Detection of proteins on blot transfer membranes.

PubMed

Sasse, Joachim; Gallagher, Sean R

2003-11-01

In the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for alkali treatment that enhances subsequent staining of bound proteins.

Single cell–resolution western blotting

PubMed Central

Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

2017-01-01

This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). the gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. to extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. once the microdevice has been fabricated, the assay can be completed in 4–6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. the technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine. PMID:27466711

Protein purification and analysis: next generation Western blotting techniques.

PubMed

Mishra, Manish; Tiwari, Shuchita; Gomes, Aldrin V

2017-11-01

Western blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results. Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized. The review discusses the most advanced western blotting techniques available and highlights the relationship between next generation western blotting techniques and its clinical relevance. Expert commentary: Over the last decade significant improvements have been made in creating more sensitive, automated, and advanced techniques by optimizing various aspects of the western blot protocol. New methods such as single cell-resolution western blot, capillary electrophoresis, DigiWest, automated microfluid western blotting and microchip electrophoresis have all been developed to reduce potential problems associated with the western blotting technique. Innovative developments in instrumentation and increased sensitivity for western blots offer novel possibilities for increasing the clinical implications of western blot.

[Better performance of Western blotting: quick vs slow protein transfer, blotting membranes and the visualization methods].

PubMed

Kong, Ling-Quan; Pu, Ying-Hui; Ma, Shi-Kun

2008-01-01

To study how the choices of the quick vs slow protein transfer, the blotting membranes and the visualization methods influence the performance of Western blotting. The cellular proteins were abstracted from human breast cell line MDA-MB-231 for analysis with Western blotting using quick (2 h) and slow (overnight) protein transfer, different blotting membranes (nitrocellulose, PVDF and nylon membranes) and different visualization methods (ECL and DAB). In Western blotting with slow and quick protein transfer, the prestained marker presented more distinct bands on nitrocellulose membrane than on the nylon and PVDF membranes, and the latter also showed clear bands on the back of the membrane to very likely cause confusion, which did not occur with nitrocellulose membrane. PVDF membrane allowed slightly clearer visualization of the proteins with DAB method as compared with nitrocellulose and nylon membranes, and on the latter two membranes, quick protein transfer was likely to result in somehow irregular bands in comparison with slow protein transfer. With slow protein transfer and chemiluminescence for visualization, all the 3 membranes showed clear background, while with quick protein transfer, nylon membrane gave rise to obvious background noise but the other two membranes did not. Different membranes should be selected for immunoblotting according to the actual needs of the experiment. Slow transfer of the proteins onto the membranes often has better effect than quick transfer, and enhanced chemiluminescence is superior to DAB for protein visualization and allows highly specific and sensitive analysis of the protein expressions.

Western Blotting of the Endocannabinoid System.

PubMed

Wager-Miller, Jim; Mackie, Ken

2016-01-01

Measuring expression levels of G protein-coupled receptors (GPCRs) is an important step for understanding the distribution, function, and regulation of these receptors. A common approach for detecting proteins from complex biological systems is Western blotting. In this chapter, we describe a general approach to Western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes, with a focus on detecting type 1 cannabinoid (CB1) receptors. When this technique is carefully used, specifically with validation of the primary antibodies, it can provide quantitative information on protein expression levels. Additional information can also be inferred from Western blotting such as potential posttranslational modifications that can be further evaluated by specific analytical techniques.

The Design of a Quantitative Western Blot Experiment

PubMed Central

Taylor, Sean C.; Posch, Anton

2014-01-01

Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting. PMID:24738055

The Dot Blot ELISA.

ERIC Educational Resources Information Center

Gerbig, Donald G., Jr.; Fenk, Christopher J.; Goodhart, Amy S.

2000-01-01

Uses two laboratory techniques, Enzyme Linked Immunosorbent Assay (ELISA) and Western Blot, to demonstrate antibody-antigen binding concepts. Includes a list of required materials and directions for the procedure, and makes suggestions for classroom applications. (Contains 13 references.) (YDS)

Multiplexed Western Blotting Using Microchip Electrophoresis.

PubMed

Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

2016-07-05

Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps.

Serological investigation of enterococcal infections using western blot.

PubMed

Sulaiman, A; Rakita, R M; Arduino, R C; Patterson, J E; Steckelberg, J M; Singh, K V; Murray, B E

1996-10-01

To assess the usefulness of Western blot in the diagnosis of enterococcal infections, a pilot study was conducted with a newly developed Western blot using sera from patients with confirmed enterococcal infections. Sera from 17 of 19 patients with enterococcal endocarditis reacted strongly to enterococcal antigens on the Western blot, and most produced specific bands at molecular weights 98 kDa and 54 kDa. Sera from patients with bacteremic cholangitis and pyelonephritis reacted frequently as well, but the pattern of bands was different from that observed with endocarditis. Eighty-five percent of 26 sera tested from patients with bacteremia and associated deep-seated infections (endocarditis, cholangitis, and pyelonephritis) were positive on Western blot, compared to 30% of sera from bacteremic patients with no clinically determined deep focus of infection (p < 0.001).

Lipid A binding proteins in macrophages detected by ligand blotting

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

Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

1987-05-01

Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessedmore » using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.« less

Multistrip western blotting to increase quantitative data output.

PubMed

Kiyatkin, Anatoly; Aksamitiene, Edita

2009-01-01

The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip western blotting increases the data output per single blotting cycle up to tenfold, allows concurrent monitoring of up to nine different proteins from the same loading of the sample, and substantially improves the data accuracy by reducing immunoblotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data, and therefore is beneficial to apply in biomedical diagnostics, systems biology, and cell signaling research.

Human thyrotropin receptor subunits characterized by thyrotropin affinity purification and western blotting.

PubMed

Leedman, P J; Newman, J D; Harrison, L C

1989-07-01

We studied the subunit structure of the human TSH receptor in thyroid tissue from patients with Graves' disease and multinodular goiter by TSH affinity chromatography, immunoprecipitation with Graves' immunoglobulins (Igs), and a modified technique of Western blotting. Human TSH receptor-binding activity was purified about 1,270-fold by sequential affinity chromatography on wheat germ lectin-agarose and TSH-agarose. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonreduced affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed three noncovalently linked subunits of 70,000, 50,000, and 35,000 mol wt. When reduced, a major subunit of 25,000 mol wt was identified. When 3 mol/L NaCl was used to elute affinity-purified receptors only the 50,000 mol wt nonreduced subunit was detected. This subunit bound [125I]bovine TSH and was precipitated by Graves' Igs. Modifications to the conventional Western blotting technique enabled thyroglobulin components (approximately 220,000 mol wt), thyroid microsomal antigen (a doublet of approximately 110,000 mol wt), and putative TSH receptor subunits of 70,000 and 50,000 mol wt to be identified in thyroid particulate membranes by Graves' Igs. Blotting of affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed subunits of either 70,000 or 50,000 mol wt, with a minority of Graves' serum samples. We conclude that the nonreduced human TSH receptor is an oligomeric complex comprising three different subunits of 70,000, 50,000, and 35,000 mol wt. The reduced receptor exists as a single subunit of 25,000 mol wt, which may be disulfide linked to form the higher mol wt forms. The 70,000 and 50,000 mol wt subunits contain epitopes that bind Graves' Igs in modified Western blots, thus directly confirming that the human TSH receptor is a target for Graves' Igs.

Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

PubMed

Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

HIV‑2 antibody detection after indeterminate or negative HIV‑1 Western blot in Cuba, 2005-2008.

PubMed

Díaz, Dervel F; Ortiz, Eva; Martín, Dayamí; Nibot, Carmen; Rizo, Adis; Silva, Eladio

2012-01-01

INTRODUCTION Differentiating between HIV-1 and HIV-2 infection is the first step to understanding HIV transmission, epidemiology and pathogenesis in geographical areas where both viruses circulate. In Cuba, positive results in mixed HIV-1/2 screening assays are confirmed by HIV-1 Western blot. Indeterminate results constitute the main limitation of this test and HIV-2 infection is among their possible causes; hence the importance of second-stage screening and confirmatory tests for HIV-2 infection. OBJECTIVE Investigate the contribution of HIV-2 antibodies to negative or indeterminate HIV-1 Western blot results in serum samples from 2005 through 2008 in Cuba. METHODS HIV-2 reactivity was studied using the ELISA DAVIH-VIH-2 diagnostic kit (Cuba) in 1723 serum samples with negative or indeterminate results for HIV-1 Western blot from January 2005 through December 2008. Duplicate sera reactive by ELISA were confirmed by HIV-2 Western blot, results interpreted according to WHO criteria. The epidemiological interview established by Cuba's National Program for Prevention and Control Sexually-Transmitted Diseases and HIV/AIDS was applied to HIV-2 Western blot-positive patients. RESULTS Among all sera studied, HIV-2 ELISA identified 12 reactive serum samples (0.70%) and 1711 non-reactive (99.30%). Western blot analysis of the 12 ELISA-reactive samples confirmed two positive samples (16.67%), 4 negative (33.33%) and 6 indeterminate (50%). Positive samples reacted against the p16, p26, gp36, p53, p56, p68 and gp105 proteins. All 12 ELISA-reactive samples belonged to the HIV-1 Western blot indeterminate group. The two HIV-2-positive samples showed well defined reactivity to gp160, p53, p55 and p34 of HIV-1. HIV-1 seroconversion was observed in all 10 remaining samples during serological followup. CONCLUSIONS Two new HIV-2 seropositive cases were diagnosed using DAVIH-VIH-2 and HIV-2 Western blot in indeterminate HIV-1 Western blot samples. Results support the recommendation

Western Blot of Stained Proteins from Dried Polyacrylamide Gels

NASA Technical Reports Server (NTRS)

Gruber, Claudia; Stan-Lotter, Helga

1996-01-01

Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

Post-staining electroblotting for efficient and reliable peptide blotting.

PubMed

Lee, Der-Yen; Chang, Geen-Dong

2015-01-01

Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.

Automated design of genomic Southern blot probes

PubMed Central

2010-01-01

Background Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments. The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample. Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. Results We have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time. We went on to experimentally validate a number

Proteomic Analyses of Corneal Tissue Subjected to Alkali Exposure

PubMed Central

Parikh, Toral; Eisner, Natalie; Venugopalan, Praseeda; Yang, Qin; Lam, Byron L.

2011-01-01

Purpose. To determine whether exposure to alkaline chemicals results in predictable changes in corneal protein profile. To determine whether protein profile changes are indicative of severity and duration of alkali exposure. Methods. Enucleated bovine and porcine (n = 59 each) eyes were used for exposure to sodium, ammonium, and calcium hydroxide, respectively. Eyes were subjected to fluorescein staining, 5-bromo-2′-deoxy-uridine (BrdU) labeling. Excised cornea was subjected to protein extraction, spectrophotometric determination of protein amount, dynamic light scattering and SDS-PAGE profiling, mass spectrometric protein identification, and iTRAQ-labeled quantification. Select identified proteins were subjected to Western blot and immunohistochemical analyses. Results. Alkali exposure resulted in lower protein extractability from corneal tissue. Elevated aggregate formation was found with strong alkali exposure (sodium hydroxide>ammonium, calcium hydroxide), even with a short duration of exposure compared with controls. The protein yield after exposure varied as a function of postexposure time. Protein profiles changed because of alkali exposure. Concentration and strength of the alkali affected the profile change significantly. Mass spectrometry identified 15 proteins from different bands with relative quantification. Plexin D1 was identified for the first time in the cornea at a protein level that was further confirmed by Western blot and immunohistochemical analyses. Conclusions. Exposure to alkaline chemicals results in predictable and reproducible changes in corneal protein profile. Stronger alkali, longer durations, or both, of exposure resulted in lower yields and significant protein profile changes compared with controls. PMID:20861482

Failure analysis of blots for diesel engine intercooler

NASA Astrophysics Data System (ADS)

Ren, Ping; Li, Zongquan; Wu, Jiangfei; Guo, Yibin; Li, Wanyou

2017-05-01

In diesel generating sets, it will lead to the abominable working condition if the fault couldn’t be recovered when the bolt of intercooler cracks. This paper aims at the fault of the blots of diesel generator intercooler and completes the analysis of the static strength and fatigue strength. Static intensity is checked considering blot preload and thermal stress. In order to obtain the thermal stress of the blot, thermodynamic of intercooler is calculated according to the measured temperature. Based on the measured vibration response and the finite element model, using dynamic load identification technique, equivalent excitation force of unit was solved. In order to obtain the force of bolt, the excitation force is loaded into the finite element model. By considering the thermal stress and preload as the average stress while the mechanical stress as the wave stress, fatigue strength analysis has been accomplished. Procedure of diagnosis is proposed in this paper. Finally, according to the result of intensity verification the fatigue failure is validation. Thereby, further studies are necessary to verification the result of the intensity analysis and put forward some improvement suggestion.

Identification of recombinant human EPO variants in greyhound plasma and urine by ELISA, LC-MS/MS and western blotting: a comparative study.

PubMed

Timms, Mark; Steel, Rohan; Vine, John

2016-02-01

The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol-epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC-MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC-MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening. Copyright © 2015 John Wiley & Sons, Ltd.

An alternative method for processing northern blots after capillary transfer.

PubMed

Nilsen, Timothy W

2015-03-02

Different laboratories use different methods for the prehybridization, hybridization, and washing steps of the northern blotting procedure. In this protocol, a northern blot is pretreated with Church and Gilbert hybridization buffer to block nonspecific probe-binding sites. The immobilized RNA is then hybridized to a DNA probe specific for the RNA of interest. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. The solutions and conditions described here may be ideal for those who prefer to use fewer ingredients in their solutions. This protocol is designed to achieve the same goals as other northern blotting approaches. It minimizes background (nonspecific adherence of probe to membrane and nonspecific hybridization) and maximizes specific hybridization to RNAs immobilized on a membrane. © 2015 Cold Spring Harbor Laboratory Press.

Prolonged incubation and stacked film exposure improve sensitivity in western blotting.

PubMed

Luo, Haitao; Rankin, Gary O; Straley, Shannon; Chen, Yi Charlie

2011-01-01

Western blotting is a basic technique for protein detection. For proteins of less abundance or antibodies of poorer quality, an increased sensitivity is often desired. Although it is commonly known that higher concentrations of antibodies and prolonged film exposure times will help improve sensitivity in western blots, both measures come with their own risks, and it is often unclear to which extent these measures should be applied. We conducted time-course studies to investigate protein-antibody interactions and primary antibody-secondary antibody interactions in western blotting. We also propose a protocol of stacked film exposure and have tested it in standard curves and cancer cell samples. Our study found that protein-primary antibody interactions and primary antibody-secondary antibody interactions could take a longer time than commonly used "one hour" or "overnight", and in some cases longer than 48h, to reach its maximum binding. We also show that the modified protocol of stacked film exposure works well for both standard curves and biological samples, reaching a maximum sensitivity in western blots without blurring target signals or increasing backgrounds. In addition to regular optimization of antibody concentrations and film exposure time, a prolonged incubation with antibodies and stacked film exposure will also help improve sensitivity and reduce background in western blotting. Copyright © 2011 Elsevier Inc. All rights reserved.

Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.

PubMed

Zellner, Maria; Babeluk, Rita; Diestinger, Michael; Pirchegger, Petra; Skeledzic, Senada; Oehler, Rudolf

2008-09-01

Since most high throughput techniques used in biomarker discovery are very time and cost intensive, highly specific and quantitative analytical alternative application methods are needed for the routine analysis. Conventional Western blotting allows detection of specific proteins to the level of single isotypes while its quantitative accuracy is rather limited. We report a novel and improved quantitative Western blotting method. The use of fluorescently labelled secondary antibodies strongly extends the dynamic range of the quantitation and improves the correlation with the protein amount (r=0.997). By an additional fluorescent staining of all proteins immediately after their transfer to the blot membrane, it is possible to visualise simultaneously the antibody binding and the total protein profile. This allows for an accurate correction for protein load. Applying this normalisation it could be demonstrated that fluorescence-based Western blotting is able to reproduce a quantitative analysis of two specific proteins in blood platelet samples from 44 subjects with different diseases as initially conducted by 2D-DIGE. These results show that the proposed fluorescence-based Western blotting is an adequate application technique for biomarker quantitation and suggest possibilities of employment that go far beyond.

Multistrip Western blotting: a tool for comparative quantitative analysis of multiple proteins.

PubMed

Aksamitiene, Edita; Hoek, Jan B; Kiyatkin, Anatoly

2015-01-01

The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip Western blotting increases data output per single blotting cycle up to tenfold; allows concurrent measurement of up to nine different total and/or posttranslationally modified protein expression obtained from the same loading of the sample; and substantially improves the data accuracy by reducing immunoblotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data and therefore is advantageous to apply in biomedical diagnostics, systems biology, and cell signaling research.

Analysis of differentially expressed genes in two immunologically distinct strains of Eimeria maxima using suppression subtractive hybridization and dot-blot hybridization

PubMed Central

2014-01-01

Background It is well known that different Eimeria maxima strains exhibit significant antigenic variation. However, the genetic basis of these phenotypes remains unclear. Methods Total RNA and mRNA were isolated from unsporulated oocysts of E. maxima strains SH and NT, which were found to have significant differences in immunogenicity in our previous research. Two subtractive cDNA libraries were constructed using suppression subtractive hybridization (SSH) and specific genes were further analyzed by dot-blot hybridization and qRT-PCR analysis. Results A total of 561 clones were selected from both cDNA libraries and the length of the inserted fragments was 0.25–1.0 kb. Dot-blot hybridization revealed a total of 86 differentially expressed clones (63 from strain SH and 23 from strain NT). Nucleotide sequencing analysis of these clones revealed ten specific contigs (six from strain SH and four from strain NT). Further analysis found that six contigs from strain SH and three from strain NT shared significant identities with previously reported proteins, and one contig was presumed to be novel. The specific differentially expressed genes were finally verified by RT-PCR and qRT-PCR analyses. Conclusions The data presented here suggest that specific genes identified between the two strains may be important molecules in the immunogenicity of E. maxima that may present potential new drug targets or vaccine candidates for coccidiosis. PMID:24894832

Chemical analyses of fossil bone.

PubMed

Zheng, Wenxia; Schweitzer, Mary Higby

2012-01-01

The preservation of microstructures consistent with soft tissues, cells, and other biological components in demineralized fragments of dinosaur bone tens of millions of years old was unexpected, and counter to current hypotheses of tissue, cellular, and molecular degradation. Although the morphological similarity of these tissues to extant counterparts was unmistakable, after at least 80 million years exposed to geochemical influences, morphological similarity is insufficient to support an endogenous source. To test this hypothesis, and to characterize these materials at a molecular level, we applied multiple independent chemical, molecular, and microscopic analyses to identify the presence of original components produced by the extinct organisms. Microscopic techniques included field emission scanning electron microscopy, analytical transmission electron microscopy, transmitted light microscopy (LM), and fluorescence microscopy (FM). The chemical and molecular techniques include enzyme-linked immunosorbant assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, western blot (immunoblot), and attenuated total reflectance infrared spectroscopy. In situ analyses performed directly on tissues included immunohistochemistry and time-of-flight secondary ion mass spectrometry. The details of sample preparation and methodology are described in detail herein.

A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

ERIC Educational Resources Information Center

Chang, Ming-Mei; Lovett, Janice

2011-01-01

Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

PubMed

Heda, Ghanshyam D; Omotola, Oluwabukola B; Heda, Rajiv P; Avery, Jamie

2016-09-01

SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.

Lipid-binding analysis using a fat blot assay.

PubMed

Munnik, Teun; Wierzchowiecka, Magdalena

2013-01-01

Protein-lipid interactions play an important role in lipid metabolism, membrane trafficking and cell -signaling by regulating protein localization, activation, and function. The Fat Blot assay is a relatively simple and inexpensive method to examine these interactions using nitrocellulose membrane-immobilized lipids. The assay is adapted from the method by Dowler et al. (Sci STKE 129:pl6, 2002) and provides qualitative and quantitative information on the relative affinity with which a protein binds to a particular lipid. To perform a Fat Blot assay, serial dilutions of different phospholipids are spotted onto a nitrocellulose membrane. These membranes are then incubated with a lipid-binding protein possessing a GST (or other epitope) tag. The membranes are washed and the protein, which is bound to the membrane by virtue of its interaction with the lipid's head group, is detected by immunoblotting with an antibody against GST (or other epitope). The procedure only requires a few micrograms of protein and is quick, simple and cheap to perform.

Treponema pallidum western blot: comparison with the FTA-ABS test as a confirmatory test for syphilis.

PubMed

Backhouse, J L; Nesteroff, S I

2001-01-01

We developed a Treponema pallidum Western blot and compared the results with Treponema pallidum particle agglutination (TPPA) and fluorescent treponemal antibody absorption (FTA-ABS) tests. The Western blot was deemed reactive if the serum reacted with at least three major antigenic bands (TpN47, TpN44.5, TpN17, TpN15). The sensitivities of the Western blot, TPPA and FTA-ABS, were all 100% and the specificities of the Western blot, TPPA and FTA-ABS were 100%, 100% and 94.5% respectively. In 52 problem sera, reactive in only one treponemal test, the agreement between the Western blot and TPPA (61.5%) was significantly better than between Western blot and FTA-ABS (38.5%). The individual sensitivities and specificities of TpN47, TpN44.5, TpN17, TpN15 were 100%, 100%, 96%, 100% and 20%, 96%, 100%, 100% respectively. We conclude that the Western blot is a useful additional confirmatory test or alternative to the FTA-ABS and that a more sensitive and specific criterion for the Western blot would be reactivity with TpN15 and two of the three other major antigens.

A Proteomics Approach to the Protein Normalization Problem: Selection of Unvarying Proteins for MS-Based Proteomics and Western Blotting.

PubMed

Wiśniewski, Jacek R; Mann, Matthias

2016-07-01

Proteomics and other protein-based analysis methods such as Western blotting all face the challenge of discriminating changes in the levels of proteins of interest from inadvertent changes in the amount loaded for analysis. Mass-spectrometry-based proteomics can now estimate the relative and absolute amounts of thousands of proteins across diverse biological systems. We reasoned that this new technology could prove useful for selection of very stably expressed proteins that could serve as better loading controls than those traditionally employed. Large-scale proteomic analyses of SDS lysates of cultured cells and tissues revealed deglycase DJ-1 as the protein with the lowest variability in abundance among different cell types in human, mouse, and amphibian cells. The protein constitutes 0.069 ± 0.017% of total cellular protein and occurs at a specific concentration of 34.6 ± 8.7 pmol/mg of total protein. Since DJ-1 is ubiquitous and therefore easily detectable with several peptides, it can be helpful in normalization of proteomic data sets. In addition, DJ-1 appears to be an advantageous loading control for Western blot that is superior to those used commonly used, allowing comparisons between tissues and cells originating from evolutionarily distant vertebrate species. Notably, this is not possible by the detection and quantitation of housekeeping proteins, which are often used in the Western blot technique. The approach introduced here can be applied to select the most appropriate loading controls for MS-based proteomics or Western blotting in any biological system.

Normalized Quantitative Western Blotting Based on Standardized Fluorescent Labeling.

PubMed

Faden, Frederik; Eschen-Lippold, Lennart; Dissmeyer, Nico

2016-01-01

Western blot (WB) analysis is the most widely used method to monitor expression of proteins of interest in protein extracts of high complexity derived from diverse experimental setups. WB allows the rapid and specific detection of a target protein, such as non-tagged endogenous proteins as well as protein-epitope tag fusions depending on the availability of specific antibodies. To generate quantitative data from independent samples within one experiment and to allow accurate inter-experimental quantification, a reliable and reproducible method to standardize and normalize WB data is indispensable. To date, it is a standard procedure to normalize individual bands of immunodetected proteins of interest from a WB lane to other individual bands of so-called housekeeping proteins of the same sample lane. These are usually detected by an independent antibody or colorimetric detection and do not reflect the real total protein of a sample. Housekeeping proteins-assumed to be constitutively expressed mostly independent of developmental and environmental states-can greatly differ in their expression under these various conditions. Therefore, they actually do not represent a reliable reference to normalize the target protein's abundance to the total amount of protein contained in each lane of a blot.Here, we demonstrate the Smart Protein Layers (SPL) technology, a combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer via WB. SPL allows a rapid and highly sensitive protein visualization and quantification with a sensitivity comparable to conventional silver staining with a 1000-fold higher dynamic range. For normalization, standardization and quantification of protein gels and WBs, a sample-dependent bi-fluorescent standard reagent is applied and, for accurate quantification of data derived from different experiments, a second calibration standard is used. Together, the precise quantification of

Lectin-Array Blotting.

PubMed

Pazos, Raquel; Echevarria, Juan; Hernandez, Alvaro; Reichardt, Niels-Christian

2017-09-01

Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate-lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots

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

Karger, A.E.; Weiss, R.; Gesteland, R.F.

A cryogenically cooled charge-coupled device (CCD) camera equipped with an area CCD array is used in a line scanning system for low-light-level imaging of chemiluminescent DNA sequencing blots. Operating the CCD camera in time-delayed integration (TDI) mode results in continuous data acquisition independent of the length of the CCD array. Scanning is possible with a resolution of 1.4 line pairs/mm at the 50% level of the modulation transfer function. High-sensitivity, low-light-level scanning of chemiluminescent direct-transfer electrophoresis (DTE) DNA sequencing blots is shown. The detection of DNA fragments on the blot involves DNA-DNA hybridization with oligonucleotide-alkaline phosphatase conjugate and 1,2-dioxetane-based chemiluminescence.more » The width of the scan allows the recording of up to four sequencing reactions (16 lanes) on one scan. The scan speed of 52 cm/h used for the sequencing blots corresponds to a data acquisition rate of 384 pixels/s. The chemiluminescence detection limit on the scanned images is 3.9 [times] 10[sup [minus]18] mol of plasmid DNA. A conditional median filter is described to remove spikes caused by cosmic ray events from the CCD images. 39 refs., 9 refs.« less

BLOTS AND ALL: A HISTORY OF THE RORSCHACH INK BLOT TEST IN BRITAIN.

PubMed

Hubbard, Katherine; Hegarty, Peter

2016-01-01

Despite the easily recognizable nature of the Rorschach ink blot test very little is known about the history of the test in Britain. We attend to the oft-ignored history of the Rorschach test in Britain and compare it to its history in the US. Prior to the Second World War, Rorschach testing in Britain had attracted advocates and critiques. Afterward, the British Rorschach Forum, a network with a high proportion of women, developed around the Tavistock Institute in London and The Rorschach Newsletter. In 1968, the International Rorschach Congress was held in London but soon after the group became less exclusive, and fell into decline. A comparative account of the Rorschach in Britain demonstrates how different national institutions invested in the 'projective hypothesis' according to the influence of psychoanalysis, the adoption of a nationalized health system, and the social positioning of 'others' throughout the twentieth century. In comparing and contrasting the history of the Rorschach in Britain and the US, we decentralize and particularize the history of North American Psychology. © 2016 Wiley Periodicals, Inc.

Blotting from PhastGel to Membranes by Ultrasound.

PubMed

Kost, Joseph; Azagury, Aharon

2015-01-01

Ultrasound based approach for enhanced protein blotting is proposed. Three minutes of ultrasound exposure (1 MHz, 2.5 W/cm(2)) was sufficient for a clear transfer of proteins from a polyacrylamide gel (PhastGel) to nitrocellulose or Nylon 66 Biotrans membrane. The proteins evaluated were prestained sodium dodecyl sulfate-polyacrylamide standards (18,500-106,000 Da) and 14C-labeled Rainbow protein molecular weight markers (14,300-200,000 Da).

Blotting from PhastGel to membranes by ultrasound.

PubMed

Kost, Joseph

2009-01-01

Ultrasound-based approach for enhanced protein blotting is proposed. Three minutes of ultrasound exposure (1 MHz, 2.5 W/cm(2)) was sufficient for a clear transfer of proteins from a polyacrylamide gel (PhastGel) to nitrocellulose or Nylon 66 Biotrans membrane. The proteins evaluated were prestained sodium dodecyl sulfate-polyacrylamide standards (18,500-106,000 Da) and (14)C-labeled Rainbow protein molecular weight markers (14,300-200,000 Da).

Protein analysis through Western blot of cells excised individually from human brain and muscle tissue

PubMed Central

Koob, A.O.; Bruns, L.; Prassler, C.; Masliah, E.; Klopstock, T.; Bender, A.

2016-01-01

Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. PMID:22402104

Protein analysis through Western blot of cells excised individually from human brain and muscle tissue.

PubMed

Koob, A O; Bruns, L; Prassler, C; Masliah, E; Klopstock, T; Bender, A

2012-06-15

Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. Copyright © 2012 Elsevier Inc. All rights reserved.

Structural identifiability analyses of candidate models for in vitro Pitavastatin hepatic uptake.

PubMed

Grandjean, Thomas R B; Chappell, Michael J; Yates, James W T; Evans, Neil D

2014-05-01

In this paper a review of the application of four different techniques (a version of the similarity transformation approach for autonomous uncontrolled systems, a non-differential input/output observable normal form approach, the characteristic set differential algebra and a recent algebraic input/output relationship approach) to determine the structural identifiability of certain in vitro nonlinear pharmacokinetic models is provided. The Organic Anion Transporting Polypeptide (OATP) substrate, Pitavastatin, is used as a probe on freshly isolated animal and human hepatocytes. Candidate pharmacokinetic non-linear compartmental models have been derived to characterise the uptake process of Pitavastatin. As a prerequisite to parameter estimation, structural identifiability analyses are performed to establish that all unknown parameters can be identified from the experimental observations available. Copyright © 2013. Published by Elsevier Ireland Ltd.

Statistical Analyses of Scatterplots to Identify Important Factors in Large-Scale Simulations

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

Kleijnen, J.P.C.; Helton, J.C.

1999-04-01

The robustness of procedures for identifying patterns in scatterplots generated in Monte Carlo sensitivity analyses is investigated. These procedures are based on attempts to detect increasingly complex patterns in the scatterplots under consideration and involve the identification of (1) linear relationships with correlation coefficients, (2) monotonic relationships with rank correlation coefficients, (3) trends in central tendency as defined by means, medians and the Kruskal-Wallis statistic, (4) trends in variability as defined by variances and interquartile ranges, and (5) deviations from randomness as defined by the chi-square statistic. The following two topics related to the robustness of these procedures are consideredmore » for a sequence of example analyses with a large model for two-phase fluid flow: the presence of Type I and Type II errors, and the stability of results obtained with independent Latin hypercube samples. Observations from analysis include: (1) Type I errors are unavoidable, (2) Type II errors can occur when inappropriate analysis procedures are used, (3) physical explanations should always be sought for why statistical procedures identify variables as being important, and (4) the identification of important variables tends to be stable for independent Latin hypercube samples.« less

In vitro detection of canine distemper virus nucleic acid with a virus-specific cDNA probe by dot-blot and in situ hybridization.

PubMed

Oglesbee, M; Jackwood, D; Perrine, K; Axthelm, M; Krakowka, S; Rice, J

1986-11-01

A cDNA library was prepared from canine distemper viral (CDV) messenger RNA (mRNA) derived from Vero cells lytically infected with the Onderstepoort strain (Ond) of CDV. A 300 base pair insert was identified which, by Northern blot analysis and Sanger sequence data, was shown to be specific to the nucleocapsid gene. The nucleocapsid (NC) clone was radiolabelled with 32P using nick translation and used to detect viral RNA in both dot-blot and in situ preparations of Vero cells lytically infected with Onderstepoort CDV (Ond-CDV) and immortalized mink lung cells persistently infected with racoon origin CDV (CCL64-RCDV). Dot-blot hybridization results paralleled immunofluorescent results in the lytically infected cells. In 18 persistently infected cell lines from the RCDV-CCL64 parental stock, 13 lines were positive and two were negative on both immunofluorescence and dot-blot hybridization analysis for CDV antigen and RNA, respectively. Viral nucleic acid was detected in these persistently infected cells, where as few as 1.9% of the members of a line were positive on immunofluorescence. A dot-blot autoradiographic signal was obtained in three lines which were negative for CDV antigen. CDV RNA was detected in both lytically and persistently infected cell lines by in situ hybridization, where decreasing probe length was important in increasing the sensitivity of this assay. Viral RNA was detected in over 90% of the lytically infected cells, where only 70% were positive for viral antigen by immunofluorescence.

Profiling EGFR activity in head and neck squamous cell carcinoma by using a novel layered membrane Western blot technology.

PubMed

Patel, Vyomesh; Ramesh, Arun; Traicoff, June L; Baibakov, Galina; Emmert-Buck, Michael R; Gutkind, J Silvio; Knezevic, Vladimir

2005-05-01

Given the role of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC), several rational approaches have now been utilized to abrogate tyrosine kinase activity and its disengagement from downstream signal transducers. Monitoring the activity of these molecules could potentially be useful to determine not only drug efficacy but also to identify HNSCC patients most likely to benefit from this type of therapy. In this study we have used a novel high throughput multi-layered Western blotting (MLWestern) method that allows the detection of multiple proteins from a single experiment in order to characterize key components in the EGFR signaling pathway in HNSCC cells. Total and activated forms of EGFR and the downstream effectors, Erk and Akt were readily detected in HNSCC cells, where in the control cells (HaCaT) these proteins could only be detected in EGF stimulated cells. Results from conventional Western blot and MLWestern were comparable. Clustering analysis of protein expression revealed similarities in cellular response between some of the cell lines indicative of similarities in their biological response. The data indicate that MLWestern can be potentially applied to identify molecular targets that could be used for rational therapeutic intervention strategies.

A new background subtraction method for Western blot densitometry band quantification through image analysis software.

PubMed

Gallo-Oller, Gabriel; Ordoñez, Raquel; Dotor, Javier

2018-06-01

Since its first description, Western blot has been widely used in molecular labs. It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex protein mixtures. Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results. Due to the technical knowledge required for densitometry analysis together with the resources availability, standard office scanners are often used for the imaging acquisition of developed Western blot films. Furthermore, the use of semi-quantitative software as ImageJ (Java-based image-processing and analysis software) is clearly increasing in different scientific fields. In this work, we describe the use of office scanner coupled with the ImageJ software together with a new image background subtraction method for accurate Western blot quantification. The proposed method represents an affordable, accurate and reproducible approximation that could be used in the presence of limited resources availability. Copyright © 2018 Elsevier B.V. All rights reserved.

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots.

PubMed

Abeyrathne, Priyanka D; Lam, Joseph S

2007-04-01

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

Automated capillary Western dot blot method for the identity of a 15-valent pneumococcal conjugate vaccine.

PubMed

Hamm, Melissa; Ha, Sha; Rustandi, Richard R

2015-06-01

Simple Western is a new technology that allows for the separation, blotting, and detection of proteins similar to a traditional Western except in a capillary format. Traditionally, identity assays for biological products are performed using either an enzyme-linked immunosorbent assay (ELISA) or a manual dot blot Western. Both techniques are usually very tedious, labor-intensive, and complicated for multivalent vaccines, and they can be difficult to transfer to other laboratories. An advantage this capillary Western technique has over the traditional manual dot blot Western method is the speed and the automation of electrophoresis separation, blotting, and detection steps performed in 96 capillaries. This article describes details of the development of an automated identity assay for a 15-valent pneumococcal conjugate vaccine, PCV15-CRM197, using capillary Western technology. Copyright © 2015 Elsevier Inc. All rights reserved.

Identifying Useful Auxiliary Variables for Incomplete Data Analyses: A Note on a Group Difference Examination Approach

ERIC Educational Resources Information Center

Raykov, Tenko; Marcoulides, George A.

2014-01-01

This research note contributes to the discussion of methods that can be used to identify useful auxiliary variables for analyses of incomplete data sets. A latent variable approach is discussed, which is helpful in finding auxiliary variables with the property that if included in subsequent maximum likelihood analyses they may enhance considerably…

Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

PubMed

Eaton, Samantha L; Roche, Sarah L; Llavero Hurtado, Maica; Oldknow, Karla J; Farquharson, Colin; Gillingwater, Thomas H; Wishart, Thomas M

2013-01-01

Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation

Fluorescent Labeling of Proteins and Its Application to SDS-PAGE and Western Blotting.

PubMed

Alba, F Javier; Bartolomé, Salvador; Bermúdez, Antonio; Daban, Joan-Ramon

2015-01-01

This chapter describes very simple fluorescent methods developed in our laboratory allowing the rapid monitoring of total protein patterns on both sodium dodecyl sulfate (SDS) polyacrylamide gels and western blots. The noncovalent dye Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) is used for the sensitive staining of proteins in SDS gels. This method is compatible with the electroblotting of protein bands and with the staining of the resulting blot with the covalent dye MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone). These staining procedures are applied sequentially; there is no need to run a duplicate unstained gel for protein blotting. Furthermore, since only the adduct formed by the reaction of MDPF with proteins is fluorescent, there is no need to destain the membrane after protein labeling. In addition, MDPF staining is compatible with further immunodetection of specific bands with polyclonal antibodies. Finally, using the adequate conditions described below, MDPF staining does not preclude the N-terminal sequence analysis of proteins in selected bands.

β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients.

PubMed

Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

2012-08-15

Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.

Quantitation of Protein Carbonylation by Dot Blot

PubMed Central

Wehr, Nancy B.; Levine, Rodney L.

2012-01-01

Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is frequently measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent, 2,4-dinitrophenylhydrazine. We developed an immunochemical dot blot method for quantitation of protein carbonylation in homogenates or purified proteins. Dimethyl sulfoxide was employed as the solvent because it very efficiently extracts proteins from tissues and keeps them soluble. It also readily dissolves 2,4-dinitrophenylhydrazine and wets PVDF membranes. The detection limit is 0.19 ± 0.04 pmol carbonyl. Sixty ng protein is sufficient to measure protein carbonyl content. This level of sensitivity allowed measurement of protein carbonylation in individual Drosophila. PMID:22326366

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro.

PubMed

Jia, Zhaofeng; Liang, Yujie; Ma, Bin; Xu, Xiao; Xiong, Jianyi; Duan, Li; Wang, Daping

2017-05-17

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation. Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC's wider application. In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N + membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

Quantum dot-based western blot for sensitive detection of pig serum antibody to actinobacillus pleuropneumoniae

NASA Astrophysics Data System (ADS)

Cişmileanu, Ana; Sima, Cornelia; Grigoriu, Constantin

2007-08-01

A quantum dot - immunoglobulin conjugate specific for pig IgG, was obtained by carbodiimide chemistry. We used a Western blot technique for detecting specific antibodies against Actinobacillus pleuropneumoniae (A. pp), which cause porcine pleuropneumonia. The antigen used in this technique was Apx haemolysin which is an important virulence factor of A. pp and it induces protective immunity in vaccined pigs. The detection on Western blot membrane was possible at 1/50 dilution of quantum dot conjugate at a dilution of pig serum till 1/6400. The results for pig serum demonstrated a higher sensitivity of QD-based Western blot technique for the presence of antibodies specific for Apx haemolysin in comparison with similar classical techniques (with coloured substrate for enzyme present in secondary antibody conjugate).

Western blotting revisited: critical perusal of underappreciated technical issues.

PubMed

Gorr, Thomas A; Vogel, Johannes

2015-04-01

The most commonly used semiquantitative analysis of protein expression still employs protein separation by denaturing SDS-PAGE with subsequent Western blotting and quantification of the resulting ODs of bands visualized with specific antibodies. However, many questions regarding this procedure are usually ignored, although still in need of answering: Does isolation or separation procedure harm the integrity or affect modifications (e.g., phosphorylation) of the protein of interest? Does denaturation reduce binding of antibodies used for detection? Should denaturation be performed or should a native gel be run? How can artificial degradations or aggregations be distinguished from biological relevant ones? If the antibody detects multiple bands (which is not uncommon), which one(s) should be taken into account for quantification and why? Which loading control protein should be chosen and is it really "housekeeping" and how can this be verified? Is the image acquisition system linear and does it come with a sufficient dynamic range? How to account and control for background staining? This article is intended to address these questions and raise the readers awareness to possible Western blot alternatives in the attempt of minimizing possible pitfalls that might loom anywhere from protein isolation to acquisition of final quantitative data. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of two commercial systems for automated processing, reading, and interpretation of Lyme borreliosis Western blots.

PubMed

Binnicker, M J; Jespersen, D J; Harring, J A; Rollins, L O; Bryant, S C; Beito, E M

2008-07-01

The diagnosis of Lyme borreliosis (LB) is commonly made by serologic testing with Western blot (WB) analysis serving as an important supplemental assay. Although specific, the interpretation of WBs for diagnosis of LB (i.e., Lyme WBs) is subjective, with considerable variability in results. In addition, the processing, reading, and interpretation of Lyme WBs are laborious and time-consuming procedures. With the need for rapid processing and more objective interpretation of Lyme WBs, we evaluated the performances of two automated interpretive systems, TrinBlot/BLOTrix (Trinity Biotech, Carlsbad, CA) and BeeBlot/ViraScan (Viramed Biotech AG, Munich, Germany), using 518 serum specimens submitted to our laboratory for Lyme WB analysis. The results of routine testing with visual interpretation were compared to those obtained by BLOTrix analysis of MarBlot immunoglobulin M (IgM) and IgG and by ViraScan analysis of ViraBlot and ViraStripe IgM and IgG assays. BLOTrix analysis demonstrated an agreement of 84.7% for IgM and 87.3% for IgG compared to visual reading and interpretation. ViraScan analysis of the ViraBlot assays demonstrated agreements of 85.7% for IgM and 94.2% for IgG, while ViraScan analysis of the ViraStripe IgM and IgG assays showed agreements of 87.1 and 93.1%, respectively. Testing by the automated systems yielded an average time savings of 64 min/run compared to processing, reading, and interpretation by our current procedure. Our findings demonstrated that automated processing and interpretive systems yield results comparable to those of visual interpretation, while reducing the subjectivity and time required for Lyme WB analysis.

Northern Blot Detection of Virus-Derived Small Interfering RNAs in Caenorhabditis elegans Using Nonradioactive Oligo Probes.

PubMed

Long, Tianyun; Lu, Rui

2017-01-01

Northern blot analysis has been widely used as a tool for detection and characterization of specific RNA molecules. When coupled with radioactive probe northern blot allows for robust detection and characterization of small RNA molecules of trace amount. Here, we describe the detection and size characterization of virus-derived small interfering RNAs (vsiRNAs) in C. elegans using nonradioactive DNA oligo probes in northern blotting. Our protocol allows for the detection and characterization of not only primary vsiRNAs but also secondary vsiRNAs, a class of single-stranded vsiRNAs that has distinct migration pattern, and can be easily adapted to the detection of vsiRNAs in other organisms.

Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

PubMed

Ng, Khong Y; Machida, Kazuya

2017-01-01

With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.

Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

NASA Astrophysics Data System (ADS)

Parra-Belky, Karlett

2002-11-01

A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

Reverse line blot hybridisation screening of Pseudallescheria/Scedosporium species in patients with cystic fibrosis.

PubMed

Lu, Q; van den Ende, A H G Gerrits; de Hoog, G S; Li, R; Accoceberry, I; Durand-Joly, I; Bouchara, J-P; Hernandez, F; Delhaes, L

2011-10-01

The PCR-RLB (reverse line blot hybridisation) was applied as a molecular technique for the detection of members of Pseudallescheria and Scedosporium from sputum of patients with cystic fibrosis (CF). Fifty-nine sputum samples were collected from 52 CF patients, which were analysed by culture and PCR-RLB. Conventional and semi-selective culture yielded five positive samples, but the PCR-RLB hybridisation assay permitted the detection of members of Pseudallescheria/Scedosporium in 32 out of 52 patients (61.5%). In total, PCR-RLB yielded 47 positives. Pseudallescheria apiosperma was detected in 20 samples, while Pseudallescheria boydii and Pseudallescheria aurantiacum were detected in 17 and eight samples, respectively. Six samples gave a positive reaction with two distinct species-specific probes and one sample with three probes. In conclusion, the PCR-RLB assay described in this study allows the detection of Scedosporium spp. in CF sputum samples and the identification of Pseudallescheria apiosperma, P. boydii, S. aurantiacum, Scedosporium prolificans and Pseudallescheria minutispora. © 2011 Blackwell Verlag GmbH.

Quantitation of protein carbonylation by dot blot.

PubMed

Wehr, Nancy B; Levine, Rodney L

2012-04-15

Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is frequently measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent, 2,4-dinitrophenylhydrazine. We developed an immunochemical dot blot method for quantitation of protein carbonylation in homogenates or purified proteins. Dimethyl sulfoxide was employed as the solvent because it very efficiently extracts proteins from tissues and keeps them soluble. It also readily dissolves 2,4-dinitrophenylhydrazine and wets polyvinylidene difluoride (PVDF) membranes. The detection limit is 0.19 ± 0.04 pmol of carbonyl, and 60 ng of protein is sufficient to measure protein carbonyl content. This level of sensitivity allowed measurement of protein carbonylation in individual Drosophila. Copyright © 2012 Elsevier Inc. All rights reserved.

Phosphinothricin Acetyltransferases Identified Using In Vivo, In Vitro, and Bioinformatic Analyses

PubMed Central

VanDrisse, Chelsey M.; Hentchel, Kristy L.

2016-01-01

ABSTRACT Acetylation of small molecules is widespread in nature, and in some cases, cells use this process to detoxify harmful chemicals. Streptomyces species utilize a Gcn5 N-acetyltransferase (GNAT), known as Bar, to acetylate and detoxify a self-produced toxin, phosphinothricin (PPT), a glutamate analogue. Bar homologues, such as MddA from Salmonella enterica, acetylate methionine analogues such as methionine sulfoximine (MSX) and methionine sulfone (MSO), but not PPT, even though Bar homologues are annotated as PPT acetyltransferases. S. enterica was used as a heterologous host to determine whether or not putative PPT acetyltransferases from various sources could acetylate PPT, MSX, and MSO. In vitro and in vivo analyses identified substrates acetylated by putative PPT acetyltransferases from Deinococcus radiodurans (DR_1057 and DR_1182) and Geobacillus kaustophilus (GK0593 and GK2920). In vivo, synthesis of DR_1182, GK0593, and GK2920 blocked the inhibitory effects of PPT, MSX, and MSO. In contrast, DR_1057 did not detoxify any of the above substrates. Results of in vitro studies were consistent with the in vivo results. In addition, phylogenetic analyses were used to predict the functionality of annotated PPT acetyltransferases in Burkholderia xenovorans, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baylyi, and Escherichia coli. IMPORTANCE The work reported here provides an example of the use of a heterologous system for the identification of enzyme function. Many members of this superfamily of proteins do not have a known function, or it has been annotated solely on the basis of sequence homology to previously characterized enzymes. The critical role of Gcn5 N-acetyltransferases (GNATs) in the modulation of central metabolic processes, and in controlling metabolic stress, necessitates approaches that can reveal their physiological role. The combination of in vivo, in vitro, and bioinformatics approaches reported here identified GNATs that can

Analyses to help identify individuals from a historical mass grave in Kassel, Germany.

PubMed

v Grumbkow, Philipp; Zipp, Anna; Grosskopf, Birgit; Fueldner, Kai; Hummel, Susanne

2012-01-01

In 2008, the skeletal remains of more than 60 human individuals were found in a mass grave on the grounds of the University of Kassel, Germany. There was no evidence helping to identify them or throwing light on the cause of their death. Mainly due to 14C age determination and initial hints on age and sex distribution, historians hypothesized that they had been soldiers of Napoleon's army who died in an epidemic in the winter of 1813/14. To test this assumption, morphological and molecular analyses were carried out on a sample. The morphological analyses comprised an age and sex determination as well as a macro- and micro-morphological inspection for pathological deviations after the commingled bones had been assembled as individuals. The molecular investigations aimed to identify the geographic origin of the remains. For this, mitochondrial and Y-chromosomal haplotypings were carried out. The results point to a group of mainly young men, some of them suffering from systemic inflammation of the periosteum. Others revealed severe aberrations in bone microstructure. The greatest similarities revealed by Y-haplogroup and -haplotype distribution were to populations that live in what are now the Benelux countries. All aspects support the thesis that these were soldiers of the Napoleonic army.

Quantitative trait loci analyses and RNA-seq identify genes affecting stress response in rainbow trout

USDA-ARS?s Scientific Manuscript database

Genomic analyses have the potential to impact aquaculture production traits by identifying markers as proxies for traits which are expensive or difficult to measure and characterizing genetic variation and biochemical mechanisms underlying phenotypic variation. One such trait is the response of rai...

A combined RT-PCR and dot-blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius).

PubMed

Lopez-Jimena, B; Cherif, N; Garcia-Rosado, E; Infante, C; Cano, I; Castro, D; Hammami, S; Borrego, J J; Alonso, M C

2010-10-01

To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin-labelled probes resulted in the identification of both genotypes separately. The application of the RT-PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46.87% of viral nervous necrosis virus carriers. The combination of RT-PCR and blot hybridization increases the detection rate up to 90.62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56.25%). This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

A Study of Rubisco through Western Blotting and Tissue Printing Techniques

ERIC Educational Resources Information Center

Ma, Zhong; Cooper, Cynthia; Kim, Hyun-Joo; Janick-Buckner, Diane

2009-01-01

We describe a laboratory exercise developed for a cell biology course for second-year undergraduate biology majors. It was designed to introduce undergraduates to the basic molecular biology techniques of Western blotting and immunodetection coupled with the technique of tissue printing in detecting the presence, relative abundance, and…

Interaction of murine intestinal mast cell proteinase with inhibitors (serpins) in blood; analysis by SDS-PAGE and western blotting.

PubMed Central

Irvine, J; Newlands, G F; Huntley, J F; Miller, H R

1990-01-01

The interaction of mouse intestinal mast cell proteinase (IMCP) with serine proteinase inhibitors (serpins) in blood was analysed: (i) by examining the capacity of the inhibitors in blood to block the binding of the irreversible serine esterase inhibitor [3H]diisopropyl fluorophosphate (DFP); (ii) by Western blotting. The binding of [3H]DFP to IMCP was blocked very rapidly by inhibitors in mouse serum and, by Western blotting, this inhibition was associated with the appearance of a 73,000 MW proteinase/inhibitor complex together with a series of higher (greater than 100,000) MW complexes. IMCP was not dissociated from these complexes when electrophoresed under reducing conditions, although prior heat treatment of mouse serum (60 for 30-160 min) abolished the formation of all proteinase/inhibitor complexes. Similarly, the activity of a 48,000 MW inhibitor of chymotrypsin was abolished by heat treatment. A titration experiment established that between 0.5 and 5 mg IMCP were inhibited per ml of serum. The properties and MW of the IMCP inhibitor complexes are typical of serpins and suggest that IMCP secreted during intestinal immunological reactions would be rapidly and irreversibly inactivated by plasma-derived inhibitors. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2312150

Differential Network Analyses of Alzheimer’s Disease Identify Early Events in Alzheimer’s Disease Pathology

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

Xia, Jing; Rocke, David M.; Perry, George

In late-onset Alzheimer’s disease (AD), multiple brain regions are not affected simultaneously. Comparing the gene expression of the affected regions to identify the differences in the biological processes perturbed can lead to greater insight into AD pathogenesis and early characteristics. We identified differentially expressed (DE) genes from single cell microarray data of four AD affected brain regions: entorhinal cortex (EC), hippocampus (HIP), posterior cingulate cortex (PCC), and middle temporal gyrus (MTG). We organized the DE genes in the four brain regions into region-specific gene coexpression networks. Differential neighborhood analyses in the coexpression networks were performed to identify genes with lowmore » topological overlap (TO) of their direct neighbors. The low TO genes were used to characterize the biological differences between two regions. Our analyses show that increased oxidative stress, along with alterations in lipid metabolism in neurons, may be some of the very early events occurring in AD pathology. Cellular defense mechanisms try to intervene but fail, finally resulting in AD pathology as the disease progresses. Furthermore, disease annotation of the low TO genes in two independent protein interaction networks has resulted in association between cancer, diabetes, renal diseases, and cardiovascular diseases.« less

Differential Network Analyses of Alzheimer’s Disease Identify Early Events in Alzheimer’s Disease Pathology

DOE PAGES

Xia, Jing; Rocke, David M.; Perry, George; ...

2014-01-01

In late-onset Alzheimer’s disease (AD), multiple brain regions are not affected simultaneously. Comparing the gene expression of the affected regions to identify the differences in the biological processes perturbed can lead to greater insight into AD pathogenesis and early characteristics. We identified differentially expressed (DE) genes from single cell microarray data of four AD affected brain regions: entorhinal cortex (EC), hippocampus (HIP), posterior cingulate cortex (PCC), and middle temporal gyrus (MTG). We organized the DE genes in the four brain regions into region-specific gene coexpression networks. Differential neighborhood analyses in the coexpression networks were performed to identify genes with lowmore » topological overlap (TO) of their direct neighbors. The low TO genes were used to characterize the biological differences between two regions. Our analyses show that increased oxidative stress, along with alterations in lipid metabolism in neurons, may be some of the very early events occurring in AD pathology. Cellular defense mechanisms try to intervene but fail, finally resulting in AD pathology as the disease progresses. Furthermore, disease annotation of the low TO genes in two independent protein interaction networks has resulted in association between cancer, diabetes, renal diseases, and cardiovascular diseases.« less

Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test.

PubMed

Gonçalves, Luciana A; Lobato, Zélia I P; Silva, Rodrigo O S; Salvarani, Felipe M; Pires, Prhiscylla S; Assis, Ronnie A; Lobato, Francisco C F

2009-11-01

Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.

Development of a nanogold slot blot inhibition assay for the detection of antibodies against bovine herpesvirus type 1.

PubMed

Japolla, Greice; Cunha-Junior, Jair Pereira; Pajuaba, Ana Claudia Arantes Marquez; Taketomi, Ernesto Akio; Bührer-Sékula, Samira; Bataus, Luiz Artur Mendes; de Souza, Guilherme Rocha Lino

2018-06-01

Bovine herpesvirus type 1 (BoHV-1) is recognized as an important pathogen causing respiratory, reproductive, and neurological disorders in cattle and is associated with economic losses to animal industry. Accurate diagnostic methods are needed for prevention of disease transmission. While the virus neutralization test is considered the gold standard method, it requires maintenance of the virus and cell cultures, which is time consuming and expensive. Serological techniques such as enzyme-linked immunosorbent assay (ELISA) are widely applied, as these are easy to perform and provide quick results. In the present study, a nanogold slot blot inhibition assay was developed for the serological diagnosis of BoHV-1 and compared with standard ELISA and horseradish peroxidase (HRP) slot blot assays. Of 42 serum samples tested by ELISA, 32 (76.2%) were positive and 10 (23.8%), were negative. The sensitivity and specificity of the nanogold slot blot inhibition assay was similar to that observed for ELISA and HRP slot blot assays, and a strong correlation was observed between the tests. Thus, the nanogold slot blot inhibition assay may serve as an efficient and rapid alternative to ELISA in settings, where plate-reading equipment is lacking.

Single-trait and multi-trait genome-wide association analyses identify novel loci for blood pressure in African-ancestry populations

PubMed Central

Liang, Jingjing; Le, Thu H.; Edwards, Digna R. Velez; Tayo, Bamidele O.; Gaulton, Kyle J.; Lu, Yingchang; Jensen, Richard A.; Chen, Guanjie; Schwander, Karen; McKenzie, Colin A.; Fox, Ervin; Nalls, Michael A.; Young, J. Hunter; Lane, Jacqueline M.; Zhou, Jie; Tang, Hua; Fornage, Myriam; Musani, Solomon K.; Wang, Heming; Forrester, Terrence; Chu, Pei-Lun; Evans, Michele K.; Morrison, Alanna C.; Martin, Lisa W.; Wiggins, Kerri L.; Hui, Qin; Zhao, Wei; Jackson, Rebecca D.; Faul, Jessica D.; Reiner, Alex P.; Bray, Michael; Denny, Joshua C.; Mosley, Thomas H.; Palmas, Walter; Guo, Xiuqing; Polak, Joseph F.; Taylor, Ken D.; Boerwinkle, Eric; Bottinger, Erwin P.; Liu, Kiang; Risch, Neil; Hunt, Steven C.; Kooperberg, Charles; Zonderman, Alan B.; Becker, Diane M.; Cai, Jianwen; Loos, Ruth J. F.; Psaty, Bruce M.; Weir, David R.; Kardia, Sharon L. R.; Arnett, Donna K.; Won, Sungho; Edwards, Todd L.; Redline, Susan; Cooper, Richard S.; Rao, D. C.; Rotimi, Charles; Levy, Daniel; Chakravarti, Aravinda

2017-01-01

Hypertension is a leading cause of global disease, mortality, and disability. While individuals of African descent suffer a disproportionate burden of hypertension and its complications, they have been underrepresented in genetic studies. To identify novel susceptibility loci for blood pressure and hypertension in people of African ancestry, we performed both single and multiple-trait genome-wide association analyses. We analyzed 21 genome-wide association studies comprised of 31,968 individuals of African ancestry, and validated our results with additional 54,395 individuals from multi-ethnic studies. These analyses identified nine loci with eleven independent variants which reached genome-wide significance (P < 1.25×10−8) for either systolic and diastolic blood pressure, hypertension, or for combined traits. Single-trait analyses identified two loci (TARID/TCF21 and LLPH/TMBIM4) and multiple-trait analyses identified one novel locus (FRMD3) for blood pressure. At these three loci, as well as at GRP20/CDH17, associated variants had alleles common only in African-ancestry populations. Functional annotation showed enrichment for genes expressed in immune and kidney cells, as well as in heart and vascular cells/tissues. Experiments driven by these findings and using angiotensin-II induced hypertension in mice showed altered kidney mRNA expression of six genes, suggesting their potential role in hypertension. Our study provides new evidence for genes related to hypertension susceptibility, and the need to study African-ancestry populations in order to identify biologic factors contributing to hypertension. PMID:28498854

An assessment of a Western blot method for the investigation of canine cutaneous adverse food reactions.

PubMed

Maina, Elisa; Matricoti, Irina; Noli, Chiara

2018-06-01

Adverse food reaction (AFR) is diagnosed with a two month elimination diet (ED), followed by challenge with the original food. To evaluate reactivity of selected EDs and performance of a Western blot serological test for the diagnosis of AFR. Twenty five food reactive (FR) and 13 non food reactive (NFR) privately owned dogs. Sera were tested for antibodies against hydrolyzed poultry feather (RCA), hydrolyzed soy (PHA), hydrolyzed fish (FUH), limited antigen horse and potato (THP), fresh horse meat and the offending food for each FR dog as documented by provocative challenge. Fourteen sera were negative and two positive to all foods. Sera from five of 13 NFR and three of 25 FR dogs were reactive to hydrolyzed foods. The RCA diet was recognized by four of 38, FUH by six of 38 and PHA by one of 28 samples. THP was recognized by 14 of 33 and fresh horse by one of ten dogs that had never eaten horse meat. The test correctly identified one of 15 dogs allergic to FUH. Twenty of 25 FR sera were negative for the dogs' respective offending foods (20% sensitivity), whereas four of 13 NFR sera were positive to the dogs' usual diets (69% specificity). Western blot analysis cannot be considered as a valid tool for the diagnosis of AFR; it may serve as an aid in selecting an ED. © 2018 ESVD and ACVD.

Improved high-throughput quantification of luminescent microplate assays using a common Western-blot imaging system.

PubMed

Hawkins, Liam J; Storey, Kenneth B

2017-01-01

Common Western-blot imaging systems have previously been adapted to measure signals from luminescent microplate assays. This can be a cost saving measure as Western-blot imaging systems are common laboratory equipment and could substitute a dedicated luminometer if one is not otherwise available. One previously unrecognized limitation is that the signals captured by the cameras in these systems are not equal for all wells. Signals are dependent on the angle of incidence to the camera, and thus the location of the well on the microplate. Here we show that: •The position of a well on a microplate significantly affects the signal captured by a common Western-blot imaging system from a luminescent assay.•The effect of well position can easily be corrected for.•This method can be applied to commercially available luminescent assays, allowing for high-throughput quantification of a wide range of biological processes and biochemical reactions.

Ferulic acid enhances IgE binding to peanut allergens in western blots.

USDA-ARS?s Scientific Manuscript database

Phenolic compounds at high concentrations are known to form insoluble complexes with proteins. We hypothesized that this complex formation could interfere with Western blot and ELISA assays for peanut allergens. To verify this, three simple phenolic compounds (ferulic, caffeic, and chlorogenic acids...

COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

EPA Science Inventory

A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

A rapid chemiluminescent slot blot immunoassay for the detection and quantification of Clostridium botulinum neurotoxin type E, in cultures.

PubMed

Cadieux, Brigitte; Blanchfield, Burke; Smith, James P; Austin, John W

2005-05-01

A simple, rapid, cost-effective in vitro slot blot immunoassay was developed for the detection and quantification of botulinum neurotoxin type E (BoNT/E) in cultures. Culture supernatants of 36 strains of clostridia, including 12 strains of Clostridium botulinum type E, 12 strains of other C. botulinum neurotoxin serotypes, and 12 strains of other clostridial species were tested. Samples containing BoNT/E were detected using affinity-purified polyclonal rabbit antisera prepared against BoNT/E with subsequent detection of secondary antibodies using chemiluminescence. All strains of C. botulinum type E tested positive, while all non C. botulinum type E strains tested negative. The sensitivity of the slot blot immunoassay for detection of BoNT/E was approximately four mouse lethal doses (MLD). The intensity of chemiluminescence was directly correlated with the concentration of BoNT/E up to 128 MLD, allowing quantification of BoNT/E between 4 and 128 MLD. The slot blot immunoassay was compared to the mouse bioassay for detection of BoNT/E using cultures derived from fish samples inoculated with C. botulinum type E, and cultures derived from naturally contaminated environmental samples. A total of 120 primary enrichment cultures derived from fish samples, of which 103 were inoculated with C. botulinum type E, and 17 were uninoculated controls, were assayed. Of the 103 primary enrichment cultures derived from inoculated fish samples, all were positive by mouse bioassay, while 94 were also positive by slot blot immunoassay, resulting in a 7.5% false-negative rate. All 17 primary enrichment cultures derived from the uninoculated fish samples were negative by both mouse bioassay and slot blot immunoassay. A total of twenty-six primary enrichment cultures derived from environmental samples were tested by mouse bioassay and slot blot immunoassay. Of 13 primary enrichment cultures positive by mouse bioassay, 12 were also positive by slot blot immunoassay, resulting in a 3

Ferulic acid enhances IgE binding to peanut allergens in western blots.

USDA-ARS?s Scientific Manuscript database

Because phenolic compounds can precipitate or complex with proteins, we postulated that interactions of phenolics with IgE antibodies help enhance IgE binding to peanut allergens in Western blots. Three different phenolics, such as, ferulic, caffeic and chlorogenic acids were examined. Each was mixe...

Application of a reverse dot blot DNA-DNA hydridization method to quantify host-feeding tendencies of two sibling species in the Anopheles gambiae complex.

PubMed

Fritz, M L; Miller, J R; Bayoh, M N; Vulule, J M; Landgraf, J R; Walker, E D

2013-12-01

A DNA-DNA hybridization method, reverse dot blot analysis (RDBA), was used to identify Anopheles gambiae s.s. and Anopheles arabiensis (Diptera: Culicidae) hosts. Of 299 blood-fed and semi-gravid An. gambiae s.l. collected from Kisian, Kenya, 244 individuals were identifiable to species; of these, 69.5% were An. arabiensis and 29.5% were An. gambiae s.s. Host identifications with RDBA were comparable with those of conventional polymerase chain reaction (PCR) followed by direct sequencing of amplicons of the vertebrate mitochondrial cytochrome b gene. Of the 174 amplicon-producing samples used to compare these two methods, 147 were identifiable by direct sequencing and 139 of these were identifiable by RDBA. Anopheles arabiensis bloodmeals were mostly (94.6%) bovine in origin, whereas An. gambiae s.s. fed upon humans more than 91.8% of the time. Tests by RDBA detected that two of 112 An. arabiensis contained blood from more than one host species, whereas PCR and direct sequencing did not. Recent use of insecticide-treated bednets in Kisian is likely to have caused the shift in the dominant vector species from An. gambiae s.s. to An. arabiensis. Reverse dot blot analysis provides an opportunity to study changes in host-feeding by members of the An. gambiae complex in response to the broadening distribution of vector control measures targeting host-selection behaviours. © 2013 The Royal Entomological Society.

Pre-study feasibility and identifying sensitivity analyses for protocol pre-specification in comparative effectiveness research.

PubMed

Girman, Cynthia J; Faries, Douglas; Ryan, Patrick; Rotelli, Matt; Belger, Mark; Binkowitz, Bruce; O'Neill, Robert

2014-05-01

The use of healthcare databases for comparative effectiveness research (CER) is increasing exponentially despite its challenges. Researchers must understand their data source and whether outcomes, exposures and confounding factors are captured sufficiently to address the research question. They must also assess whether bias and confounding can be adequately minimized. Many study design characteristics may impact on the results; however, minimal if any sensitivity analyses are typically conducted, and those performed are post hoc. We propose pre-study steps for CER feasibility assessment and to identify sensitivity analyses that might be most important to pre-specify to help ensure that CER produces valid interpretable results.

Matrix-assisted laser desorption-ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures.

PubMed

Lamer, S; Jungblut, P R

2001-03-10

In theory, peptide mass fingerprinting by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower microl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.

Serological differentiation of murine typhus and epidemic typhus using cross-adsorption and Western blotting.

PubMed

La Scola, B; Rydkina, L; Ndihokubwayo, J B; Vene, S; Raoult, D

2000-07-01

Differentiation of murine typhus due to Rickettsia typhi and epidemic typhus due to Rickettsia prowazekii is critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) against R. typhi and R. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekii and R. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers against R. typhi and R. prowazekii allowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate between R. prowazekii and R. typhi exposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use.

Serological Differentiation of Murine Typhus and Epidemic Typhus Using Cross-Adsorption and Western Blotting

PubMed Central

La Scola, Bernard; Rydkina, Lena; Ndihokubwayo, Jean-Bosco; Vene, Sirkka; Raoult, Didier

2000-01-01

Differentiation of murine typhus due to Rickettsia typhi and epidemic typhus due to Rickettsia prowazekii is critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) against R. typhi and R. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekii and R. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers against R. typhi and R. prowazekii allowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate between R. prowazekii and R. typhi exposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use. PMID:10882661

[Scientific Evaluation of Crude Drugs and Kampo Medicines Using the Eastern Blotting Method and Its Application to Biological Metabolic Studies].

PubMed

Morinaga, Osamu

2018-01-01

　The scientific evaluation of crude drugs and kampo medicines (KMs) was demonstrated using the eastern blotting method with monoclonal antibodies (MAbs) against bioactive natural compounds. Scutellariae radix is one of the most important crude drugs used in KMs. Part of its pharmaceutical properties is due to the flavone glycoside baicalin (BI). A quantitative analysis method based on eastern blotting was developed for BI using an anti-BI MAb. A rapid, simple, sensitive, specific analytical system was subsequently established for BI with the eastern blotting technique using dot-blot and chemiluminescent methods. This system was useful as a high-throughput analytical method for the determination of BI in KMs as well as HPLC and enzyme-linked immunosorbent assay systems. Furthermore, an eastern blotting method was applied to the biological metabolic study of glycyrrhizic acid (GL), the major active constituent of licorice, for investigation of metabolites of GL such as 3-monoglucuronyl-glycyrrhetinic acid (3MGA) because licorice causes pseudoaldosteronism as a side effect. This approach may make it possible to determine the pathogenic agents of licorice-induced pseudoaldosteronism.

Can hospital audit teams identify case management problems, analyse their causes, identify and implement improvements? A cross-sectional process evaluation of obstetric near-miss case reviews in Benin.

PubMed

Borchert, Matthias; Goufodji, Sourou; Alihonou, Eusèbe; Delvaux, Thérèse; Saizonou, Jacques; Kanhonou, Lydie; Filippi, Véronique

2012-10-11

Obstetric near-miss case reviews are being promoted as a quality assurance intervention suitable for hospitals in low income countries. We introduced such reviews in five district, regional and national hospitals in Benin, West Africa. In a cross-sectional study we analysed the extent to which the hospital audit teams were able to identify case management problems (CMPs), analyse their causes, agree on solutions and put these solutions into practice. We analysed case summaries, women's interview transcripts and audit minutes produced by the audit teams for 67 meetings concerning one woman with near-miss complications each. We compared the proportion of CMPs identified by an external assessment team to the number found by the audit teams. For the latter, we described the CMP causes identified, solutions proposed and implemented by the audit teams. Audit meetings were conducted regularly and were well attended. Audit teams identified half of the 714 CMPs; they were more likely to find managerial ones (71%) than the ones relating to treatment (30%). Most identified CMPs were valid. Almost all causes of CMPs were plausible, but often too superficial to be of great value for directing remedial action. Audit teams suggested solutions, most of them promising ones, for 38% of the CMPs they had identified, but recorded their implementation only for a minority (8.5%). The importance of following-up and documenting the implementation of solutions should be stressed in future audit interventions. Tools facilitating the follow-up should be made available. Near-miss case reviews hold promise, but their effectiveness to improve the quality of care sustainably and on a large scale still needs to be established.

Can hospital audit teams identify case management problems, analyse their causes, identify and implement improvements? A cross-sectional process evaluation of obstetric near-miss case reviews in Benin

PubMed Central

2012-01-01

Background Obstetric near-miss case reviews are being promoted as a quality assurance intervention suitable for hospitals in low income countries. We introduced such reviews in five district, regional and national hospitals in Benin, West Africa. In a cross-sectional study we analysed the extent to which the hospital audit teams were able to identify case management problems (CMPs), analyse their causes, agree on solutions and put these solutions into practice. Methods We analysed case summaries, women’s interview transcripts and audit minutes produced by the audit teams for 67 meetings concerning one woman with near-miss complications each. We compared the proportion of CMPs identified by an external assessment team to the number found by the audit teams. For the latter, we described the CMP causes identified, solutions proposed and implemented by the audit teams. Results Audit meetings were conducted regularly and were well attended. Audit teams identified half of the 714 CMPs; they were more likely to find managerial ones (71%) than the ones relating to treatment (30%). Most identified CMPs were valid. Almost all causes of CMPs were plausible, but often too superficial to be of great value for directing remedial action. Audit teams suggested solutions, most of them promising ones, for 38% of the CMPs they had identified, but recorded their implementation only for a minority (8.5%). Conclusions The importance of following-up and documenting the implementation of solutions should be stressed in future audit interventions. Tools facilitating the follow-up should be made available. Near-miss case reviews hold promise, but their effectiveness to improve the quality of care sustainably and on a large scale still needs to be established. PMID:23057707

The epidemiology of tick-borne haemoparasites as determined by the reverse line blot hybridization assay in an intensively studied cohort of calves in western Kenya

PubMed Central

Njiiri, Nyawira E.; Bronsvoort, B. Mark deC.; Collins, Nicola E.; Steyn, Helena C.; Troskie, Milana; Vorster, Ilse; Thumbi, S.M.; Sibeko, Kgomotso P.; Jennings, Amy; van Wyk, Ilana Conradie; Mbole-Kariuki, Mary; Kiara, Henry; Poole, E. Jane; Hanotte, Olivier; Coetzer, Koos; Oosthuizen, Marinda C.; Woolhouse, Mark; Toye, Philip

2015-01-01

The development of sensitive surveillance technologies using PCR-based detection of microbial DNA, such as the reverse line blot assay, can facilitate the gathering of epidemiological information on tick-borne diseases, which continue to hamper the productivity of livestock in many parts of Africa and elsewhere. We have employed a reverse line blot assay to detect the prevalence of tick-borne parasites in an intensively studied cohort of indigenous calves in western Kenya. The calves were recruited close to birth and monitored for the presence of infectious disease for up to 51 weeks. The final visit samples from 453 calves which survived for the study period were analyzed by RLB. The results indicated high prevalences of Theileria mutans (71.6%), T. velifera (62.8%), Anaplasma sp. Omatjenne (42.7%), A. bovis (39.9%), Theileria sp. (sable) (32.7%), T. parva (12.9%) and T. taurotragi (8.5%), with minor occurrences of eight other haemoparasites. The unexpectedly low prevalence of the pathogenic species Ehrlichia ruminantium was confirmed by a species-specific PCR targeting the pCS20 gene region. Coinfection analyses of the seven most prevalent haemoparasites indicated that they were present as coinfections in over 90% of the cases. The analyses revealed significant associations between several of the Theileria parasites, in particular T. velifera with Theileria sp. sable and T. mutans, and T. parva with T. taurotragi. There was very little coinfection of the two most common Anaplasma species, although they were commonly detected as coinfections with the Theileria parasites. The comparison of reverse line blot and serological results for four haemoparasites (T. parva, T. mutans, A. marginale and B. bigemina) indicated that, except for the mostly benign T. mutans, indigenous cattle seem capable of clearing infections of the three other, pathogenic parasites to below detectable levels. Although the study site was located across four agroecological zones, there was

Application of a Dot Blot Hybridization Platform to Assess Streptococcus uberis Population Structure in Dairy Herds

PubMed Central

Albuquerque, Pedro; Ribeiro, Niza; Almeida, Alexandre; Panschin, Irena; Porfirio, Afonso; Vales, Marta; Diniz, Francisca; Madeira, Helena; Tavares, Fernando

2017-01-01

Streptococcus uberis is considered one of the most important pathogens associated with bovine mastitis. While traditionally acknowledged as an environmental pathogen, S. uberis has been shown to adopt a contagious epidemiological pattern in several dairy herds. Since different control strategies are employed depending on the mode of transmission, in-depth studies of S. uberis populations are essential to determine the best practices to control this pathogen. In this work, we optimized and validated a dot blot platform, combined with automatic image analysis, to rapidly assess the population structure of infective S. uberis, and evaluated its efficiency when compared to multilocus sequence analysis (MLSA) genotyping. Two dairy herds with prevalent S. uberis infections were followed in a 6 month period, in order to collect and characterize isolates from cows with persistent infections. These herds, located in Portugal (Barcelos and Maia regions), had similar management practices, with the herd from Barcelos being smaller and having a better milking parlor management, since infected cow segregation was immediate. A total of 54 S. uberis isolates were obtained from 24 different cows from the two herds. To overcome operator-dependent analysis of the dot blots and increase the technique's consistency and reliability, the hybridization signals were converted into probability values, with average probabilities higher than 0.5 being considered positive results. These data allowed to confirm the isolates' identity as S. uberis using taxa-specific markers and to determine the presence of virulence- and antibiotic resistance-related genes. In addition, MLSA allowed to disclose the most prevalent S. uberis clonal lineages in both herds. Seven different clusters were identified, with Barcelos showing a high clonal diversity and Maia a dominant lineage infecting most cows, suggesting distinct epidemiological patterns, with S. uberis displaying an environmental or contagious

Proteins related to the functions of fibroblast-like synoviocytes identified by proteomic analysis.

PubMed

Zhang, Hui; Fan, Lie Ying; Zong, Ming; Sun, Li Shan; Lu, Liu

2012-01-01

It is well known that the fibroblast-like synoviocytes (FLS) play a key role in pathogenesis of rheumatoid arthritis (RA). This study was performed to separate the differentially expressed proteins of FLS from the patients with RA or osteoarthritis (OA) by two-dimensional electrophoresis (2-DE), and found proteins associated with the functions of FLS by mass spectrometry (MS). Total proteins were extracted and quantified from the primary cultured FLS from patients of RA (n=8) or OA (n=6). Proteins were separated by high-resolution 2-DE, and identified the differentially expressed proteins by MS. Western blot analyses was used to validated the expression of candidate proteins. The mRNA of these proteins was detected by semi-quantitative fluorescent PCR. There are 1147 protein spots from RA and 1324 protein spots from OA showed on 2-DE graphs, respectively. We have selected 84 protein spots for MS analysis, and 27 protein spots were successfully identified. We have found that protein isoaspartyl methyltransferase (PIMT) and pirin (iron-binding nuclear protein, PIR) with lower expression in RA, and thioredoxin 1(Trx-1) only expressed in RA may be associated with functions of FLS. Western Blot confirmed the expression of PIMT and pirin lower in RA, and Trx-1 expressed only in RA. The results of semi-quantitative fluorescent PCR are also consistent with 2-DE graphs. PIMT, pirin and Trx-1 affect the functions of FLS in some style and can be the drug targets of RA.

