RPG transcription in vivo and in vitro, consistent with the existence of a critical Rap1-TFIIA-TFIID interaction network. We also identified essential points of contact for TFIIA and Rap1 within the Rap1 binding domain of the Taf4 subunit of TFIID. These data suggest a mechanism for how interactions between TFIID, TFIIA, and Rap1 contribute to the high rate of transcription initiation seen on RPGs in vivo. PMID:23814059

Direct Broadcasting Satellite in the United States.

ERIC Educational Resources Information Center

Kim, Haeryon

The introduction of Direct Broadcasting Satellites (DBS) in the United States sparked both government's regulatory development of domestic DBS services and the communication industry's efforts to implement a commercial DBS system. J. D. Slack's symptomatic causality and technological assessment models help to explain how these practices were…

Development of a Cross-Disciplinary Investigative Model for the Introduction of Microarray Techniques at Non-R1 Undergraduate Institutions

ERIC Educational Resources Information Center

Walker, David E.; Lutz, Gary P.; Alvarez, Consuelo J.

2008-01-01

Integrating advanced biological techniques into instruction at non-R1 institutions can prove to be a challenge. Here, we report the creation of a model for the introduction of gene expression microarray technology into a research laboratory. A student assessment tool was used to evaluate: (1) technical skill development; (2) cross-disciplinary…

26 CFR 46.0-1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 16 2010-04-01 2010-04-01 true Introduction. 46.0-1 Section 46.0-1 Internal... TAX ON POLICIES ISSUED BY FOREIGN INSURERS AND OBLIGATIONS NOT IN REGISTERED FORM Introduction § 46.0-1 Introduction. The regulations in this part 46 relate to the taxes on policies issued by foreign...

Three countries' experience with Norplant introduction.

PubMed

Hardee, K; Balogh, S; Villinski, M T

1997-09-01

Despite international efforts to plan for Norplant introduction, the method has drawn the attention of critics of family planning programmes, and has raised several issues for debate since it was introduced into family planning programmes. The experiences of three countries with the introduction of Norplant highlight some of the unique features of the method that have affected its introduction. Indonesia, Bangladesh and the United States represent diverse cultural settings and systems of family planning provision. Experience in each country has highlighted the need to focus on quality of care for clients, most notably the need for good counselling and attention to removal as well as insertion. The cost of Norplant also has influenced its introduction in each country. Another issue includes the need to work with women's health advocacy groups, which is illustrated particularly in Bangladesh. Finally, the role of litigation in the United States, and its potential role in influencing Norplant introduction in other countries, is discussed. These three countries' experience illustrate the importance of understanding the programmatic context of contraceptive introduction.

Les Liaisons Dangereuses: Cancer-Related Genes and Neurodegenerative Diseases

NASA Astrophysics Data System (ADS)

Ghetti, Bernardino; Buonaguro, Franco M.

2014-07-01

The following sections are included: * INTRODUCTION * MUTATIONS IN THE CSF1R GENE ASSOCIATED WITH DIFFUSE LEUKOENCEPHALOPATHY WITH SPHEROIDS AND HUMAN CANCERS. * A SPECIAL LINK HAS BEEN SHOWN BETWEEN PTEN AND AD. * ACETYLCHOLINE DEFICIENCY AND PATHOGENESIS OF AD. * MIRNAS AND COMMON PATHWAYS IN CANCER AND NEURODEGENERATIVE DISEASE. * SUMMARY * REFERENCES

Multiple introductions boosted genetic diversity in the invasive range of black cherry (Prunus serotina; Rosaceae)

PubMed Central

Pairon, Marie; Petitpierre, Blaise; Campbell, Michael; Guisan, Antoine; Broennimann, Olivier; Baret, Philippe V.; Jacquemart, Anne-Laure; Besnard, Guillaume

2010-01-01

Background and Aims Black cherry (Prunus serotina) is a North American tree that is rapidly invading European forests. This species was introduced first as an ornamental plant then it was massively planted by foresters in many countries but its origins and the process of invasion remain poorly documented. Based on a genetic survey of both native and invasive ranges, the invasion history of black cherry was investigated by identifying putative source populations and then assessing the importance of multiple introductions on the maintenance of gene diversity. Methods Genetic variability and structure of 23 populations from the invasive range and 22 populations from the native range were analysed using eight nuclear microsatellite loci and five chloroplast DNA regions. Key Results Chloroplast DNA diversity suggests there were multiple introductions from a single geographic region (the north-eastern United States). A low reduction of genetic diversity was observed in the invasive range for both nuclear and plastid genomes. High propagule pressure including both the size and number of introductions shaped the genetic structure in Europe and boosted genetic diversity. Populations from Denmark, The Netherlands, Belgium and Germany showed high genetic diversity and low differentiation among populations, supporting the hypothesis that numerous introduction events, including multiple individuals and exchanges between sites, have taken place during two centuries of plantation. Conclusions This study postulates that the invasive black cherry has originated from east of the Appalachian Mountains (mainly the Allegheny plateau) and its invasiveness in north-western Europe is mainly due to multiple introductions containing high numbers of individuals. PMID:20400456

26 CFR 31.0-1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 15 2010-04-01 2010-04-01 false Introduction. 31.0-1 Section 31.0-1 Internal... OF INCOME TAX AT SOURCE EMPLOYMENT TAXES AND COLLECTION OF INCOME TAX AT SOURCE Introduction § 31.0-1 Introduction. (a) In general. The regulations in this part relate to the employment taxes imposed by subtitle C...

Metabolic engineering of the Chl d-dominated cyanobacterium Acaryochloris marina: production of a novel Chl species by the introduction of the chlorophyllide a oxygenase gene.

PubMed

Tsuchiya, Tohru; Mizoguchi, Tadashi; Akimoto, Seiji; Tomo, Tatsuya; Tamiaki, Hitoshi; Mimuro, Mamoru

2012-03-01

In oxygenic photosynthetic organisms, the properties of photosynthetic reaction systems primarily depend on the Chl species used. Acquisition of new Chl species with unique optical properties may have enabled photosynthetic organisms to adapt to various light environments. The artificial production of a new Chl species in an existing photosynthetic organism by metabolic engineering provides a model system to investigate how an organism responds to a newly acquired pigment. In the current study, we established a transformation system for a Chl d-dominated cyanobacterium, Acaryochloris marina, for the first time. The expression vector (constructed from a broad-host-range plasmid) was introduced into A. marina by conjugal gene transfer. The introduction of a gene for chlorophyllide a oxygenase, which is responsible for Chl b biosynthesis, into A. marina resulted in a transformant that synthesized a novel Chl species instead of Chl b. The content of the novel Chl in the transformant was approximately 10% of the total Chl, but the level of Chl a, another Chl in A. marina, did not change. The chemical structure of the novel Chl was determined to be [7-formyl]-Chl d(P) by mass spectrometry and nuclear magnetic resonance spectroscopy. [7-Formyl]-Chl d(P) is hypothesized to be produced by the combined action of chlorophyllide a oxygenase and enzyme(s) involved in Chl d biosynthesis. These results demonstrate the flexibility of the Chl biosynthetic pathway for the production of novel Chl species, indicating that a new organism with a novel Chl might be discovered in the future.

48 CFR 1846.670 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Introduction. 1846.670 Section 1846.670 Federal Acquisition Regulations System NATIONAL AERONAUTICS AND SPACE ADMINISTRATION CONTRACT MANAGEMENT QUALITY ASSURANCE Material Inspection and Receiving Reports 1846.670 Introduction. ...

20 CFR 416.1400 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... resolved. (b) Nature of the administrative review process. In making a determination or decision in your... DISABLED Determinations, Administrative Review Process, and Reopening of Determinations and Decisions Introduction, Definitions, and Initial Determinations § 416.1400 Introduction. (a) Explanation of the...

GOTree Machine (GOTM): a web-based platform for interpreting sets of interesting genes using Gene Ontology hierarchies

PubMed Central

Zhang, Bing; Schmoyer, Denise; Kirov, Stefan; Snoddy, Jay

2004-01-01

Background Microarray and other high-throughput technologies are producing large sets of interesting genes that are difficult to analyze directly. Bioinformatics tools are needed to interpret the functional information in the gene sets. Results We have created a web-based tool for data analysis and data visualization for sets of genes called GOTree Machine (GOTM). This tool was originally intended to analyze sets of co-regulated genes identified from microarray analysis but is adaptable for use with other gene sets from other high-throughput analyses. GOTree Machine generates a GOTree, a tree-like structure to navigate the Gene Ontology Directed Acyclic Graph for input gene sets. This system provides user friendly data navigation and visualization. Statistical analysis helps users to identify the most important Gene Ontology categories for the input gene sets and suggests biological areas that warrant further study. GOTree Machine is available online at . Conclusion GOTree Machine has a broad application in functional genomic, proteomic and other high-throughput methods that generate large sets of interesting genes; its primary purpose is to help users sort for interesting patterns in gene sets. PMID:14975175

Sequence variation and recognition specificity of the avirulence gene AvrPiz-t in Magnaporthe oryzae field populations

USDA-ARS?s Scientific Manuscript database

Magnaporthe oryzae, the rice blast pathogen, causes significant annual yield loss of rice worldwide. Currently, the most effective disease control approach is deployment of host resistance through introduction of resistance (R) genes into elite cultivars. The function of each R gene relies on the sp...

Directed evolution of cell size in Escherichia coli.

PubMed

Yoshida, Mari; Tsuru, Saburo; Hirata, Naoko; Seno, Shigeto; Matsuda, Hideo; Ying, Bei-Wen; Yomo, Tetsuya

2014-12-17

In bacteria, cell size affects chromosome replication, the assembly of division machinery, cell wall synthesis, membrane synthesis and ultimately growth rate. In addition, cell size can also be a target for Darwinian evolution for protection from predators. This strong coupling of cell size and growth, however, could lead to the introduction of growth defects after size evolution. An important question remains: can bacterial cell size change and/or evolve without imposing a growth burden? The directed evolution of particular cell sizes, without a growth burden, was tested with a laboratory Escherichia coli strain. Cells of defined size ranges were collected by a cell sorter and were subsequently cultured. This selection-propagation cycle was repeated, and significant changes in cell size were detected within 400 generations. In addition, the width of the size distribution was altered. The changes in cell size were unaccompanied by a growth burden. Whole genome sequencing revealed that only a few mutations in genes related to membrane synthesis conferred the size evolution. In conclusion, bacterial cell size could evolve, through a few mutations, without growth reduction. The size evolution without growth reduction suggests a rapid evolutionary change to diverse cell sizes in bacterial survival strategies.

Micro liquid chromatography-mass spectrometry with direct liquid introduction used for separation and quantitation of all-trans- and 13-cis-retinoic acids and their 4-oxo metabolites in human plasma.

PubMed

Ranalder, U B; Lausecker, B B; Huselton, C

1993-07-23

The separation and quantitation of the pentafluorobenzyl derivatives of all-trans- and 13-cis-retinoic acids and their 4-oxo metabolites in human plasma on micro high-performance liquid chromatographic columns (0.32 mm I.D.) is described. The column outlet was directly coupled to the source of a quadrupole mass spectrometer via a simple SFC-frit interface. Negative ion chemical ionization conditions were obtained by coaxial introduction of ammonia as a reagent gas. A signal-to-noise ratio well above 3 was obtained for 1 pg of each analyte injected. The limit of quantitation calculated from spiked biological plasma extracts was 0.3 ng/ml.

49 CFR 1150.11 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 8 2010-10-01 2010-10-01 false Introduction. 1150.11 Section 1150.11 Transportation Other Regulations Relating to Transportation (Continued) SURFACE TRANSPORTATION BOARD, DEPARTMENT... Designated Operators § 1150.11 Introduction. A certificate of designated operator will be issued to an...

47 CFR 2.1501 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 1 2010-10-01 2010-10-01 false Introduction. 2.1501 Section 2.1501 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL FREQUENCY ALLOCATIONS AND RADIO TREATY MATTERS; GENERAL... Radiobeacons (EPIRBs) General § 2.1501 Introduction. The procedure described herein sets forth uniform methods...

20 CFR 416.601 - Introduction.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false Introduction. 416.601 Section 416.601 Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Representative Payment § 416.601 Introduction. (a) Explanation of representative payment. This...

20 CFR 416.601 - Introduction.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 20 Employees' Benefits 2 2013-04-01 2013-04-01 false Introduction. 416.601 Section 416.601 Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Representative Payment § 416.601 Introduction. (a) Explanation of representative payment. This...

5 CFR 831.1201 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 5 Administrative Personnel 2 2010-01-01 2010-01-01 false Introduction. 831.1201 Section 831.1201 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT (CONTINUED) CIVIL SERVICE REGULATIONS (CONTINUED) RETIREMENT Disability Retirement § 831.1201 Introduction. This subpart sets out the requirements an employee...

5 CFR 831.1201 - Introduction.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 5 Administrative Personnel 2 2011-01-01 2011-01-01 false Introduction. 831.1201 Section 831.1201 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT (CONTINUED) CIVIL SERVICE REGULATIONS (CONTINUED) RETIREMENT Disability Retirement § 831.1201 Introduction. This subpart sets out the requirements an employee...

20 CFR 404.900 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

...- ) Determinations, Administrative Review Process, and Reopening of Determinations and Decisions Introduction, Definitions, and Initial Determinations § 404.900 Introduction. (a) Explanation of the administrative review... decision of the administrative law judge, you may request that the Appeals Council review the decision. (5...

HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis.

PubMed

Camilo, Cesar M; Lima, Gustavo M A; Maluf, Fernando V; Guido, Rafael V C; Polikarpov, Igor

2016-01-01

Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.

Gene: a gene-centered information resource at NCBI.

PubMed

Brown, Garth R; Hem, Vichet; Katz, Kenneth S; Ovetsky, Michael; Wallin, Craig; Ermolaeva, Olga; Tolstoy, Igor; Tatusova, Tatiana; Pruitt, Kim D; Maglott, Donna R; Murphy, Terence D

2015-01-01

The National Center for Biotechnology Information's (NCBI) Gene database (www.ncbi.nlm.nih.gov/gene) integrates gene-specific information from multiple data sources. NCBI Reference Sequence (RefSeq) genomes for viruses, prokaryotes and eukaryotes are the primary foundation for Gene records in that they form the critical association between sequence and a tracked gene upon which additional functional and descriptive content is anchored. Additional content is integrated based on the genomic location and RefSeq transcript and protein sequence data. The content of a Gene record represents the integration of curation and automated processing from RefSeq, collaborating model organism databases, consortia such as Gene Ontology, and other databases within NCBI. Records in Gene are assigned unique, tracked integers as identifiers. The content (citations, nomenclature, genomic location, gene products and their attributes, phenotypes, sequences, interactions, variation details, maps, expression, homologs, protein domains and external databases) is available via interactive browsing through NCBI's Entrez system, via NCBI's Entrez programming utilities (E-Utilities and Entrez Direct) and for bulk transfer by FTP. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Specific c-Jun target genes in malignant melanoma.

PubMed

Schummer, Patrick; Kuphal, Silke; Vardimon, Lily; Bosserhoff, Anja K; Kappelmann, Melanie

2016-05-03

A fundamental event in the development and progression of malignant melanoma is the de-regulation of cancer-relevant transcription factors. We recently showed that c-Jun is a main regulator of melanoma progression and, thus, is the most important member of the AP-1 transcription factor family in this disease. Surprisingly, no cancer-related specific c-Jun target genes in melanoma were described in the literature, so far. Therefore, we focused on pre-existing ChIP-Seq data (Encyclopedia of DNA Elements) of 3 different non-melanoma cell lines to screen direct c-Jun target genes. Here, a specific c-Jun antibody to immunoprecipitate the associated promoter DNA was used. Consequently, we identified 44 direct c-Jun targets and a detailed analysis of 6 selected genes confirmed their deregulation in malignant melanoma. The identified genes were differentially regulated comparing 4 melanoma cell lines and normal human melanocytes and we confirmed their c-Jun dependency. Direct interaction between c-Jun and the promoter/enhancer regions of the identified genes was confirmed by us via ChIP experiments. Interestingly, we revealed that the direct regulation of target gene expression via c-Jun can be independent of the existence of the classical AP-1 (5´-TGA(C/G)TCA-3´) consensus sequence allowing for the subsequent down- or up-regulation of the expression of these cancer-relevant genes. In summary, the results of this study indicate that c-Jun plays a crucial role in the development and progression of malignant melanoma via direct regulation of cancer-relevant target genes and that inhibition of direct c-Jun targets through inhibition of c-Jun is a potential novel therapeutic option for treatment of malignant melanoma.

Beta-globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy.

PubMed

Buzina, Alla; Lo, Mandy Y M; Moffett, Angela; Hotta, Akitsu; Fussner, Eden; Bharadwaj, Rikki R; Pasceri, Peter; Garcia-Martinez, J Victor; Bazett-Jones, David P; Ellis, James

2008-04-11

The Locus Control Region (LCR) requires intronic elements within beta-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional beta-globin intron 2 elements that rescue LCR activity directed by 5'HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igmu 3'MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igmu 3'MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5'HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5'HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of

Insights into the Introduction History and Population Genetic Dynamics of the Nile Monitor (Varanus niloticus) in Florida.

