WorldWideScience

Sample records for screen reveals human

  1. Genome-Wide RNAi Ionomics Screen Reveals New Genes and Regulation of Human Trace Element Metabolism

    OpenAIRE

    Malinouski, Mikalai; Hasan, Nesrin M.; Zhang, Yan; Seravalli, Javier; Lin, Jie; Avanesov, Andrei; Lutsenko, Svetlana; Gladyshev, Vadim N.

    2017-01-01

    Trace elements are essential for human metabolism and dysregulation of their homeostasis is associated with numerous disorders. Here we characterize mechanisms that regulate trace elements in human cells by designing and performing a genome-wide high-throughput siRNA/ionomics screen, and examining top hits in cellular and biochemical assays. The screen reveals high stability of the ionomes, especially the zinc ionome, and yields known regulators and novel candidates. We further uncover fundam...

  2. Genome-Wide RNAi Ionomics Screen Reveals New Genes and Regulation of Human Trace Element Metabolism

    Science.gov (United States)

    Malinouski, Mikalai; Hasan, Nesrin M.; Zhang, Yan; Seravalli, Javier; Lin, Jie; Avanesov, Andrei; Lutsenko, Svetlana; Gladyshev, Vadim N.

    2017-01-01

    Trace elements are essential for human metabolism and dysregulation of their homeostasis is associated with numerous disorders. Here we characterize mechanisms that regulate trace elements in human cells by designing and performing a genome-wide high-throughput siRNA/ionomics screen, and examining top hits in cellular and biochemical assays. The screen reveals high stability of the ionomes, especially the zinc ionome, and yields known regulators and novel candidates. We further uncover fundamental differences in the regulation of different trace elements. Specifically, selenium levels are controlled through the selenocysteine machinery and expression of abundant selenoproteins; copper balance is affected by lipid metabolism and requires machinery involved in protein trafficking and posttranslational modifications; and the iron levels are influenced by iron import and expression of the iron/heme-containing enzymes. Our approach can be applied to a variety of disease models and/or nutritional conditions, and the generated dataset opens new directions for studies of human trace element metabolism. PMID:24522796

  3. Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins

    Directory of Open Access Journals (Sweden)

    Kahlem Pascal

    2006-06-01

    Full Text Available Abstract Background Trisomy of human chromosome 21 (Chr21 results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins. Results We chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb to MCM3AP (46.6 Mb, with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His6 tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized. Conclusion The cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome.

  4. Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling

    Science.gov (United States)

    Lebensohn, Andres M; Dubey, Ramin; Neitzel, Leif R; Tacchelly-Benites, Ofelia; Yang, Eungi; Marceau, Caleb D; Davis, Eric M; Patel, Bhaven B; Bahrami-Nejad, Zahra; Travaglini, Kyle J; Ahmed, Yashi; Lee, Ethan; Carette, Jan E; Rohatgi, Rajat

    2016-01-01

    The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1α uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling β-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the β-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems. DOI: http://dx.doi.org/10.7554/eLife.21459.001 PMID:27996937

  5. Genomic library screening for viruses from the human dental plaque revealed pathogen-specific lytic phage sequences.

    Science.gov (United States)

    Al-Jarbou, Ahmed Nasser

    2012-01-01

    Bacterial pathogenesis presents an astounding arsenal of virulence factors that allow them to conquer many different niches throughout the course of infection. Principally fascinating is the fact that some bacterial species are able to induce different diseases by expression of different combinations of virulence factors. Nevertheless, studies aiming at screening for the presence of bacteriophages in humans have been limited. Such screening procedures would eventually lead to identification of phage-encoded properties that impart increased bacterial fitness and/or virulence in a particular niche, and hence, would potentially be used to reverse the course of bacterial infections. As the human oral cavity represents a rich and dynamic ecosystem for several upper respiratory tract pathogens. However, little is known about virus diversity in human dental plaque which is an important reservoir. We applied the culture-independent approach to characterize virus diversity in human dental plaque making a library from a virus DNA fraction amplified using a multiple displacement method and sequenced 80 clones. The resulting sequence showed 44% significant identities to GenBank databases by TBLASTX analysis. TBLAST homology comparisons showed that 66% was viral; 18% eukarya; 10% bacterial; 6% mobile elements. These sequences were sorted into 6 contigs and 45 single sequences in which 4 contigs and a single sequence showed significant identity to a small region of a putative prophage in the Corynebacterium diphtheria genome. These findings interestingly highlight the uniqueness of over half of the sequences, whilst the dominance of a pathogen-specific prophage sequences imply their role in virulence.

  6. Proteomic screening of human targets of viral microRNAs reveals functions associated with immune evasion and angiogenesis.

    Directory of Open Access Journals (Sweden)

    Amelia M Gallaher

    Full Text Available Kaposi's sarcoma (KS is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV. The virus expresses unique microRNAs (miRNAs, but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1 following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α, and up-regulation of a protein involved in stimulating angiogenesis (HMOX1. This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.

  7. Chemical Genomic Screening of a Saccharomyces cerevisiae Genomewide Mutant Collection Reveals Genes Required for Defense against Four Antimicrobial Peptides Derived from Proteins Found in Human Saliva

    Science.gov (United States)

    Bhatt, Sanjay; Schoenly, Nathan E.; Lee, Anna Y.; Nislow, Corey; Bobek, Libuse A.