Analysis of immunostaining and western blotting of endothelin 1 and its receptors in mitral stenosis

PubMed Central

Leão, Sydney Correia; Dashwood, Michael R.; de Andrade, Mateus Santana; Santos, Nicolas Nascimento; Teles, Olivia Regina Lins Leal; de Souza, Williasmin Batista; Rodrigues, Tania Maria de Andrade

2015-01-01

Introduction Rheumatic Fever represents a serious public health problem in developing countries, with thousands of new cases each year. It is an autoimmune disease, which occurs in response to infection by streptococcus A. Objective The aim of this study was to evaluate the immunolabeling and protein expression for endothelin-1 and 3 (ET-1, ET-3) and its receptors (ETA, ETB) in rheumatic mitral valves. Methods Immunohistochemistry was used to identify ET-1/ET-3 and ETA/ETB receptors in rheumatic and control mitral valves. Quantitative analysis of immunostaining for ET-1/ET-3 and ETA/ETB receptors was performed. In addition, western blot analysis was carried out to assess protein levels in tissue samples. Results ET-1 and ETA receptor immunostaining predominated in stenotic valves, mainly associated with fibrotic regions, inflammatory areas and neovascularization. Quantitative analysis showed that the average area with positive expression of ET-1 was 18.21±14.96%. For ETA and ETB, the mean expressed areas were respectively 15.06±13.13% and 9.20±11.09%. ET-3 did not have a significant expression. The correlation between the expression of both endothelin receptors were strongly positive (R=0.74, P=0.02), but the correlation between ET-1 and its receptor were negative for both ETA (R=-0.37, P=0.25), and ETB (R=-0.14, P=0.39). This data was supported by western blot analysis. Conclusion The strong correlation between ET-1 and its receptors suggests that both play a role in the pathophysiology of rheumatic mitral valve stenosis and may potentially act as biomarkers of this disease. PMID:26107453

Identifying and analysing protostellar disc fragments in smoothed particle hydrodynamics simulations

NASA Astrophysics Data System (ADS)

Hall, Cassandra; Forgan, Duncan; Rice, Ken

2017-09-01

We present a new method of identifying protostellar disc fragments in a simulation based on density derivatives, and analyse our data using this and the existing CLUMPFIND method, which is based on an ordered search over all particles in gravitational potential energy. Using smoothed particle hydrodynamics, we carry out nine simulations of a 0.25 M⊙ disc around a 1 M⊙ star, all of which fragment to form at least two bound objects. We find that when using all particles ordered in gravitational potential space, only fragments that survive the duration of the simulation are detected. When we use the density derivative method, all fragments are detected, so the two methods are complementary, as using the two methods together allows us to identify all fragments, and to then determine those that are likely to be destroyed. We find a tentative empirical relationship between the dominant azimuthal wavenumber in the disc m and the maximum semimajor axis a fragment may achieve in a simulation, such that amax∝1/m. We find the fragment destruction rate to be around half that predicted from population synthesis models. This is due to fragment-fragment interactions in the early gas phase of the disc, which can cause scattering and eccentricity pumping on short time-scales, and affects the fragment's internal structure. We therefore caution that measurements of eccentricity as a function of semimajor axis may not necessarily constrain the formation mechanism of giant planets and brown dwarfs.

Use of a latency-based demand assessment to identify potential demands for functional analyses.

PubMed

Call, Nathan A; Miller, Sarah J; Mintz, Joslyn Cynkus; Mevers, Joanna Lomas; Scheithauer, Mindy C; Eshelman, Julie E; Beavers, Gracie A

2016-12-01

Unlike potential tangible positive reinforcers, which are typically identified for inclusion in functional analyses empirically using preference assessments, demands are most often selected arbitrarily or based on caregiver report. The present study evaluated the use of a demand assessment with 12 participants who exhibited escape-maintained problem behavior. Participants were exposed to 10 demands, with aversiveness measured by average latency to the first instance of problem behavior. In subsequent functional analyses, results of a demand condition that included the demand with the shortest latency to problem behavior resulted in identification of an escape function for 11 of the participants. In contrast, a demand condition that included the demand with the longest latency resulted in identification of an escape function for only 5 participants. The implication of these findings is that for the remaining 7 participants, selection of the demand for the functional analysis without using the results of the demand assessment could have produced a false-negative finding. © 2016 Society for the Experimental Analysis of Behavior.

Checking transfer efficiency and equal loading via qualitative optical way in western blotting.

PubMed

Gong, Jun-Hua; Gong, Jian-Ping; Zheng, Kai-Wen

2017-11-01

The ability to determine that successful transfer and equal loading occur prior to using primary antibodies is important. And total protein staining is commonly used to check transfer efficiency and normalization, which play a crucial role in western blotting. Ponceau S and coomassie blue are commonly used, but there are disadvantages reported in recent years. Therefore, we are interested in finding another method, which is cheap, easy and fast. As we know, protein binding region of PVDF membrane is still hydrophilic when carbinol volatilizes, however, the non-protein binding region of PVDF membrane became hydrophobic again. And this different wettability between non-protein binding region and protein binding region of Polyvinylidene difluoride membrane may be used to check transfer efficiency and equal loading in western blotting. Based on the principle above, we describe an optical approach where an experimenter can observe that the proteins have been transferred to the membrane without any staining within minutes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Coronin-1C is a novel biomarker for hepatocellular carcinoma invasive progression identified by proteomics analysis and clinical validation

PubMed Central

2010-01-01

Background To better search for potential markers for hepatocellular carcinoma (HCC) invasion and metastasis, proteomic approach was applied to identify potential metastasis biomarkers associated with HCC. Methods Membrane proteins were extracted from MHCC97L and HCCLM9 cells, with a similar genetic background and remarkably different metastasis potential, and compared by SDS-PAGE and identified by ESI-MS/MS. The results were further validated by western blot analysis, immunohistochemistry (IHC) of tumor tissues from HCCLM9- and MHCC97L-nude mice, and clinical specimens. Results Membrane proteins were extracted from MHCC97L and HCCLM9 cell and compared by SDS-PAGE analyses. A total of 14 differentially expressed proteins were identified by ESI-MS/MS. Coronin-1C, a promising candidate, was found to be overexpressed in HCCLM9 cells as compared with MHCC97L cells, and validated by western blot and IHC from both nude mice tumor tissues and clinical specimens. Coronin-1C level showed an abrupt upsurge when pulmonary metastasis occurred. Increasing coronin-1C expression was found in liver cancer tissues of HCCLM9-nude mice with spontaneous pulmonary metastasis. IHC study on human HCC specimens revealed that more patients in the higher coronin-1C group had overt larger tumor and more advanced stage. Conclusions Coronin-1C could be a candidate biomarker to predict HCC invasive behavior. PMID:20181269

Coronin-1C is a novel biomarker for hepatocellular carcinoma invasive progression identified by proteomics analysis and clinical validation.

PubMed

Wu, Long; Peng, Chun-Wei; Hou, Jin-Xuan; Zhang, Yan-Hua; Chen, Chuang; Chen, Liang-Dong; Li, Yan

2010-02-24

To better search for potential markers for hepatocellular carcinoma (HCC) invasion and metastasis, proteomic approach was applied to identify potential metastasis biomarkers associated with HCC. Membrane proteins were extracted from MHCC97L and HCCLM9 cells, with a similar genetic background and remarkably different metastasis potential, and compared by SDS-PAGE and identified by ESI-MS/MS. The results were further validated by western blot analysis, immunohistochemistry (IHC) of tumor tissues from HCCLM9- and MHCC97L-nude mice, and clinical specimens. Membrane proteins were extracted from MHCC97L and HCCLM9 cell and compared by SDS-PAGE analyses. A total of 14 differentially expressed proteins were identified by ESI-MS/MS. Coronin-1C, a promising candidate, was found to be overexpressed in HCCLM9 cells as compared with MHCC97L cells, and validated by western blot and IHC from both nude mice tumor tissues and clinical specimens. Coronin-1C level showed an abrupt upsurge when pulmonary metastasis occurred. Increasing coronin-1C expression was found in liver cancer tissues of HCCLM9-nude mice with spontaneous pulmonary metastasis. IHC study on human HCC specimens revealed that more patients in the higher coronin-1C group had overt larger tumor and more advanced stage. Coronin-1C could be a candidate biomarker to predict HCC invasive behavior.

Genetic variants associated with subjective well-being, depressive symptoms and neuroticism identified through genome-wide analyses

PubMed Central

Derringer, Jaime; Gratten, Jacob; Lee, James J; Liu, Jimmy Z; de Vlaming, Ronald; Ahluwalia, Tarunveer S; Buchwald, Jadwiga; Cavadino, Alana; Frazier-Wood, Alexis C; Davies, Gail; Furlotte, Nicholas A; Garfield, Victoria; Geisel, Marie Henrike; Gonzalez, Juan R; Haitjema, Saskia; Karlsson, Robert; van der Laan, Sander W; Ladwig, Karl-Heinz; Lahti, Jari; van der Lee, Sven J; Miller, Michael B; Lind, Penelope A; Liu, Tian; Matteson, Lindsay; Mihailov, Evelin; Minica, Camelia C; Nolte, Ilja M; Mook-Kanamori, Dennis O; van der Most, Peter J; Oldmeadow, Christopher; Qian, Yong; Raitakari, Olli; Rawal, Rajesh; Realo, Anu; Rueedi, Rico; Schmidt, Börge; Smith, Albert V; Stergiakouli, Evie; Tanaka, Toshiko; Taylor, Kent; Thorleifsson, Gudmar; Wedenoja, Juho; Wellmann, Juergen; Westra, Harm-Jan; Willems, Sara M; Zhao, Wei; Amin, Najaf; Bakshi, Andrew; Bergmann, Sven; Bjornsdottir, Gyda; Boyle, Patricia A; Cherney, Samantha; Cox, Simon R; Davis, Oliver S P; Ding, Jun; Direk, Nese; Eibich, Peter; Emeny, Rebecca T; Fatemifar, Ghazaleh; Faul, Jessica D; Ferrucci, Luigi; Forstner, Andreas J; Gieger, Christian; Gupta, Richa; Harris, Tamara B; Harris, Juliette M; Holliday, Elizabeth G; Hottenga, Jouke-Jan; De Jager, Philip L; Kaakinen, Marika A; Kajantie, Eero; Karhunen, Ville; Kolcic, Ivana; Kumari, Meena; Launer, Lenore J; Franke, Lude; Li-Gao, Ruifang; Liewald, David C; Koini, Marisa; Loukola, Anu; Marques-Vidal, Pedro; Montgomery, Grant W; Mosing, Miriam A; Paternoster, Lavinia; Pattie, Alison; Petrovic, Katja E; Pulkki-Råback, Laura; Quaye, Lydia; Räikkönen, Katri; Rudan, Igor; Scott, Rodney J; Smith, Jennifer A; Sutin, Angelina R; Trzaskowski, Maciej; Vinkhuyzen, Anna E; Yu, Lei; Zabaneh, Delilah; Attia, John R; Bennett, David A; Berger, Klaus; Bertram, Lars; Boomsma, Dorret I; Snieder, Harold; Chang, Shun-Chiao; Cucca, Francesco; Deary, Ian J; van Duijn, Cornelia M; Eriksson, Johan G; Bültmann, Ute; de Geus, Eco J C; Groenen, Patrick J F; Gudnason, Vilmundur; Hansen, Torben; Hartman, Catharine A; Haworth, Claire M A; Hayward, Caroline; Heath, Andrew C; Hinds, David A; Hyppönen, Elina; Iacono, William G; Järvelin, Marjo-Riitta; Jöckel, Karl-Heinz; Kaprio, Jaakko; Kardia, Sharon L R; Keltikangas-Järvinen, Liisa; Kraft, Peter; Kubzansky, Laura D; Lehtimäki, Terho; Magnusson, Patrik K E; Martin, Nicholas G; McGue, Matt; Metspalu, Andres; Mills, Melinda; de Mutsert, Renée; Oldehinkel, Albertine J; Pasterkamp, Gerard; Pedersen, Nancy L; Plomin, Robert; Polasek, Ozren; Power, Christine; Rich, Stephen S; Rosendaal, Frits R; den Ruijter, Hester M; Schlessinger, David; Schmidt, Helena; Svento, Rauli; Schmidt, Reinhold; Alizadeh, Behrooz Z; Sørensen, Thorkild I A; Spector, Tim D; Starr, John M; Stefansson, Kari; Steptoe, Andrew; Terracciano, Antonio; Thorsteinsdottir, Unnur; Thurik, A Roy; Timpson, Nicholas J; Tiemeier, Henning; Uitterlinden, André G; Vollenweider, Peter; Wagner, Gert G; Weir, David R; Yang, Jian; Conley, Dalton C; Smith, George Davey; Hofman, Albert; Johannesson, Magnus; Laibson, David I; Medland, Sarah E; Meyer, Michelle N; Pickrell, Joseph K; Esko, Tõnu; Krueger, Robert F; Beauchamp, Jonathan P; Koellinger, Philipp D; Benjamin, Daniel J; Bartels, Meike; Cesarini, David

2016-01-01

We conducted genome-wide association studies of three phenotypes: subjective well-being (N = 298,420), depressive symptoms (N = 161,460), and neuroticism (N = 170,910). We identified three variants associated with subjective well-being, two with depressive symptoms, and eleven with neuroticism, including two inversion polymorphisms. The two depressive symptoms loci replicate in an independent depression sample. Joint analyses that exploit the high genetic correlations between the phenotypes (|ρ^| ≈ 0.8) strengthen the overall credibility of the findings, and allow us to identify additional variants. Across our phenotypes, loci regulating expression in central nervous system and adrenal/pancreas tissues are strongly enriched for association. PMID:27089181

Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation.

PubMed

Maccari, Francesca; Volpi, Nicola

2002-09-01

We describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGs) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 nug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGs was about 0.1-0.5 nug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC-treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGs and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization.

New Method for Simultaneous Species-Specific Identification of Equine Strongyles (Nematoda, Strongylida) by Reverse Line Blot Hybridization▿

PubMed Central

Traversa, Donato; Iorio, Raffaella; Klei, Thomas R.; Kharchenko, Vitaliy A.; Gawor, Jakub; Otranto, Domenico; Sparagano, Olivier A. E.

2007-01-01

The ability of a reverse line blot (RLB) assay to identify 13 common species of equine small strongyles (cyathostomins) and to discriminate them from three Strongylus spp. (large strongyles) was demonstrated. The assay relied on the specific hybridization of PCR-amplified intergenic spacer DNA fragments of the nuclear ribosomal DNA to membrane-bound species-specific probes. All cyathostomins examined were unequivocally identified and simultaneously discriminated from each other and from three large strongyles (Strongylus edentatus, Strongylus equinus, and Strongylus vulgaris). This assay will enable the accurate and rapid identification of equine cyathostomins irrespective of their life cycle stage, opening important avenues for a better understanding of their biology and epidemiology and of the pathogenesis of cyathostomin-associated disease. In particular, this RLB method promises to be a powerful diagnostic tool to determine the roles of individual species in the pathogenesis of mixed infections and to elucidate some aspects of cyathostominosis. Also, it could represent a basic step toward the development of a rapid and simple molecular test for the early detection of drug-resistant genotypes of horse strongyle species. PMID:17626168

Comparative evaluation of western blotting in hepatic and pulmonary cystic echinococcosis.

PubMed

Akisu, C; Delibas, S B; Bicmen, C; Ozkoc, S; Aksoy, U; Turgay, N

2006-12-01

Many serological tests are widely used in the diagnosis of cystic echinococcosis (CE), caused by the larval stages of Echinococcus granulosus. The present study was carried for differentiation between hepatic and pulmonary cystic echinococcosis by Western Blotting (WB). A total of 121 sera from patients with hepatic CE (37), pulmonary CE (31) and controls (53; consisting of six healthy, seven Hymenolepis nana infection, 20 hepatic and 20 pulmonary diseases other than CE) were examined. In all of the CE patients, E. gronulosus infection was confirmed by surgical intervention. Sera were previously tested using IHA and ELISA to detect the E. gronulosus specific antibodies. Sera from hepatic cases of CE reacted with 16 polypeptides of 6-116 kDa and sera from pulmonary cases of CE reacted with 14 polypeptides of 4-130 kDa by Western Blotting. The WB test enabled the detection of antibodies in the hepatic CE samples for proteins of 24, 32 34, 44-46 and 52-54 kDa in molecular weight in 78.4%, 75.7%, 78.4% and 89.2% of the patients, respectively. In the pulmonary CE samples sera WB test enabled the detection of antibodies 24, 44-46, 100, 110, 116 and 120 124 kDa in molecular weight in 81.3%, 75.0%, 87.5%, 71.9%, 84.4% and 65.6% of the patients, respectively. We indicated that the antigenic components of high molecular weight can be good candidates for differentiation of hepatic CE from pulmonary CE.

Proteomics-based approach identified differentially expressed proteins with potential roles in endometrial carcinoma.

PubMed

Li, Zhengyu; Min, Wenjiao; Huang, Canhua; Bai, Shujun; Tang, Minghai; Zhao, Xia

2010-01-01

We used proteomic approaches to identify altered expressed proteins in endometrial carcinoma, with the aim of discovering potential biomarkers or therapeutic targets for endometrial carcinoma. The global proteins extracted from endometrial carcinoma and normal endometrial tissues were separated by 2-dimensional electrophoresis and analyzed with PDQuest (Bio-Rad, Hercules, Calif) software. The differentially expressed spots were identified by mass spectrometry and searched against NCBInr protein database. Those proteins with potential roles were confirmed by Western blotting and immunohistochemical assays. Ninety-nine proteins were identified by mass spectrometry, and a cluster diagram analysis indicated that these proteins were involved in metabolism, cell transformation, protein folding, translation and modification, proliferation and apoptosis, signal transduction, cytoskeleton, and so on. In confirmatory immunoblotting and immunohistochemical analyses, overexpressions of epidermal fatty acid-binding protein, calcyphosine, and cyclophilin A were also observed in endometrial carcinoma tissues, which were consistent with the proteomic results. Our results suggested that these identified proteins, including epidermal fatty acid-binding protein, calcyphosine, and cyclophilin A, might be of potential values in the studies of endometrial carcinogenesis or investigations of diagnostic biomarkers or treatment targets for endometrial carcinoma.

Interaction of Proteins Identified in Human Thyroid Cells

PubMed Central

Pietsch, Jessica; Riwaldt, Stefan; Bauer, Johann; Sickmann, Albert; Weber, Gerhard; Grosse, Jirka; Infanger, Manfred; Eilles, Christoph; Grimm, Daniela

2013-01-01

Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains. PMID:23303277

A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

ERIC Educational Resources Information Center

Ness, Traci L.; Robinson, Rebekah L.; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H.

2015-01-01

SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes…

Preservation of pathological tissue specimens by freeze-drying for immunohistochemical staining and various molecular biological analyses.

PubMed

Matsuo, S; Sugiyama, T; Okuyama, T; Yoshikawa, K; Honda, K; Takahashi, R; Maeda, S

1999-05-01

Conditions of preserving DNA, RNA and protein in pathological specimens are of great importance as degradation of such macromolecules would critically affect results of molecular biological analysis. The feasibility of freeze-drying as a means of preserving pathological tissue samples for molecular analysis has previously been shown. In the present study, further tests on long-term storage conditions and analyses of freeze-dried samples by polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, western blotting and immunohistochemistry are reported. Rat chromosomal DNA of freeze-dried samples stored for 4 years showed slight degradation while RNA degradation was more prominently seen at an earlier stage of storage. However, these 4 year DNA and RNA samples were still able to serve as a template for some PCR and RT-PCR analyses, respectively. Overexpression of c-erbB-2 and p53 protein was demonstrated by western blotting and immunohistochemical staining using freeze-dried human breast cancer tissues. Although macromolecules in freeze-dried samples degrade to some extent during the preservation period, they should still be of value for certain molecular biological analyses and morphological examination; hence, providing more convenient and inexpensive ways of pathological tissue storage.

Dot-Blot Immunoassay of Fasciola gigantica Infection using 27 kDa and Adult Worm Regurge Antigens in Egyptian Patients

PubMed Central

Kamel, Hanan H.; Saad, Ghada A.

2013-01-01

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable. PMID:23710084

Dot-blot immunoassay of Fasciola gigantica infection using 27 kDa and adult worm regurge antigens in Egyptian patients.

PubMed

Kamel, Hanan H; Saad, Ghada A; Sarhan, Rania M

2013-04-01

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.

Male specific genes from dioecious white campion identified by fluorescent differential display.

PubMed

Scutt, Charles P; Jenkins, Tom; Furuya, Masaki; Gilmartin, Philip M

2002-05-01

Fluorescent differential display (FDD) has been used to screen for cDNAs that are differentially up-regulated in male flowers of the dioecious plant Silene latifolia in which an X/Y chromosome system of sex determination operates. To adapt FDD to the cloning of large numbers of differential cDNAs, a novel method of confirming the differential expression of these has been devised. FDD gels were Southern electro-blotted and probed with mixtures of individual cDNA clones derived from different FDD product ligation reactions. These Southern blots were then stripped and re-probed with further mixtures of individual cloned FDD products to identify the maximum number of recombinant clones carrying the true differential amplification products. Of 135 differential bands identified by FDD, 56 differential amplification products were confirmed; these represent 23 unique differentially expressed genes as determined by virtual Northern analysis and two genes expressed at or below the level of detection by virtual Northern analysis. These two low expressed genes show bands of hybridization on genomic Southern blots that are specific to male plants, indicating that they are derived from, or closely related to, Y chromosome genes.

Western blot analysis of sera from dogs with suspected food allergy.

PubMed

Favrot, Claude; Linek, Monika; Fontaine, Jacques; Beco, Luc; Rostaher, Ana; Fischer, Nina; Couturier, Nicolas; Jacquenet, Sandrine; Bihain, Bernard E

2017-04-01

Food allergy is often suspected in dogs with clinical signs of atopic dermatitis. This diagnosis is confirmed with an elimination diet and a subsequent challenge with regular food. Laboratory tests for the diagnosis of food allergy in dogs are unreliable and/or technically difficult. Cyno-DIAL ® is a Western blot method that might assist with the selection of an appropriate elimination diet. To evaluate the performance of Cyno-DIAL ® for the selection of an elimination diet and diagnosis of food allergy. Thirty eight dogs with atopic dermatitis completed an elimination diet. Combining the results of the diet trials and the challenges, 14 dogs were classified as food allergic (FA), 22 as nonfood-allergic and two as ambiguous cases. Amongst all dogs and amongst dogs with a clinical diagnosis of FA, 3% and 7% (respectively) were positive to Royal Canin Anallergenic ® , Vet-Concept Kanguru ® or Vet-Concept Dog Sana ® ; 8% and 7% to Hill's d/d Duck and Rice ® ; 8% and 21% to Hill's z/d Ultra Allergen Free ® ; 53% and 64% to Eukanuba Dermatosis FP ® ; and 32% and 43% to a home-cooked diet of horse meat, potatoes and zucchini. The specificity and sensitivity of Cyno-DIAL ® for diagnosing food allergy were 73% and 71%, respectively. Although Cyno-DIAL ® was considered potentially useful for identifying appropriate foods for elimination diet trials, it cannot be recommended for the diagnosis of food allergy. The Cyno-DIAL ® test performed better than some previously evaluated ELISA-based tests. © 2017 ESVD and ACVD.

Simultaneous Detection of Bovine Theileria and Babesia Species by Reverse Line Blot Hybridization

PubMed Central

Gubbels, J. M.; de Vos, A. P.; van der Weide, M.; Viseras, J.; Schouls, L. M.; de Vries, E.; Jongejan, F.

1999-01-01

A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species of Theileria and Babesia simultaneously. We included Theileria annulata, T. parva, T. mutans, T. taurotragi, and T. velifera in the assay, as well as parasites belonging to the T. sergenti-T. buffeli-T. orientalis group. The Babesia species included were Babesia bovis, B. bigemina, and B. divergens. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all Theileria and Babesia species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences from Bos taurus or other hemoparasites (Trypanosoma species, Cowdria ruminantium, Anaplasma marginale, and Ehrlichia species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain. T. annulata, T. orientalis, and B. bigemina were identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species. PMID:10325324

Blotting Assisted by Heating and Solvent Extraction for DESI-MS Imaging

NASA Astrophysics Data System (ADS)

Cabral, Elaine C.; Mirabelli, Mario F.; Perez, Consuelo J.; Ifa, Demian R.

2013-06-01

Imprints of potato sprout ( Solanum tuberosum L.), gingko leaves (Gingko biloba L. ) and strawberries (Fragaria x ananassa Duch. ) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces.

Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing.

PubMed

Alba, F J; Daban, J R

1997-10-01

We have studied the light emission efficiency of proteins labeled with different fluorescent dyes chemically excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction. Using this peroxyoxalate chemiluminescence system, the best results were obtained with proteins covalently labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF). Blotted proteins on polyvinylidene difluoride (PVDF) membranes can be labeled rapidly with MDPF. Our results demonstrate that energy from the excited intermediate produced in the TCPO-H2O2 reaction can be efficiently transferred to MDPF-labeled proteins in solution and on PVDF membranes. Although this nonenzymatic chemiluminescent system produces a background emission that reduces the sensitivity, the method developed in this work allows detection of 5 ng of protein in blots after 5 min exposure to X-ray film. Chemiluminescence of MDPF-labeled proteins on Western and slot blots may also be detected and quantified using a charge-coupled device (CCD) camera or a storage phosphor imaging system. This chemiluminescent method allows the staining of the total electrophoretic pattern but does not preclude further N-terminal sequencing and immunodetection of specific bands.

Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

NASA Astrophysics Data System (ADS)

Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

2007-12-01

A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

Proteomic Analyses of the Effects of Drugs of Abuse on Monocyte-Derived Mature Dendritic Cells

PubMed Central

Reynolds, Jessica L.; Mahajan, Supriya D.; Aalinkeel, Ravikunar; Nair, B.; Sykes, Donald E.; Schwartz, Stanley A.

2010-01-01

Drug abuse has become a global health concern. Understanding how drug abuse modulates the immune system and how the immune system responds to pathogens associated with drug abuse, such hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1), can be assessed by an integrated approach comparing proteomic analyses and quantitation of gene expression. Two-dimensional (2D) difference gel electrophoresis was used to determine the molecular mechanisms underlying the proteomic changes that alter normal biological processes when monocyte-derived mature dendritic cells were treated with cocaine or methamphetamine. Both drugs differentially regulated the expression of several functional classes of proteins including those that modulate apoptosis, protein folding, protein kinase activity, and metabolism and proteins that function as intracellular signal transduction molecules. Proteomic data were validated using a combination of quantitative, real-time PCR and Western blot analyses. These studies will help to identify the molecular mechanisms, including the expression of several functionally important classes of proteins that have emerged as potential mediators of pathogenesis. These proteins may predispose immunocompetent cells, including dendritic cells, to infection with viruses such as HCV and HIV-1, which are associated with drug abuse. PMID:19811410

Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting.

PubMed

Carthew, James; Karakesisoglou, Iakowos

2016-01-01

Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins.

Large-scale association analyses identify new loci influencing glycemic traits and provide insight into the underlying biological pathways.

PubMed

Scott, Robert A; Lagou, Vasiliki; Welch, Ryan P; Wheeler, Eleanor; Montasser, May E; Luan, Jian'an; Mägi, Reedik; Strawbridge, Rona J; Rehnberg, Emil; Gustafsson, Stefan; Kanoni, Stavroula; Rasmussen-Torvik, Laura J; Yengo, Loïc; Lecoeur, Cecile; Shungin, Dmitry; Sanna, Serena; Sidore, Carlo; Johnson, Paul C D; Jukema, J Wouter; Johnson, Toby; Mahajan, Anubha; Verweij, Niek; Thorleifsson, Gudmar; Hottenga, Jouke-Jan; Shah, Sonia; Smith, Albert V; Sennblad, Bengt; Gieger, Christian; Salo, Perttu; Perola, Markus; Timpson, Nicholas J; Evans, David M; Pourcain, Beate St; Wu, Ying; Andrews, Jeanette S; Hui, Jennie; Bielak, Lawrence F; Zhao, Wei; Horikoshi, Momoko; Navarro, Pau; Isaacs, Aaron; O'Connell, Jeffrey R; Stirrups, Kathleen; Vitart, Veronique; Hayward, Caroline; Esko, Tõnu; Mihailov, Evelin; Fraser, Ross M; Fall, Tove; Voight, Benjamin F; Raychaudhuri, Soumya; Chen, Han; Lindgren, Cecilia M; Morris, Andrew P; Rayner, Nigel W; Robertson, Neil; Rybin, Denis; Liu, Ching-Ti; Beckmann, Jacques S; Willems, Sara M; Chines, Peter S; Jackson, Anne U; Kang, Hyun Min; Stringham, Heather M; Song, Kijoung; Tanaka, Toshiko; Peden, John F; Goel, Anuj; Hicks, Andrew A; An, Ping; Müller-Nurasyid, Martina; Franco-Cereceda, Anders; Folkersen, Lasse; Marullo, Letizia; Jansen, Hanneke; Oldehinkel, Albertine J; Bruinenberg, Marcel; Pankow, James S; North, Kari E; Forouhi, Nita G; Loos, Ruth J F; Edkins, Sarah; Varga, Tibor V; Hallmans, Göran; Oksa, Heikki; Antonella, Mulas; Nagaraja, Ramaiah; Trompet, Stella; Ford, Ian; Bakker, Stephan J L; Kong, Augustine; Kumari, Meena; Gigante, Bruna; Herder, Christian; Munroe, Patricia B; Caulfield, Mark; Antti, Jula; Mangino, Massimo; Small, Kerrin; Miljkovic, Iva; Liu, Yongmei; Atalay, Mustafa; Kiess, Wieland; James, Alan L; Rivadeneira, Fernando; Uitterlinden, Andre G; Palmer, Colin N A; Doney, Alex S F; Willemsen, Gonneke; Smit, Johannes H; Campbell, Susan; Polasek, Ozren; Bonnycastle, Lori L; Hercberg, Serge; Dimitriou, Maria; Bolton, Jennifer L; Fowkes, Gerard R; Kovacs, Peter; Lindström, Jaana; Zemunik, Tatijana; Bandinelli, Stefania; Wild, Sarah H; Basart, Hanneke V; Rathmann, Wolfgang; Grallert, Harald; Maerz, Winfried; Kleber, Marcus E; Boehm, Bernhard O; Peters, Annette; Pramstaller, Peter P; Province, Michael A; Borecki, Ingrid B; Hastie, Nicholas D; Rudan, Igor; Campbell, Harry; Watkins, Hugh; Farrall, Martin; Stumvoll, Michael; Ferrucci, Luigi; Waterworth, Dawn M; Bergman, Richard N; Collins, Francis S; Tuomilehto, Jaakko; Watanabe, Richard M; de Geus, Eco J C; Penninx, Brenda W; Hofman, Albert; Oostra, Ben A; Psaty, Bruce M; Vollenweider, Peter; Wilson, James F; Wright, Alan F; Hovingh, G Kees; Metspalu, Andres; Uusitupa, Matti; Magnusson, Patrik K E; Kyvik, Kirsten O; Kaprio, Jaakko; Price, Jackie F; Dedoussis, George V; Deloukas, Panos; Meneton, Pierre; Lind, Lars; Boehnke, Michael; Shuldiner, Alan R; van Duijn, Cornelia M; Morris, Andrew D; Toenjes, Anke; Peyser, Patricia A; Beilby, John P; Körner, Antje; Kuusisto, Johanna; Laakso, Markku; Bornstein, Stefan R; Schwarz, Peter E H; Lakka, Timo A; Rauramaa, Rainer; Adair, Linda S; Smith, George Davey; Spector, Tim D; Illig, Thomas; de Faire, Ulf; Hamsten, Anders; Gudnason, Vilmundur; Kivimaki, Mika; Hingorani, Aroon; Keinanen-Kiukaanniemi, Sirkka M; Saaristo, Timo E; Boomsma, Dorret I; Stefansson, Kari; van der Harst, Pim; Dupuis, Josée; Pedersen, Nancy L; Sattar, Naveed; Harris, Tamara B; Cucca, Francesco; Ripatti, Samuli; Salomaa, Veikko; Mohlke, Karen L; Balkau, Beverley; Froguel, Philippe; Pouta, Anneli; Jarvelin, Marjo-Riitta; Wareham, Nicholas J; Bouatia-Naji, Nabila; McCarthy, Mark I; Franks, Paul W; Meigs, James B; Teslovich, Tanya M; Florez, Jose C; Langenberg, Claudia; Ingelsson, Erik; Prokopenko, Inga; Barroso, Inês

2012-09-01

Through genome-wide association meta-analyses of up to 133,010 individuals of European ancestry without diabetes, including individuals newly genotyped using the Metabochip, we have increased the number of confirmed loci influencing glycemic traits to 53, of which 33 also increase type 2 diabetes risk (q < 0.05). Loci influencing fasting insulin concentration showed association with lipid levels and fat distribution, suggesting impact on insulin resistance. Gene-based analyses identified further biologically plausible loci, suggesting that additional loci beyond those reaching genome-wide significance are likely to represent real associations. This conclusion is supported by an excess of directionally consistent and nominally significant signals between discovery and follow-up studies. Functional analysis of these newly discovered loci will further improve our understanding of glycemic control.

Linkage analyses in Caribbean Hispanic families identify novel loci associated with familial late-onset Alzheimer's disease.

PubMed

Barral, Sandra; Cheng, Rong; Reitz, Christiane; Vardarajan, Badri; Lee, Joseph; Kunkle, Brian; Beecham, Gary; Cantwell, Laura S; Pericak-Vance, Margaret A; Farrer, Lindsay A; Haines, Jonathan L; Goate, Alison M; Foroud, Tatiana; Boerwinkle, Eric; Schellenberg, Gerard D; Mayeux, Richard

2015-12-01

We performed linkage analyses in Caribbean Hispanic families with multiple late-onset Alzheimer's disease (LOAD) cases to identify regions that may contain disease causative variants. We selected 67 LOAD families to perform genome-wide linkage scan. Analysis of the linked regions was repeated using the entire sample of 282 families. Validated chromosomal regions were analyzed using joint linkage and association. We identified 26 regions linked to LOAD (HLOD ≥3.6). We validated 13 of the regions (HLOD ≥2.5) using the entire family sample. The strongest signal was at 11q12.3 (rs2232932: HLODmax = 4.7, Pjoint = 6.6 × 10(-6)), a locus located ∼2 Mb upstream of the membrane-spanning 4A gene cluster. We additionally identified a locus at 7p14.3 (rs10255835: HLODmax = 4.9, Pjoint = 1.2 × 10(-5)), a region harboring genes associated with the nervous system (GARS, GHRHR, and NEUROD6). Future sequencing efforts should focus on these regions because they may harbor familial LOAD causative mutations. Copyright © 2015 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

Deletion detection for diagnosis of Duchenne muscular dystrophy in the Japanese population--comparison between the polymerase chain reaction and the Southern blot analysis.

PubMed

Katayama, S; Takeshita, N; Yano, T; Ubagai, T; Qiu, X J; Katagiri, Y; Kubo, H; Hirakawa, S

1993-06-01

We compared the efficacy of the multiplex PCR with that of the cDNA analysis for detection of deletions of the DMD gene in the Japanese patients. Thirty males with DMD from 27 Japanese families were studied by the multiplex PCR, and 24 of them were also investigated by Southern blot analysis. We used five dystrophin cDNA probes for deletion analysis. A total of 19 regions were amplified by the PCR to detect deletions, 9 regions by the method of Chamberlain et al. and another 10 regions by the method of Beggs et al. Deletions were detected in 14 (52%) out of 27 DMD families by the PCR. Southern blot analysis detected deletions in 14 (64%) out of 22 families. Thirteen (93%) of the 14 DMD families with deletions detected by Southern blotting were also confirmed by the multiplex PCR. Provided care is taken in cases where the deletion is limited to a single exon, the multiplex PCR appears to be an efficient and useful alternative to conventional Southern blot analysis for detecting deletions during the prenatal and postnatal diagnosis of DMD.

Identification and characterization of Serpulina hyodysenteriae by restriction enzyme analysis and Southern blot analysis.

PubMed Central

Sotiropoulos, C; Coloe, P J; Smith, S C

1994-01-01

Chromosomal DNA restriction enzyme analysis and Southern blot hybridization were used to characterize Serpulina hyodysenteriae strains. When chromosomal DNAs from selected strains (reference serotypes) of S. hyodysenteriae were digested with the restriction endonuclease Sau3A and hybridized with a 1.1-kb S. hyodysenteriae-specific DNA probe, a common 3-kb band was always detected in S. hyodysenteriae strains but was absent from Serpulina innocens strains. When the chromosomal DNA was digested with the restriction endonuclease Asp 700 and hybridized with two S. hyodysenteriae-specific DNA probes (0.75 and 1.1 kb of DNA), distinct hybridization patterns for each S. hyodysenteriae reference strain and the Australian isolate S. hyodysenteriae 5380 were detected. Neither the 1.1-kb nor the 0.75-kb DNA probe hybridized with Asp 700- or Sau3A-digested S. innocens chromosomal DNA. The presence of the 3-kb Sau3A DNA fragment in S. hyodysenteriae reference strains from diverse geographical locations shows that this fragment is conserved among S. hyodysenteriae strains and can be used as a species-specific marker. Restriction endonuclease analysis and Southern blot hybridization with these well-defined DNA probes are reliable and accurate methods for species-specific and strain-specific identification of S. hyodysenteriae. Images PMID:7914209

Large-scale association analyses identify new loci influencing glycemic traits and provide insight into the underlying biological pathways

PubMed Central

Scott, Robert A; Lagou, Vasiliki; Welch, Ryan P; Wheeler, Eleanor; Montasser, May E; Luan, Jian’an; Mägi, Reedik; Strawbridge, Rona J; Rehnberg, Emil; Gustafsson, Stefan; Kanoni, Stavroula; Rasmussen-Torvik, Laura J; Yengo, Loïc; Lecoeur, Cecile; Shungin, Dmitry; Sanna, Serena; Sidore, Carlo; Johnson, Paul C D; Jukema, J Wouter; Johnson, Toby; Mahajan, Anubha; Verweij, Niek; Thorleifsson, Gudmar; Hottenga, Jouke-Jan; Shah, Sonia; Smith, Albert V; Sennblad, Bengt; Gieger, Christian; Salo, Perttu; Perola, Markus; Timpson, Nicholas J; Evans, David M; Pourcain, Beate St; Wu, Ying; Andrews, Jeanette S; Hui, Jennie; Bielak, Lawrence F; Zhao, Wei; Horikoshi, Momoko; Navarro, Pau; Isaacs, Aaron; O’Connell, Jeffrey R; Stirrups, Kathleen; Vitart, Veronique; Hayward, Caroline; Esko, Tönu; Mihailov, Evelin; Fraser, Ross M; Fall, Tove; Voight, Benjamin F; Raychaudhuri, Soumya; Chen, Han; Lindgren, Cecilia M; Morris, Andrew P; Rayner, Nigel W; Robertson, Neil; Rybin, Denis; Liu, Ching-Ti; Beckmann, Jacques S; Willems, Sara M; Chines, Peter S; Jackson, Anne U; Kang, Hyun Min; Stringham, Heather M; Song, Kijoung; Tanaka, Toshiko; Peden, John F; Goel, Anuj; Hicks, Andrew A; An, Ping; Müller-Nurasyid, Martina; Franco-Cereceda, Anders; Folkersen, Lasse; Marullo, Letizia; Jansen, Hanneke; Oldehinkel, Albertine J; Bruinenberg, Marcel; Pankow, James S; North, Kari E; Forouhi, Nita G; Loos, Ruth J F; Edkins, Sarah; Varga, Tibor V; Hallmans, Göran; Oksa, Heikki; Antonella, Mulas; Nagaraja, Ramaiah; Trompet, Stella; Ford, Ian; Bakker, Stephan J L; Kong, Augustine; Kumari, Meena; Gigante, Bruna; Herder, Christian; Munroe, Patricia B; Caulfield, Mark; Antti, Jula; Mangino, Massimo; Small, Kerrin; Miljkovic, Iva; Liu, Yongmei; Atalay, Mustafa; Kiess, Wieland; James, Alan L; Rivadeneira, Fernando; Uitterlinden, Andre G; Palmer, Colin N A; Doney, Alex S F; Willemsen, Gonneke; Smit, Johannes H; Campbell, Susan; Polasek, Ozren; Bonnycastle, Lori L; Hercberg, Serge; Dimitriou, Maria; Bolton, Jennifer L; Fowkes, Gerard R; Kovacs, Peter; Lindström, Jaana; Zemunik, Tatijana; Bandinelli, Stefania; Wild, Sarah H; Basart, Hanneke V; Rathmann, Wolfgang; Grallert, Harald; Maerz, Winfried; Kleber, Marcus E; Boehm, Bernhard O; Peters, Annette; Pramstaller, Peter P; Province, Michael A; Borecki, Ingrid B; Hastie, Nicholas D; Rudan, Igor; Campbell, Harry; Watkins, Hugh; Farrall, Martin; Stumvoll, Michael; Ferrucci, Luigi; Waterworth, Dawn M; Bergman, Richard N; Collins, Francis S; Tuomilehto, Jaakko; Watanabe, Richard M; de Geus, Eco J C; Penninx, Brenda W; Hofman, Albert; Oostra, Ben A; Psaty, Bruce M; Vollenweider, Peter; Wilson, James F; Wright, Alan F; Hovingh, G Kees; Metspalu, Andres; Uusitupa, Matti; Magnusson, Patrik K E; Kyvik, Kirsten O; Kaprio, Jaakko; Price, Jackie F; Dedoussis, George V; Deloukas, Panos; Meneton, Pierre; Lind, Lars; Boehnke, Michael; Shuldiner, Alan R; van Duijn, Cornelia M; Morris, Andrew D; Toenjes, Anke; Peyser, Patricia A; Beilby, John P; Körner, Antje; Kuusisto, Johanna; Laakso, Markku; Bornstein, Stefan R; Schwarz, Peter E H; Lakka, Timo A; Rauramaa, Rainer; Adair, Linda S; Smith, George Davey; Spector, Tim D; Illig, Thomas; de Faire, Ulf; Hamsten, Anders; Gudnason, Vilmundur; Kivimaki, Mika; Hingorani, Aroon; Keinanen-Kiukaanniemi, Sirkka M; Saaristo, Timo E; Boomsma, Dorret I; Stefansson, Kari; van der Harst, Pim; Dupuis, Josée; Pedersen, Nancy L; Sattar, Naveed; Harris, Tamara B; Cucca, Francesco; Ripatti, Samuli; Salomaa, Veikko; Mohlke, Karen L; Balkau, Beverley; Froguel, Philippe; Pouta, Anneli; Jarvelin, Marjo-Riitta; Wareham, Nicholas J; Bouatia-Naji, Nabila; McCarthy, Mark I; Franks, Paul W; Meigs, James B; Teslovich, Tanya M; Florez, Jose C; Langenberg, Claudia; Ingelsson, Erik; Prokopenko, Inga; Barroso, Inês

2012-01-01

Through genome-wide association meta-analyses of up to 133,010 individuals of European ancestry without diabetes, including individuals newly genotyped using the Metabochip, we have raised the number of confirmed loci influencing glycemic traits to 53, of which 33 also increase type 2 diabetes risk (q < 0.05). Loci influencing fasting insulin showed association with lipid levels and fat distribution, suggesting impact on insulin resistance. Gene-based analyses identified further biologically plausible loci, suggesting that additional loci beyond those reaching genome-wide significance are likely to represent real associations. This conclusion is supported by an excess of directionally consistent and nominally significant signals between discovery and follow-up studies. Functional follow-up of these newly discovered loci will further improve our understanding of glycemic control. PMID:22885924

Genomic analyses identify molecular subtypes of pancreatic cancer.

PubMed

Bailey, Peter; Chang, David K; Nones, Katia; Johns, Amber L; Patch, Ann-Marie; Gingras, Marie-Claude; Miller, David K; Christ, Angelika N; Bruxner, Tim J C; Quinn, Michael C; Nourse, Craig; Murtaugh, L Charles; Harliwong, Ivon; Idrisoglu, Senel; Manning, Suzanne; Nourbakhsh, Ehsan; Wani, Shivangi; Fink, Lynn; Holmes, Oliver; Chin, Venessa; Anderson, Matthew J; Kazakoff, Stephen; Leonard, Conrad; Newell, Felicity; Waddell, Nick; Wood, Scott; Xu, Qinying; Wilson, Peter J; Cloonan, Nicole; Kassahn, Karin S; Taylor, Darrin; Quek, Kelly; Robertson, Alan; Pantano, Lorena; Mincarelli, Laura; Sanchez, Luis N; Evers, Lisa; Wu, Jianmin; Pinese, Mark; Cowley, Mark J; Jones, Marc D; Colvin, Emily K; Nagrial, Adnan M; Humphrey, Emily S; Chantrill, Lorraine A; Mawson, Amanda; Humphris, Jeremy; Chou, Angela; Pajic, Marina; Scarlett, Christopher J; Pinho, Andreia V; Giry-Laterriere, Marc; Rooman, Ilse; Samra, Jaswinder S; Kench, James G; Lovell, Jessica A; Merrett, Neil D; Toon, Christopher W; Epari, Krishna; Nguyen, Nam Q; Barbour, Andrew; Zeps, Nikolajs; Moran-Jones, Kim; Jamieson, Nigel B; Graham, Janet S; Duthie, Fraser; Oien, Karin; Hair, Jane; Grützmann, Robert; Maitra, Anirban; Iacobuzio-Donahue, Christine A; Wolfgang, Christopher L; Morgan, Richard A; Lawlor, Rita T; Corbo, Vincenzo; Bassi, Claudio; Rusev, Borislav; Capelli, Paola; Salvia, Roberto; Tortora, Giampaolo; Mukhopadhyay, Debabrata; Petersen, Gloria M; Munzy, Donna M; Fisher, William E; Karim, Saadia A; Eshleman, James R; Hruban, Ralph H; Pilarsky, Christian; Morton, Jennifer P; Sansom, Owen J; Scarpa, Aldo; Musgrove, Elizabeth A; Bailey, Ulla-Maja Hagbo; Hofmann, Oliver; Sutherland, Robert L; Wheeler, David A; Gill, Anthony J; Gibbs, Richard A; Pearson, John V; Waddell, Nicola; Biankin, Andrew V; Grimmond, Sean M

2016-03-03

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-β, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.

Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions.

PubMed

Yang, Chin-Rang; Tongyoo, Pumipat; Emamian, Milad; Sandoval, Pablo C; Raghuram, Viswanathan; Knepper, Mark A

2015-12-15

The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out "deep" proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, and HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The analysis of all resulting samples generated 34.6 gigabytes of spectral data. As a result, we identified 6,766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over eight orders of magnitude of protein abundance. The data are provided to users as a public data base (https://helixweb.nih.gov/ESBL/Database/mpkFractions/). The mass spectrometry data were mapped back to their gel slices to generate "virtual Western blots" for each protein. For most of the 6,766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins was found to run aberrantly, with much higher or much lower mobilities than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE, including high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, posttranslational modifications, and expression of alternative isoforms due to alternative exon usage. Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular mass <20 kDa. Copyright © 2015 the American Physiological Society.

Genome-wide association analyses identify 18 new loci associated with serum urate concentrations

PubMed Central

Köttgen, Anna; Albrecht, Eva; Teumer, Alexander; Vitart, Veronique; Krumsiek, Jan; Hundertmark, Claudia; Pistis, Giorgio; Ruggiero, Daniela; O’Seaghdha, Conall M; Haller, Toomas; Yang, Qiong; Tanaka, Toshiko; Johnson, Andrew D; Kutalik, Zoltán; Smith, Albert V; Shi, Julia; Struchalin, Maksim; Middelberg, Rita P S; Brown, Morris J; Gaffo, Angelo L; Pirastu, Nicola; Li, Guo; Hayward, Caroline; Zemunik, Tatijana; Huffman, Jennifer; Yengo, Loic; Zhao, Jing Hua; Demirkan, Ayse; Feitosa, Mary F; Liu, Xuan; Malerba, Giovanni; Lopez, Lorna M; van der Harst, Pim; Li, Xinzhong; Kleber, Marcus E; Hicks, Andrew A; Nolte, Ilja M; Johansson, Asa; Murgia, Federico; Wild, Sarah H; Bakker, Stephan J L; Peden, John F; Dehghan, Abbas; Steri, Maristella; Tenesa, Albert; Lagou, Vasiliki; Salo, Perttu; Mangino, Massimo; Rose, Lynda M; Lehtimäki, Terho; Woodward, Owen M; Okada, Yukinori; Tin, Adrienne; Müller, Christian; Oldmeadow, Christopher; Putku, Margus; Czamara, Darina; Kraft, Peter; Frogheri, Laura; Thun, Gian Andri; Grotevendt, Anne; Gislason, Gauti Kjartan; Harris, Tamara B; Launer, Lenore J; McArdle, Patrick; Shuldiner, Alan R; Boerwinkle, Eric; Coresh, Josef; Schmidt, Helena; Schallert, Michael; Martin, Nicholas G; Montgomery, Grant W; Kubo, Michiaki; Nakamura, Yusuke; Tanaka, Toshihiro; Munroe, Patricia B; Samani, Nilesh J; Jacobs, David R; Liu, Kiang; D’Adamo, Pio; Ulivi, Sheila; Rotter, Jerome I; Psaty, Bruce M; Vollenweider, Peter; Waeber, Gerard; Campbell, Susan; Devuyst, Olivier; Navarro, Pau; Kolcic, Ivana; Hastie, Nicholas; Balkau, Beverley; Froguel, Philippe; Esko, Tõnu; Salumets, Andres; Khaw, Kay Tee; Langenberg, Claudia; Wareham, Nicholas J; Isaacs, Aaron; Kraja, Aldi; Zhang, Qunyuan; Wild, Philipp S; Scott, Rodney J; Holliday, Elizabeth G; Org, Elin; Viigimaa, Margus; Bandinelli, Stefania; Metter, Jeffrey E; Lupo, Antonio; Trabetti, Elisabetta; Sorice, Rossella; Döring, Angela; Lattka, Eva; Strauch, Konstantin; Theis, Fabian; Waldenberger, Melanie; Wichmann, H-Erich; Davies, Gail; Gow, Alan J; Bruinenberg, Marcel; Study, LifeLines Cohort; Stolk, Ronald P; Kooner, Jaspal S; Zhang, Weihua; Winkelmann, Bernhard R; Boehm, Bernhard O; Lucae, Susanne; Penninx, Brenda W; Smit, Johannes H; Curhan, Gary; Mudgal, Poorva; Plenge, Robert M; Portas, Laura; Persico, Ivana; Kirin, Mirna; Wilson, James F; Leach, Irene Mateo; van Gilst, Wiek H; Goel, Anuj; Ongen, Halit; Hofman, Albert; Rivadeneira, Fernando; Uitterlinden, Andre G; Imboden, Medea; von Eckardstein, Arnold; Cucca, Francesco; Nagaraja, Ramaiah; Piras, Maria Grazia; Nauck, Matthias; Schurmann, Claudia; Budde, Kathrin; Ernst, Florian; Farrington, Susan M; Theodoratou, Evropi; Prokopenko, Inga; Stumvoll, Michael; Jula, Antti; Perola, Markus; Salomaa, Veikko; Shin, So-Youn; Spector, Tim D; Sala, Cinzia; Ridker, Paul M; Kähönen, Mika; Viikari, Jorma; Hengstenberg, Christian; Nelson, Christopher P; Consortium, CARDIoGRAM; Consortium, DIAGRAM; Consortium, ICBP; Consortium, MAGIC; Meschia, James F; Nalls, Michael A; Sharma, Pankaj; Singleton, Andrew B; Kamatani, Naoyuki; Zeller, Tanja; Burnier, Michel; Attia, John; Laan, Maris; Klopp, Norman; Hillege, Hans L; Kloiber, Stefan; Choi, Hyon; Pirastu, Mario; Tore, Silvia; Probst-Hensch, Nicole M; Völzke, Henry; Gudnason, Vilmundur; Parsa, Afshin; Schmidt, Reinhold; Whitfield, John B; Fornage, Myriam; Gasparini, Paolo; Siscovick, David S; Polašek, Ozren; Campbell, Harry; Rudan, Igor; Bouatia-Naji, Nabila; Metspalu, Andres; Loos, Ruth J F; van Duijn, Cornelia M; Borecki, Ingrid B; Ferrucci, Luigi; Gambaro, Giovanni; Deary, Ian J; Wolffenbuttel, Bruce H R; Chambers, John C; März, Winfried; Pramstaller, Peter P; Snieder, Harold; Gyllensten, Ulf; Wright, Alan F; Navis, Gerjan; Watkins, Hugh; Witteman, Jacqueline C M; Sanna, Serena; Schipf, Sabine; Dunlop, Malcolm G; Tönjes, Anke; Ripatti, Samuli; Soranzo, Nicole; Toniolo, Daniela; Chasman, Daniel I; Raitakari, Olli; Kao, W H Linda; Ciullo, Marina; Fox, Caroline S; Caulfield, Mark; Bochud, Murielle; Gieger, Christian

2013-01-01

Elevated serum urate concentrations can cause gout, a prevalent and painful inflammatory arthritis. By combining data from >140,000 individuals of European ancestry within the Global Urate Genetics Consortium (GUGC), we identified and replicated 28 genome-wide significant loci in association with serum urate concentrations (18 new regions in or near TRIM46, INHBB, SFMBT1, TMEM171, VEGFA, BAZ1B, PRKAG2, STC1, HNF4G, A1CF, ATXN2, UBE2Q2, IGF1R, NFAT5, MAF, HLF, ACVR1B-ACVRL1 and B3GNT4). Associations for many of the loci were of similar magnitude in individuals of non-European ancestry. We further characterized these loci for associations with gout, transcript expression and the fractional excretion of urate. Network analyses implicate the inhibins-activins signaling pathways and glucose metabolism in systemic urate control. New candidate genes for serum urate concentration highlight the importance of metabolic control of urate production and excretion, which may have implications for the treatment and prevention of gout. PMID:23263486

Monoclonal Antibodies against Small Molecule Natural Products and Their Applications, Eastern Blotting and Knockout Extract

PubMed Central

Shoyama, Yukihiro

2011-01-01

To determine the hapten number in hapten-carrier protein conjugate matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry was applied. Highly specific anti-ginsenoside Rb1 and Rg1 monoclonal antibodies (MAbs) were prepared. Ginsenosides were developed on thin layer chromatography (TLC) plates which were covered by a polyvinylidene difluoride (PVDF) membrane resulting in blotting. The membrane was treated with NaIO4 solution to release the aldehyde group on the sugar moiety of the ginsenosides. By treatment of the membrane with a protein solution the ginsenoside-protein conjugation as a Schiff-base occurred, which can function to fix it to the PVDF membrane. A part of the ginsenoside aglycone was reacted with anti-ginsenoside Rb1 MAb, secondary MAb conjugated with enzyme and finally a substrate was added, resulting in a specific and highly sensitive staining that we named Eastern blotting. Furthermore, it makes one-step isolation of ginsenoside Rb1 possible using an immuno-affinity column conjugated with anti-ginsenoside Rb1 MAb. Furthermore, immunoaffinity concentration was carried out allowing high sensitivity analysis of lower concentrations of ginsenoside Rb1 so that several unknown bands could be structurally determined.

Detection of autoimmunity in early primary Epstein-Barr virus infection by Western blot analysis.

PubMed

Mascia, M T; Sandri, G; Guerzoni, C; Roncaglia, R; Mantovani, G; Ferri, C

2008-01-01

The Epstein-Barr virus (EBV) represents a potentially important factor in the pathogenesis of certain autoimmune disorders such as systemic lupus erythematosus (SLE), and Sjögren's syndrome, probably through a molecular mimicry mechanism. Several studies have focused on the relationship between previous EBV infection and clinically overt connective tissue diseases (CTDs), while the aim of this study was to investigate the immunological alterations during the early phase of primary acute EBV infection by means of ENA Western blotting (WB) analysis. This technique is able to detect a wide spectrum of anti-ENA autoantibodies, potentially directed against diverse epitopes of the same antigen. Sera from 54 subjects (F/M=24/30, mean age 17+/-6 SD years) with primary acute EBV infection were analysed using indirect immunofluorescence (IF) on Hep-2 cells for ANA, and both ELISA and WB for ENA. Only 8 ANA+ and no ENA+ were found by means of IF and ELISA techniques, respectively; however, one or more ENA autoantibodies were detected in 24/54 (44%) sera using WB. The autoantibodies were no longer present at the second evaluation. Subjects with immunological alterations had not developed any significant clinical manifestations at a 5-year follow-up. This study demonstrated the appearance of autoantibody production in a high proportion of individuals with primary acute EBV infection; interestingly, the observed serological subsets are quite similar to clinical SLE clusters. Moreover, the absence of immunological disorders during the follow-up reinforces the role of multiple genetic and/or environmental co-factors in the pathogenesis of CTDs.

Western blot (immunoblot) assay of small, round-structured virus associated with an acute gastroenteritis outbreak in Tokyo.

PubMed

Hayashi, Y; Ando, T; Utagawa, E; Sekine, S; Okada, S; Yabuuchi, K; Miki, T; Ohashi, M

1989-08-01

Small, round-structured virus (SRSV) was detected in a stool specimen of a patient during an acute gastroenteritis outbreak in Tokyo and was tentatively named SRSV-9. SRSV-9 was purified by sucrose velocity gradient centrifugation after CsCl density gradient centrifugation. The buoyant density of SRSV-9 appeared to be 1.36 g/ml in CsCl. A Western blot (immunoblot) assay using the biotin-avidin system revealed that SRSV-9 was antigenically related to the Hawaii agent but distinct from the Norwalk agent and contained a single major structural protein with a molecular size of 63.0 +/- 0.6 kilodaltons. The prevalence of SRSV-9 infection in Tokyo was surveyed by the Western blot antibody assay by using a crude virus preparation as the antigen. Seroconversion was observed in 56.5% of the patients involved in the outbreaks from which SRSV was detected by electron microscopy.

Establishment and application of a modified membrane-blot assay for Rhizomucor miehei lipases aimed at improving their methanol tolerance and thermostability.

PubMed

He, Dong; Luo, Wen; Wang, Zhiyuan; Lv, Pengmei; Yuan, Zhenhong; Huang, Shaowei; Xv, Jingliang

2017-07-01

Directed evolution has been proved an effective way to improve the stability of proteins, but high throughput screening assays for directed evolution with simultaneous improvement of two or more properties are still rare. In this study, we aimed to establish a membrane-blot assay for use in the high-throughput screening of Rhizomucor miehei lipases (RMLs). With the assistance of the membrane-blot screening assay, a mutant E47K named G10 that showed improved thermal stability was detected in the first round of error-prone PCR. Using G10 as the parent, two variants G10-11 and G10-20 that showed improved thermal stability and methanol tolerance without loss of activity compared to the wild type RML were obtained. The T 50 60 -value of G10-11 and G10-20 increased by 12°C and 6.5°C, respectively. After incubation for 1h, the remaining residual activity of G10-11 and G10-20 was 63.45% and 74.33%, respectively, in 50% methanol, and 15.98% and 30.22%, respectively, in 80% methanol. Thus, we successfully developed a membrane-blot assay that could be used for the high-throughput screening of RMLs with improved thermostability and methanol tolerance. Based on our findings, we believe that our newly developed membrane-blot assay will have potential applications in directed evolution in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of the Western blotting method for the diagnosis of congenital toxoplasmosis.

PubMed

Capobiango, Jaqueline Dario; Monica, Thaís Cabral; Ferreira, Fernanda Pinto; Mitsuka-Breganó, Regina; Navarro, Italmar Teodorico; Garcia, João Luis; Reiche, Edna Maria Vissoci

To evaluate the Western blotting method for the detection of IgG anti-Toxoplasma gondii (T. gondii) (IgG-WB) in the serum of children with suspected congenital toxoplasmosis. We accompanied 47 mothers with acquired toxoplasmosis in pregnancy and their children, between June of 2011 and June of 2014. The IgG-WB was done in house and the test was considered positive if the child had antibodies that recognized at least one band on IgG blots different from the mother's or with greater intensity than the corresponding maternal band, during the first three months of life. 15 children (15.1%) met the criteria for congenital toxoplasmosis and 32 (32.3%) had the diagnosis excluded. The symptoms were observed in 12 (80.0%) children and the most frequent were cerebral calcification in 9 (60.0%), chorioretinitis in 8 (53.3%), and hydrocephalus in 4 (26.6%). IgM antibodies anti-T. gondii detected by chemiluminescence (CL) were found in 6 (40.0%) children and the polymerase chain reaction (PCR) for detection of T. gondii DNA was positive in 5 of 7 performed (71.4%). The sensitivity of IgG-WB was of 60.0% [95% confidence interval (CI) 32.3-83.7%] and specificity 43.7% (95% CI 26.7-62.3%). The sensitivity of IgG-WB increased to 76.0 and 89.1% when associated to the research of IgM anti-T. gondii or PCR, respectively. The IgG-WB showed greater sensitivity than the detection of IgM anti-T. gondii; therefore, it can be used for the diagnosis of congenital toxoplasmosis in association with other congenital infection markers. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Retrospective Study of Hemoparasites in Cattle in Southern Italy by Reverse Line Blot Hybridization

PubMed Central

CECI, Luigi; IARUSSI, Fabrizio; GRECO, Beatrice; LACINIO, Rosanna; FORNELLI, Stefania; CARELLI, Grazia

2014-01-01

ABSTRACT Tick-borne diseases are widespread in tropical and temperate regions and are responsible for important economic losses in those areas. In order to assess the presence and prevalence of various pathogens in southern Italy, we retrospectively analyzed cattle blood samples collected for a previous study in 2000 using reverse line blot (RLB) hybridization. The study had been carried out in three regions of southern Italy on 1,500 randomly selected and apparently healthy adult cattle. RLB showed that 43.7% of the cattle were positive for nine different species of hemoparasites with either a single infection or a mixed infection. Theileria buffeli was the most common species found, being present in 27.3% of the animals, followed by Anaplasma marginale in 18.1%, Anaplasma centrale in 13.8%, Babesia bigemina and Anaplasma bovis in 4.2%, Anaplasma phagocytophilum in 1.7%, Babesia bovis in 1.6%, Babesia major in 0.2% and Babesia divergens in 0.1%. Complete blood counts showed different degrees of anemia in 363 animals (24.2%) and of these, 169 were RLB-positive for at least one pathogen. Among the ticks that were collected from the cattle, the following species were identified: Rhipicephalus bursa, Ixodes ricinus, Hyalomma marginatum, Boophilus annulatus, Dermacentor marginatus and Haemaphysalis (sulcata, parva, inermis and punctata). The results obtained confirmed the spread of endemic tick-borne pathogens in the regions studied. PMID:24614604

Retrospective study of hemoparasites in cattle in southern Italy by reverse line blot hybridization.

PubMed

Ceci, Luigi; Iarussi, Fabrizio; Greco, Beatrice; Lacinio, Rosanna; Fornelli, Stefania; Carelli, Grazia

2014-06-01

Tick-borne diseases are widespread in tropical and temperate regions and are responsible for important economic losses in those areas. In order to assess the presence and prevalence of various pathogens in southern Italy, we retrospectively analyzed cattle blood samples collected for a previous study in 2000 using reverse line blot (RLB) hybridization. The study had been carried out in three regions of southern Italy on 1,500 randomly selected and apparently healthy adult cattle. RLB showed that 43.7% of the cattle were positive for nine different species of hemoparasites with either a single infection or a mixed infection. Theileria buffeli was the most common species found, being present in 27.3% of the animals, followed by Anaplasma marginale in 18.1%, Anaplasma centrale in 13.8%, Babesia bigemina and Anaplasma bovis in 4.2%, Anaplasma phagocytophilum in 1.7%, Babesia bovis in 1.6%, Babesia major in 0.2% and Babesia divergens in 0.1%. Complete blood counts showed different degrees of anemia in 363 animals (24.2%) and of these, 169 were RLB-positive for at least one pathogen. Among the ticks that were collected from the cattle, the following species were identified: Rhipicephalus bursa, Ixodes ricinus, Hyalomma marginatum, Boophilus annulatus, Dermacentor marginatus and Haemaphysalis (sulcata, parva, inermis and punctata). The results obtained confirmed the spread of endemic tick-borne pathogens in the regions studied.

Validity of the Enzyme-linked Immunoelectrotransfer Blot (EITB) for naturally acquired porcine cysticercosis

PubMed Central

Jayashi, César M.; Gonzalez, Armando E.; Neyra, Ricardo Castillo; Rodríguez, Silvia; García, Hector H.; Lightowlers, Marshall W.

2017-01-01

The Enzyme-linked Immunoelectrotransfer Blot (EITB) has been used widely as a screening test for Taenia solium cysticercosis in swine. However, the relation between seropositivity and infection in pig populations from endemic areas has not been well defined. The aim of this study is to relate EITB seropositivity with infection and infection burden, analyse the trade-off between sensitivity and specificity with various cut-off points for the EITB assay, and finally describe the serology changes in a cohort of rural pigs raised under natural conditions. A group of 107 pigs that were used as controls during a vaccination field trial in Peru was our study population. The prevalence of porcine cysticercosis determined by necropsy examination was 16.82% (18/107) in these animals. Using EITB reactivity to ≥ 1 band as a cut-off point for the assay, the sensitivity was 88.89% (65.29-98.62, 95% CI) and the specificity was 48.31% (37.59-59.16, 95% CI). Comparing other cut-off points, involving up to as many as 7 reactive bands, a reactivity of ≥ 3 bands provided the best trade-offs in sensitivity and specificity. Using this cut-off point for the assay, the sensitivity was 77.77% (52.36 - 93.59, 95% CI) and the specificity was 76.40% (66.22 - 84.76, 95% CI). A significant association was found between cyst counts over 100 cysts and reactivity to ≥ 3 bands in the EITB assay (Fisher’s exact test, p<0.05). The results of this study suggest that the use of the EITB assay to study porcine cysticercosis may require setting different cut-offs under field and experimental conditions, and depending upon the objective of the screening process. PMID:24183647

Validation of endothelin B receptor antibodies reveals two distinct receptor-related bands on Western blot.

PubMed

Barr, Travis P; Kornberg, Daniel; Montmayeur, Jean-Pierre; Long, Melinda; Reichheld, Stephen; Strichartz, Gary R

2015-01-01

Antibodies are important tools for the study of protein expression but are often used without full validation. In this study, we used Western blots to characterize antibodies targeted to the N or C terminal (NT or CT, respectively) and the second or third intracellular loop (IL2 or IL3, respectively) of the endothelin B receptor (ETB). The IL2-targeted antibody accurately detected endogenous ETB expression in rat brain and cultured rat astrocytes by labeling a 50-kDa band, the expected weight of full-length ETB. However, this antibody failed to detect transfected ETB in HEK293 cultures. In contrast, the NT-targeted antibody accurately detected endogenous ETB in rat astrocyte cultures and transfected ETB in HEK293 cultures by labeling a 37-kDa band but failed to detect endogenous ETB in rat brain. Bands detected by the CT- or IL3-targeted antibody were found to be unrelated to ETB. Our findings show that functional ETB can be detected at 50 or 37kDa on Western blot, with drastic differences in antibody affinity for these bands. The 37-kDa band likely reflects ETB processing, which appears to be dependent on cell type and/or culture condition. Copyright © 2014 Elsevier Inc. All rights reserved.

Immunodiagnosis of Echinococcus Infections: Confirmatory Testing and Species Differentiation by a New Commercial Western Blot

PubMed Central

Liance, Martine; Janin, Veronique; Bresson-Hadni, Solange; Vuitton, Dominique-Angele; Houin, Rene; Piarroux, Renaud

2000-01-01

The Echinococcus Western Blot IgG (LDBIO Diagnostics, Lyon, France), using a whole larval antigen from Echinococcus multilocularis, was evaluated for serodiagnosis and differentiation between two human parasitic infections of worldwide importance: cystic echinococcosis, due to Echinococcus granulosus, and alveolar echinococcosis, due to E. multilocularis. Fifty and 61 serum samples from patients with cystic and alveolar echinococcosis, respectively, were used for assessing diagnostic sensitivity. The sensitivity of the assay was compared with those of screening tests used for these applications. Sera used for assessing cross-reactivities were from 154 patients with other diseases, either parasitic or not. The assay allowed the detection of serum immunoglobulin G antibodies in 97% of Echinococcus-infected patients. It had a higher sensitivity than screening assays for the detection for each echinococcosis. The assay allowed us to correctly distinguish between E. granulosus- and E. multilocularis-infected patients in 76% of cases. It did not allow us to distinguish active from inactive forms of both echinococcoses. The occurrence of cross-reactivities with neurocysticercosis indicates the necessity for retesting sera with species-specific antigens, for rare patients with neurologic disorders. This study shows the usefulness of the commercially available Echinococcus Western Blot IgG for the serological confirmation of human echinococcosis. PMID:11015390

Synthesis and Structure activity relationships of EGCG Analogues, A Recently Identified Hsp90 Inhibitor

PubMed Central

Khandelwal, Anuj; Hall, Jessica

2014-01-01

Epigallocatechin-3-gallate (EGCG), the principal polyphenol isolated from green tea, was recently shown to inhibit Hsp90, however structure-activity relationships for this natural product have not yet been produced. Herein, we report the synthesis and biological evaluation of EGCG analogues to establish structure-activity relationships between EGCG and Hsp90. All four rings as well as the linker connecting the C- and the D-rings were systematically investigated, which led to the discovery of compounds that inhibit Hs90 and display improvement in efficacy over EGCG. Anti-proliferative activity of all the analogues was determined against MCF-7 and SKBr3 cell lines and Hsp90 inhibitory activity of four most potent analogues was further evaluated by western blot analyses and degradation of Hsp90-dependent client proteins. Prenyl substituted aryl ester of 3,5-dihydroxychroman-3-ol ring system was identified as novel scaffold that exhibit Hsp90 inhibitory activity. PMID:23834230

Single-Cell Western Blotting after Whole-Cell Imaging to Assess Cancer Chemotherapeutic Response

PubMed Central

2015-01-01

Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors. PMID:25226230

A single-step simultaneous protein staining procedure for polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis.

PubMed

Pal, Jayanta K; Berwal, Sunil K; Soni, Rupali N

2012-01-01

A simple method for staining of proteins simultaneously on sodium dodecyl sulfate (SDS) polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis is described. A 5% solution of Alta, a commercially available cosmetic preparation, is added in the upper tank buffer during electrophoresis. On completion of electrophoresis, the gel is washed in distilled water and viewed on a white light plate and a transilluminator to photograph the protein profiles. The gel is processed for western blot transfer of proteins onto a nitrocellulose membrane, and upon completion, the protein profiles on the membrane are viewed and photographed as stated above. The membrane can then be processed for immunostaining as per the standard procedure. Thus, the staining procedure using Alta is simple, rapid (without any need of destaining), and cost-effective.

Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

PubMed

Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

2016-08-01

Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Genome-wide meta-analyses of multiancestry cohorts identify multiple new susceptibility loci for refractive error and myopia.

PubMed

Verhoeven, Virginie J M; Hysi, Pirro G; Wojciechowski, Robert; Fan, Qiao; Guggenheim, Jeremy A; Höhn, René; MacGregor, Stuart; Hewitt, Alex W; Nag, Abhishek; Cheng, Ching-Yu; Yonova-Doing, Ekaterina; Zhou, Xin; Ikram, M Kamran; Buitendijk, Gabriëlle H S; McMahon, George; Kemp, John P; Pourcain, Beate St; Simpson, Claire L; Mäkelä, Kari-Matti; Lehtimäki, Terho; Kähönen, Mika; Paterson, Andrew D; Hosseini, S Mohsen; Wong, Hoi Suen; Xu, Liang; Jonas, Jost B; Pärssinen, Olavi; Wedenoja, Juho; Yip, Shea Ping; Ho, Daniel W H; Pang, Chi Pui; Chen, Li Jia; Burdon, Kathryn P; Craig, Jamie E; Klein, Barbara E K; Klein, Ronald; Haller, Toomas; Metspalu, Andres; Khor, Chiea-Chuen; Tai, E-Shyong; Aung, Tin; Vithana, Eranga; Tay, Wan-Ting; Barathi, Veluchamy A; Chen, Peng; Li, Ruoying; Liao, Jiemin; Zheng, Yingfeng; Ong, Rick T; Döring, Angela; Evans, David M; Timpson, Nicholas J; Verkerk, Annemieke J M H; Meitinger, Thomas; Raitakari, Olli; Hawthorne, Felicia; Spector, Tim D; Karssen, Lennart C; Pirastu, Mario; Murgia, Federico; Ang, Wei; Mishra, Aniket; Montgomery, Grant W; Pennell, Craig E; Cumberland, Phillippa M; Cotlarciuc, Ioana; Mitchell, Paul; Wang, Jie Jin; Schache, Maria; Janmahasatian, Sarayut; Janmahasathian, Sarayut; Igo, Robert P; Lass, Jonathan H; Chew, Emily; Iyengar, Sudha K; Gorgels, Theo G M F; Rudan, Igor; Hayward, Caroline; Wright, Alan F; Polasek, Ozren; Vatavuk, Zoran; Wilson, James F; Fleck, Brian; Zeller, Tanja; Mirshahi, Alireza; Müller, Christian; Uitterlinden, André G; Rivadeneira, Fernando; Vingerling, Johannes R; Hofman, Albert; Oostra, Ben A; Amin, Najaf; Bergen, Arthur A B; Teo, Yik-Ying; Rahi, Jugnoo S; Vitart, Veronique; Williams, Cathy; Baird, Paul N; Wong, Tien-Yin; Oexle, Konrad; Pfeiffer, Norbert; Mackey, David A; Young, Terri L; van Duijn, Cornelia M; Saw, Seang-Mei; Bailey-Wilson, Joan E; Stambolian, Dwight; Klaver, Caroline C; Hammond, Christopher J

2013-03-01

Refractive error is the most common eye disorder worldwide and is a prominent cause of blindness. Myopia affects over 30% of Western populations and up to 80% of Asians. The CREAM consortium conducted genome-wide meta-analyses, including 37,382 individuals from 27 studies of European ancestry and 8,376 from 5 Asian cohorts. We identified 16 new loci for refractive error in individuals of European ancestry, of which 8 were shared with Asians. Combined analysis identified 8 additional associated loci. The new loci include candidate genes with functions in neurotransmission (GRIA4), ion transport (KCNQ5), retinoic acid metabolism (RDH5), extracellular matrix remodeling (LAMA2 and BMP2) and eye development (SIX6 and PRSS56). We also confirmed previously reported associations with GJD2 and RASGRF1. Risk score analysis using associated SNPs showed a tenfold increased risk of myopia for individuals carrying the highest genetic load. Our results, based on a large meta-analysis across independent multiancestry studies, considerably advance understanding of the mechanisms involved in refractive error and myopia.

Outbreak of larval Echinococcus multilocularis infection in Japanese monkey (Macaca fuscata) in a zoo, Hokkaido: western blotting patterns in the infected monkeys.

PubMed

Sato, Chiaki; Kawase, Shiro; Yano, Shoki; Nagano, Hideki; Fujimoto, Satoshi; Kobayashi, Nobuyuki; Miyahara, Kazuro; Yamada, Kazutaka; Sato, Motoyoshi; Kobayashi, Yoshiyasu

2005-01-01

A high prevalence of larval Echinococcus multilocularis (Em) infection was found in zoo primates in Hokkaido, Japan. In October 1997, a Japanese monkey (Macaca fuscata) died and histopathologically diagnosed as alveolar hydatidosis. Serum samples were collected from the remaining Japanese monkeys and examined for antibodies against Em by enzyme-linked immunosorbent assay and western blotting. Serological tests showed 12 more animals of the remaining 57 monkeys were possibly infected. Ultrasonography revealed that nine of these 12 animals had a cystic lesion in the liver. The band patterns of western blotting in the monkeys were very similar to those in human.

Global metabolic analyses identify key differences in metabolite levels between polymyxin-susceptible and polymyxin-resistant Acinetobacter baumannii

PubMed Central

Mahamad Maifiah, Mohd Hafidz; Cheah, Soon-Ee; Johnson, Matthew D.; Han, Mei-Ling; Boyce, John D.; Thamlikitkul, Visanu; Forrest, Alan; Kaye, Keith S.; Hertzog, Paul; Purcell, Anthony W.; Song, Jiangning; Velkov, Tony; Creek, Darren J.; Li, Jian

2016-01-01

Multidrug-resistant Acinetobacter baumannii presents a global medical crisis and polymyxins are used as the last-line therapy. This study aimed to identify metabolic differences between polymyxin-susceptible and polymyxin-resistant A. baumannii using untargeted metabolomics. The metabolome of each A. baumannii strain was measured using liquid chromatography-mass spectrometry. Multivariate and univariate statistics and pathway analyses were employed to elucidate metabolic differences between the polymyxin-susceptible and -resistant A. baumannii strains. Significant differences were identified between the metabolic profiles of the polymyxin-susceptible and -resistant A. baumannii strains. The lipopolysaccharide (LPS) deficient, polymyxin-resistant 19606R showed perturbation in specific amino acid and carbohydrate metabolites, particularly pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle intermediates. Levels of nucleotides were lower in the LPS-deficient 19606R. Furthermore, 19606R exhibited a shift in its glycerophospholipid profile towards increased abundance of short-chain lipids compared to the parent polymyxin-susceptible ATCC 19606. In contrast, in a pair of clinical isolates 03–149.1 (polymyxin-susceptible) and 03–149.2 (polymyxin-resistant, due to modification of lipid A), minor metabolic differences were identified. Notably, peptidoglycan biosynthesis metabolites were significantly depleted in both of the aforementioned polymyxin-resistant strains. This is the first comparative untargeted metabolomics study to show substantial differences in the metabolic profiles of the polymyxin-susceptible and -resistant A. baumannii. PMID:26924392

Global metabolic analyses identify key differences in metabolite levels between polymyxin-susceptible and polymyxin-resistant Acinetobacter baumannii.