PubMed

Wood, Jared P; Dowell, Stephanie A; Campbell, Todd S; Page, Robert B

2016-07-01

Invasive species are widely recognized as important drivers of the ongoing biodiversity crisis. The US state of Florida is especially susceptible to the proliferation of invasive reptiles, and nonnative lizards currently outnumber native lizard species. At present, there are 3 documented breeding populations of the Nile monitor (Varanus niloticus) in different regions of Southern Florida, and these populations are considered potential dangers to threatened, fossorial endemics, such as burrowing owls, American crocodiles, and gopher tortoises. Nevertheless, at present, both the introduction histories of these populations and the degree to which they are connected by gene flow are not known. To address these issues, we genotyped V. niloticus from Cape Coral, Homestead Air Reserve Base, and West Palm Beach at 17 microsatellite loci and conducted a variety of analyses to assess both intrapopulation genetic diversity, the degree of gene flow between populations, and the most likely introduction scenario. The results of our analyses demonstrate that all 3 populations have limited genetic diversity (mean number of effective alleles across loci in all 3 populations ~ 2.00) and are highly differentiated from one another (G ST = 0.268; G″ST = 0.628). Our results also suggest that these populations resulted from independent introduction events that occurred within the past few decades. Consequently, we advise that wildlife managers focus management efforts on containment of existing populations and intensification of monitoring efforts on potential migration corridors. © The American Genetic Association. 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

1 CFR 456.1 - Introduction.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 1 General Provisions 1 2011-01-01 2011-01-01 false Introduction. 456.1 Section 456.1 General Provisions MISCELLANEOUS AGENCIES NATIONAL CAPITAL PLANNING COMMISSION (FREEDOM OF INFORMATION ACT REGULATIONS) § 456.1 Introduction. The following regulations implement the Freedom of Information Act, as...

1 CFR 456.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 1 General Provisions 1 2010-01-01 2010-01-01 false Introduction. 456.1 Section 456.1 General Provisions MISCELLANEOUS AGENCIES NATIONAL CAPITAL PLANNING COMMISSION (FREEDOM OF INFORMATION ACT REGULATIONS) § 456.1 Introduction. The following regulations implement the Freedom of Information Act, as...

24 CFR 904.101 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Introduction. 904.101 Section 904.101 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued... LOW RENT HOUSING HOMEOWNERSHIP OPPORTUNITIES Turnkey III Program Description § 904.101 Introduction...

32 CFR 147.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 1 2010-07-01 2010-07-01 false Introduction. 147.1 Section 147.1 National Defense Department of Defense OFFICE OF THE SECRETARY OF DEFENSE PERSONNEL, MILITARY AND CIVILIAN... Introduction. The following adjudicative guidelines are established for all United States Government civilian...

8 CFR 100.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 8 Aliens and Nationality 1 2010-01-01 2010-01-01 false Introduction. 100.1 Section 100.1 Aliens and Nationality DEPARTMENT OF HOMELAND SECURITY IMMIGRATION REGULATIONS STATEMENT OF ORGANIZATION § 100.1 Introduction. The following components have been delegated authority under the Immigration and...

40 CFR 62.02 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 8 2010-07-01 2010-07-01 false Introduction. 62.02 Section 62.02 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) APPROVAL AND PROMULGATION OF STATE PLANS FOR DESIGNATED FACILITIES AND POLLUTANTS General Provisions § 62.02 Introduction...

42 CFR 493.1200 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Introduction. 493.1200 Section 493.1200 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... Introduction. (a) Each laboratory that performs nonwaived testing must establish and maintain written policies...

5 CFR 890.801 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 5 Administrative Personnel 2 2010-01-01 2010-01-01 false Introduction. 890.801 Section 890.801 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT (CONTINUED) CIVIL SERVICE REGULATIONS (CONTINUED) FEDERAL EMPLOYEES HEALTH BENEFITS PROGRAM Benefits for Former Spouses § 890.801 Introduction. This subpart sets...

40 CFR 25.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Introduction. 25.1 Section 25.1 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GENERAL PUBLIC PARTICIPATION IN PROGRAMS UNDER THE... Introduction. This part sets forth minimum requirements and suggested program elements for public participation...

32 CFR 651.28 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 4 2010-07-01 2010-07-01 true Introduction. 651.28 Section 651.28 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY (CONTINUED) ENVIRONMENTAL QUALITY ENVIRONMENTAL ANALYSIS OF ARMY ACTIONS (AR 200-2) Categorical Exclusions § 651.28 Introduction. Categorical...

32 CFR 147.28 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 1 2010-07-01 2010-07-01 false Introduction. 147.28 Section 147.28 National Defense Department of Defense OFFICE OF THE SECRETARY OF DEFENSE PERSONNEL, MILITARY AND CIVILIAN... Temporary Access § 147.28 Introduction. The following minimum investigative standards, implementing section...

29 CFR 1910.301 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 29 Labor 5 2010-07-01 2010-07-01 false Introduction. 1910.301 Section 1910.301 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR OCCUPATIONAL SAFETY AND HEALTH STANDARDS Electrical General § 1910.301 Introduction. This subpart addresses...

7 CFR 1951.851 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 14 2010-01-01 2009-01-01 true Introduction. 1951.851 Section 1951.851 Agriculture Regulations of the Department of Agriculture (Continued) RURAL HOUSING SERVICE, RURAL BUSINESS-COOPERATIVE... REGULATIONS (CONTINUED) SERVICING AND COLLECTIONS Rural Development Loan Servicing § 1951.851 Introduction. (a...

7 CFR 762.101 - Introduction.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 7 2012-01-01 2012-01-01 false Introduction. 762.101 Section 762.101 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS GUARANTEED FARM LOANS § 762.101 Introduction. (a) Scope. This subpart contains...

7 CFR 762.101 - Introduction.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 7 2014-01-01 2014-01-01 false Introduction. 762.101 Section 762.101 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS GUARANTEED FARM LOANS § 762.101 Introduction. (a) Scope. This subpart contains...

7 CFR 762.101 - Introduction.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 7 2011-01-01 2011-01-01 false Introduction. 762.101 Section 762.101 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS GUARANTEED FARM LOANS § 762.101 Introduction. (a) Scope. This subpart contains...

20 CFR 404.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

...- ) Introduction, General Provisions and Definitions § 404.1 Introduction. The regulations in this part 404... are divided into 22 subparts: (a) Subpart A contains provisions relating to general definitions and... process, and reopening of determinations and decisions. (k) Subpart K relates to employment, wages, self...

1 CFR 456.1 - Introduction.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 1 General Provisions 1 2014-01-01 2012-01-01 true Introduction. 456.1 Section 456.1 General Provisions MISCELLANEOUS AGENCIES NATIONAL CAPITAL PLANNING COMMISSION (FREEDOM OF INFORMATION ACT REGULATIONS) § 456.1 Introduction. The following regulations implement the Freedom of Information Act, as...

1 CFR 456.1 - Introduction.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 1 General Provisions 1 2012-01-01 2012-01-01 false Introduction. 456.1 Section 456.1 General Provisions MISCELLANEOUS AGENCIES NATIONAL CAPITAL PLANNING COMMISSION (FREEDOM OF INFORMATION ACT REGULATIONS) § 456.1 Introduction. The following regulations implement the Freedom of Information Act, as...

1 CFR 456.1 - Introduction.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 1 General Provisions 1 2013-01-01 2012-01-01 true Introduction. 456.1 Section 456.1 General Provisions MISCELLANEOUS AGENCIES NATIONAL CAPITAL PLANNING COMMISSION (FREEDOM OF INFORMATION ACT REGULATIONS) § 456.1 Introduction. The following regulations implement the Freedom of Information Act, as...

How Alterations in the Cdt1 Expression Lead to Gene Amplification in Breast Cancer

DTIC Science & Technology

2011-07-01

absence of extrinsic DNA damage. We measured the TLS activity by measuring the mutation frequency in a supF gene (in a shuttle vector) subjected to UV...induced DNA damage before its introduction into the cells. Error-prone TLS activity will mutate the supF gene , which is scored by a blue-white colony...Figure 4A). Sequencing of the mutant supF genes , revealed a mutation spectrum consistent with error prone TLS (Supplemental Table 1). Significantly

FunGene: the functional gene pipeline and repository.

PubMed

Fish, Jordan A; Chai, Benli; Wang, Qiong; Sun, Yanni; Brown, C Titus; Tiedje, James M; Cole, James R

2013-01-01

Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer. While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/) offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes.

7 CFR 4287.101 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 15 2010-01-01 2010-01-01 false Introduction. 4287.101 Section 4287.101 Agriculture Regulations of the Department of Agriculture (Continued) RURAL BUSINESS-COOPERATIVE SERVICE AND RURAL... § 4287.101 Introduction. (a) This subpart supplements part 4279, subparts A and B, by providing...

28 CFR 802.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Introduction. 802.1 Section 802.1 Judicial Administration COURT SERVICES AND OFFENDER SUPERVISION AGENCY FOR THE DISTRICT OF COLUMBIA DISCLOSURE OF RECORDS General § 802.1 Introduction. This part contains regulations of the Court Services and...

42 CFR 405.1801 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 2 2010-10-01 2010-10-01 false Introduction. 405.1801 Section 405.1801 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES MEDICARE PROGRAM....1801 Introduction. (a) Definitions. As used in this subpart: Administrator means the Administrator or...

29 CFR 500.0 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 29 Labor 3 2010-07-01 2010-07-01 false Introduction. 500.0 Section 500.0 Labor Regulations Relating to Labor (Continued) WAGE AND HOUR DIVISION, DEPARTMENT OF LABOR REGULATIONS MIGRANT AND SEASONAL AGRICULTURAL WORKER PROTECTION General Provisions § 500.0 Introduction. (a) The Migrant and Seasonal...

32 CFR 147.18 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 1 2010-07-01 2010-07-01 false Introduction. 147.18 Section 147.18 National Defense Department of Defense OFFICE OF THE SECRETARY OF DEFENSE PERSONNEL, MILITARY AND CIVILIAN... Standards § 147.18 Introduction. The following investigative standards are established for all United States...

7 CFR 4279.101 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 15 2010-01-01 2010-01-01 false Introduction. 4279.101 Section 4279.101 Agriculture Regulations of the Department of Agriculture (Continued) RURAL BUSINESS-COOPERATIVE SERVICE AND RURAL... Introduction. (a) Content. This subpart contains loan processing regulations for the Business and Industry (B&I...

38 CFR 21.9500 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Introduction. 21.9500 Section 21.9500 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS (CONTINUED) VOCATIONAL REHABILITATION AND EDUCATION Post-9/11 GI Bill § 21.9500 Introduction. An educational assistance...

40 CFR 52.02 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 3 2010-07-01 2010-07-01 false Introduction. 52.02 Section 52.02 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) APPROVAL AND PROMULGATION OF IMPLEMENTATION PLANS General Provisions § 52.02 Introduction. (a) This part sets forth the...

20 CFR 405.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

...-adversarial proceeding. In making a determination or decision on your claim, we conduct the administrative... CLAIMS Introduction, General Description, and Definitions § 405.1 Introduction. (a) General. This part... before an administrative law judge. If you are dissatisfied with a decision made by the Federal reviewing...

20 CFR 416.1500 - Introduction.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 20 Employees' Benefits 2 2014-04-01 2014-04-01 false Introduction. 416.1500 Section 416.1500 Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Representation of Parties § 416.1500 Introduction. You may appoint someone to represent you in any...

Ribosomal DNA stability is supported by many 'buffer genes'-introduction to the Yeast rDNA Stability Database.

PubMed

Kobayashi, Takehiko; Sasaki, Mariko

2017-01-01

The ribosomal RNA gene (rDNA) is the most abundant gene in yeast and other eukaryotic organisms. Due to its heavy transcription, repetitive structure and programmed replication fork pauses, the rDNA is one of the most unstable regions in the genome. Thus, the rDNA is the best region to study the mechanisms responsible for maintaining genome integrity. Recently, we screened a library of ∼4800 budding yeast gene knockout strains to identify mutants defective in the maintenance of rDNA stability. The results of this screen are summarized in the Yeast rDNA Stability (YRS) Database, in which the stability and copy number of rDNA in each mutant are presented. From this screen, we identified ∼700 genes that may contribute to the maintenance of rDNA stability. In addition, ∼50 mutants had abnormally high or low rDNA copy numbers. Moreover, some mutants with unstable rDNA displayed abnormalities in another chromosome. In this review, we introduce the YRS Database and discuss the roles of newly identified genes that contribute to rDNA maintenance and genome integrity. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template.

PubMed

Gouran, Hossein; Chakraborty, Sandeep; Rao, Basuthkar J; Asgeirsson, Bjarni; Dandekar, Abhaya

2014-01-01

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction.

Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template

PubMed Central

Rao, Basuthkar J.; Asgeirsson, Bjarni; Dandekar, Abhaya

2014-01-01

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction. PMID:25717364

The timing of introduction of complementary foods and later health.

PubMed

Agostoni, Carlo; Przyrembel, Hildegard

2013-01-01

Complementary food is needed when human milk (or infant formula) alone is no longer sufficient for nutritional reasons. The timing of introduction needs to be determined on an individual basis although 6 months of exclusive breastfeeding can be recommended for most healthy term infants. Solid foods are intended to 'complement' ongoing breastfeeding with those dietary items whose intake has become marginal or insufficient. Both breastfeeding and complementary feeding can have direct or later consequences on health. Possible short-term health effects concern growth velocity and infections while possible long-term effects may relate to obesity, cardiovascular disease, autoimmunity (celiac disease and type 1 diabetes) and atopic disorders. For most of these it is impossible on the basis of the available evidence to conclude on the age when risks related to the start of complementary feeding are lowest or highest, with the possible exception of infections and early growth velocity. For undesirable health consequences, whilst potential mechanisms are recognized, the evidence from mostly observational studies is insufficient and requires more and prospective research. While the 6-month goal is desirable, introduction of suitable complementary food after 4 completed months with ongoing breastfeeding can be considered without adverse health consequences for infants living in affluent countries. Even less evidence on the consequences of the timing of complementary food introduction is available for formula-fed infants. Copyright © 2013 S. Karger AG, Basel.

Development of a transgenic zebrafish model expressing GFP in the notochord, somite and liver directed by the hfe2 gene promoter.

PubMed

Bian, Yue-Hong; Xu, Cheng; Li, Junling; Xu, Jin; Zhang, Hongwei; Du, Shao Jun

2011-08-01

Hemojuvelin, also known as RGMc, is encoded by hfe2 gene that plays an important role in iron homeostasis. hfe2 is specifically expressed in the notochord, developing somite and skeletal muscles during development. The molecular regulation of hfe2 expression is, however, not clear. We reported here the characterization of hfe2 gene expression and the regulation of its tissue-specific expression in zebrafish embryos. We demonstrated that the 6 kb 5'-flanking sequence upstream of the ATG start codon in the zebrafish hfe2 gene could direct GFP specific expression in the notochord, somites, and skeletal muscle of zebrafish embryos, recapitulating the expression pattern of the endogenous gene. However, the Tg(hfe2:gfp) transgene is also expressed in the liver of fish embryos, which did not mimic the expression of the endogenous hfe2 at the early stage. Nevertheless, the Tg(hfe2:gfp) transgenic zebrafish provides a useful model to study liver development. Treating Tg(hfe2:gfp) transgenic zebrafish embryos with valproic acid, a liver development inhibitor, significantly inhibited GFP expression in zebrafish. Together, these data indicate that the tissue specific expression of hfe2 in the notochord, somites and muscles is regulated by regulatory elements within the 6 kb 5'-flanking sequence of the hfe2 gene. Moreover, the Tg(hfe2:gfp) transgenic zebrafish line provides a useful model system for analyzing liver development in zebrafish.

Long-term systemic therapy of Fabry disease in a knockout mouse by adeno-associated virus-mediated muscle-directed gene transfer

PubMed Central

Takahashi, Hiroshi; Hirai, Yukihiko; Migita, Makoto; Seino, Yoshihiko; Fukuda, Yuh; Sakuraba, Hitoshi; Kase, Ryoichi; Kobayashi, Toshihide; Hashimoto, Yasuhiro; Shimada, Takashi

2002-01-01

Fabry disease is a systemic disease caused by genetic deficiency of a lysosomal enzyme, α-galactosidase A (α-gal A), and is thought to be an important target for enzyme replacement therapy. We studied the feasibility of gene-mediated enzyme replacement for Fabry disease. The adeno-associated virus (AAV) vector containing the α-gal A gene was injected into the right quadriceps muscles of Fabry knockout mice. A time course study showed that α-gal A activity in plasma was increased to ≈25% of normal mice and that this elevated activity persisted for up to at least 30 weeks without development of anti-α-gal A antibodies. The α-gal A activity in various organs of treated Fabry mice remained 5–20% of those observed in normal mice. Accumulated globotriaosylceramide in these organs was completely cleared by 25 weeks after vector injection. Reduction of globotriaosylceramide levels was also confirmed by immunohistochemical and electronmicroscopic analyses. Echocardiographic examination of treated mice demonstrated structural improvement of cardiac hypertrophy 25 weeks after the treatment. AAV vector-mediated muscle-directed gene transfer provides an efficient and practical therapeutic approach for Fabry disease. PMID:12370426

Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker.

PubMed

Rubinstein, M; Japón, M A; Low, M J

1993-06-11

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes.

Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker.

PubMed Central

Rubinstein, M; Japón, M A; Low, M J

1993-01-01

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes. Images PMID:8392702

45 CFR 1218.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 4 2010-10-01 2010-10-01 false Introduction. 1218.1 Section 1218.1 Public Welfare Regulations Relating to Public Welfare (Continued) CORPORATION FOR NATIONAL AND COMMUNITY SERVICE VISTA VOLUNTEERS-HEARING OPPORTUNITY § 1218.1 Introduction. Section 104(d) of the Domestic Volunteer Service Act of...

22 CFR 66.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Introduction. 66.1 Section 66.1 Foreign Relations DEPARTMENT OF STATE PUBLIC DIPLOMACY AND EXCHANGES AVAILABILITY OF THE RECORDS OF THE NATIONAL ENDOWMENT FOR DEMOCRACY § 66.1 Introduction. These regulations amend the Code of Federal Regulations to...

15 CFR 734.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false Introduction. 734.1 Section 734.1 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF... ADMINISTRATION REGULATIONS § 734.1 Introduction. (a) In this part, references to the Export Administration...

15 CFR 700.2 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false Introduction. 700.2 Section 700.2 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF... PRIORITIES AND ALLOCATIONS SYSTEM Overview § 700.2 Introduction. (a) Certain national defense and energy...