    2013-01-01

    To compare the effects of four antimicrobial peptides (MUC7 12-mer, histatin 12-mer, cathelicidin KR20, and a peptide containing lactoferricin amino acids 1 to 11) on the yeast Saccharomyces cerevisiae, we employed a genomewide fitness screen of combined collections of mutants with homozygous deletions of nonessential genes and heterozygous deletions of essential genes. When an arbitrary fitness score cutoffs of 1 (indicating a fitness defect, or hypersensitivity) and −1 (indicating a fitness gain, or resistance) was used, 425 of the 5,902 mutants tested exhibited altered fitness when treated with at least one peptide. Functional analysis of the 425 strains revealed enrichment among the identified deletions in gene groups associated with the Gene Ontology (GO) terms “ribosomal subunit,” “ribosome biogenesis,” “protein glycosylation,” “vacuolar transport,” “Golgi vesicle transport,” “negative regulation of transcription,” and others. Fitness profiles of all four tested peptides were highly similar, particularly among mutant strains exhibiting the greatest fitness defects. The latter group included deletions in several genes involved in induction of the RIM101 signaling pathway, including several components of the ESCRT sorting machinery. The RIM101 signaling regulates response of yeasts to alkaline and neutral pH and high salts, and our data indicate that this pathway also plays a prominent role in regulating protective measures against all four tested peptides. In summary, the results of the chemical genomic screens of S. cerevisiae mutant collection suggest that the four antimicrobial peptides, despite their differences in structure and physical properties, share many interactions with S. cerevisiae cells and consequently a high degree of similarity between their modes of action. PMID:23208710

  8. A genome-wide screen in human embryonic stem cells reveals novel sites of allele-specific histone modification associated with known disease loci

    LENUS (Irish Health Repository)

    Prendergast, James G D

    2012-05-19

    AbstractBackgroundChromatin structure at a given site can differ between chromosome copies in a cell, and such imbalances in chromatin structure have been shown to be important in understanding the molecular mechanisms controlling several disease loci. Human genetic variation, DNA methylation, and disease have been intensely studied, uncovering many sites of allele-specific DNA methylation (ASM). However, little is known about the genome-wide occurrence of sites of allele-specific histone modification (ASHM) and their relationship to human disease. The aim of this study was to investigate the extent and characteristics of sites of ASHM in human embryonic stem cells (hESCs).ResultsUsing a statistically rigorous protocol, we investigated the genomic distribution of ASHM in hESCs, and their relationship to sites of allele-specific expression (ASE) and DNA methylation. We found that, although they were rare, sites of ASHM were substantially enriched at loci displaying ASE. Many were also found at known imprinted regions, hence sites of ASHM are likely to be better markers of imprinted regions than sites of ASM. We also found that sites of ASHM and ASE in hESCs colocalize at risk loci for developmental syndromes mediated by deletions, providing insights into the etiology of these disorders.ConclusionThese results demonstrate the potential importance of ASHM patterns in the interpretation of disease loci, and the protocol described provides a basis for similar studies of ASHM in other cell types to further our understanding of human disease susceptibility.

  9. Multiplexed quantitative high content screening reveals that cigarette smoke condensate induces changes in cell structure and function through alterations in cell signaling pathways in human bronchial cells

    International Nuclear Information System (INIS)

    Carter, Charleata A.; Hamm, Jonathan T.

    2009-01-01

    Human bronchial cells are one of the first cell types exposed to environmental toxins. Toxins often activate nuclear factor-κB (NF-κB) and protein kinase C (PKC). We evaluated the hypothesis that cigarette smoke condensate (CSC), the particulate fraction of cigarette smoke, activates PKC-α and NF-κB, and concomitantly disrupts the F-actin cytoskeleton, induces apoptosis and alters cell function in BEAS-2B human bronchial epithelial cells. Compared to controls, exposure of BEAS-2B cells to doses of 30 μg/ml CSC significantly activated PKC-α, while CSC doses above 20 μg/ml CSC significantly activated NF-κB. As NF-κB was activated, cell number decreased. CSC treatment of BEAS-2B cells induced a decrease in cell size and an increase in cell surface extensions including filopodia and lamellipodia. CSC treatment of BEAS-2B cells induced F-actin rearrangement such that stress fibers were no longer prominent at the cell periphery and throughout the cells, but relocalized to perinuclear regions. Concurrently, CSC induced an increase in the focal adhesion protein vinculin at the cell periphery. CSC doses above 30 μg/ml induced a significant increase in apoptosis in BEAS-2B cells evidenced by an increase in activated caspase 3, an increase in mitochondrial mass and a decrease in mitochondrial membrane potential. As caspase 3 increased, cell number decreased. CSC doses above 30 μg/ml also induced significant concurrent changes in cell function including decreased cell spreading and motility. CSC initiates a signaling cascade in human bronchial epithelial cells involving PKC-α, NF-κB and caspase 3, and consequently decreases cell spreading and motility. These CSC-induced alterations in cell structure likely prevent cells from performing their normal function thereby contributing to smoke-induced diseases.

  10. High-sensitivity virus and mycoplasma screening test reveals high prevalence of parvovirus B19 infection in human synovial tissues and bone marrow.