PubMed

Maifiah, Mohd Hafidz Mahamad; Cheah, Soon-Ee; Johnson, Matthew D; Han, Mei-Ling; Boyce, John D; Thamlikitkul, Visanu; Forrest, Alan; Kaye, Keith S; Hertzog, Paul; Purcell, Anthony W; Song, Jiangning; Velkov, Tony; Creek, Darren J; Li, Jian

2016-02-29

Multidrug-resistant Acinetobacter baumannii presents a global medical crisis and polymyxins are used as the last-line therapy. This study aimed to identify metabolic differences between polymyxin-susceptible and polymyxin-resistant A. baumannii using untargeted metabolomics. The metabolome of each A. baumannii strain was measured using liquid chromatography-mass spectrometry. Multivariate and univariate statistics and pathway analyses were employed to elucidate metabolic differences between the polymyxin-susceptible and -resistant A. baumannii strains. Significant differences were identified between the metabolic profiles of the polymyxin-susceptible and -resistant A. baumannii strains. The lipopolysaccharide (LPS) deficient, polymyxin-resistant 19606R showed perturbation in specific amino acid and carbohydrate metabolites, particularly pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle intermediates. Levels of nucleotides were lower in the LPS-deficient 19606R. Furthermore, 19606R exhibited a shift in its glycerophospholipid profile towards increased abundance of short-chain lipids compared to the parent polymyxin-susceptible ATCC 19606. In contrast, in a pair of clinical isolates 03-149.1 (polymyxin-susceptible) and 03-149.2 (polymyxin-resistant, due to modification of lipid A), minor metabolic differences were identified. Notably, peptidoglycan biosynthesis metabolites were significantly depleted in both of the aforementioned polymyxin-resistant strains. This is the first comparative untargeted metabolomics study to show substantial differences in the metabolic profiles of the polymyxin-susceptible and -resistant A. baumannii.

PCR colorimetric dot-blot assay and clinical pretest probability for diagnosis of Pulmonary Tuberculosis in Smear-Negative patients

PubMed Central

Scherer, Luciene Cardoso; Sperhacke, Rosa Dea; Jarczewski, Carla; Cafrune, Patrícia I; Minghelli, Simone; Ribeiro, Marta Osório; Mello, Fernanda CQ; Ruffino-Netto, Antonio; Rossetti, Maria LR; Kritski, Afrânio L

2007-01-01

Background Smear-negative pulmonary tuberculosis (SNPTB) accounts for 30% of Pulmonary Tuberculosis (PTB) cases reported annually in developing nations. Polymerase chain reaction (PCR) may provide an alternative for the rapid detection of Mycobacterium tuberculosis (MTB); however little data are available regarding the clinical utility of PCR in SNPTB, in a setting with a high burden of TB/HIV co-infection. Methods To evaluate the performance of the PCR dot-blot in parallel with pretest probability (Clinical Suspicion) in patients suspected of having SNPTB, a prospective study of 213 individuals with clinical and radiological suspicion of SNPTB was carried out from May 2003 to May 2004, in a TB/HIV reference hospital. Respiratory specialists estimated the pretest probability of active disease into high, intermediate, low categories. Expectorated sputum was examined by direct microscopy (Ziehl-Neelsen staining), culture (Lowenstein Jensen) and PCR dot-blot. Gold standard was based on culture positivity combined with the clinical definition of PTB. Results In smear-negative and HIV subjects, active PTB was diagnosed in 28.4% (43/151) and 42.2% (19/45), respectively. In the high, intermediate and low pretest probability categories active PTB was diagnosed in 67.4% (31/46), 24% (6/25), 7.5% (6/80), respectively. PCR had sensitivity of 65% (CI 95%: 50%–78%) and specificity of 83% (CI 95%: 75%–89%). There was no difference in the sensitivity of PCR in relation to HIV status. PCR sensitivity and specificity among non-previously TB treated and those treated in the past were, respectively: 69%, 43%, 85% and 80%. The high pretest probability, when used as a diagnostic test, had sensitivity of 72% (CI 95%:57%–84%) and specificity of 86% (CI 95%:78%–92%). Using the PCR dot-blot in parallel with high pretest probability as a diagnostic test, sensitivity, specificity, positive and negative predictive values were: 90%, 71%, 75%, and 88%, respectively. Among non

Cy5 total protein normalization in Western blot analysis.

PubMed

Hagner-McWhirter, Åsa; Laurin, Ylva; Larsson, Anita; Bjerneld, Erik J; Rönn, Ola

2015-10-01

Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. Copyright © 2015 Elsevier Inc. All rights reserved.

Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis.

PubMed

Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

2016-08-17

Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film.

Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis

PubMed Central

Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

2016-01-01

Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film. PMID:27531616

Genetic and physical analyses of Methylobacterium organophilum XX genes encoding methanol oxidation

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

Machlin, S.M.; Tam, P.E.; Bastien, C.A.

When allyl alcohol was used as a suicide substrate, spontaneous mutants and UV light- and nitrous acid-generated mutants of Methylobacterium organophilum XX were selected which grew on methylamine but not on methanol. There was no detectable methanol dehydrogenase (MDH) activity in crude extracts of these mutants, yet Western blots revealed that some mutants still produced MDH protein. Complementation of 50 mutants by a cosmid gene bank of M. organophilum XX demonstrated that three major regions of the genome, each of which was separated by a minimum of 40 kilobases, were required for expression of active MDH. By subcloning and Tn5more » insertion mutagenesis of subcloned fragments, at least 11 genes clustered within these three regions were subsequently identified. The identity of the MDH structural gene, which was initially determined by hybridization to the structural gene of Methylobacterium sp. strain AM1, was confirmed by Western blot analysis of an MDH-..beta..-galactosidase fusion protein.« less

An advanced dual labeled gold nanoparticles probe to detect Cryptosporidium parvum using rapid immuno-dot blot assay.

PubMed

Thiruppathiraja, Chinnasamy; Kamatchiammal, Senthilkumar; Adaikkappan, Periyakaruppan; Alagar, Muthukaruppan

2011-07-15

The zoonotic protozoan parasite Cryptosporidium parvum poses a significant risk to public health. Due to the low infectious dose of C. parvum, remarkably sensitive detection methods are required for water and food industries analysis. However PCR affirmed sensing method of the causative nucleic acid has numerous advantages, still criterion demands for simple techniques and expertise understanding to extinguish its routine use. In contrast, protein based immuno detecting techniques are simpler to perform by a commoner, but lack of sensitivity due to inadequate signal amplification. In this paper, we focused on the development of a mere sensitive immuno detection method by coupling anti-cyst antibody and alkaline phosphatase on gold nanoparticle for C. parvum is described. Outcome of the sensitivity in an immuno-dot blot assay detection is enhanced by 500 fold (using conventional method) and visually be able to detect up to 10 oocysts/mL with minimal processing period. Techniques reported in this paper substantiate the convenience of immuno-dot blot assay for the routine screening of C. parvum in water/environmental examines and most importantly, demonstrates the potential of a prototype development of simple and inexpensive diagnostic technique. Copyright © 2011 Elsevier B.V. All rights reserved.

Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results

PubMed Central

Sekizuka, Tsuyoshi; Yamochi, Tadanori; Firouzi, Sanaz; Sato, Tomoo; Umeki, Kazumi; Sasaki, Daisuke; Hasegawa, Hiroo; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Momose, Haruka; Araki, Kumiko; Saito, Masumichi; Nosaka, Kisato; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Okayama, Akihiko; Miura, Kiyonori; Satake, Masahiro; Saito, Shigeru; Itabashi, Kazuo; Yamaguchi, Kazunari; Kuroda, Makoto; Watanabe, Toshiki; Okuma, Kazu

2017-01-01

ABSTRACT Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens. PMID:28701419

Phenotypic Screening Identifies Synergistically Acting Natural Product Enhancing the Performance of Biomaterial Based Wound Healing.

PubMed

Sivasubramanian, Srinivasan; Chandrasekar, Gayathri; Svensson Akusjärvi, Sara; Thangam, Ramar; Sathuvan, Malairaj; Kumar, R B S; Hussein, Hawraa; Vincent, Savariar; Madhan, Balaraman; Gunasekaran, Palani; Kitambi, Satish S

2017-01-01

The potential of multifunctional wound heal biomaterial relies on the optimal content of therapeutic constituents as well as the desirable physical, chemical, and biological properties to accelerate the healing process. Formulating biomaterials such as amnion or collagen based scaffolds with natural products offer an affordable strategy to develop dressing material with high efficiency in healing wounds. Using image based phenotyping and quantification, we screened natural product derived bioactive compounds for modulators of types I and III collagen production from human foreskin derived fibroblast cells. The identified hit was then formulated with amnion to develop a biomaterial, and its biophysical properties, in vitro and in vivo effects were characterized. In addition, we performed functional profiling analyses by PCR array to understand the effect of individual components of these materials on various genes such as inflammatory mediators including chemokines and cytokines, growth factors, fibroblast stimulating markers for collagen secretion, matrix metalloproteinases, etc., associated with wound healing. FACS based cell cycle analyses were carried out to evaluate the potential of biomaterials for induction of proliferation of fibroblasts. Western blot analyses was done to examine the effect of biomaterial on collagen synthesis by cells and compared to cells grown in the presence of growth factors. This work demonstrated an uncomplicated way of identifying components that synergistically promote healing. Besides, we demonstrated that modulating local wound environment using biomaterials with bioactive compounds could enhance healing. This study finds that the developed biomaterials offer immense scope for healing wounds by means of their skin regenerative features such as anti-inflammatory, fibroblast stimulation for collagen secretion as well as inhibition of enzymes and markers impeding the healing, hydrodynamic properties complemented with other features

Phenotypic Screening Identifies Synergistically Acting Natural Product Enhancing the Performance of Biomaterial Based Wound Healing

PubMed Central

Sivasubramanian, Srinivasan; Chandrasekar, Gayathri; Svensson Akusjärvi, Sara; Thangam, Ramar; Sathuvan, Malairaj; Kumar, R. B. S.; Hussein, Hawraa; Vincent, Savariar; Madhan, Balaraman; Gunasekaran, Palani; Kitambi, Satish S.

2017-01-01

The potential of multifunctional wound heal biomaterial relies on the optimal content of therapeutic constituents as well as the desirable physical, chemical, and biological properties to accelerate the healing process. Formulating biomaterials such as amnion or collagen based scaffolds with natural products offer an affordable strategy to develop dressing material with high efficiency in healing wounds. Using image based phenotyping and quantification, we screened natural product derived bioactive compounds for modulators of types I and III collagen production from human foreskin derived fibroblast cells. The identified hit was then formulated with amnion to develop a biomaterial, and its biophysical properties, in vitro and in vivo effects were characterized. In addition, we performed functional profiling analyses by PCR array to understand the effect of individual components of these materials on various genes such as inflammatory mediators including chemokines and cytokines, growth factors, fibroblast stimulating markers for collagen secretion, matrix metalloproteinases, etc., associated with wound healing. FACS based cell cycle analyses were carried out to evaluate the potential of biomaterials for induction of proliferation of fibroblasts. Western blot analyses was done to examine the effect of biomaterial on collagen synthesis by cells and compared to cells grown in the presence of growth factors. This work demonstrated an uncomplicated way of identifying components that synergistically promote healing. Besides, we demonstrated that modulating local wound environment using biomaterials with bioactive compounds could enhance healing. This study finds that the developed biomaterials offer immense scope for healing wounds by means of their skin regenerative features such as anti-inflammatory, fibroblast stimulation for collagen secretion as well as inhibition of enzymes and markers impeding the healing, hydrodynamic properties complemented with other features

Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis

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

Aravalli, Rajagopal N., E-mail: aravalli@umn.edu; Park, Chang W.; Steer, Clifford J., E-mail: steer001@umn.edu

The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed amore » series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.« less

Novel application of the published kinase inhibitor set to identify therapeutic targets and pathways in triple negative breast cancer subtypes

PubMed Central

Phamduy, Theresa B.; Chrisey, Douglas B.

2017-01-01

Triple negative breast cancers (TNBCs) have high recurrence and metastasis rates. Acquisition of a mesenchymal morphology and phenotype in addition to driving migration is a consequential process that promotes metastasis. Although some kinases are known to regulate a mesenchymal phenotype, the role for a substantial portion of the human kinome remains uncharacterized. Here we evaluated the Published Kinase Inhibitor Set (PKIS) and screened a panel of TNBC cell lines to evaluate the compounds’ effects on a mesenchymal phenotype. Our screen identified 36 hits representative of twelve kinase inhibitor chemotypes based on reversal of the mesenchymal cell morphology, which was then prioritized to twelve compounds based on gene expression and migratory behavior analyses. We selected the most active compound and confirmed mesenchymal reversal on transcript and protein levels with qRT-PCR and Western Blot. Finally, we utilized a kinase array to identify candidate kinases responsible for the EMT reversal. This investigation shows the novel application to identify previously unrecognized kinase pathways and targets in acquisition of a mesenchymal TNBC phenotype that warrant further investigation. Future studies will examine specific roles of the kinases in mechanisms responsible for acquisition of the mesenchymal and/or migratory phenotype. PMID:28771473

Genome-Wide Linkage and Positional Association Analyses Identify Associations of Novel AFF3 and NTM Genes with Triglycerides: The GenSalt Study

PubMed Central

Li, Changwei; Bazzano, Lydia A.L.; Rao, Dabeeru C.; Hixson, James E.; He, Jiang; Gu, Dongfeng; Gu, Charles C.; Shimmin, Lawrence C.; Jaquish, Cashell E.; Schwander, Karen; Liu, De-Pei; Huang, Jianfeng; Lu, Fanghong; Cao, Jie; Chong, Shen; Lu, Xiangfeng; Kelly, Tanika N.

2016-01-01

We conducted a genome-wide linkage scan and positional association study to identify genes and variants influencing blood lipid levels among participants of the Genetic Epidemiology Network of Salt-Sensitivity (GenSalt) study. The GenSalt study was conducted among 1906 participants from 633 Han Chinese families. Lipids were measured from overnight fasting blood samples using standard methods. Multipoint quantitative trait genome-wide linkage scans were performed on the high-density lipoprotein, low-density lipoprotein, and log-transformed triglyceride phenotypes. Using dense panels of single nucleotide polymorphisms (SNPs), single-marker and gene-based association analyses were conducted to follow-up on promising linkage signals. Additive associations between each SNP and lipid phenotypes were tested using mixed linear regression models. Gene-based analyses were performed by combining P-values from single-marker analyses within each gene using the truncated product method (TPM). Significant associations were assessed for replication among 777 Asian participants of the Multi-ethnic Study of Atherosclerosis (MESA). Bonferroni correction was used to adjust for multiple testing. In the GenSalt study, suggestive linkage signals were identified at 2p11.2–2q12.1 [maximum multipoint LOD score (MML) = 2.18 at 2q11.2] and 11q24.3–11q25 (MML = 2.29 at 11q25) for the log-transformed triglyceride phenotype. Follow-up analyses of these two regions revealed gene-based associations of charged multivesicular body protein 3 (CHMP3), ring finger protein 103 (RNF103), AF4/FMR2 family, member 3 (AFF3), and neurotrimin (NTM ) with triglycerides (P = 4 × 10−4, 1.00 × 10−5, 2.00 × 10−5, and 1.00 × 10−7, respectively). Both the AFF3 and NTM triglyceride associations were replicated among MESA study participants (P = 1.00 × 10−7 and 8.00 × 10−5, respectively). Furthermore, NTM explained the linkage signal on chromosome 11. In conclusion, we identified novel genes

Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.

PubMed

Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S

2012-01-01

Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

PubMed

Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

2012-04-01

In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

The chemokine receptor CCR1 is identified in mast cell-derived exosomes

PubMed Central

Liang, Yuting; Qiao, Longwei; Peng, Xia; Cui, Zelin; Yin, Yue; Liao, Huanjin; Jiang, Min; Li, Li

2018-01-01

Mast cells are important effector cells of the immune system, and mast cell-derived exosomes carrying RNAs play a role in immune regulation. However, the molecular function of mast cell-derived exosomes is currently unknown, and here, we identify differentially expressed genes (DEGs) in mast cells and exosomes. We isolated mast cells derived exosomes through differential centrifugation and screened the DEGs from mast cell-derived exosomes, using the GSE25330 array dataset downloaded from the Gene Expression Omnibus database. Biochemical pathways were analyzed by Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the online tool DAVID. DEGs-associated protein-protein interaction networks (PPIs) were constructed using the STRING database and Cytoscape software. The genes identified from these bioinformatics analyses were verified by qRT-PCR and Western blot in mast cells and exosomes. We identified 2121 DEGs (843 up and 1278 down-regulated genes) in HMC-1 cell-derived exosomes and HMC-1 cells. The up-regulated DEGs were classified into two significant modules. The chemokine receptor CCR1 was screened as a hub gene and enriched in cytokine-mediated signaling pathway in module one. Seven genes, including CCR1, CD9, KIT, TGFBR1, TLR9, TPSAB1 and TPSB2 were screened and validated through qRT-PCR analysis. We have achieved a comprehensive view of the pivotal genes and pathways in mast cells and exosomes and identified CCR1 as a hub gene in mast cell-derived exosomes. Our results provide novel clues with respect to the biological processes through which mast cell-derived exosomes modulate immune responses. PMID:29511430

The chemokine receptor CCR1 is identified in mast cell-derived exosomes.

PubMed

Liang, Yuting; Qiao, Longwei; Peng, Xia; Cui, Zelin; Yin, Yue; Liao, Huanjin; Jiang, Min; Li, Li

2018-01-01

Mast cells are important effector cells of the immune system, and mast cell-derived exosomes carrying RNAs play a role in immune regulation. However, the molecular function of mast cell-derived exosomes is currently unknown, and here, we identify differentially expressed genes (DEGs) in mast cells and exosomes. We isolated mast cells derived exosomes through differential centrifugation and screened the DEGs from mast cell-derived exosomes, using the GSE25330 array dataset downloaded from the Gene Expression Omnibus database. Biochemical pathways were analyzed by Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the online tool DAVID. DEGs-associated protein-protein interaction networks (PPIs) were constructed using the STRING database and Cytoscape software. The genes identified from these bioinformatics analyses were verified by qRT-PCR and Western blot in mast cells and exosomes. We identified 2121 DEGs (843 up and 1278 down-regulated genes) in HMC-1 cell-derived exosomes and HMC-1 cells. The up-regulated DEGs were classified into two significant modules. The chemokine receptor CCR1 was screened as a hub gene and enriched in cytokine-mediated signaling pathway in module one. Seven genes, including CCR1, CD9, KIT, TGFBR1, TLR9, TPSAB1 and TPSB2 were screened and validated through qRT-PCR analysis. We have achieved a comprehensive view of the pivotal genes and pathways in mast cells and exosomes and identified CCR1 as a hub gene in mast cell-derived exosomes. Our results provide novel clues with respect to the biological processes through which mast cell-derived exosomes modulate immune responses.

Detection of human papilloma virus 16 and 18 DNA sequences by southern blot hybridization in oral leukoplakia and squamous cell carcinoma.

PubMed

Khanna, Rahul; Rao, G R K; Tiwary, S K; Rai, Ashish; Khanna, Seema; Khanna, A K

2009-04-01

The etiopathological role of human papilloma virus (HPV) in the causation of oral cancer is till a subject of speculation. We used the technique of Southern blot hybridization to detect the presence of HPV types 16 & 18 in biopsy specimens from oral cancer and leukoplakia patients as well as normal oral mucosal biopsies. The prevalence of either HPV type 16 or 18 was found in 64.5% (29/45) of oral cancer, 40%(12/30) of leukoplakia and 20%(9/45) of normal oral mucosal biopsies. No association could be demonstrated between tobacco usage habits or a history of genital warts with HPV prevalence. A significant finding was that none of the oral cancer patients were negative for both: a history of tobacco usage as well as presence of HPV infection, on Southern blot hybridization.

Recent meta-analyses neglect previous systematic reviews and meta-analyses about the same topic: a systematic examination.

PubMed

Helfer, Bartosz; Prosser, Aaron; Samara, Myrto T; Geddes, John R; Cipriani, Andrea; Davis, John M; Mavridis, Dimitris; Salanti, Georgia; Leucht, Stefan

2015-04-14

As the number of systematic reviews is growing rapidly, we systematically investigate whether meta-analyses published in leading medical journals present an outline of available evidence by referring to previous meta-analyses and systematic reviews. We searched PubMed for recent meta-analyses of pharmacological treatments published in high impact factor journals. Previous systematic reviews and meta-analyses were identified with electronic searches of keywords and by searching reference sections. We analyzed the number of meta-analyses and systematic reviews that were cited, described and discussed in each recent meta-analysis. Moreover, we investigated publication characteristics that potentially influence the referencing practices. We identified 52 recent meta-analyses and 242 previous meta-analyses on the same topics. Of these, 66% of identified previous meta-analyses were cited, 36% described, and only 20% discussed by recent meta-analyses. The probability of citing a previous meta-analysis was positively associated with its publication in a journal with a higher impact factor (odds ratio, 1.49; 95% confidence interval, 1.06 to 2.10) and more recent publication year (odds ratio, 1.19; 95% confidence interval 1.03 to 1.37). Additionally, the probability of a previous study being described by the recent meta-analysis was inversely associated with the concordance of results (odds ratio, 0.38; 95% confidence interval, 0.17 to 0.88), and the probability of being discussed was increased for previous studies that employed meta-analytic methods (odds ratio, 32.36; 95% confidence interval, 2.00 to 522.85). Meta-analyses on pharmacological treatments do not consistently refer to and discuss findings of previous meta-analyses on the same topic. Such neglect can lead to research waste and be confusing for readers. Journals should make the discussion of related meta-analyses mandatory.

Contribution of a Comparative Western Blot Method to Early Postnatal Diagnosis of Congenital Syphilis.

PubMed

Marangoni, Antonella; Foschi, Claudio; Capretti, Maria Grazia; Nardini, Paola; Compri, Monica; Corvaglia, Luigi Tommaso; Faldella, Giacomo; Cevenini, Roberto

2016-05-01

Serology has a pivotal role in the diagnosis of congenital syphilis (CS), but problems arise because of the passive transfer of IgG antibodies across the placenta. The aim of this study was to assess the diagnostic value of a comparative Western blot (WB) method finalized to match the IgG immunological profiles of mothers and their own babies at birth in order to differentiate between passively transmitted maternal antibodies and antibodies synthesized by the infants against Treponema pallidum Thirty infants born to mothers with unknown or inadequate treatment for syphilis were entered in a retrospective study, conducted at St. Orsola-Malpighi Hospital, Bologna, Italy. All of the infants underwent clinical, instrumental, and laboratory examinations, including IgM WB testing. For the retrospective study, an IgG WB assay was performed by blotting T. pallidum antigens onto nitrocellulose sheets and incubating the strips with serum specimens from mother-child pairs. CS was diagnosed in 11 out of the 30 enrolled infants; 9/11 cases received the definitive diagnosis within the first week of life, whereas the remaining two were diagnosed later because of increasing serological test titers. The use of the comparative IgG WB testing performed with serum samples from mother-child pairs allowed a correct CS diagnosis in 10/11 cases. The CS diagnosis was improved by a strategy combining comparative IgG WB results with IgM WB results, leading to a sensitivity of 100%. The comparative IgG WB test is thus a welcome addition to the conventional laboratory methods used for CS diagnosis, allowing identification and adequate treatment of infected infants and avoiding unnecessary therapy of uninfected newborns. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Large-scale analyses of common and rare variants identify 12 new loci associated with atrial fibrillation

PubMed Central

Christophersen, Ingrid E.; Rienstra, Michiel; Roselli, Carolina; Yin, Xiaoyan; Geelhoed, Bastiaan; Barnard, John; Lin, Honghuang; Arking, Dan E.; Smith, Albert V.; Albert, Christine M.; Chaffin, Mark; Tucker, Nathan R.; Li, Molong; Klarin, Derek; Bihlmeyer, Nathan A; Low, Siew-Kee; Weeke, Peter E.; Müller-Nurasyid, Martina; Smith, J. Gustav; Brody, Jennifer A.; Niemeijer, Maartje N.; Dörr, Marcus; Trompet, Stella; Huffman, Jennifer; Gustafsson, Stefan; Schurman, Claudia; Kleber, Marcus E.; Lyytikäinen, Leo-Pekka; Seppälä, Ilkka; Malik, Rainer; Horimoto, Andrea R. V. R.; Perez, Marco; Sinisalo, Juha; Aeschbacher, Stefanie; Thériault, Sébastien; Yao, Jie; Radmanesh, Farid; Weiss, Stefan; Teumer, Alexander; Choi, Seung Hoan; Weng, Lu-Chen; Clauss, Sebastian; Deo, Rajat; Rader, Daniel J.; Shah, Svati; Sun, Albert; Hopewell, Jemma C.; Debette, Stephanie; Chauhan, Ganesh; Yang, Qiong; Worrall, Bradford B.; Paré, Guillaume; Kamatani, Yoichiro; Hagemeijer, Yanick P.; Verweij, Niek; Siland, Joylene E.; Kubo, Michiaki; Smith, Jonathan D.; Van Wagoner, David R.; Bis, Joshua C.; Perz, Siegfried; Psaty, Bruce M.; Ridker, Paul M.; Magnani, Jared W.; Harris, Tamara B.; Launer, Lenore J.; Shoemaker, M. Benjamin; Padmanabhan, Sandosh; Haessler, Jeffrey; Bartz, Traci M.; Waldenberger, Melanie; Lichtner, Peter; Arendt, Marina; Krieger, Jose E.; Kähönen, Mika; Risch, Lorenz; Mansur, Alfredo J.; Peters, Annette; Smith, Blair H.; Lind, Lars; Scott, Stuart A.; Lu, Yingchang; Bottinger, Erwin B.; Hernesniemi, Jussi; Lindgren, Cecilia M.; Wong, Jorge; Huang, Jie; Eskola, Markku; Morris, Andrew P.; Ford, Ian; Reiner, Alex P.; Delgado, Graciela; Chen, Lin Y.; Chen, Yii-Der Ida; Sandhu, Roopinder K.; Li, Man; Boerwinkle, Eric; Eisele, Lewin; Lannfelt, Lars; Rost, Natalia; Anderson, Christopher D.; Taylor, Kent D.; Campbell, Archie; Magnusson, Patrik K.; Porteous, David; Hocking, Lynne J.; Vlachopoulou, Efthymia; Pedersen, Nancy L.; Nikus, Kjell; Orho-Melander, Marju; Hamsten, Anders; Heeringa, Jan; Denny, Joshua C.; Kriebel, Jennifer; Darbar, Dawood; Newton-Cheh, Christopher; Shaffer, Christian; Macfarlane, Peter W.; Heilmann, Stefanie; Almgren, Peter; Huang, Paul L.; Sotoodehnia, Nona; Soliman, Elsayed Z.; Uitterlinden, Andre G.; Hofman, Albert; Franco, Oscar H.; Völker, Uwe; Jöckel, Karl-Heinz; Sinner, Moritz F.; Lin, Henry J.; Guo, Xiuqing; Dichgans, Martin; Ingelsson, Erik; Kooperberg, Charles; Melander, Olle; Loos, Ruth J. F.; Laurikka, Jari; Conen, David; Rosand, Jonathan; van der Harst, Pim; Lokki, Marja-Liisa; Kathiresan, Sekar; Pereira, Alexandre; Jukema, J. Wouter; Hayward, Caroline; Rotter, Jerome I.; März, Winfried; Lehtimäki, Terho; Stricker, Bruno H.; Chung, Mina K.; Felix, Stephan B.; Gudnason, Vilmundur; Alonso, Alvaro; Roden, Dan M.; Kääb, Stefan; Chasman, Daniel I.; Heckbert, Susan R.; Benjamin, Emelia J.; Tanaka, Toshihiro; Lunetta, Kathryn L.; Lubitz, Steven A.; Ellinor, Patrick T.

2017-01-01

Atrial fibrillation affects more than 33 million people worldwide and increases the risk of stroke, heart failure, and death.1,2 Fourteen genetic loci have been associated with atrial fibrillation in European and Asian ancestry groups.3–7 To further define the genetic basis of atrial fibrillation, we performed large-scale, multi-racial meta-analyses of common and rare variant association studies. The genome-wide association studies (GWAS) included 18,398 individuals with atrial fibrillation and 91,536 referents; the exome-wide association studies (ExWAS) and rare variant association studies (RVAS) involved 22,806 cases and 132,612 referents. We identified 12 novel genetic loci that exceeded genome-wide significance, implicating genes involved in cardiac electrical and structural remodeling. Our results nearly double the number of known genetic loci for atrial fibrillation, provide insights into the molecular basis of atrial fibrillation, and may facilitate new potential targets for drug discovery.8 PMID:28416818

Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

PubMed

Kuramitsu, Madoka; Sekizuka, Tsuyoshi; Yamochi, Tadanori; Firouzi, Sanaz; Sato, Tomoo; Umeki, Kazumi; Sasaki, Daisuke; Hasegawa, Hiroo; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Momose, Haruka; Araki, Kumiko; Saito, Masumichi; Nosaka, Kisato; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Okayama, Akihiko; Miura, Kiyonori; Satake, Masahiro; Saito, Shigeru; Itabashi, Kazuo; Yamaguchi, Kazunari; Kuroda, Makoto; Watanabe, Toshiki; Okuma, Kazu; Hamaguchi, Isao

2017-09-01

Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens. Copyright © 2017 American Society for Microbiology.

Rapid diagnosis of pulmonary tuberculosis and detection of drug resistance by combined simultaneous amplification testing and reverse dot blot.

PubMed

Chen, Yiwen; Zhang, Lahong; Hong, Liquan; Luo, Xian; Chen, Juping; Tang, Leiming; Chen, Jiahuan; Liu, Xia; Chen, Zhaojun

2018-06-01

Making a correct and rapid diagnosis is essential for managing pulmonary tuberculosis (PTB), particularly multidrug-resistant tuberculosis. We aimed to evaluate the efficacy of the combination of simultaneous amplification testing (SAT) and reverse dot blot (RDB) for the rapid detection of Mycobacterium tuberculosis (MTB) and drug-resistant mutants in respiratory samples. 225 suspected PTB and 32 non-TB pulmonary disease samples were collected. All sputum samples were sent for acid-fast bacilli smear, SAT, culture and drug susceptibility testing (DST) by the BACTEC TM MGIT TM 960 system. 53 PTB samples were tested by both RDB and DNA sequencing to identify drug resistance genes and mutated sites. The SAT positive rate (64.9%) was higher than the culture positive rate (55.1%), with a coincidence rate of 83.7%. The sensitivity and specificity of SAT for diagnosing PTB were 66.7% and 100%, respectively, while those for culture were 53.9% and 84.2%, respectively. RDB has high sensitivity and specificity in identifying drug resistance genes and mutated sites. The results of RDB correlated well with those of DST and DNA sequencing, with coincidence rates of 92.5% and 98.1%, respectively. The combination of SAT and RDB is promising for rapidly detecting PTB and monitoring drug resistance in clinical laboratories. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Analyses of expression and localization of two mammalian-type transglutaminases in Physarum polycephalum, an acellular slime mold.

PubMed

Wada, Fumitaka; Ogawa, Atsuko; Hanai, Yuko; Nakamura, Akio; Maki, Masatoshi; Hitomi, Kiyotaka

2004-11-01

Transglutaminase (TGase) is an enzyme that modifies proteins by crosslinking or polyamination. Physarum polycephalum, an acellular slime mold, is the evolutionally lowest organism that has a mammalian-type transglutaminase. We have cloned a cDNA for Physarum polycephalum TGase (PpTGB), homologous to a previously identified TGase (PpTGA), whose sequence is similar to that of mammalian TGases. PpTGB encodes a primary sequence identical to that of PpTGA except for 11 amino acid residues at the N-terminus. Reverse transcription-PCR and Western blotting analyses showed that both PpTGA and PpTGB are expressed in microplasmodia and macroplasmodia during their life cycle, except for in sporangia. For biochemical characterization, we carried out the ectopical expressions of PpTGA and PpTGB in Dictyostelium discoideum. Subcellular fractionation of these Dictyostelium cells showed that the expressed PpTGA, but not PpTGB, localizes to the membrane fraction. Furthermore, in Physarum, subcellular fractionation and immunostaining indicated specific localization at the plasma membrane in macroplasmodia, while the localization was entirely cytoplasmic in microplasmodia.

Epigenomic Elements Analyses for Promoters Identify ESRRG as a New Susceptibility Gene for Obesity-related Traits

PubMed Central

Dong, Shan-Shan; Guo, Yan; Zhu, Dong-Li; Chen, Xiao-Feng; Wu, Xiao-Ming; Shen, Hui; Chen, Xiang-Ding; Tan, Li-Jun; Tian, Qing; Deng, Hong-Wen; Yang, Tie-Lin

2016-01-01

OBJECTIVES With ENCODE epigenomic data and results from published genome-wide association studies (GWASs), we aimed to find regulatory signatures of obesity genes and discover novel susceptibility genes. METHODS Obesity genes were obtained from public GWASs databases and their promoters were annotated based on the regulatory elements information. Significantly enriched or depleted epigenomic elements in the promoters of obesity genes were evaluated and all human genes were then prioritized according to the existence of the selected elements to predict new candidate genes. Top ranked genes were subsequently applied to validate their associations with obesity-related traits in three independent in-house GWASs samples. RESULTS We identified RAD21 and EZH2 as over-represented, STAT2 and IRF3 as depleted transcription factors. Histone modification of H3K9me3 and chromatin state segmentation of “poised promoter” and “repressed” were overrepresented. All genes were prioritized and we selected the top five genes for validation at population level. Combined results from the three GWASs samples, rs7522101 in ESRRG remained significantly associated with BMI after multiple testing corrections (P = 7.25 × 10−5). It was also associated with β-cell function (P = 1.99 × 10−3) and fasting glucose level (P < 0.05) in the meta-analyses of glucose and insulin-related traits consortium (MAGIC) dataset. CONCLUSIONS In summary, we identified epigenomic characteristics for obesity genes and suggested ESRRG as a novel obesity susceptibility gene. PMID:27113491

Longitudinal analyses of the DNA methylome in deployed military servicemen identify susceptibility loci for post-traumatic stress disorder.

PubMed

Rutten, B P F; Vermetten, E; Vinkers, C H; Ursini, G; Daskalakis, N P; Pishva, E; de Nijs, L; Houtepen, L C; Eijssen, L; Jaffe, A E; Kenis, G; Viechtbauer, W; van den Hove, D; Schraut, K G; Lesch, K-P; Kleinman, J E; Hyde, T M; Weinberger, D R; Schalkwyk, L; Lunnon, K; Mill, J; Cohen, H; Yehuda, R; Baker, D G; Maihofer, A X; Nievergelt, C M; Geuze, E; Boks, M P M

2018-05-01

In order to determine the impact of the epigenetic response to traumatic stress on post-traumatic stress disorder (PTSD), this study examined longitudinal changes of genome-wide blood DNA methylation profiles in relation to the development of PTSD symptoms in two prospective military cohorts (one discovery and one replication data set). In the first cohort consisting of male Dutch military servicemen (n=93), the emergence of PTSD symptoms over a deployment period to a combat zone was significantly associated with alterations in DNA methylation levels at 17 genomic positions and 12 genomic regions. Evidence for mediation of the relation between combat trauma and PTSD symptoms by longitudinal changes in DNA methylation was observed at several positions and regions. Bioinformatic analyses of the reported associations identified significant enrichment in several pathways relevant for symptoms of PTSD. Targeted analyses of the significant findings from the discovery sample in an independent prospective cohort of male US marines (n=98) replicated the observed relation between decreases in DNA methylation levels and PTSD symptoms at genomic regions in ZFP57, RNF39 and HIST1H2APS2. Together, our study pinpoints three novel genomic regions where longitudinal decreases in DNA methylation across the period of exposure to combat trauma marks susceptibility for PTSD.

A Western blot-based investigation of the yeast secretory pathway designed for an intermediate-level undergraduate cell biology laboratory.

PubMed

Hood-Degrenier, Jennifer K

2008-01-01

The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor.

Immunoproteomic Analysis To Identify Shiga Toxin-Producing Escherichia coli Outer Membrane Proteins Expressed during Human Infection

PubMed Central

Montero, David; Orellana, Paz; Gutiérrez, Daniela; Araya, Daniela; Salazar, Juan Carlos; Prado, Valeria; Oñate, Ángel; del Canto, Felipe

2014-01-01

Shiga-toxin producing Escherichia coli (STEC) is the etiologic agent of acute diarrhea, dysentery, and hemolytic-uremic syndrome (HUS). There is no approved vaccine for STEC infection in humans, and antibiotic use is contraindicated, as it promotes Shiga toxin production. In order to identify STEC-associated antigens and immunogenic proteins, outer membrane proteins (OMPs) were extracted from STEC O26:H11, O103, O113:H21, and O157:H7 strains, and commensal E. coli strain HS was used as a control. SDS-PAGE, two-dimensional-PAGE analysis, Western blot assays using sera from pediatric HUS patients and controls, and matrix-assisted laser desorption ionization–tandem time of flight analyses were used to identify 12 immunogenic OMPs, some of which were not reactive with control sera. Importantly, seven of these proteins have not been previously reported to be immunogenic in STEC strains. Among these seven proteins, OmpT and Cah displayed IgG and IgA reactivity with sera from HUS patients. Genes encoding these two proteins were present in a majority of STEC strains. Knowledge of the antigens produced during infection of the host and the immune response to those antigens will be important for future vaccine development. PMID:25156722

The Analysing Children's Creative Thinking Framework: Development of an Observation-Led Approach to Identifying and Analysing Young Children's Creative Thinking

ERIC Educational Resources Information Center

Robson, Sue

2014-01-01

Increased international recognition of the value of supporting creative thinking suggests the value of development of approaches to its identification in children. Development of an observation-led framework, the Analysing Children's Creative Thinking (ACCT) framework, is described, and a case made for the validity of inferring creative thinking…

Epigenomic elements analyses for promoters identify ESRRG as a new susceptibility gene for obesity-related traits.

PubMed

Dong, S-S; Guo, Y; Zhu, D-L; Chen, X-F; Wu, X-M; Shen, H; Chen, X-D; Tan, L-J; Tian, Q; Deng, H-W; Yang, T-L

2016-07-01

With ENCODE epigenomic data and results from published genome-wide association studies (GWASs), we aimed to find regulatory signatures of obesity genes and discover novel susceptibility genes. Obesity genes were obtained from public GWAS databases and their promoters were annotated based on the regulatory element information. Significantly enriched or depleted epigenomic elements in the promoters of obesity genes were evaluated and all human genes were then prioritized according to the existence of the selected elements to predict new candidate genes. Top-ranked genes were subsequently applied to validate their associations with obesity-related traits in three independent in-house GWAS samples. We identified RAD21 and EZH2 as over-represented, and STAT2 (signal transducer and activator of transcription 2) and IRF3 (interferon regulatory transcription factor 3) as depleted transcription factors. Histone modification of H3K9me3 and chromatin state segmentation of 'poised promoter' and 'repressed' were over-represented. All genes were prioritized and we selected the top five genes for validation at the population level. Combining results from the three GWAS samples, rs7522101 in ESRRG (estrogen-related receptor-γ) remained significantly associated with body mass index after multiple testing corrections (P=7.25 × 10(-5)). It was also associated with β-cell function (P=1.99 × 10(-3)) and fasting glucose level (P<0.05) in the meta-analyses of glucose and insulin-related traits consortium (MAGIC) data set.Cnoclusions:In summary, we identified epigenomic characteristics for obesity genes and suggested ESRRG as a novel obesity-susceptibility gene.

Genome-Wide Linkage, Exome Sequencing and Functional Analyses Identify ABCB6 as the Pathogenic Gene of Dyschromatosis Universalis Hereditaria

PubMed Central

Wang, Na; Wang, Chuan; Chen, Xuechao; Sheng, Donglai; Fu, Xi’an; See, Kelvin; Foo, Jia Nee; Low, Huiqi; Liany, Herty; Irwan, Ishak Darryl; Liu, Jian; Yang, Baoqi; Chen, Mingfei; Yu, Yongxiang; Yu, Gongqi; Niu, Guiye; You, Jiabao; Zhou, Yan; Ma, Shanshan; Wang, Ting; Yan, Xiaoxiao; Goh, Boon Kee; Common, John E. A.; Lane, Birgitte E.; Sun, Yonghu; Zhou, Guizhi; Lu, Xianmei; Wang, Zhenhua; Tian, Hongqing; Cao, Yuanhua; Chen, Shumin; Liu, Qiji; Liu, Jianjun; Zhang, Furen

2014-01-01

Background As a genetic disorder of abnormal pigmentation, the molecular basis of dyschromatosis universalis hereditaria (DUH) had remained unclear until recently when ABCB6 was reported as a causative gene of DUH. Methodology We performed genome-wide linkage scan using Illumina Human 660W-Quad BeadChip and exome sequencing analyses using Agilent SureSelect Human All Exon Kits in a multiplex Chinese DUH family to identify the pathogenic mutations and verified the candidate mutations using Sanger sequencing. Quantitative RT-PCR and Immunohistochemistry was performed to verify the expression of the pathogenic gene, Zebrafish was also used to confirm the functional role of ABCB6 in melanocytes and pigmentation. Results Genome-wide linkage (assuming autosomal dominant inheritance mode) and exome sequencing analyses identified ABCB6 as the disease candidate gene by discovering a coding mutation (c.1358C>T; p.Ala453Val) that co-segregates with the disease phenotype. Further mutation analysis of ABCB6 in four other DUH families and two sporadic cases by Sanger sequencing confirmed the mutation (c.1358C>T; p.Ala453Val) and discovered a second, co-segregating coding mutation (c.964A>C; p.Ser322Lys) in one of the four families. Both mutations were heterozygous in DUH patients and not present in the 1000 Genome Project and dbSNP database as well as 1,516 unrelated Chinese healthy controls. Expression analysis in human skin and mutagenesis interrogation in zebrafish confirmed the functional role of ABCB6 in melanocytes and pigmentation. Given the involvement of ABCB6 mutations in coloboma, we performed ophthalmological examination of the DUH carriers of ABCB6 mutations and found ocular abnormalities in them. Conclusion Our study has advanced our understanding of DUH pathogenesis and revealed the shared pathological mechanism between pigmentary DUH and ocular coloboma. PMID:24498303

Prediction of the optimum hybridization conditions of dot-blot-SNP analysis using estimated melting temperature of oligonucleotide probes.

PubMed

Shiokai, Sachiko; Kitashiba, Hiroyasu; Nishio, Takeshi

2010-08-01

Although the dot-blot-SNP technique is a simple cost-saving technique suitable for genotyping of many plant individuals, optimization of hybridization and washing conditions for each SNP marker requires much time and labor. For prediction of the optimum hybridization conditions for each probe, we compared T (m) values estimated from nucleotide sequences using the DINAMelt web server, measured T (m) values, and hybridization conditions yielding allele-specific signals. The estimated T (m) values were comparable to the measured T (m) values with small differences of less than 3 degrees C for most of the probes. There were differences of approximately 14 degrees C between the specific signal detection conditions and estimated T (m) values. Change of one level of SSC concentrations of 0.1, 0.2, 0.5, and 1.0x SSC corresponded to a difference of approximately 5 degrees C in optimum signal detection temperature. Increasing the sensitivity of signal detection by shortening the exposure time to X-ray film changed the optimum hybridization condition for specific signal detection. Addition of competitive oligonucleotides to the hybridization mixture increased the suitable hybridization conditions by 1.8. Based on these results, optimum hybridization conditions for newly produced dot-blot-SNP markers will become predictable.

Agreement between coding schemas used to identify bleeding-related hospitalizations in claims analyses of nonvalvular atrial fibrillation patients.

PubMed

Coleman, Craig I; Vaitsiakhovich, Tatsiana; Nguyen, Elaine; Weeda, Erin R; Sood, Nitesh A; Bunz, Thomas J; Schaefer, Bernhard; Meinecke, Anna-Katharina; Eriksson, Daniel

2018-01-01

Schemas to identify bleeding-related hospitalizations in claims data differ in billing codes used and coding positions allowed. We assessed agreement across bleeding-related hospitalization coding schemas for claims analyses of nonvalvular atrial fibrillation (NVAF) patients on oral anticoagulation (OAC). We hypothesized that prior coding schemas used to identify bleeding-related hospitalizations in claim database studies would provide varying levels of agreement in incidence rates. Within MarketScan data, we identified adults, newly started on OAC for NVAF from January 2012 to June 2015. Billing code schemas developed by Cunningham et al., the US Food and Drug Administration (FDA) Mini-Sentinel program, and Yao et al. were used to identify bleeding-related hospitalizations as a surrogate for major bleeding. Bleeds were subcategorized as intracranial hemorrhage (ICH), gastrointestinal (GI), or other. Schema agreement was assessed by comparing incidence, rates of events/100 person-years (PYs), and Cohen's kappa statistic. We identified 151 738 new-users of OAC with NVAF (CHA2DS2-VASc score = 3, [interquartile range = 2-4] and median HAS-BLED score = 3 [interquartile range = 2-3]). The Cunningham, FDA Mini-Sentinel, and Yao schemas identified any bleeding-related hospitalizations in 1.87% (95% confidence interval [CI]: 1.81-1.94), 2.65% (95% CI: 2.57-2.74), and 4.66% (95% CI: 4.55-4.76) of patients (corresponding rates = 3.45, 4.90, and 8.65 events/100 PYs). Kappa agreement across schemas was weak-to-moderate (κ = 0.47-0.66) for any bleeding hospitalization. Near-perfect agreement (κ = 0.99) was observed with the FDA Mini-Sentinel and Yao schemas for ICH-related hospitalizations, but agreement was weak when comparing Cunningham to FDA Mini-Sentinel or Yao (κ = 0.52-0.53). FDA Mini-Sentinel and Yao agreement was moderate (κ = 0.62) for GI bleeding, but agreement was weak when comparing Cunningham to FDA Mini-Sentinel or Yao (κ�

Comparative gene expression analysis between coronary arteries and internal mammary arteries identifies a role for the TES gene in endothelial cell functions relevant to coronary artery disease.

PubMed

Archacki, Stephen R; Angheloiu, George; Moravec, Christine S; Liu, Hui; Topol, Eric J; Wang, Qing Kenneth

2012-03-15

Coronary artery disease (CAD) is the leading cause of death worldwide. It has been established that internal mammary arteries (IMA) are resistant to the development of atherosclerosis, whereas left anterior descending (LAD) coronary arteries are athero-prone. The contrasting properties of these two arteries provide an innovative strategy to identify the genes that play important roles in the development of atherosclerosis. We carried out microarray analysis to identify genes differentially expressed between IMA and LAD. Twenty-nine genes showed significant differences in their expression levels between IMA and LAD, which included the TES gene encoding Testin. The role of TES in the cardiovascular system is unknown. Here we show that TES is involved in endothelial cell (EC) functions relevant to atherosclerosis. Western blot analysis showed higher TES expression in IMA than in LAD. Reverse transcription polymerase chain reaction and western blot analyses showed that TES was consistently and markedly down-regulated by more than 6-fold at both mRNA and protein levels in patients with CAD compared with controls without CAD (P= 0.000049). The data suggest that reduced TES expression is associated with the development of CAD. Knockdown of TES expression by small-interfering RNA promoted oxidized-LDL-mediated monocyte adhesion to ECs, EC migration and the transendothelial migration of monocytes, while the over-expression of TES in ECs blunted these processes. These results demonstrate association between reduced TES expression and CAD, establish a novel role for TES in EC functions and raise the possibility that reduced TES expression increases susceptibility to the development of CAD.

Use of a reverse line blot assay to survey small strongyle (Strongylida: Cyathostominae) populations in horses before and after treatment with ivermectin.

PubMed

Ionita, Mariana; Howe, Daniel K; Lyons, Eugene T; Tolliver, Sharon C; Kaplan, Ray M; Mitrea, Ioan Liviu; Yeargan, Michelle

2010-03-25

A sensitive and specific PCR hybridization assay was applied for species-specific monitoring of the small strongyle (Strongylida: Cyathostominae) populations in horses in a herd before and after treatment with the anthelmintic drug ivermectin. Fecal samples were collected pre- and post-treatment weekly from eight individual horses (four foals and four yearlings) for 6 weeks to determine counts of strongyle eggs per gram of feces (EPGs). Additionally, one foal and one yearling were nontreated controls. Also, one horse, from another herd known to be infected with Strongylus spp., was a positive control for these parasites. Genomic DNA was obtained from eggs in groups of approximately 6000-7000 eggs except for two samples containing low EPGs in which 450 eggs were used. Amplification of the intergenic spacers (IGSs) of ribosomal DNA (rDNA) of small and large strongyles followed by reverse line blot (RLB) assay were performed to identify the presence of the 12 most common equine small strongyle species and to discriminate them from Strongylus spp. Overall, 11 small strongyle species were identified in pretreatment samples. In the samples collected at 4 weeks after ivermectin treatment, eight small strongyle species were identified and four of them were predominant (Cylicocyclus nassatus, Cylicostephanus longibursatus, Cylicostephanus calicatus and Cylicostephanus minutus). At 5 and 6 weeks post-treatment, the RLB assay analysis showed almost the same composition in the small strongyle population as before treatment. Strongylus spp. were identified only in samples collected from the positive control horse for these parasites. These data confirm the ability of the PCR-RLB technique for simultaneous species-specific differentiation of equine strongyle eggs, indicating a valuable way of furthering drug-resistance studies.

Characterization of Nora Virus Structural Proteins via Western Blot Analysis.

PubMed

Ericson, Brad L; Carlson, Darby J; Carlson, Kimberly A

2016-01-01

Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

Characterization of Nora Virus Structural Proteins via Western Blot Analysis

PubMed Central

Ericson, Brad L.; Carlson, Darby J.

2016-01-01

Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses. PMID:27298753

Using multivariate analyses and GIS to identify pollutants and their spatial patterns in urban soils in Galway, Ireland.

PubMed

Zhang, Chaosheng

2006-08-01

Galway is a small but rapidly growing tourism city in western Ireland. To evaluate its environmental quality, a total of 166 surface soil samples (0-10 cm depth) were collected from parks and grasslands at the density of 1 sample per 0.25 km2 at the end of 2004. All samples were analysed using ICP-AES for the near-total concentrations of 26 chemical elements. Multivariate statistics and GIS techniques were applied to classify the elements and to identify elements influenced by human activities. Cluster analysis (CA) and principal component analysis (PCA) classified the elements into two groups: the first group predominantly derived from natural sources, the second being influenced by human activities. GIS mapping is a powerful tool in identifying the possible sources of pollutants. Relatively high concentrations of Cu, Pb and Zn were found in the city centre, old residential areas, and along major traffic routes, showing significant effects of traffic pollution. The element As is enriched in soils of the old built-up areas, which can be attributed to coal and peat combustion for home heating. Such significant spatial patterns of pollutants displayed by urban soils may imply potential health threat to residents of the contaminated areas of the city.

Integrated genomic analyses identify WEE1 as a critical mediator of cell fate and novel therapeutic target in acute myeloid leukemia

PubMed Central

Porter, Christopher C.; Kim, Jihye; Fosmire, Susan; Gearheart, Christy M.; van Linden, Annemie; Baturin, Dmitry; Zaberezhnyy, Vadym; Patel, Purvi R.; Gao, Dexiang; Tan, Aik Choon; DeGregori, James

2011-01-01

Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge about the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational approaches, we performed genome-wide shRNA screens to identify proteins that mediate AML cell fate after cytarabine exposure, gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context, and examination of existing gene expression data from primary patient samples. The integration of these independent analyses strongly implicates cell cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell survival after cytarabine exposure. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival. Pharmacologic inhibition of WEE1 in AML cell lines and primary cells is synergistic with cytarabine. Further experiments demonstrate that inhibition of WEE1 prevents S-phase arrest induced by cytarabine, broadening the functions of WEE1 that may be exploited therapeutically. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as potential therapeutic target in AML. PMID:22289989

The combination of quantitative PCR and western blot detecting CP4-EPSPS component in Roundup Ready soy plant tissues and commercial soy-related foodstuffs.

PubMed

Xiao, Xiao; Wu, Honghong; Zhou, Xinghu; Xu, Sheng; He, Jian; Shen, Wenbiao; Zhou, Guanghong; Huang, Ming

2012-06-01

With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods

Comparison of Multispot EIA with Western blot for confirmatory serodiagnosis of HIV.

PubMed

Torian, Lucia V; Forgione, Lisa A; Punsalang, Amado E; Pirillo, Robert E; Oleszko, William R

2011-12-01

Recent improvements in the sensitivity of immunoassays (IA) used for HIV screening, coupled with increasing recognition of the importance of rapid point-of-care testing, have led to proposals to adjust the algorithm for serodiagnosis of HIV so that screening and confirmation can be performed using a dual or triple IA sequence that does not require Western blotting for confirmation. One IA that has been proposed as a second or confirmatory test is the Bio-Rad Multispot(®) Rapid HIV-1/HIV-2 Test. This test would have the added advantage of differentiating between HIV-1 and HIV-2 antibodies. To compare the sensitivity and type-specificity of an algorithm combining a 3rd generation enzyme immunoassay (EIA) followed by a confirmatory Multispot with the conventional algorithm that combines a 3rd generation EIA (Bio-Rad GS HIV-1/HIV-2 Plus O EIA) followed by confirmatory Western blot (Bio-Rad GS HIV-1 WB). 8760 serum specimens submitted for HIV testing to the New York City Public Health Laboratory between May 22, 2007, and April 30, 2010, tested repeatedly positive on 3rd generation HIV-1-2+O EIA screening and received parallel confirmatory testing by WB and Multispot (MS). 8678/8760 (99.1%) specimens tested WB-positive; 82 (0.9%) tested WB-negative or indeterminate (IND). 8690/8760 specimens (99.2%) tested MS-positive, of which 14 (17.1%) had been classified as negative or IND by WB. Among the HIV-1 WB-positive specimens, MS classified 26 (0.29%) as HIV-2. Among the HIV-1 WB negative and IND, MS detected 12 HIV-2. MS detected an additional 14 HIV-1 infections among WB negative or IND specimens, differentiated 26 HIV-1 WB positives as HIV-2, and detected 12 additional HIV-2 infections among WB negative/IND. A dual 3rd generation EIA algorithm incorporating MS had equivalent HIV-1 sensitivity to the 3rd generation EIA-WB algorithm and had the added advantage of detecting 12 HIV-2 specimens that were not HIV-1 WB cross-reactors. In this series an algorithm using EIA

Contribution of dot-blot assay to the diagnosis and management of myositis: a three-year practice at a university hospital centre.

PubMed

Martel, Clothilde; Vignaud, Guillaume; Liozon, Eric; Magy, Laurent; Gallouedec, Gael; Ly, Kim; Bezanahary, Holly; Cypierre, Anne; Lapébie, François-Xavier; Palat, Sylvain; Gondran, Guillaume; Jauberteau, Marie-Odile; Fauchais, Anne-Laure

2016-01-01

Idiopathic inflammatory myopathies (IIM) are heterogeneous autoimmune diseases with wide clinical spectrum that may lead to delayed diagnosis. The aim of this study was to examine the impact of IIM-specific dot-blot assay on diagnostic process of patients presenting with muscular or systemic symptoms evocating of IIM. We collected all the prescriptions of an IIM specific dot-blot assay (8 autoantigens including Jo-1, PL-7, PL-12, SRP, Mi-2, Ku, PM/Scl and Scl-70) over a 38-month period. 316 myositis dot-blot assays (MSD) were performed in 274 patients (156 women, mean age 53±10.6 years) referring for muscular and/or systemic symptoms suggesting IIM. The timing of dot prescription through the diagnostic process was highly variable: without (35%), concomitantly (16%) or after electromyographic studies (35%). Fifty-nine patients (22%) had IIM according to Bohan and Peter's criteria. Among them, 29 (49%) had positive dot (8 Jo-1, 6 PM-Scl, 5 PL-12, 5 SRP, 2 Mi-2, 2 PL-7 and 1 Ku). Various other diagnoses were performed including 35 autoimmune disease or granulomatosis (12%), 19 inflammatory rheumatic disease (7%), 16 non inflammatory muscular disorders (6%), 10 drug-induced myalgia (4%), 11 infectious myositis (4%). Except 11 borderline SRP results and one transient PM-Scl, MSD was positive only in one case of IIM. Dot allowed clinicians to correct diagnosis in 4 cases and improved the diagnosis of IIM subtypes in 4 cases. This study reflects the interest of myositis dot in the rapid diagnosis process of patients with non-specific muscular symptoms leading to various diagnoses including IIM.

Epidemiology of Babesia, Anaplasma and Trypanosoma species using a new expanded reverse line blot hybridization assay.

PubMed

Paoletta, Martina Soledad; López Arias, Ludmila; de la Fournière, Sofía; Guillemi, Eliana Carolina; Luciani, Carlos; Sarmiento, Néstor Fabián; Mosqueda, Juan; Farber, Marisa Diana; Wilkowsky, Silvina Elizabeth

2018-02-01

Vector-borne hemoparasitic infections are a major problem that affects livestock industries worldwide, particularly in tropical and subtropical regions. In this work, a reverse line blot (RLB) hybridization assay was developed for the simultaneous detection and identification of Anaplasma, Babesia and bovine trypanosomes, encompassing in this way the most relevant hemoparasites that affect cattle. A total of 186 bovine blood samples collected from two different ecoepidemiological regions of northeast Argentina, with and without tick control, were analyzed with this new RLB. High diversity of parasites, such as Babesia bovis, B. bigemina, Anaplasma marginale and three different Trypanosoma species, was found. High rates of coinfections were also detected, and significant differences were observed not only in the prevalence of parasites but also in the level of coinfections between the two analyzed areas. Regarding the Trypanosoma genus, we provide molecular evidence of the presence of T. vivax and T. theileri for the first time in Argentina. Besides, since the RLB is a prospective tool, it allowed the identification of a yet unknown bovine trypanosome which could not be assigned to any of the bovine species known so far. In the present study we provide new insights on the prevalence of several pathogens that directly impact on livestock production in Argentina. The RLB assay developed here allows to identify simultaneously numerous pathogenic species which can also be easily expanded to detect other blood borne pathogens. These characteristics make the RLB hybridization assay an essential tool for epidemiological survey of all vector-borne pathogens. Copyright © 2017 Elsevier GmbH. All rights reserved.

Differential staining of Western blots of human secreted glycoproteins from serum, milk, saliva, and seminal fluid using lectins displaying diverse sugar specificities.

PubMed

Gilboa-Garber, Nechama; Lerrer, Batya; Lesman-Movshovich, Efrat; Dgani, Orly

2005-12-01

Human milk, serum, saliva, and seminal fluid glycoproteins (gps) nourish and protect newborn and adult tissues. Their saccharides, which resemble cell membrane components, may block pathogen adhesion and infection. In the present study, they were examined by a battery of lectins from plants, animals, and bacteria, using hemagglutination inhibition and Western blot analyses. The lectins included galactophilic ones from Aplysia gonad, Erythrina corallodendron, Maclura pomifera (MPL), peanut, and Pseudomonas aeruginosa (PA-IL); fucose-binding lectins from Pseudomonas aeruginosa (PA-IIL), Ralstonia solanacearum (RSL), and Ulex europaeus (UEA-I), and mannose/glucose-binding Con A. The results demonstrated the chosen lectin efficiency for differential analysis of human secreted gps as compared to CBB staining. They unveiled the diversity of these body fluid gp glycans (those of the milk and seminal fluid being highest): the milk gps interacted most strongly with PA-IIL, followed by RSL; the saliva gps with RSL, followed by PA-IIL and MPL; the serum gps with Con A and MPL, followed by PA-IIL and RSL, and the seminal plasma gps with RSL and MPL, followed by UEA-I and PA-IIL. The potential usage of these lectins as probes for scientific, industrial, and medical purposes, and for quality control of the desired gps is clearly indicated.

Comparative analyses identify molecular signature of MRI-classified SVZ-associated glioblastoma

PubMed Central

Lin, Chin-Hsing Annie; Rhodes, Christopher T.; Lin, ChenWei; Phillips, Joanna J.; Berger, Mitchel S.

2017-01-01

ABSTRACT Glioblastoma (GBM) is a highly aggressive brain cancer with limited therapeutic options. While efforts to identify genes responsible for GBM have revealed mutations and aberrant gene expression associated with distinct types of GBM, patients with GBM are often diagnosed and classified based on MRI features. Therefore, we seek to identify molecular representatives in parallel with MRI classification for group I and group II primary GBM associated with the subventricular zone (SVZ). As group I and II GBM contain stem-like signature, we compared gene expression profiles between these 2 groups of primary GBM and endogenous neural stem progenitor cells to reveal dysregulation of cell cycle, chromatin status, cellular morphogenesis, and signaling pathways in these 2 types of MRI-classified GBM. In the absence of IDH mutation, several genes associated with metabolism are differentially expressed in these subtypes of primary GBM, implicating metabolic reprogramming occurs in tumor microenvironment. Furthermore, histone lysine methyltransferase EZH2 was upregulated while histone lysine demethylases KDM2 and KDM4 were downregulated in both group I and II primary GBM. Lastly, we identified 9 common genes across large data sets of gene expression profiles among MRI-classified group I/II GBM, a large cohort of GBM subtypes from TCGA, and glioma stem cells by unsupervised clustering comparison. These commonly upregulated genes have known functions in cell cycle, centromere assembly, chromosome segregation, and mitotic progression. Our findings highlight altered expression of genes important in chromosome integrity across all GBM, suggesting a common mechanism of disrupted fidelity of chromosome structure in GBM. PMID:28278055

Genomic Analyses Yield Markers for Identifying Agronomically Important Genes in Potato

USDA-ARS?s Scientific Manuscript database

This study explores the genetic architecture underling the potato evolution through a comprehensive assessment of wild and cultivated potato species based on the re-sequencing of 201 accessions of Solanum section Petota with >12 × genome coverage. We identified 450 domesticated genes, which showed e...

Comparison of a serum indirect fluorescent antibody test with two Western blot tests for the diagnosis of equine protozoal myeloencephalitis.

PubMed

Duarte, Paulo C; Daft, Barbara M; Conrad, Patricia A; Packham, Andrea E; Gardner, Ian A

2003-01-01

A serum indirect fluorescent antibody test (IFAT) was compared with a Western blot (WB) and a modified Western blot (mWB) for diagnosis of equine protozoal myeloencephalitis (EPM). Using receiver-operating characteristic (ROC) analysis, the area under the curve of the IFAT was greater than the areaunder the curves of the WB and the mWB (P = 0.025 and P = 0.044, respectively). There was no statistically significant difference between the areas under the curves of the WBs (P > 0.05). On the basis of an arbitrarily chosen cut-off titer for a positive test result of 1:80 for the IFAT and interpreting weak positive WB results as positive test results, the sensitivities and 95% confidence intervals (CI) of all 3 tests were identical and equal to 88.9% (51.8-99.7%). The specificities and 95% CIs of the IFAT, WB, and mWB test were 100% (91-100%), 87.2% (72.6-95.7%), and 69.2% (52.4-83%), respectively. The overall accuracy of the IFAT was shown to be better than that of the WBs and, therefore, the test has potential for use in the diagnosis of EPM caused by Sarcocystis neurona.

Genomic analyses identify hundreds of variants associated with age at menarche and support a role for puberty timing in cancer risk.

PubMed

Day, Felix R; Thompson, Deborah J; Helgason, Hannes; Chasman, Daniel I; Finucane, Hilary; Sulem, Patrick; Ruth, Katherine S; Whalen, Sean; Sarkar, Abhishek K; Albrecht, Eva; Altmaier, Elisabeth; Amini, Marzyeh; Barbieri, Caterina M; Boutin, Thibaud; Campbell, Archie; Demerath, Ellen; Giri, Ayush; He, Chunyan; Hottenga, Jouke J; Karlsson, Robert; Kolcic, Ivana; Loh, Po-Ru; Lunetta, Kathryn L; Mangino, Massimo; Marco, Brumat; McMahon, George; Medland, Sarah E; Nolte, Ilja M; Noordam, Raymond; Nutile, Teresa; Paternoster, Lavinia; Perjakova, Natalia; Porcu, Eleonora; Rose, Lynda M; Schraut, Katharina E; Segrè, Ayellet V; Smith, Albert V; Stolk, Lisette; Teumer, Alexander; Andrulis, Irene L; Bandinelli, Stefania; Beckmann, Matthias W; Benitez, Javier; Bergmann, Sven; Bochud, Murielle; Boerwinkle, Eric; Bojesen, Stig E; Bolla, Manjeet K; Brand, Judith S; Brauch, Hiltrud; Brenner, Hermann; Broer, Linda; Brüning, Thomas; Buring, Julie E; Campbell, Harry; Catamo, Eulalia; Chanock, Stephen; Chenevix-Trench, Georgia; Corre, Tanguy; Couch, Fergus J; Cousminer, Diana L; Cox, Angela; Crisponi, Laura; Czene, Kamila; Davey Smith, George; de Geus, Eco J C N; de Mutsert, Renée; De Vivo, Immaculata; Dennis, Joe; Devilee, Peter; Dos-Santos-Silva, Isabel; Dunning, Alison M; Eriksson, Johan G; Fasching, Peter A; Fernández-Rhodes, Lindsay; Ferrucci, Luigi; Flesch-Janys, Dieter; Franke, Lude; Gabrielson, Marike; Gandin, Ilaria; Giles, Graham G; Grallert, Harald; Gudbjartsson, Daniel F; Guénel, Pascal; Hall, Per; Hallberg, Emily; Hamann, Ute; Harris, Tamara B; Hartman, Catharina A; Heiss, Gerardo; Hooning, Maartje J; Hopper, John L; Hu, Frank; Hunter, David J; Ikram, M Arfan; Im, Hae Kyung; Järvelin, Marjo-Riitta; Joshi, Peter K; Karasik, David; Kellis, Manolis; Kutalik, Zoltan; LaChance, Genevieve; Lambrechts, Diether; Langenberg, Claudia; Launer, Lenore J; Laven, Joop S E; Lenarduzzi, Stefania; Li, Jingmei; Lind, Penelope A; Lindstrom, Sara; Liu, YongMei; Luan, Jian'an; Mägi, Reedik; Mannermaa, Arto; Mbarek, Hamdi; McCarthy, Mark I; Meisinger, Christa; Meitinger, Thomas; Menni, Cristina; Metspalu, Andres; Michailidou, Kyriaki; Milani, Lili; Milne, Roger L; Montgomery, Grant W; Mulligan, Anna M; Nalls, Mike A; Navarro, Pau; Nevanlinna, Heli; Nyholt, Dale R; Oldehinkel, Albertine J; O'Mara, Tracy A; Padmanabhan, Sandosh; Palotie, Aarno; Pedersen, Nancy; Peters, Annette; Peto, Julian; Pharoah, Paul D P; Pouta, Anneli; Radice, Paolo; Rahman, Iffat; Ring, Susan M; Robino, Antonietta; Rosendaal, Frits R; Rudan, Igor; Rueedi, Rico; Ruggiero, Daniela; Sala, Cinzia F; Schmidt, Marjanka K; Scott, Robert A; Shah, Mitul; Sorice, Rossella; Southey, Melissa C; Sovio, Ulla; Stampfer, Meir; Steri, Maristella; Strauch, Konstantin; Tanaka, Toshiko; Tikkanen, Emmi; Timpson, Nicholas J; Traglia, Michela; Truong, Thérèse; Tyrer, Jonathan P; Uitterlinden, André G; Edwards, Digna R Velez; Vitart, Veronique; Völker, Uwe; Vollenweider, Peter; Wang, Qin; Widen, Elisabeth; van Dijk, Ko Willems; Willemsen, Gonneke; Winqvist, Robert; Wolffenbuttel, Bruce H R; Zhao, Jing Hua; Zoledziewska, Magdalena; Zygmunt, Marek; Alizadeh, Behrooz Z; Boomsma, Dorret I; Ciullo, Marina; Cucca, Francesco; Esko, Tõnu; Franceschini, Nora; Gieger, Christian; Gudnason, Vilmundur; Hayward, Caroline; Kraft, Peter; Lawlor, Debbie A; Magnusson, Patrik K E; Martin, Nicholas G; Mook-Kanamori, Dennis O; Nohr, Ellen A; Polasek, Ozren; Porteous, David; Price, Alkes L; Ridker, Paul M; Snieder, Harold; Spector, Tim D; Stöckl, Doris; Toniolo, Daniela; Ulivi, Sheila; Visser, Jenny A; Völzke, Henry; Wareham, Nicholas J; Wilson, James F; Spurdle, Amanda B; Thorsteindottir, Unnur; Pollard, Katherine S; Easton, Douglas F; Tung, Joyce Y; Chang-Claude, Jenny; Hinds, David; Murray, Anna; Murabito, Joanne M; Stefansson, Kari; Ong, Ken K; Perry, John R B

2017-06-01

The timing of puberty is a highly polygenic childhood trait that is epidemiologically associated with various adult diseases. Using 1000 Genomes Project-imputed genotype data in up to ∼370,000 women, we identify 389 independent signals (P < 5 × 10 -8 ) for age at menarche, a milestone in female pubertal development. In Icelandic data, these signals explain ∼7.4% of the population variance in age at menarche, corresponding to ∼25% of the estimated heritability. We implicate ∼250 genes via coding variation or associated expression, demonstrating significant enrichment in neural tissues. Rare variants near the imprinted genes MKRN3 and DLK1 were identified, exhibiting large effects when paternally inherited. Mendelian randomization analyses suggest causal inverse associations, independent of body mass index (BMI), between puberty timing and risks for breast and endometrial cancers in women and prostate cancer in men. In aggregate, our findings highlight the complexity of the genetic regulation of puberty timing and support causal links with cancer susceptibility.

Genomic analyses identify hundreds of variants associated with age at menarche and support a role for puberty timing in cancer risk

PubMed Central

Day, Felix R; Thompson, Deborah J; Helgason, Hannes; Chasman, Daniel I; Finucane, Hilary; Sulem, Patrick; Ruth, Katherine S; Whalen, Sean; Sarkar, Abhishek K; Albrecht, Eva; Altmaier, Elisabeth; Amini, Marzyeh; Barbieri, Caterina M; Boutin, Thibaud; Campbell, Archie; Demerath, Ellen; Giri, Ayush; He, Chunyan; Hottenga, Jouke J; Karlsson, Robert; Kolcic, Ivana; Loh, Po-Ru; Lunetta, Kathryn L; Mangino, Massimo; Marco, Brumat; McMahon, George; Medland, Sarah E; Nolte, Ilja M; Noordam, Raymond; Nutile, Teresa; Paternoster, Lavinia; Perjakova, Natalia; Porcu, Eleonora; Rose, Lynda M; Schraut, Katharina E; Segrè, Ayellet V; Smith, Albert V; Stolk, Lisette; Teumer, Alexander; Andrulis, Irene L; Bandinelli, Stefania; Beckmann, Matthias W; Benitez, Javier; Bergmann, Sven; Bochud, Murielle; Boerwinkle, Eric; Bojesen, Stig E; Bolla, Manjeet K; Brand, Judith S; Brauch, Hiltrud; Brenner, Hermann; Broer, Linda; Brüning, Thomas; Buring, Julie E; Campbell, Harry; Catamo, Eulalia; Chanock, Stephen; Chenevix-Trench, Georgia; Corre, Tanguy; Couch, Fergus J; Cousminer, Diana L; Cox, Angela; Crisponi, Laura; Czene, Kamila; Smith, George Davey; de Geus, Eco JCN; de Mutsert, Renée; De Vivo, Immaculata; Dennis, Joe; Devilee, Peter; dos-Santos-Silva, Isabel; Dunning, Alison M; Eriksson, Johan G; Fasching, Peter A; Fernández-Rhodes, Lindsay; Ferrucci, Luigi; Flesch-Janys, Dieter; Franke, Lude; Gabrielson, Marike; Gandin, Ilaria; Giles, Graham G; Grallert, Harald; Gudbjartsson, Daniel F; Guénel, Pascal; Hall, Per; Hallberg, Emily; Hamann, Ute; Harris, Tamara B; Hartman, Catharina A; Heiss, Gerardo; Hooning, Maartje J; Hopper, John L; Hu, Frank; Hunter, David J; Ikram, M Arfan; Im, Hae Kyung; Järvelin, Marjo-Riitta; Joshi, Peter K; Karasik, David; Kellis, Manolis; Kutalik, Zoltan; LaChance, Genevieve; Lambrechts, Diether; Langenberg, Claudia; Launer, Lenore J; Laven, Joop S E; Lenarduzzi, Stefania; Li, Jingmei; Lind, Penelope A; Lindstrom, Sara; Liu, YongMei; Luan, Jian’an; Mägi, Reedik; Mannermaa, Arto; Mbarek, Hamdi; McCarthy, Mark I; Meisinger, Christa; Meitinger, Thomas; Menni, Cristina; Metspalu, Andres; Michailidou, Kyriaki; Milani, Lili; Milne, Roger L; Montgomery, Grant W; Mulligan, Anna M; Nalls, Mike A; Navarro, Pau; Nevanlinna, Heli; Nyholt, Dale R; Oldehinkel, Albertine J; O’Mara, Tracy A; Padmanabhan, Sandosh; Palotie, Aarno; Pedersen, Nancy; Peters, Annette; Peto, Julian; Pharoah, Paul D P; Pouta, Anneli; Radice, Paolo; Rahman, Iffat; Ring, Susan M; Robino, Antonietta; Rosendaal, Frits R; Rudan, Igor; Rueedi, Rico; Ruggiero, Daniela; Sala, Cinzia F; Schmidt, Marjanka K; Scott, Robert A; Shah, Mitul; Sorice, Rossella; Southey, Melissa C; Sovio, Ulla; Stampfer, Meir; Steri, Maristella; Strauch, Konstantin; Tanaka, Toshiko; Tikkanen, Emmi; Timpson, Nicholas J; Traglia, Michela; Truong, Thérèse; Tyrer, Jonathan P; Uitterlinden, André G; Velez Edwards, Digna R; Vitart, Veronique; Völker, Uwe; Vollenweider, Peter; Wang, Qin; Widen, Elisabeth; van Dijk, Ko Willems; Willemsen, Gonneke; Winqvist, Robert; Wolffenbuttel, Bruce H R; Zhao, Jing Hua; Zoledziewska, Magdalena; Zygmunt, Marek; Alizadeh, Behrooz Z; Boomsma, Dorret I; Ciullo, Marina; Cucca, Francesco; Esko, Tõnu; Franceschini, Nora; Gieger, Christian; Gudnason, Vilmundur; Hayward, Caroline; Kraft, Peter; Lawlor, Debbie A; Magnusson, Patrik K E; Martin, Nicholas G; Mook-Kanamori, Dennis O; Nohr, Ellen A; Polasek, Ozren; Porteous, David; Price, Alkes L; Ridker, Paul M; Snieder, Harold; Spector, Tim D; Stöckl, Doris; Toniolo, Daniela; Ulivi, Sheila; Visser, Jenny A; Völzke, Henry; Wareham, Nicholas J; Wilson, James F; Spurdle, Amanda B; Thorsteindottir, Unnur; Pollard, Katherine S; Easton, Douglas F; Tung, Joyce Y; Chang-Claude, Jenny; Hinds, David; Murray, Anna; Murabito, Joanne M; Stefansson, Kari; Ong, Ken K; Perry, John R B

2018-01-01

The timing of puberty is a highly polygenic childhood trait that is epidemiologically associated with various adult diseases. Using 1000 Genomes Project–imputed genotype data in up to ~370,000 women, we identify 389 independent signals (P < 5 × 10−8) for age at menarche, a milestone in female pubertal development. In Icelandic data, these signals explain ~7.4% of the population variance in age at menarche, corresponding to ~25% of the estimated heritability. We implicate ~250 genes via coding variation or associated expression, demonstrating significant enrichment in neural tissues. Rare variants near the imprinted genes MKRN3 and DLK1 were identified, exhibiting large effects when paternally inherited. Mendelian randomization analyses suggest causal inverse associations, independent of body mass index (BMI), between puberty timing and risks for breast and endometrial cancers in women and prostate cancer in men. In aggregate, our findings highlight the complexity of the genetic regulation of puberty timing and support causal links with cancer susceptibility. PMID:28436984

Density-based clustering analyses to identify heterogeneous cellular sub-populations

NASA Astrophysics Data System (ADS)

Heaster, Tiffany M.; Walsh, Alex J.; Landman, Bennett A.; Skala, Melissa C.

2017-02-01

Autofluorescence microscopy of NAD(P)H and FAD provides functional metabolic measurements at the single-cell level. Here, density-based clustering algorithms were applied to metabolic autofluorescence measurements to identify cell-level heterogeneity in tumor cell cultures. The performance of the density-based clustering algorithm, DENCLUE, was tested in samples with known heterogeneity (co-cultures of breast carcinoma lines). DENCLUE was found to better represent the distribution of cell clusters compared to Gaussian mixture modeling. Overall, DENCLUE is a promising approach to quantify cell-level heterogeneity, and could be used to understand single cell population dynamics in cancer progression and treatment.

Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.

PubMed

Sørensen, M S; Duch, M; Paludan, K; Jørgensen, P; Pedersen, F S

1992-03-15

Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.

Application of a reverse dot blot, DNA-DNA hydridization method to quantify host-feeding tendencies of two sibling species in the Anopheles gambiae complex

PubMed Central

Fritz, Megan L; Miller, James R; Bayoh, M Nabie; Vulule, John M; Landgraf, Jeffrey R; Walker, Edward D

2012-01-01

A DNA-DNA hybridization method, reverse dot blot analysis (RDBA), was used for identification of Anopheles gambiae s.s. and An. arabiensis hosts. Of 299 blood fed and half gravid An. gambiae s.l. collected from Kisian, Kenya, 244 individuals were identifiable to species; 69.5% were An. arabiensis, and 29.5% were An. gambiae s.s. Host identifications with RDBA were comparable to conventional PCR followed by direct sequencing of amplicons of the vertebrate mitochondrial cytochrome B gene. Of the 174 amplicon-producing samples used for comparison of these two methods, 147 were identifiable by direct sequencing, and 139 of these same by RDBA. An. arabiensis blood meals were mostly (>90%) bovine in origin, whereas An. gambiae s.s. fed upon humans > 90% of the time. RDBA detected that 2 of 112 An. arabiensis had blood from more than one host species, whereas PCR and direct sequencing did not. Recent insecticide-treated bednet (ITN) use in Kisian has likely caused the shift in the dominant vector species from An. gambiae s.s. to An. arabiensis. RDBA provides an opportunity to study changes in host-feeding by members of the An. gambiae complex as a response to the broadening distribution of vector control measures targeting host-selection behaviors. PMID:24188164

Exposure to Sarcocystis spp. in horses from Spain determined by Western blot analysis using Sarcocystis neurona merozoites as heterologous antigen.