45 CFR 1219.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 4 2010-10-01 2010-10-01 false Introduction. 1219.1 Section 1219.1 Public Welfare Regulations Relating to Public Welfare (Continued) CORPORATION FOR NATIONAL AND COMMUNITY SERVICE COMPETITIVE SERVICE ELIGIBILITY § 1219.1 Introduction. Section 415(d), Title IV, of the Domestic Volunteer Service Act...

29 CFR 501.0 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 29 Labor 3 2010-07-01 2010-07-01 false Introduction. 501.0 Section 501.0 Labor Regulations Relating to Labor (Continued) WAGE AND HOUR DIVISION, DEPARTMENT OF LABOR REGULATIONS ENFORCEMENT OF... AND NATIONALITY ACT General Provisions § 501.0 Introduction. The regulations in this part cover the...

20 CFR 416.200 - Introduction.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 20 Employees' Benefits 2 2013-04-01 2013-04-01 false Introduction. 416.200 Section 416.200 Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Eligibility General § 416.200 Introduction. You are eligible for SSI benefits if you meet all the...

20 CFR 416.200 - Introduction.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 20 Employees' Benefits 2 2012-04-01 2012-04-01 false Introduction. 416.200 Section 416.200 Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Eligibility General § 416.200 Introduction. You are eligible for SSI benefits if you meet all the...

20 CFR 416.200 - Introduction.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 20 Employees' Benefits 2 2014-04-01 2014-04-01 false Introduction. 416.200 Section 416.200 Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Eligibility General § 416.200 Introduction. You are eligible for SSI benefits if you meet all the...

20 CFR 416.200 - Introduction.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false Introduction. 416.200 Section 416.200 Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Eligibility General § 416.200 Introduction. You are eligible for SSI benefits if you meet all the...

22 CFR 301.1 - Introduction.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 22 Foreign Relations 2 2014-04-01 2014-04-01 false Introduction. 301.1 Section 301.1 Foreign Relations PEACE CORPS PUBLIC ACCESS TO CLASSIFIED MATERIAL § 301.1 Introduction. The following regulations implement Executive Order 12356 and provide guidance for members of the public desiring a review for...

22 CFR 301.1 - Introduction.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 22 Foreign Relations 2 2011-04-01 2009-04-01 true Introduction. 301.1 Section 301.1 Foreign Relations PEACE CORPS PUBLIC ACCESS TO CLASSIFIED MATERIAL § 301.1 Introduction. The following regulations implement Executive Order 12356 and provide guidance for members of the public desiring a review for...

20 CFR 416.200 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Introduction. 416.200 Section 416.200 Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Eligibility General § 416.200 Introduction. You are eligible for SSI benefits if you meet all the...

32 CFR 516.69 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 3 2010-07-01 2010-07-01 true Introduction. 516.69 Section 516.69 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY AID OF CIVIL AUTHORITIES AND PUBLIC RELATIONS LITIGATION Cooperation With the Office of Special Counsel § 516.69 Introduction. This subpart...

32 CFR 516.69 - Introduction.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 32 National Defense 3 2011-07-01 2009-07-01 true Introduction. 516.69 Section 516.69 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY AID OF CIVIL AUTHORITIES AND PUBLIC RELATIONS LITIGATION Cooperation With the Office of Special Counsel § 516.69 Introduction. This subpart...

18 CFR 401.0 - Introduction.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 18 Conservation of Power and Water Resources 2 2011-04-01 2011-04-01 false Introduction. 401.0 Section 401.0 Conservation of Power and Water Resources DELAWARE RIVER BASIN COMMISSION ADMINISTRATIVE MANUAL RULES OF PRACTICE AND PROCEDURE § 401.0 Introduction. (a) The Delaware River Basin Compact...

22 CFR 301.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 22 Foreign Relations 2 2010-04-01 2010-04-01 true Introduction. 301.1 Section 301.1 Foreign Relations PEACE CORPS PUBLIC ACCESS TO CLASSIFIED MATERIAL § 301.1 Introduction. The following regulations implement Executive Order 12356 and provide guidance for members of the public desiring a review for...

20 CFR 416.601 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Introduction. 416.601 Section 416.601 Employees' Benefits SOCIAL SECURITY ADMINISTRATION SUPPLEMENTAL SECURITY INCOME FOR THE AGED, BLIND, AND DISABLED Representative Payment § 416.601 Introduction. (a) Explanation of representative payment. This subpart explains the principles and procedures...

Updating the landscape of direct-to-consumer pharmacogenomic testing.

PubMed

Filipski, Kelly K; Murphy, John D; Helzlsouer, Kathy J

2017-01-01

Pharmacogenomics has identified important drug-gene interactions that affect the safety and efficacy of medications. Direct-to-consumer genetic testing, when first introduced, included some pharmacogenomic-related genes. The current landscape of pharmacogenomic direct-to-consumer testing is reviewed. Prior published reviews of the literature were updated through February 2017 and a scan of the current availability of direct-to-consumer genomic testing by companies was conducted. Results of the review demonstrate a shift toward physician-approved ordering.

Nicotiana tabacum EIL2 directly regulates expression of at least one tobacco gene induced by sulphur starvation.

PubMed

Wawrzyńska, Anna; Lewandowska, Małgorzata; Sirko, Agnieszka

2010-03-01

Sulphur deficiency severely affects plant growth and their agricultural productivity leading to diverse changes in development and metabolisms. Molecular mechanisms regulating gene expression under low sulphur conditions remain largely unknown. AtSLIM1, a member of the EIN3-like (EIL) family was reported to be a central transcriptional regulator of the plant sulphur response, however, no direct interaction of this protein with any sulphur-responsive promoters was demonstrated. The focus of this study was on the analysis of a promoter region of UP9C, a tobacco gene strongly induced by sulphur limitation. Cloning and subsequent examination of this promoter resulted in the identification of a 20-nt sequence (UPE-box), also present in the promoters of several Arabidopsis genes, including three out of four homologues of UP9C. The UPE-box, consisting of two parallel tebs sequences (TEIL binding site), proved to be necessary to bind the transcription factors belonging to the EIL family and of a 5-nt conserved sequence at the 3'-end. The yeast one-hybrid analysis resulted in the identification of one transcription factor (NtEIL2) capable of binding to the UPE-box. The interactions of NtEIL2, and its homologue from Arabidopsis, AtSLIM1, with DNA were affected by mutations within the UPE-box. Transient expression assays in Nicotiana benthamiana have further shown that both factors, NtEIL2 and AtSLIM1, activate the UP9C promoter. Interestingly, activation by NtEIL2, but not by AtSLIM1, was dependent on the sulphur-deficiency of the plants.

Direct Detection and Differentiation of Pathogenic Leptospira Species Using a Multi-Gene Targeted Real Time PCR Approach

PubMed Central

Ferreira, Ana Sofia; Costa, Pedro; Rocha, Teresa; Amaro, Ana; Vieira, Maria Luísa; Ahmed, Ahmed; Thompson, Gertrude; Hartskeerl, Rudy A.; Inácio, João

2014-01-01

Leptospirosis is a growing public and veterinary health concern caused by pathogenic species of Leptospira. Rapid and reliable laboratory tests for the direct detection of leptospiral infections in animals are in high demand not only to improve diagnosis but also for understanding the epidemiology of the disease. In this work we describe a novel and simple TaqMan-based multi-gene targeted real-time PCR approach able to detect and differentiate Leptospira interrogans, L. kirschneri, L. borgpeteresenii and L. noguchii, which constitute the veterinary most relevant pathogenic species of Leptospira. The method uses sets of species-specific probes, and respective flanking primers, designed from ompL1 and secY gene sequences. To monitor the presence of inhibitors, a duplex amplification assay targeting both the mammal β-actin and the leptospiral lipL32 genes was implemented. The analytical sensitivity of all primer and probe sets was estimated to be <10 genome equivalents (GE) in the reaction mixture. Application of the amplification reactions on genomic DNA from a variety of pathogenic and non-pathogenic Leptospira strains and other non-related bacteria revealed a 100% analytical specificity. Additionally, pathogenic leptospires were successfully detected in five out of 29 tissue samples from animals (Mus spp., Rattus spp., Dolichotis patagonum and Sus domesticus). Two samples were infected with L. borgpetersenii, two with L. interrogans and one with L. kirschneri. The possibility to detect and identify these pathogenic agents to the species level in domestic and wildlife animals reinforces the diagnostic information and will enhance our understanding of the epidemiology of leptopirosis. PMID:25398140

Host-Induced Gene Silencing of Rice Blast Fungus Magnaporthe oryzae Pathogenicity Genes Mediated by the Brome Mosaic Virus.

PubMed

Zhu, Lin; Zhu, Jian; Liu, Zhixue; Wang, Zhengyi; Zhou, Cheng; Wang, Hong

2017-09-26

Magnaporthe oryzae is a devastating plant pathogen, which has a detrimental impact on rice production worldwide. Despite its agronomical importance, some newly-emerging pathotypes often overcome race-specific disease resistance rapidly. It is thus desirable to develop a novel strategy for the long-lasting resistance of rice plants to ever-changing fungal pathogens. Brome mosaic virus (BMV)-induced RNA interference (RNAi) has emerged as a useful tool to study host-resistance genes for rice blast protection. Planta-generated silencing of targeted genes inside biotrophic pathogens can be achieved by expression of M. oryzae -derived gene fragments in the BMV-mediated gene silencing system, a technique termed host-induced gene silencing (HIGS). In this study, the effectiveness of BMV-mediated HIGS in M. oryzae was examined by targeting three predicted pathogenicity genes, MoABC1, MoMAC1 and MoPMK1 . Systemic generation of fungal gene-specific small interfering RNA (siRNA) molecules induced by inoculation of BMV viral vectors inhibited disease development and reduced the transcription of targeted fungal genes after subsequent M. oryzae inoculation. Combined introduction of fungal gene sequences in sense and antisense orientation mediated by the BMV silencing vectors significantly enhanced the efficiency of this host-generated trans-specific RNAi, implying that these fungal genes played crucial roles in pathogenicity. Collectively, our results indicated that BMV-HIGS system was a great strategy for protecting host plants against the invasion of pathogenic fungi.

GeneTopics - interpretation of gene sets via literature-driven topic models

PubMed Central

2013-01-01

Background Annotation of a set of genes is often accomplished through comparison to a library of labelled gene sets such as biological processes or canonical pathways. However, this approach might fail if the employed libraries are not up to date with the latest research, don't capture relevant biological themes or are curated at a different level of granularity than is required to appropriately analyze the input gene set. At the same time, the vast biomedical literature offers an unstructured repository of the latest research findings that can be tapped to provide thematic sub-groupings for any input gene set. Methods Our proposed method relies on a gene-specific text corpus and extracts commonalities between documents in an unsupervised manner using a topic model approach. We automatically determine the number of topics summarizing the corpus and calculate a gene relevancy score for each topic allowing us to eliminate non-specific topics. As a result we obtain a set of literature topics in which each topic is associated with a subset of the input genes providing directly interpretable keywords and corresponding documents for literature research. Results We validate our method based on labelled gene sets from the KEGG metabolic pathway collection and the genetic association database (GAD) and show that the approach is able to detect topics consistent with the labelled annotation. Furthermore, we discuss the results on three different types of experimentally derived gene sets, (1) differentially expressed genes from a cardiac hypertrophy experiment in mice, (2) altered transcript abundance in human pancreatic beta cells, and (3) genes implicated by GWA studies to be associated with metabolite levels in a healthy population. In all three cases, we are able to replicate findings from the original papers in a quick and semi-automated manner. Conclusions Our approach provides a novel way of automatically generating meaningful annotations for gene sets that are directly

49 CFR 1103.10 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 8 2010-10-01 2010-10-01 false Introduction. 1103.10 Section 1103.10... OF TRANSPORTATION RULES OF PRACTICE PRACTITIONERS Canons of Ethics § 1103.10 Introduction. The... practice law and (b) others who have fulfilled the requirements set forth in § 1103.3. The former are bound...

15 CFR 736.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false Introduction. 736.1 Section 736.1....1 Introduction. In this part, references to the EAR are references to 15 CFR chapter VII, subchapter... the EAR regarding export clearance requirements. Also note that for short supply controls all...

45 CFR 1217.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 4 2010-10-01 2010-10-01 false Introduction. 1217.1 Section 1217.1 Public Welfare Regulations Relating to Public Welfare (Continued) CORPORATION FOR NATIONAL AND COMMUNITY SERVICE VISTA VOLUNTEER LEADER § 1217.1 Introduction. Section 105(a)(1), Part A, of the Domestic Volunteer Service Act of...

7 CFR 6.20 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 1 2010-01-01 2010-01-01 false Introduction. 6.20 Section 6.20 Agriculture Office of the Secretary of Agriculture IMPORT QUOTAS AND FEES Dairy Tariff-Rate Import Quota Licensing § 6.20 Introduction. (a) Presidential Proclamation 6763 of December 23, 1994, modified the Harmonized Tariff Schedule...

29 CFR 75.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 29 Labor 1 2010-07-01 2010-07-01 true Introduction. 75.1 Section 75.1 Labor Office of the Secretary of Labor DEPARTMENT OF LABOR REVIEW AND CERTIFICATION PROCEDURES FOR RURAL INDUSTRIALIZATION LOAN AND GRANT PROGRAMS UNDER THE CONSOLIDATED FARM AND RURAL DEVELOPMENT ACT OF 1972 § 75.1 Introduction...

15 CFR 30.15 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Introduction. (a) For export shipments to foreign countries, the EEI is used both for statistical and for... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Introduction. 30.15 Section 30.15... control regulations, as well as the requirements of the statistical regulations of this part. For...

Senescence of immortal human fibroblasts by the introduction of normal human chromosome 6

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

Sandhu, A.K.; Hubbard, K.; Kaur, G.P.

1994-06-07

In these studies the authors show that introduction of a normal human chromosome 6 or 6q can suppress the immortal phenotype of simian virus 40-transformed human fibroblasts (SV/HF). Normal human fibroblasts have a limited life span in culture. Immortal clones of SV/HF displayed nonrandom rearrangements in chromosome 6. Single human chromosomes present in mouse/human monochromosomal hybrids were introduced into SV/HF via microcell fusion and maintained by selection for a dominant selectable marker gpt, previously integrated into the human chromosome. Clones of SV/HF cells bearing chromosome 6 displayed limited potential for cell division and morphological characteristics of senescent cells. The lossmore » of chromosome 6 from the suppressed clones correlated with the reappearance of immortal clones. Introduced chromosome 6 in the senescing cells was distinguished from those of parental cells by analysis for DNA sequences specific for the donor chromosome. The results further show that suppression of immortal phenotype in SV/HF is specific to chromosome 6. Introduction of individual human chromosomes 2, 8, or 19 did not impart cellular senescence in SV/HF. In addition, introduction of chromosome 6 into human glioblastoma cells did not lead to senescence. Based upon these results the authors propose that at least one of the genes (SEN6) for cellular senescence in human fibroblasts is present on the long arm of chromosome 6.« less

Establishment and characterization of scleroderma fibroblast clonal cell lines by introduction of the hTERT gene

PubMed Central

Kapanadze, Bagrat; Morris, Erin; Smith, Edwin; Trojanowska, Maria

2010-01-01

Abstract Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-βRI indicative of altered TGF-β signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis. PMID:19432820

Establishment and characterization of scleroderma fibroblast clonal cell lines by introduction of the hTERT gene.

PubMed

Kapanadze, Bagrat; Morris, Erin; Smith, Edwin; Trojanowska, Maria

2010-05-01

Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-betaRI indicative of altered TGF-beta signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis.

Overexpression of MIC-3 indicates a direct role for the MIC gene family in mediating Upland cotton (Gossypium hirsutum) resistance to root-knot nematode (Meloidogyne incognita).

PubMed

Wubben, Martin J; Callahan, Franklin E; Velten, Jeff; Burke, John J; Jenkins, Johnie N

2015-02-01

Transgene-based analysis of the MIC-3 gene provides the first report of a cotton gene having a direct role in mediating cotton resistance to root-knot nematode. Major quantitative trait loci have been mapped to Upland cotton (Gossypium hirsutum L.) chromosomes 11 and 14 that govern the highly resistant phenotype in response to infection by root-knot nematode (RKN; Meloidogyne incognita); however, nearly nothing is known regarding the underlying molecular determinants of this RKN-resistant phenotype. Multiple lines of circumstantial evidence have strongly suggested that the MIC (Meloidogyne Induced Cotton) gene family plays an integral role in mediating cotton resistance to RKN. In this report, we demonstrate that overexpression of MIC-3 in the RKN-susceptible genetic background Coker 312 reduces RKN egg production by ca. 60-75 % compared to non-transgenic controls and transgene-null sibling lines. MIC-3 transcript and protein overexpression were confirmed in root tissues of multiple independent transgenic lines with each line showing a similar level of increased resistance to RKN. In contrast to RKN fecundity, transgenic lines showed RKN-induced root galling similar to the susceptible controls. In addition, we determined that this effect of MIC-3 overexpression was specific to RKN as no effect was observed on reniform nematode (Rotylenchulus reniformis) reproduction. Transgenic lines did not show obvious alterations in growth, morphology, flowering, or fiber quality traits. Gene expression analyses showed that MIC-3 transcript levels in uninfected transgenic roots exceeded levels observed in RKN-infected roots of naturally resistant plants and that overexpression did not alter the regulation of native MIC genes in the genome. These results are the first report describing a direct role for a specific gene family in mediating cotton resistance to a plant-parasitic nematode.

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

PubMed

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

PubMed

Pyeon, Hye-Rim; Nah, Hee-Ju; Kang, Seung-Hoon; Choi, Si-Sun; Kim, Eung-Soo

2017-05-31

Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem

An Introduction to Business German.

ERIC Educational Resources Information Center

Ambacher, Robert

At Millersville University (Pennsylvania), business German is taught in the German section in a two-semester introduction at the sophomore level, a junior-level advanced course, and a senior-level translation course. These four courses are augmented by introductions to business and economics, both taught in English outside the German section.…

15 CFR 740.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false Introduction. 740.1 Section 740.1... Introduction. In this part, references to the EAR are references to 15 CFR chapter VII, subchapter C. (a) Scope... availability of any License Exceptions. Special commodity controls apply to short supply items. License...