    Science.gov (United States)

    Watanabe, Ken; Otabe, Koji; Shimizu, Norio; Komori, Keiichirou; Mizuno, Mitsuru; Katano, Hisako; Koga, Hideyuki; Sekiya, Ichiro

    2018-03-27

    Latent microorganism infection is a safety concern for the clinical application of mesenchymal stem cells (MSCs). The aim of this study is to investigate the frequencies and sensitivities of the latent virus and mycoplasma infections in synovium, bone marrow, peripheral blood cells, and blood plasma and cultured synovial MSCs. Total DNA and RNA of the synovium (n = 124), bone marrow (n = 123), peripheral blood cells (n = 121), plasma (n = 121), and 14-day cultured synovial MSCs (n = 63) were collected from patients who underwent total knee arthroplasty or anterior ligament reconstruction after written informed consents were obtained. The multiplex polymerase chain reaction (PCR) primers were designed to quantitatively measure the representative genomes of 13 DNA viruses, 6 RNA viruses, and 9 mycoplasmas. Multi-spliced mRNA detection and virus spike test were also performed to demonstrate the sensitivity of synovial MSCs to the candidate pathogens. In synovium and bone marrow, the positive rates of parvovirus B19 genome were significantly higher than in peripheral blood cells (18.7% and 22% vs. 0.8%, respectively). Multi-alignment analysis of amplified and sequenced viral target genes showed the proximity of the parvovirus B19 gene from different tissue in the same patients. Synovial MSCs cultured for 14 days were positive for virus infection only in two patients (2/62 = 3%). Parvovirus B19 multi-spliced mRNAs were not detected in these two samples. Virus spike test demonstrated the sensitivity of synovial MSCs to herpes simplex virus (HSV)1 and cytomegalovirus (CMV), but not to parvovirus B19. This study revealed a relatively high incidence of latent parvovirus B19 in synovium and bone marrow tissue.

  11. Alkylation sensitivity screens reveal a conserved cross-species functionome

    Science.gov (United States)

    Svilar, David; Dyavaiah, Madhu; Brown, Ashley R.; Tang, Jiang-bo; Li, Jianfeng; McDonald, Peter R.; Shun, Tong Ying; Braganza, Andrea; Wang, Xiao-hong; Maniar, Salony; St Croix, Claudette M.; Lazo, John S.; Pollack, Ian F.; Begley, Thomas J.; Sobol, Robert W.

    2013-01-01

    To identify genes that contribute to chemotherapy resistance in glioblastoma, we conducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma derived cell line with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored towards “druggable” targets. Select DNA repair genes in the screen were validated independently, confirming the DNA glycosylases UNG and MYH as well as MPG to be involved in the response to high dose TMZ. The involvement of UNG and MYH is likely the result of a TMZ-induced burst of reactive oxygen species. We then compared the human TMZ sensitizing genes identified in our screen with those previously identified from alkylator screens conducted in E. coli and S. cerevisiae. The conserved biological processes across all three species composes an Alkylation Functionome that includes many novel proteins not previously thought to impact alkylator resistance. This high-throughput screen, validation and cross-species analysis was then followed by a mechanistic analysis of two essential nodes: base excision repair (BER) DNA glycosylases (UNG, human and mag1, S. cerevisiae) and protein modification systems, including UBE3B and ICMT in human cells or pby1, lip22, stp22 and aim22 in S. cerevisiae. The conserved processes of BER and protein modification were dual targeted and yielded additive sensitization to alkylators in S. cerevisiae. In contrast, dual targeting of BER and protein modification genes in human cells did not increase sensitivity, suggesting an epistatic relationship. Importantly, these studies provide potential new targets to overcome alkylating agent resistance. PMID:23038810

  12. Natural Product Screening Reveals Naphthoquinone Complex I Bypass Factors.

    Directory of Open Access Journals (Sweden)

    Scott B Vafai

    Full Text Available Deficiency of mitochondrial complex I is encountered in both rare and common diseases, but we have limited therapeutic options to treat this lesion to the oxidative phosphorylation system (OXPHOS. Idebenone and menadione are redox-active molecules capable of rescuing OXPHOS activity by engaging complex I-independent pathways of entry, often referred to as "complex I bypass." In the present study, we created a cellular model of complex I deficiency by using CRISPR genome editing to knock out Ndufa9 in mouse myoblasts, and utilized this cell line to develop a high-throughput screening platform for novel complex I bypass factors. We screened a library of ~40,000 natural product extracts and performed bioassay-guided fractionation on a subset of the top scoring hits. We isolated four plant-derived 1,4-naphthoquinone complex I bypass factors with structural similarity to menadione: chimaphilin and 3-chloro-chimaphilin from Chimaphila umbellata and dehydro-α-lapachone and dehydroiso-α-lapachone from Stereospermum euphoroides. We also tested a small number of structurally related naphthoquinones from commercial sources and identified two additional compounds with complex I bypass activity: 2-methoxy-1,4-naphthoquinone and 2-methoxy-3-methyl-1,4,-naphthoquinone. The six novel complex I bypass factors reported here expand this class of molecules and will be useful as tool compounds for investigating complex I disease biology.