PubMed

Arias, M; Yeargan, M; Francisco, I; Dangoudoubiyam, S; Becerra, P; Francisco, R; Sánchez-Andrade, R; Paz-Silva, A; Howe, D K

2012-04-30

Horses serve as an intermediate host for several species of Sarcocystis, all of which utilize canids as the definitive host. Sarcocystis spp. infection and formation of latent sarcocysts in horses often appears to be subclinical, but morbidity can occur, especially when the parasite burden is large. A serological survey was conducted to determine the presence of antibodies against Sarcocystis spp. in seemingly healthy horses from the Galicia region of Spain. Western blot analyses using Sarcocystis neurona merozoites as heterologous antigen suggested greater than 80% seroprevalance of Sarcocystis spp. in a sample set of 138 horses. The serum samples were further tested with enzyme-linked immunosorbent assays (ELISAs) based on recombinant S. neurona-specific surface antigens (rSnSAGs). As expected for horses from the Eastern Hemisphere, less than 4% of the serum samples were positive when analyzed with either the rSnSAG2 or the rSnSAG4/3 ELISAs. An additional 246 horses were tested using the rSnSAG2 ELISA, which revealed that less than 3% of the 384 samples were seropositive. Collectively, the results of this serologic study suggested that a large proportion of horses from this region of Spain are exposed to Sarcocystis spp. Furthermore, the anti-Sarcocystis seroreactivity in these European horses could be clearly distinguished from anti-S. neurona antibodies using the rSnSAG2 and rSnSAG4/3 ELISAs. Copyright © 2011 Elsevier B.V. All rights reserved.

Personalized genomic analyses for cancer mutation discovery and interpretation

PubMed Central

Jones, Siân; Anagnostou, Valsamo; Lytle, Karli; Parpart-Li, Sonya; Nesselbush, Monica; Riley, David R.; Shukla, Manish; Chesnick, Bryan; Kadan, Maura; Papp, Eniko; Galens, Kevin G.; Murphy, Derek; Zhang, Theresa; Kann, Lisa; Sausen, Mark; Angiuoli, Samuel V.; Diaz, Luis A.; Velculescu, Victor E.

2015-01-01

Massively parallel sequencing approaches are beginning to be used clinically to characterize individual patient tumors and to select therapies based on the identified mutations. A major question in these analyses is the extent to which these methods identify clinically actionable alterations and whether the examination of the tumor tissue alone is sufficient or whether matched normal DNA should also be analyzed to accurately identify tumor-specific (somatic) alterations. To address these issues, we comprehensively evaluated 815 tumor-normal paired samples from patients of 15 tumor types. We identified genomic alterations using next-generation sequencing of whole exomes or 111 targeted genes that were validated with sensitivities >95% and >99%, respectively, and specificities >99.99%. These analyses revealed an average of 140 and 4.3 somatic mutations per exome and targeted analysis, respectively. More than 75% of cases had somatic alterations in genes associated with known therapies or current clinical trials. Analyses of matched normal DNA identified germline alterations in cancer-predisposing genes in 3% of patients with apparently sporadic cancers. In contrast, a tumor-only sequencing approach could not definitively identify germline changes in cancer-predisposing genes and led to additional false-positive findings comprising 31% and 65% of alterations identified in targeted and exome analyses, respectively, including in potentially actionable genes. These data suggest that matched tumor-normal sequencing analyses are essential for precise identification and interpretation of somatic and germline alterations and have important implications for the diagnostic and therapeutic management of cancer patients. PMID:25877891

Ugene, a newly identified protein that is commonly over-expressed in cancer, and that binds uracil DNA-glycosylase

PubMed Central

Guo, Chunguang; Zhang, Xiaodong; Fink, Stephen P; Platzer, Petra; Wilson, Keith; Willson, James K. V.; Wang, Zhenghe; Markowitz, Sanford D

2008-01-01

Expression microarrays identified a novel transcript, designated as Ugene, whose expression is absent in normal colon and colon adenomas, but that is commonly induced in malignant colon cancers. These findings were validated by real-time PCR and Northern blot analysis in an independent panel of colon cancer cases. In addition, Ugene expression was found to be elevated in many other common cancer types, including, breast, lung, uterus, and ovary. Immunofluorescence of V5-tagged Ugene revealed it to have a nuclear localization. In a pull-down assay, uracil DNA-glycosylase 2 (UNG2), an important enzyme in the base excision repair pathway, was identified as a partner protein that binds to Ugene. Co-immunoprecipitation and Western blot analysis confirmed the binding between the endogenous Ugene and UNG2 proteins. Using deletion constructs, we find that Ugene binds to the first 25 amino acids of the UNG2 NH2-terminus. We suggest Ugene induction in cancer may contribute to the cancer phenotype by interacting with the base excision repair pathway. PMID:18676834

Identifying patterns of adaptation in breast cancer patients with cancer-related fatigue using response shift analyses at subgroup level.

PubMed

Salmon, Maxime; Blanchin, Myriam; Rotonda, Christine; Guillemin, Francis; Sébille, Véronique

2017-11-01

Fatigue is the most prevalent symptom in breast cancer. It might be perceived differently among patients over time as a consequence of the differing patients' adaptation and psychological adjustment to their cancer experience which can be related to response shift (RS). RS analyses can provide important insights on patients' adaptation to cancer but it is usually assumed that RS occurs in the same way in all individuals which is unrealistic. This study aimed to identify patients' subgroups in which different RS effects on self-reported fatigue could occur over time using a combination of methods for manifest and latent variables. The FATSEIN study comprised 466 breast cancer patients followed over a 2-year period. Fatigue was measured with the Multidimensional Fatigue Inventory questionnaire (MFI-20) during 10 visits. A novel combination of Mixed Models, Growth Mixture Modeling, and Structural Equation Modeling was used to assess the occurrence of RS in fatigue changes to identify subgroups displaying different RS patterns over time. An increase in fatigue was evidenced over the 8-month follow-up, followed by a decrease between the 8- and 24-month. Four latent classes of patients were identified. Different RS patterns were detected in all latent classes between the inclusion and 8 months (last cycle of chemotherapy). No RS was evidenced between 8- and 24-month. Several RS effects were evidenced in different groups of patients. Women seemed to adapt differently to their treatment and breast cancer experience possibly indicating differing needs for medical/psychological support. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Eighteen Years of Molecular Genotyping the Hemophilia Inversion Hotspot: From Southern Blot to Inverse Shifting-PCR

PubMed Central

Rossetti, Liliana C.; Radic, Claudia P.; Abelleyro, Miguel M.; Larripa, Irene B.; De Brasi, Carlos D.

2011-01-01

The factor VIII gene (F8) intron 22 inversion (Inv22) is a paradigmatic duplicon-mediated rearrangement, found in about one half of patients with severe hemophilia A worldwide. The identification of this prevalent cause of hemophilia was delayed for nine years after the F8 characterization in 1984. The aim of this review is to present the wide diversity of practical approaches that have been developed for genotyping the Inv22 (and related int22h rearrangements) since discovery in 1993. The sequence— Southern blot, long distance-PCR and inverse shifting-PCR—for Inv22 genotyping is an interesting example of scientific ingenuity and evolution in order to resolve challenging molecular diagnostic problems. PMID:22072947

Characterization of the groEL and groES loci in Bifidobacterium breve UCC 2003: genetic, transcriptional, and phylogenetic analyses.

PubMed

Ventura, Marco; Canchaya, Carlos; Zink, Ralf; Fitzgerald, Gerald F; van Sinderen, Douwe

2004-10-01

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes, including the GroEL and GroES proteins. The groES and groEL genes are highly conserved among eubacteria and are typically arranged as an operon. Genome analysis of Bifidobacterium breve UCC 2003 revealed that the groES and groEL genes are located in different chromosomal regions. The heat inducibility of the groEL and groES genes of B. breve UCC 2003 was verified by slot blot analysis. Northern blot analyses showed that the cspA gene is cotranscribed with the groEL gene, while the groES gene is transcribed as a monocistronic unit. The transcription initiation sites of these two mRNAs were determined by primer extension. Sequence and transcriptional analyses of the region flanking the groEL and groES genes of various bifidobacteria revealed similar groEL-cspA and groES gene units, suggesting a novel genetic organization of these chaperones. Phylogenetic analysis of the available bifidobacterial groES and groEL genes suggested that these genes evolved differently. Discrepancies in the phylogenetic positioning of groES-based trees make this gene an unreliable molecular marker. On the other hand, the bifidobacterial groEL gene sequences can be used as an alternative to current methods for tracing Bifidobacterium species, particularly because they allow a high level of discrimination between closely related species of this genus.

Estimation of hepatitis E virus (HEV) pig seroprevalence using ELISA and Western blot and comparison between human and pig HEV sequences in Belgium.

PubMed

Thiry, Damien; Mauroy, Axel; Saegerman, Claude; Thomas, Isabelle; Wautier, Magali; Miry, Cora; Czaplicki, Guy; Berkvens, Dirk; Praet, Nicolas; van der Poel, Wim; Cariolet, Roland; Brochier, Bernard; Thiry, Etienne

2014-08-27

Zoonotic transmission of hepatitis E virus (HEV) is of special concern, particularly in high income countries were waterborne infections are less frequent than in developing countries. High HEV seroprevalences can be found in European pig populations. The aims of this study were to obtain prevalence data on HEV infection in swine in Belgium and to phylogenetically compare Belgian human HEV sequences with those obtained from swine. An ELISA screening prevalence of 73% (95% CI 68.8-77.5) was determined in Belgian pigs and a part of the results were re-evaluated by Western blot (WB). A receiver operating characteristic curve analysis was performed and scenarios varying the ELISA specificity relative to WB were analysed. The seroprevalences estimated by the different scenarios ranged between 69 and 81% and are in agreement with the high exposure of the European pig population to HEV. Pig HEV sequences were genetically compared to those detected in humans in Belgium and a predominance of genotype 3 subtype f was shown in both swine and humans. The high HEV seroprevalence in swine and the close phylogenetic relationships between pig and human HEV sequences further support the risk for zoonotic transmission of HEV between humans and pigs. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel isoprenylated proteins identified by an expression library screen.

PubMed

Biermann, B J; Morehead, T A; Tate, S E; Price, J R; Randall, S K; Crowell, D N

1994-10-14

Isoprenylated proteins are involved in eukaryotic cell growth and signal transduction. The protein determinant for prenylation is a short carboxyl-terminal motif containing a cysteine, to which the isoprenoid is covalently attached via thioether linkage. To date, isoprenylated proteins have almost all been identified by demonstrating the attachment of an isoprenoid to previously known proteins. Thus, many isoprenylated proteins probably remain undiscovered. To identify novel isoprenylated proteins for subsequent biochemical study, colony blots of a Glycine max cDNA expression library were [3H]farnesyl-labeled in vitro. Proteins identified by this screen contained several different carboxyl termini that conform to consensus farnesylation motifs. These proteins included known farnesylated proteins (DnaJ homologs) and several novel proteins, two of which contained six or more tandem repeats of a hexapeptide having the consensus sequence (E/G)(G/P)EK(P/K)K. Thus, plants contain a diverse array of genes encoding farnesylated proteins, and our results indicate that fundamental differences in the identities of farnesylated proteins may exist between plants and other eukaryotes. Expression library screening by direct labeling can be adapted to identify isoprenylated proteins from other organisms, as well as proteins with other post-translational modifications.

High throughput microscopy identifies bisphenol AP, a bisphenol A analog, as a novel AR down-regulator.

PubMed

Stossi, Fabio; Dandekar, Radhika D; Bolt, Michael J; Newberg, Justin Y; Mancini, Maureen G; Kaushik, Akash K; Putluri, Vasanta; Sreekumar, Arun; Mancini, Michael A

2016-03-29

Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.

Descriptive and Experimental Analyses of Potential Precursors to Problem Behavior

PubMed Central

Borrero, Carrie S.W; Borrero, John C

2008-01-01

We conducted descriptive observations of severe problem behavior for 2 individuals with autism to identify precursors to problem behavior. Several comparative probability analyses were conducted in addition to lag-sequential analyses using the descriptive data. Results of the descriptive analyses showed that the probability of the potential precursor was greater given problem behavior compared to the unconditional probability of the potential precursor. Results of the lag-sequential analyses showed a marked increase in the probability of a potential precursor in the 1-s intervals immediately preceding an instance of problem behavior, and that the probability of problem behavior was highest in the 1-s intervals immediately following an instance of the precursor. We then conducted separate functional analyses of problem behavior and the precursor to identify respective operant functions. Results of the functional analyses showed that both problem behavior and the precursor served the same operant functions. These results replicate prior experimental analyses on the relation between problem behavior and precursors and extend prior research by illustrating a quantitative method to identify precursors to more severe problem behavior. PMID:18468281

Electron microscopic, genetic and protein expression analyses of Helicobacter acinonychis strains from a Bengal tiger.

PubMed

Tegtmeyer, Nicole; Rivas Traverso, Francisco; Rohde, Manfred; Oyarzabal, Omar A; Lehn, Norbert; Schneider-Brachert, Wulf; Ferrero, Richard L; Fox, James G; Berg, Douglas E; Backert, Steffen

2013-01-01

Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5-6 µm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections.

Electron Microscopic, Genetic and Protein Expression Analyses of Helicobacter acinonychis Strains from a Bengal Tiger

PubMed Central

Tegtmeyer, Nicole; Rivas Traverso, Francisco; Rohde, Manfred; Oyarzabal, Omar A.; Lehn, Norbert; Schneider-Brachert, Wulf; Ferrero, Richard L.; Fox, James G.; Berg, Douglas E.; Backert, Steffen

2013-01-01

Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5–6 µm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections. PMID:23940723

ANALYSES OF RESPONSE–STIMULUS SEQUENCES IN DESCRIPTIVE OBSERVATIONS

PubMed Central

Samaha, Andrew L; Vollmer, Timothy R; Borrero, Carrie; Sloman, Kimberly; Pipkin, Claire St. Peter; Bourret, Jason

2009-01-01

Descriptive observations were conducted to record problem behavior displayed by participants and to record antecedents and consequences delivered by caregivers. Next, functional analyses were conducted to identify reinforcers for problem behavior. Then, using data from the descriptive observations, lag-sequential analyses were conducted to examine changes in the probability of environmental events across time in relation to occurrences of problem behavior. The results of the lag-sequential analyses were interpreted in light of the results of functional analyses. Results suggested that events identified as reinforcers in a functional analysis followed behavior in idiosyncratic ways: after a range of delays and frequencies. Thus, it is possible that naturally occurring reinforcement contingencies are arranged in ways different from those typically evaluated in applied research. Further, these complex response–stimulus relations can be represented by lag-sequential analyses. However, limitations to the lag-sequential analysis are evident. PMID:19949537

The Rural Wings Project: Bridging the Digital Divide with Satellite-Provided Internet. Phase I--Identifying and Analysing the Learning Needs of 31 Communities in 10 Countries

ERIC Educational Resources Information Center

Hansson, Henrik; Mihailidis, Paul; Larsson, Ken; Sotiriou, Menelaos; Sotiriou, Sofoklis; Uzunoglu, Nikolaos; Gargalakos, Michail

2007-01-01

The digitally marginalised communities are in focus in the EU-funded Rural Wings project 2006-2008. The aim is to identify and analyse the user learning needs in non-connected communities and to meet these needs by providing satellite Internet broadband connections, education and tools. This article reports the findings of the user needs…

Clinical application of a rapid method using agarose gel electrophoresis and Western blotting to evaluate von Willebrand factor protease activity.

PubMed

Kirzek, D M; Rick, M E

2001-03-01

A method for evaluating the activity of the von Willebrand factor (vWF) protease is described, and a clinical application is illustrated. The procedure utilizes gel electrophoresis, Western blotting, and luminographic detection methods to evaluate the distribution of vWF multimers before and after incubation of clinical samples under conditions that favor proteolysis by this enzyme. Physiologically, the high-molecular-weight multimers of vWF are cleaved by the vWF protease under conditions of high shear stress in parts of the arterial circulation; cleavage of vWF multimers is also observed after exposure of vWF to denaturing agents in vitro and thus can serve as a laboratory test for the activity of the protease. vWF protease activity is decreased or absent in patients with thrombotic thrombocytopenic purpura due to an inhibiting autoantibody, and this leads to high levels of noncleaved vWF and to life-threatening thrombosis, thrombocytopenia and anemia. The assay evaluates the activity of the protease by assessing the cleavage of vWF multimers after patient plasmas are incubated in vitro under denaturing conditions. With the use of these electrophoresis and Western blotting techniques, patient plasmas can be rapidly assessed for the activity of the vWF protease which may aid in the treatment strategy for these patients.

Comparative Methylome Analyses Identify Epigenetic Regulatory Loci of Human Brain Evolution

PubMed Central

Mendizabal, Isabel; Shi, Lei; Keller, Thomas E.; Konopka, Genevieve; Preuss, Todd M.; Hsieh, Tzung-Fu; Hu, Enzhi; Zhang, Zhe; Su, Bing; Yi, Soojin V.

2016-01-01

How do epigenetic modifications change across species and how do these modifications affect evolution? These are fundamental questions at the forefront of our evolutionary epigenomic understanding. Our previous work investigated human and chimpanzee brain methylomes, but it was limited by the lack of outgroup data which is critical for comparative (epi)genomic studies. Here, we compared whole genome DNA methylation maps from brains of humans, chimpanzees and also rhesus macaques (outgroup) to elucidate DNA methylation changes during human brain evolution. Moreover, we validated that our approach is highly robust by further examining 38 human-specific DMRs using targeted deep genomic and bisulfite sequencing in an independent panel of 37 individuals from five primate species. Our unbiased genome-scan identified human brain differentially methylated regions (DMRs), irrespective of their associations with annotated genes. Remarkably, over half of the newly identified DMRs locate in intergenic regions or gene bodies. Nevertheless, their regulatory potential is on par with those of promoter DMRs. An intriguing observation is that DMRs are enriched in active chromatin loops, suggesting human-specific evolutionary remodeling at a higher-order chromatin structure. These findings indicate that there is substantial reprogramming of epigenomic landscapes during human brain evolution involving noncoding regions. PMID:27563052

Integrating multiple ‘omics’ analyses identifies serological protein biomarkers for preeclampsia

PubMed Central

2013-01-01

Background Preeclampsia (PE) is a pregnancy-related vascular disorder which is the leading cause of maternal morbidity and mortality. We sought to identify novel serological protein markers to diagnose PE with a multi-’omics’ based discovery approach. Methods Seven previous placental expression studies were combined for a multiplex analysis, and in parallel, two-dimensional gel electrophoresis was performed to compare serum proteomes in PE and control subjects. The combined biomarker candidates were validated with available ELISA assays using gestational age-matched PE (n=32) and control (n=32) samples. With the validated biomarkers, a genetic algorithm was then used to construct and optimize biomarker panels in PE assessment. Results In addition to the previously identified biomarkers, the angiogenic and antiangiogenic factors (soluble fms-like tyrosine kinase (sFlt-1) and placental growth factor (PIGF)), we found 3 up-regulated and 6 down-regulated biomakers in PE sera. Two optimal biomarker panels were developed for early and late onset PE assessment, respectively. Conclusions Both early and late onset PE diagnostic panels, constructed with our PE biomarkers, were superior over sFlt-1/PIGF ratio in PE discrimination. The functional significance of these PE biomarkers and their associated pathways were analyzed which may provide new insights into the pathogenesis of PE. PMID:24195779

A qualitative method for analysing multivoicedness

PubMed Central

Aveling, Emma-Louise; Gillespie, Alex; Cornish, Flora

2015-01-01

‘Multivoicedness’ and the ‘multivoiced Self’ have become important theoretical concepts guiding research. Drawing on the tradition of dialogism, the Self is conceptualised as being constituted by a multiplicity of dynamic, interacting voices. Despite the growth in literature and empirical research, there remains a paucity of established methodological tools for analysing the multivoiced Self using qualitative data. In this article, we set out a systematic, practical ‘how-to’ guide for analysing multivoicedness. Using theoretically derived tools, our three-step method comprises: identifying the voices of I-positions within the Self’s talk (or text), identifying the voices of ‘inner-Others’, and examining the dialogue and relationships between the different voices. We elaborate each step and illustrate our method using examples from a published paper in which data were analysed using this method. We conclude by offering more general principles for the use of the method and discussing potential applications. PMID:26664292

Analyses of Sporocarps, Morphotyped Ectomycorrhizae, Environmental ITS and LSU Sequences Identify Common Genera that Occur at a Periglacial Site

PubMed Central

Jumpponen, Ari; Brown, Shawn P.; Trappe, James M.; Cázares, Efrén; Strömmer, Rauni

2015-01-01

Periglacial substrates exposed by retreating glaciers represent extreme and sensitive environments defined by a variety of abiotic stressors that challenge organismal establishment and survival. The simple communities often residing at these sites enable their analyses in depth. We utilized existing data and mined published sporocarp, morphotyped ectomycorrhizae (ECM), as well as environmental sequence data of internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal RNA gene to identify taxa that occur at a glacier forefront in the North Cascades Mountains in Washington State in the USA. The discrete data types consistently identified several common and widely distributed genera, perhaps best exemplified by Inocybe and Laccaria. Although we expected low diversity and richness, our environmental sequence data included 37 ITS and 26 LSU operational taxonomic units (OTUs) that likely form ECM. While environmental surveys of metabarcode markers detected large numbers of targeted ECM taxa, both the fruiting body and the morphotype datasets included genera that were undetected in either of the metabarcode datasets. These included hypogeous (Hymenogaster) and epigeous (Lactarius) taxa, some of which may produce large sporocarps but may possess small and/or spatially patchy genets. We highlight the importance of combining various data types to provide a comprehensive view of a fungal community, even in an environment assumed to host communities of low species richness and diversity. PMID:29376900

Reducing Missed Laboratory Results: Defining Temporal Responsibility, Generating User Interfaces for Test Process Tracking, and Retrospective Analyses to Identify Problems

PubMed Central

Tarkan, Sureyya; Plaisant, Catherine; Shneiderman, Ben; Hettinger, A. Zachary

2011-01-01

Researchers have conducted numerous case studies reporting the details on how laboratory test results of patients were missed by the ordering medical providers. Given the importance of timely test results in an outpatient setting, there is limited discussion of electronic versions of test result management tools to help clinicians and medical staff with this complex process. This paper presents three ideas to reduce missed results with a system that facilitates tracking laboratory tests from order to completion as well as during follow-up: (1) define a workflow management model that clarifies responsible agents and associated time frame, (2) generate a user interface for tracking that could eventually be integrated into current electronic health record (EHR) systems, (3) help identify common problems in past orders through retrospective analyses. PMID:22195201

Patterns of Limnohabitans Microdiversity across a Large Set of Freshwater Habitats as Revealed by Reverse Line Blot Hybridization

PubMed Central

Jezbera, Jan; Jezberová, Jitka; Kasalický, Vojtěch; Šimek, Karel; Hahn, Martin W.

2013-01-01

Among abundant freshwater Betaproteobacteria, only few groups are considered to be of central ecological importance. One of them is the well-studied genus Limnohabitans and mainly its R-BT subcluster, investigated previously mainly by fluorescence in situ hybridization methods. We designed, based on sequences from a large Limnohabitans culture collection, 18 RLBH (Reverse Line Blot Hybridization) probes specific for different groups within the genus Limnohabitans by targeting diagnostic sequences on their 16 S–23 S rRNA ITS regions. The developed probes covered in sum 92% of the available isolates. This set of probes was applied to environmental DNA originating from 161 different European standing freshwater habitats to reveal the microdiversity (intra-genus) patterns of the Limnohabitans genus along a pH gradient. Investigated habitats differed in various physicochemical parameters, and represented a very broad range of standing freshwater habitats. The Limnohabitans microdiversity, assessed as number of RLBH-defined groups detected, increased significantly along the gradient of rising pH of habitats. 14 out of 18 probes returned detection signals that allowed predictions on the distribution of distinct Limnohabitans groups. Most probe-defined Limnohabitans groups showed preferences for alkaline habitats, one for acidic, and some seemed to lack preferences. Complete niche-separation was indicated for some of the probe-targeted groups. Moreover, bimodal distributions observed for some groups of Limnohabitans, suggested further niche separation between genotypes within the same probe-defined group. Statistical analyses suggested that different environmental parameters such as pH, conductivity, oxygen and altitude influenced the distribution of distinct groups. The results of our study do not support the hypothesis that the wide ecological distribution of Limnohabitans bacteria in standing freshwater habitats results from generalist adaptations of these bacteria

SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence.

PubMed

Mazzatti, Dawn J; Pawelec, Graham; Longdin, Robin; Powell, Jonathan R; Forsey, Rosalyn J

2007-06-05

The adaptive immune response requires waves of T-cell clonal expansion on contact with pathogen and elimination after clearance of the source of antigen. However, lifelong persistent infections with common viruses cause chronic antigenic stimulation which takes its toll on adaptive immunity in late life. Chronic antigenic stress results in deregulation of the T-cell response and accumulation of anergic cells. Longitudinal studies of the elderly show that this impacts on survival. Identifying the nature of the defects in chronically-stimulated T-cells and protein bio-markers of these dysfunctional cells would help to understand age-associated compromised T-cell function (immunosenescence) and facilitate the development of targeted intervention strategies.The purpose of this work was to use surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to analyse proteins associated with T-cell senescence in order to identify potential bio-markers. Clonal populations of T-cells isolated from elderly octogenarian and centenarian donors were grown in vitro until senescence, and early passage and late passage (pre-senescent) cells were analysed using SELDI-TOF-MS ProteinChip arrays. Discriminant analysis identified several protein or peptide peaks in the region of 14.5-16.5 kDa that were associated with T-cell clone senescence. Human profilin-1, a ubiquitous protein associated with actin remodelling and cellular motility was unambiguously identified. Altered expression of profilin-1 in senescent T-cell clones was confirmed by Western blot analysis. Due to the proposed roles of profilin-1 in cellular survival, cytoskeleton remodelling, motility, and proliferation, it is hypothesised that differential expression of profilin-1 in ageing may contribute directly to immunosenescence.

Comparative Methylome Analyses Identify Epigenetic Regulatory Loci of Human Brain Evolution.

PubMed

Mendizabal, Isabel; Shi, Lei; Keller, Thomas E; Konopka, Genevieve; Preuss, Todd M; Hsieh, Tzung-Fu; Hu, Enzhi; Zhang, Zhe; Su, Bing; Yi, Soojin V

2016-11-01

How do epigenetic modifications change across species and how do these modifications affect evolution? These are fundamental questions at the forefront of our evolutionary epigenomic understanding. Our previous work investigated human and chimpanzee brain methylomes, but it was limited by the lack of outgroup data which is critical for comparative (epi)genomic studies. Here, we compared whole genome DNA methylation maps from brains of humans, chimpanzees and also rhesus macaques (outgroup) to elucidate DNA methylation changes during human brain evolution. Moreover, we validated that our approach is highly robust by further examining 38 human-specific DMRs using targeted deep genomic and bisulfite sequencing in an independent panel of 37 individuals from five primate species. Our unbiased genome-scan identified human brain differentially methylated regions (DMRs), irrespective of their associations with annotated genes. Remarkably, over half of the newly identified DMRs locate in intergenic regions or gene bodies. Nevertheless, their regulatory potential is on par with those of promoter DMRs. An intriguing observation is that DMRs are enriched in active chromatin loops, suggesting human-specific evolutionary remodeling at a higher-order chromatin structure. These findings indicate that there is substantial reprogramming of epigenomic landscapes during human brain evolution involving noncoding regions. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes.

PubMed

Lin, J C; Pagano, J S

1986-08-01

A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.

Epitope presentation is an important determinant of the utility of antigens identified from protein arrays in the development of autoantibody diagnostic assays.

PubMed

Murphy, Mairead A; O'Connell, David J; O'Kane, Sara L; O'Brien, John K; O'Toole, Sharon; Martin, Cara; Sheils, Orla; O'Leary, John J; Cahill, Dolores J

2012-08-03

Autoantibodies represent an attractive biomarker for diagnostic assays principally due to the stability of immunoglobulin in patient serum facilitating measurement with conventional assays. Immune responses to tumorigenesis may facilitate detection of ovarian cancer in the early stages of the disease with identification of a panel of tumour specific autoantibodies. Despite the reporting of many tumour associated autoantibodies using arrays of tumour antigens, this has not led to the advance in diagnostic capability as rapidly as was initially expected. Here we examine the potential diagnostic utility of candidate autoantibody biomarkers identified via screening of serum samples on a high content human protein array from a unique cohort of early stage and late stage ovarian cancer patients. We analyse the performance of autoantibodies to the tumour suppressor protein p53 and the novel autoantigens alpha adducin and endosulfine alpha identified in our array screen. Each antigen has different performance characteristics using conventional ELISA format and Western blot immunoassay. The high attrition rate of promising autoantigens identified by array screening can in part be explained by the presentation of the epitope of the antigen in the subsequent method of validation and this study provides directions on maximising the potential of candidate biomarkers. This article is part of a Special Issue entitled: Translational Proteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

[Western Blot diagnostic yield for simultaneous antibody-detection in patients with human cysticercosis, hydatidosis, and human fascioliasis].

PubMed

Davelois, Kelly; Escalante, Hermes; Jara, César

2016-01-01

. To determine the diagnostic yield using western blotting to simultaneously detect antibodies in patients with human cysticercosis, hydatidosis, and human fascioliasis. Materials and methods . Cross-sectional study of diagnostic yield assessment. Excretory/secretory antigens were obtained from Taenia solium larvae, Echinococcus granulosus cysts, and the adult flukes of Fasciola hepática, which were then separated using the polyacrylamide gel electrophoresis technique, transferred, and attached to a nitrocellulose membrane to be probed with sera from the patient infected with the three parasites. The sensitivity of the technique was assessed using 300 individual serum samples, 60 pools of two parasites, and 20 pools of three parasites with 75 sera from patients with other parasites, 10 from patients with other diseases, and 15 from patients without parasites. Results . The technique revealed 13 glycoproteins (GP): GP 35, 31, 24, 23, 18, 17, 14, and 13 kDa for cysticercosis; GP 8, 16, and 21 kDa for hydatidosis; and GP 17 and 23 kDa for fascioliasis. The test detected the presence of antibodies with a sensitivity of 96% (95% confidence interval [CI] = 94.62-98.54%) in the detection of one or the thirteen bands, a specificity of 100% (95% CI = 99.50-100.00%); individually, there was a sensitivity for cysticercosis of 97% (95% CI = 93.16-100.00%), for hydatidosis of 94% (95% CI = 88.85-99.15%) and for fascioliasis of 96% (95% CI = 91.66-100.00%). Conclusions . Western blotting is effective in the simultaneous detection of antibodies in patients with human cysticercosis, hydatidosis, and fascioliasis, and it can be used as a diagnostic test to either rule out or confirm the presence of antibodies in endemic areas.

Label-free DNA quantification via a 'pipette, aggregate and blot' (PAB) approach with magnetic silica particles on filter paper.

PubMed

Li, Jingyi; Liu, Qian; Alsamarri, Hussein; Lounsbury, Jenny A; Haversitick, Doris M; Landers, James P

2013-03-07

Reliable measurement of DNA concentration is essential for a broad range of applications in biology and molecular biology, and for many of these, quantifying the nucleic acid content is inextricably linked to obtaining optimal results. In its most simplistic form, quantitative analysis of nucleic acids can be accomplished by UV-Vis absorbance and, in more sophisticated format, by fluorimetry. A recently reported new concept, the 'pinwheel assay', involves a label-free approach for quantifying DNA through aggregation of paramagnetic beads in a rotating magnetic field. Here, we describe a simplified version of that assay adapted for execution using only a pipet and filter paper. The 'pipette, aggregate, and blot' (PAB) approach allows DNA to induce bead aggregation in a pipette tip through exposure to a magnetic field, followed by dispensing (blotting) onto filter paper. The filter paper immortalises the extent of aggregation, and digital images of the immortalized bead conformation, acquired with either a document scanner or a cell phone camera, allows for DNA quantification using a noncomplex algorithm. Human genomic DNA samples extracted from blood are quantified with the PAB approach and the results utilized to define the volume of sample used in a PCR reaction that is sensitive to input mass of template DNA. Integrating the PAB assay with paper-based DNA extraction and detection modalities has the potential to yield 'DNA quant-on-paper' devices that may be useful for point-of-care testing.

Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues.

PubMed

Banks, Rosamonde E; Craven, Rachel A; Harnden, Patricia A; Selby, Peter J

2003-04-01

Western blotting remains a central technique in confirming identities of proteins, their quantitation and analysis of various isoforms. The biotin-avidin/streptavidin system is often used as an amplification step to increase sensitivity but in some tissues such as kidney, "nonspecific" interactions may be a problem due to high levels of endogenous biotin-containing proteins. The EnVision system, developed for immunohistochemical applications, relies on binding of a polymeric conjugate consisting of up to 100 peroxidase molecules and 20 secondary antibody molecules linked directly to an activated dextran backbone, to the primary antibody. This study demonstrates that it is also a viable and sensitive alternative detection system in Western blotting applications.

Genome-Wide Association Analyses in 128,266 Individuals Identifies New Morningness and Sleep Duration Loci.

PubMed

Jones, Samuel E; Tyrrell, Jessica; Wood, Andrew R; Beaumont, Robin N; Ruth, Katherine S; Tuke, Marcus A; Yaghootkar, Hanieh; Hu, Youna; Teder-Laving, Maris; Hayward, Caroline; Roenneberg, Till; Wilson, James F; Del Greco, Fabiola; Hicks, Andrew A; Shin, Chol; Yun, Chang-Ho; Lee, Seung Ku; Metspalu, Andres; Byrne, Enda M; Gehrman, Philip R; Tiemeier, Henning; Allebrandt, Karla V; Freathy, Rachel M; Murray, Anna; Hinds, David A; Frayling, Timothy M; Weedon, Michael N

2016-08-01

Disrupted circadian rhythms and reduced sleep duration are associated with several human diseases, particularly obesity and type 2 diabetes, but until recently, little was known about the genetic factors influencing these heritable traits. We performed genome-wide association studies of self-reported chronotype (morning/evening person) and self-reported sleep duration in 128,266 white British individuals from the UK Biobank study. Sixteen variants were associated with chronotype (P<5x10-8), including variants near the known circadian rhythm genes RGS16 (1.21 odds of morningness, 95% CI [1.15, 1.27], P = 3x10-12) and PER2 (1.09 odds of morningness, 95% CI [1.06, 1.12], P = 4x10-10). The PER2 signal has previously been associated with iris function. We sought replication using self-reported data from 89,283 23andMe participants; thirteen of the chronotype signals remained associated at P<5x10-8 on meta-analysis and eleven of these reached P<0.05 in the same direction in the 23andMe study. We also replicated 9 additional variants identified when the 23andMe study was used as a discovery GWAS of chronotype (all P<0.05 and meta-analysis P<5x10-8). For sleep duration, we replicated one known signal in PAX8 (2.6 minutes per allele, 95% CI [1.9, 3.2], P = 5.7x10-16) and identified and replicated two novel associations at VRK2 (2.0 minutes per allele, 95% CI [1.3, 2.7], P = 1.2x10-9; and 1.6 minutes per allele, 95% CI [1.1, 2.2], P = 7.6x10-9). Although we found genetic correlation between chronotype and BMI (rG = 0.056, P = 0.05); undersleeping and BMI (rG = 0.147, P = 1x10-5) and oversleeping and BMI (rG = 0.097, P = 0.04), Mendelian Randomisation analyses, with limited power, provided no consistent evidence of causal associations between BMI or type 2 diabetes and chronotype or sleep duration. Our study brings the total number of loci associated with chronotype to 22 and with sleep duration to three, and provides new insights into the biology of sleep and circadian

Myostatin inhibitors in sports drug testing: Detection of myostatin-neutralizing antibodies in plasma/serum by affinity purification and Western blotting.

PubMed

Walpurgis, Katja; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

2016-02-01

Myostatin is a key regulator of skeletal muscle growth and inhibition of its signaling pathway results in an increased muscle mass and function. The aim of this study was to develop a qualitative detection assay for myostatin-neutralizing antibodies for doping control purposes by using immunological approaches. To detect different types of myostatin-neutralizing antibodies irrespective of their amino acid sequence, an immunological assay specific for antibodies directed against myostatin and having a human Fc domain was established. Affinity purification and Western blotting strategies were combined to allow extracting and identifying relevant analytes from 200 μL of plasma/serum in a non-targeted approach. The assay was characterized regarding specificity, linearity, precision, robustness, and recovery. The assay was found to be highly specific, robust, and linear from 0.1 to 1 μg/mL. The precision was successfully specified at three different concentrations and the recovery of the affinity purification was 58%. Within this study, an immunological detection assay for myostatin-neutralizing antibodies present in plasma/serum specimens was developed and successfully characterized. The presented approach can easily be modified to include other therapeutic antibodies and serves as proof-of-concept for the detection of antibody-based myostatin inhibitors in doping control samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

HPLC-MS and GC-MS analyses combined with orthogonal partial least squares to identify cytotoxic constituents from turmeric (Curcuma longa L.).

PubMed

Jiang, Jianlan; Zhang, Huan; Li, Zidan; Zhang, Xiaohang; Su, Xin; Li, Yan; Qiao, Bin; Yuan, Yingjin

2013-08-01

We investigated the fingerprints of 48 batches of turmeric total extracts (TTE) by HPLC-MS-MS and GC-MS analyses and 43 characteristic peaks (22 constituents from HPLC-MS-MS; 21 from GC-MS) were analyzed qualitatively and quantitatively. An MTT {3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide} assay was implemented to measure the cytotoxicity of the TTE against HeLa cells. Then we utilized orthogonal partial least squares analysis, which correlated the chemical composition of the TTE to its cytotoxic activity, to identify potential cytotoxic constituents from turmeric. The result showed that 19 constituents contributed significantly to the cytotoxicity. The obtained result was verified by canonical correlation analysis. Comparison with previous reports also indicated some interaction between the curcuminoids and sesquiterpenoids in turmeric.

A research design for the quantification of the neuropeptides substance p and calcitonin gene-related Peptide in rat skin using Western blot analysis.

PubMed

Lapin, Guilherme Abbud Franco; Hochman, Bernardo; Nishioka, Michele Akemi; Maximino, Jessica Ruivo; Chadi, Gerson; Ferreira, Lydia Masako

2015-06-01

To describe and standardize a protocol that overcomes the technical limitations of Western blot (WB) analysis in the quantification of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) following nociceptive stimuli in rat skin. Male Wistar rats (Rattus norvegicus albinus) weighing 250 to 350 g were used in this study. Elements of WB analysis were adapted by using specific manipulation of samples, repeated cycles of freezing and thawing, more thorough maceration, and a more potent homogenizer; increasing lytic reagents; promoting greater inhibition of protease activity; and using polyvinylidene fluoride membranes as transfer means for skin-specific protein. Other changes were also made to adapt the WB analysis to a rat model. University research center. Western blot analysis adapted to a rat model. This research design has proven effective in collecting and preparing skin samples to quantify SP and CGRP using WB analysis in rat skin. This study described a research design that uses WB analysis as a reproducible, technically accessible, and cost-effective method for the quantification of SP and CGRP in rat skin that overcomes technical biases.

Application of FTA sample collection and DNA purification system on the determination of CTG trinucleotide repeat size by PCR-based Southern blotting.

PubMed

Hsiao, K M; Lin, H M; Pan, H; Li, T C; Chen, S S; Jou, S B; Chiu, Y L; Wu, M F; Lin, C C; Li, S Y

1999-01-01

Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein kinase gene. The clinical severity of this disease correlates with the length of the CTG trinucleotide repeats. Determination of the CTG repeat length has been primarily relied on by Southern blot analysis of restriction enzyme-digested genomic DNA. The development of PCR-based Southern blotting methodology provides a much more sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the use of PCR on the diagnosis of DM. A modified PCR protocol to amplify different lengths of CTG repeat region using various concentrations of 7deaza-dGTP has been reported (1). Here we describe a procedure including sample collection, DNA purification, and PCR analysis of CTG repeat length without using 7-deaza-dGTP. This protocol is very sensitive and convenient because only a small number of nucleate cells are needed for detection of CTG expansion. Therefore, it could be very useful in clinical and prenatal diagnosis as well as in prevalence study of DM.

Western blotting method (TESAcruzi) as a supplemental test for confirming the presence of anti-Trypanosoma cruzi antibodies in finger prick blood samples from children aged 0-5 years in Brazil.

PubMed

Frade, Amanda Farage; Luquetti, Alejandro O; Prata, Aluísio; Ferreira, Antonio Walter

2011-01-01

Some Latin American countries have plans for total control and/or eradication of Chagas disease by the main vector (Triatoma infestans) and by blood transfusion. To achieve this, patients with Chagas disease must be identified. A Western blotting test, TESAcruzi, is described as a supplemental test for diagnosis of Chagas disease using samples collected from children <5 years living in different states of Brazil. Blood samples collected by finger prick on filter paper were sent to the test laboratory by a central laboratory to confirm results obtained previously. Ten percent of negative samples, all doubtful and all positive samples were received. Commercial reagents, IgG indirect immunofluorescence, enzyme immunoassay, and a recently introduced TESAcruzi test were used. From 8788 samples, 163 (1.85%) were reactive by IgG-ELISA and 312 (3.55%) by IgG IIF. From these, 77 (0.87%) were reactive in the TESAcruzi test. The results had high clinical value to identify those truly infected. Copyright © 2010 Elsevier B.V. All rights reserved.

Analyses of Response-Stimulus Sequences in Descriptive Observations

ERIC Educational Resources Information Center

Samaha, Andrew L.; Vollmer, Timothy R.; Borrero, Carrie; Sloman, Kimberly; Pipkin, Claire St. Peter; Bourret, Jason

2009-01-01

Descriptive observations were conducted to record problem behavior displayed by participants and to record antecedents and consequences delivered by caregivers. Next, functional analyses were conducted to identify reinforcers for problem behavior. Then, using data from the descriptive observations, lag-sequential analyses were conducted to examine…

Association analyses of East Asian individuals and trans-ancestry analyses with European individuals reveal new loci associated with cholesterol and triglyceride levels.

PubMed

Spracklen, Cassandra N; Chen, Peng; Kim, Young Jin; Wang, Xu; Cai, Hui; Li, Shengxu; Long, Jirong; Wu, Ying; Wang, Ya Xing; Takeuchi, Fumihiko; Wu, Jer-Yuarn; Jung, Keum-Ji; Hu, Cheng; Akiyama, Koichi; Zhang, Yonghong; Moon, Sanghoon; Johnson, Todd A; Li, Huaixing; Dorajoo, Rajkumar; He, Meian; Cannon, Maren E; Roman, Tamara S; Salfati, Elias; Lin, Keng-Hung; Guo, Xiuqing; Sheu, Wayne H H; Absher, Devin; Adair, Linda S; Assimes, Themistocles L; Aung, Tin; Cai, Qiuyin; Chang, Li-Ching; Chen, Chien-Hsiun; Chien, Li-Hsin; Chuang, Lee-Ming; Chuang, Shu-Chun; Du, Shufa; Fan, Qiao; Fann, Cathy S J; Feranil, Alan B; Friedlander, Yechiel; Gordon-Larsen, Penny; Gu, Dongfeng; Gui, Lixuan; Guo, Zhirong; Heng, Chew-Kiat; Hixson, James; Hou, Xuhong; Hsiung, Chao Agnes; Hu, Yao; Hwang, Mi Yeong; Hwu, Chii-Min; Isono, Masato; Juang, Jyh-Ming Jimmy; Khor, Chiea-Chuen; Kim, Yun Kyoung; Koh, Woon-Puay; Kubo, Michiaki; Lee, I-Te; Lee, Sun-Ju; Lee, Wen-Jane; Liang, Kae-Woei; Lim, Blanche; Lim, Sing-Hui; Liu, Jianjun; Nabika, Toru; Pan, Wen-Harn; Peng, Hao; Quertermous, Thomas; Sabanayagam, Charumathi; Sandow, Kevin; Shi, Jinxiu; Sun, Liang; Tan, Pok Chien; Tan, Shu-Pei; Taylor, Kent D; Teo, Yik-Ying; Toh, Sue-Anne; Tsunoda, Tatsuhiko; van Dam, Rob M; Wang, Aili; Wang, Feijie; Wang, Jie; Wei, Wen Bin; Xiang, Yong-Bing; Yao, Jie; Yuan, Jian-Min; Zhang, Rong; Zhao, Wanting; Chen, Yii-Der Ida; Rich, Stephen S; Rotter, Jerome I; Wang, Tzung-Dau; Wu, Tangchun; Lin, Xu; Han, Bok-Ghee; Tanaka, Toshihiro; Cho, Yoon Shin; Katsuya, Tomohiro; Jia, Weiping; Jee, Sun-Ha; Chen, Yuan-Tsong; Kato, Norihiro; Jonas, Jost B; Cheng, Ching-Yu; Shu, Xiao-Ou; He, Jiang; Zheng, Wei; Wong, Tien-Yin; Huang, Wei; Kim, Bong-Jo; Tai, E-Shyong; Mohlke, Karen L; Sim, Xueling

2017-05-01

Large-scale meta-analyses of genome-wide association studies (GWAS) have identified >175 loci associated with fasting cholesterol levels, including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG). With differences in linkage disequilibrium (LD) structure and allele frequencies between ancestry groups, studies in additional large samples may detect new associations. We conducted staged GWAS meta-analyses in up to 69,414 East Asian individuals from 24 studies with participants from Japan, the Philippines, Korea, China, Singapore, and Taiwan. These meta-analyses identified (P < 5 × 10-8) three novel loci associated with HDL-C near CD163-APOBEC1 (P = 7.4 × 10-9), NCOA2 (P = 1.6 × 10-8), and NID2-PTGDR (P = 4.2 × 10-8), and one novel locus associated with TG near WDR11-FGFR2 (P = 2.7 × 10-10). Conditional analyses identified a second signal near CD163-APOBEC1. We then combined results from the East Asian meta-analysis with association results from up to 187,365 European individuals from the Global Lipids Genetics Consortium in a trans-ancestry meta-analysis. This analysis identified (log10Bayes Factor ≥6.1) eight additional novel lipid loci. Among the twelve total loci identified, the index variants at eight loci have demonstrated at least nominal significance with other metabolic traits in prior studies, and two loci exhibited coincident eQTLs (P < 1 × 10-5) in subcutaneous adipose tissue for BPTF and PDGFC. Taken together, these analyses identified multiple novel lipid loci, providing new potential therapeutic targets. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Genomic and Transcriptomic Analyses to Identify Pathways Involved in Nanoparticle Generation in the Ubiquitous Marine Bacterium Alteromonas macleodii Under Elevated Copper Conditions

NASA Astrophysics Data System (ADS)

Cusick, K. D.; Dale, J.; Little, B.; Cockrell, A.; Biffinger, J.

2016-02-01

Alteromonas macleodii is a ubiquitous marine bacterium that clusters by molecular analyses into two ecotypes: surface and deep-water. Our group isolated a marine bacterium from copper coupons that generates nanoparticles (NPs) at elevated copper concentrations. Sequencing of the 16S rRNA gene identified it as an A. macleodii strain. In phylogenetic analyses based on the gyrB gene, it clustered with other surface isolates; however, it formed a unique cluster separate from that of other surface isolates based on rpoB gene sequences. Copper is commonly employed as an antifouling agent on the hulls of ships, and so copper tolerance and NP generation is under investigation in this strain. The overall goals of this study were: (1) to determine if copper tolerance is the result of changes at the genetic or transcriptional level and (2) to identify the genes involved in NP formation. Sub-cultures were established from the initial isolate in which copper concentrations were increased in .25 mM increments through multiple generations. These sub-cultures were assayed for NP formation in seawater medium supplemented with 3-4 mM copper. Scanning electron microscopy revealed large aggregates of NPs on the exterior surface of all sub-cultures. Additionally, a portion of the cells in all sub-cultures displayed an elongated morphology in comparison to the wild-type. No NPs were observed in wild-type controls grown without the addition of increased copper. Metagenomic sequencing of natural populations of A. macleodii revealed extreme divergence in several large genomic regions whose content includes genes coding for exopolysaccharide production and metal resistance. High-throughput sequencing is being used to determine whether copper tolerance and NP generation is the result of genetic or transcriptional changes. These results will be extended to natural communities to gain insights into the role of bacterial NPs during conditions of elevated metal concentrations in coastal systems.

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene.

PubMed

Nicolas, Aude; Kenna, Kevin P; Renton, Alan E; Ticozzi, Nicola; Faghri, Faraz; Chia, Ruth; Dominov, Janice A; Kenna, Brendan J; Nalls, Mike A; Keagle, Pamela; Rivera, Alberto M; van Rheenen, Wouter; Murphy, Natalie A; van Vugt, Joke J F A; Geiger, Joshua T; Van der Spek, Rick A; Pliner, Hannah A; Shankaracharya; Smith, Bradley N; Marangi, Giuseppe; Topp, Simon D; Abramzon, Yevgeniya; Gkazi, Athina Soragia; Eicher, John D; Kenna, Aoife; Mora, Gabriele; Calvo, Andrea; Mazzini, Letizia; Riva, Nilo; Mandrioli, Jessica; Caponnetto, Claudia; Battistini, Stefania; Volanti, Paolo; La Bella, Vincenzo; Conforti, Francesca L; Borghero, Giuseppe; Messina, Sonia; Simone, Isabella L; Trojsi, Francesca; Salvi, Fabrizio; Logullo, Francesco O; D'Alfonso, Sandra; Corrado, Lucia; Capasso, Margherita; Ferrucci, Luigi; Moreno, Cristiane de Araujo Martins; Kamalakaran, Sitharthan; Goldstein, David B; Gitler, Aaron D; Harris, Tim; Myers, Richard M; Phatnani, Hemali; Musunuri, Rajeeva Lochan; Evani, Uday Shankar; Abhyankar, Avinash; Zody, Michael C; Kaye, Julia; Finkbeiner, Steven; Wyman, Stacia K; LeNail, Alex; Lima, Leandro; Fraenkel, Ernest; Svendsen, Clive N; Thompson, Leslie M; Van Eyk, Jennifer E; Berry, James D; Miller, Timothy M; Kolb, Stephen J; Cudkowicz, Merit; Baxi, Emily; Benatar, Michael; Taylor, J Paul; Rampersaud, Evadnie; Wu, Gang; Wuu, Joanne; Lauria, Giuseppe; Verde, Federico; Fogh, Isabella; Tiloca, Cinzia; Comi, Giacomo P; Sorarù, Gianni; Cereda, Cristina; Corcia, Philippe; Laaksovirta, Hannu; Myllykangas, Liisa; Jansson, Lilja; Valori, Miko; Ealing, John; Hamdalla, Hisham; Rollinson, Sara; Pickering-Brown, Stuart; Orrell, Richard W; Sidle, Katie C; Malaspina, Andrea; Hardy, John; Singleton, Andrew B; Johnson, Janel O; Arepalli, Sampath; Sapp, Peter C; McKenna-Yasek, Diane; Polak, Meraida; Asress, Seneshaw; Al-Sarraj, Safa; King, Andrew; Troakes, Claire; Vance, Caroline; de Belleroche, Jacqueline; Baas, Frank; Ten Asbroek, Anneloor L M A; Muñoz-Blanco, José Luis; Hernandez, Dena G; Ding, Jinhui; Gibbs, J Raphael; Scholz, Sonja W; Floeter, Mary Kay; Campbell, Roy H; Landi, Francesco; Bowser, Robert; Pulst, Stefan M; Ravits, John M; MacGowan, Daniel J L; Kirby, Janine; Pioro, Erik P; Pamphlett, Roger; Broach, James; Gerhard, Glenn; Dunckley, Travis L; Brady, Christopher B; Kowall, Neil W; Troncoso, Juan C; Le Ber, Isabelle; Mouzat, Kevin; Lumbroso, Serge; Heiman-Patterson, Terry D; Kamel, Freya; Van Den Bosch, Ludo; Baloh, Robert H; Strom, Tim M; Meitinger, Thomas; Shatunov, Aleksey; Van Eijk, Kristel R; de Carvalho, Mamede; Kooyman, Maarten; Middelkoop, Bas; Moisse, Matthieu; McLaughlin, Russell L; Van Es, Michael A; Weber, Markus; Boylan, Kevin B; Van Blitterswijk, Marka; Rademakers, Rosa; Morrison, Karen E; Basak, A Nazli; Mora, Jesús S; Drory, Vivian E; Shaw, Pamela J; Turner, Martin R; Talbot, Kevin; Hardiman, Orla; Williams, Kelly L; Fifita, Jennifer A; Nicholson, Garth A; Blair, Ian P; Rouleau, Guy A; Esteban-Pérez, Jesús; García-Redondo, Alberto; Al-Chalabi, Ammar; Rogaeva, Ekaterina; Zinman, Lorne; Ostrow, Lyle W; Maragakis, Nicholas J; Rothstein, Jeffrey D; Simmons, Zachary; Cooper-Knock, Johnathan; Brice, Alexis; Goutman, Stephen A; Feldman, Eva L; Gibson, Summer B; Taroni, Franco; Ratti, Antonia; Gellera, Cinzia; Van Damme, Philip; Robberecht, Wim; Fratta, Pietro; Sabatelli, Mario; Lunetta, Christian; Ludolph, Albert C; Andersen, Peter M; Weishaupt, Jochen H; Camu, William; Trojanowski, John Q; Van Deerlin, Vivianna M; Brown, Robert H; van den Berg, Leonard H; Veldink, Jan H; Harms, Matthew B; Glass, Jonathan D; Stone, David J; Tienari, Pentti; Silani, Vincenzo; Chiò, Adriano; Shaw, Christopher E; Traynor, Bryan J; Landers, John E

2018-03-21

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS. Copyright © 2018 Elsevier Inc. All rights reserved.

Sarcoidosis Related Novel Candidate Genes Identified by Multi-Omics Integrative Analyses.

PubMed

Hočevar, Keli; Maver, Aleš; Kunej, Tanja; Peterlin, Borut

2018-05-01

Sarcoidosis is a multifactorial systemic disease characterized by granulomatous inflammation and greatly impacting on global public health. The etiology and mechanisms of sarcoidosis are not fully understood. Recent high-throughput biological research has generated vast amounts of multi-omics big data on sarcoidosis, but their significance remains to be determined. We sought to identify novel candidate regions, and genes consistently altered in heterogeneous omics studies so as to reveal the underlying molecular mechanisms. We conducted a comprehensive integrative literature analysis on global data on sarcoidosis, including genomic, transcriptomic, proteomic, and phenomic studies. We performed positional integration analysis of 38 eligible datasets originating from 17 different biological layers. Using the integration interval length of 50 kb, we identified 54 regions reaching significance value p ≤ 0.0001 and 15 regions with significance value p ≤ 0.00001, when applying more stringent criteria. Secondary literature analysis of the top 20 regions, with the most significant accumulation of signals, revealed several novel candidate genes for which associations with sarcoidosis have not yet been established, but have considerable support for their involvement based on omic data. These new plausible candidate genes include NELFE, CFB, EGFL7, AGPAT2, FKBPL, NRC3, and NEU1. Furthermore, annotated data were prepared to enable custom visualization and browsing of these sarcoidosis related omics evidence in the University of California Santa Cruz (UCSC) Genome Browser. Further multi-omics approaches are called for sarcoidosis biomarkers and diagnostic and therapeutic innovation. Our approach for harnessing multi-omics data and the findings presented herein reflect important steps toward understanding the etiology and underlying pathological mechanisms of sarcoidosis.

Proteomics of Aspergillus fumigatus Conidia-containing Phagolysosomes Identifies Processes Governing Immune Evasion.

PubMed

Schmidt, Hella; Vlaic, Sebastian; Krüger, Thomas; Schmidt, Franziska; Balkenhol, Johannes; Dandekar, Thomas; Guthke, Reinhard; Kniemeyer, Olaf; Heinekamp, Thorsten; Brakhage, Axel A

2018-06-01

Invasive infections by the human pathogenic fungus Aspergillus fumigatus start with the outgrowth of asexual, airborne spores (conidia) into the lung tissue of immunocompromised patients. The resident alveolar macrophages phagocytose conidia, which end up in phagolysosomes. However, A. fumigatus conidia resist phagocytic degradation to a certain degree. This is mainly attributable to the pigment 1,8-dihydroxynaphthalene (DHN) melanin located in the cell wall of conidia, which manipulates the phagolysosomal maturation and prevents their intracellular killing. To get insight in the underlying molecular mechanisms, we comparatively analyzed proteins of mouse macrophage phagolysosomes containing melanized wild-type (wt) or nonmelanized pksP mutant conidia. For this purpose, a protocol to isolate conidia-containing phagolysosomes was established and a reference protein map of phagolysosomes was generated. We identified 637 host and 22 A. fumigatus proteins that were differentially abundant in the phagolysosome. 472 of the host proteins were overrepresented in the pksP mutant and 165 in the wt conidia-containing phagolysosome. Eight of the fungal proteins were produced only in pksP mutant and 14 proteins in wt conidia-containing phagolysosomes. Bioinformatical analysis compiled a regulatory module, which indicates host processes affected by the fungus. These processes include vATPase-driven phagolysosomal acidification, Rab5 and Vamp8-dependent endocytic trafficking, signaling pathways, as well as recruitment of the Lamp1 phagolysosomal maturation marker and the lysosomal cysteine protease cathepsin Z. Western blotting and immunofluorescence analyses confirmed the proteome data and moreover showed differential abundance of the major metabolic regulator mTOR. Taken together, with the help of a protocol optimized to isolate A. fumigatus conidia-containing phagolysosomes and a potent bioinformatics algorithm, we were able to confirm A. fumigatus conidia

Perturbation analyses of intermolecular interactions

NASA Astrophysics Data System (ADS)

Koyama, Yohei M.; Kobayashi, Tetsuya J.; Ueda, Hiroki R.

2011-08-01

Conformational fluctuations of a protein molecule are important to its function, and it is known that environmental molecules, such as water molecules, ions, and ligand molecules, significantly affect the function by changing the conformational fluctuations. However, it is difficult to systematically understand the role of environmental molecules because intermolecular interactions related to the conformational fluctuations are complicated. To identify important intermolecular interactions with regard to the conformational fluctuations, we develop herein (i) distance-independent and (ii) distance-dependent perturbation analyses of the intermolecular interactions. We show that these perturbation analyses can be realized by performing (i) a principal component analysis using conditional expectations of truncated and shifted intermolecular potential energy terms and (ii) a functional principal component analysis using products of intermolecular forces and conditional cumulative densities. We refer to these analyses as intermolecular perturbation analysis (IPA) and distance-dependent intermolecular perturbation analysis (DIPA), respectively. For comparison of the IPA and the DIPA, we apply them to the alanine dipeptide isomerization in explicit water. Although the first IPA principal components discriminate two states (the α state and PPII (polyproline II) + β states) for larger cutoff length, the separation between the PPII state and the β state is unclear in the second IPA principal components. On the other hand, in the large cutoff value, DIPA eigenvalues converge faster than that for IPA and the top two DIPA principal components clearly identify the three states. By using the DIPA biplot, the contributions of the dipeptide-water interactions to each state are analyzed systematically. Since the DIPA improves the state identification and the convergence rate with retaining distance information, we conclude that the DIPA is a more practical method compared with the

Perturbation analyses of intermolecular interactions.

PubMed

Koyama, Yohei M; Kobayashi, Tetsuya J; Ueda, Hiroki R

2011-08-01

Conformational fluctuations of a protein molecule are important to its function, and it is known that environmental molecules, such as water molecules, ions, and ligand molecules, significantly affect the function by changing the conformational fluctuations. However, it is difficult to systematically understand the role of environmental molecules because intermolecular interactions related to the conformational fluctuations are complicated. To identify important intermolecular interactions with regard to the conformational fluctuations, we develop herein (i) distance-independent and (ii) distance-dependent perturbation analyses of the intermolecular interactions. We show that these perturbation analyses can be realized by performing (i) a principal component analysis using conditional expectations of truncated and shifted intermolecular potential energy terms and (ii) a functional principal component analysis using products of intermolecular forces and conditional cumulative densities. We refer to these analyses as intermolecular perturbation analysis (IPA) and distance-dependent intermolecular perturbation analysis (DIPA), respectively. For comparison of the IPA and the DIPA, we apply them to the alanine dipeptide isomerization in explicit water. Although the first IPA principal components discriminate two states (the α state and PPII (polyproline II) + β states) for larger cutoff length, the separation between the PPII state and the β state is unclear in the second IPA principal components. On the other hand, in the large cutoff value, DIPA eigenvalues converge faster than that for IPA and the top two DIPA principal components clearly identify the three states. By using the DIPA biplot, the contributions of the dipeptide-water interactions to each state are analyzed systematically. Since the DIPA improves the state identification and the convergence rate with retaining distance information, we conclude that the DIPA is a more practical method compared with the

Genomic analyses identify recurrent MEF2D fusions in acute lymphoblastic leukaemia

PubMed Central

Gu, Zhaohui; Churchman, Michelle; Roberts, Kathryn; Li, Yongjin; Liu, Yu; Harvey, Richard C.; McCastlain, Kelly; Reshmi, Shalini C.; Payne-Turner, Debbie; Iacobucci, Ilaria; Shao, Ying; Chen, I-Ming; Valentine, Marcus; Pei, Deqing; Mungall, Karen L.; Mungall, Andrew J.; Ma, Yussanne; Moore, Richard; Marra, Marco; Stonerock, Eileen; Gastier-Foster, Julie M.; Devidas, Meenakshi; Dai, Yunfeng; Wood, Brent; Borowitz, Michael; Larsen, Eric E.; Maloney, Kelly; Mattano Jr, Leonard A.; Angiolillo, Anne; Salzer, Wanda L.; Burke, Michael J.; Gianni, Francesca; Spinelli, Orietta; Radich, Jerald P.; Minden, Mark D.; Moorman, Anthony V.; Patel, Bella; Fielding, Adele K.; Rowe, Jacob M.; Luger, Selina M.; Bhatia, Ravi; Aldoss, Ibrahim; Forman, Stephen J.; Kohlschmidt, Jessica; Mrózek, Krzysztof; Marcucci, Guido; Bloomfield, Clara D.; Stock, Wendy; Kornblau, Steven; Kantarjian, Hagop M.; Konopleva, Marina; Paietta, Elisabeth; Willman, Cheryl L.; L. Loh, Mignon; P. Hunger, Stephen; Mullighan, Charles G.

2016-01-01

Chromosomal rearrangements are initiating events in acute lymphoblastic leukaemia (ALL). Here using RNA sequencing of 560 ALL cases, we identify rearrangements between MEF2D (myocyte enhancer factor 2D) and five genes (BCL9, CSF1R, DAZAP1, HNRNPUL1 and SS18) in 22 B progenitor ALL (B-ALL) cases with a distinct gene expression profile, the most common of which is MEF2D-BCL9. Examination of an extended cohort of 1,164 B-ALL cases identified 30 cases with MEF2D rearrangements, which include an additional fusion partner, FOXJ2; thus, MEF2D-rearranged cases comprise 5.3% of cases lacking recurring alterations. MEF2D-rearranged ALL is characterized by a distinct immunophenotype, DNA copy number alterations at the rearrangement sites, older diagnosis age and poor outcome. The rearrangements result in enhanced MEF2D transcriptional activity, lymphoid transformation, activation of HDAC9 expression and sensitive to histone deacetylase inhibitor treatment. Thus, MEF2D-rearranged ALL represents a distinct form of high-risk leukaemia, for which new therapeutic approaches should be considered. PMID:27824051

Identifying the Research Process to Analyse the Adoption of the International Baccalaureate's Diploma Programme in England

ERIC Educational Resources Information Center

Outhwaite, Deborah

2018-01-01

This article analyses the flow-line around the methodology used inside an educational research process that was originally established to examine the expansion of the International Baccalaureate's Diploma Programme (IBDP) in England. This article analyses the research question, then assesses the research focus, aims and objectives. The article…

A 15-year-long Southern blotting analysis of FMR1 to detect female carriers and for prenatal diagnosis of fragile X syndrome in Taiwan.

PubMed

Tzeng, C-C; Tsai, L-P; Chang, Y-K; Hung, Y-J; Chang, Y-Y; Su, Y-P; Jiang, J-J; Liang, H-M

2017-08-01

Here, we review the results of Southern blotting analyses of the FMR1 gene performed in our reference laboratory in Taiwan over a 15-year period. In total, 725 high-risk women with a family history of fragile X syndrome (FXS) or idiopathic intellectual disability, 3911 low-risk pregnant women without such family history, and prenatal diagnosis data for 32 foetuses from 24 carrier mothers were included. Only 2 carriers were in the low-risk group, which indicated a prevalence of 1 of 1955 women (95% confidence interval: 1/7156-1/539). A total of 100 carriers were found to be in the high-risk group, thus revealing a significantly higher frequency than the low-risk group (100/725 vs 2/3911, P<0.0001). Eight of the 14 foetuses that inherited the maternal mutant allele were verified to have a full mutation, with the smallest maternal pre-mutation allele carrying 56 CGG repeats. The overall findings confirmed that the carrier prevalence among low-risk women in Taiwan is significantly lower than that reported in western countries. Therefore, the most important step for preventing FXS in Taiwan would be to focus on high-risk women by promoting general awareness of this disease and spreading knowledge regarding the benefits of carrier screening and prenatal testing. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Comparison of Antibacterial Activity of Selected Thyme (Thymus) Species by Means of the Dot Blot Test with Direct Bioautographic Detection.

PubMed

Orłowska, Marta; Kowalska, Teresa; Sajewicz, Mieczysław; Jesionek, Wioleta; Choma, Irena M; Majer-Dziedzic, Barbara; Szymczak, Grażyna; Waksmundzka-Hajnos, Monika

2015-01-01

Bioautography carried out with the aid of thin-layer chromatographic adsorbents can be used to assess antibacterial activity in samples of different origin. It can either be used as a simple and cost-effective detection method applied to a developed chromatogram, or to the dot blot test performed on a chromatographic plate, where total antibacterial activity of a sample is scrutinized. It was an aim of this study to compare antibacterial activity of 18 thyme (Thymus) specimens and species (originating from the same gardening plot and harvested in the same period of time) by means of a dot blot test with direct bioautography. A two-step extraction of herbal material was applied, and at step two the polar fraction of secondary metabolites was obtained under the earlier optimized extraction conditions [methanol-water (27+73, v/v), 130°C]. This fraction was then tested for its antibacterial activity against Bacillus subtilis bacteria. It was established that all investigated extracts exhibited antibacterial activity, yet distinct differences were perceived in the size of the bacterial growth inhibition zones among the compared thyme species. Based on the results obtained, T. citriodorus "golden dwarf" (sample No. 5) and T. marschallianus (sample No. 6) were selected as promising targets for further investigations and possible inclusion in a herbal pharmacopeia, which is an essential scientific novelty of this study.

A Review of Empirical Analyses of Disinvestment Initiatives.

PubMed

Chambers, James D; Salem, Mark N; D'Cruz, Brittany N; Subedi, Prasun; Kamal-Bahl, Sachin J; Neumann, Peter J

Disinvesting in low-value health care services provides opportunities for investment in higher value care and thus an increase in health care efficiency. To identify international experience with disinvestment initiatives and to review empirical analyses of disinvestment initiatives. We performed a literature search using the PubMed database to identify international experience with disinvestment initiatives. We also reviewed empirical analyses of disinvestment initiatives. We identified 26 unique disinvestment initiatives implemented across 11 countries. Nineteen addressed multiple intervention types, six addressed only drugs, and one addressed only devices. We reviewed 18 empirical analyses of disinvestment initiatives: 7 reported that the initiative was successful, 8 reported that the initiative was unsuccessful, and 3 reported that findings were mixed; that is, the study considered multiple services and reported a decrease in the use of some but not others. Thirty-seven low-value services were evaluated across the 18 empirical analyses, for 14 (38%) of which the disinvestment initiative led to a decline in use. Six of the seven studies that reported the disinvestment initiative to be successful included an attempt to promote the disinvestment initiative among participating clinicians. The success of disinvestment initiatives has been mixed, with fewer than half the identified empirical studies reporting that use of the low-value service was reduced. Our findings suggest that promotion of the disinvestment initiative among clinicians is a key component to the success of the disinvestment initiative. Copyright © 2017 International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc. All rights reserved.

Analyses of germline variants associated with ovarian cancer survival identify functional candidates at the 1q22 and 19p12 outcome loci

PubMed Central

Glubb, Dylan M.; Johnatty, Sharon E.; Quinn, Michael C.J.; O’Mara, Tracy A.; Tyrer, Jonathan P.; Gao, Bo; Fasching, Peter A.; Beckmann, Matthias W.; Lambrechts, Diether; Vergote, Ignace; Velez Edwards, Digna R.; Beeghly-Fadiel, Alicia; Benitez, Javier; Garcia, Maria J.; Goodman, Marc T.; Thompson, Pamela J.; Dörk, Thilo; Dürst, Matthias; Modungo, Francesmary; Moysich, Kirsten; Heitz, Florian; du Bois, Andreas; Pfisterer, Jacobus; Hillemanns, Peter; Karlan, Beth Y.; Lester, Jenny; Goode, Ellen L.; Cunningham, Julie M.; Winham, Stacey J.; Larson, Melissa C.; McCauley, Bryan M.; Kjær, Susanne Krüger; Jensen, Allan; Schildkraut, Joellen M.; Berchuck, Andrew; Cramer, Daniel W.; Terry, Kathryn L.; Salvesen, Helga B.; Bjorge, Line; Webb, Penny M.; Grant, Peter; Pejovic, Tanja; Moffitt, Melissa; Hogdall, Claus K.; Hogdall, Estrid; Paul, James; Glasspool, Rosalind; Bernardini, Marcus; Tone, Alicia; Huntsman, David; Woo, Michelle; Group, AOCS; deFazio, Anna; Kennedy, Catherine J.; Pharoah, Paul D.P.; MacGregor, Stuart; Chenevix-Trench, Georgia

2017-01-01

We previously identified associations with ovarian cancer outcome at five genetic loci. To identify putatively causal genetic variants and target genes, we prioritized two ovarian outcome loci (1q22 and 19p12) for further study. Bioinformatic and functional genetic analyses indicated that MEF2D and ZNF100 are targets of candidate outcome variants at 1q22 and 19p12, respectively. At 19p12, the chromatin interaction of a putative regulatory element with the ZNF100 promoter region correlated with candidate outcome variants. At 1q22, putative regulatory elements enhanced MEF2D promoter activity and haplotypes containing candidate outcome variants modulated these effects. In a public dataset, MEF2D and ZNF100 expression were both associated with ovarian cancer progression-free or overall survival time. In an extended set of 6,162 epithelial ovarian cancer patients, we found that functional candidates at the 1q22 and 19p12 loci, as well as other regional variants, were nominally associated with patient outcome; however, no associations reached our threshold for statistical significance (p<1×10-5). Larger patient numbers will be needed to convincingly identify any true associations at these loci. PMID:29029385

Confirmation of the immunoreactivity of monoclonal anti-human C-terminal EGFR antibodies in bronze Corydoras Corydoras aeneus (Callichthyidae Teleostei) by Western Blot method.

PubMed

Mytych, Jennifer; Satora, Leszek; Kozioł, Katarzyna

2018-02-01

Bronze corydoras (Corydoras aeneus) uses the distal part of the intestine as accessory respiratory organ. Our previous study showed the presence of epidermal growth factor receptor (EGFR) cytoplasmic domain in the digestive tract of the bronze corydoras. In this study, using Western Blot method, we validated the results presented in the previous research. In detail, results of Western Blot analysis on digestive and respiratory part of bronze corydoras intestine homogenates confirmed the immunoreactivity of anti-cytoplasmic domain (C-terminal) human EGFR antibodies with protein band of approximately 180kDa (EGFR molecular weight). This indicates a high homology of EGFR domain between these species and the possibility of such antibody use in bronze corydoras. Statistically significantly higher EGFR expression was observed in the respiratory part of intestine when compared to the digestive part. This implies higher proliferation activity and angiogenesis of epithelium in this part of intestine, creating conditions for air respiration. Therefore, Corydoras aeneus may be considered as a model organism for the molecular studies of the mechanisms of epithelial proliferation initiation and inhibition depending on hypoxia and normoxia. Copyright © 2017. Published by Elsevier GmbH.

Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

PubMed

Wang, Hye-young; Kim, Hyunjung; Kim, Yeun; Bang, Hyeeun; Kim, Jong-Pill; Hwang, Joo Hwan; Cho, Sang-Nae; Kim, Tae Ue; Lee, Hyeyoung

2015-10-01

Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

Exosomal Fetuin-A identified by proteomics: a novel urinary biomarker for detecting acute kidney injury

PubMed Central

Zhou, Hua; Pisitkun, Trairak; Aponte, Angel; Yuen, Peter S.T.; Hoffert, Jason D.; Yasuda, Hideo; Hu, Xuzhen; Chawla, Lakhmir; Shen, Rong-Fong; Knepper, Mark A.; Star., Robert A.

2008-01-01

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We aimed to discover biomarkers in urinary exosomes to detect acute kidney injury (AKI) which has a high mortality and morbidity. Animals were injected intravenously with cisplatin. Urinary exosomes were isolated by differential centrifugation. Protein changes were evaluated by two-dimensional difference in gel electrophoresis and changed proteins were identified by MALDI-TOF-TOF or LC-MS/MS. The identified candidate biomarkers were validated by western blotting in individual urine samples from rats subjected to cisplatin injection; bilateral ischemia and reperfusion (I/R); volume depletion (VD); and ICU patients with and without AKI. We identified 18 proteins that were increased and 9 proteins that were decreased 8 hr after cisplatin. Most of the candidates could not be validated by western blotting. However, exosomal Fetuin-A increased 52.5-fold at day 2 (1 day before serum creatinine increase and tubule damage) and remained elevated 51.5-fold at day 5 (peak renal injury) after cisplatin injection. By immuno-electron microscopy and elution studies, Fetuin-A was located inside urinary exosomes. Urinary Fetuin-A was increased 31.6-fold in the early phase (2~8hr) of ischemia/reperfusion, but not in prerenal azotemia. Urinary exosomal Fetuin-A also increased in three ICU patients with AKI compared to the patients without AKI. We conclude that 1) Proteomic analysis of urinary exosomes can provide biomarker candidates for the diagnosis of AKI; 2) Urinary Fetuin-A might be a predictive biomarker of structural renal injury. PMID:17021608

How individual participant data meta-analyses have influenced trial design, conduct, and analysis.

PubMed

Tierney, Jayne F; Pignon, Jean-Pierre; Gueffyier, Francois; Clarke, Mike; Askie, Lisa; Vale, Claire L; Burdett, Sarah

2015-11-01

To demonstrate how individual participant data (IPD) meta-analyses have impacted directly on the design and conduct of trials and highlight other advantages IPD might offer. Potential examples of the impact of IPD meta-analyses on trials were identified at an international workshop, attended by individuals with experience in the conduct of IPD meta-analyses and knowledge of trials in their respective clinical areas. Experts in the field who did not attend were asked to provide any further examples. We then examined relevant trial protocols, publications, and Web sites to verify the impacts of the IPD meta-analyses. A subgroup of workshop attendees sought further examples and identified other aspects of trial design and conduct that may inform IPD meta-analyses. We identified 52 examples of IPD meta-analyses thought to have had a direct impact on the design or conduct of trials. After screening relevant trial protocols and publications, we identified 28 instances where IPD meta-analyses had clearly impacted on trials. They have influenced the selection of comparators and participants, sample size calculations, analysis and interpretation of subsequent trials, and the conduct and analysis of ongoing trials, sometimes in ways that would not possible with systematic reviews of aggregate data. We identified additional potential ways that IPD meta-analyses could be used to influence trials. IPD meta-analysis could be better used to inform the design, conduct, analysis, and interpretation of trials. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

How individual participant data meta-analyses have influenced trial design, conduct, and analysis

PubMed Central

Tierney, Jayne F.; Pignon, Jean-Pierre; Gueffyier, Francois; Clarke, Mike; Askie, Lisa; Vale, Claire L.; Burdett, Sarah; Alderson, P.; Askie, L.; Bennett, D.; Burdett, S.; Clarke, M.; Dias, S.; Emberson, J.; Gueyffier, F.; Iorio, A.; Macleod, M.; Mol, B.W.; Moons, C.; Parmar, M.; Perera, R.; Phillips, R.; Pignon, J.P.; Rees, J.; Reitsma, H.; Riley, R.; Rovers, M.; Rydzewska, L.; Schmid, C.; Shepperd, S.; Stenning, S.; Stewart, L.; Tierney, J.; Tudur Smith, C.; Vale, C.; Welge, J.; White, I.; Whiteley, W.