15 CFR 918.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 3 2010-01-01 2010-01-01 false Introduction. 918.1 Section 918.1 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) NATIONAL OCEANIC... Introduction. Pursuant to section 207 of the National Sea Grant College Program Act, as amended (Pub. L. 94-461...

15 CFR 12.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Introduction. 12.1 Section 12.1 Commerce and Foreign Trade Office of the Secretary of Commerce FAIR PACKAGING AND LABELING § 12.1 Introduction. (a) These procedures apply to the discharge of the responsibility given to the Secretary of...

15 CFR 764.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false Introduction. 764.1 Section 764.1 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF... MEASURES § 764.1 Introduction. In this part, references to the EAR are references to 15 CFR chapter VII...

15 CFR 770.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false Introduction. 770.1 Section 770.1 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF... Introduction. In this part, references to the EAR are references to 15 CFR chapter VII, subchapter C. This part...

15 CFR 746.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false Introduction. 746.1 Section 746.1 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF... CONTROLS § 746.1 Introduction. In this part, references to the EAR are references to 15 CFR chapter VII...

20 CFR 410.101 - Introduction.

Code of Federal Regulations, 2011 CFR

2011-04-01

...-BLACK LUNG BENEFITS (1969- ) Introduction, General Provisions, and Definitions § 410.101 Introduction... the provisions of part B (Black Lung Benefits) of title IV of the Federal Coal Mine Health and Safety Act of 1969, as enacted December 30, 1969, as amended by the Black Lung Benefits Act of 1972, and as...

22 CFR 5.1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Introduction. 5.1 Section 5.1 Foreign Relations DEPARTMENT OF STATE GENERAL ORGANIZATION § 5.1 Introduction. The sections in this part 5 are issued pursuant to section 3 of the Administrative Procedure Act, 5 U.S.C. 552, effective July 4, 1967. ...

32 CFR 651.32 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 4 2010-07-01 2010-07-01 true Introduction. 651.32 Section 651.32 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY (CONTINUED) ENVIRONMENTAL QUALITY ENVIRONMENTAL ANALYSIS OF ARMY ACTIONS (AR 200-2) Environmental Assessment § 651.32 Introduction. (a) An EA is intended to facilitate agency planning and...

Analysis of reliability of professor recommendation letters based on concordance with self-introduction letter.

PubMed

Kim, Sang Hyun

2013-12-01

The purpose of this study was to examine the concordance between a checklist's categories of professor recommendation letters and characteristics of the self-introduction letter. Checklists of professor recommendation letters were analyzed and classified into cognitive, social, and affective domains. Simple correlation was performed to determine whether the characteristics of the checklists were concordant with those of the self-introduction letter. The difference in ratings of the checklists by pass or fail grades was analyzed by independent sample t-test. Logistic regression analysis was performed to determine whether a pass or fail grade was influenced by ratings on the checklists. The Cronbach alpha value of the checklists was 0.854. Initiative, as an affective domain, in the professor's recommendation letter was highly ranked among the six checklist categories. Self-directed learning in the self-introduction letter was influenced by a pass or fail grade by logistic regression analysis (p<0.05). Successful applicants received higher ratings than those who failed in every checklist category, particularly in problem-solving ability, communication skills, initiative, and morality (p<0.05). There was a strong correlation between cognitive and affective characteristics in the professor recommendation letters and the sum of all characteristics in the self-introduction letter.

Reconstructing Asian faunal introductions to eastern Africa from multi-proxy biomolecular and archaeological datasets

PubMed Central

Buckley, Michael; Crowther, Alison; Frantz, Laurent; Eager, Heidi; Lebrasseur, Ophélie; Hutterer, Rainer; Hulme-Beaman, Ardern; Van Neer, Wim; Douka, Katerina; Veall, Margaret-Ashley; Quintana Morales, Eriéndira M.; Schuenemann, Verena J.; Reiter, Ella; Allen, Richard; Dimopoulos, Evangelos A.; Helm, Richard M.; Shipton, Ceri; Mwebi, Ogeto; Denys, Christiane; Horton, Mark; Wynne-Jones, Stephanie; Fleisher, Jeffrey; Radimilahy, Chantal; Wright, Henry; Searle, Jeremy B.; Krause, Johannes; Larson, Greger; Boivin, Nicole L.

2017-01-01

Human-mediated biological exchange has had global social and ecological impacts. In sub-Saharan Africa, several domestic and commensal animals were introduced from Asia in the pre-modern period; however, the timing and nature of these introductions remain contentious. One model supports introduction to the eastern African coast after the mid-first millennium CE, while another posits introduction dating back to 3000 BCE. These distinct scenarios have implications for understanding the emergence of long-distance maritime connectivity, and the ecological and economic impacts of introduced species. Resolution of this longstanding debate requires new efforts, given the lack of well-dated fauna from high-precision excavations, and ambiguous osteomorphological identifications. We analysed faunal remains from 22 eastern African sites spanning a wide geographic and chronological range, and applied biomolecular techniques to confirm identifications of two Asian taxa: domestic chicken (Gallus gallus) and black rat (Rattus rattus). Our approach included ancient DNA (aDNA) analysis aided by BLAST-based bioinformatics, Zooarchaeology by Mass Spectrometry (ZooMS) collagen fingerprinting, and direct AMS (accelerator mass spectrometry) radiocarbon dating. Our results support a late, mid-first millennium CE introduction of these species. We discuss the implications of our findings for models of biological exchange, and emphasize the applicability of our approach to tropical areas with poor bone preservation. PMID:28817590

Volume 1 - Introduction

EPA Pesticide Factsheets

An introduction to the Emissions Inventory Improvement Program (EIIP) materials. Describes EIIP development, use of EIIP, inventory staff training, and planning, development, documentation, and reporting of inventories.

A thousand bites - Insect introductions and late Holocene environments

NASA Astrophysics Data System (ADS)

Panagiotakopulu, Eva; Buckland, Paul C.

2017-01-01

The impact of insect species directly associated with man-made habitats and human dispersal has been, and remains globally significant. Their early expansion from their original niches into Europe is intrinsically related to discussions of climate change, origins of domesticated plants and animals, the spread of agriculture and infectious diseases. The Holocene fossil records of the dispersal of three storage pest species, Sitophilus granarius, Oryzaephilus surinamensis, and Tribolium castaneum, the housefly, Musca domestica, and the human flea, Pulex irritans from 221 sites have been mapped ranging from the Near East to Europe and from the Neolithic to the post medieval period. The importance of human induced change as a driver for the spread of synanthropic faunas and the potential for the spread of disease during this process are discussed. The results show links between mobility of farming groups and distribution of synanthropic insect species and produce a roadmap for the different cultural periods of the Late Holocene based on dispersal of these synanthropic insects. During the Neolithic, the first wave of insect introductions shows the northern European frontiers of storage of cereals, introduction of domestic animals and pastoralism and exchange. Pest introductions, linked with the itinerary of the Roman army, reached the most northerly parts of the Empire. During the medieval period, the insect records indicate further expansion and changes which parallel the spread of epidemic diseases like Plague. Understanding the timing and the rates of change of synanthropic insects provides key information about the development of the homogenised and highly anthropogenic environments in which we live today.

Computer Assisted Introduction to Mechanics.

ERIC Educational Resources Information Center

Huggins, Elisha R.

These six chapters provide an introduction to Newtonian mechanics, based on a coordinated use of text material, laboratory work, and the computer. The material is essentially self-contained so that it can serve as a short text on mechanics or as a text supplement in a regular physics course. Chapter 1 is a brief introduction to the computer…

EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos

PubMed Central

Auclair, Ghislain; Borgel, Julie; Sanz, Lionel A.; Vallet, Judith; Guibert, Sylvain; Dumas, Michael; Cavelier, Patricia; Girardot, Michael; Forné, Thierry; Feil, Robert; Weber, Michael

2016-01-01

The extent to which histone modifying enzymes contribute to DNA methylation in mammals remains unclear. Previous studies suggested a link between the lysine methyltransferase EHMT2 (also known as G9A and KMT1C) and DNA methylation in the mouse. Here, we used a model of knockout mice to explore the role of EHMT2 in DNA methylation during mouse embryogenesis. The Ehmt2 gene is expressed in epiblast cells but is dispensable for global DNA methylation in embryogenesis. In contrast, EHMT2 regulates DNA methylation at specific sequences that include CpG-rich promoters of germline-specific genes. These loci are bound by EHMT2 in embryonic cells, are marked by H3K9 dimethylation, and have strongly reduced DNA methylation in Ehmt2−/− embryos. EHMT2 also plays a role in the maintenance of germline-derived DNA methylation at one imprinted locus, the Slc38a4 gene. Finally, we show that DNA methylation is instrumental for EHMT2-mediated gene silencing in embryogenesis. Our findings identify EHMT2 as a critical factor that facilitates repressive DNA methylation at specific genomic loci during mammalian development. PMID:26576615

Transcriptome reprogramming due to the introduction of a barley telosome into bread wheat affects more barley genes than wheat.

PubMed

Rey, Elodie; Abrouk, Michael; Keeble-Gagnère, Gabriel; Karafiátová, Miroslava; Vrána, Jan; Balzergue, Sandrine; Soubigou-Taconnat, Ludivine; Brunaud, Véronique; Martin-Magniette, Marie-Laure; Endo, Takashi R; Bartoš, Jan; Appels, Rudi; Doležel, Jaroslav

2018-03-06

Despite a long history, the production of useful alien introgression lines in wheat remains difficult mainly due to linkage drag and incomplete genetic compensation. In addition, little is known about the molecular mechanisms underlying the impact of foreign chromatin on plant phenotype. Here, a comparison of the transcriptomes of barley, wheat and a wheat-barley 7HL addition line allowed the transcriptional impact both on 7HL genes of a non-native genetic background and on the wheat gene complement as a result of the presence of 7HL to be assessed. Some 42% (389/923) of the 7HL genes assayed were differentially transcribed, which was the case for only 3% (960/35 301) of the wheat gene complement. The absence of any transcript in the addition line of a suite of chromosome 7A genes implied the presence of a 36 Mbp deletion at the distal end of the 7AL arm; this deletion was found to be in common across the full set of Chinese Spring/Betzes barley addition lines. The remaining differentially transcribed wheat genes were distributed across the whole genome. The up-regulated barley genes were mostly located in the proximal part of the 7HL arm, while the down-regulated ones were concentrated in the distal part; as a result, genes encoding basal cellular functions tended to be transcribed, while those encoding specific functions were suppressed. An insight has been gained into gene transcription in an alien introgression line, thereby providing a basis for understanding the interactions between wheat and exotic genes in introgression materials. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Novel Directional Solidification Processing of Hypermonotectic Alloys

NASA Technical Reports Server (NTRS)

Grugel, Richard N.

1999-01-01

Gravity driven separation precludes uniform microstructural development during controlled directional solidification (DS) processing of hypermonotectic alloys. It is well established that liquid/liquid suspensions, in which the respective components are immiscible and have significant density differences, can be established and maintained by utilizing ultrasound. A historical introduction to this work is presented with the intent of establishing the basis for applying the phenomena to promote microstructural uniformity during controlled directional solidification processing of immiscible mixtures. Experimental work based on transparent organics, as well as salt systems, will be presented in view of the processing parameters.

The Receptor Tyrosine Kinase EphA2 Is a Direct Target Gene of Hypermethylated in Cancer 1 (HIC1)*

PubMed Central

Foveau, Bénédicte; Boulay, Gaylor; Pinte, Sébastien; Van Rechem, Capucine; Rood, Brian R.; Leprince, Dominique

2012-01-01

The tumor suppressor gene hypermethylated in cancer 1 (HIC1), which encodes a transcriptional repressor, is epigenetically silenced in many human tumors. Here, we show that ectopic expression of HIC1 in the highly malignant MDA-MB-231 breast cancer cell line severely impairs cell proliferation, migration, and invasion in vitro. In parallel, infection of breast cancer cell lines with a retrovirus expressing HIC1 also induces decreased mRNA and protein expression of the tyrosine kinase receptor EphA2. Moreover, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments demonstrate that endogenous HIC1 proteins are bound, together with the MTA1 corepressor, to the EphA2 promoter in WI38 cells. Taken together, our results identify EphA2 as a new direct target gene of HIC1. Finally, we observe that inactivation of endogenous HIC1 through RNA interference in normal breast epithelial cells results in the up-regulation of EphA2 and is correlated with increased cellular migration. To conclude, our results involve the tumor suppressor HIC1 in the transcriptional regulation of the tyrosine kinase receptor EphA2, whose ligand ephrin-A1 is also a HIC1 target gene. Thus, loss of the regulation of this Eph pathway through HIC1 epigenetic silencing could be an important mechanism in the pathogenesis of epithelial cancers. PMID:22184117

From gene engineering to gene modulation and manipulation: can we prevent or detect gene doping in sports?

PubMed

Fischetto, Giuseppe; Bermon, Stéphane

2013-10-01

During the last 2 decades, progress in deciphering the human gene map as well as the discovery of specific defective genes encoding particular proteins in some serious human diseases have resulted in attempts to treat sick patients with gene therapy. There has been considerable focus on human recombinant proteins which were gene-engineered and produced in vitro (insulin, growth hormone, insulin-like growth factor-1, erythropoietin). Unfortunately, these substances and methods also became improper tools for unscrupulous athletes. Biomedical research has focused on the possible direct insertion of gene material into the body, in order to replace some defective genes in vivo and/or to promote long-lasting endogenous synthesis of deficient proteins. Theoretically, diabetes, anaemia, muscular dystrophies, immune deficiency, cardiovascular diseases and numerous other illnesses could benefit from such innovative biomedical research, though much work remains to be done. Considering recent findings linking specific genotypes and physical performance, it is tempting to submit the young athletic population to genetic screening or, alternatively, to artificial gene expression modulation. Much research is already being conducted in order to achieve a safe transfer of genetic material to humans. This is of critical importance since uncontrolled production of the specifically coded protein, with serious secondary adverse effects (polycythaemia, acute cardiovascular problems, cancer, etc.), could occur. Other unpredictable reactions (immunogenicity of vectors or DNA-vector complex, autoimmune anaemia, production of wild genetic material) also remain possible at the individual level. Some new substances (myostatin blockers or anti-myostatin antibodies), although not gene material, might represent a useful and well-tolerated treatment to prevent progression of muscular dystrophies. Similarly, other molecules, in the roles of gene or metabolic activators [5-aminoimidazole-4

Introduction to Carpentry. Introduction to Construction Series. Instructor Edition.

ERIC Educational Resources Information Center

Oklahoma State Dept. of Vocational and Technical Education, Stillwater. Curriculum and Instructional Materials Center.

This competency-based curriculum guide on the specialty area of carpentry is part of the Introduction to Construction series. The series is designed with the flexible training requirements of open shop contractors, preapprenticeship programs, multicraft high school programs, technology education programs, and cooperative education programs in…

Introduction to Bricklaying. Introduction to Construction Series. Instructor Edition.

ERIC Educational Resources Information Center

Oklahoma State Dept. of Vocational and Technical Education, Stillwater. Curriculum and Instructional Materials Center.

This competency-based curriculum guide on the specialty area of bricklaying is part of the Introduction to Construction series. The series is designed with the flexible training requirements of open shop contractors, preapprenticeship programs, multicraft high school programs, technology education programs, and cooperative education programs in…

Multiple Introductions of Dengue 2 Virus Strains into Saudi Arabia from 1992 to 2014

PubMed Central

El-Kafrawy, Sherif A.; Sohrab, Sayed S.; Ela, Said Abol; Abd-Alla, Adly M.M.; Alhabbab, Rowa; Farraj, Suha A.; Othman, Norah A.; Hassan, Ahmed M.; Bergoin, Max; Klitting, Raphaelle; Charrel, Remi N.; Hashem, Anwar M.; Madani, Tariq A.

2016-01-01

Abstract Introduction: Dengue is a significant arboviral infection that represents a major public health concern worldwide. The infection is endemic in most parts of South East Asia, sub-Saharan Africa, and Latin America. Among the four dengue virus (DENV) serotypes, DENV-2 has been reported to be the predominant serotype in Saudi Arabia since 1992. However, virological and epidemiological data of DENV-2 from Saudi Arabia are severely deficient and require further investigations. Methods: Full genome sequencing of a recent DENV-2 isolate and phylogenetic analysis of all available DENV-2 sequences from Saudi Arabia. Results: Based on full genome and envelope (E) gene sequence, we show that a recent isolate (DENV-2-Jeddah-2014) belongs to the Indian subcontinent lineage of the Cosmopolitan genotype with close similarity to recent strains from Pakistan. Interestingly, the E gene sequence of DENV-2-Jeddah-2014 isolate was slightly divergent from those previously identified in Saudi Arabia between 1992 and 2004 with three to nine amino acid (aa) substitutions. While our data show that the Cosmopolitan genotype is still circulating in Saudi Arabia, they highlight four distinct genetic groups suggesting at least four independent introductions into the Kingdom. Conclusions: The close clustering of DENV-2 isolates reported from Saudi Arabia between 1992 and 2014 with strains from countries providing the highest numbers of pilgrims attending either Hajj or Umrah pilgrimages (Indonesia, Pakistan, India) clearly suggests a role for pilgrims or expatriates coming from DENV endemic countries in DENV-2 importation into Saudi Arabia. Accordingly, continuous monitoring of the circulation of DENVs in Saudi Arabia must be implemented to undertake effective control and management strategies in the Kingdom. Screening of the pilgrims coming to perform Hajj and Umrah might help prevent the introduction of new DENV strains, which is expected to increase the burden of the disease not only

Apoptosis Gene Information System--AGIS.