  13. A genomewide screen for suppressors of Alu-mediated rearrangements reveals a role for PIF1.

    Directory of Open Access Journals (Sweden)

    Karen M Chisholm

    Full Text Available Alu-mediated rearrangement of tumor suppressor genes occurs frequently during carcinogenesis. In breast cancer, this mechanism contributes to loss of the wild-type BRCA1 allele in inherited disease and to loss of heterozygosity in sporadic cancer. To identify genes required for suppression of Alu-mediated recombination we performed a genomewide screen of a collection of 4672 yeast gene deletion mutants using a direct repeat recombination assay. The primary screen and subsequent analysis identified 12 candidate genes including TSA, ELG1, and RRM3, which are known to play a significant role in maintaining genomic stability. Genetic analysis of the corresponding human homologs was performed in sporadic breast tumors and in inherited BRCA1-associated carcinomas. Sequencing of these genes in high risk breast cancer families revealed a potential role for the helicase PIF1 in cancer predisposition. PIF1 variant L319P was identified in three breast cancer families; importantly, this variant, which is predicted to be functionally damaging, was not identified in a large series of controls nor has it been reported in either dbSNP or the 1000 Genomes Project. In Schizosaccharomyces pombe, Pfh1 is required to maintain both mitochondrial and nuclear genomic integrity. Functional studies in yeast of human PIF1 L319P revealed that this variant cannot complement the essential functions of Pfh1 in either the nucleus or mitochondria. Our results provide a global view of nonessential genes involved in suppressing Alu-mediated recombination and implicate variation in PIF1 in breast cancer predisposition.

  14. Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

    Science.gov (United States)

    Potter, Paul K; Bowl, Michael R; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E; Simon, Michelle M; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V; Law, Gemma; MacLaren, Robert E; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H; Foster, Russell G; Jackson, Ian J; Peirson, Stuart N; Thakker, Rajesh V; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D M

    2016-08-18

    Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

  15. False-positive Human Papillomavirus DNA tests in cervical screening

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Pribac, Igor; Lynge, Elsebeth

    2011-01-01

    Based on data from randomised controlled trials (RCT) on primary cervical screening, it has been reported that the problem of more frequent false-positive tests in Human Papillomavirus (HPV) DNA screening compared to cytology could be overcome. However, these reports predominantly operated...

  16. Human language reveals a universal positivity bias.

    Science.gov (United States)

    Dodds, Peter Sheridan; Clark, Eric M; Desu, Suma; Frank, Morgan R; Reagan, Andrew J; Williams, Jake Ryland; Mitchell, Lewis; Harris, Kameron Decker; Kloumann, Isabel M; Bagrow, James P; Megerdoomian, Karine; McMahon, Matthew T; Tivnan, Brian F; Danforth, Christopher M

    2015-02-24

    Using human evaluation of 100,000 words spread across 24 corpora in 10 languages diverse in origin and culture, we present evidence of a deep imprint of human sociality in language, observing that (i) the words of natural human language possess a universal positivity bias, (ii) the estimated emotional content of words is consistent between languages under translation, and (iii) this positivity bias is strongly independent of frequency of word use. Alongside these general regularities, we describe interlanguage variations in the emotional spectrum of languages that allow us to rank corpora. We also show how our word evaluations can be used to construct physical-like instruments for both real-time and offline measurement of the emotional content of large-scale texts.

  17. MYSTERIES OF THE HUMAN FETUS REVEALED.

    Science.gov (United States)

    Sandman, Curt A

    2015-09-01

    The impressive program of research from the DiPietro laboratory succeeds in its aim to document the ontogeny of human fetal neurobehavioral development. From studies of great depth and breadth, and wielding creative methods of assessment, DiPietro et al. open a window into the largely inaccessible developing human fetal brain. This commentary, with reference to the seminal cardiovascular studies of the Laceys, supports the measures of the fetal heart to index fetal well-being and to provide evidence of stimulus processing. A separate case is made that the DiPietro program provides unique and invaluable information for assessing the influential Developmental Origins of Health and Disease or Fetal Programming Models. The goal of these models, to predict or understand the influences of early experience or response patterns on later postnatal life, is identical to the ultimate goal of the DiPietro program. Because human fetal behavior is uncontaminated by socialization or parenting or peers, it may be the best reflection of fetal exposures. The remarkable neurobehavioral profiles generated by the DiPietro program can make a critical contribution to the Fetal Programming Model in terms of sensitive and critical periods of nervous system vulnerability and to specify gestational periods of neurobehavioral risk. © 2015 The Society for Research in Child Development, Inc.

  18. Human tears reveal insights into corneal neovascularization.

    Science.gov (United States)

    Zakaria, Nadia; Van Grasdorff, Sigi; Wouters, Kristien; Rozema, Jos; Koppen, Carina; Lion, Eva; Cools, Nathalie; Berneman, Zwi; Tassignon, Marie-José

    2012-01-01

    Corneal neovascularization results from the encroachment of blood vessels from the surrounding conjunctiva onto the normally avascular cornea. The aim of this study is to identify factors in human tears that are involved in development and/or maintenance of corneal neovascularization in humans. This could allow development of diagnostic tools for monitoring corneal neovascularization and combination monoclonal antibody therapies for its treatment. In an observational case-control study we enrolled a total of 12 patients with corneal neovascularization and 10 healthy volunteers. Basal tears along with reflex tears from the inferior fornix, superior fornix and using a corneal bath were collected along with blood serum samples. From all patients, ocular surface photographs were taken. Concentrations of the pro-angiogenic cytokines interleukin (IL)-6, IL-8, Vascular Endothelial Growth Factor (VEGF), Monocyte Chemoattractant Protein 1 (MCP-1) and Fas Ligand (FasL) were determined in blood and tear samples using a flow cytometric multiplex assay. Our results show that the concentration of pro-angiogenic cytokines in human tears are significantly higher compared to their concentrations in serum, with highest levels found in basal tears. Interestingly, we could detect a significantly higher concentration of IL- 6, IL-8 and VEGF in localized corneal tears of patients with neovascularized corneas when compared to the control group. This is the first study of its kind demonstrating a significant difference of defined factors in tears from patients with neovascularized corneas as compared to healthy controls. These results provide the basis for future research using animal models to further substantiate the role of these cytokines in the establishment and maintenance of corneal neovascularization.