2015-01-01

Objectives To demonstrate how individual participant data (IPD) meta-analyses have impacted directly on the design and conduct of trials and highlight other advantages IPD might offer. Study Design and Setting Potential examples of the impact of IPD meta-analyses on trials were identified at an international workshop, attended by individuals with experience in the conduct of IPD meta-analyses and knowledge of trials in their respective clinical areas. Experts in the field who did not attend were asked to provide any further examples. We then examined relevant trial protocols, publications, and Web sites to verify the impacts of the IPD meta-analyses. A subgroup of workshop attendees sought further examples and identified other aspects of trial design and conduct that may inform IPD meta-analyses. Results We identified 52 examples of IPD meta-analyses thought to have had a direct impact on the design or conduct of trials. After screening relevant trial protocols and publications, we identified 28 instances where IPD meta-analyses had clearly impacted on trials. They have influenced the selection of comparators and participants, sample size calculations, analysis and interpretation of subsequent trials, and the conduct and analysis of ongoing trials, sometimes in ways that would not possible with systematic reviews of aggregate data. We identified additional potential ways that IPD meta-analyses could be used to influence trials. Conclusions IPD meta-analysis could be better used to inform the design, conduct, analysis, and interpretation of trials. PMID:26186982

Comparative proteomic analyses reveal that FlbA down-regulates gliT expression and SOD activity in Aspergillus fumigatus.

PubMed

Shin, Kwang-Soo; Park, Hee-Soo; Kim, Young-Hwan; Yu, Jae-Hyuk

2013-07-11

FlbA is a regulator of G-protein signaling protein that plays a central role in attenuating heterotrimeric G-protein mediated vegetative growth signaling in Aspergillus. The deletion of flbA (∆flbA) in the opportunistic human pathogen Aspergillus fumigatus results in accelerated cell death and autolysis in submerged culture. To further investigate the effects of ∆flbA on intracellular protein levels we carried out 2-D proteome analyses of 2-day old submerged cultures of ∆flbA and wild type (WT) strains and observed 160 differentially expressed proteins. Via nano-LC-ESI-MS/MS analyses, we revealed the identity of 10 and 2 proteins exhibiting high and low level accumulation, respectively, in ∆flbA strain. Notably, the GliT protein is accumulated at about 1800-fold higher levels in ∆flbA than WT. Moreover, GliT is secreted at high levels from ∆flbA strain, whereas Sod1 (superoxide dismutase) is secreted at a higher level in WT. Northern blot analyses reveal that ∆flbA results in elevated accumulation of gliT mRNA. Consequently, ∆flbA strain exhibits enhanced tolerance to gliotoxin toxicity. Finally, ∆flbA strain displayed enhanced SOD activity and elevated resistance to menadione and paraquat. In summary, FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and of exogenous gliotoxin in A. fumigatus. Regulator of G protein Signaling (RGS) proteins play crucial roles in fundamental biological processes in filamentous fungi. FlbA is the first studied filamentous fungal RGS protein, yet much remains to be understood about its roles in the opportunistic human pathogen Aspergillus fumigatus. In the present study, we examined the effects of the deletion of flbA using comprehensive analyses of the intra- and extracellular proteomes of A. fumigatus wild type and the flbA deletion mutant. Via MS analyses, we identified 10 proteins exhibiting high level accumulation in the flbA deletion

Target gene analyses of 39 amelogenesis imperfecta kindreds

PubMed Central

Chan, Hui-Chen; Estrella, Ninna M. R. P.; Milkovich, Rachel N.; Kim, Jung-Wook; Simmer, James P.; Hu, Jan C-C.

2012-01-01

Previously, mutational analyses identified six disease-causing mutations in 24 amelogenesis imperfecta (AI) kindreds. We have since expanded the number of AI kindreds to 39, and performed mutation analyses covering the coding exons and adjoining intron sequences for the six proven AI candidate genes [amelogenin (AMELX), enamelin (ENAM), family with sequence similarity 83, member H (FAM83H), WD repeat containing domain 72 (WDR72), enamelysin (MMP20), and kallikrein-related peptidase 4 (KLK4)] and for ameloblastin (AMBN) (a suspected candidate gene). All four of the X-linked AI families (100%) had disease-causing mutations in AMELX, suggesting that AMELX is the only gene involved in the aetiology of X-linked AI. Eighteen families showed an autosomal-dominant pattern of inheritance. Disease-causing mutations were identified in 12 (67%): eight in FAM83H, and four in ENAM. No FAM83H coding-region or splice-junction mutations were identified in three probands with autosomal-dominant hypocalcification AI (ADHCAI), suggesting that a second gene may contribute to the aetiology of ADHCAI. Six families showed an autosomal-recessive pattern of inheritance, and disease-causing mutations were identified in three (50%): two in MMP20, and one in WDR72. No disease-causing mutations were found in 11 families with only one affected member. We conclude that mutation analyses of the current candidate genes for AI have about a 50% chance of identifying the disease-causing mutation in a given kindred. PMID:22243262

Detection of Rickettsia in Rhipicephalus sanguineus Ticks and Ctenocephalides felis Fleas from Southeastern Tunisia by Reverse Line Blot Assay

PubMed Central

Khrouf, Fatma; M'Ghirbi, Youmna; Znazen, Abir; Ben Jemaa, Mounir; Hammami, Adnene

2014-01-01

Ticks (n = 663) and fleas (n = 470) collected from domestic animals from southeastern Tunisia were screened for Rickettsia infection using reverse line blot assay. Evidence of spotted fever group Rickettsia was obtained. We detected Rickettsia felis in fleas, Rickettsia massiliae Bar 29 and the Rickettsia conorii Israeli spotted fever strain in ticks, and Rickettsia conorii subsp. conorii and Rickettsia spp. in both arthropods. The sensitivity of the adopted technique allowed the identification of a new association between fleas and R. conorii subsp. conorii species. The presence of these vector-borne Rickettsia infections should be considered when diagnosing this disease in humans in Tunisia. PMID:24226919

Reporting completeness and transparency of meta-analyses of depression screening tool accuracy: A comparison of meta-analyses published before and after the PRISMA statement.

PubMed

Rice, Danielle B; Kloda, Lorie A; Shrier, Ian; Thombs, Brett D

2016-08-01

Meta-analyses that are conducted rigorously and reported completely and transparently can provide accurate evidence to inform the best possible healthcare decisions. Guideline makers have raised concerns about the utility of existing evidence on the diagnostic accuracy of depression screening tools. The objective of our study was to evaluate the transparency and completeness of reporting in meta-analyses of the diagnostic accuracy of depression screening tools using the PRISMA tool adapted for diagnostic test accuracy meta-analyses. We searched MEDLINE and PsycINFO from January 1, 2005 through March 13, 2016 for recent meta-analyses in any language on the diagnostic accuracy of depression screening tools. Two reviewers independently assessed the transparency in reporting using the PRISMA tool with appropriate adaptations made for studies of diagnostic test accuracy. We identified 21 eligible meta-analyses. Twelve of 21 meta-analyses complied with at least 50% of adapted PRISMA items. Of 30 adapted PRISMA items, 11 were fulfilled by ≥80% of included meta-analyses, 3 by 50-79% of meta-analyses, 7 by 25-45% of meta-analyses, and 9 by <25%. On average, post-PRISMA meta-analyses complied with 17 of 30 items compared to 13 of 30 items pre-PRISMA. Deficiencies in the transparency of reporting in meta-analyses of the diagnostic test accuracy of depression screening tools of meta-analyses were identified. Authors, reviewers, and editors should adhere to the PRISMA statement to improve the reporting of meta-analyses of the diagnostic accuracy of depression screening tools. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

PubMed Central

Nebl, Thomas; Prieto, Judith Helena; Kapp, Eugene; Smith, Brian J.; Williams, Melanie J.; Yates, John R.; Cowman, Alan F.; Tonkin, Christopher J.

2011-01-01

Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca2+-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of 32[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca2+-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component. PMID:21980283

Functional Analyses of the Crohn's Disease Risk Gene LACC1.

PubMed

Assadi, Ghazaleh; Vesterlund, Liselotte; Bonfiglio, Ferdinando; Mazzurana, Luca; Cordeddu, Lina; Schepis, Danika; Mjösberg, Jenny; Ruhrmann, Sabrina; Fabbri, Alessia; Vukojevic, Vladana; Percipalle, Piergiorgio; Salomons, Florian A; Laurencikiene, Jurga; Törkvist, Leif; Halfvarson, Jonas; D'Amato, Mauro

2016-01-01

Genetic variation in the Laccase (multicopper oxidoreductase) domain-containing 1 (LACC1) gene has been shown to affect the risk of Crohn's disease, leprosy and, more recently, ulcerative colitis and juvenile idiopathic arthritis. LACC1 function appears to promote fatty-acid oxidation, with concomitant inflammasome activation, reactive oxygen species production, and anti-bacterial responses in macrophages. We sought to contribute to elucidating LACC1 biological function by extensive characterization of its expression in human tissues and cells, and through preliminary analyses of the regulatory mechanisms driving such expression. We implemented Western blot, quantitative real-time PCR, immunofluorescence microscopy, and flow cytometry analyses to investigate fatty acid metabolism-immune nexus (FAMIN; the LACC1 encoded protein) expression in subcellular compartments, cell lines and relevant human tissues. Gene-set enrichment analyses were performed to initially investigate modulatory mechanisms of LACC1 expression. A small-interference RNA knockdown in vitro model system was used to study the effect of FAMIN depletion on peroxisome function. FAMIN expression was detected in macrophage-differentiated THP-1 cells and several human tissues, being highest in neutrophils, monocytes/macrophages, myeloid and plasmacytoid dendritic cells among peripheral blood cells. Subcellular co-localization was exclusively confined to peroxisomes, with some additional positivity for organelle endomembrane structures. LACC1 co-expression signatures were enriched for genes involved in peroxisome proliferator-activated receptors (PPAR) signaling pathways, and PPAR ligands downregulated FAMIN expression in in vitro model systems. FAMIN is a peroxisome-associated protein with primary role(s) in macrophages and other immune cells, where its metabolic functions may be modulated by PPAR signaling events. However, the precise molecular mechanisms through which FAMIN exerts its biological effects

Semiquantitative Dot Blot with the GRA8 antigen to differentiate the stages of toxoplasmosis infection.

PubMed

Costa, Juan Gabriel; Vilariño, María Julia

2018-06-01

In this work we present a novel methodology to differentiate the phases of toxoplasmosis infection: the "semiquantitative Dot Blot". It is a simple technique that does not require expensive equipment, does not involve a long technique development, and can be used in a low-complexity laboratory. In this study, two recombinant sequences of Toxoplasma gondii GRA8 antigen were used, and specific IgG antibodies were detected in selected patient samples. This method makes it possible to obtain a score for each serum and define whether the patient is in the acute or chronic phase of the infection. The sensitivity and specificity results varied depending on the antigenic sequence used. With GRA8A, 62.1% and 72.7% were obtained, while with GRA8B, 82.8% and 72.1% were obtained, respectively. Although the sensitivity and specificity values were not close to 100%, they were similar to those reported with the same antigens in ELISA. Therefore, this quantitative technique would be a good alternative to ELISA. Copyright © 2018 Elsevier B.V. All rights reserved.

Microarray and differential display identify genes involved in jasmonate-dependent anther development.

PubMed

Mandaokar, Ajin; Kumar, V Dinesh; Amway, Matt; Browse, John

2003-07-01

Jasmonate (JA) is a signaling compound essential for anther development and pollen fertility in Arabidopsis. Mutations that block the pathway of JA synthesis result into male sterility. To understand the processes of anther and pollen maturation, we used microarray and differential display approaches to compare gene expression pattern in anthers of wild-type Arabidopsis and the male-sterile mutant, opr3. Microarray experiment revealed 25 genes that were up-regulated more than 1.8-fold in wild-type anthers as compared to mutant anthers. Experiments based on differential display identified 13 additional genes up-regulated in wild-type anthers compared to opr3 for a total of 38 differentially expressed genes. Searches of the Arabidopsis and non-redundant databases disclosed known or likely functions for 28 of the 38 genes identified, while 10 genes encode proteins of unknown function. Northern blot analysis of eight representative clones as probes confirmed low expression in opr3 anthers compared with wild-type anthers. JA responsiveness of these same genes was also investigated by northern blot analysis of anther RNA isolated from wild-type and opr3 plants, In these experiments, four genes were induced in opr3 anthers within 0.5-1 h of JA treatment while the remaining genes were up-regulated only 1-8 h after JA application. None of these genes was induced by JA in anthers of the coil mutant that is deficient in JA responsiveness. The four early-induced genes in opr3 encode lipoxygenase, a putative bHLH transcription factor, epithiospecifier protein and an unknown protein. We propose that these and other early components may be involved in JA signaling and in the initiation of developmental processes. The four late genes encode an extensin-like protein, a peptide transporter and two unknown proteins, which may represent components required later in anther and pollen maturation. Transcript profiling has provided a successful approach to identify genes involved in

Novel diagnostic procedure for determining metastasis to sentinel lymph nodes in breast cancer using a semi-dry dot-blot method.

PubMed

Otsubo, Ryota; Oikawa, Masahiro; Hirakawa, Hiroshi; Shibata, Kenichiro; Abe, Kuniko; Hayashi, Tomayoshi; Kinoshita, Naoe; Shigematsu, Kazuto; Hatachi, Toshiko; Yano, Hiroshi; Matsumoto, Megumi; Takagi, Katsunori; Tsuchiya, Tomoshi; Tomoshige, Koichi; Nakashima, Masahiro; Taniguchi, Hideki; Omagari, Takeyuki; Itoyanagi, Noriaki; Nagayasu, Takeshi

2014-02-15

We developed an easy, quick and cost-effective detection method for lymph node metastasis called the semi-dry dot-blot (SDB) method, which visualizes the presence of cancer cells with washing of sectioned lymph nodes by anti-pancytokeratin antibody, modifying dot-blot technology. We evaluated the validity and efficacy of the SDB method for the diagnosis of lymph node metastasis in a clinical setting (Trial 1). To evaluate the validity of the SDB method in clinical specimens, 180 dissected lymph nodes from 29 cases, including breast, gastric and colorectal cancer, were examined. Each lymph node was sliced at the maximum diameter and the sensitivity, specificity and accuracy of the SDB method were determined and compared with the final pathology report. Metastasis was detected in 32 lymph nodes (17.8%), and the sensitivity, specificity and accuracy of the SDB method were 100, 98.0 and 98.3%, respectively (Trial 2). To evaluate the efficacy of the SDB method in sentinel lymph node (SLN) biopsy, 174 SLNs from 100 cases of clinically node-negative breast cancer were analyzed. Each SLN was longitudinally sliced at 2-mm intervals and the sensitivity, specificity, accuracy and time required for the SDB method were determined and compared with the intraoperative pathology report. Metastasis was detected in 15 SLNs (8.6%), and the sensitivity, specificity, accuracy and mean required time of the SDB method were 93.3, 96.9, 96.6 and 43.3 min, respectively. The SDB method is a novel and reliable modality for the intraoperative diagnosis of SLN metastasis. © 2013 UICC.

A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

PubMed

Lin, Wen-Wei; Chen, I-Ju; Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R; Cheng, Tian-Lu

2016-01-01

Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

Combined linkage and association analyses identify a novel locus for obesity near PROX1 in Asians.

PubMed

Kim, Hyun-Jin; Yoo, Yun Joo; Ju, Young Seok; Lee, Seungbok; Cho, Sung-Il; Sung, Joohon; Kim, Jong-Il; Seo, Jeong-Sun

2013-11-01

Although genome-wide association studies (GWAS) have substantially contributed to understanding the genetic architecture, unidentified variants for complex traits remain an issue. One of the efficient approaches is the improvement of the power of GWAS scan by weighting P values with prior linkage signals. Our objective was to identify the novel candidates for obesity in Asian populations by using genemapping strategies that combine linkage and association analyses. To obtain linkage information for body mass index (BMI) and waist circumference (WC), we performed a multipoint genome-wide linkage study in an isolated Mongolian sample of 1,049 individuals from 74 families. Next, a family-based GWAS, which integrates within- and between-family components, was performed using the genotype data of 756 individuals of the Mongolian sample, and P values for association were weighted using linkage information obtained previously. For both BMI (LOD = 3.3) and WC (LOD = 2.6), the highest linkage peak was discovered at chromosome 10q11.22. In family-based GWAS combined with linkage information, six single-nucleotide polymorphisms (SNPs) for BMI and five SNPs for WC reached a significant level of association (linkage weighted P < 1 × 10(-5) ). Of these, only one of the SNPs associated with WC (rs1704198) was replicated in 327 Korean families comprising 1,301 individuals. This SNP was located in the proximity of the prosperorelated homeobox 1 (PROX1) gene, the function of which was validated previously in a mouse model. Our powerful strategic analysis enabled the discovery of a novel candidate gene, PROX1, associated with WC in an Asian population. Copyright © 2012 The Obesity Society.

Multi-country health surveys: are the analyses misleading?

PubMed

Masood, Mohd; Reidpath, Daniel D

2014-05-01

The aim of this paper was to review the types of approaches currently utilized in the analysis of multi-country survey data, specifically focusing on design and modeling issues with a focus on analyses of significant multi-country surveys published in 2010. A systematic search strategy was used to identify the 10 multi-country surveys and the articles published from them in 2010. The surveys were selected to reflect diverse topics and foci; and provide an insight into analytic approaches across research themes. The search identified 159 articles appropriate for full text review and data extraction. The analyses adopted in the multi-country surveys can be broadly classified as: univariate/bivariate analyses, and multivariate/multivariable analyses. Multivariate/multivariable analyses may be further divided into design- and model-based analyses. Of the 159 articles reviewed, 129 articles used model-based analysis, 30 articles used design-based analyses. Similar patterns could be seen in all the individual surveys. While there is general agreement among survey statisticians that complex surveys are most appropriately analyzed using design-based analyses, most researchers continued to use the more common model-based approaches. Recent developments in design-based multi-level analysis may be one approach to include all the survey design characteristics. This is a relatively new area, however, and there remains statistical, as well as applied analytic research required. An important limitation of this study relates to the selection of the surveys used and the choice of year for the analysis, i.e., year 2010 only. There is, however, no strong reason to believe that analytic strategies have changed radically in the past few years, and 2010 provides a credible snapshot of current practice.

Comparative analyses of Legionella species identifies genetic features of strains causing Legionnaires' disease.

PubMed

Gomez-Valero, Laura; Rusniok, Christophe; Rolando, Monica; Neou, Mario; Dervins-Ravault, Delphine; Demirtas, Jasmin; Rouy, Zoe; Moore, Robert J; Chen, Honglei; Petty, Nicola K; Jarraud, Sophie; Etienne, Jerome; Steinert, Michael; Heuner, Klaus; Gribaldo, Simonetta; Médigue, Claudine; Glöckner, Gernot; Hartland, Elizabeth L; Buchrieser, Carmen

2014-01-01

The genus Legionella comprises over 60 species. However, L. pneumophila and L. longbeachae alone cause over 95% of Legionnaires’ disease. To identify the genetic bases underlying the different capacities to cause disease we sequenced and compared the genomes of L. micdadei, L. hackeliae and L. fallonii (LLAP10), which are all rarely isolated from humans. We show that these Legionella species possess different virulence capacities in amoeba and macrophages, correlating with their occurrence in humans. Our comparative analysis of 11 Legionella genomes belonging to five species reveals highly heterogeneous genome content with over 60% representing species-specific genes; these comprise a complete prophage in L. micdadei, the first ever identified in a Legionella genome. Mobile elements are abundant in Legionella genomes; many encode type IV secretion systems for conjugative transfer, pointing to their importance for adaptation of the genus. The Dot/Icm secretion system is conserved, although the core set of substrates is small, as only 24 out of over 300 described Dot/Icm effector genes are present in all Legionella species. We also identified new eukaryotic motifs including thaumatin, synaptobrevin or clathrin/coatomer adaptine like domains. Legionella genomes are highly dynamic due to a large mobilome mainly comprising type IV secretion systems, while a minority of core substrates is shared among the diverse species. Eukaryotic like proteins and motifs remain a hallmark of the genus Legionella. Key factors such as proteins involved in oxygen binding, iron storage, host membrane transport and certain Dot/Icm substrates are specific features of disease-related strains.

Integrating Genetic, Transcriptional, and Functional Analyses to Identify Five Novel Genes for Atrial Fibrillation

PubMed Central

Sinner, Moritz F.; Tucker, Nathan R.; Lunetta, Kathryn L.; Ozaki, Kouichi; Smith, J. Gustav; Trompet, Stella; Bis, Joshua C.; Lin, Honghuang; Chung, Mina K.; Nielsen, Jonas B.; Lubitz, Steven A.; Krijthe, Bouwe P.; Magnani, Jared W.; Ye, Jiangchuan; Gollob, Michael H.; Tsunoda, Tatsuhiko; Müller-Nurasyid, Martina; Lichtner, Peter; Peters, Annette; Dolmatova, Elena; Kubo, Michiaki; Smith, Jonathan D.; Psaty, Bruce M.; Smith, Nicholas L.; Jukema, J. Wouter; Chasman, Daniel I.; Albert, Christine M.; Ebana, Yusuke; Furukawa, Tetsushi; MacFarlane, Peter; Harris, Tamara B.; Darbar, Dawood; Dörr, Marcus; Holst, Anders G.; Svendsen, Jesper H.; Hofman, Albert; Uitterlinden, Andre G.; Gudnason, Vilmundur; Isobe, Mitsuaki; Malik, Rainer; Dichgans, Martin; Rosand, Jonathan; Van Wagoner, David R.; Benjamin, Emelia J.; Milan, David J.; Melander, Olle; Heckbert, Susan R.; Ford, Ian; Liu, Yongmei; Barnard, John; Olesen, Morten S.; Stricker, Bruno H.C.; Tanaka, Toshihiro; Kääb, Stefan; Ellinor, Patrick T.

2014-01-01

Background Atrial fibrillation (AF) affects over 30 million individuals worldwide and is associated with an increased risk of stroke, heart failure, and death. AF is highly heritable, yet the genetic basis for the arrhythmia remains incompletely understood. Methods & Results To identify new AF-related genes, we utilized a multifaceted approach, combining large-scale genotyping in two ethnically distinct populations, cis-eQTL mapping, and functional validation. Four novel loci were identified in individuals of European descent near the genes NEURL (rs12415501, RR=1.18, 95%CI 1.13 – 1.23, p=6.5×10−16), GJA1 (rs13216675, RR=1.10, 95%CI 1.06 – 1.14, p=2.2×10−8), TBX5 (rs10507248, RR=1.12, 95%CI 1.08 – 1.16, p=5.7×10−11), and CAND2 (rs4642101, RR=1.10, 95%CI 1.06 – 1.14, p=9.8×10−9). In Japanese, novel loci were identified near NEURL (rs6584555, RR=1.32, 95%CI 1.26–1.39, p=2.0×10−25) and CUX2 (rs6490029, RR=1.12, 95%CI 1.08–1.16, p=3.9×10−9). The top SNPs or their proxies were identified as cis-eQTLs for the genes CAND2 (p=2.6×10−19), GJA1 (p=2.66×10−6), and TBX5 (p=1.36×10−05). Knockdown of the zebrafish orthologs of NEURL and CAND2 resulted in prolongation of the atrial action potential duration (17% and 45%, respectively). Conclusions We have identified five novel loci for AF. Our results further expand the diversity of genetic pathways implicated in AF and provide novel molecular targets for future biological and pharmacological investigation. PMID:25124494

Functional Analyses and Treatment of Precursor Behavior

PubMed Central

Najdowski, Adel C; Wallace, Michele D; Ellsworth, Carrie L; MacAleese, Alicia N; Cleveland, Jackie M

2008-01-01

Functional analysis has been demonstrated to be an effective method to identify environmental variables that maintain problem behavior. However, there are cases when conducting functional analyses of severe problem behavior may be contraindicated. The current study applied functional analysis procedures to a class of behavior that preceded severe problem behavior (precursor behavior) and evaluated treatments based on the outcomes of the functional analyses of precursor behavior. Responding for all participants was differentiated during the functional analyses, and individualized treatments eliminated precursor behavior. These results suggest that functional analysis of precursor behavior may offer an alternative, indirect method to assess the operant function of severe problem behavior. PMID:18468282

Analyses of Mobilization Manpower Supply and Demand.

DTIC Science & Technology

1982-03-01

7AD-AI30 148 ANALYSES OF MOBIL ZATION MANPOWER SUPPLY AND DEMAND U) l1 . ADMINISTRATIVE SCIENCES CORP SPRINOFIELD VA BREAU EAL MAR82 ASCR134...79-C-0527 for use in identifying and quantifying issues in the CPAM process, and to employ the model for selected quantitative ard qualitative analyses...nurses and corpsmen) to operate on a Commander FX Microcomputer, to be used by 2 the Bureau of Medicine and Surgery to develop inputs for Navy-wide

Contamination analyses of technology mirror assembly optical surfaces

NASA Technical Reports Server (NTRS)

Germani, Mark S.

1991-01-01

Automated electron microprobe analyses were performed on tape lift samples from the Technology Mirror Assembly (TMA) optical surfaces. Details of the analyses are given, and the contamination of the mirror surfaces is discussed. Based on the automated analyses of the tape lifts from the TMA surfaces and the control blank, we can conclude that the particles identified on the actual samples were not a result of contamination due to the handling or sampling process itself and that the particles reflect the actual contamination on the surface of the mirror.

Anthocyanin analyses of Vaccinium fruit dietary supplements

USDA-ARS?s Scientific Manuscript database

Vaccinium fruit ingredients within dietary supplements were identified by comparisons with anthocyanin analyses of known Vaccinium profiles (demonstration of anthocyanin fingerprinting). Available Vaccinium supplements were purchased and analyzed; their anthocyanin profiles (based on HPLC separation...

Clinical usefulness of Western blotting and ELISA avidity for the diagnosis of human toxocariasis.

PubMed

Rudzińska, M; Kowalewska, B; Sikorska, K

2017-01-01

The serodiagnosis of human toxocariasis is difficult. Specific IgGs detected routinely with ELISA based on Toxocara excretory-secretory (TES) antigens often persist for years at an elevated level, which does not allow either the differentiation between an active and persistent infection or monitoring of the effect of treatment. Additionally, false-positive results may occur in co-infections with other helminths due to cross-reactions. We evaluated the usefulness of an IgG avidity index (AI) and a Western blotting (WB) IgG in the diagnosis of patients suspected of Toxocara infection. We studied 138 subjects who were submitted to serological testing two or more times. Confirmation of an infection by WB was achieved in 73.2% of patients. A high AI was obtained in 89.1% of patients, and low AI and borderline AI were found in only 10.9%. Low and borderline values of AI remained at similar levels in subsequent studies over 2-3 years. The results showed the necessity of obligatory verification of all ELISA IgG positive and questionable results by WB. The index of IgG avidity may be helpful in excluding recent infection, but its usefulness in detecting an active phase of invasion requires further research. © 2016 John Wiley & Sons Ltd.

Evaluation of a new eastern blotting technique for the analysis of ginsenoside Re in American ginseng berry pulp extracts.

PubMed

Morinaga, Osamu; Uto, Takuhiro; Yuan, Chun-Su; Tanaka, Hiroyuki; Shoyama, Yukihiro

2010-06-01

A new eastern blotting technique has been established for ginsenoside Re (G-Re) contained in American ginseng berry pulp extracts. G-Re in American ginseng berry pulp was extracted using 100% methanol, 100% ethanol, 50% aqueous methanol, and 50% aqueous ethanol. The combined crude extracts were applied onto a polyethersulfone membrane and developed using the methanol-water-acetic acid solvent system (45:55:1 v/v). Separated components were immunostained using anti-G-Re monoclonal antibody. G-Re was first specifically detected and then quantitatively analyzed using NIH Imaging software. We also confirmed that the most suitable solvent was 50% aqueous methanol for extracting G-Re from American ginseng berry pulp. (c) 2009 Elsevier B.V. All rights reserved.

Novel actin crosslinker superfamily member identified by a two step degenerate PCR procedure.

PubMed

Byers, T J; Beggs, A H; McNally, E M; Kunkel, L M

1995-07-24

Actin-crosslinking proteins link F-actin into the bundles and networks that constitute the cytoskeleton. Dystrophin, beta-spectrin, alpha-actinin, ABP-120, ABP-280, and fimbrin share homologous actin-binding domains and comprise an actin crosslinker superfamily. We have identified a novel member of this superfamily (ACF7) using a degenerate primer-mediated PCR strategy that was optimized to resolve less-abundant superfamily sequences. The ACF7 gene is on human chromosome 1 and hybridizes to high molecular weight bands on northern blots. Sequence comparisons argue that ACF7 does not fit into one of the existing families, but represents a new class within the superfamily.

Diagnostic potential of Western blot analysis of sera from dogs with leishmaniasis in endemic areas and significance of the pattern.

PubMed

Aisa, M J; Castillejo, S; Gallego, M; Fisa, R; Riera, M C; de Colmenares, M; Torras, S; Roura, X; Sentis, J; Portus, M

1998-02-01

Serum samples collected from 237 dogs in Catalonia (northeastern Spain) were screened by Western blot analysis to detect the presence of antibodies specific to different Leishmania infantum polypeptide fractions. Leishmaniasis was confirmed in 72 of these dogs by direct examination and/or culture. Another 165 animals from the Priorat region were studied periodically for 2-8 years between 1987 and 1995, giving a total of 565 determinations. A control group of 93 dogs from nonendemic areas was also studied. Sera from dogs with leishmaniasis recognized antigens with molecular weights ranging from 12 to 85 kD. The most sensitive antigens were those of 70, 65, 46, 30, 28, 14, and 12 kD, which were recognized by 75%, 75%, 78%, 75%, 81%, 79%, and 75%, respectively, of the sera from dogs with positive parasitologic examination results. Antigens of 70 and 65 kD were also recognized by two dogs from nonendemic areas. Antigens of 14 and 12 kD were the first to be recognized by sera of asymptomatic dogs with titers less than the cut-off value of the dot-ELISA that increased during the longitudinal study, and the presence of antibodies specific for these fractions was observed for up to six years before seroconversion observed by dot-ELISA. These antibodies were also the first to disappear in dogs in which the disease was self-limited. The study corroborates the high sensitivity and specificity of Western blots in the diagnosis of canine leishmaniasis when the bands of low molecular weight (less than 46 kD) are considered, and indicates that fractions of 14 and 12 kD are useful in detecting early forms of the disease.

A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis

PubMed Central

Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R.; Cheng, Tian-Lu

2016-01-01

Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15–120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis. PMID:27494183

Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

PubMed

Wang, Guan-Feng; Ji, Jiabing; El-Kasmi, Farid; Dangl, Jeffery L; Johal, Guri; Balint-Kurti, Peter J

2015-02-01

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

Reporting and methodological quality of meta-analyses in urological literature.

PubMed

Xia, Leilei; Xu, Jing; Guzzo, Thomas J

2017-01-01

To assess the overall quality of published urological meta-analyses and identify predictive factors for high quality. We systematically searched PubMed to identify meta-analyses published from January 1st, 2011 to December 31st, 2015 in 10 predetermined major paper-based urology journals. The characteristics of the included meta-analyses were collected, and their reporting and methodological qualities were assessed by the PRISMA checklist (27 items) and AMSTAR tool (11 items), respectively. Descriptive statistics were used for individual items as a measure of overall compliance, and PRISMA and AMSTAR scores were calculated as the sum of adequately reported domains. Logistic regression was used to identify predictive factors for high qualities. A total of 183 meta-analyses were included. The mean PRISMA and AMSTAR scores were 22.74 ± 2.04 and 7.57 ± 1.41, respectively. PRISMA item 5, protocol and registration, items 15 and 22, risk of bias across studies, items 16 and 23, additional analysis had less than 50% adherence. AMSTAR item 1, " a priori " design, item 5, list of studies and item 10, publication bias had less than 50% adherence. Logistic regression analyses showed that funding support and " a priori " design were associated with superior reporting quality, following PRISMA guideline and " a priori " design were associated with superior methodological quality. Reporting and methodological qualities of recently published meta-analyses in major paper-based urology journals are generally good. Further improvement could potentially be achieved by strictly adhering to PRISMA guideline and having " a priori " protocol.

Integrative analyses of miRNA and proteomics identify potential biological pathways associated with onset of pulmonary fibrosis in the bleomycin rat model

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

Fukunaga, Satoki; Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 3-1-98 Kasugade-Naka, Konohana-ku, Osaka 554-8558; Kakehashi, Anna

To determine miRNAs and their predicted target proteins regulatory networks which are potentially involved in onset of pulmonary fibrosis in the bleomycin rat model, we conducted integrative miRNA microarray and iTRAQ-coupled LC-MS/MS proteomic analyses, and evaluated the significance of altered biological functions and pathways. We observed that alterations of miRNAs and proteins are associated with the early phase of bleomycin-induced pulmonary fibrosis, and identified potential target pairs by using ingenuity pathway analysis. Using the data set of these alterations, it was demonstrated that those miRNAs, in association with their predicted target proteins, are potentially involved in canonical pathways reflective ofmore » initial epithelial injury and fibrogenic processes, and biofunctions related to induction of cellular development, movement, growth, and proliferation. Prediction of activated functions suggested that lung cells acquire proliferative, migratory, and invasive capabilities, and resistance to cell death especially in the very early phase of bleomycin-induced pulmonary fibrosis. The present study will provide new insights for understanding the molecular pathogenesis of idiopathic pulmonary fibrosis. - Highlights: • We analyzed bleomycin-induced pulmonary fibrosis in the rat. • Integrative analyses of miRNA microarray and proteomics were conducted. • We determined the alterations of miRNAs and their potential target proteins. • The alterations may control biological functions and pathways in pulmonary fibrosis. • Our result may provide new insights of pulmonary fibrosis.« less

Genome-wide meta-analyses identify novel loci associated with n-3 and n-6 polyunsaturated fatty acid levels in Chinese and European-ancestry populations.

PubMed

Hu, Yao; Li, Huaixing; Lu, Ling; Manichaikul, Ani; Zhu, Jingwen; Chen, Yii-Der I; Sun, Liang; Liang, Shuang; Siscovick, David S; Steffen, Lyn M; Tsai, Michael Y; Rich, Stephen S; Lemaitre, Rozenn N; Lin, Xu

2016-03-15

Epidemiological studies suggest that levels of n-3 and n-6 long-chain polyunsaturated fatty acids are associated with risk of cardio-metabolic outcomes across different ethnic groups. Recent genome-wide association studies in populations of European ancestry have identified several loci associated with plasma and/or erythrocyte polyunsaturated fatty acids. To identify additional novel loci, we carried out a genome-wide association study in two population-based cohorts consisting of 3521 Chinese participants, followed by a trans-ethnic meta-analysis with meta-analysis results from 8962 participants of European ancestry. Four novel loci (MYB, AGPAT4, DGAT2 and PPT2) reached genome-wide significance in the trans-ethnic meta-analysis (log10(Bayes Factor) ≥ 6). Of them, associations of MYB and AGPAT4 with docosatetraenoic acid (log10(Bayes Factor) = 11.5 and 8.69, respectively) also reached genome-wide significance in the Chinese-specific genome-wide association analyses (P = 4.15 × 10(-14) and 4.30 × 10(-12), respectively), while associations of DGAT2 with gamma-linolenic acid (log10(Bayes Factor) = 6.16) and of PPT2 with docosapentaenoic acid (log10(Bayes Factor) = 6.24) were nominally significant in both Chinese- and European-specific genome-wide association analyses (P ≤ 0.003). We also confirmed previously reported loci including FADS1, NTAN1, NRBF2, ELOVL2 and GCKR. Different effect sizes in FADS1 and independent association signals in ELOVL2 were observed. These results provide novel insight into the genetic background of polyunsaturated fatty acids and their differences between Chinese and European populations. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza.

PubMed

Mell, Joshua Chang; Viadas, Cristina; Moleres, Javier; Sinha, Sunita; Fernández-Calvet, Ariadna; Porsch, Eric A; St Geme, Joseph W; Nislow, Corey; Redfield, Rosemary J; Garmendia, Junkal

2016-04-01

Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe "transformed recombinant enrichment profiling" (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2AHi375 by hmw1A86-028NP resulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1A86-028NP was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1A86-028NP might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1A86-028NP bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation.

Comprehensive multi-stage linkage analyses identify a locus for adult height on chromosome 3p in a healthy Caucasian population.

PubMed

Ellis, Justine A; Scurrah, Katrina J; Duncan, Anna E; Lamantia, Angela; Byrnes, Graham B; Harrap, Stephen B

2007-04-01

There have been a number of genome-wide linkage studies for adult height in recent years. These studies have yielded few well-replicated loci, and none have been further confirmed by the identification of associated gene variants. The inconsistent results may be attributable to the fact that few studies have combined accurate phenotype measures with informative statistical modelling in healthy populations. We have performed a multi-stage genome-wide linkage analysis for height in 275 adult sibling pairs drawn randomly from the Victorian Family Heart Study (VFHS), a healthy population-based Caucasian cohort. Height was carefully measured in a standardised fashion on regularly calibrated equipment. Following genome-wide identification of a peak Z-score of 3.14 on chromosome 3 at 69 cM, we performed a fine-mapping analysis of this region in an extended sample of 392 two-generation families. We used a number of variance components models that incorporated assortative mating and shared environment effects, and we observed a peak LOD score of approximately 3.5 at 78 cM in four of the five models tested. We also demonstrated that the most prevalent model in the literature gave the worst fit, and the lowest LOD score (2.9) demonstrating the importance of appropriate modelling. The region identified in this study replicates the results of other genome-wide scans of height and bone-related phenotypes, strongly suggesting the presence of a gene important in bone growth on chromosome 3p. Association analyses of relevant candidate genes should identify the genetic variants responsible for the chromosome 3p linkage signal in our population.

Comprehensive Analyses of Ventricular Myocyte Models Identify Targets Exhibiting Favorable Rate Dependence

PubMed Central

Bugana, Marco; Severi, Stefano; Sobie, Eric A.

2014-01-01

Reverse rate dependence is a problematic property of antiarrhythmic drugs that prolong the cardiac action potential (AP). The prolongation caused by reverse rate dependent agents is greater at slow heart rates, resulting in both reduced arrhythmia suppression at fast rates and increased arrhythmia risk at slow rates. The opposite property, forward rate dependence, would theoretically overcome these parallel problems, yet forward rate dependent (FRD) antiarrhythmics remain elusive. Moreover, there is evidence that reverse rate dependence is an intrinsic property of perturbations to the AP. We have addressed the possibility of forward rate dependence by performing a comprehensive analysis of 13 ventricular myocyte models. By simulating populations of myocytes with varying properties and analyzing population results statistically, we simultaneously predicted the rate-dependent effects of changes in multiple model parameters. An average of 40 parameters were tested in each model, and effects on AP duration were assessed at slow (0.2 Hz) and fast (2 Hz) rates. The analysis identified a variety of FRD ionic current perturbations and generated specific predictions regarding their mechanisms. For instance, an increase in L-type calcium current is FRD when this is accompanied by indirect, rate-dependent changes in slow delayed rectifier potassium current. A comparison of predictions across models identified inward rectifier potassium current and the sodium-potassium pump as the two targets most likely to produce FRD AP prolongation. Finally, a statistical analysis of results from the 13 models demonstrated that models displaying minimal rate-dependent changes in AP shape have little capacity for FRD perturbations, whereas models with large shape changes have considerable FRD potential. This can explain differences between species and between ventricular cell types. Overall, this study provides new insights, both specific and general, into the determinants of AP duration

Comprehensive analyses of ventricular myocyte models identify targets exhibiting favorable rate dependence.

PubMed

Cummins, Megan A; Dalal, Pavan J; Bugana, Marco; Severi, Stefano; Sobie, Eric A

2014-03-01

Reverse rate dependence is a problematic property of antiarrhythmic drugs that prolong the cardiac action potential (AP). The prolongation caused by reverse rate dependent agents is greater at slow heart rates, resulting in both reduced arrhythmia suppression at fast rates and increased arrhythmia risk at slow rates. The opposite property, forward rate dependence, would theoretically overcome these parallel problems, yet forward rate dependent (FRD) antiarrhythmics remain elusive. Moreover, there is evidence that reverse rate dependence is an intrinsic property of perturbations to the AP. We have addressed the possibility of forward rate dependence by performing a comprehensive analysis of 13 ventricular myocyte models. By simulating populations of myocytes with varying properties and analyzing population results statistically, we simultaneously predicted the rate-dependent effects of changes in multiple model parameters. An average of 40 parameters were tested in each model, and effects on AP duration were assessed at slow (0.2 Hz) and fast (2 Hz) rates. The analysis identified a variety of FRD ionic current perturbations and generated specific predictions regarding their mechanisms. For instance, an increase in L-type calcium current is FRD when this is accompanied by indirect, rate-dependent changes in slow delayed rectifier potassium current. A comparison of predictions across models identified inward rectifier potassium current and the sodium-potassium pump as the two targets most likely to produce FRD AP prolongation. Finally, a statistical analysis of results from the 13 models demonstrated that models displaying minimal rate-dependent changes in AP shape have little capacity for FRD perturbations, whereas models with large shape changes have considerable FRD potential. This can explain differences between species and between ventricular cell types. Overall, this study provides new insights, both specific and general, into the determinants of AP duration

Correlation Functions Aid Analyses Of Spectra

NASA Technical Reports Server (NTRS)

Beer, Reinhard; Norton, Robert H., Jr.

1989-01-01

New uses found for correlation functions in analyses of spectra. In approach combining elements of both pattern-recognition and traditional spectral-analysis techniques, spectral lines identified in data appear useless at first glance because they are dominated by noise. New approach particularly useful in measurement of concentrations of rare species of molecules in atmosphere.

Identifying gene networks underlying the neurobiology of ethanol and alcoholism.

PubMed

Wolen, Aaron R; Miles, Michael F

2012-01-01

For complex disorders such as alcoholism, identifying the genes linked to these diseases and their specific roles is difficult. Traditional genetic approaches, such as genetic association studies (including genome-wide association studies) and analyses of quantitative trait loci (QTLs) in both humans and laboratory animals already have helped identify some candidate genes. However, because of technical obstacles, such as the small impact of any individual gene, these approaches only have limited effectiveness in identifying specific genes that contribute to complex diseases. The emerging field of systems biology, which allows for analyses of entire gene networks, may help researchers better elucidate the genetic basis of alcoholism, both in humans and in animal models. Such networks can be identified using approaches such as high-throughput molecular profiling (e.g., through microarray-based gene expression analyses) or strategies referred to as genetical genomics, such as the mapping of expression QTLs (eQTLs). Characterization of gene networks can shed light on the biological pathways underlying complex traits and provide the functional context for identifying those genes that contribute to disease development.

Challenges and solutions to pre- and post-randomization subgroup analyses.

PubMed

Desai, Manisha; Pieper, Karen S; Mahaffey, Ken

2014-01-01

Subgroup analyses are commonly performed in the clinical trial setting with the purpose of illustrating that the treatment effect was consistent across different patient characteristics or identifying characteristics that should be targeted for treatment. There are statistical issues involved in performing subgroup analyses, however. These have been given considerable attention in the literature for analyses where subgroups are defined by a pre-randomization feature. Although subgroup analyses are often performed with subgroups defined by a post-randomization feature--including analyses that estimate the treatment effect among compliers--discussion of these analyses has been neglected in the clinical literature. Such analyses pose a high risk of presenting biased descriptions of treatment effects. We summarize the challenges of doing all types of subgroup analyses described in the literature. In particular, we emphasize issues with post-randomization subgroup analyses. Finally, we provide guidelines on how to proceed across the spectrum of subgroup analyses.

Integrative analyses of transcriptome sequencing identify novel functional lncRNAs in esophageal squamous cell carcinoma.

PubMed

Li, C-Q; Huang, G-W; Wu, Z-Y; Xu, Y-J; Li, X-C; Xue, Y-J; Zhu, Y; Zhao, J-M; Li, M; Zhang, J; Wu, J-Y; Lei, F; Wang, Q-Y; Li, S; Zheng, C-P; Ai, B; Tang, Z-D; Feng, C-C; Liao, L-D; Wang, S-H; Shen, J-H; Liu, Y-J; Bai, X-F; He, J-Z; Cao, H-H; Wu, B-L; Wang, M-R; Lin, D-C; Koeffler, H P; Wang, L-D; Li, X; Li, E-M; Xu, L-Y

2017-02-13

Long non-coding RNAs (lncRNAs) have a critical role in cancer initiation and progression, and thus may mediate oncogenic or tumor suppressing effects, as well as be a new class of cancer therapeutic targets. We performed high-throughput sequencing of RNA (RNA-seq) to investigate the expression level of lncRNAs and protein-coding genes in 30 esophageal samples, comprised of 15 esophageal squamous cell carcinoma (ESCC) samples and their 15 paired non-tumor tissues. We further developed an integrative bioinformatics method, denoted URW-LPE, to identify key functional lncRNAs that regulate expression of downstream protein-coding genes in ESCC. A number of known onco-lncRNA and many putative novel ones were effectively identified by URW-LPE. Importantly, we identified lncRNA625 as a novel regulator of ESCC cell proliferation, invasion and migration. ESCC patients with high lncRNA625 expression had significantly shorter survival time than those with low expression. LncRNA625 also showed specific prognostic value for patients with metastatic ESCC. Finally, we identified E1A-binding protein p300 (EP300) as a downstream executor of lncRNA625-induced transcriptional responses. These findings establish a catalog of novel cancer-associated functional lncRNAs, which will promote our understanding of lncRNA-mediated regulation in this malignancy.

Reporting and methodological quality of meta-analyses in urological literature

PubMed Central

Xu, Jing

2017-01-01

Purpose To assess the overall quality of published urological meta-analyses and identify predictive factors for high quality. Materials and Methods We systematically searched PubMed to identify meta-analyses published from January 1st, 2011 to December 31st, 2015 in 10 predetermined major paper-based urology journals. The characteristics of the included meta-analyses were collected, and their reporting and methodological qualities were assessed by the PRISMA checklist (27 items) and AMSTAR tool (11 items), respectively. Descriptive statistics were used for individual items as a measure of overall compliance, and PRISMA and AMSTAR scores were calculated as the sum of adequately reported domains. Logistic regression was used to identify predictive factors for high qualities. Results A total of 183 meta-analyses were included. The mean PRISMA and AMSTAR scores were 22.74 ± 2.04 and 7.57 ± 1.41, respectively. PRISMA item 5, protocol and registration, items 15 and 22, risk of bias across studies, items 16 and 23, additional analysis had less than 50% adherence. AMSTAR item 1, “a priori” design, item 5, list of studies and item 10, publication bias had less than 50% adherence. Logistic regression analyses showed that funding support and “a priori” design were associated with superior reporting quality, following PRISMA guideline and “a priori” design were associated with superior methodological quality. Conclusions Reporting and methodological qualities of recently published meta-analyses in major paper-based urology journals are generally good. Further improvement could potentially be achieved by strictly adhering to PRISMA guideline and having “a priori” protocol. PMID:28439452

Accurate and Practical Identification of 20 Fusarium Species by Seven-Locus Sequence Analysis and Reverse Line Blot Hybridization, and an In Vitro Antifungal Susceptibility Study▿†

PubMed Central

Wang, He; Xiao, Meng; Kong, Fanrong; Chen, Sharon; Dou, Hong-Tao; Sorrell, Tania; Li, Ruo-Yu; Xu, Ying-Chun

2011-01-01

Eleven reference and 25 clinical isolates of Fusarium were subject to multilocus DNA sequence analysis to determine the species and haplotypes of the fusarial isolates from Beijing and Shandong, China. Seven loci were analyzed: the translation elongation factor 1 alpha gene (EF-1α); the nuclear rRNA internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions; the second largest subunit of the RNA polymerase gene (RPB2); the calmodulin gene (CAM); and the mitochondrial small subunit (mtSSU) rRNA gene. We also evaluated an IGS-targeted PCR/reverse line blot (RLB) assay for species/haplotype identification of Fusarium. Twenty Fusarium species and seven species complexes were identified. Of 25 clinical isolates (10 species), the Gibberella (Fusarium) fujikuroi species complex was the commonest (40%) and was followed by the Fusarium solani species complex (FSSC) (36%) and the F. incarnatum-F. equiseti species complex (12%). Six FSSC isolates were identified to the species level as FSSC-3+4, and three as FSSC-5. Twenty-nine IGS, 27 EF-1α, 26 RPB2, 24 CAM, 18 ITS, 19 LSU, and 18 mtSSU haplotypes were identified; 29 were unique, and haplotypes for 24 clinical strains were novel. By parsimony informative character analysis, the IGS locus was the most phylogenetically informative, and the rRNA gene regions were the least. Results by RLB were concordant with multilocus sequence analysis for all isolates. Amphotericin B was the most active drug against all species. Voriconazole MICs were high (>8 μg/ml) for 15 (42%) isolates, including FSSC. Analysis of larger numbers of isolates is required to determine the clinical utility of the seven-locus sequence analysis and RLB assay in species classification of fusaria. PMID:21389150

Performing statistical analyses on quantitative data in Taverna workflows: an example using R and maxdBrowse to identify differentially-expressed genes from microarray data.

PubMed

Li, Peter; Castrillo, Juan I; Velarde, Giles; Wassink, Ingo; Soiland-Reyes, Stian; Owen, Stuart; Withers, David; Oinn, Tom; Pocock, Matthew R; Goble, Carole A; Oliver, Stephen G; Kell, Douglas B

2008-08-07

There has been a dramatic increase in the amount of quantitative data derived from the measurement of changes at different levels of biological complexity during the post-genomic era. However, there are a number of issues associated with the use of computational tools employed for the analysis of such data. For example, computational tools such as R and MATLAB require prior knowledge of their programming languages in order to implement statistical analyses on data. Combining two or more tools in an analysis may also be problematic since data may have to be manually copied and pasted between separate user interfaces for each tool. Furthermore, this transfer of data may require a reconciliation step in order for there to be interoperability between computational tools. Developments in the Taverna workflow system have enabled pipelines to be constructed and enacted for generic and ad hoc analyses of quantitative data. Here, we present an example of such a workflow involving the statistical identification of differentially-expressed genes from microarray data followed by the annotation of their relationships to cellular processes. This workflow makes use of customised maxdBrowse web services, a system that allows Taverna to query and retrieve gene expression data from the maxdLoad2 microarray database. These data are then analysed by R to identify differentially-expressed genes using the Taverna RShell processor which has been developed for invoking this tool when it has been deployed as a service using the RServe library. In addition, the workflow uses Beanshell scripts to reconcile mismatches of data between services as well as to implement a form of user interaction for selecting subsets of microarray data for analysis as part of the workflow execution. A new plugin system in the Taverna software architecture is demonstrated by the use of renderers for displaying PDF files and CSV formatted data within the Taverna workbench. Taverna can be used by data analysis

Performing statistical analyses on quantitative data in Taverna workflows: An example using R and maxdBrowse to identify differentially-expressed genes from microarray data

PubMed Central

Li, Peter; Castrillo, Juan I; Velarde, Giles; Wassink, Ingo; Soiland-Reyes, Stian; Owen, Stuart; Withers, David; Oinn, Tom; Pocock, Matthew R; Goble, Carole A; Oliver, Stephen G; Kell, Douglas B

2008-01-01

Background There has been a dramatic increase in the amount of quantitative data derived from the measurement of changes at different levels of biological complexity during the post-genomic era. However, there are a number of issues associated with the use of computational tools employed for the analysis of such data. For example, computational tools such as R and MATLAB require prior knowledge of their programming languages in order to implement statistical analyses on data. Combining two or more tools in an analysis may also be problematic since data may have to be manually copied and pasted between separate user interfaces for each tool. Furthermore, this transfer of data may require a reconciliation step in order for there to be interoperability between computational tools. Results Developments in the Taverna workflow system have enabled pipelines to be constructed and enacted for generic and ad hoc analyses of quantitative data. Here, we present an example of such a workflow involving the statistical identification of differentially-expressed genes from microarray data followed by the annotation of their relationships to cellular processes. This workflow makes use of customised maxdBrowse web services, a system that allows Taverna to query and retrieve gene expression data from the maxdLoad2 microarray database. These data are then analysed by R to identify differentially-expressed genes using the Taverna RShell processor which has been developed for invoking this tool when it has been deployed as a service using the RServe library. In addition, the workflow uses Beanshell scripts to reconcile mismatches of data between services as well as to implement a form of user interaction for selecting subsets of microarray data for analysis as part of the workflow execution. A new plugin system in the Taverna software architecture is demonstrated by the use of renderers for displaying PDF files and CSV formatted data within the Taverna workbench. Conclusion Taverna can

Incorporating cost-benefit analyses into software assurance planning

NASA Technical Reports Server (NTRS)

Feather, M. S.; Sigal, B.; Cornford, S. L.; Hutchinson, P.

2001-01-01

The objective is to use cost-benefit analyses to identify, for a given project, optimal sets of software assurance activities. Towards this end we have incorporated cost-benefit calculations into a risk management framework.

Accident Analyses in Support of the Sludge Water System Safety Analysis

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

FINFROCK, S.H.

This document quantifies the potential health effects of the unmitigated hazards identified Hey (2002) for retrieval of sludge from the KE basin. It also identifies potential controls and any supporting mitigative analyses.

Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins.

PubMed

Barkla, Bronwyn J; Vera-Estrella, Rosario; Raymond, Carolyn

2016-05-10

Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance. This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.

Proteomic Analyses of the Unexplored Sea Anemone Bunodactis verrucosa

PubMed Central

Campos, Alexandre; Turkina, Maria V.; Ribeiro, Tiago; Osorio, Hugo; Vasconcelos, Vítor; Antunes, Agostinho

2018-01-01

Cnidarian toxic products, particularly peptide toxins, constitute a promising target for biomedicine research. Indeed, cnidarians are considered as the largest phylum of generally toxic animals. However, research on peptides and toxins of sea anemones is still limited. Moreover, most of the toxins from sea anemones have been discovered by classical purification approaches. Recently, high-throughput methodologies have been used for this purpose but in other Phyla. Hence, the present work was focused on the proteomic analyses of whole-body extract from the unexplored sea anemone Bunodactis verrucosa. The proteomic analyses applied were based on two methods: two-dimensional gel electrophoresis combined with MALDI-TOF/TOF and shotgun proteomic approach. In total, 413 proteins were identified, but only eight proteins were identified from gel-based analyses. Such proteins are mainly involved in basal metabolism and biosynthesis of antibiotics as the most relevant pathways. In addition, some putative toxins including metalloproteinases and neurotoxins were also identified. These findings reinforce the significance of the production of antimicrobial compounds and toxins by sea anemones, which play a significant role in defense and feeding. In general, the present study provides the first proteome map of the sea anemone B. verrucosa stablishing a reference for future studies in the discovery of new compounds. PMID:29364843

Proteomic Analyses of the Unexplored Sea Anemone Bunodactis verrucosa.

PubMed

Domínguez-Pérez, Dany; Campos, Alexandre; Alexei Rodríguez, Armando; Turkina, Maria V; Ribeiro, Tiago; Osorio, Hugo; Vasconcelos, Vítor; Antunes, Agostinho

2018-01-24

Cnidarian toxic products, particularly peptide toxins, constitute a promising target for biomedicine research. Indeed, cnidarians are considered as the largest phylum of generally toxic animals. However, research on peptides and toxins of sea anemones is still limited. Moreover, most of the toxins from sea anemones have been discovered by classical purification approaches. Recently, high-throughput methodologies have been used for this purpose but in other Phyla. Hence, the present work was focused on the proteomic analyses of whole-body extract from the unexplored sea anemone Bunodactis verrucosa . The proteomic analyses applied were based on two methods: two-dimensional gel electrophoresis combined with MALDI-TOF/TOF and shotgun proteomic approach. In total, 413 proteins were identified, but only eight proteins were identified from gel-based analyses. Such proteins are mainly involved in basal metabolism and biosynthesis of antibiotics as the most relevant pathways. In addition, some putative toxins including metalloproteinases and neurotoxins were also identified. These findings reinforce the significance of the production of antimicrobial compounds and toxins by sea anemones, which play a significant role in defense and feeding. In general, the present study provides the first proteome map of the sea anemone B. verrucosa stablishing a reference for future studies in the discovery of new compounds.

Meta-analyses with industry involvement are massively published and report no caveats for antidepressants.

PubMed

Ebrahim, Shanil; Bance, Sheena; Athale, Abha; Malachowski, Cindy; Ioannidis, John P A

2016-02-01

To identify the impact of industry involvement in the publication and interpretation of meta-analyses of antidepressant trials in depression. Using MEDLINE, we identified all meta-analyses evaluating antidepressants for depression published in January 2007-March 2014. We extracted data pertaining to author affiliations, conflicts of interest, and whether the conclusion of the abstract included negative statements on whether the antidepressant(s) were effective or safe. We identified 185 eligible meta-analyses. Fifty-four meta-analyses (29%) had authors who were employees of the assessed drug manufacturer, and 147 (79%) had some industry link (sponsorship or authors who were industry employees and/or had conflicts of interest). Only 58 meta-analyses (31%) had negative statements in the concluding statement of the abstract. Meta-analyses including an author who were employees of the manufacturer of the assessed drug were 22-fold less likely to have negative statements about the drug than other meta-analyses [1/54 (2%) vs. 57/131 (44%); P < 0.001]. There is a massive production of meta-analyses of antidepressants for depression authored by or linked to the industry, and they almost never report any caveats about antidepressants in their abstracts. Our findings add a note of caution for meta-analyses with ties to the manufacturers of the assessed products. Copyright © 2016 Elsevier Inc. All rights reserved.

Functional Analyses and Treatment of Precursor Behavior

ERIC Educational Resources Information Center

Najdowski, Adel C.; Wallace, Michele D.; Ellsworth, Carrie L.; MacAleese, Alicia N.; Cleveland, Jackie

2008-01-01

Functional analysis has been demonstrated to be an effective method to identify environmental variables that maintain problem behavior. However, there are cases when conducting functional analyses of severe problem behavior may be contraindicated. The current study applied functional analysis procedures to a class of behavior that preceded severe…

Genome-Wide and Gene-Based Meta-Analyses Identify Novel Loci Influencing Blood Pressure Response to Hydrochlorothiazide.

PubMed

Salvi, Erika; Wang, Zhiying; Rizzi, Federica; Gong, Yan; McDonough, Caitrin W; Padmanabhan, Sandosh; Hiltunen, Timo P; Lanzani, Chiara; Zaninello, Roberta; Chittani, Martina; Bailey, Kent R; Sarin, Antti-Pekka; Barcella, Matteo; Melander, Olle; Chapman, Arlene B; Manunta, Paolo; Kontula, Kimmo K; Glorioso, Nicola; Cusi, Daniele; Dominiczak, Anna F; Johnson, Julie A; Barlassina, Cristina; Boerwinkle, Eric; Cooper-DeHoff, Rhonda M; Turner, Stephen T

2017-01-01

This study aimed to identify novel loci influencing the antihypertensive response to hydrochlorothiazide monotherapy. A genome-wide meta-analysis of blood pressure (BP) response to hydrochlorothiazide was performed in 1739 white hypertensives from 6 clinical trials within the International Consortium for Antihypertensive Pharmacogenomics Studies, making it the largest study to date of its kind. No signals reached genome-wide significance (P<5×10 - 8 ), and the suggestive regions (P<10 -5 ) were cross-validated in 2 black cohorts treated with hydrochlorothiazide. In addition, a gene-based analysis was performed on candidate genes with previous evidence of involvement in diuretic response, in BP regulation, or in hypertension susceptibility. Using the genome-wide meta-analysis approach, with validation in blacks, we identified 2 suggestive regulatory regions linked to gap junction protein α1 gene (GJA1) and forkhead box A1 gene (FOXA1), relevant for cardiovascular and kidney function. With the gene-based approach, we identified hydroxy-delta-5-steroid dehydrogenase, 3 β- and steroid δ-isomerase 1 gene (HSD3B1) as significantly associated with BP response (P<2.28×10 - 4 ). HSD3B1 encodes the 3β-hydroxysteroid dehydrogenase enzyme and plays a crucial role in the biosynthesis of aldosterone and endogenous ouabain. By amassing all of the available pharmacogenomic studies of BP response to hydrochlorothiazide, and using 2 different analytic approaches, we identified 3 novel loci influencing BP response to hydrochlorothiazide. The gene-based analysis, never before applied to pharmacogenomics of antihypertensive drugs to our knowledge, provided a powerful strategy to identify a locus of interest, which was not identified in the genome-wide meta-analysis because of high allelic heterogeneity. These data pave the way for future investigations on new pathways and drug targets to enhance the current understanding of personalized antihypertensive treatment. © 2016

Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

PubMed

Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

2012-12-27

L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

Identifying gender specific risk/need areas for male and female juvenile offenders: Factor analyses with the Structured Assessment of Violence Risk in Youth (SAVRY).

PubMed

Hilterman, Ed L B; Bongers, Ilja; Nicholls, Tonia L; van Nieuwenhuizen, Chijs

2016-02-01

By constructing risk assessment tools in which the individual items are organized in the same way for male and female juvenile offenders it is assumed that these items and subscales have similar relevance across males and females. The identification of criminogenic needs that vary in relevance for 1 of the genders, could contribute to more meaningful risk assessments, especially for female juvenile offenders. In this study, exploratory factor analyses (EFA) on a construction sample of male (n = 3,130) and female (n = 466) juvenile offenders were used to aggregate the 30 items of the Structured Assessment of Violence Risk in Youth (SAVRY) into empirically based risk/need factors and explore differences between genders. The factor models were cross-validated through confirmatory factor analyses (CFA) on a validation sample of male (n = 2,076) and female (n = 357) juvenile offenders. In both the construction sample and the validation sample, 5 factors were identified: (a) Antisocial behavior; (b) Family functioning; (c) Personality traits; (d) Social support; and (e) Treatability. The male and female models were significantly different and the internal consistency of the factors was good, both in the construction sample and the validation sample. Clustering risk/need items for male and female juvenile offenders into meaningful factors may guide clinicians in the identification of gender-specific treatment interventions. PsycINFO Database Record (c) 2016 APA, all rights reserved.

Significance of functional disease-causal/susceptible variants identified by whole-genome analyses for the understanding of human diseases.

PubMed

Hitomi, Yuki; Tokunaga, Katsushi

2017-01-01

Human genome variation may cause differences in traits and disease risks. Disease-causal/susceptible genes and variants for both common and rare diseases can be detected by comprehensive whole-genome analyses, such as whole-genome sequencing (WGS), using next-generation sequencing (NGS) technology and genome-wide association studies (GWAS). Here, in addition to the application of an NGS as a whole-genome analysis method, we summarize approaches for the identification of functional disease-causal/susceptible variants from abundant genetic variants in the human genome and methods for evaluating their functional effects in human diseases, using an NGS and in silico and in vitro functional analyses. We also discuss the clinical applications of the functional disease causal/susceptible variants to personalized medicine.

Identifying Breeding Priorities for Blueberry Flavor Using Biochemical, Sensory, and Genotype by Environment Analyses

PubMed Central

Gilbert, Jessica L.; Guthart, Matthew J.; Gezan, Salvador A.; Pisaroglo de Carvalho, Melissa; Schwieterman, Michael L.; Colquhoun, Thomas A.; Bartoshuk, Linda M.; Sims, Charles A.; Clark, David G.; Olmstead, James W.

2015-01-01

Breeding for a subjective goal such as flavor is challenging, as many blueberry cultivars are grown worldwide, and identifying breeding targets relating to blueberry flavor biochemistry that have a high degree of genetic control and low environmental variability are priorities. A variety of biochemical compounds and physical characters induce the sensory responses of taste, olfaction, and somatosensation, all of which interact to create what is perceived flavor. The goal of this study was to identify the flavor compounds with a larger genetic versus environmental component regulating their expression over an array of cultivars, locations, and years. Over the course of three years, consumer panelists rated overall liking, texture, sweetness, sourness, and flavor intensity of 19 southern highbush blueberry (Vaccinium corymbosum hybrids) genotypes in 30 sensory panels. Significant positive correlations to overall liking of blueberry fruit (P<0.001) were found with sweetness (R2 = 0.70), texture (R2 = 0.68), and flavor (R2 = 0.63). Sourness had a significantly negative relationship with overall liking (R2 = 0.55). The relationship between flavor and texture liking was also linear (R2 = 0.73, P<0.0001) demonstrating interaction between olfaction and somatosensation. Partial least squares analysis was used to identify sugars, acids, and volatile compounds contributing to liking and sensory intensities, and revealed strong effects of fructose, pH, and several volatile compounds upon all sensory parameters measured. To assess the feasibility of breeding for flavor components, a three year study was conducted to compare genetic and environmental influences on flavor biochemistry. Panelists could discern genotypic variation in blueberry sensory components, and many of the compounds affecting consumer favor of blueberries, such as fructose, pH, β-caryophyllene oxide and 2-heptanone, were sufficiently genetically controlled that allocating resources for their breeding is

Identifying Breeding Priorities for Blueberry Flavor Using Biochemical, Sensory, and Genotype by Environment Analyses.

PubMed

Gilbert, Jessica L; Guthart, Matthew J; Gezan, Salvador A; Pisaroglo de Carvalho, Melissa; Schwieterman, Michael L; Colquhoun, Thomas A; Bartoshuk, Linda M; Sims, Charles A; Clark, David G; Olmstead, James W

2015-01-01

Breeding for a subjective goal such as flavor is challenging, as many blueberry cultivars are grown worldwide, and identifying breeding targets relating to blueberry flavor biochemistry that have a high degree of genetic control and low environmental variability are priorities. A variety of biochemical compounds and physical characters induce the sensory responses of taste, olfaction, and somatosensation, all of which interact to create what is perceived flavor. The goal of this study was to identify the flavor compounds with a larger genetic versus environmental component regulating their expression over an array of cultivars, locations, and years. Over the course of three years, consumer panelists rated overall liking, texture, sweetness, sourness, and flavor intensity of 19 southern highbush blueberry (Vaccinium corymbosum hybrids) genotypes in 30 sensory panels. Significant positive correlations to overall liking of blueberry fruit (P<0.001) were found with sweetness (R2 = 0.70), texture (R2 = 0.68), and flavor (R2 = 0.63). Sourness had a significantly negative relationship with overall liking (R2 = 0.55). The relationship between flavor and texture liking was also linear (R2 = 0.73, P<0.0001) demonstrating interaction between olfaction and somatosensation. Partial least squares analysis was used to identify sugars, acids, and volatile compounds contributing to liking and sensory intensities, and revealed strong effects of fructose, pH, and several volatile compounds upon all sensory parameters measured. To assess the feasibility of breeding for flavor components, a three year study was conducted to compare genetic and environmental influences on flavor biochemistry. Panelists could discern genotypic variation in blueberry sensory components, and many of the compounds affecting consumer favor of blueberries, such as fructose, pH, β-caryophyllene oxide and 2-heptanone, were sufficiently genetically controlled that allocating resources for their breeding is

Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

PubMed

Buschmann, H; Green, P; Sambade, A; Doonan, J H; Lloyd, C W

2011-04-01

Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

Applying isozyme analyses in tree-breeding programs

Treesearch

W. T. Adams

1981-01-01

Four examples illustrate the potential for practical use of isozyme analyses in applied breeding programs. These include identifying parent trees and clones, seed sources, and parentage of controlled crosses, and evaluating the effectiveness of different procedures involving open-pollination to produce seed of specific crosses. The improved ability to assess the true...

A Review of Meta-Analyses in Education: Methodological Strengths and Weaknesses

ERIC Educational Resources Information Center

Ahn, Soyeon; Ames, Allison J.; Myers, Nicholas D.

2012-01-01

The current review addresses the validity of published meta-analyses in education that determines the credibility and generalizability of study findings using a total of 56 meta-analyses published in education in the 2000s. Our objectives were to evaluate the current meta-analytic practices in education, identify methodological strengths and…

High-resolution linkage analyses to identify genes that influence Varroa sensitive hygiene behavior in honey bees.

PubMed

Tsuruda, Jennifer M; Harris, Jeffrey W; Bourgeois, Lanie; Danka, Robert G; Hunt, Greg J

2012-01-01

Varroa mites (V. destructor) are a major threat to honey bees (Apis melilfera) and beekeeping worldwide and likely lead to colony decline if colonies are not treated. Most treatments involve chemical control of the mites; however, Varroa has evolved resistance to many of these miticides, leaving beekeepers with a limited number of alternatives. A non-chemical control method is highly desirable for numerous reasons including lack of chemical residues and decreased likelihood of resistance. Varroa sensitive hygiene behavior is one of two behaviors identified that are most important for controlling the growth of Varroa populations in bee hives. To identify genes influencing this trait, a study was conducted to map quantitative trait loci (QTL). Individual workers of a backcross family were observed and evaluated for their VSH behavior in a mite-infested observation hive. Bees that uncapped or removed pupae were identified. The genotypes for 1,340 informative single nucleotide polymorphisms were used to construct a high-resolution genetic map and interval mapping was used to analyze the association of the genotypes with the performance of Varroa sensitive hygiene. We identified one major QTL on chromosome 9 (LOD score = 3.21) and a suggestive QTL on chromosome 1 (LOD = 1.95). The QTL confidence interval on chromosome 9 contains the gene 'no receptor potential A' and a dopamine receptor. 'No receptor potential A' is involved in vision and olfaction in Drosophila, and dopamine signaling has been previously shown to be required for aversive olfactory learning in honey bees, which is probably necessary for identifying mites within brood cells. Further studies on these candidate genes may allow for breeding bees with this trait using marker-assisted selection.

Rapid detection of rifampin-resistant clinical isolates of Mycobacterium tuberculosis by reverse dot blot hybridization.

PubMed

Guo, Qian; Yu, Yan; Zhu, Yan Ling; Zhao, Xiu Qin; Liu, Zhi Guang; Zhang, Yuan Yuan; Li, Gui Lian; Wei, Jian Hao; Wu, Yi Mou; Wan, Kang Lin

2015-01-01

A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis). 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Genome-wide methylomic and transcriptomic analyses identify subtype-specific epigenetic signatures commonly dysregulated in glioma stem cells and glioblastoma.

PubMed

Pangeni, Rajendra P; Zhang, Zhou; Alvarez, Angel A; Wan, Xuechao; Sastry, Namratha; Lu, Songjian; Shi, Taiping; Huang, Tianzhi; Lei, Charles X; James, C David; Kessler, John A; Brennan, Cameron W; Nakano, Ichiro; Lu, Xinghua; Hu, Bo; Zhang, Wei; Cheng, Shi-Yuan

2018-06-21

Glioma stem cells (GSCs), a subpopulation of tumor cells, contribute to tumor heterogeneity and therapy resistance. Gene expression profiling classified glioblastoma (GBM) and GSCs into four transcriptomically-defined subtypes. Here, we determined the DNA methylation signatures in transcriptomically pre-classified GSC and GBM bulk tumors subtypes. We hypothesized that these DNA methylation signatures correlate with gene expression and are uniquely associated either with only GSCs or only GBM bulk tumors. Additional methylation signatures may be commonly associated with both GSCs and GBM bulk tumors, i.e., common to non-stem-like and stem-like tumor cell populations and correlating with the clinical prognosis of glioma patients. We analyzed Illumina 450K methylation array and expression data from a panel of 23 patient-derived GSCs. We referenced these results with The Cancer Genome Atlas (TCGA) GBM datasets to generate methylomic and transcriptomic signatures for GSCs and GBM bulk tumors of each transcriptomically pre-defined tumor subtype. Survival analyses were carried out for these signature genes using publicly available datasets, including from TCGA. We report that DNA methylation signatures in proneural and mesenchymal tumor subtypes are either unique to GSCs, unique to GBM bulk tumors, or common to both. Further, dysregulated DNA methylation correlates with gene expression and clinical prognoses. Additionally, many previously identified transcriptionally-regulated markers are also dysregulated due to DNA methylation. The subtype-specific DNA methylation signatures described in this study could be useful for refining GBM sub-classification, improving prognostic accuracy, and making therapeutic decisions.

Using Meta-analyses for Comparative Effectiveness Research

PubMed Central

Ruppar, Todd M.; Phillips, Lorraine J.; Chase, Jo-Ana D.

2012-01-01

Comparative effectiveness research seeks to identify the most effective interventions for particular patient populations. Meta-analysis is an especially valuable form of comparative effectiveness research because it emphasizes the magnitude of intervention effects rather than relying on tests of statistical significance among primary studies. Overall effects can be calculated for diverse clinical and patient-centered variables to determine the outcome patterns. Moderator analyses compare intervention characteristics among primary studies by determining if effect sizes vary among studies with different intervention characteristics. Intervention effectiveness can be linked to patient characteristics to provide evidence for patient-centered care. Moderator analyses often answer questions never posed by primary studies because neither multiple intervention characteristics nor populations are compared in single primary studies. Thus meta-analyses provide unique contributions to knowledge. Although meta-analysis is a powerful comparative effectiveness strategy, methodological challenges and limitations in primary research must be acknowledged to interpret findings. PMID:22789450

Lectin array and glycogene expression analyses of ovarian cancer cell line A2780 and its cisplatin-resistant derivate cell line A2780-cp.

PubMed

Zhao, Ran; Qin, Wenjun; Qin, Ruihuan; Han, Jing; Li, Can; Wang, Yisheng; Xu, Congjian

2017-01-01

Ovarian cancer is one of the most lethal gynecological malignancies, in which platinum resistance is a common cause of its relapse and death. Glycosylation has been reported to be involved in drug resistance, and glycomic analyses of ovarian cancer may improve our understanding of the mechanisms underlying cancer cell drug resistance and provide potential biomarkers and therapeutic targets. The serous ovarian cancer cell line A2780 and its platinum-resistant counterpart A2780-cp were used in this study. We performed a lectin array analysis to compare the glycosylation patterns of the two cell lines, a gene expression array was employed to probe the differences in glycogenes. Furthermore, the results were verified by lectin blots. A2780-cp cell exhibited stronger intensities of Lens culinaris (LCA) Canavalia ensiformis (ConA), and Lycopersicon esculentum (LEL) and weaker intensities of Sambucus nigra (SNA) lectins. The gene expression array analysis revealed increased expression of Fut8, B3gnt4, B3gnt5, B4galt2 and decreased expression of Fut1 and ST6GalNAc 6 expression were evident in the A2780-cp cells. The lectin blot confirmed the differences in LCA, ConA, SNA and LEL between the A2780 and A2780-cp cells. The combination of the lectin and gene expression analyses showed that the levels of core fucosylation and poly-LacNAc were increased in the A2780-cp cells and the levels of Fuc α1-2(gal β1-4) GlcNAc and α2-6-linked sialic structures were decreased in the A2780-cp cells. These glycans represent potential biomarkers and might be involved in the mechanism of drug resistance in ovarian cancer.

Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

PubMed

Mishima, Eikan; Jinno, Daisuke; Akiyama, Yasutoshi; Itoh, Kunihiko; Nankumo, Shinnosuke; Shima, Hisato; Kikuchi, Koichi; Takeuchi, Yoichi; Elkordy, Alaa; Suzuki, Takehiro; Niizuma, Kuniyasu; Ito, Sadayoshi; Tomioka, Yoshihisa; Abe, Takaaki

2015-01-01

The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.

Establishment of CMab-43, a Sensitive and Specific Anti-CD133 Monoclonal Antibody, for Immunohistochemistry.

PubMed

Itai, Shunsuke; Fujii, Yuki; Nakamura, Takuro; Chang, Yao-Wen; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Suzuki, Hiroyoshi; Harada, Hiroyuki; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2017-10-01

CD133, also known as prominin-1, was first described as a cell surface marker on early progenitor and hematopoietic stem cells. It is a five-domain transmembrane protein composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites, and a short C-terminal intracellular tail. CD133 has been used as a marker to identify cancer stem cells derived from primary solid tumors and as a prognostic marker of gliomas. Herein, we developed novel anti-CD133 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We expressed the full length of CD133 in LN229 glioblastoma cells, immunized mice with LN229/CD133 cells, and performed the first screening using flow cytometry. After limiting dilution, we established 100 anti-CD133 mAbs, reacting with LN229/CD133 cells but not with LN229 cells. Subsequently, we performed the second and third screening with Western blot and immunohistochemical analyses, respectively. Among 100 mAbs, 11 strongly reacted with CD133 in Western blot analysis. One of 11 clones, CMab-43 (IgG 2a , kappa), showed a sensitive and specific reaction against colon cancer cells, warranting the use of CMab-43 in detecting CD133 in pathological analyses of CD133-expressing cancers.

Identifying familiar strangers in human encounter networks

NASA Astrophysics Data System (ADS)

Liang, Di; Li, Xiang; Zhang, Yi-Qing

2016-10-01

Familiar strangers, pairs of individuals who encounter repeatedly but never know each other, have been discovered for four decades yet lack an effective method to identify. Here we propose a novel method called familiar stranger classifier (FSC) to identify familiar strangers from three empirical datasets, and classify human relationships into four types, i.e., familiar stranger (FS), in-role (IR), friend (F) and stranger (S). The analyses of the human encounter networks show that the average number of FS one may encounter is finite but larger than the Dunbar Number, and their encounters are structurally more stable and denser than those of S, indicating the encounters of FS are not limited by the social capacity, and more robust than the random scenario. Moreover, the temporal statistics of encounters between FS over the whole time span show strong periodicity, which are diverse from the bursts of encounters within one day, suggesting the significance of longitudinal patterns of human encounters. The proposed method to identify FS in this paper provides a valid framework to understand human encounter patterns and analyse complex human social behaviors.

High-Resolution Linkage Analyses to Identify Genes That Influence Varroa Sensitive Hygiene Behavior in Honey Bees

PubMed Central

Tsuruda, Jennifer M.; Harris, Jeffrey W.; Bourgeois, Lanie; Danka, Robert G.; Hunt, Greg J.