PubMed

Sakharkar, Kishore R; Clement, Marie V; Chow, Vincent T K; Pervaiz, Shazib

2006-05-01

Genes implicated in apoptosis have great relevance to biology, medicine and oncology. Here, we describe a unique resource, Apoptosis Gene Information System (AGIS) that provides data for over 2400 genes involved directly or indirectly, in apoptotic pathways of more than 350 different organisms. The organization of this information system is based on the principle of one-gene, one record. AGIS will be updated on a six monthly basis as new information becomes available. AGIS can be accessed at: http://www.cellfate.org/AGIS/.

Gene encoding plant asparagine synthetase

DOEpatents

Coruzzi, Gloria M.; Tsai, Fong-Ying

1993-10-26

The identification and cloning of the gene(s) for plant asparagine synthetase (AS), an important enzyme involved in the formation of asparagine, a major nitrogen transport compound of higher plants is described. Expression vectors constructed with the AS coding sequence may be utilized to produce plant AS; to engineer herbicide resistant plants, salt/drought tolerant plants or pathogen resistant plants; as a dominant selectable marker; or to select for novel herbicides or compounds useful as agents that synchronize plant cells in culture. The promoter for plant AS, which directs high levels of gene expression and is induced in an organ specific manner and by darkness, is also described. The AS promoter may be used to direct the expression of heterologous coding sequences in appropriate hosts.

Direct Activation of Amidohydrolase Domain-Containing 1 Gene by Thyroid Hormone Implicates a Role in the Formation of Adult Intestinal Stem Cells During Xenopus Metamorphosis

PubMed Central

Okada, Morihiro; Miller, Thomas C.; Fu, Liezhen

2015-01-01

The T3-dependent anuran metamorphosis resembles postembryonic development in mammals, the period around birth when plasma T3 levels peak. In particular, the remodeling of the intestine during metamorphosis mimics neonatal intestinal maturation in mammals when the adult intestinal epithelial self-renewing system is established. We have been using intestinal metamorphosis to investigate how the organ-specific adult stem cells are formed during vertebrate development. Early studies in Xenopus laevis have shown that this process involves complete degeneration of the larval epithelium and de novo formation of adult stem cells. A tissue-specific microarray analysis of intestinal gene expression during Xenopus laevis metamorphosis has identified a number of candidate stem cell genes. Here we have carried out detailed analyses of one such gene, amidohydrolase domain containing 1 (AMDHD1) gene, which encodes an enzyme in the histidine catabolic pathway. We show that AMDHD1 is exclusively expressed in the proliferating adult epithelial stem cells during metamorphosis with little expression in other intestinal tissues. We further provide evidence that T3 activates AMDHD1 gene expression directly at the transcription level through T3 receptor binding to the AMDHD1 gene in the intestine. In addition, we have reported earlier that histidine ammonia-lyase gene, another gene in histidine catabolic pathway, is similarly regulated by T3 in the intestine. These results together suggest that histidine catabolism plays a critical role in the formation and/or proliferation of adult intestinal stem cells during metamorphosis. PMID:26086244

Direct Activation of Amidohydrolase Domain-Containing 1 Gene by Thyroid Hormone Implicates a Role in the Formation of Adult Intestinal Stem Cells During Xenopus Metamorphosis.

PubMed

Okada, Morihiro; Miller, Thomas C; Fu, Liezhen; Shi, Yun-Bo

2015-09-01

The T3-dependent anuran metamorphosis resembles postembryonic development in mammals, the period around birth when plasma T3 levels peak. In particular, the remodeling of the intestine during metamorphosis mimics neonatal intestinal maturation in mammals when the adult intestinal epithelial self-renewing system is established. We have been using intestinal metamorphosis to investigate how the organ-specific adult stem cells are formed during vertebrate development. Early studies in Xenopus laevis have shown that this process involves complete degeneration of the larval epithelium and de novo formation of adult stem cells. A tissue-specific microarray analysis of intestinal gene expression during Xenopus laevis metamorphosis has identified a number of candidate stem cell genes. Here we have carried out detailed analyses of one such gene, amidohydrolase domain containing 1 (AMDHD1) gene, which encodes an enzyme in the histidine catabolic pathway. We show that AMDHD1 is exclusively expressed in the proliferating adult epithelial stem cells during metamorphosis with little expression in other intestinal tissues. We further provide evidence that T3 activates AMDHD1 gene expression directly at the transcription level through T3 receptor binding to the AMDHD1 gene in the intestine. In addition, we have reported earlier that histidine ammonia-lyase gene, another gene in histidine catabolic pathway, is similarly regulated by T3 in the intestine. These results together suggest that histidine catabolism plays a critical role in the formation and/or proliferation of adult intestinal stem cells during metamorphosis.

Introduction to Drywall. Introduction to Construction Series. Instructor Edition.

ERIC Educational Resources Information Center

Oklahoma State Dept. of Vocational and Technical Education, Stillwater. Curriculum and Instructional Materials Center.

This competency-based curriculum guide on the specialty area of drywall is part of the Introduction to Construction series. The series is designed with the flexible training requirements of open shop contractors, preapprenticeship programs, multicraft high school programs, technology education programs, and cooperative education programs in mind.…

Introduction to Plumbing. Introduction to Construction Series. Instructor Edition.

ERIC Educational Resources Information Center

Oklahoma State Dept. of Vocational and Technical Education, Stillwater. Curriculum and Instructional Materials Center.

This competency-based curriculum guide on the specialty area of plumbing is part of the Introduction to Construction series. The series is designed with the flexible training requirements of open shop contractors, preapprenticeship programs, multicraft high school programs, technology education programs, and cooperative education programs in mind.…

Introduction of direct-to-consumer advertising of prescription drugs in Canada: an opinion survey on regulatory policy.

PubMed

Mintzes, Barbara; Barer, Morris; Lexchin, Joel; Bassett, Ken L

2005-06-01

Canada is strongly influenced by US cross-border direct-to-consumer advertising (DTCA) and has held consultations to discuss introduction of DTCA since 1996. This article describes a survey of Canadian drug policy experts carried out in 2001, during one such legislative review. The survey results are compared to more recent DTCA policy developments. We recruited key informants on pharmaceutical policy to complete a faxed questionnaire that queried their opinions on DTCA information quality, effects on drug and health care use, and regulatory issues. Respondents were asked about the evidence they had used to back their opinions. Analysis was descriptive. Of 79 identified potential participants, 60 (76%) participated, 40% of whom were from federal and provincial government; 3% were private insurers; 18%, 15%, and 8% were from health professional groups, consumer groups, and patient groups, respectively; 8% and 7% were from pharmaceutical and advertising industries, respectively. Opinions were highly polarized on the effects of DTCA on drug and health care use. Advertising and pharmaceutical industry respondents were generally positive, public sector, health professional and consumer groups generally negative. Over 80% believed DTCA leads to higher private and public drug costs and more frequent physician visits. Fewer judged billboards or television to be appropriate media for DTCA than magazines or the Internet, and most believed that children and adolescents should not be targeted. Given the polarization observed within this survey, we examined how DTCA policy has evolved in Canada since 2001. The federal government has legislative authority over DTCA, but bears few of the additional costs potentially incurred through policy change. These fall to the provinces, which provide an eroding patchwork of public coverage for prescription drugs in the face of rapidly increasing costs. No new federal legislation has been tabled since 2001. However, considerable shifts in

[Gene therapy for inherited retinal dystrophies].

PubMed

Côco, Monique; Han, Sang Won; Sallum, Juliana Maria Ferraz

2009-01-01

The inherited retinal dystrophies comprise a large number of disorders characterized by a slow and progressive retinal degeneration. They are the result of mutations in genes that express in either the photoreceptor cells or the retinal pigment epithelium. The mode of inheritance can be autosomal dominant, autosomal recessive, X linked recessive, digenic or mitochondrial DNA inherited. At the moment, there is no treatment for these conditions and the patients can expect a progressive loss of vision. Accurate genetic counseling and support for rehabilitation are indicated. Research into the molecular and genetic basis of disease is continually expanding and improving the prospects for rational treatments. In this way, gene therapy, defined as the introduction of exogenous genetic material into human cells for therapeutic purposes, may ultimately offer the greatest treatment for the inherited retinal dystrophies. The eye is an attractive target for gene therapy because of its accessibility, immune privilege and translucent media. A number of retinal diseases affecting the eye have known gene defects. Besides, there is a well characterized animal model for many of these conditions. Proposals for clinical trials of gene therapy for inherited retinal degenerations owing to defects in the gene RPE65, have recently received ethical approval and the obtained preliminary results brought large prospects in the improvement on patient's quality of life.

Production of gamma-aminobutyric acid from glucose by introduction of synthetic scaffolds between isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase in recombinant Escherichia coli.

PubMed

Pham, Van Dung; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

2015-08-10

Escherichia coli were engineered for the direct production of gamma-aminobutyric acid from glucose by introduction of synthetic protein scaffold. In this study, three enzymes consisting GABA pathway (isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase) were connected via synthetic protein scaffold. By introduction of scaffold, 0.92g/L of GABA was produced from 10g/L of glucose while no GABA was produced in wild type E. coli. The optimum pH and temperature for GABA production were 4.5 and 30°C, respectively. When competing metabolic network was inactivated by knockout mutation, maximum GABA concentration of 1.3g/L was obtained from 10g/L glucose. The recombinant E. coli strain which produces GABA directly from glucose was successfully constructed by introduction of protein scaffold. Copyright © 2015 Elsevier B.V. All rights reserved.

Revealing networks from dynamics: an introduction

NASA Astrophysics Data System (ADS)

Timme, Marc; Casadiego, Jose

2014-08-01

What can we learn from the collective dynamics of a complex network about its interaction topology? Taking the perspective from nonlinear dynamics, we briefly review recent progress on how to infer structural connectivity (direct interactions) from accessing the dynamics of the units. Potential applications range from interaction networks in physics, to chemical and metabolic reactions, protein and gene regulatory networks as well as neural circuits in biology and electric power grids or wireless sensor networks in engineering. Moreover, we briefly mention some standard ways of inferring effective or functional connectivity.

Cystic fibrosis gene modifier SLC26A9 modulates airway response to CFTR-directed therapeutics.

PubMed

Strug, Lisa J; Gonska, Tanja; He, Gengming; Keenan, Katherine; Ip, Wan; Boëlle, Pierre-Yves; Lin, Fan; Panjwani, Naim; Gong, Jiafen; Li, Weili; Soave, David; Xiao, Bowei; Tullis, Elizabeth; Rabin, Harvey; Parkins, Michael D; Price, April; Zuberbuhler, Peter C; Corvol, Harriet; Ratjen, Felix; Sun, Lei; Bear, Christine E; Rommens, Johanna M

2016-10-15

Cystic fibrosis is realizing the promise of personalized medicine. Recent advances in drug development that target the causal CFTR directly result in lung function improvement, but variability in response is demanding better prediction of outcomes to improve management decisions. The genetic modifier SLC26A9 contributes to disease severity in the CF pancreas and intestine at birth and here we assess its relationship with disease severity and therapeutic response in the airways. SLC26A9 association with lung disease was assessed in individuals from the Canadian and French CF Gene Modifier consortia with CFTR-gating mutations and in those homozygous for the common Phe508del mutation. Variability in response to a CFTR-directed therapy attributed to SLC26A9 genotype was assessed in Canadian patients with gating mutations. A primary airway model system determined if SLC26A9 shows modification of Phe508del CFTR function upon treatment with a CFTR corrector. In those with gating mutations that retain cell surface-localized CFTR we show that SLC26A9 modifies lung function while this is not the case in individuals homozygous for Phe508del where cell surface expression is lacking. Treatment response to ivacaftor, which aims to improve CFTR-channel opening probability in patients with gating mutations, shows substantial variability in response, 28% of which can be explained by rs7512462 in SLC26A9 (P = 0.0006). When homozygous Phe508del primary bronchial cells are treated to restore surface CFTR, SLC26A9 likewise modifies treatment response (P = 0.02). Our findings indicate that SLC26A9 airway modification requires CFTR at the cell surface, and that a common variant in SLC26A9 may predict response to CFTR-directed therapeutics.

Identification of Genes Expressed in Premalignant Breast Disease by Microscopy-Directed Cloning

NASA Astrophysics Data System (ADS)

Jensen, Roy A.; Page, David L.; Holt, Jeffrey T.

1994-09-01

Histopathologic study of human breast biopsy samples has identified specific lesions which are associated with a high risk of development of invasive breast cancer. Presumably, these lesions (collectively termed premalignant breast disease) represent the earliest recognizable morphologic expression of fundamental molecular events that lead to the development of invasive breast cancer. To study molecular events underlying premalignant breast disease, we have developed a method for isolating RNA from histologically identified lesions from frozen human breast tissue. This method specifically obtains mRNA from breast epithelial cells and has identified three genes which are differentially expressed in premalignant breast epithelial lesions. One gene identified by this method is overexpressed in four of five noncomedo ductal carcinoma in situ lesions and appears to be the human homologue of the gene encoding the M2 subunit of ribonucleotide reductase, an enzyme involved in DNA synthesis.

Genome mining-directed activation of a silent angucycline biosynthetic gene cluster in Streptomyces chattanoogensis.

PubMed

Zhou, Zhenxing; Xu, Qingqing; Bu, Qingting; Guo, Yuanyang; Liu, Shuiping; Liu, Yu; Du, Yiling; Li, Yongquan

2015-02-09

Genomic sequencing of actinomycetes has revealed the presence of numerous gene clusters seemingly capable of natural product biosynthesis, yet most clusters are cryptic under laboratory conditions. Bioinformatics analysis of the completely sequenced genome of Streptomyces chattanoogensis L10 (CGMCC 2644) revealed a silent angucycline biosynthetic gene cluster. The overexpression of a pathway-specific activator gene under the constitutive ermE* promoter successfully triggered the expression of the angucycline biosynthetic genes. Two novel members of the angucycline antibiotic family, chattamycins A and B, were further isolated and elucidated. Biological activity assays demonstrated that chattamycin B possesses good antitumor activities against human cancer cell lines and moderate antibacterial activities. The results presented here provide a feasible method to activate silent angucycline biosynthetic gene clusters to discover potential new drug leads. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Matching genetics with oceanography: directional gene flow in a Mediterranean fish species.

PubMed

Schunter, C; Carreras-Carbonell, J; Macpherson, E; Tintoré, J; Vidal-Vijande, E; Pascual, A; Guidetti, P; Pascual, M

2011-12-01

Genetic connectivity and geographic fragmentation are two opposing mechanisms determining the population structure of species. While the first homogenizes the genetic background across populations the second one allows their differentiation. Therefore, knowledge of processes affecting dispersal of marine organisms is crucial to understand their genetic distribution patterns and for the effective management of their populations. In this study, we use genetic analyses of eleven microsatellites in combination with oceanographic satellite and dispersal simulation data to determine distribution patterns for Serranus cabrilla, a ubiquitous demersal broadcast spawner, in the Mediterranean Sea. Pairwise population F(ST) values ranged between -0.003 and 0.135. Two genetically distinct clusters were identified, with a clear division located between the oceanographic discontinuities at the Ibiza Channel (IC) and the Almeria-Oran Front (AOF), revealing an admixed population in between. The Balearic Front (BF) also appeared to dictate population structure. Directional gene flow on the Spanish coast was observed as S. cabrilla dispersed from west to east over the AOF, from north to south on the IC and from south of the IC towards the Balearic Islands. Correlations between genetic and oceanographic data were highly significant. Seasonal changes in current patterns and the relationship between ocean circulation patterns and spawning season may also play an important role in population structure around oceanographic fronts. © 2011 Blackwell Publishing Ltd.

Multiple introductions of a reassortant H5N1 avian influenza virus of clade 2.3.2.1c with PB2 gene of H9N2 subtype into Indian poultry.

PubMed

Tosh, Chakradhar; Nagarajan, Shanmugasundaram; Kumar, Manoj; Murugkar, Harshad V; Venkatesh, Govindarajulu; Shukla, Shweta; Mishra, Amit; Mishra, Pranav; Agarwal, Sonam; Singh, Bharati; Dubey, Prashant; Tripathi, Sushil; Kulkarni, Diwakar D

2016-09-01

Highly pathogenic avian influenza (HPAI) H5N1 viruses are a threat to poultry in Asia, Europe, Africa and North America. Here, we report isolation and characterization of H5N1 viruses isolated from ducks and turkeys in Kerala, Chandigarh and Uttar Pradesh, India between November 2014 and March 2015. Genetic and phylogenetic analyses of haemagglutinin gene identified that the virus belonged to a new clade 2.3.2.1c which has not been detected earlier in Indian poultry. The virus possessed molecular signature for high pathogenicity to chickens, which was corroborated by intravenous pathogenicity index of 2.96. The virus was a reassortant which derives its PB2 gene from H9N2 virus isolated in China during 2007-2013. However, the neuraminidase and internal genes are of H5N1 subtype. Phylogenetic and network analysis revealed that after detection in China in 2013/2014, the virus moved to Europe, West Africa and other Asian countries including India. The analyses further indicated multiple introductions of H5N1 virus in Indian poultry and internal spread in Kerala. One of the outbreaks in ducks in Kerala is linked to the H5N1 virus isolated from wild birds in Dubai suggesting movement of virus probably through migration of wild birds. However, the outbreaks in ducks in Chandigarh and Uttar Pradesh were from an unknown source in Asia which also contributed gene pools to the outbreaks in Europe and West Africa. The widespread incidence of the novel H5N1 HPAI is similar to the spread of clade 2.2 ("Qinghai-like") virus in 2005, and should be monitored to avoid threat to animal and public health. Copyright © 2016 Elsevier B.V. All rights reserved.

Uncoordinated MAC for Adaptive Multi Beam Directional Networks: Analysis and Evaluation

DTIC Science & Technology

2016-08-01

control (MAC) policies for emerging systems that are equipped with fully digital antenna arrays which are capable of adaptive multi-beam directional...Adaptive Beam- forming, Multibeam, Directional Networking, Random Access, Smart Antennas I. INTRODUCTION Fully digital beamforming antenna arrays that...are capable of adaptive multi-beam communications are quickly becoming a reality. These antenna arrays allow users to form multiple simultaneous

Phylogenetic analysis reveals multiple introductions of Cynodon species in Australia.