  19. Elevated phenylalanine on newborn screening: follow-up testing may reveal undiagnosed galactosaemia.

    Science.gov (United States)

    Shakespeare, Lynette; Downing, Melanie; Allen, Joyce; Casbolt, Ann-Marie; Ellin, Sheila; Maloney, Martin; Race, Gillian; Bonham, Jim

    2010-11-01

    Introduction Newborn screening for phenylketonuria (PKU) can reveal other conditions which lead to an increased blood spot phenylalanine (Phe) concentration. We have investigated the proportion of blood spot samples that gave a positive screen due to clinically significant conditions other than PKU, compared the positive predictive value (PPV) of our referral Phe cut-off with that recommended by the UK Newborn Screening Programme Centre (UKNSPC) (>210 and >240 μmol/L, respectively) and evaluated the effectiveness of reflex testing for galactosaemia using a lower blood spot Phe cut-off concentration of 130 μmol/L. All blood spot samples that screened positive, for an increased Phe concentration, between April 2001 and March 2008, were identified from the records of the Sheffield Newborn Screening Laboratory and the diagnoses noted. In addition, all cases of galactosaemia detected in or notified to our screening laboratory within this time were also examined and the screened Phe concentrations compared. Out of 438,674 babies who were screened, 67 had Phe concentration >210 μmol/L (15 per 100,000). Of these, 40 had PKU or persistent hyperphenylalaninaemia with a Phe concentration identified by screening between 270 and 2350 μmol/L. A further 11 were diagnosed with another clinically significant disorder: galactosaemia (n = 8), biopterin defects (n = 2), tyrosinaemia Type 1 (n = 1). In addition, 16 had transient elevations in Phe. In total, nine cases of galactosaemia were identified, of whom, three had Phe concentrations 240 μmol/L) will not affect the detection rate of classical PKU, but will improve the PPV from 76% to 80%. The use of a lower cut-off (130 μmol/L) for reflex galactosaemia testing enables the timely identification of asymptomatic cases that benefit particularly from early treatment, without prompting any unnecessary clinical referrals or delaying any referrals. This intervention may reduce mortality in this vulnerable group.

  20. High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling

    Directory of Open Access Journals (Sweden)

    Du Peige

    2008-10-01

    Full Text Available Abstract Background Estrogen receptor α (ERα is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERα, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE with a reporter gene. This allowed the cellular activity of ERα, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2. Results From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131 as a repressor of ERα mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERα in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERα interrupts or prevents ERα binding to the estrogen response element (ERE. In addition, ZNF131 was able to suppress the expression of pS2, an ERα target gene. Conclusion We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERα-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERα regulation in mammalian cells.

  1. A functional genomics screen in planarians reveals regulators of whole-brain regeneration

    Science.gov (United States)

    Roberts-Galbraith, Rachel H; Brubacher, John L; Newmark, Phillip A

    2016-01-01

    Planarians regenerate all body parts after injury, including the central nervous system (CNS). We capitalized on this distinctive trait and completed a gene expression-guided functional screen to identify factors that regulate diverse aspects of neural regeneration in Schmidtea mediterranea. Our screen revealed molecules that influence neural cell fates, support the formation of a major connective hub, and promote reestablishment of chemosensory behavior. We also identified genes that encode signaling molecules with roles in head regeneration, including some that are produced in a previously uncharacterized parenchymal population of cells. Finally, we explored genes downregulated during planarian regeneration and characterized, for the first time, glial cells in the planarian CNS that respond to injury by repressing several transcripts. Collectively, our studies revealed diverse molecules and cell types that underlie an animal’s ability to regenerate its brain. DOI: http://dx.doi.org/10.7554/eLife.17002.001 PMID:27612384

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

    Science.gov (United States)

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

  3. Direct Lineage Reprogramming Reveals Disease-Specific Phenotypes of Motor Neurons from Human ALS Patients

    Directory of Open Access Journals (Sweden)

    Meng-Lu Liu

    2016-01-01

    Full Text Available Subtype-specific neurons obtained from adult humans will be critical to modeling neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS. Here, we show that adult human skin fibroblasts can be directly and efficiently converted into highly pure motor neurons without passing through an induced pluripotent stem cell stage. These adult human induced motor neurons (hiMNs exhibit the cytological and electrophysiological features of spinal motor neurons and form functional neuromuscular junctions (NMJs with skeletal muscles. Importantly, hiMNs converted from ALS patient fibroblasts show disease-specific degeneration manifested through poor survival, soma shrinkage, hypoactivity, and an inability to form NMJs. A chemical screen revealed that the degenerative features of ALS hiMNs can be remarkably rescued by the small molecule kenpaullone. Taken together, our results define a direct and efficient strategy to obtain disease-relevant neuronal subtypes from adult human patients and reveal their promising value in disease modeling and drug identification.