2012-01-01

Varroa mites (V. destructor) are a major threat to honey bees (Apis melilfera) and beekeeping worldwide and likely lead to colony decline if colonies are not treated. Most treatments involve chemical control of the mites; however, Varroa has evolved resistance to many of these miticides, leaving beekeepers with a limited number of alternatives. A non-chemical control method is highly desirable for numerous reasons including lack of chemical residues and decreased likelihood of resistance. Varroa sensitive hygiene behavior is one of two behaviors identified that are most important for controlling the growth of Varroa populations in bee hives. To identify genes influencing this trait, a study was conducted to map quantitative trait loci (QTL). Individual workers of a backcross family were observed and evaluated for their VSH behavior in a mite-infested observation hive. Bees that uncapped or removed pupae were identified. The genotypes for 1,340 informative single nucleotide polymorphisms were used to construct a high-resolution genetic map and interval mapping was used to analyze the association of the genotypes with the performance of Varroa sensitive hygiene. We identified one major QTL on chromosome 9 (LOD score = 3.21) and a suggestive QTL on chromosome 1 (LOD = 1.95). The QTL confidence interval on chromosome 9 contains the gene ‘no receptor potential A’ and a dopamine receptor. ‘No receptor potential A’ is involved in vision and olfaction in Drosophila, and dopamine signaling has been previously shown to be required for aversive olfactory learning in honey bees, which is probably necessary for identifying mites within brood cells. Further studies on these candidate genes may allow for breeding bees with this trait using marker-assisted selection. PMID:23133626

Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues

PubMed Central

Tulloch, Lindsay B.; Menzies, Stefanie K.; Fraser, Andrew L.; Gould, Eoin R.; King, Elizabeth F.; Zacharova, Marija K.; Florence, Gordon J.

2017-01-01

Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or

Multivariate analyses of tinnitus complaint and change in tinnitus complaint: a masker study.

PubMed

Jakes, S; Stephens, S D

1987-11-01

Multivariate statistical techniques were used to re-analyse the data from the recent DHSS multi-centre masker study. These analyses were undertaken to three ends. First, to clarify and attempt to replicate the previously found factor structure of complaints about tinnitus. Secondly, to attempt to identify common factors in the change or improvement measures pre- and post-masker treatment. Thirdly, to identify predictors of any such outcome factors. Two complaint factors were identified; 'Distress' and 'intrusiveness'. A series of analyses were conducted on change measures using different numbers of subjects and variables. When only semantic differential scales were used, the change factors were very similar to the complaint factors noted above. When variables measuring other aspects of improvement were included, several other factors were identified. These included; 'tinnitus helped', 'masking effects', 'residual inhibition' and 'matched loudness'. Twenty-five conceptually distinct predictors of outcome were identified. These predictor variables were quite different for different outcome factors. For example, high-frequency hearing loss was a predictor of tinnitus being helped by the masker, and a low frequency match and a low masking threshold predicted therapeutic success on residual inhibition. Decrease in matched loudness was predicted by louder tinnitus initially.

Genome-wide association analyses identify new risk variants and the genetic architecture of amyotrophic lateral sclerosis

PubMed Central

van Rheenen, Wouter; Shatunov, Aleksey; Dekker, Annelot M; McLaughlin, Russell L; Diekstra, Frank P; Pulit, Sara L; van der Spek, Rick A A; Võsa, Urmo; de Jong, Simone; Robinson, Matthew R; Yang, Jian; Fogh, Isabella; van Doormaal, Perry TC; Tazelaar, Gijs H P; Koppers, Max; Blokhuis, Anna M; Sproviero, William; Jones, Ashley R; Kenna, Kevin P; van Eijk, Kristel R; Harschnitz, Oliver; Schellevis, Raymond D; Brands, William J; Medic, Jelena; Menelaou, Androniki; Vajda, Alice; Ticozzi, Nicola; Lin, Kuang; Rogelj, Boris; Vrabec, Katarina; Ravnik-Glavač, Metka; Koritnik, Blaž; Zidar, Janez; Leonardis, Lea; Grošelj, Leja Dolenc; Millecamps, Stéphanie; Salachas, François; Meininger, Vincent; de Carvalho, Mamede; Pinto, Susana; Mora, Jesus S; Rojas-García, Ricardo; Polak, Meraida; Chandran, Siddharthan; Colville, Shuna; Swingler, Robert; Morrison, Karen E; Shaw, Pamela J; Hardy, John; Orrell, Richard W; Pittman, Alan; Sidle, Katie; Fratta, Pietro; Malaspina, Andrea; Topp, Simon; Petri, Susanne; Abdulla, Susanne; Drepper, Carsten; Sendtner, Michael; Meyer, Thomas; Ophoff, Roel A; Staats, Kim A; Wiedau-Pazos, Martina; Lomen-Hoerth, Catherine; Van Deerlin, Vivianna M; Trojanowski, John Q; Elman, Lauren; McCluskey, Leo; Basak, A Nazli; Tunca, Ceren; Hamzeiy, Hamid; Parman, Yesim; Meitinger, Thomas; Lichtner, Peter; Radivojkov-Blagojevic, Milena; Andres, Christian R; Maurel, Cindy; Bensimon, Gilbert; Landwehrmeyer, Bernhard; Brice, Alexis; Payan, Christine A M; Saker-Delye, Safaa; Dürr, Alexandra; Wood, Nicholas W; Tittmann, Lukas; Lieb, Wolfgang; Franke, Andre; Rietschel, Marcella; Cichon, Sven; Nöthen, Markus M; Amouyel, Philippe; Tzourio, Christophe; Dartigues, Jean-François; Uitterlinden, Andre G; Rivadeneira, Fernando; Estrada, Karol; Hofman, Albert; Curtis, Charles; Blauw, Hylke M; van der Kooi, Anneke J; de Visser, Marianne; Goris, An; Weber, Markus; Shaw, Christopher E; Smith, Bradley N; Pansarasa, Orietta; Cereda, Cristina; Bo, Roberto Del; Comi, Giacomo P; D’Alfonso, Sandra; Bertolin, Cinzia; Sorarù, Gianni; Mazzini, Letizia; Pensato, Viviana; Gellera, Cinzia; Tiloca, Cinzia; Ratti, Antonia; Calvo, Andrea; Moglia, Cristina; Brunetti, Maura; Arcuti, Simona; Capozzo, Rosa; Zecca, Chiara; Lunetta, Christian; Penco, Silvana; Riva, Nilo; Padovani, Alessandro; Filosto, Massimiliano; Muller, Bernard; Stuit, Robbert Jan; Blair, Ian; Zhang, Katharine; McCann, Emily P; Fifita, Jennifer A; Nicholson, Garth A; Rowe, Dominic B; Pamphlett, Roger; Kiernan, Matthew C; Grosskreutz, Julian; Witte, Otto W; Ringer, Thomas; Prell, Tino; Stubendorff, Beatrice; Kurth, Ingo; Hübner, Christian A; Leigh, P Nigel; Casale, Federico; Chio, Adriano; Beghi, Ettore; Pupillo, Elisabetta; Tortelli, Rosanna; Logroscino, Giancarlo; Powell, John; Ludolph, Albert C; Weishaupt, Jochen H; Robberecht, Wim; Van Damme, Philip; Franke, Lude; Pers, Tune H; Brown, Robert H; Glass, Jonathan D; Landers, John E; Hardiman, Orla; Andersen, Peter M; Corcia, Philippe; Vourc’h, Patrick; Silani, Vincenzo; Wray, Naomi R; Visscher, Peter M; de Bakker, Paul I W; van Es, Michael A; Pasterkamp, R Jeroen; Lewis, Cathryn M; Breen, Gerome; Al-Chalabi, Ammar; van den Berg, Leonard H; Veldink, Jan H

2017-01-01

To elucidate the genetic architecture of amyotrophic lateral sclerosis (ALS) and find associated loci, we assembled a custom imputation reference panel from whole-genome-sequenced patients with ALS and matched controls (n = 1,861). Through imputation and mixed-model association analysis in 12,577 cases and 23,475 controls, combined with 2,579 cases and 2,767 controls in an independent replication cohort, we fine-mapped a new risk locus on chromosome 21 and identified C21orf2 as a gene associated with ALS risk. In addition, we identified MOBP and SCFD1 as new associated risk loci. We established evidence of ALS being a complex genetic trait with a polygenic architecture. Furthermore, we estimated the SNP-based heritability at 8.5%, with a distinct and important role for low-frequency variants (frequency 1–10%). This study motivates the interrogation of larger samples with full genome coverage to identify rare causal variants that underpin ALS risk. PMID:27455348

Selection of DNA aptamers against Human Cardiac Troponin I for colorimetric sensor based dot blot application.

PubMed

Dorraj, Ghamar Soltan; Rassaee, Mohammad Javad; Latifi, Ali Mohammad; Pishgoo, Bahram; Tavallaei, Mahmood

2015-08-20

Troponin T and I are ideal markers which are highly sensitive and specific for myocardial injury and have shown better efficacy than earlier markers. Since aptamers are ssDNA or RNA that bind to a wide variety of target molecules, the purpose of this research was to select an aptamer from a 79bp single-stranded DNA (ssDNA) random library that was used to bind the Human Cardiac Troponin I from a synthetic nucleic acids library by systematic evolution of ligands exponential enrichment (Selex) based on several selection and amplification steps. Human Cardiac Troponin I protein was coated onto the surface of streptavidin magnetic beads to extract specific aptamer from a large and diverse random ssDNA initial oligonucleotide library. As a result, several aptamers were selected and further examined for binding affinity and specificity. Finally TnIApt 23 showed beast affinity in nanomolar range (2.69nM) toward the target protein. A simple and rapid colorimetric detection assay for Human Cardiac Troponin I using the novel and specific aptamer-AuNPs conjugates based on dot blot assay was developed. The detection limit for this protein using aptamer-AuNPs-based assay was found to be 5ng/ml. Copyright © 2015 Elsevier B.V. All rights reserved.

Demonstration of monoclonal anti-carcinoembryonic antigen (CEA) antibody internalization by electron microscopy, western blotting and radioimmunoassay.

PubMed

Tsaltas, G; Ford, C H; Gallant, M

1992-01-01

One of the important factors affecting the action of monoclonal antibodies (Mabs) or immunoconjugates on tumour sites depends on whether the Mab is internalized by the cancer cells in question. The underexplored subject of internalization is discussed in this paper, and a number of in vitro techniques for investigating internalization are evaluated, using a model which consists of a well characterized anti-carcinoembryonic antigen (anti-CEA) Mab and a number of CEA expressing human cancer cell lines. Employing two alternative radiolabeling assays, evidence for internalization of the anti-CEA Mab by a CEA-positive colorectal cancer cell line (LS174T) was obtained throughout the time intervals examined (5 min to 150 min). Electronmicroscopy employing horseradish-peroxidase labeled anti-CEA Mab and control antibody permitted direct visualization of anti-CEA Mab-related staining in intracellular compartments of a high CEA-expressor human colorectal cell line (SKCO1). Finally Western blots of samples derived from cytosolic and membrane components of solubilized cells from lung and colonic cancer cell lines provided evidence for internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 minutes. Internalized anti-CEA was detected in all CEA expressing cell lines (LS174T, SKCO1, BENN) but not in the case of a very low CEA expressor line (COLO 320).

Phosphotyrosine enrichment identifies focal adhesion kinase and other tyrosine kinases for targeting in canine hemangiosarcoma.

PubMed

Marley, K; Maier, C S; Helfand, S C

2012-09-01

Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival. © 2012 Blackwell Publishing Ltd.

Proteins analysed as virtual knots

NASA Astrophysics Data System (ADS)

Alexander, Keith; Taylor, Alexander J.; Dennis, Mark R.

2017-02-01

Long, flexible physical filaments are naturally tangled and knotted, from macroscopic string down to long-chain molecules. The existence of knotting in a filament naturally affects its configuration and properties, and may be very stable or disappear rapidly under manipulation and interaction. Knotting has been previously identified in protein backbone chains, for which these mechanical constraints are of fundamental importance to their molecular functionality, despite their being open curves in which the knots are not mathematically well defined; knotting can only be identified by closing the termini of the chain somehow. We introduce a new method for resolving knotting in open curves using virtual knots, which are a wider class of topological objects that do not require a classical closure and so naturally capture the topological ambiguity inherent in open curves. We describe the results of analysing proteins in the Protein Data Bank by this new scheme, recovering and extending previous knotting results, and identifying topological interest in some new cases. The statistics of virtual knots in protein chains are compared with those of open random walks and Hamiltonian subchains on cubic lattices, identifying a regime of open curves in which the virtual knotting description is likely to be important.

Proteins analysed as virtual knots

PubMed Central

Alexander, Keith; Taylor, Alexander J.; Dennis, Mark R.

2017-01-01

Long, flexible physical filaments are naturally tangled and knotted, from macroscopic string down to long-chain molecules. The existence of knotting in a filament naturally affects its configuration and properties, and may be very stable or disappear rapidly under manipulation and interaction. Knotting has been previously identified in protein backbone chains, for which these mechanical constraints are of fundamental importance to their molecular functionality, despite their being open curves in which the knots are not mathematically well defined; knotting can only be identified by closing the termini of the chain somehow. We introduce a new method for resolving knotting in open curves using virtual knots, which are a wider class of topological objects that do not require a classical closure and so naturally capture the topological ambiguity inherent in open curves. We describe the results of analysing proteins in the Protein Data Bank by this new scheme, recovering and extending previous knotting results, and identifying topological interest in some new cases. The statistics of virtual knots in protein chains are compared with those of open random walks and Hamiltonian subchains on cubic lattices, identifying a regime of open curves in which the virtual knotting description is likely to be important. PMID:28205562

Semi-quantitative analyses of hippocampal heat shock protein-70 expression based on the duration of ischemia and the volume of cerebral infarction in mice.

PubMed

Choi, Jong-Il; Kim, Sang-Dae; Kim, Se-Hoon; Lim, Dong-Jun; Ha, Sung-Kon

2014-06-01

We investigated the expression of hippocampal heat shock protein 70 (HSP-70) infarction volume after different durations of experimental ischemic stroke in mice. Focal cerebral ischemia was induced in mice by occluding the middle cerebral artery with the modified intraluminal filament technique. Twenty-four hours after ischemia induction, both hippocampi were extracted for HSP-70 protein analyses. Slices from each hemisphere were stained with 2,3,5-triphenyltetrazolium chloride (2%), and infarction volumes were calculated. HSP-70 levels were evaluated using western blot and enzyme-linked immunosorbent assay (ELISA). HSP-70 subtype (hsp70.1, hspa1a, hspa1b) mRNA levels in the hippocampus were measured using reverse transcription-polymerase chain reaction (RT-PCR). Cerebral infarctions were found ipsilateral to the occlusion in 10 mice exposed to transient ischemia (5 each in the 30-min and 60-min occlusion groups), whereas no focal infarctions were noted in any of the sham mice. The average infarct volumes of the 2 ischemic groups were 22.28±7.31 mm(3) [30-min group±standard deviation (SD)] and 38.06±9.53 mm(3) (60-min group±SD). Western blot analyses and ELISA showed that HSP-70 in hippocampal tissues increased in the infarction groups than in the sham group. However, differences in HSP-70 levels between the 2 infarction groups were statistically insignificant. Moreover, RT-PCR results demonstrated no relationship between the mRNA expression of HSP-70 subtypes and occlusion time or infarction volume. Our results indicated no significant difference in HSP-70 expression between the 30- and 60-min occlusion groups despite the statistical difference in infarction volumes. Furthermore, HSP-70 subtype mRNA expression was independent of both occlusion duration and cerebral infarction volume.

Detection and characterization of latent HSV RNA by in situ and northern blot hybridization in guinea pigs.

PubMed

Burke, R L; Hartog, K; Croen, K D; Ostrove, J M

1991-04-01

Following intravaginal infection of guinea pigs, herpes simplex virus establishes a latent infection in the sensory lumbosacral ganglia. Using the techniques of in situ and Northern blot hybridization, we have characterized this latent HSV-2 virus and compared it to latent HSV-1 at the same anatomical site. For HSV-2, a single 1.8-kb latency-associated transcript (LAT) was detected. In contrast, as described for latent HSV-1 in the trigeminal ganglia of rabbits and mice, two HSV-1 LAT species were detected in the lumbosacral ganglia, an abundant transcript of 1.8 kb and a less abundant transcript of 1.55 kb. Despite these differences in LAT expression, the clinical course of the acute and recurrent genital disease was similar for both viruses. LAT was detected in 0.3-6.0% of the sensory neurons of sacral but not in lumbar ganglia. The abundance of LAT correlated with the severity of the initial infection, but not with the frequency of recurrent disease. Thus, vaccination strategies that substantially reduced or eliminated symptomatic disease following challenge infection appeared to block the establishment of a latent infection.

An image processing and analysis tool for identifying and analysing complex plant root systems in 3D soil using non-destructive analysis: Root1.

PubMed

Flavel, Richard J; Guppy, Chris N; Rabbi, Sheikh M R; Young, Iain M

2017-01-01

The objective of this study was to develop a flexible and free image processing and analysis solution, based on the Public Domain ImageJ platform, for the segmentation and analysis of complex biological plant root systems in soil from x-ray tomography 3D images. Contrasting root architectures from wheat, barley and chickpea root systems were grown in soil and scanned using a high resolution micro-tomography system. A macro (Root1) was developed that reliably identified with good to high accuracy complex root systems (10% overestimation for chickpea, 1% underestimation for wheat, 8% underestimation for barley) and provided analysis of root length and angle. In-built flexibility allowed the user interaction to (a) amend any aspect of the macro to account for specific user preferences, and (b) take account of computational limitations of the platform. The platform is free, flexible and accurate in analysing root system metrics.

AG 85, a major secretion protein of Mycobacterium tuberculosis, can be identified in ancient bone.

PubMed

Schmidt-Schultz, Tyede H; Schultz, Michael

2015-06-01

For the confirmation of Ag 85 in ancient and recent ECM of native macerated human bone, five cases were investigated. In three individuals, highly positive results for Ag 85 were identified in Western blot: 1) a male from Arzhan, South Siberia, dating from the 7th century BC, 2) a male from Kirchberg in Hesse, Germany, dating from the 10th - 12th century AD and 3) a recent female with a proven diagnosis of TB. As a negative control, a recent male is presented who did not suffer from TB. In another recent male, Ag 85 could be identified only very weakly. From cases in the literature it is well-known that highly positive results for Ag 85 indicate active TB, however, weakly positive results indicate a silent initial infection with Mtb. Thus, apparently, also in ancient individuals, it might well be possible to differentiate between diseased persons and disease carriers using paleoproteomic techniques. Copyright © 2015 Elsevier Ltd. All rights reserved.

Algorithm for Identifying Erroneous Rain-Gauge Readings

NASA Technical Reports Server (NTRS)

Rickman, Doug

2005-01-01

An algorithm analyzes rain-gauge data to identify statistical outliers that could be deemed to be erroneous readings. Heretofore, analyses of this type have been performed in burdensome manual procedures that have involved subjective judgements. Sometimes, the analyses have included computational assistance for detecting values falling outside of arbitrary limits. The analyses have been performed without statistically valid knowledge of the spatial and temporal variations of precipitation within rain events. In contrast, the present algorithm makes it possible to automate such an analysis, makes the analysis objective, takes account of the spatial distribution of rain gauges in conjunction with the statistical nature of spatial variations in rainfall readings, and minimizes the use of arbitrary criteria. The algorithm implements an iterative process that involves nonparametric statistics.

Root Cause Analyses of Suicides of Mental Health Clients: Identifying Systematic Processes and Service-Level Prevention Strategies.

PubMed

Gillies, Donna; Chicop, David; O'Halloran, Paul

2015-01-01

The ability to predict imminent risk of suicide is limited, particularly among mental health clients. Root cause analysis (RCA) can be used by health services to identify service-wide approaches to suicide prevention. To (a) develop a standardized taxonomy for RCAs; (b) to quantitate service-related factors associated with suicides; and (c) to identify service-related suicide prevention strategies. The RCAs of all people who died by suicide within 1 week of contact with the mental health service over 5 years were thematically analyzed using a data collection tool. Data were derived from RCAs of all 64 people who died by suicide between 2008 and 2012. Major themes were categorized as individual, situational, and care-related factors. The most common factor was that clients had recently denied suicidality. Reliance on carers, recent changes in medication, communication problems, and problems in follow-through were also commonly identified. Given the difficulty in predicting suicide in people whose expressions of suicidal ideation change so rapidly, services may consider the use of strategies aimed at improving the individual, stressor, support, and care factors identified in this study.

Antibodies against the mono-methylated arginine-glycine repeat (MMA-RG) of the Epstein-Barr virus nuclear antigen 2 (EBNA2) identify potential cellular proteins targeted in viral transformation.

PubMed

Ayoubian, Hiresh; Fröhlich, Thomas; Pogodski, Dagmar; Flatley, Andrew; Kremmer, Elisabeth; Schepers, Aloys; Feederle, Regina; Arnold, Georg J; Grässer, Friedrich A

2017-08-01

The Epstein-Barr virus is a human herpes virus with oncogenic potential. The virus-encoded nuclear antigen 2 (EBNA2) is a key mediator of viral tumorigenesis. EBNA2 features an arginine-glycine (RG) repeat at amino acids (aa)339-354 that is essential for the transformation of lymphocytes and contains symmetrically (SDMA) and asymmetrically (ADMA) di-methylated arginine residues. The SDMA-modified EBNA2 binds the survival motor neuron protein (SMN), thus mimicking SMD3, a cellular SDMA-containing protein that interacts with SMN. Accordingly, a monoclonal antibody (mAb) specific for the SDMA-modified RG repeat of EBNA2 also binds to SMD3. With the novel mAb 19D4 we now show that EBNA2 contains mono-methylated arginine (MMA) residues within the RG repeat. Using 19D4, we immune-precipitated and analysed by mass spectrometry cellular proteins in EBV-transformed B-cells that feature MMA motifs that are similar to the one in EBNA2. Among the cellular proteins identified, we confirmed by immunoprecipitation and/or Western blot analyses Aly/REF, Coilin, DDX5, FXR1, HNRNPK, LSM4, MRE11, NRIP, nucleolin, PRPF8, RBM26, SMD1 (SNRDP1) and THRAP3 proteins that are either known to contain MMA residues or feature RG repeat sequences that probably serve as methylation substrates. The identified proteins are involved in splicing, tumorigenesis, transcriptional activation, DNA stability and RNA processing or export. Furthermore, we found that several proteins involved in energy metabolism are associated with MMA-modified proteins. Interestingly, the viral EBNA1 protein that features methylated RG repeat motifs also reacted with the antibodies. Our results indicate that the region between aa 34-52 of EBNA1 contains ADMA or SDMA residues, while the region between aa 328-377 mainly contains MMA residues.

Evaluation of the Aspergillus Western Blot IgG Kit for Diagnosis of Chronic Aspergillosis

PubMed Central

Oliva, A.; Flori, P.; Hennequin, C.; Dubus, J.-C.; Reynaud-Gaubert, M.; Charpin, D.; Vergnon, J. M.; Gay, P.; Colly, A.; Piarroux, R.; Pelloux, H.

2014-01-01

Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method. PMID:25392351

Porcine Cysticercosis: Possible Cross-Reactivity of Taenia hydatigena to GP50 Antigen in the Enzyme-Linked Immunoelectrotransfer Blot Assay.

PubMed

Muro, Claudio; Gomez-Puerta, Luis A; Flecker, Robert H; Gamboa, Ricardo; Barreto, Percy Vilchez; Dorny, Pierre; Tsang, Victor C W; Gilman, Robert H; Gonzalez, Armando E; Garcia, Hector H; O'Neal, Seth E; For The Cysticercosis Working Group In Peru

2017-12-01

The lentil lectin glycoprotein enzyme-linked immunoelectrotransfer blot (LLGP EITB, reported sensitivity 99% and specificity 100%) is used as a serologic marker of exposure to Taenia solium in pigs. However, only a limited number of parasites have been evaluated for cross reactivity. Pigs may host other related cestode infections, including Taenia hydatigena, which have not been formally evaluated for cross-reactions. We investigated a corral in Tumbes, Peru, a region where a cysticercosis elimination demonstration project was completed in 2012. In this corral, 14/19 (73.7%) 6-8-week-old piglets were reactive to GP50 on LLGP EITB, and all had circulating Taenia sp. antigens. From eight necropsied piglets; four were infected with T. hydatigena metacestodes whereas none had evidence of T. solium infection. Two resident dogs were subsequently confirmed to have T. hydatigena taeniasis. These results suggest GP50 cross-reactivity in T. hydatigena- infected pigs, although controlled experimental infection is needed to confirm this hypothesis.

Descriptive and experimental analyses of variables maintaining self-injurious behavior.

PubMed Central

Lerman, D C; Iwata, B A

1993-01-01

Independent descriptive (correlational) and functional (experimental) analyses were conducted to determine the extent to which the two methods would yield data supporting similar conclusions about variables maintaining the self-injurious behavior (SIB) of 6 subjects. For the descriptive analyses, subjects were observed in their residences and at training sites at various times each day while observers recorded naturally occurring sequences of specified subject and staff behaviors. The subjects also participated in a day program for the assessment and treatment of SIB, in which they were exposed to functional analyses that manipulated potential maintaining variables in multielement designs. Both sets of data were analyzed via conditional probabilities to identify relevant antecedent and consequent events for subjects' SIB. Using outcomes of the experimental analysis as the standard for comparison, results indicated that the descriptive analysis was useful in identifying the extent to which SIB was related to social versus nonsocial contingencies, but was limited in its ability to distinguish between positive and negative reinforcement (i.e., attention versus escape). PMID:8407680

Identifying scales of pattern in ecological data: a comparison of lacunarity, spectral and wavelet analyses

Treesearch

Sari C. Saunders; Jiquan Chen; Thomas D. Drummer; Eric J. Gustafson; Kimberley D. Brosofske

2005-01-01

Identifying scales of pattern in ecological systems and coupling patterns to processes that create them are ongoing challenges. We examined the utility of three techniques (lacunarity, spectral, and wavelet analysis) for detecting scales of pattern of ecological data. We compared the information obtained using these methods for four datasets, including: surface...

Network meta-analyses performed by contracting companies and commissioned by industry.

PubMed

Schuit, Ewoud; Ioannidis, John Pa

2016-11-25

Industry commissions contracting companies to perform network meta-analysis for health technology assessment (HTA) and reimbursement submissions. Our objective was to estimate the number of network meta-analyses performed by consulting companies contracted by industry, to assess whether they were published, and to explore reasons for non-publication. We searched MEDLINE for network meta-analyses of randomized trials. Papers were included if they had authors affiliated with any contracting company. All identified contracting companies as well as additional ones from the list of the exhibitors at the International Society for Pharmacoeconomics and Outcomes Research, an annual meeting that representatives from many contracting companies attend and exhibit at, were surveyed regarding conduct and publication of network meta-analyses. In 162 of 822 (20%) network meta-analysis papers, authors were affiliated to 66 contracting companies. Another 36 contracting companies were identified by the exhibitors list. Three companies had no contact information and six merged with others, therefore 93 companies were contacted. Thirty seven out of ninety three (40%) companies responded, and 19 indicated that they had performed a total of 476 network meta-analyses, but only 102 (21%) papers were published. Thirteen companies that disclosed to have conducted 174 network meta-analyses (45 published) provided reasons for non-publication. Of the 129 still unpublished meta-analyses, for 40 there were plans for future publication, for 37 the sponsor did not allow publication, for 16 the contracting companies did not plan to publish the meta-analysis, for another 23 plans were unclear, and the remaining 13 were used as HTA submission. The protocol of the network meta-analysis was publically available from 11/162 (6.8%) network meta-analyses published by authors affiliated with contracting companies. There is a prolific sector of professional contracting companies that perform network meta-analyses

Far-western blotting as a solution to the non-specificity of the anti-erythropoietin receptor antibody

PubMed Central

Fecková, Barbora; Kimáková, Patrícia; Ilkovičová, Lenka; Szentpéteriová, Erika; Debeljak, Nataša; Solárová, Zuzana; Sačková, Veronika; Šemeláková, Martina; Bhide, Mangesh; Solár, Peter

2016-01-01

The erythropoietin receptor (EpoR) is a member of the cytokine receptor family. The interaction between erythropoietin (Epo) and EpoR is important for the production and maturation of erythroid cells, resulting in the stimulation of hematopoiesis. The fact that EpoR was also detected in neoplastic cells has opened the question about the relevance of anemia treatment with recombinant Epo in cancer patients. Numerous studies have reported pro-stimulating and anti-apoptotic effects of Epo in cancer cells, thus demonstrating EpoR functionality in these cells. By contrast, a previous study claims the absence of EpoR in tumor cells. This apparent discrepancy is based, according to certain authors, on the use of non-specific anti-EpoR antibodies. With the aim of bypassing the direct detection of EpoR with an anti-EpoR antibody, the present authors propose a far-western blot methodology, which in addition, confirms the interaction of Epo with EpoR. Applying this technique, the presence of EpoR and its interaction with Epo in human ovarian adenocarcinoma A2780 and normal human umbilical vein endothelial cells was confirmed. Furthermore, modified immunoprecipitation of EpoR followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis confirmed a 57 kDa protein as a human Epo-interacting protein in both cell lines. PMID:27446474

Detection of 98. 5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene

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

Cuppens, H.; Marynen, P.; Cassiman, J.J.

1993-12-01

The authors have previously shown that about 85% of the mutations in 194 Belgian cystic fibrosis alleles could be detected by a reverse dot-blot assay. In the present study, 50 Belgian chromosomes were analyzed for mutations in the cystic fibrosis transmembrane conductance regulator gene by means of direct solid phase automatic sequencing of PCR products of individual exons. Twenty-six disease mutations and 14 polymorphisms were found. Twelve of these mutations and 3 polymorphisms were not described before. With the exception of one mutant allele carrying two mutations, these mutations were the only mutations found in the complete coding region andmore » their exon/intron boundaries. The total sensitivity of mutant CF alleles that could be identified was 98.5%. Given the heterogeneity of these mutations, most of them very rare, CFTR mutation screening still remains rather complex in the population, and population screening, whether desirable or not, does not appear to be technically feasible with the methods currently available. 24 refs., 1 fig., 2 tabs.« less

How distributed processing produces false negatives in voxel-based lesion-deficit analyses.

PubMed

Gajardo-Vidal, Andrea; Lorca-Puls, Diego L; Crinion, Jennifer T; White, Jitrachote; Seghier, Mohamed L; Leff, Alex P; Hope, Thomas M H; Ludersdorfer, Philipp; Green, David W; Bowman, Howard; Price, Cathy J

2018-07-01

In this study, we hypothesized that if the same deficit can be caused by damage to one or another part of a distributed neural system, then voxel-based analyses might miss critical lesion sites because preservation of each site will not be consistently associated with preserved function. The first part of our investigation used voxel-based multiple regression analyses of data from 359 right-handed stroke survivors to identify brain regions where lesion load is associated with picture naming abilities after factoring out variance related to object recognition, semantics and speech articulation so as to focus on deficits arising at the word retrieval level. A highly significant lesion-deficit relationship was identified in left temporal and frontal/premotor regions. Post-hoc analyses showed that damage to either of these sites caused the deficit of interest in less than half the affected patients (76/162 = 47%). After excluding all patients with damage to one or both of the identified regions, our second analysis revealed a new region, in the anterior part of the left putamen, which had not been previously detected because many patients had the deficit of interest after temporal or frontal damage that preserved the left putamen. The results illustrate how (i) false negative results arise when the same deficit can be caused by different lesion sites; (ii) some of the missed effects can be unveiled by adopting an iterative approach that systematically excludes patients with lesions to the areas identified in previous analyses, (iii) statistically significant voxel-based lesion-deficit mappings can be driven by a subset of patients; (iv) focal lesions to the identified regions are needed to determine whether the deficit of interest is the consequence of focal damage or much more extensive damage that includes the identified region; and, finally, (v) univariate voxel-based lesion-deficit mappings cannot, in isolation, be used to predict outcome in other patients

Application of Sigma Metrics and Performance Comparison Between Two Biochemistry Analyser and a Blood Gas Analyser for the Determination of Electrolytes.

PubMed

Ustundag-Budak, Yasemin; Huysal, Kagan

2017-02-01

Electrolytes have a narrow range of biological variation and small changes are clinically significant. It is important to select the best method for clinical decision making and patient monitoring in the emergency room. The sigma metrics model provides an objective method to evaluate the performance of a method. To calculate sigma metrics for electrolytes measured with one arterial blood gas analyser including two auto-analysers that use different technologies. To identify the best approach for electrolyte monitoring in an emergency setting and the context of routine emergency room workflow. The Coefficient of Variation (CV) was determined from Internal Quality Control (IQC). Data was measured from July 2015 to January 2016 for all three analysers. The records of KBUD external quality data (Association of Clinical Biochemists, Istanbul, Turkey) for both Mindray BS-2000M analyser (Mindray, Shenzhen, China) and Architect C16000 (Abbott Diagnostics, Abbott Park, IL) and MLE clinical laboratory evaluation program (Washington, DC, USA) for Radiometer ABL 700 (Radiometer Trading, Copenhagen, Denmark) during the study period were used to determine the bias. The calculated average sigma values for sodium (-1.1), potassium (3.3), and chloride (0.06) were with the Radiometer ABL700. All calculated sigma values were better than the auto-analysers. The sigma values obtained from all analysers suggest that running more controls and increasing the calibration frequency for electrolytes is necessary for quality assurance.

Integrative genomic analyses identify LIN28 and OLIG2 as markers of survival and metastatic potential in childhood central nervous system primitive neuro-ectodermal brain tumours

PubMed Central

Picard, Daniel; Miller, Suzanne; Hawkins, Cynthia E; Bouffet, Eric; Rogers, Hazel A; Chan, Tiffany SY; Kim, Seung-Ki; Ra, Young-Shin; Fangusaro, Jason; Korshunov, Andrey; Toledano, Helen; Nakamura, Hideo; Hayden, James T; Chan, Jennifer; Lafay-Cousin, Lucie; Hu, Ping X; Fan, Xing; Muraszko, Karin M; Pomeroy, Scott L; Lau, Ching C; Ng, Ho-Keung; Jones, Chris; Meter, Timothy Van; Clifford, Steven C; Eberhart, Charles; Gajjar, Amar; Pfister, Stefan M; Grundy, Richard G; Huang, Annie

2013-01-01

Background Childhood Central Nervous System Primitive Neuro-Ectodermal brain Tumours (CNS-PNETs) are highly aggressive brain tumours for which molecular features and best therapeutic strategy remains unknown. We interrogated a large cohort of these rare tumours in order to identify molecular markers that will enhance clinical management of CNS-PNET. Methods Transcriptional and copy number profiles from primary hemispheric CNS-PNETs were examined using clustering, gene and pathways enrichment analyses to discover tumour sub-groups and group-specific molecular markers. Immuno-histochemical and/or gene expression analyses were used to validate and examine the clinical significance of novel sub-group markers in 123 primary CNS-PNETs. Findings Three molecular sub-groups of CNS-PNETs distinguished by primitive neural (Group 1), oligo-neural (Group 2) and mesenchymal lineage (Group 3) gene expression signature were identified. Tumour sub-groups exhibited differential expression of cell lineage markers, LIN28 and OLIG2, and correlated with distinct demographics, survival and metastatic incidence. Group 1 tumours affected primarily younger females; male: female ratios were respectively 0.61 (median age 2.9 years; 95% CI: 2.4–5.2; p≤ 0.005), 1.25 (median age 7.9 years; 95% CI: 6–9.7) and 1.63 (median age 5.9 years; 95% CI: 4.9–7.8) for group 1, 2 and 3 patients. Overall outcome was poorest in group 1 patients which had a median survival of 0.8 years (95% CI: 0.47–1.2; p=0.019) as compared to 1.8 years (95% CI: 1.4–2.3) and 4.3 years; (95% CI: 0.82–7.8) respectively for group 2 and 3 patients. Group 3 tumours had the highest incidence of metastases at diagnosis; M0: M+ ratio were respectively 0.9 and 3.9 for group 3, versus group 1 and 2 tumours combined (p=0.037). Interpretation LIN28 and OLIG2 represent highly promising, novel diagnostic and prognostic molecular markers for CNS PNET that warrants further evaluation in prospective clinical trials. PMID:22691720

Performance of Stratified and Subgrouped Disproportionality Analyses in Spontaneous Databases.

PubMed

Seabroke, Suzie; Candore, Gianmario; Juhlin, Kristina; Quarcoo, Naashika; Wisniewski, Antoni; Arani, Ramin; Painter, Jeffery; Tregunno, Philip; Norén, G Niklas; Slattery, Jim

2016-04-01

Disproportionality analyses are used in many organisations to identify adverse drug reactions (ADRs) from spontaneous report data. Reporting patterns vary over time, with patient demographics, and between different geographical regions, and therefore subgroup analyses or adjustment by stratification may be beneficial. The objective of this study was to evaluate the performance of subgroup and stratified disproportionality analyses for a number of key covariates within spontaneous report databases of differing sizes and characteristics. Using a reference set of established ADRs, signal detection performance (sensitivity and precision) was compared for stratified, subgroup and crude (unadjusted) analyses within five spontaneous report databases (two company, one national and two international databases). Analyses were repeated for a range of covariates: age, sex, country/region of origin, calendar time period, event seriousness, vaccine/non-vaccine, reporter qualification and report source. Subgroup analyses consistently performed better than stratified analyses in all databases. Subgroup analyses also showed benefits in both sensitivity and precision over crude analyses for the larger international databases, whilst for the smaller databases a gain in precision tended to result in some loss of sensitivity. Additionally, stratified analyses did not increase sensitivity or precision beyond that associated with analytical artefacts of the analysis. The most promising subgroup covariates were age and region/country of origin, although this varied between databases. Subgroup analyses perform better than stratified analyses and should be considered over the latter in routine first-pass signal detection. Subgroup analyses are also clearly beneficial over crude analyses for larger databases, but further validation is required for smaller databases.

Identifying the Deleterious Effect of Rare LHX4 Allelic Variants, a Challenging Issue

PubMed Central

Rochette, Claire; Jullien, Nicolas; Saveanu, Alexandru; Caldagues, Emmanuelle; Bergada, Ignacio; Braslavsky, Debora; Pfeifer, Marija; Reynaud, Rachel; Herman, Jean-Paul; Barlier, Anne; Brue, Thierry; Enjalbert, Alain; Castinetti, Frederic

2015-01-01

LHX4 is a LIM homeodomain transcription factor involved in the early steps of pituitary ontogenesis. To date, 8 heterozygous LHX4 mutations have been reported as responsible of combined pituitary hormone deficiency (CPHD) in Humans. We identified 4 new LHX4 heterozygous allelic variants in patients with congenital hypopituitarism: W204X, delK242, N271S and Q346R. Our objective was to determine the role of LHX4 variants in patients’ phenotypes. Heterologous HEK293T cells were transfected with plasmids encoding for wild-type or mutant LHX4. Protein expression was analysed by Western Blot, and DNA binding by electro-mobility shift assay experiments. Target promoters of LHX4 were cotransfected with wild type or mutant LHX4 to test the transactivating abilities of each variant. Our results show that the W204X mutation was associated with early GH and TSH deficiencies and later onset ACTH deficiency. It led to a truncated protein unable to bind to alpha-Gsu promoter binding consensus sequence. W204X was not able to activate target promoters in vitro. Cotransfection experiments did not favour a dominant negative effect. In contrast, all other mutants were able to bind the promoters and led to an activation similar as that observed with wild type LHX4, suggesting that they were likely polymorphisms. To conclude, our study underlines the need for functional in vitro studies to ascertain the role of rare allelic variants of LHX4 in disease phenotypes. It supports the causative role of the W204X mutation in CPHD and adds up childhood onset ACTH deficiency to the clinical spectrum of the various phenotypes related to LHX4 mutations. PMID:25955177

Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis.

PubMed

Oliva, A; Flori, P; Hennequin, C; Dubus, J-C; Reynaud-Gaubert, M; Charpin, D; Vergnon, J M; Gay, P; Colly, A; Piarroux, R; Pelloux, H; Ranque, S

2015-01-01

Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Large-scale association analyses identifies 13 new susceptibility loci for coronary artery disease

PubMed Central

Schunkert, Heribert; König, Inke R.; Kathiresan, Sekar; Reilly, Muredach P.; Assimes, Themistocles L.; Holm, Hilma; Preuss, Michael; Stewart, Alexandre F. R.; Barbalic, Maja; Gieger, Christian; Absher, Devin; Aherrahrou, Zouhair; Allayee, Hooman; Altshuler, David; Anand, Sonia S.; Andersen, Karl; Anderson, Jeffrey L.; Ardissino, Diego; Ball, Stephen G.; Balmforth, Anthony J.; Barnes, Timothy A.; Becker, Diane M.; Becker, Lewis C.; Berger, Klaus; Bis, Joshua C.; Boekholdt, S. Matthijs; Boerwinkle, Eric; Braund, Peter S.; Brown, Morris J.; Burnett, Mary Susan; Buysschaert, Ian; Carlquist, Cardiogenics, John F.; Chen, Li; Cichon, Sven; Codd, Veryan; Davies, Robert W.; Dedoussis, George; Dehghan, Abbas; Demissie, Serkalem; Devaney, Joseph M.; Do, Ron; Doering, Angela; Eifert, Sandra; El Mokhtari, Nour Eddine; Ellis, Stephen G.; Elosua, Roberto; Engert, James C.; Epstein, Stephen E.; Faire, Ulf de; Fischer, Marcus; Folsom, Aaron R.; Freyer, Jennifer; Gigante, Bruna; Girelli, Domenico; Gretarsdottir, Solveig; Gudnason, Vilmundur; Gulcher, Jeffrey R.; Halperin, Eran; Hammond, Naomi; Hazen, Stanley L.; Hofman, Albert; Horne, Benjamin D.; Illig, Thomas; Iribarren, Carlos; Jones, Gregory T.; Jukema, J.Wouter; Kaiser, Michael A.; Kaplan, Lee M.; Kastelein, John J.P.; Khaw, Kay-Tee; Knowles, Joshua W.; Kolovou, Genovefa; Kong, Augustine; Laaksonen, Reijo; Lambrechts, Diether; Leander, Karin; Lettre, Guillaume; Li, Mingyao; Lieb, Wolfgang; Linsel-Nitschke, Patrick; Loley, Christina; Lotery, Andrew J.; Mannucci, Pier M.; Maouche, Seraya; Martinelli, Nicola; McKeown, Pascal P.; Meisinger, Christa; Meitinger, Thomas; Melander, Olle; Merlini, Pier Angelica; Mooser, Vincent; Morgan, Thomas; Mühleisen, Thomas W.; Muhlestein, Joseph B.; Münzel, Thomas; Musunuru, Kiran; Nahrstaedt, Janja; Nelson, Christopher P.; Nöthen, Markus M.; Olivieri, Oliviero; Patel, Riyaz S.; Patterson, Chris C.; Peters, Annette; Peyvandi, Flora; Qu, Liming; Quyyumi, Arshed A.; Rader, Daniel J.; Rallidis, Loukianos S.; Rice, Catherine; Rosendaal, Frits R.; Rubin, Diana; Salomaa, Veikko; Sampietro, M. Lourdes; Sandhu, Manj S.; Schadt, Eric; Schäfer, Arne; Schillert, Arne; Schreiber, Stefan; Schrezenmeir, Jürgen; Schwartz, Stephen M.; Siscovick, David S.; Sivananthan, Mohan; Sivapalaratnam, Suthesh; Smith, Albert; Smith, Tamara B.; Snoep, Jaapjan D.; Soranzo, Nicole; Spertus, John A.; Stark, Klaus; Stirrups, Kathy; Stoll, Monika; Tang, W. H. Wilson; Tennstedt, Stephanie; Thorgeirsson, Gudmundur; Thorleifsson, Gudmar; Tomaszewski, Maciej; Uitterlinden, Andre G.; van Rij, Andre M.; Voight, Benjamin F.; Wareham, Nick J.; Wells, George A.; Wichmann, H.-Erich; Wild, Philipp S.; Willenborg, Christina; Witteman, Jaqueline C. M.; Wright, Benjamin J.; Ye, Shu; Zeller, Tanja; Ziegler, Andreas; Cambien, Francois; Goodall, Alison H.; Cupples, L. Adrienne; Quertermous, Thomas; März, Winfried; Hengstenberg, Christian; Blankenberg, Stefan; Ouwehand, Willem H.; Hall, Alistair S.; Deloukas, Panos; Thompson, John R.; Stefansson, Kari; Roberts, Robert; Thorsteinsdottir, Unnur; O’Donnell, Christopher J.; McPherson, Ruth; Erdmann, Jeanette; Samani, Nilesh J.

2011-01-01

We performed a meta-analysis of 14 genome-wide association studies of coronary artery disease (CAD) comprising 22,233 cases and 64,762 controls of European descent, followed by genotyping of top association signals in 60,738 additional individuals. This genomic analysis identified 13 novel loci harboring one or more SNPs that were associated with CAD at P<5×10−8 and confirmed the association of 10 of 12 previously reported CAD loci. The 13 novel loci displayed risk allele frequencies ranging from 0.13 to 0.91 and were associated with a 6 to 17 percent increase in the risk of CAD per allele. Notably, only three of the novel loci displayed significant association with traditional CAD risk factors, while the majority lie in gene regions not previously implicated in the pathogenesis of CAD. Finally, five of the novel CAD risk loci appear to have pleiotropic effects, showing strong association with various other human diseases or traits. PMID:21378990

On meta- and mega-analyses for gene–environment interactions

PubMed Central

Huang, Jing; Liu, Yulun; Vitale, Steve; Penning, Trevor M.; Whitehead, Alexander S.; Blair, Ian A.; Vachani, Anil; Clapper, Margie L.; Muscat, Joshua E.; Lazarus, Philip; Scheet, Paul; Moore, Jason H.; Chen, Yong

2017-01-01

Gene-by-environment (G × E) interactions are important in explaining the missing heritability and understanding the causation of complex diseases, but a single, moderately sized study often has limited statistical power to detect such interactions. With the increasing need for integrating data and reporting results from multiple collaborative studies or sites, debate over choice between mega- versus meta-analysis continues. In principle, data from different sites can be integrated at the individual level into a “mega” data set, which can be fit by a joint “mega-analysis.” Alternatively, analyses can be done at each site, and results across sites can be combined through a “meta-analysis” procedure without integrating individual level data across sites. Although mega-analysis has been advocated in several recent initiatives, meta-analysis has the advantages of simplicity and feasibility, and has recently led to several important findings in identifying main genetic effects. In this paper, we conducted empirical and simulation studies, using data from a G × E study of lung cancer, to compare the mega- and meta-analyses in four commonly used G × E analyses under the scenario that the number of studies is small and sample sizes of individual studies are relatively large. We compared the two data integration approaches in the context of fixed effect models and random effects models separately. Our investigations provide valuable insights in understanding the differences between mega- and meta-analyses in practice of combining small number of studies in identifying G × E interactions. PMID:29110346

On meta- and mega-analyses for gene-environment interactions.

PubMed

Huang, Jing; Liu, Yulun; Vitale, Steve; Penning, Trevor M; Whitehead, Alexander S; Blair, Ian A; Vachani, Anil; Clapper, Margie L; Muscat, Joshua E; Lazarus, Philip; Scheet, Paul; Moore, Jason H; Chen, Yong

2017-12-01

Gene-by-environment (G × E) interactions are important in explaining the missing heritability and understanding the causation of complex diseases, but a single, moderately sized study often has limited statistical power to detect such interactions. With the increasing need for integrating data and reporting results from multiple collaborative studies or sites, debate over choice between mega- versus meta-analysis continues. In principle, data from different sites can be integrated at the individual level into a "mega" data set, which can be fit by a joint "mega-analysis." Alternatively, analyses can be done at each site, and results across sites can be combined through a "meta-analysis" procedure without integrating individual level data across sites. Although mega-analysis has been advocated in several recent initiatives, meta-analysis has the advantages of simplicity and feasibility, and has recently led to several important findings in identifying main genetic effects. In this paper, we conducted empirical and simulation studies, using data from a G × E study of lung cancer, to compare the mega- and meta-analyses in four commonly used G × E analyses under the scenario that the number of studies is small and sample sizes of individual studies are relatively large. We compared the two data integration approaches in the context of fixed effect models and random effects models separately. Our investigations provide valuable insights in understanding the differences between mega- and meta-analyses in practice of combining small number of studies in identifying G × E interactions. © 2017 WILEY PERIODICALS, INC.

Identifying Children At Risk for Being Bullies in the US

PubMed Central

Shetgiri, Rashmi; Lin, Hua; Flores, Glenn

2012-01-01

Objective To identify risk factors associated with the highest and lowest prevalence of bullying perpetration among US children. Methods Using the 2001–2002 Health Behavior in School-Aged Children, a nationally-representative survey of US children in 6th–10th grades, bivariate analyses were conducted to identify factors associated with any (≥ once or twice), moderate (≥ two-three times/month), and frequent (≥ weekly) bullying. Stepwise multivariable analyses identified risk factors associated with bullying. Recursive partitioning analysis (RPA) identified risk factors which, in combination, identify students with the highest and lowest bullying prevalence. Results The prevalence of any bullying in the 13,710 students was 37.3%, moderate bullying was 12.6%, and frequent bullying was 6.6%. Characteristics associated with bullying were similar in the multivariable analyses and RPA clusters. In RPA, the highest prevalence of any bullying (67%) accrued in children with a combination of fighting and weapon-carrying. Students who carry weapons, smoke, and drink alcohol more than 5–6 days weekly were at highest risk for moderate bullying (61%). Those who carry weapons, smoke, drink > once daily, have above-average academic performance, moderate/high family affluence, and feel irritable or bad-tempered daily were at highest risk for frequent bullying (68%). Conclusions Risk clusters for any, moderate, and frequent bullying differ. Children who fight and carry weapons are at highest risk of any bullying. Weapon-carrying, smoking, and alcohol use are included in the highest risk clusters for moderate and frequent bullying. Risk-group categories may be useful to providers in identifying children at highest risks for bullying and in targeting interventions. PMID:22989731

Association between Adult Height and Risk of Colorectal, Lung, and Prostate Cancer: Results from Meta-analyses of Prospective Studies and Mendelian Randomization Analyses.

PubMed

Khankari, Nikhil K; Shu, Xiao-Ou; Wen, Wanqing; Kraft, Peter; Lindström, Sara; Peters, Ulrike; Schildkraut, Joellen; Schumacher, Fredrick; Bofetta, Paolo; Risch, Angela; Bickeböller, Heike; Amos, Christopher I; Easton, Douglas; Eeles, Rosalind A; Gruber, Stephen B; Haiman, Christopher A; Hunter, David J; Chanock, Stephen J; Pierce, Brandon L; Zheng, Wei

2016-09-01

Observational studies examining associations between adult height and risk of colorectal, prostate, and lung cancers have generated mixed results. We conducted meta-analyses using data from prospective cohort studies and further carried out Mendelian randomization analyses, using height-associated genetic variants identified in a genome-wide association study (GWAS), to evaluate the association of adult height with these cancers. A systematic review of prospective studies was conducted using the PubMed, Embase, and Web of Science databases. Using meta-analyses, results obtained from 62 studies were summarized for the association of a 10-cm increase in height with cancer risk. Mendelian randomization analyses were conducted using summary statistics obtained for 423 genetic variants identified from a recent GWAS of adult height and from a cancer genetics consortium study of multiple cancers that included 47,800 cases and 81,353 controls. For a 10-cm increase in height, the summary relative risks derived from the meta-analyses of prospective studies were 1.12 (95% CI 1.10, 1.15), 1.07 (95% CI 1.05, 1.10), and 1.06 (95% CI 1.02, 1.11) for colorectal, prostate, and lung cancers, respectively. Mendelian randomization analyses showed increased risks of colorectal (odds ratio [OR] = 1.58, 95% CI 1.14, 2.18) and lung cancer (OR = 1.10, 95% CI 1.00, 1.22) associated with each 10-cm increase in genetically predicted height. No association was observed for prostate cancer (OR = 1.03, 95% CI 0.92, 1.15). Our meta-analysis was limited to published studies. The sample size for the Mendelian randomization analysis of colorectal cancer was relatively small, thus affecting the precision of the point estimate. Our study provides evidence for a potential causal association of adult height with the risk of colorectal and lung cancers and suggests that certain genetic factors and biological pathways affecting adult height may also affect the risk of these cancers.

Association between Adult Height and Risk of Colorectal, Lung, and Prostate Cancer: Results from Meta-analyses of Prospective Studies and Mendelian Randomization Analyses

PubMed Central

Khankari, Nikhil K.; Shu, Xiao-Ou; Wen, Wanqing; Kraft, Peter; Lindström, Sara; Peters, Ulrike; Schildkraut, Joellen; Schumacher, Fredrick; Bofetta, Paolo; Risch, Angela; Bickeböller, Heike; Amos, Christopher I.; Easton, Douglas; Gruber, Stephen B.; Haiman, Christopher A.; Hunter, David J.; Chanock, Stephen J.; Pierce, Brandon L.; Zheng, Wei

2016-01-01

Background Observational studies examining associations between adult height and risk of colorectal, prostate, and lung cancers have generated mixed results. We conducted meta-analyses using data from prospective cohort studies and further carried out Mendelian randomization analyses, using height-associated genetic variants identified in a genome-wide association study (GWAS), to evaluate the association of adult height with these cancers. Methods and Findings A systematic review of prospective studies was conducted using the PubMed, Embase, and Web of Science databases. Using meta-analyses, results obtained from 62 studies were summarized for the association of a 10-cm increase in height with cancer risk. Mendelian randomization analyses were conducted using summary statistics obtained for 423 genetic variants identified from a recent GWAS of adult height and from a cancer genetics consortium study of multiple cancers that included 47,800 cases and 81,353 controls. For a 10-cm increase in height, the summary relative risks derived from the meta-analyses of prospective studies were 1.12 (95% CI 1.10, 1.15), 1.07 (95% CI 1.05, 1.10), and 1.06 (95% CI 1.02, 1.11) for colorectal, prostate, and lung cancers, respectively. Mendelian randomization analyses showed increased risks of colorectal (odds ratio [OR] = 1.58, 95% CI 1.14, 2.18) and lung cancer (OR = 1.10, 95% CI 1.00, 1.22) associated with each 10-cm increase in genetically predicted height. No association was observed for prostate cancer (OR = 1.03, 95% CI 0.92, 1.15). Our meta-analysis was limited to published studies. The sample size for the Mendelian randomization analysis of colorectal cancer was relatively small, thus affecting the precision of the point estimate. Conclusions Our study provides evidence for a potential causal association of adult height with the risk of colorectal and lung cancers and suggests that certain genetic factors and biological pathways affecting adult height may also affect the

Identifying Frequent Users of an Urban Emergency Medical Service Using Descriptive Statistics and Regression Analyses.

PubMed

Norman, Chenelle; Mello, Michael; Choi, Bryan

2016-01-01

This retrospective cohort study provides a descriptive analysis of a population that frequently uses an urban emergency medical service (EMS) and identifies factors that contribute to use among all frequent users. For purposes of this study we divided frequent users into the following groups: low- frequent users (4 EMS transports in 2012), medium-frequent users (5 to 6 EMS transports in 2012), high-frequent users (7 to 10 EMS transports in 2012) and super-frequent users (11 or more EMS transports in 2012). Overall, we identified 539 individuals as frequent users. For all groups of EMS frequent users (i.e. low, medium, high and super) one or more hospital admissions, receiving a referral for follow-up care upon discharge, and having no insurance were found to be statistically significant with frequent EMS use (P<0.05). Within the diagnostic categories, 41.61% of super-frequent users had a diagnosis of "primarily substance abuse/misuse" and among low-frequent users a majority, 53.33%, were identified as having a "reoccurring (medical) diagnosis." Lastly, relative risk ratios for the highest group of users, super-frequent users, were 3.34 (95% CI [1.90-5.87]) for obtaining at least one referral for follow-up care, 13.67 (95% CI [5.60-33.34]) for having four or more hospital admissions and 5.95 (95% CI [1.80-19.63]) for having a diagnoses of primarily substance abuse/misuse. Findings from this study demonstrate that among low- and medium-frequent users a majority of patients are using EMS for reoccurring medical conditions. This could potentially be avoided with better care management. In addition, this study adds to the current literature that illustrates a strong correlation between substance abuse/misuse and high/super-frequent EMS use. For the subgroup analysis among individuals 65 years of age and older, we did not find any of the independent variables included in our model to be statistically significant with frequent EMS use.

Low Reporting Quality of the Meta-Analyses in Diagnostic Pathology.

PubMed

Liu, Xulei; Kinzler, Michael; Yuan, Jiangfan; He, Guozhong; Zhang, Lanjing

2017-03-01

- Little is known regarding the reporting quality of meta-analyses in diagnostic pathology. - To compare reporting quality of meta-analyses in diagnostic pathology and medicine and to examine factors associated with reporting quality of diagnostic pathology meta-analyses. - Meta-analyses were identified in 12 major diagnostic pathology journals without specifying years and 4 major medicine journals in 2006 and 2011 using PubMed. Reporting quality of meta-analyses was evaluated using the 27-item checklist of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement published in 2009. A higher PRISMA score indicates higher reporting quality. - Forty-one diagnostic pathology meta-analyses and 118 medicine meta-analyses were included. Overall, reporting quality of meta-analyses in diagnostic pathology was lower than that in medicine (median [interquartile range] = 22 [15, 25] versus 27 [23, 28], P < .001). Compared with medicine meta-analyses, diagnostic pathology meta-analyses less likely reported 23 of the 27 items (85.2%) on the PRISMA checklist, but more likely reported the data items. Higher reporting quality of diagnostic pathology meta-analyses was associated with recent publication years (later than 2009 versus 2009 or earlier, P = .002) and non-North American first authors (versus North American, P = .001), but not journal publisher's location (P = .11). Interestingly, reporting quality was not associated with adjusted citation ratio for meta-analyses in either diagnostic pathology or medicine (P = .40 and P = .09, respectively). - Meta-analyses in diagnostic pathology had lower reporting quality than those in medicine. Reporting quality of diagnostic pathology meta-analyses is linked to publication year and first author's location, but not to journal publisher's location or article's adjusted citation ratios. More research and education on meta-analysis methodology may improve the reporting quality of diagnostic pathology meta-analyses.

Molecular and functional analyses of the contractile apparatus in lymphatic muscle

NASA Technical Reports Server (NTRS)

Muthuchamy, Mariappan; Gashev, Anatoliy; Boswell, Niven; Dawson, Nancy; Zawieja, David; Delp, Z. (Principal Investigator)

2003-01-01

Lymphatics are necessary for the generation and regulation of lymph flow. Lymphatics use phasic contractions and extrinsic compressions to generate flow; tonic contractions alter resistance. Lymphatic muscle exhibits important differences from typical vascular smooth muscle. In this study, the thoracic duct exhibited significant functional differences from mesenteric lymphatics. To understand the molecular basis for these differences, we examined the profiles of contractile proteins and their messages in mesenteric lymphatics, thoracic duct, and arterioles. Results demonstrated that mesenteric lymphatics express only SMB smooth muscle myosin heavy chain (SM-MHC), whereas thoracic duct and arterioles expressed both SMA and SMB isoforms. Both SM1 and SM2 isoforms of SM-MHC were detected in arterioles and mesenteric and thoracic lymphatics. In addition, the fetal cardiac/skeletal slow-twitch muscle-specific beta-MHC message was detected only in mesenteric lymphatics. All four actin messages, cardiac alpha-actin, vascular alpha-actin, enteric gamma-actin, and skeletal alpha-actin, were present in both mesenteric lymphatics and arterioles. However, in thoracic duct, predominantly cardiac alpha-actin and vascular alpha-actin were found. Western blot and immunohistochemical analyses corroborated the mRNA studies. However, in arterioles only vascular alpha-actin protein was detected. These data indicate that lymphatics display genotypic and phenotypic characteristics of vascular, cardiac, and visceral myocytes, which are needed to fulfill the unique roles of the lymphatic system.

Data Identifiers and Citations Enable Reproducible Science

NASA Astrophysics Data System (ADS)

Tilmes, C.

2011-12-01

Modern science often involves data processing with tremendous volumes of data. Keeping track of that data has been a growing challenge for data center. Researchers who access and use that data don't always reference and cite their data sources adequately for consumers of their research to follow their methodology or reproduce their analyses or experiments. Recent research has led to recommendations for good identifiers and citations that can help address this problem. This paper will describe some of the best practices in data identifiers, reference and citation. Using a simplified example scenario based on a long term remote sensing satellite mission, it will explore issues in identifying dynamic data sets and the importance of good data citations for reproducibility. It will describe the difference between granule and collection level identifiers, using UUIDs and DOIs to illustrate some recommendations for developing identifiers and assigning them during data processing. As data processors create data products, the provenance of the input products and precise steps that led to their creation are recorded and published for users of the data to see. As researchers access the data from an archive, they can use the provenance to help understand the genesis of the data, which could have effects on their usage of the data. By citing the data on publishing their research, others can retrieve the precise data used in their research and reproduce the analyses and experiments to confirm the results. Describing the experiment to a sufficient extent to reproduce the research enforces a formal approach that lends credibility to the results, and ultimately, to the policies of decision makers depending on that research.

Identifying genes affectng stress response in rainbow trout

USDA-ARS?s Scientific Manuscript database

Genomic analyses have the potential to impact aquaculture production traits by identifying markers as proxies for traits which are expensive or difficult to measure and characterizing genetic variation and biochemical mechanisms underlying phenotypic variation. One such set of traits are the respon...

Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva

PubMed Central

Yasmin, Rubina; Barber, Cheryl A.; Castro, Talita; Malamud, Daniel; Kim, Beum Jun; Zhu, Hui; Montagna, Richard A.; Abrams, William R.

2018-01-01

In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co) in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP) and reverse dot-blot for detection (RDB) and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV) RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease. PMID:29401479

Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva.

PubMed

Sabalza, Maite; Yasmin, Rubina; Barber, Cheryl A; Castro, Talita; Malamud, Daniel; Kim, Beum Jun; Zhu, Hui; Montagna, Richard A; Abrams, William R

2018-01-01

In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co) in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP) and reverse dot-blot for detection (RDB) and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV) RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease.

Two-Phase Contiguous Supported Lipid Bilayer Model for Membrane Rafts via Polymer Blotting and Stenciling.

PubMed

Richards, Mark J; Daniel, Susan

2017-02-07

The supported lipid bilayer has been portrayed as a useful model of the cell membrane compatible with many biophysical tools and techniques that demonstrate its appeal in learning about the basic features of the plasma membrane. However, some of its potential has yet to be realized, particularly in the area of bilayer patterning and phase/composition heterogeneity. In this work, we generate contiguous bilayer patterns as a model system that captures the general features of membrane domains and lipid rafts. Micropatterned polymer templates of two types are investigated for generating patterned bilayer formation: polymer blotting and polymer lift-off stenciling. While these approaches have been used previously to create bilayer arrays by corralling bilayers patches with various types of boundaries impenetrable to bilayer diffusion, unique to the methods presented here, there are no physical barriers to diffusion. In this work, interfaces between contiguous lipid phases define the pattern shapes, with continuity between them allowing transfer of membrane-bound biomolecules between the phases. We examine effectors of membrane domain stability including temperature and cholesterol content to investigate domain dynamics. Contiguous patterning of supported bilayers as a model of lipid rafts expands the application of the SLB to an area with current appeal and brings with it a useful toolset for characterization and analysis. These combined tools should be helpful to researchers investigating lipid raft dynamics and function and biomolecule partitioning studies. Additionally, this patterning technique may be useful for applications such as bioseparations that exploit differences in lipid phase partitioning or creation of membranes that bind species like viruses preferentially at lipid phase boundaries, to name a few.

Genome-Wide Search Identifies 1.9 Mb from the Polar Bear Y Chromosome for Evolutionary Analyses

PubMed Central

Bidon, Tobias; Schreck, Nancy; Hailer, Frank; Nilsson, Maria A.; Janke, Axel

2015-01-01

The male-inherited Y chromosome is the major haploid fraction of the mammalian genome, rendering Y-linked sequences an indispensable resource for evolutionary research. However, despite recent large-scale genome sequencing approaches, only a handful of Y chromosome sequences have been characterized to date, mainly in model organisms. Using polar bear (Ursus maritimus) genomes, we compare two different in silico approaches to identify Y-linked sequences: 1) Similarity to known Y-linked genes and 2) difference in the average read depth of autosomal versus sex chromosomal scaffolds. Specifically, we mapped available genomic sequencing short reads from a male and a female polar bear against the reference genome and identify 112 Y-chromosomal scaffolds with a combined length of 1.9 Mb. We verified the in silico findings for the longer polar bear scaffolds by male-specific in vitro amplification, demonstrating the reliability of the average read depth approach. The obtained Y chromosome sequences contain protein-coding sequences, single nucleotide polymorphisms, microsatellites, and transposable elements that are useful for evolutionary studies. A high-resolution phylogeny of the polar bear patriline shows two highly divergent Y chromosome lineages, obtained from analysis of the identified Y scaffolds in 12 previously published male polar bear genomes. Moreover, we find evidence of gene conversion among ZFX and ZFY sequences in the giant panda lineage and in the ancestor of ursine and tremarctine bears. Thus, the identification of Y-linked scaffold sequences from unordered genome sequences yields valuable data to infer phylogenomic and population-genomic patterns in bears. PMID:26019166

Localization and Distribution of 'Candidatus Liberibacter asiaticus’ in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody

PubMed Central

Ding, Fang; Duan, Yongping; Paul, Cristina; Brlansky, Ronald H.; Hartung, John S.

2015-01-01

‘Candidatus Liberibacter asiaticus’ (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant. PMID:25946013

Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

PubMed

Ding, Fang; Duan, Yongping; Paul, Cristina; Brlansky, Ronald H; Hartung, John S

2015-01-01

'Candidatus Liberibacter asiaticus' (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.

Precise Chemical Analyses of Planetary Surfaces

NASA Technical Reports Server (NTRS)

Kring, David; Schweitzer, Jeffrey; Meyer, Charles; Trombka, Jacob; Freund, Friedemann; Economou, Thanasis; Yen, Albert; Kim, Soon Sam; Treiman, Allan H.; Blake, David;

Genome-wide association analyses identify new susceptibility loci for oral cavity and pharyngeal cancer.

PubMed

Lesseur, Corina; Diergaarde, Brenda; Olshan, Andrew F; Wünsch-Filho, Victor; Ness, Andrew R; Liu, Geoffrey; Lacko, Martin; Eluf-Neto, José; Franceschi, Silvia; Lagiou, Pagona; Macfarlane, Gary J; Richiardi, Lorenzo; Boccia, Stefania; Polesel, Jerry; Kjaerheim, Kristina; Zaridze, David; Johansson, Mattias; Menezes, Ana M; Curado, Maria Paula; Robinson, Max; Ahrens, Wolfgang; Canova, Cristina; Znaor, Ariana; Castellsagué, Xavier; Conway, David I; Holcátová, Ivana; Mates, Dana; Vilensky, Marta; Healy, Claire M; Szeszenia-Dąbrowska, Neonila; Fabiánová, Eleonóra; Lissowska, Jolanta; Grandis, Jennifer R; Weissler, Mark C; Tajara, Eloiza H; Nunes, Fabio D; de Carvalho, Marcos B; Thomas, Steve; Hung, Rayjean J; Peters, Wilbert H M; Herrero, Rolando; Cadoni, Gabriella; Bueno-de-Mesquita, H Bas; Steffen, Annika; Agudo, Antonio; Shangina, Oxana; Xiao, Xiangjun; Gaborieau, Valérie; Chabrier, Amélie; Anantharaman, Devasena; Boffetta, Paolo; Amos, Christopher I; McKay, James D; Brennan, Paul

2016-12-01

We conducted a genome-wide association study of oral cavity and pharyngeal cancer in 6,034 cases and 6,585 controls from Europe, North America and South America. We detected eight significantly associated loci (P < 5 × 10 -8 ), seven of which are new for these cancer sites. Oral and pharyngeal cancers combined were associated with loci at 6p21.32 (rs3828805, HLA-DQB1), 10q26.13 (rs201982221, LHPP) and 11p15.4 (rs1453414, OR52N2-TRIM5). Oral cancer was associated with two new regions, 2p23.3 (rs6547741, GPN1) and 9q34.12 (rs928674, LAMC3), and with known cancer-related loci-9p21.3 (rs8181047, CDKN2B-AS1) and 5p15.33 (rs10462706, CLPTM1L). Oropharyngeal cancer associations were limited to the human leukocyte antigen (HLA) region, and classical HLA allele imputation showed a protective association with the class II haplotype HLA-DRB1*1301-HLA-DQA1*0103-HLA-DQB1*0603 (odds ratio (OR) = 0.59, P = 2.7 × 10 -9 ). Stratified analyses on a subgroup of oropharyngeal cases with information available on human papillomavirus (HPV) status indicated that this association was considerably stronger in HPV-positive (OR = 0.23, P = 1.6 × 10 -6 ) than in HPV-negative (OR = 0.75, P = 0.16) cancers.

Genome-Wide Search Identifies 1.9 Mb from the Polar Bear Y Chromosome for Evolutionary Analyses.

PubMed

Bidon, Tobias; Schreck, Nancy; Hailer, Frank; Nilsson, Maria A; Janke, Axel

2015-05-27

The male-inherited Y chromosome is the major haploid fraction of the mammalian genome, rendering Y-linked sequences an indispensable resource for evolutionary research. However, despite recent large-scale genome sequencing approaches, only a handful of Y chromosome sequences have been characterized to date, mainly in model organisms. Using polar bear (Ursus maritimus) genomes, we compare two different in silico approaches to identify Y-linked sequences: 1) Similarity to known Y-linked genes and 2) difference in the average read depth of autosomal versus sex chromosomal scaffolds. Specifically, we mapped available genomic sequencing short reads from a male and a female polar bear against the reference genome and identify 112 Y-chromosomal scaffolds with a combined length of 1.9 Mb. We verified the in silico findings for the longer polar bear scaffolds by male-specific in vitro amplification, demonstrating the reliability of the average read depth approach. The obtained Y chromosome sequences contain protein-coding sequences, single nucleotide polymorphisms, microsatellites, and transposable elements that are useful for evolutionary studies. A high-resolution phylogeny of the polar bear patriline shows two highly divergent Y chromosome lineages, obtained from analysis of the identified Y scaffolds in 12 previously published male polar bear genomes. Moreover, we find evidence of gene conversion among ZFX and ZFY sequences in the giant panda lineage and in the ancestor of ursine and tremarctine bears. Thus, the identification of Y-linked scaffold sequences from unordered genome sequences yields valuable data to infer phylogenomic and population-genomic patterns in bears. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Differential proteomic and tissue expression analyses identify valuable diagnostic biomarkers of hepatocellular differentiation and hepatoid adenocarcinomas.

PubMed

Reis, Henning; Padden, Juliet; Ahrens, Maike; Pütter, Carolin; Bertram, Stefanie; Pott, Leona L; Reis, Anna-Carinna; Weber, Frank; Juntermanns, Benjamin; Hoffmann, Andreas-C; Eisenacher, Martin; Schlaak, Joörg F; Canbay, Ali; Meyer, Helmut E; Sitek, Barbara; Baba, Hideo A

2015-10-01

The exact discrimination of lesions with true hepatocellular differentiation from secondary tumours and neoplasms with hepatocellular histomorphology like hepatoid adenocarcinomas (HAC) is crucial. Therefore, we aimed to identify ancillary protein biomarkers by using complementary proteomic techniques (2D-DIGE, label-free MS). The identified candidates were immunohistochemically validated in 14 paired samples of hepatocellular carcinoma (HCC) and non-tumourous liver tissue (NT). The candidates and HepPar1/Arginase1 were afterwards tested for consistency in a large cohort of hepatocellular lesions and NT (n = 290), non-hepatocellular malignancies (n = 383) and HAC (n = 13). Eight non-redundant, differentially expressed proteins were suitable for further immunohistochemical validation and four (ABAT, BHMT, FABP1, HAOX1) for further evaluation. Sensitivity and specificity rates for HCC/HAC were as follows: HepPar1 80.2%, 94.3% / 80.2%, 46.2%; Arginase1 82%, 99.4% / 82%, 69.2%; BHMT 61.4%, 93.8% / 61.4%, 100%; ABAT 84.4%, 33.7% / 84.4%, 30.8%; FABP1 87.2%, 95% / 87.2%, 69.2%; HAOX1 95.5%, 36.3% / 95.5%, 46.2%. The best 2×/3× biomarker panels for the diagnosis of HCC consisted of Arginase1/HAOX1 and BHMT/Arginase1/HAOX1 and for HAC consisted of Arginase1/FABP1 and BHMT/Arginase1/FABP1. In summary, we successfully identified, validated and benchmarked protein biomarker candidates of hepatocellular differentiation. BHMT in particular exhibited superior diagnostic characteristics in hepatocellular lesions and specifically in HAC. BHMT is therefore a promising (panel based) biomarker candidate in the differential diagnostic process of lesions with hepatocellular aspect.

Linkage and related analyses of Barrett's esophagus and its associated adenocarcinomas.

PubMed

Sun, Xiangqing; Elston, Robert; Falk, Gary W; Grady, William M; Faulx, Ashley; Mittal, Sumeet K; Canto, Marcia I; Shaheen, Nicholas J; Wang, Jean S; Iyer, Prasad G; Abrams, Julian A; Willis, Joseph E; Guda, Kishore; Markowitz, Sanford; Barnholtz-Sloan, Jill S; Chandar, Apoorva; Brock, Wendy; Chak, Amitabh

2016-07-01

Familial aggregation and segregation analysis studies have provided evidence of a genetic basis for esophageal adenocarcinoma (EAC) and its premalignant precursor, Barrett's esophagus (BE). We aim to demonstrate the utility of linkage analysis to identify the genomic regions that might contain the genetic variants that predispose individuals to this complex trait (BE and EAC). We genotyped 144 individuals in 42 multiplex pedigrees chosen from 1000 singly ascertained BE/EAC pedigrees, and performed both model-based and model-free linkage analyses, using S.A.G.E. and other software. Segregation models were fitted, from the data on both the 42 pedigrees and the 1000 pedigrees, to determine parameters for performing model-based linkage analysis. Model-based and model-free linkage analyses were conducted in two sets of pedigrees: the 42 pedigrees and a subset of 18 pedigrees with female affected members that are expected to be more genetically homogeneous. Genome-wide associations were also tested in these families. Linkage analyses on the 42 pedigrees identified several regions consistently suggestive of linkage by different linkage analysis methods on chromosomes 2q31, 12q23, and 4p14. A linkage on 15q26 is the only consistent linkage region identified in the 18 female-affected pedigrees, in which the linkage signal is higher than in the 42 pedigrees. Other tentative linkage signals are also reported. Our linkage study of BE/EAC pedigrees identified linkage regions on chromosomes 2, 4, 12, and 15, with some reported associations located within our linkage peaks. Our linkage results can help prioritize association tests to delineate the genetic determinants underlying susceptibility to BE and EAC.

Overlapping meta-analyses on the same topic: survey of published studies.

PubMed

Siontis, Konstantinos C; Hernandez-Boussard, Tina; Ioannidis, John P A

2013-07-19

To assess how common it is to have multiple overlapping meta-analyses of randomized trials published on the same topic. Survey of published meta-analyses. PubMed. Meta-analyses published in 2010 were identified, and 5% of them were randomly selected. We further selected those that included randomized trials and examined effectiveness of any medical intervention. For eligible meta-analyses, we searched for other meta-analyses on the same topic (covering the same comparisons, indications/settings, and outcomes or overlapping subsets of them) published until February 2013. Of 73 eligible meta-analyses published in 2010, 49 (67%) had at least one other overlapping meta-analysis (median two meta-analyses per topic, interquartile range 1-4, maximum 13). In 17 topics at least one author was involved in at least two of the overlapping meta-analyses. No characteristics of the index meta-analyses were associated with the potential for overlapping meta-analyses. Among pairs of overlapping meta-analyses in 20 randomly selected topics, 13 of the more recent meta-analyses did not include any additional outcomes. In three of the four topics with eight or more published meta-analyses, many meta-analyses examined only a subset of the eligible interventions or indications/settings covered by the index meta-analysis. Conversely, for statins in the prevention of atrial fibrillation after cardiac surgery, 11 meta-analyses were published with similar eligibility criteria for interventions and setting: there was still variability on which studies were included, but the results were always similar or even identical across meta-analyses. While some independent replication of meta-analyses by different teams is possibly useful, the overall picture suggests that there is a waste of efforts with many topics covered by multiple overlapping meta-analyses.

Assessment of the Impact of Scheduled Postmarketing Safety Summary Analyses on Regulatory Actions

PubMed Central

Sekine, S; Pinnow, EE; Wu, E; Kurtzig, R; Hall, M; Dal Pan, GJ

2016-01-01

In addition to standard postmarketing drug safety monitoring, Section 915 of the Food and Drug Administration Amendments Act of 2007 (FDAAA) requires the US Food and Drug Administration (FDA) to conduct a summary analysis of adverse event reports to identify risks of a drug or biologic product 18 months after product approval, or after 10,000 patients have used the product, whichever is later. We assessed the extent to which these analyses identified new safety signals and resultant safety-related label changes. Among 458 newly approved products, 300 were the subjects of a scheduled analysis; a new safety signal that resulted in a safety-related label change was found for 11 of these products. Less than 2% of 713 safety-related label changes were based on the scheduled analyses. Our study suggests that the safety summary analyses provide only marginal value over other pharmacovigilance activities. PMID:26853718

Association analyses of more than 140,000 men identify 63 new prostate cancer susceptibility loci.