PubMed

Jewell, M; Frère, C H; Harris-Shultz, K; Anderson, W F; Godwin, I D; Lambrides, C J

2012-11-01

The distinction between native and introduced flora within isolated land masses presents unique challenges. The geological and colonisation history of Australia, the world's largest island, makes it a valuable system for studying species endemism, introduction, and phylogeny. Using this strategy we investigated Australian cosmopolitan grasses belonging to the genus Cynodon. While it is believed that seven species of Cynodon are present in Australia, no genetic analyses have investigated the origin, diversity and phylogenetic history of Cynodon within Australia. To address this gap, 147 samples (92 from across Australia and 55 representing global distribution) were sequenced for a total of 3336bp of chloroplast DNA spanning six genes. Data showed the presence of at least six putatively introduced Cynodon species (C. transvaalensis, C. incompletus, C. hirsutus, C. radiatus, C. plectostachyus and C. dactylon) in Australia and suggested multiple recent introductions. C. plectostachyus, a species often confused with C. nlemfuensis, was not previously considered to be present in Australia. Most significantly, we identified two common haplotypes that formed a monophyletic clade diverging from previously identified Cynodon species. We hypothesise that these two haplotypes may represent a previously undescribed species of Cynodon. We provide further evidence that two Australian native species, Brachyachne tenella and B. convergens belong in the genus Cynodon and, therefore, argue for the taxonomic revision of the genus Cynodon. Copyright © 2012 Elsevier Inc. All rights reserved.

Nano-scale gene delivery systems; current technology, obstacles, and future directions.

PubMed

Garcia-Guerra, Antonio; Dunwell, Thomas L; Trigueros, Sonia

2018-01-07

Within the different applications of nanomedicine currently being developed, nano-gene delivery is appearing as an exciting new technique with the possibility to overcome recognised hurdles and fulfill several biological and medical needs. The central component of all delivery systems is the requirement for the delivery of genetic material into cells, and for them to eventually reside in the nucleus where their desired function will be exposed. However, genetic material does not passively enter cells; thus, a delivery system is necessary. The emerging field of nano-gene delivery exploits the use of new materials and the properties that arise at the nanometre-scale to produce delivery vectors that can effectively deliver genetic material into a variety of different types of cells. The novel physicochemical properties of the new delivery vectors can be used to address the current challenges existing in nucleic acid delivery in vitro and in vivo. While there is a growing interest in nanostructure-based gene delivery, the field is still in its infancy, and there is yet much to discover about nanostructures and their physicochemical properties in a biological context. We carry out an organized and focused search of bibliographic databases. Our results suggest that despite new breakthroughs in nanostructure synthesis and advanced characterization techniques, we still face many barriers in producing highly efficient and non-toxic delivery systems. In this review, we overview the types of systems currently used for clinical and biomedical research applications along with their advantages and disadvantages, as well as discussing barriers that arise from nano-scale interactions with biological material. In conclusion, we hope that by bringing the far reaching multidisciplinary nature of nano-gene delivery to light, new targeted nanotechnology-bases strategies are developed to overcome the major challenges covered in this review. Copyright© Bentham Science Publishers; For

PCR-based detection of gene transfer vectors: application to gene doping surveillance.

PubMed

Perez, Irene C; Le Guiner, Caroline; Ni, Weiyi; Lyles, Jennifer; Moullier, Philippe; Snyder, Richard O

2013-12-01

Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.

Gene-Environment Interactions in Genome-Wide Association Studies: Current Approaches and New Directions

ERIC Educational Resources Information Center

Winham, Stacey J.; Biernacka, Joanna M.

2013-01-01

Background: Complex psychiatric traits have long been thought to be the result of a combination of genetic and environmental factors, and gene-environment interactions are thought to play a crucial role in behavioral phenotypes and the susceptibility and progression of psychiatric disorders. Candidate gene studies to investigate hypothesized…

Comparison of gene delivery techniques for therapeutic angiogenesis ultrasound-mediated destruction of carrier microbubbles versus direct intramuscular injection.

PubMed

Kobulnik, Jeremy; Kuliszewski, Michael A; Stewart, Duncan J; Lindner, Jonathan R; Leong-Poi, Howard

2009-10-27

This study was designed to compare the efficacy of angiogenic gene delivery by ultrasound-mediated (UM) destruction of intravenous carrier microbubbles to direct intramuscular (IM) injections. Current trials of gene therapy for angiogenesis remain limited by suboptimal, invasive delivery techniques. Hind-limb ischemia was produced by iliac artery ligation in 99 rats. In 32 rats, UM delivery of green fluorescent protein (GFP)/vascular endothelial growth factor-165 (VEGF(165)) plasmid deoxyribonucleic acid was performed. Thirty-five animals received IM injections of VEGF(165)/GFP plasmid. Remaining rats received no treatment. Before delivery (day 14 after ligation) and at days 17, 21, and 28 and week 8 after ligation, microvascular blood volume and microvascular blood flow to the proximal hind limbs were assessed by contrast-enhanced ultrasound (n = 8 per group). Total transfection was assessed by reverse transcriptase-polymerase chain reaction, and localization of transfection was determined by immunohistochemistry. By day 28, both IM and UM delivery of VEGF(165) produced significant increases in microvascular blood volume and microvascular blood flow. Whereas increases in microvascular blood volume were similar between treatment groups, microvascular blood flow was greater (p < 0.005) in UM-treated animals as compared with IM-treated animals, persisting to week 8. The VEGF(165)/GFP messenger ribonucleic acid expression was greater (p < 0.05) for IM-treated animals. A strong GFP signal was detected for both groups and was localized to focal perivascular regions and myocytes around injection sites for IM and to the vascular endothelium of arterioles/capillaries in a wider distribution for UM delivery. Despite lower transfection levels, UM delivery of VEGF(165) is as effective as IM injections. The UM delivery results in directed vascular transfection over a wider distribution, which may account for the more efficient angiogenesis.

The Argumentative Introduction in Oral Interpretation.

ERIC Educational Resources Information Center

Mills, Daniel; Gaer, David C.

A study examined introductions used in competitive oral interpretation events. A total of 97 introductions (from four oral interpretation events at a nationally recognized Midwestern intercollegiate forensic tournament) were analyzed using four categories: Descriptive, Simple Theme, Descriptive and Simple Theme, and Argumentative Theme. Results…

Maternal breastfeeding, early introduction of non-breast milk, and excess weight in preschoolers.

PubMed

Nascimento, Viviane Gabriela; da Silva, Janaína Paula Costa; Ferreira, Patrícia Calesco; Bertoli, Ciro João; Leone, Claudio

2016-12-01

Investigate associations between excess weight in preschool children, breastfeeding duration and age of non-breast milk introduction. Cross-sectional study of a representative sample of 817 preschool children, aged 2 to 4 years, attending municipal day care centers in the city of Taubaté. The weight and height of children were measured in the day care centers in 2009, 2010 and 2011. The body mass index z-score (BMIz) was calculated and children were classified as risk of overweight (BMIz≥1 to<2) or excess weight (BMIz≥2). Data analysis was carried out by comparison of proportions, coefficient of correlation and multivariate linear regression. The prevalence of risk of overweight was 18.9% and of excess weight (overweight or obesity) was 9.3%. The median duration of breastfeeding and age of introduction of non-breast milk was 6 months. The child's BMIz showed direct correlation with birth weight (r=0.154; p<0.001) and maternal Body Mass Index (BMI) (r=0.113; p=0.002). The correlation was inverse with the total duration of breastfeeding (r=-0.099; p=0.006) and age at non-breast milk introduction (r=-0.112; p=0.002). There was no correlation between the child's BMIz with birth length, duration of exclusive breastfeeding and mother's age. The earlier the introduction of non-breast milk, the higher the correlation with excess weight at preschool age. Copyright © 2016. Publicado por Elsevier Editora Ltda.

Maternal breastfeeding, early introduction of non-breast milk, and excess weight in preschoolers

PubMed Central

Nascimento, Viviane Gabriela; da Silva, Janaína Paula Costa; Ferreira, Patrícia Calesco; Bertoli, Ciro João; Leone, Claudio

2016-01-01

Abstract Objective: Investigate associations between excess weight in preschool children, breastfeeding duration and age of non-breast milk introduction. Methods: Cross-sectional study of a representative sample of 817 preschool children, aged 2-4 years, attending municipal day care centers in the city of Taubaté. The weight and height of children were measured in the day care centers in 2009, 2010 and 2011. The body mass index z-score (BMIz) was calculated and children were classified as risk of overweight (BMIz≥1 to<2) or excess weight (BMIz≥2). Data analysis was carried out by comparison of proportions, coefficient of correlation and multivariate linear regression. Results: The prevalence of risk of overweight was 18.9% and of excess weight (overweight or obesity) was 9.3%. The median duration of breastfeeding and age of introduction of non-breast milk was 6 months. The child's BMIz showed direct correlation with birth weight (r=0.154; p<0.001) and maternal body mass index (BMI) (r=0.113; p=0.002). The correlation was inverse with the total duration of breastfeeding (r=−0.099; p=0.006) and age at non-breast milk introduction (r=−0.112; p=0.002). There was no correlation between the child's BMIz with birth length, duration of exclusive breastfeeding and mother's age. Conclusions: The earlier the introduction of non-breast milk, the higher the correlation with excess weight at preschool age. PMID:27452430

Introduction to metallic nanoparticles

PubMed Central

Mody, Vicky V.; Siwale, Rodney; Singh, Ajay; Mody, Hardik R.

2010-01-01

Metallic nanoparticles have fascinated scientist for over a century and are now heavily utilized in biomedical sciences and engineering. They are a focus of interest because of their huge potential in nanotechnology. Today these materials can be synthesized and modified with various chemical functional groups which allow them to be conjugated with antibodies, ligands, and drugs of interest and thus opening a wide range of potential applications in biotechnology, magnetic separation, and preconcentration of target analytes, targeted drug delivery, and vehicles for gene and drug delivery and more importantly diagnostic imaging. Moreover, various imaging modalities have been developed over the period of time such as MRI, CT, PET, ultrasound, SERS, and optical imaging as an aid to image various disease states. These imaging modalities differ in both techniques and instrumentation and more importantly require a contrast agent with unique physiochemical properties. This led to the invention of various nanoparticulated contrast agent such as magnetic nanoparticles (Fe3O4), gold, and silver nanoparticles for their application in these imaging modalities. In addition, to use various imaging techniques in tandem newer multifunctional nanoshells and nanocages have been developed. Thus in this review article, we aim to provide an introduction to magnetic nanoparticles (Fe3O4), gold nanoparticles, nanoshells and nanocages, and silver nanoparticles followed by their synthesis, physiochemical properties, and citing some recent applications in the diagnostic imaging and therapy of cancer. PMID:21180459

The global regulator of pathogenesis PnCon7 positively regulates Tox3 effector gene expression through direct interaction in the wheat pathogen Parastagonospora nodorum.

PubMed

Lin, Shao-Yu; Chooi, Yit-Heng; Solomon, Peter S

2018-05-03

To investigate effector gene regulation in the wheat pathogenic fungus Parastagonospora nodorum, the promoter and expression of Tox3 was characterised through a series of complementary approaches. Promoter deletion and DNase I footprinting experiments identified a 25 bp region in the Tox3 promoter as being required for transcription. Subsequent yeast one-hybrid analysis using the DNA sequence as bait identified that interacting partner as the C2H2 zinc finger transcription factor PnCon7, a putative master regulator of pathogenesis. Silencing of PnCon7 resulted in the down-regulation of Tox3 demonstrating that the transcription factor has a positive regulatory role on gene expression. Analysis of Tox3 expression in the PnCon7 silenced strains revealed a strong correlation with PnCon7 transcript levels, supportive of a direct regulatory role. Subsequent pathogenicity assays using PnCon7-silenced isolates revealed that the transcription factor was required for Tox3-mediated disease. The expression of two other necrotrophic effectors (ToxA and Tox1) was also affected but in a non-dose dependent manner suggesting that the regulatory role of PnCon7 on these genes was indirect. Collectively, these data have advanced our fundamental understanding of the Con7 master regulator of pathogenesis by demonstrating its positive regulatory role on the Tox3 effector in P. nodorum through direct interaction. This article is protected by copyright. All rights reserved. © 2018 John Wiley & Sons Ltd.

Fall Protection Introduction, #33462

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

Chochoms, Michael

The proper use of fall prevention and fall protection controls can reduce the risk of deaths and injuries caused by falls. This course, Fall Protection Introduction (#33462), is designed as an introduction to various types of recognized fall prevention and fall protection systems at Los Alamos National Laboratory (LANL), including guardrail systems, safety net systems, fall restraint systems, and fall arrest systems. Special emphasis is given to the components, inspection, care, and storage of personal fall arrest systems (PFASs). This course also presents controls for falling object hazards and emergency planning considerations for persons who have fallen.

Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium.

PubMed

Die, Jose V; Baldwin, Ransom L; Rowland, Lisa J; Li, Robert; Oh, Sunghee; Li, Congjun; Connor, Erin E; Ranilla, Maria-Jose

2017-01-01

The rumen is lined on the luminal side by a stratified squamous epithelium that is responsible for not only absorption, but also transport, extensive short-chain fatty acid (SCFA) metabolism and protection. Butyrate has been demonstrated to initiate the differentiation of the tissue following introduction of solid feed to the weaning neonate as well as affecting the metabolism of other nutrients and absorption of nutrients in in vitro experiments. The objective of the present study was to validate expression stability of eight putative reference genes bovine rumen, considering the intrinsic heterogeneity of bovine rumen with regard to different luminal characteristics due to direct infusion of butyrate to double the intra-ruminal content of the rumen liquor. Our focus was on identifying stable reference genes which are suitable to normalize real-time RT-qPCR experiments from rumen samples collected from clinical assays, irrespective of localization within the organ and the across physiological state. The most stably expressed genes included: ACTB, UXT, DBNDD2, RPS9, DDX54 and HMBS. Their high stability values suggest these reference genes will facilitate better evaluation of variation of across an array of conditions including: localization within the rumen, differences among cattle fed an array of rations, as well as response to development in the weaning animal. Moreover, we anticipate these reference genes may be useful for expression studies in other ruminants.

Reduced risk of peanut sensitization following exposure through breast-feeding and early peanut introduction.

PubMed

Pitt, Tracy J; Becker, Allan B; Chan-Yeung, Moira; Chan, Edmond S; Watson, Wade T A; Chooniedass, Rishma; Azad, Meghan B

2018-02-01

Recent trials have shown that avoiding peanuts during infancy increases the risk of peanut allergy; however, these studies did not address maternal peanut consumption. We sought to investigate the relationship between maternal peanut consumption while breast-feeding, timing of direct peanut introduction, and peanut sensitization at age 7 years. Secondary analysis of a nested cohort within the 1995 Canadian Asthma Primary Prevention Study intervention study was performed. Breast-feeding and maternal and infant peanut consumption were captured by repeated questionnaires during infancy. Skin prick testing for peanut sensitization was performed at age 7 years. Overall, 58.2% of mothers consumed peanuts while breast-feeding and 22.5% directly introduced peanuts to their infant by 12 months. At 7 years, 9.4% of children were sensitized to peanuts. The lowest incidence (1.7%) was observed among children whose mothers consumed peanuts while breast-feeding and directly introduced peanuts before 12 months. Incidence was significantly higher (P < .05) if mothers consumed peanuts while breast-feeding but delayed introducing peanuts to their infant beyond 12 months (15.1%), or if mothers avoided peanuts themselves but directly introduced peanuts by 12 months (17.6%). Interaction analyses controlling for study group and maternal atopy confirmed that maternal peanut consumption while breast-feeding and infant peanut consumption by 12 months were protective in combination, whereas either exposure in isolation was associated with an increased risk of sensitization (P interaction = .003). In this secondary analysis, maternal peanut consumption while breast-feeding paired with direct introduction of peanuts in the first year of life was associated with the lowest risk of peanut sensitization, compared with all other combinations of maternal and infant peanut consumption. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All

Direct reaction theories for exotic nuclei: An introduction via semi-classical methods

NASA Astrophysics Data System (ADS)

Bonaccorso, Angela

2018-07-01

The structure of exotic nuclei has only been studied from around 1985, because they are very short lived and because before that, it was not possible to produce and deliver them as beams on a target. They have large N / Z or Z / N ratios, are weakly bound and quite extended most of the time. Thus breakup, transfer and/or inelastic excitations of the surface are some of their most common reaction mechanisms. Direct reactions, for their simplicity, have played a fundamental role in the last thirty years in the process of understanding such "new" type of structures. On the other hand, direct reactions have been studied and understood for a much longer time, starting with the pioneering experiments in the early '50 on deuteron-induced reactions and the reaction models developed by S.T. Butler and collaborators. Both subjects are extremely vast and there is a large literature available of books, review articles and original papers. I will discuss here only a few selected examples of the many interesting problems that have been encountered and solved in all those years. I consider them breakthroughs in the field and as such I hope they can inspire young generations of researchers.

Site-directed introduction of disulfide groups on antibodies for highly sensitive immunosensors.

PubMed

Acero Sánchez, Josep Ll; Fragoso, Alex; Joda, Hamdi; Suárez, Guillaume; McNeil, Calum J; O'Sullivan, Ciara K

2016-07-01

The interface between the sample and the transducer surface is critical to the performance of a biosensor. In this work, we compared different strategies for covalent self-assembly of antibodies onto bare gold substrates by introducing disulfide groups into the immunoglobulin structure, which acted as anchor molecules able to chemisorb spontaneously onto clean gold surfaces. The disulfide moieties were chemically introduced to the antibody via the primary amines, carboxylic acids, and carbohydrates present in its structure. The site-directed modification via the carbohydrate chains exhibited the best performance in terms of analyte response using a model system for the detection of the stroke marker neuron-specific enolase. SPR measurements clearly showed the potential for creating biologically active densely packed self-assembled monolayers (SAMs) in a one-step protocol compared to both mixed SAMs of alkanethiol compounds and commercial immobilization layers. The ability of the carbohydrate strategy to construct an electrochemical immunosensor was investigated using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) transduction. Graphical Abstract Left: Functionalization strategies of bare gold substrates via direct bio-SAM using disulfide-containing antibody chemically modified via their primary amines (A), carbohydrates (B) and carboxylic acids (C). Right: Dependence of the peak height with NSE concentration at NSE21-CHO modified electrochemical immunosensor. Inset: Logarithmic calibration plot.