  4. Screening of plant extracts for human tyrosinase inhibiting effects.

    Science.gov (United States)

    Kim, M; Park, J; Song, K; Kim, H G; Koh, J-S; Boo, Y C

    2012-04-01

    Screening for tyrosinase (TYR) inhibitors potentially useful for control of skin pigmentation has been hampered by the limited availability of human TYR. To overcome this hurdle, we have established human embryonic kidney (HEK293)-TYR cells that constitutively express human TYR. In the current study, we assayed human TYR inhibition activities of 50 plant extracts using the lysates of transformed HEK293-TYR cells. The strongest inhibition of human TYR was shown by the extract of Vaccinium bracteatum Thunberg, followed by the extract of Morus bombycis Koidzumi. The former extract did not inhibit mushroom TYR activity whereas significant inhibition was observed with the latter extract, demonstrating the importance of using human TYR in the screening for human TYR inhibitors. Upon liquid-liquid partitioning of the extract from V. bracteatum, the active constituents were enriched in the ethyl acetate fraction, and the subsequent preparatory thin-layer chromatography identified p-coumaric acid (PCA) as the main active constituent. The hypo-pigmentation of PCA was verified in the MelanoDerm™ Skin Model. This study demonstrates that transformed HEK293-TYR cells could expedite the discovery of human TYR-specific inhibitors from natural sources which might be useful in the control of skin pigmentation. © 2012 The Authors. ICS © 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  5. Human papilloma virus prevalence in a multiethnic screening population.

    Science.gov (United States)

    Chen, Kang Mei; Stephen, Josena K; Ghanem, Tamer; Stachler, Robert; Gardner, Glendon; Jones, Lamont; Schweitzer, Vanessa P; Hall, Francis; Divine, George; Worsham, Maria J

    2013-03-01

    The goal was to determine the prevalence of high-risk HPV16 using saliva in a screening population in Detroit, Michigan. Real-time quantitative polymerase chain reaction was applied to detect HPV16 in saliva DNA from 349 screening subjects without head and neck cancer (HNC), 156 with HNC, and 19 controls. Cut points for human papilloma virus (HPV) positivity were >0 and >0.001 copy/cell. Proportions were compared between groups using exact χ(2) or Fisher exact tests (P 0, each group had an overall HPV prevalence of more than 5%, with a higher prevalence of 30.8% in the HNC patient group. At a cut point >0.001, the prevalence was lower: 0% in the control, 1.2% in the screening, and 16.7% in the HNC group. In the latter, for both cut points, HPV prevalence was different across sites (0, women in the screening group had a higher prevalence of HPV than did men (P = .010), and at >0.001, the prevalence was higher for men in the HNC group than for women (P = .035). In the screening group, at >0, only African Americans had a higher prevalence than Caucasian Americans (P = .025). In the screening group, a 6.9% and 1.2% screening rate was noted at cut points >0 and >0.001, respectively. The results provide data to inform public health considerations of the feasibility of saliva as a screening tool in at-risk populations with the long-term goal of prophylactic vaccination against oral HPV.

  6. Human factors engineering review for CRT screen design

    International Nuclear Information System (INIS)

    Yi, S. M.; Joo, C. Y.; Ra, J. C.

    1999-01-01

    The information interface between man and machine may be more important than hardware and workplace layout considerations. Transmitting and receiving data through this information interface can be characterized as a communication or interface problem. Management of man-machine interface is essential for the enhancement of the information processing and decision-making capability of computer users working in real time, demanding task. The design of human-computer interface is not a rigid and static procedure. The content and context of each interface varies according to the specific application. So, the purpose of this study is to review the human factor design process for interfaces, to make human factor guidelines for CRT screen and to apply these to CRT screen design. (author)

  7. Human papillomavirus testing and genotyping in cervical screening

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Lynge, Elsebeth; Bonde, Jesper

    2011-01-01

    the incidence of cervical cancer, but has a low sensitivity for high-grade cervical intraepithelial neoplasia (CIN) and requires frequent testing. Several HPV tests have become available commercially. They appear to be more sensitive for high-grade CIN, and may further reduce the incidence of cervical cancer......Mass vaccination against human papillomavirus (HPV) genotypes 16 and 18 will, in the long term, reduce the incidence of cervical cancer, but screening will remain an important cancer control measure in both vaccinated and unvaccinated women. Since the 1960s, cytology screening has helped to reduce...

  8. Combining chemical genomics screens in yeast to reveal spectrum of effects of chemical inhibition of sphingolipid biosynthesis

    Directory of Open Access Journals (Sweden)

    Giaever Guri

    2009-01-01

    Full Text Available Abstract Background Single genome-wide screens for the effect of altered gene dosage on drug sensitivity in the model organism Saccharomyces cerevisiae provide only a partial picture of the mechanism of action of a drug. Results Using the example of the tumor cell invasion inhibitor dihydromotuporamine C, we show that a more complete picture of drug action can be obtained by combining different chemical genomics approaches – analysis of the sensitivity of ρ0 cells lacking mitochondrial DNA, drug-induced haploinsufficiency, suppression of drug sensitivity by gene overexpression and chemical-genetic synthetic lethality screening using strains deleted of nonessential genes. Killing of yeast by this chemical requires a functional mitochondrial electron-transport chain and cytochrome c heme lyase function. However, we find that it does not require genes associated with programmed cell death in yeast. The chemical also inhibits endocytosis and intracellular vesicle trafficking and interferes with vacuolar acidification in yeast and in human cancer cells. These effects can all be ascribed to inhibition of sphingolipid biosynthesis by dihydromotuporamine C. Conclusion Despite their similar conceptual basis, namely altering drug sensitivity by modifying gene dosage, each of the screening approaches provided a distinct set of information that, when integrated, revealed a more complete picture of the mechanism of action of a drug on cells.