PubMed

Schumacher, Fredrick R; Al Olama, Ali Amin; Berndt, Sonja I; Benlloch, Sara; Ahmed, Mahbubl; Saunders, Edward J; Dadaev, Tokhir; Leongamornlert, Daniel; Anokian, Ezequiel; Cieza-Borrella, Clara; Goh, Chee; Brook, Mark N; Sheng, Xin; Fachal, Laura; Dennis, Joe; Tyrer, Jonathan; Muir, Kenneth; Lophatananon, Artitaya; Stevens, Victoria L; Gapstur, Susan M; Carter, Brian D; Tangen, Catherine M; Goodman, Phyllis J; Thompson, Ian M; Batra, Jyotsna; Chambers, Suzanne; Moya, Leire; Clements, Judith; Horvath, Lisa; Tilley, Wayne; Risbridger, Gail P; Gronberg, Henrik; Aly, Markus; Nordström, Tobias; Pharoah, Paul; Pashayan, Nora; Schleutker, Johanna; Tammela, Teuvo L J; Sipeky, Csilla; Auvinen, Anssi; Albanes, Demetrius; Weinstein, Stephanie; Wolk, Alicja; Håkansson, Niclas; West, Catharine M L; Dunning, Alison M; Burnet, Neil; Mucci, Lorelei A; Giovannucci, Edward; Andriole, Gerald L; Cussenot, Olivier; Cancel-Tassin, Géraldine; Koutros, Stella; Beane Freeman, Laura E; Sorensen, Karina Dalsgaard; Orntoft, Torben Falck; Borre, Michael; Maehle, Lovise; Grindedal, Eli Marie; Neal, David E; Donovan, Jenny L; Hamdy, Freddie C; Martin, Richard M; Travis, Ruth C; Key, Tim J; Hamilton, Robert J; Fleshner, Neil E; Finelli, Antonio; Ingles, Sue Ann; Stern, Mariana C; Rosenstein, Barry S; Kerns, Sarah L; Ostrer, Harry; Lu, Yong-Jie; Zhang, Hong-Wei; Feng, Ninghan; Mao, Xueying; Guo, Xin; Wang, Guomin; Sun, Zan; Giles, Graham G; Southey, Melissa C; MacInnis, Robert J; FitzGerald, Liesel M; Kibel, Adam S; Drake, Bettina F; Vega, Ana; Gómez-Caamaño, Antonio; Szulkin, Robert; Eklund, Martin; Kogevinas, Manolis; Llorca, Javier; Castaño-Vinyals, Gemma; Penney, Kathryn L; Stampfer, Meir; Park, Jong Y; Sellers, Thomas A; Lin, Hui-Yi; Stanford, Janet L; Cybulski, Cezary; Wokolorczyk, Dominika; Lubinski, Jan; Ostrander, Elaine A; Geybels, Milan S; Nordestgaard, Børge G; Nielsen, Sune F; Weischer, Maren; Bisbjerg, Rasmus; Røder, Martin Andreas; Iversen, Peter; Brenner, Hermann; Cuk, Katarina; Holleczek, Bernd; Maier, Christiane; Luedeke, Manuel; Schnoeller, Thomas; Kim, Jeri; Logothetis, Christopher J; John, Esther M; Teixeira, Manuel R; Paulo, Paula; Cardoso, Marta; Neuhausen, Susan L; Steele, Linda; Ding, Yuan Chun; De Ruyck, Kim; De Meerleer, Gert; Ost, Piet; Razack, Azad; Lim, Jasmine; Teo, Soo-Hwang; Lin, Daniel W; Newcomb, Lisa F; Lessel, Davor; Gamulin, Marija; Kulis, Tomislav; Kaneva, Radka; Usmani, Nawaid; Singhal, Sandeep; Slavov, Chavdar; Mitev, Vanio; Parliament, Matthew; Claessens, Frank; Joniau, Steven; Van den Broeck, Thomas; Larkin, Samantha; Townsend, Paul A; Aukim-Hastie, Claire; Dominguez, Manuela Gago; Castelao, Jose Esteban; Martinez, Maria Elena; Roobol, Monique J; Jenster, Guido; van Schaik, Ron H N; Menegaux, Florence; Truong, Thérèse; Koudou, Yves Akoli; Xu, Jianfeng; Khaw, Kay-Tee; Cannon-Albright, Lisa; Pandha, Hardev; Michael, Agnieszka; Thibodeau, Stephen N; McDonnell, Shannon K; Schaid, Daniel J; Lindstrom, Sara; Turman, Constance; Ma, Jing; Hunter, David J; Riboli, Elio; Siddiq, Afshan; Canzian, Federico; Kolonel, Laurence N; Le Marchand, Loic; Hoover, Robert N; Machiela, Mitchell J; Cui, Zuxi; Kraft, Peter; Amos, Christopher I; Conti, David V; Easton, Douglas F; Wiklund, Fredrik; Chanock, Stephen J; Henderson, Brian E; Kote-Jarai, Zsofia; Haiman, Christopher A; Eeles, Rosalind A

2018-06-11

Genome-wide association studies (GWAS) and fine-mapping efforts to date have identified more than 100 prostate cancer (PrCa)-susceptibility loci. We meta-analyzed genotype data from a custom high-density array of 46,939 PrCa cases and 27,910 controls of European ancestry with previously genotyped data of 32,255 PrCa cases and 33,202 controls of European ancestry. Our analysis identified 62 novel loci associated (P < 5.0 × 10 -8 ) with PrCa and one locus significantly associated with early-onset PrCa (≤55 years). Our findings include missense variants rs1800057 (odds ratio (OR) = 1.16; P = 8.2 × 10 -9 ; G>C, p.Pro1054Arg) in ATM and rs2066827 (OR = 1.06; P = 2.3 × 10 -9 ; T>G, p.Val109Gly) in CDKN1B. The combination of all loci captured 28.4% of the PrCa familial relative risk, and a polygenic risk score conferred an elevated PrCa risk for men in the ninetieth to ninety-ninth percentiles (relative risk = 2.69; 95% confidence interval (CI): 2.55-2.82) and first percentile (relative risk = 5.71; 95% CI: 5.04-6.48) risk stratum compared with the population average. These findings improve risk prediction, enhance fine-mapping, and provide insight into the underlying biology of PrCa 1 .

Fourier Transform Mass Spectrometry: The Transformation of Modern Environmental Analyses

PubMed Central

Lim, Lucy; Yan, Fangzhi; Bach, Stephen; Pihakari, Katianna; Klein, David

2016-01-01

Unknown compounds in environmental samples are difficult to identify using standard mass spectrometric methods. Fourier transform mass spectrometry (FTMS) has revolutionized how environmental analyses are performed. With its unsurpassed mass accuracy, high resolution and sensitivity, researchers now have a tool for difficult and complex environmental analyses. Two features of FTMS are responsible for changing the face of how complex analyses are accomplished. First is the ability to quickly and with high mass accuracy determine the presence of unknown chemical residues in samples. For years, the field has been limited by mass spectrometric methods that were based on knowing what compounds of interest were. Secondly, by utilizing the high resolution capabilities coupled with the low detection limits of FTMS, analysts also could dilute the sample sufficiently to minimize the ionization changes from varied matrices. PMID:26784175

Novel cytosolic allergens of Aspergillus fumigatus identified from germinating conidia.

PubMed

Singh, Bharat; Sharma, Gainda L; Oellerich, Michael; Kumar, Ram; Singh, Seema; Bhadoria, Dharam P; Katyal, Anju; Reichard, Utz; Asif, Abdul R

2010-11-05

Aspergillus fumigatus is the common cause of allergic broncho-pulmonary aspergillosis (ABPA) and most of the allergens have been described from its secreted fraction. In the present investigation, germinating conidial cytosolic proteins of A. fumigatus were extracted from a 16 h culture. The proteome from this fraction was developed, and immuno-blots were generated using pooled ABPA patients' sera. Well separated Immunoglobulin-E (IgE) and Immunoglobulin-G (IgG) reactive spots were picked from corresponding 2DE gels and subjected to mass spectrometric analysis. As a result, 66 immuno-reactive proteins were identified from two geographically different strains (190/96 and DAYA) of A. fumigatus. Only 3 out of 66 proteins reacted with IgG, and the remaining 63 proteins were found to be IgE reactive. These 63 IgE-reactive cytosolic proteins from germinating conidia included 2 already known (Asp f12 and Asp f22) and 4 predicted allergens (Hsp88, Hsp70, malate dehydrogenase, and alcohol dehydrogenase) based on their homology with other known fungal allergens. In view of this, the panel of presently identified IgE-reactive novel proteins holds the potential of providing a basis for the wider diagnostic application in assay for allergic aspergillosis. We could demonstrate that recombinantly expressed proteins from this panel showed consistent reactivity with IgE of individual sera of ABPA patients. The recombinantly expressed proteins may also be useful in desensitization therapy of allergic disorders including ABPA.

Predictors of Post-School Success: A Systematic Review of NLTS2 Secondary Analyses

ERIC Educational Resources Information Center

Mazzotti, Valerie L.; Rowe, Dawn A.; Sinclair, James; Poppen, Marcus; Woods, William E.; Shearer, Mackenzie L.

2016-01-01

The purpose of this systematic review was to (a) systematically review the literature to identify National Longitudinal Transition Study-2 secondary analyses articles published since 2009 that met the quality indicators for correlational research, (b) further extend the findings of Test et al. by identifying additional evidence to support the…

Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA*

PubMed Central

Schoenherr, Regine M.; Saul, Richard G.; Whiteaker, Jeffrey R.; Yan, Ping; Whiteley, Gordon R.; Paulovich, Amanda G.

2015-01-01

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first

Serum detection of IgG antibodies against Demodex canis by western blot in healthy dogs and dogs with juvenile generalized demodicosis.

PubMed

Ravera, Ivan; Ferreira, Diana; Gallego, Laia Solano; Bardagí, Mar; Ferrer, Lluís

2015-08-01

The aim of this study was to investigate the presence of canine immunoglobulins (Ig) G against Demodex proteins in the sera of healthy dogs and of dogs with juvenile generalized demodicosis (CanJGD) with or without secondary pyoderma. Demodex mites were collected from dogs with CanJGD. Protein concentration was measured and a western blot technique was performed. Pooled sera from healthy dogs reacted mainly with antigen bands ranging from 55 to 72 kDa. Pooled sera from dogs with CanJGD without secondary pyoderma reacted either with 10 kDa antigen band or 55 to 72 kDa bands. Pooled sera from dogs with CanJGD with secondary pyoderma reacted only with a 10 kDa antigen band. The results of this study suggest that both healthy dogs and dogs with CanJGD develop a humoral response against different proteins of Demodex canis. Copyright © 2015 Elsevier Ltd. All rights reserved.

A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes.

PubMed

Fonseca, M E; Marcellino, L H; Gander, E

1996-04-05

A rapid and sensitive dot-blot hybridization assay using in vitro-transcribed digoxigenin-labelled RNA probes (riboprobes) was developed aiming at detection of citrus exocortis viroid (CEVd) in crude sap of infected Citrus medica plants. The protocol includes a very quick and simple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is highly recommended because the hybridization signals in formaldehyde-denatured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be sensitive enough to detect 0.1 ng of purified CEVd RNA and was able to detect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditions, avoiding non-specific background.

A Simple Method to Avoid Nonspecific Signal When Using Monoclonal Anti-Tau Antibodies in Western Blotting of Mouse Brain Proteins.

PubMed

Petry, Franck R; Nicholls, Samantha B; Hébert, Sébastien S; Planel, Emmanuel

2017-01-01

In Alzheimer's disease and other tauopathies, tau displays several abnormal post-translation modifications such as hyperphosphorylation, truncation, conformation, and oligomerization. Mouse monoclonal antibodies have been raised against such tau modifications for research, diagnostic, and therapeutic purposes. However, many of these primary antibodies are at risk of giving nonspecific signals in common Western blotting procedures. Not because they are unspecific, but because the secondary antibodies used to detect them will also detect the heavy chain of endogenous mouse immunoglobulins (Igs), and give a nonspecific signal at the same molecular weight than tau protein (around 50 kDa). Here, we propose the use of anti-light chain secondary antibodies as a simple and efficient technique to prevent nonspecific Igs signals at around 50 kDa. We demonstrate the efficacy of this method by removing artifactual signals when using monoclonal antibodies directed at tau phosphorylation (AT100, 12E8, AT270), tau truncation (TauC3), tau oligomerization (TOMA), or tau abnormal conformation (Alz50), in wild-type, 3×Tg-AD, and tau knockout mice.

Identifying sighting clusters of endangered taxa with historical records.

PubMed

Duffy, Karl J

2011-04-01

The probability and time of extinction of taxa is often inferred from statistical analyses of historical records. Many of these analyses require the exclusion of multiple records within a unit of time (i.e., a month or a year). Nevertheless, spatially explicit, temporally aggregated data may be useful for identifying clusters of sightings (i.e., sighting clusters) in space and time. Identification of sighting clusters highlights changes in the historical recording of endangered taxa. I used two methods to identify sighting clusters in historical records: the Ederer-Myers-Mantel (EMM) test and the space-time permutation scan (STPS). I applied these methods to the spatially explicit sighting records of three species of orchids that are listed as endangered in the Republic of Ireland under the Wildlife Act (1976): Cephalanthera longifolia, Hammarbya paludosa, and Pseudorchis albida. Results with the EMM test were strongly affected by the choice of the time interval, and thus the number of temporal samples, used to examine the records. For example, sightings of P. albida clustered when the records were partitioned into 20-year temporal samples, but not when they were partitioned into 22-year temporal samples. Because the statistical power of EMM was low, it will not be useful when data are sparse. Nevertheless, the STPS identified regions that contained sighting clusters because it uses a flexible scanning window (defined by cylinders of varying size that move over the study area and evaluate the likelihood of clustering) to detect them, and it identified regions with high and regions with low rates of orchid sightings. The STPS analyses can be used to detect sighting clusters of endangered species that may be related to regions of extirpation and may assist in the categorization of threat status. ©2010 Society for Conservation Biology.

Comparative Biochemical and Proteomic Analyses of Soybean Seed Cultivars Differing in Protein and Oil Content.

PubMed

Min, Chul Woo; Gupta, Ravi; Kim, So Wun; Lee, So Eui; Kim, Yong Chul; Bae, Dong Won; Han, Won Young; Lee, Byong Won; Ko, Jong Min; Agrawal, Ganesh Kumar; Rakwal, Randeep; Kim, Sun Tae

2015-08-19

This study develops differential protein profiles of soybean (Glycine max) seeds (cv. Saedanbaek and Daewon) varying in protein (47.9 and 39.2%) and oil (16.3 and 19.7%) content using protamine sulfate (PS) precipitation method coupled with a 2D gel electrophoresis (2DGE) approach. Of 71 detected differential spots between Daewon and Saedanbaek, 48 were successfully identified by MALDI-TOF/TOF. Gene ontology analysis revealed that up-regulated proteins in Saedanbaek were largely associated with nutrient reservoir activity (42.6%), which included mainly seed-storage proteins (SSPs; subunits of glycinin and β-conglycinin). Similar results were also obtained in two cultivars of wild soybean (G. soja cv. WS22 and WS15) differing in protein content. Western blots confirmed higher accumulation of SSPs in protein-rich Saedanbaek. Findings presented and discussed in this study highlight a possible involvement of the urea cycle for increased accumulation of SSPs and hence the higher protein content in soybean seeds.

Functional analyses of rare genetic variants in complement component C9 identified in patients with age-related macular degeneration.

PubMed

Kremlitzka, Mariann; Geerlings, Maartje J; de Jong, Sarah; Bakker, Bjorn; Nilsson, Sara C; Fauser, Sascha; Hoyng, Carel B; de Jong, Eiko K; den Hollander, Anneke I; Blom, Anna M

2018-05-14

Age-related macular degeneration (AMD) is a progressive disease of the central retina and the leading cause of irreversible vision loss in the western world. The involvement of abnormal complement activation in AMD has been suggested by association of variants in genes encoding complement proteins with disease development. A low-frequency variant (p.P167S) in the complement component C9 (C9) gene was recently shown to be highly associated with AMD, however its functional outcome remains largely unexplored. In this study, we reveal five novel rare genetic variants (p.M45L, p.F62S, p.G126R, p.T170I and p.A529T) in C9 in AMD patients, and evaluate their functional effects in vitro together with the previously identified (p.R118W and p.P167S) C9 variants.Our results demonstrate that the concentration of C9 is significantly elevated in patients' sera carrying the p.M45L, p.F62S, p.P167S and p.A529T variants compared to non-carrier controls. However, no difference can be observed in soluble terminal complement complex levels between the carrier and non-carrier groups. Comparing the polymerization of the C9 variants we reveal that the p.P167S mutant spontaneously aggregates, while the other mutant proteins (except for C9 p.A529T) fail to polymerize in the presence of zinc. Altered polymerization of the p.F62S and p.P167S proteins associated with decreased lysis of sheep erythrocytes and ARPE-19 cells by carriers' sera. Our data suggest that the analysed C9 variants affect only the secretion and polymerization of C9, without influencing its classical lytic activity. Future studies need to be performed to understand the implications of the altered polymerization of C9 in AMD pathology.

Genome-wide association analysis identifies 30 new susceptibility loci for schizophrenia.

PubMed

Li, Zhiqiang; Chen, Jianhua; Yu, Hao; He, Lin; Xu, Yifeng; Zhang, Dai; Yi, Qizhong; Li, Changgui; Li, Xingwang; Shen, Jiawei; Song, Zhijian; Ji, Weidong; Wang, Meng; Zhou, Juan; Chen, Boyu; Liu, Yahui; Wang, Jiqiang; Wang, Peng; Yang, Ping; Wang, Qingzhong; Feng, Guoyin; Liu, Benxiu; Sun, Wensheng; Li, Baojie; He, Guang; Li, Weidong; Wan, Chunling; Xu, Qi; Li, Wenjin; Wen, Zujia; Liu, Ke; Huang, Fang; Ji, Jue; Ripke, Stephan; Yue, Weihua; Sullivan, Patrick F; O'Donovan, Michael C; Shi, Yongyong

2017-11-01

We conducted a genome-wide association study (GWAS) with replication in 36,180 Chinese individuals and performed further transancestry meta-analyses with data from the Psychiatry Genomics Consortium (PGC2). Approximately 95% of the genome-wide significant (GWS) index alleles (or their proxies) from the PGC2 study were overrepresented in Chinese schizophrenia cases, including ∼50% that achieved nominal significance and ∼75% that continued to be GWS in the transancestry analysis. The Chinese-only analysis identified seven GWS loci; three of these also were GWS in the transancestry analyses, which identified 109 GWS loci, thus yielding a total of 113 GWS loci (30 novel) in at least one of these analyses. We observed improvements in the fine-mapping resolution at many susceptibility loci. Our results provide several lines of evidence supporting candidate genes at many loci and highlight some pathways for further research. Together, our findings provide novel insight into the genetic architecture and biological etiology of schizophrenia.

Job Hazard Analyses for Musculoskeletal Disorder Risk Factors in Pressing Operations of Dry-cleaning Establishments.

PubMed

Park, Jung-Keun

2016-12-01

Job hazard analyses were conducted to assess exposure to musculoskeletal disorder (MSD) risk factors in seven workers of three dry-cleaning establishments. In accordance with the Washington State Ergonomics Rule, the analyses were performed in two separate steps: (1) observation and checklist approaches were made to identify a "caution zone job" in the seven workers' pressing operations across the three shops; and (2) detailed posture and motion analyses were undertaken to determine a "MSD hazard" in one worker's operation using a video technique. One "caution zone job" was identified and it was the pressing operation job in which five physical risk factors were found in the pressing operations. The detailed analyses confirmed that one "MSD hazard", i.e., awkward posture in shoulders, was prevalent in the pressing operations of the three dry-cleaning facilities. It would be desirable to reduce MSD risk factors including awkward shoulder posture in the dry-cleaning industry.

Identifying children at risk for being bullies in the United States.

PubMed

Shetgiri, Rashmi; Lin, Hua; Flores, Glenn

2012-01-01

To identify risk factors associated with the greatest and lowest prevalence of bullying perpetration among U.S. children. Using the 2001-2002 Health Behavior in School-Aged Children, a nationally representative survey of U.S. children in 6th-10th grades, bivariate analyses were conducted to identify factors associated with any (once or twice or more), moderate (two to three times/month or more), and frequent (weekly or more) bullying. Stepwise multivariable analyses identified risk factors associated with bullying. Recursive partitioning analysis (RPA) identified risk factors which, in combination, identify students with the highest and lowest bullying prevalence. The prevalence of any bullying in the 13,710 students was 37.3%, moderate bullying was 12.6%, and frequent bullying was 6.6%. Characteristics associated with bullying were similar in the multivariable analyses and RPA clusters. In RPA, the highest prevalence of any bullying (67%) accrued in children with a combination of fighting and weapon-carrying. Students who carry weapons, smoke, and drink alcohol more than 5 to 6 days/week were at greatest risk for moderate bullying (61%). Those who carry weapons, smoke, have more than one alcoholic drink per day, have above-average academic performance, moderate/high family affluence, and feel irritable or bad-tempered daily were at greatest risk for frequent bullying (68%). Risk clusters for any, moderate, and frequent bullying differ. Children who fight and carry weapons are at greatest risk of any bullying. Weapon-carrying, smoking, and alcohol use are included in the greatest risk clusters for moderate and frequent bullying. Risk-group categories may be useful to providers in identifying children at the greatest risk for bullying and in targeting interventions. Copyright © 2012 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.

Longitudinal and cross-sectional analyses of visual field progression in participants of the Ocular Hypertension Treatment Study.

PubMed

Artes, Paul H; Chauhan, Balwantray C; Keltner, John L; Cello, Kim E; Johnson, Chris A; Anderson, Douglas R; Gordon, Mae O; Kass, Michael A

2010-12-01

To assess agreement between longitudinal and cross-sectional analyses for determining visual field progression in data from the Ocular Hypertension Treatment Study. Visual field data from 3088 eyes of 1570 participants (median follow-up, 7 years) were analyzed. Longitudinal analyses were performed using change probability with total and pattern deviation, and cross-sectional analyses were performed using the glaucoma hemifield test, corrected pattern standard deviation, and mean deviation. The rates of mean deviation and general height change were compared to estimate the degree of diffuse loss in emerging glaucoma. Agreement on progression in longitudinal and cross-sectional analyses ranged from 50% to 61% and remained nearly constant across a wide range of criteria. In contrast, agreement on absence of progression ranged from 97.0% to 99.7%, being highest for the stricter criteria. Analyses of pattern deviation were more conservative than analyses of total deviation, with a 3 to 5 times lesser incidence of progression. Most participants developing field loss had both diffuse and focal changes. Despite considerable overall agreement, 40% to 50% of eyes identified as having progressed with either longitudinal or cross-sectional analyses were identified with only one of the analyses. Because diffuse change is part of early glaucomatous damage, pattern deviation analyses may underestimate progression in patients with ocular hypertension.

Twenty years of meta-analyses in orthopaedic surgery: has quality kept up with quantity?

PubMed

Dijkman, Bernadette G; Abouali, Jihad A K; Kooistra, Bauke W; Conter, Henry J; Poolman, Rudolf W; Kulkarni, Abhaya V; Tornetta, Paul; Bhandari, Mohit

2010-01-01

As the number of studies in the literature is increasing, orthopaedic surgeons highly depend on meta-analyses as their primary source of scientific evidence. The objectives of this review were to assess the scientific quality and number of published meta-analyses on orthopaedics-related topics over time. We conducted, in duplicate and independently, a systematic review of published meta-analyses in orthopaedics in the years 2005 and 2008 and compared them with a previous systematic review of meta-analyses from 1969 to 1999. A search of electronic databases (MEDLINE, EMBASE, and the Cochrane Database of Systematic Reviews) was performed to identify meta-analyses published in 2005 and 2008. We searched bibliographies and contacted content experts to identify additional relevant studies. Two investigators independently assessed the quality of the studies, using the Oxman and Guyatt index, and abstracted relevant data. We included forty-five and forty-four meta-analyses from 2005 and 2008, respectively. While the number of meta-analyses increased fivefold from 1999 to 2008, the mean quality score did not change significantly over time (p = 0.067). In the later years, a significantly lower proportion of meta-analyses had methodological flaws (56% in 2005 and 68% in 2008) compared with meta-analyses published prior to 2000 (88%) (p = 0.006). In 2005 and 2008, respectively, 18% and 30% of the meta-analyses had major to extensive flaws in their methodology. Studies from 2008 with positive conclusions used and described appropriate criteria for the validity assessment less often than did those with negative results. The use of random-effects and fixed-effects models as pooling methods became more popular toward 2008. Although the methodological quality of orthopaedic meta-analyses has increased in the past twenty years, a substantial proportion continues to show major to extensive flaws. As the number of published meta-analyses is increasing, a routine checklist for

Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa).

PubMed

Cuttell, Leigh; Gómez-Morales, Maria Angeles; Cookson, Beth; Adams, Peter J; Reid, Simon A; Vanderlinde, Paul B; Jackson, Louise A; Gray, C; Traub, Rebecca J

2014-01-31

Trichinella surveillance in wildlife relies on muscle digestion of large samples which are logistically difficult to store and transport in remote and tropical regions as well as labour-intensive to process. Serological methods such as enzyme-linked immunosorbent assays (ELISAs) offer rapid, cost-effective alternatives for surveillance but should be paired with additional tests because of the high false-positive rates encountered in wildlife. We investigated the utility of ELISAs coupled with Western blot (WB) in providing evidence of Trichinella exposure or infection in wild boar. Serum samples were collected from 673 wild boar from a high- and low-risk region for Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were examined using both an 'in-house' and a commercially available indirect-ELISA that used excretory-secretory (E/S) antigens. Cut-off values for positive results were determined using sera from the low-risk population. All wild boar from the high-risk region (352) and 139/321 (43.3%) of the wild boar from the low-risk region were tested by artificial digestion. Testing by Western blot using E/S antigens, and a Trichinella-specific real-time PCR was also carried out on all ELISA-positive samples. The two ELISAs correctly classified all positive controls as well as one naturally infected wild boar from Gabba Island in the Torres Strait. In both the high- and low-risk populations, the ELISA results showed substantial agreement (k-value=0.66) that increased to very good (k-value=0.82) when WB-positive only samples were compared. The results of testing sera collected from the Australian mainland showed the Trichinella seroprevalence was 3.5% (95% C.I. 0.0-8.0) and 2.3% (95% C.I. 0.0-5.6) using the in-house and commercial ELISA coupled with WB respectively. These estimates were significantly higher (P<0.05) than the artificial digestion estimate of 0.0% (95% C.I. 0.0-1.1). Real-time PCR testing of muscle from

GeNets: a unified web platform for network-based genomic analyses.

PubMed

Li, Taibo; Kim, April; Rosenbluh, Joseph; Horn, Heiko; Greenfeld, Liraz; An, David; Zimmer, Andrew; Liberzon, Arthur; Bistline, Jon; Natoli, Ted; Li, Yang; Tsherniak, Aviad; Narayan, Rajiv; Subramanian, Aravind; Liefeld, Ted; Wong, Bang; Thompson, Dawn; Calvo, Sarah; Carr, Steve; Boehm, Jesse; Jaffe, Jake; Mesirov, Jill; Hacohen, Nir; Regev, Aviv; Lage, Kasper

2018-06-18

Functional genomics networks are widely used to identify unexpected pathway relationships in large genomic datasets. However, it is challenging to compare the signal-to-noise ratios of different networks and to identify the optimal network with which to interpret a particular genetic dataset. We present GeNets, a platform in which users can train a machine-learning model (Quack) to carry out these comparisons and execute, store, and share analyses of genetic and RNA-sequencing datasets.

Nonindependence and sensitivity analyses in ecological and evolutionary meta-analyses.

PubMed

Noble, Daniel W A; Lagisz, Malgorzata; O'dea, Rose E; Nakagawa, Shinichi

2017-05-01

Meta-analysis is an important tool for synthesizing research on a variety of topics in ecology and evolution, including molecular ecology, but can be susceptible to nonindependence. Nonindependence can affect two major interrelated components of a meta-analysis: (i) the calculation of effect size statistics and (ii) the estimation of overall meta-analytic estimates and their uncertainty. While some solutions to nonindependence exist at the statistical analysis stages, there is little advice on what to do when complex analyses are not possible, or when studies with nonindependent experimental designs exist in the data. Here we argue that exploring the effects of procedural decisions in a meta-analysis (e.g. inclusion of different quality data, choice of effect size) and statistical assumptions (e.g. assuming no phylogenetic covariance) using sensitivity analyses are extremely important in assessing the impact of nonindependence. Sensitivity analyses can provide greater confidence in results and highlight important limitations of empirical work (e.g. impact of study design on overall effects). Despite their importance, sensitivity analyses are seldom applied to problems of nonindependence. To encourage better practice for dealing with nonindependence in meta-analytic studies, we present accessible examples demonstrating the impact that ignoring nonindependence can have on meta-analytic estimates. We also provide pragmatic solutions for dealing with nonindependent study designs, and for analysing dependent effect sizes. Additionally, we offer reporting guidelines that will facilitate disclosure of the sources of nonindependence in meta-analyses, leading to greater transparency and more robust conclusions. © 2017 John Wiley & Sons Ltd.

Identifying and characterizing hepatitis C virus hotspots in Massachusetts: a spatial epidemiological approach.

PubMed

Stopka, Thomas J; Goulart, Michael A; Meyers, David J; Hutcheson, Marga; Barton, Kerri; Onofrey, Shauna; Church, Daniel; Donahue, Ashley; Chui, Kenneth K H

2017-04-20

Hepatitis C virus (HCV) infections have increased during the past decade but little is known about geographic clustering patterns. We used a unique analytical approach, combining geographic information systems (GIS), spatial epidemiology, and statistical modeling to identify and characterize HCV hotspots, statistically significant clusters of census tracts with elevated HCV counts and rates. We compiled sociodemographic and HCV surveillance data (n = 99,780 cases) for Massachusetts census tracts (n = 1464) from 2002 to 2013. We used a five-step spatial epidemiological approach, calculating incremental spatial autocorrelations and Getis-Ord Gi* statistics to identify clusters. We conducted logistic regression analyses to determine factors associated with the HCV hotspots. We identified nine HCV clusters, with the largest in Boston, New Bedford/Fall River, Worcester, and Springfield (p < 0.05). In multivariable analyses, we found that HCV hotspots were independently and positively associated with the percent of the population that was Hispanic (adjusted odds ratio [AOR]: 1.07; 95% confidence interval [CI]: 1.04, 1.09) and the percent of households receiving food stamps (AOR: 1.83; 95% CI: 1.22, 2.74). HCV hotspots were independently and negatively associated with the percent of the population that were high school graduates or higher (AOR: 0.91; 95% CI: 0.89, 0.93) and the percent of the population in the "other" race/ethnicity category (AOR: 0.88; 95% CI: 0.85, 0.91). We identified locations where HCV clusters were a concern, and where enhanced HCV prevention, treatment, and care can help combat the HCV epidemic in Massachusetts. GIS, spatial epidemiological and statistical analyses provided a rigorous approach to identify hotspot clusters of disease, which can inform public health policy and intervention targeting. Further studies that incorporate spatiotemporal cluster analyses, Bayesian spatial and geostatistical models, spatially weighted regression

Genome-Wide Meta-Analyses of Breast, Ovarian, and Prostate Cancer Association Studies Identify Multiple New Susceptibility Loci Shared by at Least Two Cancer Types.

PubMed

Kar, Siddhartha P; Beesley, Jonathan; Amin Al Olama, Ali; Michailidou, Kyriaki; Tyrer, Jonathan; Kote-Jarai, ZSofia; Lawrenson, Kate; Lindstrom, Sara; Ramus, Susan J; Thompson, Deborah J; Kibel, Adam S; Dansonka-Mieszkowska, Agnieszka; Michael, Agnieszka; Dieffenbach, Aida K; Gentry-Maharaj, Aleksandra; Whittemore, Alice S; Wolk, Alicja; Monteiro, Alvaro; Peixoto, Ana; Kierzek, Andrzej; Cox, Angela; Rudolph, Anja; Gonzalez-Neira, Anna; Wu, Anna H; Lindblom, Annika; Swerdlow, Anthony; Ziogas, Argyrios; Ekici, Arif B; Burwinkel, Barbara; Karlan, Beth Y; Nordestgaard, Børge G; Blomqvist, Carl; Phelan, Catherine; McLean, Catriona; Pearce, Celeste Leigh; Vachon, Celine; Cybulski, Cezary; Slavov, Chavdar; Stegmaier, Christa; Maier, Christiane; Ambrosone, Christine B; Høgdall, Claus K; Teerlink, Craig C; Kang, Daehee; Tessier, Daniel C; Schaid, Daniel J; Stram, Daniel O; Cramer, Daniel W; Neal, David E; Eccles, Diana; Flesch-Janys, Dieter; Edwards, Digna R Velez; Wokozorczyk, Dominika; Levine, Douglas A; Yannoukakos, Drakoulis; Sawyer, Elinor J; Bandera, Elisa V; Poole, Elizabeth M; Goode, Ellen L; Khusnutdinova, Elza; Høgdall, Estrid; Song, Fengju; Bruinsma, Fiona; Heitz, Florian; Modugno, Francesmary; Hamdy, Freddie C; Wiklund, Fredrik; Giles, Graham G; Olsson, Håkan; Wildiers, Hans; Ulmer, Hans-Ulrich; Pandha, Hardev; Risch, Harvey A; Darabi, Hatef; Salvesen, Helga B; Nevanlinna, Heli; Gronberg, Henrik; Brenner, Hermann; Brauch, Hiltrud; Anton-Culver, Hoda; Song, Honglin; Lim, Hui-Yi; McNeish, Iain; Campbell, Ian; Vergote, Ignace; Gronwald, Jacek; Lubiński, Jan; Stanford, Janet L; Benítez, Javier; Doherty, Jennifer A; Permuth, Jennifer B; Chang-Claude, Jenny; Donovan, Jenny L; Dennis, Joe; Schildkraut, Joellen M; Schleutker, Johanna; Hopper, John L; Kupryjanczyk, Jolanta; Park, Jong Y; Figueroa, Jonine; Clements, Judith A; Knight, Julia A; Peto, Julian; Cunningham, Julie M; Pow-Sang, Julio; Batra, Jyotsna; Czene, Kamila; Lu, Karen H; Herkommer, Kathleen; Khaw, Kay-Tee; Matsuo, Keitaro; Muir, Kenneth; Offitt, Kenneth; Chen, Kexin; Moysich, Kirsten B; Aittomäki, Kristiina; Odunsi, Kunle; Kiemeney, Lambertus A; Massuger, Leon F A G; Fitzgerald, Liesel M; Cook, Linda S; Cannon-Albright, Lisa; Hooning, Maartje J; Pike, Malcolm C; Bolla, Manjeet K; Luedeke, Manuel; Teixeira, Manuel R; Goodman, Marc T; Schmidt, Marjanka K; Riggan, Marjorie; Aly, Markus; Rossing, Mary Anne; Beckmann, Matthias W; Moisse, Matthieu; Sanderson, Maureen; Southey, Melissa C; Jones, Michael; Lush, Michael; Hildebrandt, Michelle A T; Hou, Ming-Feng; Schoemaker, Minouk J; Garcia-Closas, Montserrat; Bogdanova, Natalia; Rahman, Nazneen; Le, Nhu D; Orr, Nick; Wentzensen, Nicolas; Pashayan, Nora; Peterlongo, Paolo; Guénel, Pascal; Brennan, Paul; Paulo, Paula; Webb, Penelope M; Broberg, Per; Fasching, Peter A; Devilee, Peter; Wang, Qin; Cai, Qiuyin; Li, Qiyuan; Kaneva, Radka; Butzow, Ralf; Kopperud, Reidun Kristin; Schmutzler, Rita K; Stephenson, Robert A; MacInnis, Robert J; Hoover, Robert N; Winqvist, Robert; Ness, Roberta; Milne, Roger L; Travis, Ruth C; Benlloch, Sara; Olson, Sara H; McDonnell, Shannon K; Tworoger, Shelley S; Maia, Sofia; Berndt, Sonja; Lee, Soo Chin; Teo, Soo-Hwang; Thibodeau, Stephen N; Bojesen, Stig E; Gapstur, Susan M; Kjær, Susanne Krüger; Pejovic, Tanja; Tammela, Teuvo L J; Dörk, Thilo; Brüning, Thomas; Wahlfors, Tiina; Key, Tim J; Edwards, Todd L; Menon, Usha; Hamann, Ute; Mitev, Vanio; Kosma, Veli-Matti; Setiawan, Veronica Wendy; Kristensen, Vessela; Arndt, Volker; Vogel, Walther; Zheng, Wei; Sieh, Weiva; Blot, William J; Kluzniak, Wojciech; Shu, Xiao-Ou; Gao, Yu-Tang; Schumacher, Fredrick; Freedman, Matthew L; Berchuck, Andrew; Dunning, Alison M; Simard, Jacques; Haiman, Christopher A; Spurdle, Amanda; Sellers, Thomas A; Hunter, David J; Henderson, Brian E; Kraft, Peter; Chanock, Stephen J; Couch, Fergus J; Hall, Per; Gayther, Simon A; Easton, Douglas F; Chenevix-Trench, Georgia; Eeles, Rosalind; Pharoah, Paul D P; Lambrechts, Diether

2016-09-01

Breast, ovarian, and prostate cancers are hormone-related and may have a shared genetic basis, but this has not been investigated systematically by genome-wide association (GWA) studies. Meta-analyses combining the largest GWA meta-analysis data sets for these cancers totaling 112,349 cases and 116,421 controls of European ancestry, all together and in pairs, identified at P < 10(-8) seven new cross-cancer loci: three associated with susceptibility to all three cancers (rs17041869/2q13/BCL2L11; rs7937840/11q12/INCENP; rs1469713/19p13/GATAD2A), two breast and ovarian cancer risk loci (rs200182588/9q31/SMC2; rs8037137/15q26/RCCD1), and two breast and prostate cancer risk loci (rs5013329/1p34/NSUN4; rs9375701/6q23/L3MBTL3). Index variants in five additional regions previously associated with only one cancer also showed clear association with a second cancer type. Cell-type-specific expression quantitative trait locus and enhancer-gene interaction annotations suggested target genes with potential cross-cancer roles at the new loci. Pathway analysis revealed significant enrichment of death receptor signaling genes near loci with P < 10(-5) in the three-cancer meta-analysis. We demonstrate that combining large-scale GWA meta-analysis findings across cancer types can identify completely new risk loci common to breast, ovarian, and prostate cancers. We show that the identification of such cross-cancer risk loci has the potential to shed new light on the shared biology underlying these hormone-related cancers. Cancer Discov; 6(9); 1052-67. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 932. ©2016 American Association for Cancer Research.

Stable isotope analyses of feather amino acids identify penguin migration strategies at ocean basin scales.

PubMed

Polito, Michael J; Hinke, Jefferson T; Hart, Tom; Santos, Mercedes; Houghton, Leah A; Thorrold, Simon R

2017-08-01

Identifying the at-sea distribution of wide-ranging marine predators is critical to understanding their ecology. Advances in electronic tracking devices and intrinsic biogeochemical markers have greatly improved our ability to track animal movements on ocean-wide scales. Here, we show that, in combination with direct tracking, stable carbon isotope analysis of essential amino acids in tail feathers provides the ability to track the movement patterns of two, wide-ranging penguin species over ocean basin scales. In addition, we use this isotopic approach across multiple breeding colonies in the Scotia Arc to evaluate migration trends at a regional scale that would be logistically challenging using direct tracking alone. © 2017 The Author(s).

Conserved Regulators of Nucleolar Size Revealed by Global Phenotypic Analyses

PubMed Central

Neumüller, Ralph A.; Gross, Thomas; Samsonova, Anastasia A.; Vinayagam, Arunachalam; Buckner, Michael; Founk, Karen; Hu, Yanhui; Sharifpoor, Sara; Rosebrock, Adam P.; Andrews, Brenda; Winston, Fred; Perrimon, Norbert

2014-01-01

Regulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intracellular information. A central player in this process is RNA polymerase I (Pol I), which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I–mediated transcription and, consequently, nucleolar hypertrophy. To map the genetic network underlying the regulation of nucleolar size and of Pol I–mediated transcription, we performed comparative, genome-wide loss-of-function analyses of nucleolar size in Saccharomyces cerevisiae and Drosophila melanogaster coupled with mass spectrometry–based analyses of the ribosomal DNA (rDNA) promoter. With this approach, we identified a set of conserved and nonconserved molecular complexes that control nucleolar size. Furthermore, we characterized a direct role of the histone information regulator (HIR) complex in repressing rRNA transcription in yeast. Our study provides a full-genome, cross-species analysis of a nuclear subcompartment and shows that this approach can identify conserved molecular modules. PMID:23962978

Structural and antigenic identification of the ORF12 protein (alpha TIF) of equine herpesvirus 1.

PubMed

Lewis, J B; Thompson, Y G; Feng, X; Holden, V R; O'Callaghan, D; Caughman, G B

1997-04-14

The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, alpha TIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12) and shown to transactivate immediate early (IE) gene promoters. However, a specific virion protein corresponding to the ORF12 product has not been identified definitively. In the present study the ORF12 protein, designated ETIF, was identified as a 60-kDa virion component on the basis of protein fingerprint analyses in which the limited proteolysis profiles of the major 60-kDa in vitro transcription/ translation product of an ORF12 expression vector (pT7-12) were compared to those of purified virion proteins of similar size. ETIF was localized to the viral tegument in Western blot assays of EHV-1 virions and subvirion fractions using polyclonal antiserum and monoclonal antibodies generated against a glutathione-S-transferase-ETIF fusion protein. Northern and Western blot analyses of EHV-1-infected cell lysates prepared under various metabolic blocks indicated that ORF12 is expressed as a late gene, and cross reaction of polyclonal anti-GST-ETIF with a 63.5-kDa HSV-1 protein species suggested that ETIF and HSV-1 alpha TIF are antigenically related. Last, DNA band shift assays used to assess ETIF-specific complex formation indicated that ETIF participates in an infected cell protein complex with the EHV-1 IE promoter TAATGARAT motif.

Purification and analyses of the specificity of two putative diagnostic antigens for larval cyathostomin infection in horses.

PubMed

Dowdall, S M J; Proudman, C J; Love, S; Klei, T R; Matthews, J B

2003-12-01

Cyathostomins are important equine gastrointestinal parasites. Mass emergence of mucosal stage larvae causes a potentially fatal colitis. Mucosal stages are undetectable non-invasively. An assay that would estimate mucosal larval stage infection would greatly assist in treatment, control and prognosis. Previously, we identified two putative diagnostic antigens (20 and 25 kDa) in somatic larval preparations. Here, we describe their purification and antigen-specific IgG(T) responses to them. Western blots confirmed the purity of the antigens and showed that epitopes in the 20 kDa complex were specific to larval cyathostomins. No cross-reactive antigens appeared to be present in Parascaris equorum or Strongyloides westeri species. Low levels of cross-reactivity were observed in Strongylus edentatus and Strongylus vulgaris species. Use of purified antigens greatly reduced background binding in equine sera. These results indicate that both antigen complexes may be of use in a diagnostic assay.

Multicollinearity in spatial genetics: separating the wheat from the chaff using commonality analyses.

PubMed

Prunier, J G; Colyn, M; Legendre, X; Nimon, K F; Flamand, M C

2015-01-01

Direct gradient analyses in spatial genetics provide unique opportunities to describe the inherent complexity of genetic variation in wildlife species and are the object of many methodological developments. However, multicollinearity among explanatory variables is a systemic issue in multivariate regression analyses and is likely to cause serious difficulties in properly interpreting results of direct gradient analyses, with the risk of erroneous conclusions, misdirected research and inefficient or counterproductive conservation measures. Using simulated data sets along with linear and logistic regressions on distance matrices, we illustrate how commonality analysis (CA), a detailed variance-partitioning procedure that was recently introduced in the field of ecology, can be used to deal with nonindependence among spatial predictors. By decomposing model fit indices into unique and common (or shared) variance components, CA allows identifying the location and magnitude of multicollinearity, revealing spurious correlations and thus thoroughly improving the interpretation of multivariate regressions. Despite a few inherent limitations, especially in the case of resistance model optimization, this review highlights the great potential of CA to account for complex multicollinearity patterns in spatial genetics and identifies future applications and lines of research. We strongly urge spatial geneticists to systematically investigate commonalities when performing direct gradient analyses. © 2014 John Wiley & Sons Ltd.

Homeopathy: meta-analyses of pooled clinical data.

PubMed

Hahn, Robert G

2013-01-01

In the first decade of the evidence-based era, which began in the mid-1990s, meta-analyses were used to scrutinize homeopathy for evidence of beneficial effects in medical conditions. In this review, meta-analyses including pooled data from placebo-controlled clinical trials of homeopathy and the aftermath in the form of debate articles were analyzed. In 1997 Klaus Linde and co-workers identified 89 clinical trials that showed an overall odds ratio of 2.45 in favor of homeopathy over placebo. There was a trend toward smaller benefit from studies of the highest quality, but the 10 trials with the highest Jadad score still showed homeopathy had a statistically significant effect. These results challenged academics to perform alternative analyses that, to demonstrate the lack of effect, relied on extensive exclusion of studies, often to the degree that conclusions were based on only 5-10% of the material, or on virtual data. The ultimate argument against homeopathy is the 'funnel plot' published by Aijing Shang's research group in 2005. However, the funnel plot is flawed when applied to a mixture of diseases, because studies with expected strong treatments effects are, for ethical reasons, powered lower than studies with expected weak or unclear treatment effects. To conclude that homeopathy lacks clinical effect, more than 90% of the available clinical trials had to be disregarded. Alternatively, flawed statistical methods had to be applied. Future meta-analyses should focus on the use of homeopathy in specific diseases or groups of diseases instead of pooling data from all clinical trials. © 2013 S. Karger GmbH, Freiburg.

Topographic ERP analyses: a step-by-step tutorial review.

PubMed

Murray, Micah M; Brunet, Denis; Michel, Christoph M

2008-06-01

In this tutorial review, we detail both the rationale for as well as the implementation of a set of analyses of surface-recorded event-related potentials (ERPs) that uses the reference-free spatial (i.e. topographic) information available from high-density electrode montages to render statistical information concerning modulations in response strength, latency, and topography both between and within experimental conditions. In these and other ways these topographic analysis methods allow the experimenter to glean additional information and neurophysiologic interpretability beyond what is available from canonical waveform analyses. In this tutorial we present the example of somatosensory evoked potentials (SEPs) in response to stimulation of each hand to illustrate these points. For each step of these analyses, we provide the reader with both a conceptual and mathematical description of how the analysis is carried out, what it yields, and how to interpret its statistical outcome. We show that these topographic analysis methods are intuitive and easy-to-use approaches that can remove much of the guesswork often confronting ERP researchers and also assist in identifying the information contained within high-density ERP datasets.

Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer

PubMed Central

2013-01-01

Introduction Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations. Methods Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively. Results Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively. Conclusion Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the

Systematic Review of Health Economic Analyses of Measles and Rubella Immunization Interventions.

PubMed

Thompson, Kimberly M; Odahowski, Cassie L

2016-07-01

Economic analyses for vaccine-preventable diseases provide important insights about the value of prevention. We reviewed the literature to identify all of the peer-reviewed, published economic analyses of interventions related to measles and rubella immunization options to assess the different types of analyses performed and characterize key insights. We searched PubMed, the Science Citation Index, and references from relevant articles for studies in English and found 67 analyses that reported primary data and quantitative estimates of benefit-cost or cost-effectiveness analyses for measles and/or rubella immunization interventions. We removed studies that we characterized as cost-minimization analyses from this sample because they generally provide insights that focused on more optimal strategies to achieve the same health outcome. The 67 analyses we included demonstrate the large economic benefits associated with preventing measles and rubella infections using vaccines and the benefit of combining measles and rubella antigens into a formulation that saves the costs associated with injecting the vaccines separately. Despite the importance of population immunity and dynamic viral transmission, most of the analyses used static models to estimate cases prevented and characterize benefits, although the use of dynamic models continues to increase. Many of the analyses focused on characterizing the most significant adverse outcomes (e.g., mortality for measles, congenital rubella syndrome for rubella) and/or only direct costs, and the most complete analyses present data from high-income countries. © 2014 Society for Risk Analysis.

In-depth analyses of paleolithic pigments in cave climatic conditions

NASA Astrophysics Data System (ADS)

Touron, Stéphanie; Trichereau, Barbara; Syvilay, Delphine

2017-07-01

Painted caves are a specific environment which preservation needs multidisciplinary studies carried out within the different actors. The actions set-up must follow national and European ethics and treaties and be as less invasive as possible to preserve the integrity of the site. Studying colorants in caves should meet these expectations and take into account on-field conditions: high humidity rate, reduced access to electricity, etc. Therefore, non-invasive analyses should be preferred. However, their limits restrict the field of application and sometimes sampling and laboratory analyses must be used to answer the problematic. It is especially true when the pigment is covered by calcite. For this purpose, the Laser-Induced Breakdown Spectroscopy (LIBS) has been assessed to identify the composition with stratigraphic analyses. This study carries out in-depth profile on laboratory samples in conditions close to the ones meet in caves. Samples were prepared on a calcareous substrate using three pigments: red ochre, manganese black and carbon black and two binding media: water and saliva. All samples have been covered by calcite. Four sets of measurements have then been done using the LIBS instrument. The in-depth profiles were obtained using the Standard Normal Variate (SNV) normalization. For all the samples, the pigment layer was identified in the second or third shot, the calcite layer being quite thin. However, the results remain promising with the carbon black pigment but not really conclusive, the carbon being generally quite difficult to quantify.

Genome-wide Meta-analyses of Breast, Ovarian and Prostate Cancer Association Studies Identify Multiple New Susceptibility Loci Shared by At Least Two Cancer Types

PubMed Central

Kar, Siddhartha P.; Beesley, Jonathan; Al Olama, Ali Amin; Michailidou, Kyriaki; Tyrer, Jonathan; Kote-Jarai, ZSofia; Lawrenson, Kate; Lindstrom, Sara; Ramus, Susan J.; Thompson, Deborah J.; Kibel, Adam S.; Dansonka-Mieszkowska, Agnieszka; Michael, Agnieszka; Dieffenbach, Aida K.; Gentry-Maharaj, Aleksandra; Whittemore, Alice S.; Wolk, Alicja; Monteiro, Alvaro; Peixoto, Ana; Kierzek, Andrzej; Cox, Angela; Rudolph, Anja; Gonzalez-Neira, Anna; Wu, Anna H.; Lindblom, Annika; Swerdlow, Anthony; Ziogas, Argyrios; Ekici, Arif B.; Burwinkel, Barbara; Karlan, Beth Y.; Nordestgaard, Børge G.; Blomqvist, Carl; Phelan, Catherine; McLean, Catriona; Pearce, Celeste Leigh; Vachon, Celine; Cybulski, Cezary; Slavov, Chavdar; Stegmaier, Christa; Maier, Christiane; Ambrosone, Christine B.; Høgdall, Claus K.; Teerlink, Craig C.; Kang, Daehee; Tessier, Daniel C.; Schaid, Daniel J.; Stram, Daniel O.; Cramer, Daniel W.; Neal, David E.; Eccles, Diana; Flesch-Janys, Dieter; Velez Edwards, Digna R.; Wokozorczyk, Dominika; Levine, Douglas A.; Yannoukakos, Drakoulis; Sawyer, Elinor J.; Bandera, Elisa V.; Poole, Elizabeth M.; Goode, Ellen L.; Khusnutdinova, Elza; Høgdall, Estrid; Song, Fengju; Bruinsma, Fiona; Heitz, Florian; Modugno, Francesmary; Hamdy, Freddie C.; Wiklund, Fredrik; Giles, Graham G.; Olsson, Håkan; Wildiers, Hans; Ulmer, Hans-Ulrich; Pandha, Hardev; Risch, Harvey A.; Darabi, Hatef; Salvesen, Helga B.; Nevanlinna, Heli; Gronberg, Henrik; Brenner, Hermann; Brauch, Hiltrud; Anton-Culver, Hoda; Song, Honglin; Lim, Hui-Yi; McNeish, Iain; Campbell, Ian; Vergote, Ignace; Gronwald, Jacek; Lubiński, Jan; Stanford, Janet L.; Benítez, Javier; Doherty, Jennifer A.; Permuth, Jennifer B.; Chang-Claude, Jenny; Donovan, Jenny L.; Dennis, Joe; Schildkraut, Joellen M.; Schleutker, Johanna; Hopper, John L.; Kupryjanczyk, Jolanta; Park, Jong Y.; Figueroa, Jonine; Clements, Judith A.; Knight, Julia A.; Peto, Julian; Cunningham, Julie M.; Pow-Sang, Julio; Batra, Jyotsna; Czene, Kamila; Lu, Karen H.; Herkommer, Kathleen; Khaw, Kay-Tee; Matsuo, Keitaro; Muir, Kenneth; Offitt, Kenneth; Chen, Kexin; Moysich, Kirsten B.; Aittomäki, Kristiina; Odunsi, Kunle; Kiemeney, Lambertus A.; Massuger, Leon F.A.G.; Fitzgerald, Liesel M.; Cook, Linda S.; Cannon-Albright, Lisa; Hooning, Maartje J.; Pike, Malcolm C.; Bolla, Manjeet K.; Luedeke, Manuel; Teixeira, Manuel R.; Goodman, Marc T.; Schmidt, Marjanka K.; Riggan, Marjorie; Aly, Markus; Rossing, Mary Anne; Beckmann, Matthias W.; Moisse, Matthieu; Sanderson, Maureen; Southey, Melissa C.; Jones, Michael; Lush, Michael; Hildebrandt, Michelle A. T.; Hou, Ming-Feng; Schoemaker, Minouk J.; Garcia-Closas, Montserrat; Bogdanova, Natalia; Rahman, Nazneen; Le, Nhu D.; Orr, Nick; Wentzensen, Nicolas; Pashayan, Nora; Peterlongo, Paolo; Guénel, Pascal; Brennan, Paul; Paulo, Paula; Webb, Penelope M.; Broberg, Per; Fasching, Peter A.; Devilee, Peter; Wang, Qin; Cai, Qiuyin; Li, Qiyuan; Kaneva, Radka; Butzow, Ralf; Kopperud, Reidun Kristin; Schmutzler, Rita K.; Stephenson, Robert A.; MacInnis, Robert J.; Hoover, Robert N.; Winqvist, Robert; Ness, Roberta; Milne, Roger L.; Travis, Ruth C.; Benlloch, Sara; Olson, Sara H.; McDonnell, Shannon K.; Tworoger, Shelley S.; Maia, Sofia; Berndt, Sonja; Lee, Soo Chin; Teo, Soo-Hwang; Thibodeau, Stephen N.; Bojesen, Stig E.; Gapstur, Susan M.; Kjær, Susanne Krüger; Pejovic, Tanja; Tammela, Teuvo L.J.; Dörk, Thilo; Brüning, Thomas; Wahlfors, Tiina; Key, Tim J.; Edwards, Todd L.; Menon, Usha; Hamann, Ute; Mitev, Vanio; Kosma, Veli-Matti; Setiawan, Veronica Wendy; Kristensen, Vessela; Arndt, Volker; Vogel, Walther; Zheng, Wei; Sieh, Weiva; Blot, William J.; Kluzniak, Wojciech; Shu, Xiao-Ou; Gao, Yu-Tang; Schumacher, Fredrick; Freedman, Matthew L.; Berchuck, Andrew; Dunning, Alison M.; Simard, Jacques; Haiman, Christopher A.; Spurdle, Amanda; Sellers, Thomas A.; Hunter, David J.; Henderson, Brian E.; Kraft, Peter; Chanock, Stephen J.; Couch, Fergus J.; Hall, Per; Gayther, Simon A.; Easton, Douglas F.; Chenevix-Trench, Georgia; Eeles, Rosalind; Pharoah, Paul D.P.; Lambrechts, Diether

2016-01-01

Breast, ovarian, and prostate cancers are hormone-related and may have a shared genetic basis but this has not been investigated systematically by genome-wide association (GWA) studies. Meta-analyses combining the largest GWA meta-analysis data sets for these cancers totaling 112,349 cases and 116,421 controls of European ancestry, all together and in pairs, identified at P < 10−8 seven new cross-cancer loci: three associated with susceptibility to all three cancers (rs17041869/2q13/BCL2L11; rs7937840/11q12/INCENP; rs1469713/19p13/GATAD2A), two breast and ovarian cancer risk loci (rs200182588/9q31/SMC2; rs8037137/15q26/RCCD1), and two breast and prostate cancer risk loci (rs5013329/1p34/NSUN4; rs9375701/6q23/L3MBTL3). Index variants in five additional regions previously associated with only one cancer also showed clear association with a second cancer type. Cell-type specific expression quantitative trait locus and enhancer-gene interaction annotations suggested target genes with potential cross-cancer roles at the new loci. Pathway analysis revealed significant enrichment of death receptor signaling genes near loci with P < 10−5 in the three-cancer meta-analysis. PMID:27432226

Identifying geographic hot spots of reassortment in a multipartite plant virus

PubMed Central

Savory, Fiona R; Varma, Varun; Ramakrishnan, Uma

2014-01-01

Reassortment between different species or strains plays a key role in the evolution of multipartite plant viruses and can have important epidemiological implications. Identifying geographic locations where reassortant lineages are most likely to emerge could be a valuable strategy for informing disease management and surveillance efforts. We developed a predictive framework to identify potential geographic hot spots of reassortment based upon spatially explicit analyses of genome constellation diversity. To demonstrate the utility of this approach, we examined spatial variation in the potential for reassortment among Cardamom bushy dwarf virus (CBDV; Nanoviridae, Babuvirus) isolates in Northeast India. Using sequence data corresponding to six discrete genome components for 163 CBDV isolates, a quantitative measure of genome constellation diversity was obtained for locations across the sampling region. Two key areas were identified where viruses with highly distinct genome constellations cocirculate, and these locations were designated as possible geographic hot spots of reassortment, where novel reassortant lineages could emerge. Our study demonstrates that the potential for reassortment can be spatially dependent in multipartite plant viruses and highlights the use of evolutionary analyses to identify locations which could be actively managed to facilitate the prevention of outbreaks involving novel reassortant strains. PMID:24944570

DMINDA: an integrated web server for DNA motif identification and analyses

PubMed Central

Ma, Qin; Zhang, Hanyuan; Mao, Xizeng; Zhou, Chuan; Liu, Bingqiang; Chen, Xin; Xu, Ying

2014-01-01

DMINDA (DNA motif identification and analyses) is an integrated web server for DNA motif identification and analyses, which is accessible at http://csbl.bmb.uga.edu/DMINDA/. This web site is freely available to all users and there is no login requirement. This server provides a suite of cis-regulatory motif analysis functions on DNA sequences, which are important to elucidation of the mechanisms of transcriptional regulation: (i) de novo motif finding for a given set of promoter sequences along with statistical scores for the predicted motifs derived based on information extracted from a control set, (ii) scanning motif instances of a query motif in provided genomic sequences, (iii) motif comparison and clustering of identified motifs, and (iv) co-occurrence analyses of query motifs in given promoter sequences. The server is powered by a backend computer cluster with over 150 computing nodes, and is particularly useful for motif prediction and analyses in prokaryotic genomes. We believe that DMINDA, as a new and comprehensive web server for cis-regulatory motif finding and analyses, will benefit the genomic research community in general and prokaryotic genome researchers in particular. PMID:24753419

How often do sensitivity analyses for economic parameters change cost-utility analysis conclusions?

PubMed

Schackman, Bruce R; Gold, Heather Taffet; Stone, Patricia W; Neumann, Peter J

2004-01-01

There is limited evidence about the extent to which sensitivity analysis has been used in the cost-effectiveness literature. Sensitivity analyses for health-related QOL (HR-QOL), cost and discount rate economic parameters are of particular interest because they measure the effects of methodological and estimation uncertainties. To investigate the use of sensitivity analyses in the pharmaceutical cost-utility literature in order to test whether a change in economic parameters could result in a different conclusion regarding the cost effectiveness of the intervention analysed. Cost-utility analyses of pharmaceuticals identified in a prior comprehensive audit (70 articles) were reviewed and further audited. For each base case for which sensitivity analyses were reported (n = 122), up to two sensitivity analyses for HR-QOL (n = 133), cost (n = 99), and discount rate (n = 128) were examined. Article mentions of thresholds for acceptable cost-utility ratios were recorded (total 36). Cost-utility ratios were denominated in US dollars for the year reported in each of the original articles in order to determine whether a different conclusion would have been indicated at the time the article was published. Quality ratings from the original audit for articles where sensitivity analysis results crossed the cost-utility ratio threshold above the base-case result were compared with those that did not. The most frequently mentioned cost-utility thresholds were $US20,000/QALY, $US50,000/QALY, and $US100,000/QALY. The proportions of sensitivity analyses reporting quantitative results that crossed the threshold above the base-case results (or where the sensitivity analysis result was dominated) were 31% for HR-QOL sensitivity analyses, 20% for cost-sensitivity analyses, and 15% for discount-rate sensitivity analyses. Almost half of the discount-rate sensitivity analyses did not report quantitative results. Articles that reported sensitivity analyses where results crossed the cost

DATA AND ANALYSES

EPA Science Inventory

In order to promote transparency and clarity of the analyses performed in support of EPA's Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens, the data and the analyses are now available on this web site. The data is presented in two diffe...

VCP gene analyses in Japanese patients with sporadic amyotrophic lateral sclerosis identify a new mutation.

PubMed

Hirano, Makito; Nakamura, Yusaku; Saigoh, Kazumasa; Sakamoto, Hikaru; Ueno, Shuichi; Isono, Chiharu; Mitsui, Yoshiyuki; Kusunoki, Susumu

2015-03-01

Accumulating evidence has proven that mutations in the VCP gene encoding valosin-containing protein (VCP) cause inclusion body myopathy with Paget disease of the bone and frontotemporal dementia. This gene was later found to be causative for amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, occurring typically in elderly persons. We thus sequenced the VCP gene in 75 Japanese patients with sporadic ALS negative for mutations in other genes causative for ALS and found a novel mutation, p.Arg487His, in 1 patient. The newly identified mutant as well as known mutants rendered neuronal cells susceptible to oxidative stress. The presence of the mutation in the Japanese population extends the geographic region for involvement of the VCP gene in sporadic ALS to East Asia. Copyright © 2015 Elsevier Inc. All rights reserved.

Characteristics and Methodological Quality of Meta-Analyses on Hypertension Treatments-A Cross-Sectional Study.

PubMed

Wu, Xin Yin; Du, Xin Jian; Ho, Robin S T; Lee, Clarence C Y; Yip, Benjamin H K; Wong, Martin C S; Wong, Samuel Y S; Chung, Vincent C H

2017-02-01

Methodological quality of meta-analyses on hypertension treatments can affect treatment decision-making. The authors conducted a cross-sectional study to investigate the methodological quality of meta-analyses on hypertension treatments. One hundred and fifty-eight meta-analyses were identified. Overall, methodological quality was unsatisfactory in the following aspects: comprehensive reporting of financial support (1.9%), provision of included and excluded lists of studies (22.8%), inclusion of grey literature (27.2%), and inclusion of protocols (32.9%). The 126 non-Cochrane meta-analyses had poor performance on almost all the methodological items. Non-Cochrane meta-analyses focused on nonpharmacologic treatments were more likely to consider scientific quality of included studies when making conclusions. The 32 Cochrane meta-analyses generally had good methodological quality except for comprehensive reporting of the sources of support. These results highlight the need for cautious interpretation of these meta-analyses, especially among physicians and policy makers when guidelines are formulated. Future meta-analyses should pay attention to improving these methodological aspects. ©2016 Wiley Periodicals, Inc.

Newly identified allatostatin Bs and their receptor in the two-spotted cricket, Gryllus bimaculatus.

PubMed

Tsukamoto, Yusuke; Nagata, Shinji

2016-06-01

A cDNA encoding allatostatin Bs (ASTBs) containing the W(X)6W motif was identified using a database generated by a next generation sequencer (NGS) in the two-spotted cricket, Gryllus bimaculatus. The contig sequence revealed the presence of five novel putative ASTBs (GbASTBs) in addition to GbASTBs previously identified in G. bimaculatus. MALDI-TOF MS analyses revealed the presence of these novel and previously identified GbASTBs with three missing GbASTBs. We also identified a cDNA encoding G. bimaculatus GbASTB receptor (GbASTBR) in the NGS data. Phylogenetic analysis demonstrated that this receptor was highly conserved with other insect ASTBRs, including the sex peptide receptor of Drosophila melanogaster. Calcium imaging analyses indicated that the GbASTBR heterologously expressed in HEK293 cells exhibited responses to all identified GbASTBs at a concentration range of 10(-10)-10(-5)M. Copyright © 2016 Elsevier Inc. All rights reserved.

Sumoylated α-skeletal muscle actin in the skeletal muscle of adult rats.

PubMed

Uda, Munehiro; Kawasaki, Hiroaki; Iizumi, Kyoichi; Shigenaga, Ayako; Baba, Takeshi; Naito, Hisashi; Yoshioka, Toshitada; Yamakura, Fumiyuki

2015-11-01

Skeletal muscles are composed of two major muscle fiber types: slow-twitch oxidative fibers and fast-twitch glycolytic fibers. The proteins in these muscle fibers are known to differ in their expression, relative abundance, and post-translational modifications. In this study, we report a previously unreported post-translational modification of α-skeletal muscle actin in the skeletal muscles of adult male F344 rats in vivo. Using two-dimensional electrophoresis (2D-PAGE), we first examined the differences in the protein expression profiles between the soleus and plantaris muscles. We found higher intensity protein spots at approximately 60 kDa and pH 9 on 2D-PAGE for the soleus muscle compared with the plantaris muscle. These spots were identified as α-skeletal muscle actin by liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry and western blot analyses. In addition, we found that the 60 kDa α-skeletal muscle actin is modified by small ubiquitin-like modifier (SUMO) 1, using 2D-PAGE and western blot analyses. Furthermore, we found that α-skeletal muscle actin with larger molecular weight was localized in the nuclear and cytosol of the skeletal muscle, but not in the myofibrillar fraction by the combination of subcellular fractionation and western blot analyses. These results suggest that α-skeletal muscle actin is modified by SUMO-1 in the skeletal muscles, localized in nuclear and cytosolic fractions, and the extent of this modification is much higher in the slow muscles than in the fast muscles. This is the first study to show the presence of SUMOylated actin in animal tissues.

Detection of Y chromosome sequences in a 45,X/46,XXq - patient by Southern blot analysis of PCR-amplified DNA and fluorescent in situ hybridization (FISH)

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

Kocova, M.; Siegel, S.F.; Wenger, S.L.

1995-02-13

In some cases of gonadal dysgenesis, cytogenetic analysis seems to be discordant with the phenotype of the patients. We have applied techniques such as Southern blot analysis and fluorescent in situ hybridization (FISH) to resolve the phenotype/genotype discrepancy in a patient with ambiguous genitalia in whom the peripheral blood karotype was 45,X. Gonadectomy at age 7 months showed the gonadal tissue to be prepubertal testis on the left side and a streak gonad on the right. The karyotype obtained from the left gonad was 45,X/46,XXq- and that from the right gonad was 45,X. Three different techniques, PCR amplification, FISH, andmore » chromosome painting for X and Y chromosomes, confirmed the presence of Y chromosome sequences. Five different tissues were evaluated. The highest percentage of Y chromosome positive cells were detected in the left gonad, followed by the peripheral blood lymphocytes, skin fibroblasts, and buccal mucosa. No Y chromosomal material could be identified in the right gonad. Since the Xq- chromosome is present in the left gonad (testis), it is likely that the Xq- contains Y chromosomal material. Sophisticated analysis in this patient showed that she has at least 2 cell lines, one of which contains Y chromosomal material. These techniques elucidated the molecular basis of the genital ambiguity for this patient. When Y chromosome sequences are present in patients with Ullrich-Turner syndrome or gonadal dysgenesis, the risk for gonadal malignancy is significantly increased. Hence, molecular diagnostic methods to ascertain for the presence of Y chromosome sequences may expedite the evaluation of patients with the ambiguous genitalia. 21 refs., 4 figs., 2 tabs.« less

FusionAnalyser: a new graphical, event-driven tool for fusion rearrangements discovery

PubMed Central

Piazza, Rocco; Pirola, Alessandra; Spinelli, Roberta; Valletta, Simona; Redaelli, Sara; Magistroni, Vera; Gambacorti-Passerini, Carlo

2012-01-01

Gene fusions are common driver events in leukaemias and solid tumours; here we present FusionAnalyser, a tool dedicated to the identification of driver fusion rearrangements in human cancer through the analysis of paired-end high-throughput transcriptome sequencing data. We initially tested FusionAnalyser by using a set of in silico randomly generated sequencing data from 20 known human translocations occurring in cancer and subsequently using transcriptome data from three chronic and three acute myeloid leukaemia samples. in all the cases our tool was invariably able to detect the presence of the correct driver fusion event(s) with high specificity. In one of the acute myeloid leukaemia samples, FusionAnalyser identified a novel, cryptic, in-frame ETS2–ERG fusion. A fully event-driven graphical interface and a flexible filtering system allow complex analyses to be run in the absence of any a priori programming or scripting knowledge. Therefore, we propose FusionAnalyser as an efficient and robust graphical tool for the identification of functional rearrangements in the context of high-throughput transcriptome sequencing data. PMID:22570408

FusionAnalyser: a new graphical, event-driven tool for fusion rearrangements discovery.

PubMed

Piazza, Rocco; Pirola, Alessandra; Spinelli, Roberta; Valletta, Simona; Redaelli, Sara; Magistroni, Vera; Gambacorti-Passerini, Carlo

2012-09-01

Gene fusions are common driver events in leukaemias and solid tumours; here we present FusionAnalyser, a tool dedicated to the identification of driver fusion rearrangements in human cancer through the analysis of paired-end high-throughput transcriptome sequencing data. We initially tested FusionAnalyser by using a set of in silico randomly generated sequencing data from 20 known human translocations occurring in cancer and subsequently using transcriptome data from three chronic and three acute myeloid leukaemia samples. in all the cases our tool was invariably able to detect the presence of the correct driver fusion event(s) with high specificity. In one of the acute myeloid leukaemia samples, FusionAnalyser identified a novel, cryptic, in-frame ETS2-ERG fusion. A fully event-driven graphical interface and a flexible filtering system allow complex analyses to be run in the absence of any a priori programming or scripting knowledge. Therefore, we propose FusionAnalyser as an efficient and robust graphical tool for the identification of functional rearrangements in the context of high-throughput transcriptome sequencing data.

Sperm flagella protein components: Human meichroacidin constructed by the membrane occupation and recognition nexus motif

PubMed Central

MATSUOKA, YASUHIRO; NISHIMURA, HIROMI; NUMAZAWA, KAHORI; TSUCHIDA, JUNJI; MIYAGAWA, YASUSHI; TSUJIMURA, AKIRA; MATSUMIYA, KIYOMI; OKUYAMA, AKIHIKO; NISHIMUNE, YOSHITAKE

2005-01-01

Background and Aims:   In a previous study, the authors of the present study cloned mouse meichroacidin (MCA), which is expressed in stages of spermatogenesis from pachytene spermatocytes through round spermatid germ cells. MCA protein contains the membrane occupation and recognition nexus (MORN) motif and localizes to a male meiotic metaphase chromosome. Recently, a MCA homolog of carp (Cyprinus carpio), MORN motif‐containing sperm‐specific axonemal protein (MSAP), was reportedly identified and localized in sperm flagella. Present knowledge of human spermiogenesis requires the identification of proteins in human sperm. The present study identified the human orthologue of MCA. Methods:   Colony hybridization using a human testis plasmid cDNA library was carried out to clone human MCA (h‐MCA) cDNA. Northern blot, Western blot, and immunohistochemical analyses were carried out. Results:   h‐MCA was found to be specifically expressed in the testes. The h‐MCA amino acid sequence shared 79.8% identity with mouse MCA and contained MORN motifs. h‐MCA localized in the sperm flagellum and basal body, as does MSAP in carp. Conclusion:   Expression and localization analyses showed that h‐MCA is a component of the sperm flagellum and basal body and might play an important role in the development of the sperm flagellum in humans. (Reprod Med Biol 2005; 4: 213–219) PMID:29699225

Systemic approaches identify a garlic-derived chemical, Z-ajoene, as a glioblastoma multiforme cancer stem cell-specific targeting agent.

PubMed

Jung, Yuchae; Park, Heejoo; Zhao, Hui-Yuan; Jeon, Raok; Ryu, Jae-Ha; Kim, Woo-Young

2014-07-01

Glioblastoma multiforme (GBM) is one of the most common brain malignancies and has a very poor prognosis. Recent evidence suggests that the presence of cancer stem cells (CSC) in GBM and the rare CSC subpopulation that is resistant to chemotherapy may be responsible for the treatment failure and unfavorable prognosis of GBM. A garlic-derived compound, Z-ajoene, has shown a range of biological activities, including anti-proliferative effects on several cancers. Here, we demonstrated for the first time that Z-ajoene specifically inhibits the growth of the GBM CSC population. CSC sphere-forming inhibition was achieved at a concentration that did not exhibit a cytotoxic effect in regular cell culture conditions. The specificity of this inhibitory effect on the CSC population was confirmed by detecting CSC cell surface marker CD133 expression and biochemical marker ALDH activity. In addition, stem cell-related mRNA profiling and real-time PCR revealed the differential expression of CSC-specific genes, including Notch, Wnt, and Hedgehog, upon treatment with Z-ajoene. A proteomic approach, i.e., reverse-phase protein array (RPPA) and Western blot analysis, showed decreased SMAD4, p-AKT, 14.3.3 and FOXO3A expression. The protein interaction map (http://string-db.org/) of the identified molecules suggested that the AKT, ERK/p38 and TGFβ signaling pathways are key mediators of Z-ajoene's action, which affects the transcriptional network that includes FOXO3A. These biological and bioinformatic analyses collectively demonstrate that Z-ajoene is a potential candidate for the treatment of GBM by specifically targeting GBM CSCs. We also show how this systemic approach strengthens the identification of new therapeutic agents that target CSCs.

[Optimization of the immunoelectrophoresis technic (western blot) for the confirmation of human immunodeficiency virus infection (HIV) in Panama].

PubMed

Pascale, J M; de Austin, E; de Moreno, N O; Ledezma, C; de Márquez, E; Blanco, R; Quiroz, E; Calzada, J; Vincensini, A R; de Martin, M C

1995-01-01

The purpose of this study is to report the results of the authors' investigation to apply the western blot technique (WB UP-LCS) in the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection. To do this, the authors separated the proteins of the HIV-1 virus by electrophoresis, based on their molecular weight, in poliacilamide gel with SDS (SDS-PAGE) during 3 hours at 200 volts. Then they electrotransferred these proteins to nitrocellulose paper during four hours at 200 milliamperes, with the aid of external cooling. The nitrocellulose strips were evaluated considering the incubation time (1 and 16 hours), two conjugates (human anti IgG with Peroxidase and human anti IgG Biotin plus Streptatividine with Peroxidase) and two dilutions of the patients' sera (1/50 and 1/100). Based on their results the Authors conclude that, in the first place, the optimal conditions for the test include a dilution of 1/100 of the patients serum, incubation of the serum for 16 hours and the use of the conjugate of anti human IgG with Biotin and Streptavidine with Peroxidase; secondary, that the immunologic reactivity against proteins p24 and gp 160/120 is the most important diagnostic criterion for the confirmation of infection with HIV-1 and that they obtained a diagnostic correlation of 100% at a cost which was 5 to 7 times less than that of the commercial system.

ISAAC - InterSpecies Analysing Application using Containers.

PubMed

Baier, Herbert; Schultz, Jörg

2014-01-15

Information about genes, transcripts and proteins is spread over a wide variety of databases. Different tools have been developed using these databases to identify biological signals in gene lists from large scale analysis. Mostly, they search for enrichments of specific features. But, these tools do not allow an explorative walk through different views and to change the gene lists according to newly upcoming stories. To fill this niche, we have developed ISAAC, the InterSpecies Analysing Application using Containers. The central idea of this web based tool is to enable the analysis of sets of genes, transcripts and proteins under different biological viewpoints and to interactively modify these sets at any point of the analysis. Detailed history and snapshot information allows tracing each action. Furthermore, one can easily switch back to previous states and perform new analyses. Currently, sets can be viewed in the context of genomes, protein functions, protein interactions, pathways, regulation, diseases and drugs. Additionally, users can switch between species with an automatic, orthology based translation of existing gene sets. As todays research usually is performed in larger teams and consortia, ISAAC provides group based functionalities. Here, sets as well as results of analyses can be exchanged between members of groups. ISAAC fills the gap between primary databases and tools for the analysis of large gene lists. With its highly modular, JavaEE based design, the implementation of new modules is straight forward. Furthermore, ISAAC comes with an extensive web-based administration interface including tools for the integration of third party data. Thus, a local installation is easily feasible. In summary, ISAAC is tailor made for highly explorative interactive analyses of gene, transcript and protein sets in a collaborative environment.

Developmental trajectories of paediatric headache - sex-specific analyses and predictors.

PubMed

Isensee, Corinna; Fernandez Castelao, Carolin; Kröner-Herwig, Birgit

2016-01-01

Headache is the most common pain disorder in children and adolescents and is associated with diverse dysfunctions and psychological symptoms. Several studies evidenced sex-specific differences in headache frequency. Until now no study exists that examined sex-specific patterns of change in paediatric headache across time and included pain-related somatic and (socio-)psychological predictors. Latent Class Growth Analysis (LCGA) was used in order to identify different trajectory classes of headache across four annual time points in a population-based sample (n = 3 227; mean age 11.34 years; 51.2 % girls). In multinomial logistic regression analyses the influence of several predictors on the class membership was examined. For girls, a four-class model was identified as the best fitting model. While the majority of girls reported no (30.5 %) or moderate headache frequencies (32.5 %) across time, one class with a high level of headache days (20.8 %) and a class with an increasing headache frequency across time (16.2 %) were identified. For boys a two class model with a 'no headache class' (48.6 %) and 'moderate headache class' (51.4 %) showed the best model fit. Regarding logistic regression analyses, migraine and parental headache proved to be stable predictors across sexes. Depression/anxiety was a significant predictor for all pain classes in girls. Life events, dysfunctional stress coping and school burden were also able to differentiate at least between some classes in both sexes. The identified trajectories reflect sex-specific differences in paediatric headache, as seen in the number and type of classes extracted. The documented risk factors can deliver ideas for preventive actions and considerations for treatment programmes.