Regulation of gene expression in plasmid ColE1: delayed expression of the kil gene.

PubMed Central

Zhang, S P; Yan, L F; Zubay, G

1988-01-01

cea, imm, and kil are a cluster of three functionally related genes of the plasmid ColE1. The cea and kil genes are in the same inducible operon, with transcription being initiated from a promoter adjacent to the cea gene. The imm gene is located between the cea and kil genes, but it is transcribed in the opposite direction. Complementary interaction between the imm mRNA and the anti-imm sequences in the middle of the cea-kil transcript causes a pronounced delay in expression of the kil gene when the cea-kil operon is induced. A segment in the overlapping region between the cea and imm genes causes delayed expression of the kil gene in the absence of imm gene transcription. This delay effect increases the yields of colicin synthesized in induced cells. Images PMID:3142845

An introduction to metabolomics and its potential application in veterinary science.

PubMed

Jones, Oliver A H; Cheung, Victoria L

2007-10-01

Metabolomics has been found to be applicable to a wide range of fields, including the study of gene function, toxicology, plant sciences, environmental analysis, clinical diagnostics, nutrition, and the discrimination of organism genotypes. This approach combines high-throughput sample analysis with computer-assisted multivariate pattern-recognition techniques. It is increasingly being deployed in toxico- and pharmacokinetic studies in the pharmaceutical industry, especially during the safety assessment of candidate drugs in human medicine. However, despite the potential of this technique to reduce both costs and the numbers of animals used for research, examples of the application of metabolomics in veterinary research are, thus far, rare. Here we give an introduction to metabolomics and discuss its potential in the field of veterinary science.

Visual gene developer: a fully programmable bioinformatics software for synthetic gene optimization.

PubMed

Jung, Sang-Kyu; McDonald, Karen

2011-08-16

Direct gene synthesis is becoming more popular owing to decreases in gene synthesis pricing. Compared with using natural genes, gene synthesis provides a good opportunity to optimize gene sequence for specific applications. In order to facilitate gene optimization, we have developed a stand-alone software called Visual Gene Developer. The software not only provides general functions for gene analysis and optimization along with an interactive user-friendly interface, but also includes unique features such as programming capability, dedicated mRNA secondary structure prediction, artificial neural network modeling, network & multi-threaded computing, and user-accessible programming modules. The software allows a user to analyze and optimize a sequence using main menu functions or specialized module windows. Alternatively, gene optimization can be initiated by designing a gene construct and configuring an optimization strategy. A user can choose several predefined or user-defined algorithms to design a complicated strategy. The software provides expandable functionality as platform software supporting module development using popular script languages such as VBScript and JScript in the software programming environment. Visual Gene Developer is useful for both researchers who want to quickly analyze and optimize genes, and those who are interested in developing and testing new algorithms in bioinformatics. The software is available for free download at http://www.visualgenedeveloper.net.

26 CFR 52.0-1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 17 2010-04-01 2010-04-01 false Introduction. 52.0-1 Section 52.0-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) MISCELLANEOUS EXCISE TAXES (CONTINUED) ENVIRONMENTAL TAXES § 52.0-1 Introduction. The regulations in this part 52 are designated...

26 CFR 1.614-0 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 7 2010-04-01 2010-04-01 true Introduction. 1.614-0 Section 1.614-0 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Natural Resources § 1.614-0 Introduction. Section 614 relates to the definition of...

26 CFR 1.614-0 - Introduction.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 7 2011-04-01 2009-04-01 true Introduction. 1.614-0 Section 1.614-0 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) Natural Resources § 1.614-0 Introduction. Section 614 relates to the definition of...

26 CFR 48.0-1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... MANUFACTURERS AND RETAILERS EXCISE TAXES Introduction § 48.0-1 Introduction. The regulations in this part 48 are designated “Manufacturers and Retailers Excise Tax Regulations.” The regulations relate to the excise taxes... automobiles, highway-type tires, taxable fuel, aviation fuel, coal, certain vaccines, and sporting goods...

A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria.

PubMed

Choi, Goro; Jung, Jae Hwan; Park, Byung Hyun; Oh, Seung Jun; Seo, Ji Hyun; Choi, Jong Seob; Kim, Do Hyun; Seo, Tae Seok

2016-06-21

In this study, we developed a centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria contaminated milk samples. The microdevice was designed to contain identical triplicate functional units and each unit has four reaction chambers, thereby making it possible to perform twelve direct-RPA reactions simultaneously. The integrated microdevice consisted of two layers: RPA reagents were injected in the top layer, while spiked milk samples with food poisoning bacteria were loaded into sample reservoirs in the bottom layer. For multiplex bacterial detection, the target gene-specific primers and probes were dried in each reaction chamber. The introduced samples and reagents could be equally aliquoted and dispensed into each reaction chamber by centrifugal force, and then the multiplex direct-RPA reaction was executed. The target genes of bacteria spiked in milk could be amplified at 39 °C without a DNA extraction step by using the direct-RPA cocktails, which were a combination of a direct PCR buffer and RPA enzymes. As the target gene amplification proceeded, the increased fluorescence signals coming from the reaction chambers were recorded in real-time at an interval of 2 min. The entire process, including the sample distribution, the direct-RPA reaction, and the real-time analysis, was accomplished with a custom-made portable genetic analyzer and a miniaturized optical detector. Monoplex, duplex, and triplex food poisoning bacteria (Salmonella enterica, Escherichia coli O157:H7, and Vibrio parahaemolyticus) detection was successfully performed with a detection sensitivity of 4 cells per 3.2 μL of milk samples within 30 min. By implementing the direct-PRA on the miniaturized centrifugal microsystem, the on-site food poisoning bacteria analysis would be feasible with high speed, sensitivity, and multiplicity.

Cas9-Guide RNA Directed Genome Editing in Soybean[OPEN

PubMed Central

Li, Zhongsen; Liu, Zhan-Bin; Xing, Aiqiu; Moon, Bryan P.; Koellhoffer, Jessica P.; Huang, Lingxia; Ward, R. Timothy; Clifton, Elizabeth; Falco, S. Carl; Cigan, A. Mark

2015-01-01

Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing. PMID:26294043

The metabolic regulator CodY links L. monocytogenes metabolism to virulence by directly activating the virulence regulatory gene, prfA

PubMed Central

Lobel, Lior; Sigal, Nadejda; Borovok, Ilya; Belitsky, Boris R.; Sonenshein, Abraham L.; Herskovits, Anat A.

2015-01-01

Summary Metabolic adaptations are critical to the ability of bacterial pathogens to grow within host cells and are normally preceded by sensing of host-specific metabolic signals, which in turn can influence the pathogen's virulence state. Previously, we reported that the intracellular bacterial pathogen Listeria monocytogenes responds to low availability of branched-chain amino acids (BCAA) within mammalian cells by up-regulating both BCAA biosynthesis and virulence genes. The induction of virulence genes required the BCAA-responsive transcription regulator, CodY, but the molecular mechanism governing this mode of regulation was unclear. In this report, we demonstrate that CodY directly binds the coding sequence of the L. monocytogenes master virulence activator gene, prfA, 15 nt downstream of its start codon, and that this binding results in up-regulation of prfA transcription specifically under low concentrations of BCAA. Mutating this site abolished CodY binding and reduced prfA transcription in macrophages, and attenuated bacterial virulence in mice. Notably, the mutated binding site did not alter prfA transcription or PrfA activity under other conditions that are known to activate PrfA, such as during growth in the presence of glucose-1-phosphate. This study highlights the tight crosstalk between L. monocytogenes metabolism and virulence' while revealing novel features of CodY-mediated regulation. PMID:25430920

Introduction to Concrete Masonry. Introduction to Construction Series. Instructor Edition.

ERIC Educational Resources Information Center

Oklahoma State Dept. of Vocational and Technical Education, Stillwater. Curriculum and Instructional Materials Center.

This competency-based curriculum guide on the specialty area of concrete masonry is part of the Introduction to Construction series. The series is designed with the flexible training requirements of open shop contractors, preapprenticeship programs, multicraft high school programs, technology education programs, and cooperative education programs…

Introduction to Sheet Metal. Introduction to Construction Series. Instructor Edition.

ERIC Educational Resources Information Center

Oklahoma State Dept. of Vocational and Technical Education, Stillwater. Curriculum and Instructional Materials Center.

This competency-based curriculum guide on the specialty area of sheet metal is part of the Introduction to Construction series. The series is designed with the flexible training requirements of open shop contractors, preapprenticeship programs, multicraft high school programs, technology education programs, and cooperative education programs in…

26 CFR 43.0-1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 16 2010-04-01 2010-04-01 true Introduction. 43.0-1 Section 43.0-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) MISCELLANEOUS EXCISE TAXES EXCISE TAX ON TRANSPORTATION BY WATER § 43.0-1 Introduction. The regulations in this part 43 are designated...

26 CFR 40.0-1 - Introduction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 16 2010-04-01 2010-04-01 true Introduction. 40.0-1 Section 40.0-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) MISCELLANEOUS EXCISE TAXES EXCISE TAX PROCEDURAL REGULATIONS § 40.0-1 Introduction. (a) In general. The regulations in this part 40 are...

Direct sequencing of FAH gene in Pakistani tyrosinemia type 1 families reveals a novel mutation.

PubMed

Ijaz, Sadaqat; Zahoor, Muhammad Yasir; Imran, Muhammad; Afzal, Sibtain; Bhinder, Munir A; Ullah, Ihsan; Cheema, Huma Arshad; Ramzan, Khushnooda; Shehzad, Wasim

2016-03-01

Hereditary tyrosinemia type 1 (HT1) is a rare inborn error of tyrosine catabolism with a worldwide prevalence of one out of 100,000 live births. HT1 is clinically characterized by hepatic and renal dysfunction resulting from the deficiency of fumarylacetoacetate hydrolase (FAH) enzyme, caused by recessive mutations in the FAH gene. We present here the first report on identification of FAH mutations in HT1 patients from Pakistan with a novel one. Three Pakistani families, each having one child affected with HT1, were enrolled over a period of 1.5 years. Two of the affected children had died as they were presented late with acute form. All regions of the FAH gene spanning exons and splicing sites were amplified by polymerase chain reaction (PCR) and mutation analysis was carried out by direct sequencing. Results of sequencing were confirmed by restriction fragment length polymorphism (PCR-RFLP) analysis. Three different FAH mutations, one in each family, were found to co-segregate with the disease phenotype. Two of these FAH mutations have been known (c.192G>T and c.1062+5G>A [IVS12+5G>A]), while c.67T>C (p.Ser23Pro) was a novel mutation. The novel variant was not detected in any of 120 chromosomes from normal ethnically matched individuals. Most of the HT1 patients die before they present to hospitals in Pakistan, as is indicated by enrollment of only three families in 1.5 years. Most of those with late clinical presentation do not survive due to delayed diagnosis followed by untimely treatment. This tragic condition advocates the establishment of expanded newborn screening program for HT1 within Pakistan.

Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development

PubMed Central

Tricker, Penny J.; Gibbings, J. George; Rodríguez López, Carlos M.; Hadley, Paul; Wilkinson, Mike J.

2012-01-01

Environmental cues influence the development of stomata on the leaf epidermis, and allow plants to exert plasticity in leaf stomatal abundance in response to the prevailing growing conditions. It is reported that Arabidopsis thaliana ‘Landsberg erecta’ plants grown under low relative humidity have a reduced stomatal index and that two genes in the stomatal development pathway, SPEECHLESS and FAMA, become de novo cytosine methylated and transcriptionally repressed. These environmentally-induced epigenetic responses were abolished in mutants lacking the capacity for de novo DNA methylation, for the maintenance of CG methylation, and in mutants for the production of short-interfering non-coding RNAs (siRNAs) in the RNA-directed DNA methylation pathway. Induction of methylation was quantitatively related to the induction of local siRNAs under low relative humidity. Our results indicate the involvement of both transcriptional and post-transcriptional gene suppression at these loci in response to environmental stress. Thus, in a physiologically important pathway, a targeted epigenetic response to a specific environmental stress is reported and several of its molecular, mechanistic components are described, providing a tractable platform for future epigenetics experiments. Our findings suggest epigenetic regulation of stomatal development that allows for anatomical and phenotypic plasticity, and may help to explain at least some of the plant’s resilience to fluctuating relative humidity. PMID:22442411

Deontic introduction: A theory of inference from is to ought.

PubMed

Elqayam, Shira; Thompson, Valerie A; Wilkinson, Meredith R; Evans, Jonathan St B T; Over, David E

2015-09-01

Humans have a unique ability to generate novel norms. Faced with the knowledge that there are hungry children in Somalia, we easily and naturally infer that we ought to donate to famine relief charities. Although a contentious and lively issue in metaethics, such inference from "is" to "ought" has not been systematically studied in the psychology of reasoning. We propose that deontic introduction is the result of a rich chain of pragmatic inference, most of it implicit; specifically, when an action is causally linked to a valenced goal, valence transfers to the action and bridges into a deontic conclusion. Participants in 5 experiments were presented with utility conditionals in which an action results in a benefit, a cost, or neutral outcome (e.g., "If Lisa buys the booklet, she will pass the exam") and asked to evaluate how strongly deontic conclusions (e.g., "Lisa should buy the booklet") follow from the premises. Findings show that the direction of the conclusions was determined by outcome valence (Experiments 1a and 1b), whereas their strength was determined by the strength of the causal link between action and outcome (Experiments 1, 2a, and 2b). We also found that deontic introduction is defeasible and can be suppressed by additional premises that interfere with any of the links in the implicit chain of inference (Experiments 2a, 2b, and 3). We propose that deontic introduction is a species-specific generative capacity whose function is to regulate future behavior. (c) 2015 APA, all rights reserved).

Genes, dreams, and cancer.

PubMed Central

Sikora, K.

1994-01-01

There have been tremendous advances in our understanding of cancer from the application of molecular biology over the past decade. The disease is caused by a series of defects in the genes that accelerate growth--oncogenes--and those that slow down cellular turnover--tumour suppressor genes. The proteins they encode provide a promising hunting ground in which to design and test new anticancer drugs. Several treatment strategies are now under clinical trial entailing direct gene transfer. These include the use of gene marking to detect minimal residual disease, the production of novel cancer vaccines by the insertion of genes which uncloak cancer cells so making them visible to the host's immune system, the isolation and coupling of cancer specific molecular switches upstream of drug activating genes, and the correction of aberrant oncogenes or tumour suppressor genes. The issues in these approaches are likely to have a profound impact on the management of cancer patients as we enter the next century. Images p1221-a PMID:8180542

The ethical introduction of genome-based information and technologies into public health.

PubMed

Howard, H C; Swinnen, E; Douw, K; Vondeling, H; Cassiman, J-J; Cambon-Thomsen, A; Borry, P

2013-01-01

With the human genome project running from 1989 until its completion in 2003, and the incredible advances in sequencing technology and in bioinformatics during the last decade, there has been a shift towards an increase focus on studying common complex disorders which develop due to the interplay of many different genes as well as environmental factors. Although some susceptibility genes have been identified in some populations for disorders such as cancer, diabetes and cardiovascular diseases, the integration of this information into the health care system has proven to be much more problematic than for single gene disorders. Furthermore, with the 1000$ genome supposedly just around the corner, and whole genome sequencing gradually being integrated into research protocols as well as in the clinical context, there is a strong push for the uptake of additional genomic testing. Indeed, the advent of public health genomics, wherein genomics would be integrated in all aspects of health care and public health, should be taken seriously. Although laudable, these advances also bring with them a slew of ethical and social issues that challenge the normative frameworks used in clinical genetics until now. With this in mind, we highlight herein 5 principles that are used as a primer to discuss the ethical introduction of genome-based information and genome-based technologies into public health. Copyright © 2013 S. Karger AG, Basel.

Quantitative trait locus gene mapping: a new method for locating alcohol response genes.

PubMed

Crabbe, J C

1996-01-01

Alcoholism is a multigenic trait with important non-genetic determinants. Studies with genetic animal models of susceptibility to several of alcohol's effects suggest that several genes contributing modest effects on susceptibility (Quantitative Trait Loci, or QTLs) are important. A new technique of QTL gene mapping has allowed the identification of the location in mouse genome of several such QTLs. The method is described, and the locations of QTLs affecting the acute alcohol withdrawal reaction are described as an example of the method. Verification of these QTLs in ancillary studies is described and the strengths, limitations, and future directions to be pursued are discussed. QTL mapping is a promising method for identifying genes in rodents with the hope of directly extrapolating the results to the human genome. This review is based on a paper presented at the First International Congress of the Latin American Society for Biomedical Research on Alcoholism, Santiago, Chile, November 1994.

Evolution of genetic architecture under directional selection.

PubMed

Hansen, Thomas F; Alvarez-Castro, José M; Carter, Ashley J R; Hermisson, Joachim; Wagner, Günter P

2006-08-01

We investigate the multilinear epistatic model under mutation-limited directional selection. We confirm previous results that only directional epistasis, in which genes on average reinforce or diminish each other's effects, contribute to the initial evolution of mutational effects. Thus, either canalization or decanalization can occur under directional selection, depending on whether positive or negative epistasis is prevalent. We then focus on the evolution of the epistatic coefficients themselves. In the absence of higher-order epistasis, positive pairwise epistasis will tend to weaken relative to additive effects, while negative pairwise epistasis will tend to become strengthened. Positive third-order epistasis will counteract these effects, while negative third-order epistasis will reinforce them. More generally, gene interactions of all orders have an inherent tendency for negative changes under directional selection, which can only be modified by higher-order directional epistasis. We identify three types of nonadditive quasi-equilibrium architectures that, although not strictly stable, can be maintained for an extended time: (1) nondirectional epistatic architectures; (2) canalized architectures with strong epistasis; and (3) near-additive architectures in which additive effects keep increasing relative to epistasis.