  9. Cognitive-behavioral screening reveals prevalent impairment in a large multicenter ALS cohort.

    Science.gov (United States)

    Murphy, Jennifer; Factor-Litvak, Pam; Goetz, Raymond; Lomen-Hoerth, Catherine; Nagy, Peter L; Hupf, Jonathan; Singleton, Jessica; Woolley, Susan; Andrews, Howard; Heitzman, Daragh; Bedlack, Richard S; Katz, Jonathan S; Barohn, Richard J; Sorenson, Eric J; Oskarsson, Björn; Fernandes Filho, J Americo M; Kasarskis, Edward J; Mozaffar, Tahseen; Rollins, Yvonne D; Nations, Sharon P; Swenson, Andrea J; Koczon-Jaremko, Boguslawa A; Mitsumoto, Hiroshi

    2016-03-01

    To characterize the prevalence of cognitive and behavioral symptoms using a cognitive/behavioral screening battery in a large prospective multicenter study of amyotrophic lateral sclerosis (ALS). Two hundred seventy-four patients with ALS completed 2 validated cognitive screening tests and 2 validated behavioral interviews with accompanying caregivers. We examined the associations between cognitive and behavioral performance, demographic and clinical data, and C9orf72 mutation data. Based on the ALS Cognitive Behavioral Screen cognitive score, 6.5% of the sample scored below the cutoff score for frontotemporal lobar dementia, 54.2% scored in a range consistent with ALS with mild cognitive impairment, and 39.2% scored in the normal range. The ALS Cognitive Behavioral Screen behavioral subscale identified 16.5% of the sample scoring below the dementia cutoff score, with an additional 14.1% scoring in the ALS behavioral impairment range, and 69.4% scoring in the normal range. This investigation revealed high levels of cognitive and behavioral impairment in patients with ALS within 18 months of symptom onset, comparable to prior investigations. This investigation illustrates the successful use and scientific value of adding a cognitive-behavioral screening tool in studies of motor neuron diseases, to provide neurologists with an efficient method to measure these common deficits and to understand how they relate to key clinical variables, when extensive neuropsychological examinations are unavailable. These tools, developed specifically for patients with motor impairment, may be particularly useful in patient populations with multiple sclerosis and Parkinson disease, who are known to have comorbid cognitive decline. © 2016 American Academy of Neurology.

  10. Deciphering CAPTCHAs: what a Turing test reveals about human cognition.

    Directory of Open Access Journals (Sweden)

    Thomas Hannagan

    Full Text Available Turning Turing's logic on its head, we used widespread letter-based Turing Tests found on the internet (CAPTCHAs to shed light on human cognition. We examined the basis of the human ability to solve CAPTCHAs, where machines fail. We asked whether this is due to our use of slow-acting inferential processes that would not be available to machines, or whether fast-acting automatic orthographic processing in humans has superior robustness to shape variations. A masked priming lexical decision experiment revealed efficient processing of CAPTCHA words in conditions that rule out the use of slow inferential processing. This shows that the human superiority in solving CAPTCHAs builds on a high degree of invariance to location and continuous transforms, which is achieved during the very early stages of visual word recognition in skilled readers.

  11. Human factors guidelines for large-screen displays

    International Nuclear Information System (INIS)

    Collier, Steve

    2005-09-01

    Any control-room project (including upgrades or evolutionary improvements to existing control-rooms) is well advised at the outset first to gather and update related background material for the design. This information-gathering exercise should also take into account experience from similar projects and operating experience. For these reasons, we decided to use our research, and experience in large-screen display design with several clients to update human factors guidance for large-screen displays, to take into account new ergonomics guidelines, operating experience, and work from similar projects. To write the updated guidelines, we drew on much of our experience across several departments at IFE, including research funded by the HRP programme, and experience with individual clients. Guidance here is accordingly focused mainly on recent areas of technical and human innovations in the man-machine interface. One particular area of focus was on the increasing use of large-screen display systems in modern control-rooms, and on how guidelines could be adapted and supplemented for their design. Guidance or reference to recommended sources is also given for control suite arrangement and layout, control-room layout, workstation layout, design of displays and controls, and design of the work environment, especially insofar as these ergonomic issues interact with the effectiveness of modern displays, in particular large screen displays. The work shows that there can be synergy between HRP research and bilateral activities: the one side offers a capability to develop tools and guidelines, while the other side gives an opportunity to test and refine these in practice, to the benefit of both parties. (Author)

  12. Human factors guidelines for large-screen displays

    Energy Technology Data Exchange (ETDEWEB)

    Collier, Steve

    2005-09-15

    Any control-room project (including upgrades or evolutionary improvements to existing control-rooms) is well advised at the outset first to gather and update related background material for the design. This information-gathering exercise should also take into account experience from similar projects and operating experience. For these reasons, we decided to use our research, and experience in large-screen display design with several clients to update human factors guidance for large-screen displays, to take into account new ergonomics guidelines, operating experience, and work from similar projects. To write the updated guidelines, we drew on much of our experience across several departments at IFE, including research funded by the HRP programme, and experience with individual clients. Guidance here is accordingly focused mainly on recent areas of technical and human innovations in the man-machine interface. One particular area of focus was on the increasing use of large-screen display systems in modern control-rooms, and on how guidelines could be adapted and supplemented for their design. Guidance or reference to recommended sources is also given for control suite arrangement and layout, control-room layout, workstation layout, design of displays and controls, and design of the work environment, especially insofar as these ergonomic issues interact with the effectiveness of modern displays, in particular large screen displays. The work shows that there can be synergy between HRP research and bilateral activities: the one side offers a capability to develop tools and guidelines, while the other side gives an opportunity to test and refine these in practice, to the benefit of both parties. (Author)

  13. RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization.

    Directory of Open Access Journals (Sweden)

    Julia F Pielage

    2008-03-01

    Full Text Available Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of approximately 80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa-induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.