Structural analyses to identify selective inhibitors of glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme

PubMed Central

Danshina, Polina V.; Qu, Weidong; Temple, Brenda R.; Rojas, Rafael J.; Miley, Michael J.; Machius, Mischa; Betts, Laurie; O'Brien, Deborah A.

2016-01-01

STUDY HYPOTHESIS Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development. STUDY FINDING This study identified a small-molecule GAPDHS inhibitor with micromolar potency and >10-fold selectivity that exerts the expected inhibitory effects on sperm glycolysis and motility. WHAT IS KNOWN ALREADY Glycolytic ATP production is required for sperm motility and male fertility in many mammalian species. Selective inhibition of GAPDHS, one of the glycolytic isozymes with restricted expression during spermatogenesis, is a potential strategy for the development of a non-hormonal contraceptive that directly blocks sperm function. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Homology modeling and x-ray crystallography were used to identify structural features that are conserved in GAPDHS orthologs in mouse and human sperm, but distinct from the GAPDH orthologs present in somatic tissues. We identified three binding pockets surrounding the substrate and cofactor in these isozymes and conducted a virtual screen to identify small-molecule compounds predicted to bind more tightly to GAPDHS than to GAPDH. Following the production of recombinant human and mouse GAPDHS, candidate compounds were tested in dose–response enzyme assays to identify inhibitors that blocked the activity of GAPDHS more effectively than GAPDH. The effects of a selective inhibitor on the motility of mouse and human sperm were monitored by computer-assisted sperm analysis, and sperm lactate production was measured to assess inhibition of glycolysis in the target cell. MAIN RESULTS AND THE ROLE OF CHANCE Our studies produced the first apoenzyme crystal structures for human and mouse GAPDHS and a 1.73 Å crystal structure for NAD+-bound human GAPDHS, facilitating the identification of unique

Differential expression of two glutathione S-transferases identified from the American dog tick, Dermacentor variabilis.

PubMed

Dreher-Lesnick, S M; Mulenga, A; Simser, J A; Azad, A F

2006-08-01

Reciprocal signalling and gene expression play a cardinal role during pathogen-host molecular interactions and are prerequisite to the maintenance of balanced homeostasis. Gene expression repertoire changes during rickettsial infection and glutathione-S-transferases (GSTs) were among the genes found up-regulated in Rickettsia-infected Dermacentor variabilis. GSTs are well known to play an important part in cellular stress responses in the host. We have cloned two full-length GSTs from D. variabilis (DvGST1 and DvGST2). Comparison of these two DvGST molecules with those of other species indicate that DvGST1 is related to the mammalian class theta and insect class delta GSTs, while DvGST2 does not seem to fall in the same family. Northern blotting analyses revealed differential expression patterns, where DvGST1 and DvGST2 transcripts are found in the tick gut, with DvGST2 transcripts also present in the ovaries. Both DvGST transcripts are up-regulated upon tick feeding. Challenge of fed adult ticks with Escherichia coli injection showed decreased transcript amounts compared with ticks injected with phosphate-buffered saline (sham) and naïve ticks.

Financial relationships in economic analyses of targeted therapies in oncology.

PubMed

Valachis, Antonis; Polyzos, Nikolaos P; Nearchou, Andreas; Lind, Pehr; Mauri, Davide

2012-04-20

A potential financial relationship between investigators and pharmaceutical manufacturers has been associated with an increased likelihood of reporting favorable conclusions about a sponsor's proprietary agent in pharmacoeconomic studies. The purpose of this study is to investigate whether there is an association between financial relationships and outcome in economic analyses of new targeted therapies in oncology. We searched PubMed (last update June 2011) for economic analyses of targeted therapies (including monoclonal antibodies, tyrosine-kinase inhibitors, and mammalian target of rapamycin inhibitors) in oncology. The trials were qualitatively rated regarding the cost assessment as favorable, neutral, or unfavorable on the basis of prespecified criteria. Overall, 81 eligible studies were identified. Economic analyses that were funded by pharmaceutical companies were more likely to report favorable qualitative cost estimates (28 [82%] of 34 v 21 [45%] of 47; P = .003). The presence of an author affiliated with manufacturer was not associated with study outcome. Furthermore, if only studies including a conflict of interest statement were included (66 of 81), studies that reported any financial relationship with manufacturers (author affiliation and/or funding and/or other financial relationship) were more likely to report favorable results of targeted therapies compared with studies without financial relationship (32 [71%] of 45 v nine [43%] of 21; P = .025). Our study reveals a potential threat for industry-related bias in economic analyses of targeted therapies in oncology in favor of analyses with financial relationships between authors and manufacturers. A more balanced funding of economic analyses from other sources may allow greater confidence in the interpretation of their results.

Enhanced approaches for identifying Amadori products: application to peanut allergens

USDA-ARS?s Scientific Manuscript database

The dry roasting of peanuts is suggested to influence allergenic sensitization due to formation of advanced glycation end products (AGE) on peanut proteins. Identifying AGEs is technically challenging. The AGE composition of peanut proteins was probed with nanoLC-ESI-MS and MS/MS analyses. Amadori ...

Novel Autism Subtype-Dependent Genetic Variants Are Revealed by Quantitative Trait and Subphenotype Association Analyses of Published GWAS Data

PubMed Central

Hu, Valerie W.; Addington, Anjene; Hyman, Alexander

2011-01-01

The heterogeneity of symptoms associated with autism spectrum disorders (ASDs) has presented a significant challenge to genetic analyses. Even when associations with genetic variants have been identified, it has been difficult to associate them with a specific trait or characteristic of autism. Here, we report that quantitative trait analyses of ASD symptoms combined with case-control association analyses using distinct ASD subphenotypes identified on the basis of symptomatic profiles result in the identification of highly significant associations with 18 novel single nucleotide polymorphisms (SNPs). The symptom categories included deficits in language usage, non-verbal communication, social development, and play skills, as well as insistence on sameness or ritualistic behaviors. Ten of the trait-associated SNPs, or quantitative trait loci (QTL), were associated with more than one subtype, providing partial replication of the identified QTL. Notably, none of the novel SNPs is located within an exonic region, suggesting that these hereditary components of ASDs are more likely related to gene regulatory processes (or gene expression) than to structural or functional changes in gene products. Seven of the QTL reside within intergenic chromosomal regions associated with rare copy number variants that have been previously reported in autistic samples. Pathway analyses of the genes associated with the QTL identified in this study implicate neurological functions and disorders associated with autism pathophysiology. This study underscores the advantage of incorporating both quantitative traits as well as subphenotypes into large-scale genome-wide analyses of complex disorders. PMID:21556359

Assessing Plausibility of Tentative Chemical Identifications from Suspect Screening Analyses via Chemical Function

EPA Science Inventory

Suspect screening (SSA) and non-targeted analysis (NTA) have become increasingly useful methods for identifying chemicals in indoor environments, which is where many chemical exposures occur. However, the tentative chemical identifications from these analyses must be confirmed. T...

Analyses of shuttle orbiter approach and landing conditions

NASA Technical Reports Server (NTRS)

Teper, G. L.; Dimarco, R. J.; Ashkenas, I. L.; Hoh, R. H.

1981-01-01

A study of one shuttle orbiter approach and landing conditions are summarized. Causes of observed PIO like flight deficiencies are identified and potential cures are examined. Closed loop pilot/vehicle analyses are described and path/attitude stability boundaries defined. The latter novel technique proved of great value in delineating and illustrating the basic causes of this multiloop pilot control problem. The analytical results are shown to be consistent with flight test and fixed base simulation. Conclusions are drawn relating to possible improvements of the shuttle orbiter/digital flight control system.

Process improvement for regulatory analyses of custom-blend fertilizers.

PubMed

Wegner, Keith A

2014-01-01

Chemical testing of custom-blend fertilizers is essential to ensure that the products meet the formulation requirements. For purposes of proper crop nutrition and consumer protection, regulatory oversight promotes compliance and particular attention to blending and formulation specifications. Analyses of custom-blend fertilizer products must be performed and reported within a very narrow window in order to be effective. The Colorado Department of Agriculture's Biochemistry Laboratory is an ISO 17025 accredited facility and conducts analyses of custom-blend fertilizer products primarily during the spring planting season. Using the Lean Six Sigma (LSS) process, the Biochemistry Laboratory has reduced turnaround times from as much as 45 days to as little as 3 days. The LSS methodology focuses on waste reduction through identifying: non-value-added steps, unneeded process reviews, optimization of screening and confirmatory analyses, equipment utilization, nonessential reporting requirements, and inefficient personnel deployment. Eliminating these non-value-added activities helped the laboratory significantly shorten turnaround time and reduce costs. Key improvement elements discovered during the LSS process included: focused sample tracking, equipment redundancy, strategic supply stocking, batch size optimization, critical sample paths, elimination of nonessential QC reviews, and more efficient personnel deployment.

DMINDA: an integrated web server for DNA motif identification and analyses.

PubMed

Ma, Qin; Zhang, Hanyuan; Mao, Xizeng; Zhou, Chuan; Liu, Bingqiang; Chen, Xin; Xu, Ying

2014-07-01

DMINDA (DNA motif identification and analyses) is an integrated web server for DNA motif identification and analyses, which is accessible at http://csbl.bmb.uga.edu/DMINDA/. This web site is freely available to all users and there is no login requirement. This server provides a suite of cis-regulatory motif analysis functions on DNA sequences, which are important to elucidation of the mechanisms of transcriptional regulation: (i) de novo motif finding for a given set of promoter sequences along with statistical scores for the predicted motifs derived based on information extracted from a control set, (ii) scanning motif instances of a query motif in provided genomic sequences, (iii) motif comparison and clustering of identified motifs, and (iv) co-occurrence analyses of query motifs in given promoter sequences. The server is powered by a backend computer cluster with over 150 computing nodes, and is particularly useful for motif prediction and analyses in prokaryotic genomes. We believe that DMINDA, as a new and comprehensive web server for cis-regulatory motif finding and analyses, will benefit the genomic research community in general and prokaryotic genome researchers in particular. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structural Identifiability of Dynamic Systems Biology Models

PubMed Central

Villaverde, Alejandro F.

2016-01-01

A powerful way of gaining insight into biological systems is by creating a nonlinear differential equation model, which usually contains many unknown parameters. Such a model is called structurally identifiable if it is possible to determine the values of its parameters from measurements of the model outputs. Structural identifiability is a prerequisite for parameter estimation, and should be assessed before exploiting a model. However, this analysis is seldom performed due to the high computational cost involved in the necessary symbolic calculations, which quickly becomes prohibitive as the problem size increases. In this paper we show how to analyse the structural identifiability of a very general class of nonlinear models by extending methods originally developed for studying observability. We present results about models whose identifiability had not been previously determined, report unidentifiabilities that had not been found before, and show how to modify those unidentifiable models to make them identifiable. This method helps prevent problems caused by lack of identifiability analysis, which can compromise the success of tasks such as experiment design, parameter estimation, and model-based optimization. The procedure is called STRIKE-GOLDD (STRuctural Identifiability taKen as Extended-Generalized Observability with Lie Derivatives and Decomposition), and it is implemented in a MATLAB toolbox which is available as open source software. The broad applicability of this approach facilitates the analysis of the increasingly complex models used in systems biology and other areas. PMID:27792726

Combining gene expression and genetic analyses to identify candidate genes involved in cold responses in pea.

PubMed

Legrand, Sylvain; Marque, Gilles; Blassiau, Christelle; Bluteau, Aurélie; Canoy, Anne-Sophie; Fontaine, Véronique; Jaminon, Odile; Bahrman, Nasser; Mautord, Julie; Morin, Julie; Petit, Aurélie; Baranger, Alain; Rivière, Nathalie; Wilmer, Jeroen; Delbreil, Bruno; Lejeune-Hénaut, Isabelle

2013-09-01

Cold stress affects plant growth and development. In order to better understand the responses to cold (chilling or freezing tolerance), we used two contrasted pea lines. Following a chilling period, the Champagne line becomes tolerant to frost whereas the Terese line remains sensitive. Four suppression subtractive hybridisation libraries were obtained using mRNAs isolated from pea genotypes Champagne and Terese. Using quantitative polymerase chain reaction (qPCR) performed on 159 genes, 43 and 54 genes were identified as differentially expressed at the initial time point and during the time course study, respectively. Molecular markers were developed from the differentially expressed genes and were genotyped on a population of 164 RILs derived from a cross between Champagne and Terese. We identified 5 candidate genes colocalizing with 3 different frost damage quantitative trait loci (QTL) intervals and a protein quantity locus (PQL) rich region previously reported. This investigation revealed the role of constitutive differences between both genotypes in the cold responses, in particular with genes related to glycine degradation pathway that could confer to Champagne a better frost tolerance. We showed that freezing tolerance involves a decrease of expression of genes related to photosynthesis and the expression of a gene involved in the production of cysteine and methionine that could act as cryoprotectant molecules. Although it remains to be confirmed, this study could also reveal the involvement of the jasmonate pathway in the cold responses, since we observed that two genes related to this pathway were mapped in a frost damage QTL interval and in a PQL rich region interval, respectively. Copyright © 2013 Elsevier GmbH. All rights reserved.

Analysing the hidden curriculum: use of a cultural web

PubMed Central

Mossop, Liz; Dennick, Reg; Hammond, Richard; Robbé, Iain

2013-01-01

CONTEXT Major influences on learning about medical professionalism come from the hidden curriculum. These influences can contribute positively or negatively towards the professional enculturation of clinical students. The fact that there is no validated method for identifying the components of the hidden curriculum poses problems for educators considering professionalism. The aim of this study was to analyse whether a cultural web, adapted from a business context, might assist in the identification of elements of the hidden curriculum at a UK veterinary school. METHODS A qualitative approach was used. Seven focus groups consisting of three staff groups and four student groups were organised. Questioning was framed using the cultural web, which is a model used by business owners to assess their environment and consider how it affects their employees and customers. The focus group discussions were recorded, transcribed and analysed thematically using a combination of a priori and emergent themes. RESULTS The cultural web identified elements of the hidden curriculum for both students and staff. These included: core assumptions; routines; rituals; control systems; organisational factors; power structures, and symbols. Discussions occurred about how and where these issues may affect students’ professional identity development. CONCLUSIONS The cultural web framework functioned well to help participants identify elements of the hidden curriculum. These aspects aligned broadly with previously described factors such as role models and institutional slang. The influence of these issues on a student’s development of a professional identity requires discussion amongst faculty staff, and could be used to develop learning opportunities for students. The framework is promising for the analysis of the hidden curriculum and could be developed as an instrument for implementation in other clinical teaching environments. PMID:23323652

Survey of the Methods and Reporting Practices in Published Meta-analyses of Test Performance: 1987 to 2009

ERIC Educational Resources Information Center

Dahabreh, Issa J.; Chung, Mei; Kitsios, Georgios D.; Terasawa, Teruhiko; Raman, Gowri; Tatsioni, Athina; Tobar, Annette; Lau, Joseph; Trikalinos, Thomas A.; Schmid, Christopher H.

2013-01-01

We performed a survey of meta-analyses of test performance to describe the evolution in their methods and reporting. Studies were identified through MEDLINE (1966-2009), reference lists, and relevant reviews. We extracted information on clinical topics, literature review methods, quality assessment, and statistical analyses. We reviewed 760…

A systems biology strategy to identify molecular mechanisms of action and protein indicators of traumatic brain injury.

PubMed

Yu, Chenggang; Boutté, Angela; Yu, Xueping; Dutta, Bhaskar; Feala, Jacob D; Schmid, Kara; Dave, Jitendra; Tawa, Gregory J; Wallqvist, Anders; Reifman, Jaques

2015-02-01

The multifactorial nature of traumatic brain injury (TBI), especially the complex secondary tissue injury involving intertwined networks of molecular pathways that mediate cellular behavior, has confounded attempts to elucidate the pathology underlying the progression of TBI. Here, systems biology strategies are exploited to identify novel molecular mechanisms and protein indicators of brain injury. To this end, we performed a meta-analysis of four distinct high-throughput gene expression studies involving different animal models of TBI. By using canonical pathways and a large human protein-interaction network as a scaffold, we separately overlaid the gene expression data from each study to identify molecular signatures that were conserved across the different studies. At 24 hr after injury, the significantly activated molecular signatures were nonspecific to TBI, whereas the significantly suppressed molecular signatures were specific to the nervous system. In particular, we identified a suppressed subnetwork consisting of 58 highly interacting, coregulated proteins associated with synaptic function. We selected three proteins from this subnetwork, postsynaptic density protein 95, nitric oxide synthase 1, and disrupted in schizophrenia 1, and hypothesized that their abundance would be significantly reduced after TBI. In a penetrating ballistic-like brain injury rat model of severe TBI, Western blot analysis confirmed our hypothesis. In addition, our analysis recovered 12 previously identified protein biomarkers of TBI. The results suggest that systems biology may provide an efficient, high-yield approach to generate testable hypotheses that can be experimentally validated to identify novel mechanisms of action and molecular indicators of TBI. © 2014 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

One Step at a Time: Using Task Analyses to Teach Skills

ERIC Educational Resources Information Center

Snodgrass, Melinda R.; Meadan, Hedda; Ostrosky, Michaelene M.; Cheung, W. Catherine

2017-01-01

Task analyses are useful when teaching children how to complete tasks by breaking the tasks into small steps, particularly when children struggle to learn a skill during typical classroom instruction. We describe how to create a task analysis by identifying the steps a child needs to independently perform the task, how to assess what steps a child…

Best Practice in Developing Critical Analyses: An Empirical Study of Self-Efficacy from an Interdisciplinary Perspective

ERIC Educational Resources Information Center

Sams, Coleen; Sams, Doreen

2011-01-01

Despite substantial usage of case study analyses as learning tools in diverse fields of study, business graduate students often arrive with little to no prior knowledge in how to effectively use this tool. Using the Sams efficacy model (2009) strategic to business education this study identified a dearth in the usage of case study analyses in…

Whole-genome sequencing and analyses identify high genetic heterogeneity, diversity and endemicity of rotavirus genotype P[6] strains circulating in Africa.

PubMed

Nyaga, Martin M; Tan, Yi; Seheri, Mapaseka L; Halpin, Rebecca A; Akopov, Asmik; Stucker, Karla M; Fedorova, Nadia B; Shrivastava, Susmita; Duncan Steele, A; Mwenda, Jason M; Pickett, Brett E; Das, Suman R; Jeffrey Mphahlele, M

2018-05-18

Rotavirus A (RVA) exhibits a wide genotype diversity globally. Little is known about the genetic composition of genotype P[6] from Africa. This study investigated possible evolutionary mechanisms leading to genetic diversity of genotype P[6] VP4 sequences. Phylogenetic analyses on 167 P[6] VP4 full-length sequences were conducted, which included six porcine-origin sequences. Of the 167 sequences, 57 were newly acquired through whole genome sequencing as part of this study. The other 110 sequences were all publicly-available global P[6] VP4 full-length sequences downloaded from GenBank. The strength of association between the phenotypic features and the phylogeny was also determined. A number of reassortment and mixed infections of RVA genotype P[6] strains were observed in this study. Phylogenetic analyses demostrated the extensive genetic diversity that exists among human P[6] strains, porcine-like strains, their concomitant clades/subclades and estimated that P[6] VP4 gene has a higher substitution rate with the mean of 1.05E-3 substitutions/site/year. Further, the phylogenetic analyses indicated that genotype P[6] strains were endemic in Africa, characterised by an extensive genetic diversity and long-time local evolution of the viruses. This was also supported by phylogeographic clustering and G-genotype clustering of the P[6] strains when Bayesian Tip-association Significance testing (BaTS) was applied, clearly supporting that the viruses evolved locally in Africa instead of spatial mixing among different regions. Overall, the results demonstrated that multiple mechanisms such as reassortment events, various mutations and possibly interspecies transmission account for the enormous diversity of genotype P[6] strains in Africa. These findings highlight the need for continued global surveillance of rotavirus diversity. Copyright © 2018 Elsevier B.V. All rights reserved.

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

DOE PAGES

Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric; ...

2015-03-30

Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

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

Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric

Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

Methods to systematically review and meta-analyse observational studies: a systematic scoping review of recommendations.

PubMed

Mueller, Monika; D'Addario, Maddalena; Egger, Matthias; Cevallos, Myriam; Dekkers, Olaf; Mugglin, Catrina; Scott, Pippa

2018-05-21

Systematic reviews and meta-analyses of observational studies are frequently performed, but no widely accepted guidance is available at present. We performed a systematic scoping review of published methodological recommendations on how to systematically review and meta-analyse observational studies. We searched online databases and websites and contacted experts in the field to locate potentially eligible articles. We included articles that provided any type of recommendation on how to conduct systematic reviews and meta-analyses of observational studies. We extracted and summarised recommendations on pre-defined key items: protocol development, research question, search strategy, study eligibility, data extraction, dealing with different study designs, risk of bias assessment, publication bias, heterogeneity, statistical analysis. We summarised recommendations by key item, identifying areas of agreement and disagreement as well as areas where recommendations were missing or scarce. The searches identified 2461 articles of which 93 were eligible. Many recommendations for reviews and meta-analyses of observational studies were transferred from guidance developed for reviews and meta-analyses of RCTs. Although there was substantial agreement in some methodological areas there was also considerable disagreement on how evidence synthesis of observational studies should be conducted. Conflicting recommendations were seen on topics such as the inclusion of different study designs in systematic reviews and meta-analyses, the use of quality scales to assess the risk of bias, and the choice of model (e.g. fixed vs. random effects) for meta-analysis. There is a need for sound methodological guidance on how to conduct systematic reviews and meta-analyses of observational studies, which critically considers areas in which there are conflicting recommendations.

Biomarkers and Attention-Deficit/Hyperactivity Disorder: A Systematic Review and Meta-Analyses

ERIC Educational Resources Information Center

Scassellati, Catia; Bonvicini, Cristian; Faraone, Stephen V.; Gennarelli, Massimo

2012-01-01

Objective: To determine whether peripheral biochemical markers (biomarkers) might differentiate patients with attention-deficit/hyperactivity disorder (ADHD) from non-ADHD individuals. Method: We conducted a systematic search and a series of meta-analyses of case-control studies comprising studies from 1969 to 2011. Results: We identified 210…

Challenges and opportunities in analysing students modelling

NASA Astrophysics Data System (ADS)

Blanco-Anaya, Paloma; Justi, Rosária; Díaz de Bustamante, Joaquín

2017-02-01

Modelling-based teaching activities have been designed and analysed from distinct theoretical perspectives. In this paper, we use one of them - the model of modelling diagram (MMD) - as an analytical tool in a regular classroom context. This paper examines the challenges that arise when the MMD is used as an analytical tool to characterise the modelling process experienced by students working in small groups aiming at creating and testing a model of a sedimentary basin from the information provided. The study was conducted in a regular Biology and Geology classroom (16-17 years old students). Data was collected through video recording of the classes, along with written reports and the material models made by each group. The results show the complexity of adapting MMD at two levels: the group modelling and the actual requirements for the activity. Our main challenges were to gather the modelling process of each individual and the group, as well as to identify, from students' speech, which stage of modelling they were performing at a given time. When facing such challenges, we propose some changes in the MMD so that it can be properly used to analyse students performing modelling activities in groups.

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis

DOE PAGES

Weyman, Philip D.; Beeri, Karen; Lefebvre, Stephane C.; ...

2014-10-10

Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow onmore » urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.« less

Aquaporins 7 and 11 in boar spermatozoa: detection, localisation and relationship with sperm quality.

PubMed

Prieto-Martínez, Noelia; Vilagran, Ingrid; Morató, Roser; Rodríguez-Gil, Joan E; Yeste, Marc; Bonet, Sergi

2016-04-01

Aquaporins (AQPs) are integral membrane water channels that allow transport of water and small solutes across cell membranes. Although water permeability is known to play a critical role in mammalian cells, including spermatozoa, little is known about their localisation in boar spermatozoa. Two aquaporins, AQP7 and AQP11, in boar spermatozoa were identified by western blotting and localised through immunocytochemistry analyses. Western blot results showed that boar spermatozoa expressed AQP7 (25kDa) and AQP11 (50kDa). Immunocytochemistry analyses demonstrated that AQP7 was localised in the connecting piece of boar spermatozoa, while AQP11 was found in the head and mid-piece and diffuse labelling was also seen along the tail. Despite differences in AQP7 and AQP11 content between boar ejaculates, these differences were not found to be correlated with sperm quality in the case of AQP7. Conversely, AQP11 content showed a significant correlation (P<0.05) with sperm membrane integrity and fluidity and sperm motility. In conclusion, boar spermatozoa express AQP7 and AQP11, and the amounts of AQP11 but not those of AQP7 are correlated with sperm motility and membrane integrity.

The moral economy of austerity: analysing UK welfare reform.

PubMed

Morris, Lydia

2016-03-01

This paper notes the contemporary emergence of 'morality' in both sociological argument and political rhetoric, and analyses its significance in relation to ongoing UK welfare reforms. It revisits the idea of 'moral economy' and identifies two strands in its contemporary application; that all economies depend on an internal moral schema, and that some external moral evaluation is desirable. UK welfare reform is analysed as an example of the former, with reference to three distinct orientations advanced in the work of Freeden (1996), Laclau (2014), and Lockwood (1996). In this light, the paper then considers challenges to the reform agenda, drawn from third sector and other public sources. It outlines the forms of argument present in these challenges, based respectively on rationality, legality, and morality, which together provide a basis for evaluation of the welfare reforms and for an alternative 'moral economy'. © London School of Economics and Political Science 2016.

A framework for identifying carbon hotspots and forest management drivers

Treesearch

Nilesh Timilsina; Francisco J. Escobedo; Wendell P. Cropper; Amr Abd-Elrahman; Thomas Brandeis; Sonia Delphin; Samuel Lambert

2013-01-01

Spatial analyses of ecosystem system services that are directly relevant to both forest management decision making and conservation in the subtropics are rare. Also, frameworks that identify and map carbon stocks and corresponding forest management drivers using available regional, national, and international-level forest inventory datasets could provide insights into...

Systematic MicroRNA Analysis Identifies ATP6V0C as an Essential Host Factor for Human Cytomegalovirus Replication

PubMed Central

Pavelin, Jon; Reynolds, Natalie; Chiweshe, Stephen; Wu, Guanming; Tiribassi, Rebecca; Grey, Finn

2013-01-01

Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions. PMID:24385903

Gene sequence analyses and other DNA-based methods for yeast species recognition

USDA-ARS?s Scientific Manuscript database

DNA sequence analyses, as well as other DNA-based methodologies, have transformed the way in which yeasts are identified. The focus of this chapter will be on the resolution of species using various types of DNA comparisons. In other chapters in this book, Rozpedowska, Piškur and Wolfe discuss mul...

Gene Expression Analyses of Subchondral Bone in Early Experimental Osteoarthritis by Microarray

PubMed Central

Chen, YuXian; Shen, Jun; Lu, HuaDing; Zeng, Chun; Ren, JianHua; Zeng, Hua; Li, ZhiFu; Chen, ShaoMing; Cai, DaoZhang; Zhao, Qing

2012-01-01

Osteoarthritis (OA) is a degenerative joint disease that affects both cartilage and bone. A better understanding of the early molecular changes in subchondral bone may help elucidate the pathogenesis of OA. We used microarray technology to investigate the time course of molecular changes in the subchondral bone in the early stages of experimental osteoarthritis in a rat model. We identified 2,234 differentially expressed (DE) genes at 1 week, 1,944 at 2 weeks and 1,517 at 4 weeks post-surgery. Further analyses of the dysregulated genes indicated that the events underlying subchondral bone remodeling occurred sequentially and in a time-dependent manner at the gene expression level. Some of the identified dysregulated genes that were identified have suspected roles in bone development or remodeling; these genes include Alp, Igf1, Tgf β1, Postn, Mmp3, Tnfsf11, Acp5, Bmp5, Aspn and Ihh. The differences in the expression of these genes were confirmed by real-time PCR, and the results indicated that our microarray data accurately reflected gene expression patterns characteristic of early OA. To validate the results of our microarray analysis at the protein level, immunohistochemistry staining was used to investigate the expression of Mmp3 and Aspn protein in tissue sections. These analyses indicate that Mmp3 protein expression completely matched the results of both the microarray and real-time PCR analyses; however, Aspn protein expression was not observed to differ at any time. In summary, our study demonstrated a simple method of separation of subchondral bone sample from the knee joint of rat, which can effectively avoid bone RNA degradation. These findings also revealed the gene expression profiles of subchondral bone in the rat OA model at multiple time points post-surgery and identified important DE genes with known or suspected roles in bone development or remodeling. These genes may be novel diagnostic markers or therapeutic targets for OA. PMID:22384228

Subgroup analyses in confirmatory clinical trials: time to be specific about their purposes.

PubMed

Tanniou, Julien; van der Tweel, Ingeborg; Teerenstra, Steven; Roes, Kit C B

2016-02-18

It is well recognized that treatment effects may not be homogeneous across the study population. Subgroup analyses constitute a fundamental step in the assessment of evidence from confirmatory (Phase III) clinical trials, where conclusions for the overall study population might not hold. Subgroup analyses can have different and distinct purposes, requiring specific design and analysis solutions. It is relevant to evaluate methodological developments in subgroup analyses against these purposes to guide health care professionals and regulators as well as to identify gaps in current methodology. We defined four purposes for subgroup analyses: (1) Investigate the consistency of treatment effects across subgroups of clinical importance, (2) Explore the treatment effect across different subgroups within an overall non-significant trial, (3) Evaluate safety profiles limited to one or a few subgroup(s), (4) Establish efficacy in the targeted subgroup when included in a confirmatory testing strategy of a single trial. We reviewed the methodology in line with this "purpose-based" framework. The review covered papers published between January 2005 and April 2015 and aimed to classify them in none, one or more of the aforementioned purposes. In total 1857 potentially eligible papers were identified. Forty-eight papers were selected and 20 additional relevant papers were identified from their references, leading to 68 papers in total. Nineteen were dedicated to purpose 1, 16 to purpose 4, one to purpose 2 and none to purpose 3. Seven papers were dedicated to more than one purpose, the 25 remaining could not be classified unambiguously. Purposes of the methods were often not specifically indicated, methods for subgroup analysis for safety purposes were almost absent and a multitude of diverse methods were developed for purpose (1). It is important that researchers developing methodology for subgroup analysis explicitly clarify the objectives of their methods in terms that can be

Identifying Balance Measures Most Likely to Identify Recent Falls.

PubMed

Criter, Robin E; Honaker, Julie A

2016-01-01

Falls sustained by older adults are an increasing health care issue. Early identification of those at risk for falling can lead to successful prevention of falls. Balance complaints are common among individuals who fall or are at risk for falling. The purpose of this study was to evaluate the clinical utility of a multifaceted balance protocol used for fall risk screening, with the hypothesis that this protocol would successfully identify individuals who had a recent fall (within the previous 12 months). This is a retrospective review of 30 individuals who self-referred for a free fall risk screening. Measures included case history, Activities-Specific Balance Confidence Scale, modified Clinical Test of Sensory Interaction on Balance, Timed Up and Go test, and Dynamic Visual Acuity. Statistical analyses were focused on the ability of the test protocol to identify a fall within the past 12 months and included descriptive statistics, clinical utility indices, logistic regression, receiver operating characteristic curve, area under the curve analysis, effect size (Cohen d), and Spearman correlation coefficients. All individuals who self-referred for this free screening had current imbalance complaints, and were typically women (70%), had a mean age of 77.2 years, and had a fear of falling (70%). Almost half (46.7%) reported at least 1 lifetime fall and 40.0% within the past 12 months. Regression analysis suggested that the Timed Up and Go test was the most important indicator of a recent fall. A cutoff score of 12 or more seconds was optimal (sensitivity: 83.3%; specificity: 61.1%). Older adults with current complaints of imbalance have a higher rate of falls, fall-related injury, and fear of falling than the general community-dwelling public. The Timed Up and Go test is useful for determining recent fall history in individuals with imbalance.

Stable isotope analyses reveal the importance of seagrass beds as feeding areas for juvenile Myrophic punctatus in Florida

EPA Science Inventory

The feeding habits and habitats of the speckled worm eel Myrophis punctatus were studied on the mangrove edge of the Indian River Lagoon (Florida) using stomach contents and stable isotope analyses of carbon (δ13C) and nitrogen (δ15N). Stomach dietary analyses identified four tax...

Cooper's Taxonomy of Literature Reviews Applied to Meta-Analyses in Educational Achievement.

ERIC Educational Resources Information Center

Sipe, Theresa Ann; Stallings, William M.

H. M. Cooper (1988) has developed a taxonomy that classified literature reviews based on six characteristics: (1) focus of attention; (2) goal of the synthesis; (3) perspective on the literature; (4) coverage of the literature; (5) organization of the perspective; and (6) intended audience. One hundred and three meta-analyses identified from the…

Selecting Effective Interventions to Increase Math Computation Fluency via Brief Experimental Analyses

ERIC Educational Resources Information Center

Reisener, Carmen D.; Dufrene, Brad A.; Clark, Chelsi R.; Olmi, D. Joe; Tingstrom, Daniel H.

2016-01-01

In a response to intervention RtI paradigm, the use of brief experimental analyses (BEAs) for identifying effective interventions for elementary and middle school students struggling with math is a relatively new area of research. This investigation includes two studies, both of which employed a brief multielement design and an extended analysis…

Using software agents to preserve individual health data confidentiality in micro-scale geographical analyses.

PubMed

Kamel Boulos, Maged N; Cai, Qiang; Padget, Julian A; Rushton, Gerard

2006-04-01

Confidentiality constraints often preclude the release of disaggregate data about individuals, which limits the types and accuracy of the results of geographical health analyses that could be done. Access to individually geocoded (disaggregate) data often involves lengthy and cumbersome procedures through review boards and committees for approval (and sometimes is not possible). Moreover, current data confidentiality-preserving solutions compatible with fine-level spatial analyses either lack flexibility or yield less than optimal results (because of confidentiality-preserving changes they introduce to disaggregate data), or both. In this paper, we present a simulation case study to illustrate how some analyses cannot be (or will suffer if) done on aggregate data. We then quickly review some existing data confidentiality-preserving techniques, and move on to explore a solution based on software agents with the potential of providing flexible, controlled (software-only) access to unmodified confidential disaggregate data and returning only results that do not expose any person-identifiable details. The solution is thus appropriate for micro-scale geographical analyses where no person-identifiable details are required in the final results (i.e., only aggregate results are needed). Our proposed software agent technique also enables post-coordinated analyses to be designed and carried out on the confidential database(s), as needed, compared to a more conventional solution based on the Web Services model that would only support a rigid, pre-coordinated (pre-determined) and rather limited set of analyses. The paper also provides an exploratory discussion of mobility, security, and trust issues associated with software agents, as well as possible directions/solutions to address these issues, including the use of virtual organizations. Successful partnerships between stakeholder organizations, proper collaboration agreements, clear policies, and unambiguous interpretations

2-tiered antibody testing for early and late Lyme disease using only an immunoglobulin G blot with the addition of a VlsE band as the second-tier test.

PubMed

Branda, John A; Aguero-Rosenfeld, Maria E; Ferraro, Mary Jane; Johnson, Barbara J B; Wormser, Gary P; Steere, Allen C

2010-01-01

Standard 2-tiered immunoglobulin G (IgG) testing has performed well in late Lyme disease (LD), but IgM testing early in the illness has been problematic. IgG VlsE antibody testing, by itself, improves early sensitivity, but may lower specificity. We studied whether elements of the 2 approaches could be combined to produce a second-tier IgG blot that performs well throughout the infection. Separate serum sets from LD patients and control subjects were tested independently at 2 medical centers using whole-cell enzyme immunoassays and IgM and IgG immunoblots, with recombinant VlsE added to the IgG blots. The results from both centers were combined, and a new second-tier IgG algorithm was developed. With standard 2-tiered IgM and IgG testing, 31% of patients with active erythema migrans (stage 1), 63% of those with acute neuroborreliosis or carditis (stage 2), and 100% of those with arthritis or late neurologic involvement (stage 3) had positive results. Using new IgG criteria, in which only the VlsE band was scored as a second-tier test among patients with early LD (stage 1 or 2) and 5 of 11 IgG bands were required in those with stage 3 LD, 34% of patients with stage 1, 96% of those with stage 2, and 100% of those with stage 3 infection had positive responses. Both new and standard testing achieved 100% specificity. Compared with standard IgM and IgG testing, the new IgG algorithm (with VlsE band) eliminates the need for IgM testing; it provides comparable or better sensitivity, and it maintains high specificity.

Protecting Confidentiality in Cancer Registry Data With Geographic Identifiers.

PubMed

Yu, Mandi; Reiter, Jerome Phillip; Zhu, Li; Liu, Benmei; Cronin, Kathleen A; Feuer, Eric J Rocky

2017-07-01

The National Cancer Institute's Surveillance, Epidemiology, and End Results Program releases research files of cancer registry data. These files include geographic information at the county level, but no finer. Access to finer geography, such as census tract identifiers, would enable richer analyses-for example, examination of health disparities across neighborhoods. To date, tract identifiers have been left off the research files because they could compromise the confidentiality of patients' identities. We present an approach to inclusion of tract identifiers based on multiply imputed, synthetic data. The idea is to build a predictive model of tract locations, given patient and tumor characteristics, and randomly simulate the tract of each patient by sampling from this model. For the predictive model, we use multivariate regression trees fitted to the latitude and longitude of the population centroid of each tract. We implement the approach in the registry data from California. The method results in synthetic data that reproduce a wide range (but not all) of analyses of census tract socioeconomic cancer disparities and have relatively low disclosure risks, which we assess by comparing individual patients' actual and synthetic tract locations. We conclude with a discussion of how synthetic data sets can be used by researchers with cancer registry data. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Meta-Analyses of Predictors of Hope in Adolescents.

PubMed

Yarcheski, Adela; Mahon, Noreen E

2016-03-01

The purposes of this study were to identify predictors of hope in the literature reviewed, to use meta-analysis to determine the mean effect size (ES) across studies between each predictor and hope, and to examine four moderators on each predictor-hope relationship. Using preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines for the literature reviewed, 77 published studies or doctoral dissertations completed between 1990 and 2012 met the inclusion criteria. Eleven predictors of hope were identified and each predictor in relation to hope was subjected to meta-analysis. Five predictors (positive affect, life satisfaction, optimism, self-esteem, and social support) of hope had large mean ESs, 1 predictor (depression) had a medium ES, 4 predictors (negative affect, stress, academic achievement, and violence) had small ESs, and 1 predictor (gender) had a trivial ES. Findings are interpreted for the 11 predictors in relation to hope. Limitations and conclusions are addressed; future studies are recommended. © The Author(s) 2014.

A Primer on Systematic Reviews and Meta-Analyses.

PubMed

Nguyen, Nghia H; Singh, Siddharth

2018-05-01

With the rapid growth of biomedical literature, there is increasing need to make meaningful inferences from a comprehensive and complex body of evidence. Systematic reviews with or without meta-analyses offer an objective and summative approach to synthesize knowledge and critically appraise evidence to inform clinical practice. Systematic reviews also help identify key knowledge gaps for future investigation. In this review, the authors provide a step-by-step approach to conducting a systematic review. These include: (1) formulating a focused and clinically-relevant question; (2) designing a detailed review protocol with explicit inclusion and exclusion criteria; (3) performing a systematic literature search of multiple databases and unpublished data, in consultation with a medical librarian, to identify relevant studies; (4) meticulous data abstraction by at least two sets of investigators independently; (5) assessing risk of bias in individual studies; (6) quantitative synthesis with meta-analysis; and (7) critically and transparently ascertaining quality of evidence. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Upgrading Existing Buildings to Universal Design. What Cost-Benefit Analyses Can Tell Us.

PubMed

Aslaksen, Finn

2016-01-01

This article is based on a project aimed at finding the benefits of different measures to upgrade existing public buildings and outdoor areas to be accessible for all. The study was initiated by The Ministry of Children and Equality. The ministry asked for a study of benefits based on a stated preferences (SP) method and an easy-to-complete calculation tool for CBA. In the project 18 commonly used measures and their typical costs were identified. The benefits of each measure were analysed in a stated preference study. The SP analyses included 9 multiple choices in 4 different sequences in an Internet based survey with 800 respondents. The project concluded that it is possible to use stated preferences survey to identify the respondent's valuation of measures to improve accessibility in existing buildings. Some of the measures have a high cost-/benefit ratio. The project report including the calculation manual is based on the average valuation for each measure. But in the background analyses (not referred in the report) there are also some analyses of valuations for target groups for the various measures. The target groups were defined for each measure based on information about the respondents' abilities and use of technical aids. The analyses presented in this paper indicate how valuation varies between the target groups and the average population. This is named the measures profile. Some measures have benefits for the target group that are only twice as high as for the average citizen while another type of measures has high benefits only the target group. The first type which has a wide profile often has high overall socioeconomic benefits, while the last group with a narrow profile more often has low overall socioeconomic benefits, but may be very important for certain user groups and therefore essential for the elimination of discrimination and exclusion of those groups.

Uncertainty in Operational Atmospheric Analyses and Re-Analyses

NASA Astrophysics Data System (ADS)

Langland, R.; Maue, R. N.

2016-12-01

This talk will describe uncertainty in atmospheric analyses of wind and temperature produced by operational forecast models and in re-analysis products. Because the "true" atmospheric state cannot be precisely quantified, there is necessarily error in every atmospheric analysis, and this error can be estimated by computing differences ( variance and bias) between analysis products produced at various centers (e.g., ECMWF, NCEP, U.S Navy, etc.) that use independent data assimilation procedures, somewhat different sets of atmospheric observations and forecast models with different resolutions, dynamical equations, and physical parameterizations. These estimates of analysis uncertainty provide a useful proxy to actual analysis error. For this study, we use a unique multi-year and multi-model data archive developed at NRL-Monterey. It will be shown that current uncertainty in atmospheric analyses is closely correlated with the geographic distribution of assimilated in-situ atmospheric observations, especially those provided by high-accuracy radiosonde and commercial aircraft observations. The lowest atmospheric analysis uncertainty is found over North America, Europe and Eastern Asia, which have the largest numbers of radiosonde and commercial aircraft observations. Analysis uncertainty is substantially larger (by factors of two to three times) in most of the Southern hemisphere, the North Pacific ocean, and under-developed nations of Africa and South America where there are few radiosonde or commercial aircraft data. It appears that in regions where atmospheric analyses depend primarily on satellite radiance observations, analysis uncertainty of both temperature and wind remains relatively high compared to values found over North America and Europe.

Purification of infectious human herpesvirus 6A virions and association of host cell proteins

PubMed Central

Hammarstedt, Maria; Ahlqvist, Jenny; Jacobson, Steven; Garoff, Henrik; Fogdell-Hahn, Anna

2007-01-01

Background Viruses that are incorporating host cell proteins might trigger autoimmune diseases. It is therefore of interest to identify possible host proteins associated with viruses, especially for enveloped viruses that have been suggested to play a role in autoimmune diseases, like human herpesvirus 6A (HHV-6A) in multiple sclerosis (MS). Results We have established a method for rapid and morphology preserving purification of HHV-6A virions, which in combination with parallel analyses with background control material released from mock-infected cells facilitates qualitative and quantitative investigations of the protein content of HHV-6A virions. In our iodixanol gradient purified preparation, we detected high levels of viral DNA by real-time PCR and viral proteins by metabolic labelling, silver staining and western blots. In contrast, the background level of cellular contamination was low in the purified samples as demonstrated by the silver staining and metabolic labelling analyses. Western blot analyses showed that the cellular complement protein CD46, the receptor for HHV-6A, is associated with the purified and infectious virions. Also, the cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions. Conclusion Cellular proteins are associated with HHV-6A virions. The relevance of the association in disease and especially in autoimmunity will be further investigated. PMID:17949490

A multi-criteria evaluation system for marine litter pollution based on statistical analyses of OSPAR beach litter monitoring time series.

PubMed

Schulz, Marcus; Neumann, Daniel; Fleet, David M; Matthies, Michael

2013-12-01

During the last decades, marine pollution with anthropogenic litter has become a worldwide major environmental concern. Standardized monitoring of litter since 2001 on 78 beaches selected within the framework of the Convention for the Protection of the Marine Environment of the North-East Atlantic (OSPAR) has been used to identify temporal trends of marine litter. Based on statistical analyses of this dataset a two-part multi-criteria evaluation system for beach litter pollution of the North-East Atlantic and the North Sea is proposed. Canonical correlation analyses, linear regression analyses, and non-parametric analyses of variance were used to identify different temporal trends. A classification of beaches was derived from cluster analyses and served to define different states of beach quality according to abundances of 17 input variables. The evaluation system is easily applicable and relies on the above-mentioned classification and on significant temporal trends implied by significant rank correlations. Copyright © 2013 Elsevier Ltd. All rights reserved.

Search Strategy to Identify Dental Survival Analysis Articles Indexed in MEDLINE.

PubMed

Layton, Danielle M; Clarke, Michael

2016-01-01

Articles reporting survival outcomes (time-to-event outcomes) in patients over time are challenging to identify in the literature. Research shows the words authors use to describe their dental survival analyses vary, and that allocation of medical subject headings by MEDLINE indexers is inconsistent. Together, this undermines accurate article identification. The present study aims to develop and validate a search strategy to identify dental survival analyses indexed in MEDLINE (Ovid). A gold standard cohort of articles was identified to derive the search terms, and an independent gold standard cohort of articles was identified to test and validate the proposed search strategies. The first cohort included all 6,955 articles published in the 50 dental journals with the highest impact factors in 2008, of which 95 articles were dental survival articles. The second cohort included all 6,514 articles published in the 50 dental journals with the highest impact factors for 2012, of which 148 were dental survival articles. Each cohort was identified by a systematic hand search. Performance parameters of sensitivity, precision, and number needed to read (NNR) for the search strategies were calculated. Sensitive, precise, and optimized search strategies were developed and validated. The performances of the search strategy maximizing sensitivity were 92% sensitivity, 14% precision, and 7.11 NNR; the performances of the strategy maximizing precision were 93% precision, 10% sensitivity, and 1.07 NNR; and the performances of the strategy optimizing the balance between sensitivity and precision were 83% sensitivity, 24% precision, and 4.13 NNR. The methods used to identify search terms were objective, not subjective. The search strategies were validated in an independent group of articles that included different journals and different publication years. Across the three search strategies, dental survival articles can be identified with sensitivity up to 92%, precision up to 93

A bioinformatics approach for identifying transgene insertion sites using whole genome sequencing data.

PubMed

Park, Doori; Park, Su-Hyun; Ban, Yong Wook; Kim, Youn Shic; Park, Kyoung-Cheul; Kim, Nam-Soo; Kim, Ju-Kon; Choi, Ik-Young

2017-08-15

Genetically modified crops (GM crops) have been developed to improve the agricultural traits of modern crop cultivars. Safety assessments of GM crops are of paramount importance in research at developmental stages and before releasing transgenic plants into the marketplace. Sequencing technology is developing rapidly, with higher output and labor efficiencies, and will eventually replace existing methods for the molecular characterization of genetically modified organisms. To detect the transgenic insertion locations in the three GM rice gnomes, Illumina sequencing reads are mapped and classified to the rice genome and plasmid sequence. The both mapped reads are classified to characterize the junction site between plant and transgene sequence by sequence alignment. Herein, we present a next generation sequencing (NGS)-based molecular characterization method, using transgenic rice plants SNU-Bt9-5, SNU-Bt9-30, and SNU-Bt9-109. Specifically, using bioinformatics tools, we detected the precise insertion locations and copy numbers of transfer DNA, genetic rearrangements, and the absence of backbone sequences, which were equivalent to results obtained from Southern blot analyses. NGS methods have been suggested as an effective means of characterizing and detecting transgenic insertion locations in genomes. Our results demonstrate the use of a combination of NGS technology and bioinformatics approaches that offers cost- and time-effective methods for assessing the safety of transgenic plants.

Transcriptome-wide analyses indicate mitochondrial responses to particulate air pollution exposure.

PubMed

Winckelmans, Ellen; Nawrot, Tim S; Tsamou, Maria; Den Hond, Elly; Baeyens, Willy; Kleinjans, Jos; Lefebvre, Wouter; Van Larebeke, Nicolas; Peusens, Martien; Plusquin, Michelle; Reynders, Hans; Schoeters, Greet; Vanpoucke, Charlotte; de Kok, Theo M; Vrijens, Karen

2017-08-18

Due to their lack of repair capacity mitochondria are critical targets for environmental toxicants. We studied genes and pathways reflecting mitochondrial responses to short- and medium-term PM 10 exposure. Whole genome gene expression was measured in peripheral blood of 98 adults (49% women). We performed linear regression analyses stratified by sex and adjusted for individual and temporal characteristics to investigate alterations in gene expression induced by short-term (week before blood sampling) and medium-term (month before blood sampling) PM 10 exposure. Overrepresentation analyses (ConsensusPathDB) were performed to identify enriched mitochondrial associated pathways and gene ontology sets. Thirteen Human MitoCarta genes were measured by means of quantitative real-time polymerase chain reaction (qPCR) along with mitochondrial DNA (mtDNA) content in an independent validation cohort (n = 169, 55.6% women). Overrepresentation analyses revealed significant pathways (p-value <0.05) related to mitochondrial genome maintenance and apoptosis for short-term exposure and to the electron transport chain (ETC) for medium-term exposure in women. For men, medium-term PM 10 exposure was associated with the Tri Carbonic Acid cycle. In an independent study population, we validated several ETC genes, including UQCRH and COX7C (q-value <0.05), and some genes crucial for the maintenance of the mitochondrial genome, including LONP1 (q-value: 0.07) and POLG (q-value: 0.04) in women. In this exploratory study, we identified mitochondrial genes and pathways associated with particulate air pollution indicating upregulation of energy producing pathways as a potential mechanism to compensate for PM-induced mitochondrial damage.

Enhanced Lipidome Coverage in Shotgun Analyses by using Gas-Phase Fractionation

NASA Astrophysics Data System (ADS)

Nazari, Milad; Muddiman, David C.

2016-11-01

A high resolving power shotgun lipidomics strategy using gas-phase fractionation and data-dependent acquisition (DDA) was applied toward comprehensive characterization of lipids in a hen ovarian tissue in an untargeted fashion. Using this approach, a total of 822 unique lipids across a diverse range of lipid categories and classes were identified based on their MS/MS fragmentation patterns. Classes of glycerophospholipids and glycerolipids, such as glycerophosphocholines (PC), glycerophosphoethanolamines (PE), and triglycerides (TG), are often the most abundant peaks observed in shotgun lipidomics analyses. These ions suppress the signal from low abundance ions and hinder the chances of characterizing low abundant lipids when DDA is used. These issues were circumvented by utilizing gas-phase fractionation, where DDA was performed on narrow m/z ranges instead of a broad m/z range. Employing gas-phase fractionation resulted in an increase in sensitivity by more than an order of magnitude in both positive- and negative-ion modes. Furthermore, the enhanced sensitivity increased the number of lipids identified by a factor of ≈4, and facilitated identification of low abundant lipids from classes such as cardiolipins that are often difficult to observe in untargeted shotgun analyses and require sample-specific preparation steps prior to analysis. This method serves as a resource for comprehensive profiling of lipids from many different categories and classes in an untargeted manner, as well as for targeted and quantitative analyses of individual lipids. Furthermore, this comprehensive analysis of the lipidome can serve as a species- and tissue-specific database for confident identification of other MS-based datasets, such as mass spectrometry imaging.

Does the inclusion of grey literature influence estimates of intervention effectiveness reported in meta-analyses?

PubMed

McAuley, L; Pham, B; Tugwell, P; Moher, D

2000-10-07

The inclusion of only a subset of all available evidence in a meta-analysis may introduce biases and threaten its validity; this is particularly likely if the subset of included studies differ from those not included, which may be the case for published and grey literature (unpublished studies, with limited distribution). We set out to examine whether exclusion of grey literature, compared with its inclusion in meta-analysis, provides different estimates of the effectiveness of interventions assessed in randomised trials. From a random sample of 135 meta-analyses, we identified and retrieved 33 publications that included both grey and published primary studies. The 33 publications contributed 41 separate meta-analyses from several disease areas. General characteristics of the meta-analyses and associated studies and outcome data at the trial level were collected. We explored the effects of the inclusion of grey literature on the quantitative results using logistic-regression analyses. 33% of the meta-analyses were found to include some form of grey literature. The grey literature, when included, accounts for between 4.5% and 75% of the studies in a meta-analysis. On average, published work, compared with grey literature, yielded significantly larger estimates of the intervention effect by 15% (ratio of odds ratios=1.15 [95% CI 1.04-1.28]). Excluding abstracts from the analysis further compounded the exaggeration (1.33 [1.10-1.60]). The exclusion of grey literature from meta-analyses can lead to exaggerated estimates of intervention effectiveness. In general, meta-analysts should attempt to identify, retrieve, and include all reports, grey and published, that meet predefined inclusion criteria.

Comparisons of several aerodynamic methods for application to dynamic loads analyses

NASA Technical Reports Server (NTRS)

Kroll, R. I.; Miller, R. D.

1976-01-01

The results of a study are presented in which the applicability at subsonic speeds of several aerodynamic methods for predicting dynamic gust loads on aircraft, including active control systems, was examined and compared. These aerodynamic methods varied from steady state to an advanced unsteady aerodynamic formulation. Brief descriptions of the structural and aerodynamic representations and of the motion and load equations are presented. Comparisons of numerical results achieved using the various aerodynamic methods are shown in detail. From these results, aerodynamic representations for dynamic gust analyses are identified. It was concluded that several aerodynamic methods are satisfactory for dynamic gust analyses of configurations having either controls fixed or active control systems that primarily affect the low frequency rigid body aircraft response.

Storage and stability of IgG and IgM monoclonal antibodies dried on filter paper and utility in Neisseria meningitidis serotyping by Dot-blot ELISA.

PubMed

Ferraz, Aline S; Belo, Elza F T; Coutinho, Ligia M C C; Oliveira, Ana P; Carmo, Andréia M S; Franco, Daniele L; Ferreira, Tatiane; Yto, André Y; Machado, Marta S F; Scola, Monica C G; De Gaspari, Elizabeth

2008-03-06

A simple filter paper method was developed for, the transport and storage of monoclonal antibodies (Mabs) at room temperature or -20 degrees C after spotting on filter paper, for subsequent serotyping of outer membrane antigens of N.meningitidis by dot-blot ELISA. Monoclonal antibodies (Mabs) were spotted within a 0.5-1 cm diameter area of Whatman grade 903 paper, which were stored individually at room temperature or at -20 degrees C. These MAbs were stored and analyzed after periods of one week, 4 weeks, 12 months, or 13 years in the case of frozen Mab aliquots, or after 4 weeks at -20 degrees C or at room temperature (RT) in the case of Mabs dried on filter paper strips. Assays were performed in parallel using dot-blot ELISA. In addition to the MAbs specific for serotyping class 1, 2 or 3, we used a larger number of Mabs for polysaccharides, lipooligosaccharides (LOS), class 5 and cross-reactive antigens for native outer membrane of N.meningitidis. The Mabs dried on filter paper were eluted with phosphate-buffered saline (PBS) containing 0.2% gelatin. Mabs of the isotypes IgG and IgM dried on filter papers were not affected by duration of storage. The detection by serotyping Mabs was generally consistent for dried filter paper MAb samples stored frozen for over 1 year at -20 degrees C, and although decreased reactive antibody titers were found after storage, this did not interfere with the specificity of the Mabs used after 13 years as dry spots on filter paper. The use of filter paper is an inexpensive and convenient method for collecting, storing, and transporting Mab samples for serotyping studies. In addition, the samples occupy little space and can be readily transported without freezing. The efficiency of using immunoglobulin G (IgG) or M (IgM) eluted was found to be consistent with measurement of IgG or IgM titers in most corresponding, ascites Mabs stored frozen for over 1 year. The application of meningococcal typing methods and designations depend on

IgG western blot for confirmatory diagnosis of equivocal cases of toxoplasmosis by EIA-IgG and fluorescent antibody test.

PubMed

Khammari, Imen; Saghrouni, Fatma; Yaacoub, Alia; Gaied Meksi, Sondoss; Ach, Hinda; Garma, Lamia; Fathallah, Akila; Ben Saïd, Moncef

2013-08-01

The performance values of available techniques used in serodiagnosis of toxoplasmosis are satisfactory but they raise problems of equivocal and discordant results for very low IgG titers. Recently marketed, LDBio-Toxo II IgG Western blot (IB) showed an excellent correlation with the dye test. We estimated the proportion of equivocal and discordant results between the enzyme immunoassay Platelia Toxo IgG (EIA-IgG) and fluorescent antibody test (FAT) and assessed the usefulness of the IB as a confirmatory test. Out of 2,136 sera collected from pregnant women, 1,644 (77.0%) tested unequivocally positive and 407 (19.0%) were negative in both EIA-IgG and FAT. The remaining 85 (4%) sera showed equivocal or discordant results. Among them, 73 (85.9%) were positive and 12 (14.1%) were negative in IB. Forty-one (89.1%) equivocal sera in EIA-IgG and 46 (86.8%) equivocal sera in FAT were positive in IB. Reducing the cut-off values of both screening techniques improved significantly their sensitivity in detecting very low IgG titers at the expense of their specificity. In conclusion, equivocal results in routine-used techniques and their discordance in determination of the immune status in pregnancy women were not uncommon. IB test appeard to be highly useful in these situations as a confirmatory technique.

A quantitative image cytometry technique for time series or population analyses of signaling networks.

PubMed

Ozaki, Yu-ichi; Uda, Shinsuke; Saito, Takeshi H; Chung, Jaehoon; Kubota, Hiroyuki; Kuroda, Shinya

2010-04-01

Modeling of cellular functions on the basis of experimental observation is increasingly common in the field of cellular signaling. However, such modeling requires a large amount of quantitative data of signaling events with high spatio-temporal resolution. A novel technique which allows us to obtain such data is needed for systems biology of cellular signaling. We developed a fully automatable assay technique, termed quantitative image cytometry (QIC), which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification. With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. The signaling activities quantified by the assay system showed good correlation with, as well as comparable reproducibility to, western blot analysis. Taking advantage of the high spatio-temporal resolution, we investigated the signaling dynamics of the ERK pathway in PC12 cells. The QIC technique appears as a highly quantitative and versatile technique, which can be a convenient replacement for the most conventional techniques including western blot, flow cytometry and live cell imaging. Thus, the QIC technique can be a powerful tool for investigating the systems biology of cellular signaling.

Black carp vasa identifies embryonic and gonadal germ cells.

PubMed

Xue, Ting; Yu, Miao; Pan, Qihua; Wang, Yizhou; Fang, Jian; Li, Lingyu; Deng, Yu; Chen, Kai; Wang, Qian; Chen, Tiansheng

2017-07-01

Identification of molecular markers is an essential step in the study of germ cells. Vasa is an RNA helicase and a well-known germ cell marker that plays a crucial role in germ cell development. Here, we identified the Vasa homolog termed Mpvasa as the first germ cell marker in black carp (Mylopharyngodon piceus). First, a 2819-bp full-length Mpvasa complementary DNA (cDNA) was cloned by PCR using degenerated primers of conserved sequences and gene-specific primers. The Mpvasa cDNA sequence encodes a 637-amino acid protein that contains eight conserved characteristic motifs of the DEAD box protein family, and shares high identity to grass carp (81%) and zebrafish (74%) vasa homologs. Second, Mpvasa expression was restricted to the gonad in adulthood by RT-PCR and Western blot analysis. The dynamic patterns of temporal-spatial expression of Mpvasa during gametogenesis were examined by in situ hybridization, and Mpvasa transcripts were strictly detected in gonadal germ cells throughout oogenesis, predominantly in immature oocytes (stage I, II, and III oocytes). Third, Mpvasa transcripts were highly detected in unfertilized eggs and early embryos, and the signal indicated a dynamic migration of the primordial germ cells during embryogenesis, suggesting that Mpvasa transcripts were maternally inherited and specifically distributed in germ cells. Taken together, these results demonstrated that Mpvasa is an applicable molecular marker for identification of gonadal and embryonic germ cells, which facilitates the isolation and utilization of germ cells in black carp.

Systematic overview and critical appraisal of meta-analyses of interventions in intensive care medicine.

PubMed

Koster, T M; Wetterslev, J; Gluud, C; Keus, F; van der Horst, I C C

2018-05-24

Meta-analysed intervention effect estimates are perceived to represent the highest level of evidence. However, such effects and the randomized clinical trials which are included in them need critical appraisal before the effects can be trusted. Critical appraisal of a predefined set of all meta-analyses on interventions in intensive care medicine to assess their quality and assessed the risks of bias in those meta-analyses having the best quality. We conducted a systematic search to select all meta-analyses of randomized clinical trials on interventions used in intensive care medicine. Selected meta-analyses were critically appraised for basic scientific criteria, (1) presence of an available protocol, (2) report of a full search strategy, and (3) use of any bias risk assessment of included trials. All meta-analyses which qualified these criteria were scrutinized by full "Risk of Bias in Systematic Reviews" ROBIS evaluation of 4 domains of risks of bias, and a "Preferred Reporting Items for Systematic Reviews and Meta-Analyses" PRISMA evaluation. We identified 467 meta-analyses. A total of 56 meta-analyses complied with these basic scientific criteria. We scrutinized the risks of bias in the 56 meta-analyses by full ROBIS evaluation and a PRISMA evaluation. Only 4 meta-analyses scored low risk of bias in all the 4 ROBIS domains and 41 meta-analyses reported all 27 items of the PRISMA checklist. In contrast with what might be perceived as the highest level of evidence only 0.9% of all meta-analyses were judged to have overall low risk of bias. © 2018 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Sequence and Transcriptional Analyses of the Fish Retroviruses Walleye Epidermal Hyperplasia Virus Types 1 and 2: Evidence for a Gene Duplication

PubMed Central

LaPierre, Lorie A.; Holzschu, Donald L.; Bowser, Paul R.; Casey, James W.

1999-01-01

Walleye epidermal hyperplasia virus types 1 and 2 (WEHV1 and WEHV2, respectively) are associated with a hyperproliferative skin lesion on walleyes that appears and regresses seasonally. We have determined the complete nucleotide sequences and transcriptional profiles of these viruses. WEHV1 and WEHV2 are large, complex retroviruses of 12,999 and 13,125 kb in length, respectively, that are closely related to one another and to walleye dermal sarcoma virus (WDSV). These walleye retroviruses contain three open reading frames, orfA, orfB, and orfC, in addition to gag, pol, and env. orfA and orfB are adjacent to one another and located downstream of env. The OrfA proteins were previously identified as cyclin D homologs that may contribute to the induction of cell proliferation leading to epidermal hyperplasia and dermal sarcoma. The sequence analysis of WEHV1 and WEHV2 revealed that the OrfB proteins are distantly related to the OrfA proteins, suggesting that orfB arose by gene duplication. Presuming that the precursor of orfA and orfB was derived from a cellular cyclin, these genes are the first accessory genes of complex retroviruses that can be traced to a cellular origin. WEHV1, WEHV2, and WDSV are the only retroviruses that have an open reading frame, orfC, of considerable size (ca. 130 amino acids) in the leader region preceding gag. While we were unable to predict a function for the OrfC proteins, they are more conserved than OrfA and OrfB, suggesting that they may be biologically important to the viruses. The transcriptional profiles of WEHV1 and WEHV2 were also similar to that of WDSV; Northern blot analyses detected only low levels of the orfA transcripts in developing lesions, whereas abundant levels of genomic, env, orfA, and orfB transcripts were detected in regressing lesions. The splice donors and acceptors of individual transcripts were identified by reverse transcriptase PCR. The similarities of WEHV1, WEHV2, and WDSV suggest that these viruses use

Phylogenetic relationship and species delimitation of matsutake and allied species based on multilocus phylogeny and haplotype analyses.

PubMed

Ota, Yuko; Yamanaka, Takashi; Murata, Hitoshi; Neda, Hitoshi; Ohta, Akira; Kawai, Masataka; Yamada, Akiyoshi; Konno, Miki; Tanaka, Chihiro

2012-01-01

Tricholoma matsutake (S. Ito & S. Imai) Singer and its allied species are referred to as matsutake worldwide and are the most economically important edible mushrooms in Japan. They are widely distributed in the northern hemisphere and established an ectomycorrhizal relationship with conifer and broadleaf trees. To clarify relationships among T. matsutake and its allies, and to delimit phylogenetic species, we analyzed multilocus datasets (ITS, megB1, tef, gpd) with samples that were correctly identified based on morphological characteristics. Phylogenetic analyses clearly identified four major groups: matsutake, T. bakamatsutake, T. fulvocastaneum and T. caligatum; the latter three species were outside the matsutake group. The haplotype analyses and median-joining haplotype network analyses showed that the matsutake group included four closely related but clearly distinct taxa (T. matsutake, T. anatolicum, Tricholoma sp. from Mexico and T. magnivelare) from different geographical regions; these were considered to be distinct phylogenetic species.

A method for identifying EMI critical circuits during development of a large C3

NASA Astrophysics Data System (ADS)

Barr, Douglas H.

The circuit analysis methods and process Boeing Aerospace used on a large, ground-based military command, control, and communications (C3) system are described. This analysis was designed to help identify electromagnetic interference (EMI) critical circuits. The methodology used the MIL-E-6051 equipment criticality categories as the basis for defining critical circuits, relational database technology to help sort through and account for all of the approximately 5000 system signal cables, and Macintosh Plus personal computers to predict critical circuits based on safety margin analysis. The EMI circuit analysis process systematically examined all system circuits to identify which ones were likely to be EMI critical. The process used two separate, sequential safety margin analyses to identify critical circuits (conservative safety margin analysis, and detailed safety margin analysis). These analyses used field-to-wire and wire-to-wire coupling models using both worst-case and detailed circuit parameters (physical and electrical) to predict circuit safety margins. This process identified the predicted critical circuits that could then be verified by test.

SPARQL-enabled identifier conversion with Identifiers.org

PubMed Central

Wimalaratne, Sarala M.; Bolleman, Jerven; Juty, Nick; Katayama, Toshiaki; Dumontier, Michel; Redaschi, Nicole; Le Novère, Nicolas; Hermjakob, Henning; Laibe, Camille

2015-01-01

Motivation: On the semantic web, in life sciences in particular, data is often distributed via multiple resources. Each of these sources is likely to use their own International Resource Identifier for conceptually the same resource or database record. The lack of correspondence between identifiers introduces a barrier when executing federated SPARQL queries across life science data. Results: We introduce a novel SPARQL-based service to enable on-the-fly integration of life science data. This service uses the identifier patterns defined in the Identifiers.org Registry to generate a plurality of identifier variants, which can then be used to match source identifiers with target identifiers. We demonstrate the utility of this identifier integration approach by answering queries across major producers of life science Linked Data. Availability and implementation: The SPARQL-based identifier conversion service is available without restriction at http://identifiers.org/services/sparql. Contact: sarala@ebi.ac.uk PMID:25638809

SPARQL-enabled identifier conversion with Identifiers.org.

PubMed

Wimalaratne, Sarala M; Bolleman, Jerven; Juty, Nick; Katayama, Toshiaki; Dumontier, Michel; Redaschi, Nicole; Le Novère, Nicolas; Hermjakob, Henning; Laibe, Camille

2015-06-01

On the semantic web, in life sciences in particular, data is often distributed via multiple resources. Each of these sources is likely to use their own International Resource Identifier for conceptually the same resource or database record. The lack of correspondence between identifiers introduces a barrier when executing federated SPARQL queries across life science data. We introduce a novel SPARQL-based service to enable on-the-fly integration of life science data. This service uses the identifier patterns defined in the Identifiers.org Registry to generate a plurality of identifier variants, which can then be used to match source identifiers with target identifiers. We demonstrate the utility of this identifier integration approach by answering queries across major producers of life science Linked Data. The SPARQL-based identifier conversion service is available without restriction at http://identifiers.org/services/sparql. © The Author 2015. Published by Oxford University Press.

A Novel Quantitative Method for Diabetic Cardiac Autonomic Neuropathy Assessment in Type 1 Diabetic Mice

PubMed Central

Yang, Bufan; Posada-Quintero, Hugo F.; Siu, Kin L.; Rolle, Marsha; Brink, Peter; Birzgalis, Aija; Moore, Leon C.

2014-01-01

In this work, we used a sensitive and noninvasive computational method to assess diabetic cardiovascular autonomic neuropathy (DCAN) from pulse oximeter (photoplethysmographic; PPG) recordings from mice. The method, which could be easily applied to humans, is based on principal dynamic mode (PDM) analysis of heart rate variability (HRV). Unlike the power spectral density, PDM has been shown to be able to separately identify the activities of the parasympathetic and sympathetic nervous systems without pharmacological intervention. HRV parameters were measured by processing PPG signals from conscious 1.5- to 5-month-old C57/BL6 control mice and in Akita mice, a model of insulin-dependent type 1 diabetes, and compared with the gold-standard Western blot and immunohistochemical analyses. The PDM results indicate significant cardiac autonomic impairment in the diabetic mice in comparison to the controls. When tail-cuff PPG recordings were collected and analyzed starting from 1.5 months of age in both C57/Bl6 controls and Akita mice, onset of DCAN was seen at 3 months in the Akita mice, which persisted up to the termination of the recording at 5 months. Western blot and immunohistochemical analyses also showed a reduction in nerve density in Akita mice at 3 and 4 months as compared to the control mice, thus, corroborating our PDM data analysis of HRV records. Western blot analysis of autonomic nerve proteins corroborated the PPG-based HRV analysis via the PDM approach. In contrast, traditional HRV analysis (based on either the power spectral density or time-domain measures) failed to detect the nerve rarefaction. PMID:25097056

Sequence and structural analyses of nuclear export signals in the NESdb database

PubMed Central

Xu, Darui; Farmer, Alicia; Collett, Garen; Grishin, Nick V.; Chook, Yuh Min

2012-01-01

We compiled >200 nuclear export signal (NES)–containing CRM1 cargoes in a database named NESdb. We analyzed the sequences and three-dimensional structures of natural, experimentally identified NESs and of false-positive NESs that were generated from the database in order to identify properties that might distinguish the two groups of sequences. Analyses of amino acid frequencies, sequence logos, and agreement with existing NES consensus sequences revealed strong preferences for the Φ1-X3-Φ2-X2-Φ3-X-Φ4 pattern and for negatively charged amino acids in the nonhydrophobic positions of experimentally identified NESs but not of false positives. Strong preferences against certain hydrophobic amino acids in the hydrophobic positions were also revealed. These findings led to a new and more precise NES consensus. More important, three-dimensional structures are now available for 68 NESs within 56 different cargo proteins. Analyses of these structures showed that experimentally identified NESs are more likely than the false positives to adopt α-helical conformations that transition to loops at their C-termini and more likely to be surface accessible within their protein domains or be present in disordered or unobserved parts of the structures. Such distinguishing features for real NESs might be useful in future NES prediction efforts. Finally, we also tested CRM1-binding of 40 NESs that were found in the 56 structures. We found that 16 of the NES peptides did not bind CRM1, hence illustrating how NESs are easily misidentified. PMID:22833565

A side-effect free method for identifying cancer drug targets.

PubMed

Ashraf, Md Izhar; Ong, Seng-Kai; Mujawar, Shama; Pawar, Shrikant; More, Pallavi; Paul, Somnath; Lahiri, Chandrajit

2018-04-27

Identifying effective drug targets, with little or no side effects, remains an ever challenging task. A potential pitfall of failing to uncover the correct drug targets, due to side effect of pleiotropic genes, might lead the potential drugs to be illicit and withdrawn. Simplifying disease complexity, for the investigation of the mechanistic aspects and identification of effective drug targets, have been done through several approaches of protein interactome analysis. Of these, centrality measures have always gained importance in identifying candidate drug targets. Here, we put forward an integrated method of analysing a complex network of cancer and depict the importance of k-core, functional connectivity and centrality (KFC) for identifying effective drug targets. Essentially, we have extracted the proteins involved in the pathways leading to cancer from the pathway databases which enlist real experimental datasets. The interactions between these proteins were mapped to build an interactome. Integrative analyses of the interactome enabled us to unearth plausible reasons for drugs being rendered withdrawn, thereby giving future scope to pharmaceutical industries to potentially avoid them (e.g. ESR1, HDAC2, F2, PLG, PPARA, RXRA, etc). Based upon our KFC criteria, we have shortlisted ten proteins (GRB2, FYN, PIK3R1, CBL, JAK2, LCK, LYN, SYK, JAK1 and SOCS3) as effective candidates for drug development.

Secretome analysis of rat osteoblasts during icariin treatment induced osteogenesis

PubMed Central

Qian, Weiqing; Su, Yan; Zhang, Yajie; Yao, Nianwei; Gu, Nin; Zhang, Xu; Yin, Hong

2018-01-01

Osteoporosis is a serious public health problem and icariin (ICA) is the active component of the Epimedium sagittatum, a traditional Chinese medicinal herb. The present study aimed to investigate the effects and underlying mechanisms of ICA as a potential therapy for osteoporosis. Calvaria osteoblasts were isolated from newborn rats and treated with ICA. Cell viability, apoptosis, alkaline phosphatase activity and calcium deposition were analyzed. Bioinformatics analyses were performed to identify differentially expressed proteins (DEPs) in response to ICA treatment. Western blot analysis was performed to validate the expression of DEPs. ICA administration promoted osteoblast viability, alkaline phosphatase activity, calcium deposition and inhibited osteoblast apoptosis. Secretome analysis of ICA-treated cells was performed using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A total of 56 DEPs were identified, including serpin family F member 1 (PEDF), protein disulfide isomerase family A, member 3 (PDIA3), nuclear protein, co-activator of histone transcription (NPAT), c-Myc and heat shock protein 70 (HSP70). These proteins were associated with signaling pathways, including Fas and p53. Bioinformatics and western blot analyses confirmed that the expression levels of the six DEPs were upregulated following ICA treatment. These genes may be directly or indirectly involved in ICA-mediated osteogenic differentiation and osteogenesis. It was demonstrated that ICA treatment promoted osteogenesis by modulating the expression of PEDF, PDIA3, NPAT and HSP70 through signaling pathways, including Fas and p53. PMID:29532868

Electron Microprobe Analyses of Lithic Fragments and Their Minerals from Luna 20 Fines

NASA Technical Reports Server (NTRS)

Conrad, G. H.; Hlava, P. F.; Green, J. A.; Moore, R. B.; Moreland, G.; Dowty, E.; Prinz, M.; Keil, K.; Nehru, C. E.; Bunch, T. E.

1973-01-01

The bulk analyses (determined with the broad beam electron microprobe technique) of lithic fragments are given in weight percentages and are arranged according to the rock classification. Within each rock group the analyses are arranged in order of increasing FeO content. Thin section and lithic fragment numbers are given at the top of each column of analysis and correspond to the numbers recorded on photo mosaics on file in the Institute of Meteoritics. CIPW molecular norms are given for each analysis. Electron microprobe mineral analyses (given in oxide weight percentages), structural formulae and molecular end member values are presented for plagioclase, olivine, pyroxene and K-feldspar. The minerals are selected mostly from lithic fragments that were also analyzed for bulk composition. Within each mineral group the analyses are presented according to the section number and lithic fragment number. Within each lithic fragment the mineral analyses are arranged as follows: Plagioclase in order of increasing CaO; olivine and pyroexene in order of increasing FeO; and K-feldspar in order of increasing K2O. The mineral grains are identified at the top of each column of analysis by grain number and lithic fragment number.

Integrative analyses of leprosy susceptibility genes indicate a common autoimmune profile.

PubMed

Zhang, Deng-Feng; Wang, Dong; Li, Yu-Ye; Yao, Yong-Gang

2016-04-01

Leprosy is an ancient chronic infection in the skin and peripheral nerves caused by Mycobacterium leprae. The development of leprosy depends on genetic background and the immune status of the host. However, there is no systematic view focusing on the biological pathways, interaction networks and overall expression pattern of leprosy-related immune and genetic factors. To identify the hub genes in the center of leprosy genetic network and to provide an insight into immune and genetic factors contributing to leprosy. We retrieved all reported leprosy-related genes and performed integrative analyses covering gene expression profiling, pathway analysis, protein-protein interaction network, and evolutionary analyses. A list of 123 differentially expressed leprosy related genes, which were enriched in activation and regulation of immune response, was obtained in our analyses. Cross-disorder analysis showed that the list of leprosy susceptibility genes was largely shared by typical autoimmune diseases such as lupus erythematosus and arthritis, suggesting that similar pathways might be affected in leprosy and autoimmune diseases. Protein-protein interaction (PPI) and positive selection analyses revealed a co-evolution network of leprosy risk genes. Our analyses showed that leprosy associated genes constituted a co-evolution network and might undergo positive selection driven by M. leprae. We suggested that leprosy may be a kind of autoimmune disease and the development of leprosy is a matter of defect or over-activation of body immunity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Biomarkers for identifying the early phases of osteoarthritis secondary to medial patellar luxation in dogs.