INTRODUCTING A THERMAL DISSIPATION PROBE SYSTEM FOR MEASURING BI-DIRECTIONAL ROOT WATER FLUX

EPA Science Inventory

The interest in measuring the direction and magnitude of root sapflow has accelerated in the past few years because of interest in the redistribution of water in the soil by roots. Plant roots have been shown to redistribute water between areas of soil with differing water conte...

Identification of Primary Transcriptional Regulation of Cell Cycle-Regulated Genes upon DNA Damage

PubMed Central

Zhou, Tong; Chou, Jeff; Mullen, Thomas E.; Elkon, Rani; Zhou, Yingchun; Simpson, Dennis A.; Bushel, Pierre R.; Paules, Richard S.; Lobenhofer, Edward K.; Hurban, Patrick; Kaufmann, William K.

2007-01-01

The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization. PMID:17404513

Self-excising Cre/mutant lox marker recycling system for multiple gene integrations and consecutive gene deletions in Aspergillus oryzae.

PubMed

Zhang, Silai; Ban, Akihiko; Ebara, Naoki; Mizutani, Osamu; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2017-04-01

In this study, we developed a self-excising Cre/loxP-mediated marker recycling system with mutated lox sequences to introduce a number of biosynthetic genes into Aspergillus oryzae. To construct the self-excising marker cassette, both the selectable marker, the Aspergillus nidulans adeA gene, and the Cre recombinase gene (cre), conditionally expressed by the xylanase-encoding gene promoter, were designed to be located between the mutant lox sequences, lox66 and lox71. However, construction of the plasmid failed, possibly owing to a slight expression of cre downstream of the fungal gene promoter in Escherichia coli. Hence, to avoid the excision of the cassette in E. coli, a 71-bp intron of the A. oryzae xynG2 gene was inserted into the cre gene. The A. oryzae adeA deletion mutant was transformed with the resulting plasmid in the presence of glucose, and the transformants were cultured in medium containing xylose as the sole carbon source. PCR analysis of genomic DNA from resultant colonies revealed the excision of both the marker and Cre expression construct, indicating that the self-excising marker cassette was efficient at removing the selectable marker. Using the marker recycling system, hyperproduction of kojic acid could be achieved in A. oryzae by the introduction of two genes that encode oxidoreductase and transporter. Furthermore, we also constructed an alternative marker recycling cassette bearing the A. nidulans pyrithiamine resistant gene (ptrA) as a dominant selectable marker. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Self-Directed Learning Needs, Patterns, and Outcomes among General Surgeons

ERIC Educational Resources Information Center

Gagliardi, Anna R.; Wright, Frances C.; Victor, J. Charles; Brouwers, Melissa C.; Silver, Ivan L.

2009-01-01

Introduction: To explore the relationship between self-directed learning (SDL) needs, patterns, barriers, and outcomes among nonacademic general surgeons. Methods: Participants dictated details of SDL episodes associated with cancer patient management from October 2007 to March 2008. Transcripts were coded thematically. Frequencies were calculated…

Selection of recombinant MVA by rescue of the essential D4R gene.

PubMed

Ricci, Patricia S; Schäfer, Birgit; Kreil, Thomas R; Falkner, Falko G; Holzer, Georg W

2011-12-12

Modified vaccinia virus Ankara (MVA) has become a promising vaccine vector due to its immunogenicity and its proven safety in humans. As a general approach for stringent and rapid selection of recombinant MVA, we assessed marker rescue of the essential viral D4R gene in an engineered deletion mutant that is fully replication defective in wild-type cells. Recombinant, replicating virus was obtained by re-introduction of the deleted viral gene as a dominant selection marker into the deletion mutant.

BOOK REVIEW: Introduction to Black Hole Physics Introduction to Black Hole Physics

NASA Astrophysics Data System (ADS)

Tanaka, Takahiro

2012-07-01

Introduction to Black Hole Physics is a large volume (504 pages), and yet despite this it is still really an introductory text. The book gives an introduction to general relativity, but most of the text is dedicated to attracting the reader's attention to the interesting world of black hole physics. In this sense, the book is very distinct from other textbooks on general relativity. We are told that it was based on the lectures given by Professor Frolov, one of the authors, over the last 30 years. One can obtain the basic ideas about black holes, and also the necessary tips to understand general relativity at a very basic level. For example, in the discussion about particle motion in curved space, the authors start with a brief review on analytical mechanics. The book does not require its readers to have a great deal of knowledge in advance. If you are familiar with such a basic subject, you can simply omit that section. The reason why I especially picked up on this topic as an example is that the book devotes a significant number of pages to geodesic motions in black hole spacetime. One of the main motivations to study black holes is related to how they will actually be observed, once we develop the ability to observe them clearly. The book does explain such discoveries as, for instance, how the motion of a particle is related to a beautiful mathematical structure arising from the hidden symmetry of spacetime, which became transparent via the recent progress in the exploration of black holes in higher dimensions; a concise introduction to this latest topic is deferred to Appendix D, so as not to distract the reader with its mathematical complexities. It should be also mentioned that the book is not limited to general relativistic aspects: quantum fields on a black hole background and Hawking radiation are also covered. Also included are current hot topics, for instance the gravitational waves from a system including black holes, whose first direct detection is

Pulsed Irradiation Improves Target Selectivity of Infrared Laser-Evoked Gene Operator for Single-Cell Gene Induction in the Nematode C. elegans

PubMed Central

Suzuki, Motoshi; Toyoda, Naoya; Takagi, Shin

2014-01-01

Methods for turning on/off gene expression at the experimenter’s discretion would be useful for various biological studies. Recently, we reported on a novel microscope system utilizing an infrared laser-evoked gene operator (IR-LEGO) designed for inducing heat shock response efficiently in targeted single cells in living organisms without cell damage, thereby driving expression of a transgene under the control of a heat shock promoter. Although the original IR-LEGO can be successfully used for gene induction, several limitations hinder its wider application. Here, using the nematode Caenorhabditis elegans (C. elegans) as a subject, we have made improvements in IR-LEGO. For better spatial control of heating, a pulsed irradiation method using an optical chopper was introduced. As a result, single cells of C. elegans embryos as early as the 2-cell stage and single neurons in ganglia can be induced to express genes selectively. In addition, the introduction of site-specific recombination systems to IR-LEGO enables the induction of gene expression controlled by constitutive and cell type-specific promoters. The strategies adopted here will be useful for future applications of IR-LEGO to other organisms. PMID:24465705

Visual gene developer: a fully programmable bioinformatics software for synthetic gene optimization

PubMed Central

2011-01-01

Background Direct gene synthesis is becoming more popular owing to decreases in gene synthesis pricing. Compared with using natural genes, gene synthesis provides a good opportunity to optimize gene sequence for specific applications. In order to facilitate gene optimization, we have developed a stand-alone software called Visual Gene Developer. Results The software not only provides general functions for gene analysis and optimization along with an interactive user-friendly interface, but also includes unique features such as programming capability, dedicated mRNA secondary structure prediction, artificial neural network modeling, network & multi-threaded computing, and user-accessible programming modules. The software allows a user to analyze and optimize a sequence using main menu functions or specialized module windows. Alternatively, gene optimization can be initiated by designing a gene construct and configuring an optimization strategy. A user can choose several predefined or user-defined algorithms to design a complicated strategy. The software provides expandable functionality as platform software supporting module development using popular script languages such as VBScript and JScript in the software programming environment. Conclusion Visual Gene Developer is useful for both researchers who want to quickly analyze and optimize genes, and those who are interested in developing and testing new algorithms in bioinformatics. The software is available for free download at http://www.visualgenedeveloper.net. PMID:21846353

Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor.

PubMed

Bahadur, Urvashi; Ganjam, Goutham K; Vasudevan, Nandini; Kondaiah, Paturu

2005-02-28

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-alpha) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ERalpha antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the

Introduction of new technology in vascular surgery.

PubMed

Bergqvist, D

2008-01-01

In this review paper introduction of new technologies in vascular surgery is discussed. The difficulties compared to introduction of pharmacological treatment are analyzed. Pros and cons with randomized controlled trials and observational studies are listed.

Clinical trials of GMP products in the gene therapy field.

PubMed

Bamford, Kathleen B

2011-01-01

Advances in gene therapy are increasingly leading to clinical assessment in many fields of medicine with diverse approaches. The basic science stems from approaches aimed at different functions such as correcting a missing/abnormal gene, altering the proportion or expression of normal genes to augment a physiological process or using this principle to destroy malignant or infected cells. As the technology advances, it is increasingly important to ensure that clinical trials answer the questions that need to be asked. In this chapter we review examples of published clinical trials, resources for accessing information about registered trials, the process of regulating trials, good clinical practice, and good manufacturing practice as well as summarising the approach taken by regulatory authorities in reviewing applications for the introduction of products for use in the clinic.

Patterns of genetic diversity reveal multiple introductions and recurrent founder effects during range expansion in invasive populations of Geranium carolinianum (Geraniaceae)

PubMed Central

Shirk, R Y; Hamrick, J L; Zhang, C; Qiang, S

2014-01-01

Genetic diversity, and thus the adaptive potential of invasive populations, is largely based on three factors: patterns of genetic diversity in the species' native range, the number and location of introductions and the number of founding individuals per introduction. Specifically, reductions in genetic diversity (‘founder effects') should be stronger for species with low within-population diversity in their native range and few introductions of few individuals to the invasive range. We test these predictions with Geranium carolinianum, a winter annual herb native to North America and invasive in China. We measure the extent of founder effects using allozymes and microsatellites, and ask whether this is consistent with its colonization history and patterns of diversity in the native range. In the native range, genetic diversity is higher and structure is lower than expected based on life history traits. In China, our results provide evidence for multiple introductions near Nanjing, Jiangsu province, with subsequent range expansion to the west and south. Patterns of genetic diversity across China reveal weak founder effects that are driven largely by low-diversity populations at the expansion front, away from the introduction location. This suggests that reduced diversity in China has resulted from successive founder events during range expansion, and that the loss of genetic diversity in the Nanjing area was mitigated by multiple introductions from diverse source populations. This has implications for the future of G. carolinianum in China, as continued gene flow among populations should eventually increase genetic diversity within the more recently founded populations. PMID:24346497

Direct observation of interface instability during crystal growth

NASA Technical Reports Server (NTRS)

Tiller, W. A.; Feigelson, R. S.; Elwell, D.

1982-01-01

The general aim of this investigation was to study interface stability and solute segregation phenomena during crystallization of a model system. Emphasis was to be placed on direct observational studies partly because this offered the possibility at a later stage of performing related experiments under substantially convection-free conditions in the space shuttle. The major achievements described in this report are: (1) the development of a new model system for fundamental studies of crystal growth from the melt and the measurement of a range of material parameters necessary for comparison of experiment with theory. (2) The introduction of a new method of measuring segregation coefficient using absorption of a laser beam by the liquid phase. (3) The comparison of segregation in crystals grown by gradient freezing and by pulling from the melt. (4) The introduction into the theory of solute segregation of an interface field term and comparison with experiment. (5) The introduction of the interface field term into the theories of constitutional supercooling and morphological stability and assessment of its importance.

Evaluating the consistency of gene sets used in the analysis of bacterial gene expression data.

PubMed

Tintle, Nathan L; Sitarik, Alexandra; Boerema, Benjamin; Young, Kylie; Best, Aaron A; Dejongh, Matthew

2012-08-08

Statistical analyses of whole genome expression data require functional information about genes in order to yield meaningful biological conclusions. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) are common sources of functionally grouped gene sets. For bacteria, the SEED and MicrobesOnline provide alternative, complementary sources of gene sets. To date, no comprehensive evaluation of the data obtained from these resources has been performed. We define a series of gene set consistency metrics directly related to the most common classes of statistical analyses for gene expression data, and then perform a comprehensive analysis of 3581 Affymetrix® gene expression arrays across 17 diverse bacteria. We find that gene sets obtained from GO and KEGG demonstrate lower consistency than those obtained from the SEED and MicrobesOnline, regardless of gene set size. Despite the widespread use of GO and KEGG gene sets in bacterial gene expression data analysis, the SEED and MicrobesOnline provide more consistent sets for a wide variety of statistical analyses. Increased use of the SEED and MicrobesOnline gene sets in the analysis of bacterial gene expression data may improve statistical power and utility of expression data.

Genomic Analysis Reveals Contrasting PIFq Contribution to Diurnal Rhythmic Gene Expression in PIF-Induced and -Repressed Genes.

PubMed

Martin, Guiomar; Soy, Judit; Monte, Elena

2016-01-01

Members of the PIF quartet (PIFq; PIF1, PIF3, PIF4, and PIF5) collectively contribute to induce growth in Arabidopsis seedlings under short day (SD) conditions, specifically promoting elongation at dawn. Their action involves the direct regulation of growth-related and hormone-associated genes. However, a comprehensive definition of the PIFq-regulated transcriptome under SD is still lacking. We have recently shown that SD and free-running (LL) conditions correspond to "growth" and "no growth" conditions, respectively, correlating with greater abundance of PIF protein in SD. Here, we present a genomic analysis whereby we first define SD-regulated genes at dawn compared to LL in the wild type, followed by identification of those SD-regulated genes whose expression depends on the presence of PIFq. By using this sequential strategy, we have identified 349 PIF/SD-regulated genes, approximately 55% induced and 42% repressed by both SD and PIFq. Comparison with available databases indicates that PIF/SD-induced and PIF/SD-repressed sets are differently phased at dawn and mid-morning, respectively. In addition, we found that whereas rhythmicity of the PIF/SD-induced gene set is lost in LL, most PIF/SD-repressed genes keep their rhythmicity in LL, suggesting differential regulation of both gene sets by the circadian clock. Moreover, we also uncovered distinct overrepresented functions in the induced and repressed gene sets, in accord with previous studies in other examined PIF-regulated processes. Interestingly, promoter analyses showed that, whereas PIF/SD-induced genes are enriched in direct PIF targets, PIF/SD-repressed genes are mostly indirectly regulated by the PIFs and might be more enriched in ABA-regulated genes.

The evolution of milk casein genes from tooth genes before the origin of mammals.

PubMed

Kawasaki, Kazuhiko; Lafont, Anne-Gaelle; Sire, Jean-Yves

2011-07-01

Caseins are among cardinal proteins that evolved in the lineage leading to mammals. In milk, caseins and calcium phosphate (CaP) form a huge complex called casein micelle. By forming the micelle, milk maintains high CaP concentrations, which help altricial mammalian neonates to grow bone and teeth. Two types of caseins are known. Ca-sensitive caseins (α(s)- and β-caseins) bind Ca but precipitate at high Ca concentrations, whereas Ca-insensitive casein (κ-casein) does not usually interact with Ca but instead stabilizes the micelle. Thus, it is thought that these two types of caseins are both necessary for stable micelle formation. Both types of caseins show high substitution rates, which make it difficult to elucidate the evolution of caseins. Yet, recent studies have revealed that all casein genes belong to the secretory calcium-binding phosphoprotein (SCPP) gene family that arose by gene duplication. In the present study, we investigated exon-intron structures and phylogenetic distributions of casein and other SCPP genes, particularly the odontogenic ameloblast-associated (ODAM) gene, the SCPP-Pro-Gln-rich 1 (SCPPPQ1) gene, and the follicular dendritic cell secreted peptide (FDCSP) gene. The results suggest that contemporary Ca-sensitive casein genes arose from a putative common ancestor, which we refer to as CSN1/2. The six putative exons comprising CSN1/2 are all found in SCPPPQ1, although ODAM also shares four of these exons. By contrast, the five exons of the Ca-insensitive casein gene are all reminiscent of FDCSP. The phylogenetic distribution of these genes suggests that both SCPPPQ1 and FDCSP arose from ODAM. We thus argue that all casein genes evolved from ODAM via two different pathways; Ca-sensitive casein genes likely originated directly from SCPPPQ1, whereas the Ca-insensitive casein genes directly differentiated from FDCSP. Further, expression of ODAM, SCPPPQ1, and FDCSP was detected in dental tissues, supporting the idea that both types of caseins

Directed evolution of polymerase function by compartmentalized self-replication.

PubMed

Ghadessy, F J; Ong, J L; Holliger, P

2001-04-10

We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication.

Expression kinetics of key genes in the early innate immune response to Great Lakes viral hemorrhagic septicemia virus IVb infection in yellow perch (Perca flavescens)

USGS Publications Warehouse

Olson, Wendy; Emmenegger, Eveline; Glenn, Jolene; Simchick, Crystal; Winton, Jim; Goetz, Frederick

2013-01-01

The recently discovered strain of viral hemorrhagic septicemia virus, VHSV-IVb, represents an example of the introduction of an extremely pathogenic rhabdovirus capable of infecting a wide variety of new fish species in a new host-environment. The goal of the present study was to delineate the expression kinetics of key genes in the innate immune response relative to the very early stages of VHSV-IVb infection using the yellow perch (Perca flavescens) as a model. Administration of VHSV-IVb by IP-injection into juvenile yellow perch resulted in 84% cumulative mortality, indicating their high susceptibility to this disease. In fish sampled in the very early stages of infection, a significant up-regulation of Mx gene expression in the liver, as well as IL-1β and SAA activation in the head kidney, spleen, and liver was directly correlated to viral load. The potential down-regulation of Mx in the hematopoietic tissues, head kidney and spleen, may represent a strategy utilized by the virus to increase replication.

Gene Expression Studies in Lygus lineolaris

USDA-ARS?s Scientific Manuscript database