  14. A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium tetraurelia

    Science.gov (United States)

    Marker, Simone; Carradec, Quentin; Tanty, Véronique; Arnaiz, Olivier; Meyer, Eric

    2014-01-01

    In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction. These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia. PMID:24860163

  15. Functional Genomic Screening Reveals Core Modulators of Echinocandin Stress Responses in Candida albicans

    Directory of Open Access Journals (Sweden)

    Tavia Caplan

    2018-05-01

    Full Text Available Summary: Candida albicans is a leading cause of death due to fungal infection. Treatment of systemic candidiasis often relies on echinocandins, which disrupt cell wall synthesis. Resistance is readily acquired via mutations in the drug target gene, FKS1. Both basal tolerance and resistance to echinocandins require cellular stress responses. We performed a systematic analysis of 3,030 C. albicans mutants to define circuitry governing cellular responses to echinocandins. We identified 16 genes for which deletion or transcriptional repression enhanced echinocandin susceptibility, including components of the Pkc1-MAPK signaling cascade. We discovered that the molecular chaperone Hsp90 is required for the stability of Pkc1 and Bck1, establishing key mechanisms through which Hsp90 mediates echinocandin resistance. We also discovered that perturbation of the CCT chaperonin complex causes enhanced echinocandin sensitivity, altered cell wall architecture, and aberrant septin localization. Thus, we provide insights into the mechanisms by which cellular chaperones enable crucial responses to echinocandin-induced stress. : Caplan et al. screen 3,030 Candida albicans mutants to define circuitry governing cellular responses to echinocandins, the first-line therapy for systemic candidiasis. They reveal that the molecular chaperone Hsp90 is required for stability of Pkc1 and Bck1 and that the CCT chaperonin complex is a key modulator of echinocandin susceptibility. Keywords: fungal pathogen, Candida albicans, echinocandins, Hsp90, Pkc1, CCT complex, client protein, stress response, functional genomic screen, drug resistance

  16. Genetic Screen Reveals the Role of Purine Metabolism in Staphylococcus aureus Persistence to Rifampicin

    Directory of Open Access Journals (Sweden)

    Rebecca Yee

    2015-12-01

    Full Text Available Chronic infections with Staphylococcus aureus such as septicemia, osteomyelitis, endocarditis, and biofilm infections are difficult to treat because of persisters. Despite many efforts in understanding bacterial persistence, the mechanisms of persister formation in S. aureus remain elusive. Here, we performed a genome-wide screen of a transposon mutant library to study the molecular mechanisms involved in persistence of community-acquired S. aureus. Screening of the library for mutants defective in persistence or tolerance to rifampicin revealed many genes involved in metabolic pathways that are important for antibiotic persistence. In particular, the identified mutants belonged to metabolic pathways involved in carbohydrate, amino acid, lipid, vitamin and purine biosynthesis. Five mutants played a role in purine biosynthesis and two mutants, purB, an adenylosuccinate lyase, and purM, a phosphoribosylaminoimidazole synthetase, were selected for further confirmation. Mutants purB and purM showed defective persistence compared to the parental strain USA300 in multiple stress conditions including various antibiotics, low pH, and heat stress. The defect in persistence was restored by complementation with the wildtype purB and purM gene in the respective mutants. These findings provide new insights into the mechanisms of persistence in S. aureus and provide novel therapeutic targets for developing more effective treatment for persistent infections due to S. aureus.

  17. Signature-tagged mutagenesis screening revealed a novel smooth-to-rough transition determinant of Salmonella enterica serovar Enteritidis.

    Science.gov (United States)

    Jiao, Yang; Guo, Rongxian; Tang, Peipei; Kang, Xilong; Yin, Junlei; Wu, Kaiyue; Geng, Shizhong; Li, Qiuchun; Sun, Jun; Xu, Xiulong; Zhou, Xiaohui; Gan, Junji; Jiao, Xinan; Liu, Xiufan; Pan, Zhiming

    2017-03-03

    Salmonella enterica serovar Enteritidis (S. Enteritidis) has emerged as one of the most important food-borne pathogens for humans. Lipopolysaccharide (LPS), as a component of the outer membrane, is responsible for the virulence and smooth-to-rough transition in S. Enteritidis. In this study, we screened S. Enteritidis signature-tagged transposon mutant library using monoclonal antibody against somatic O 9 antigen (O 9 MAb) and O 9 factor rabbit antiserum to identify novel genes that are involved in smooth-to-rough transition. A total of 480 mutants were screened and one mutant with transposon insertion in rfbG gene had smooth-to-rough transition phenotype. In order to verify the role of rfbG gene, an rfbG insertion or deletion mutant was constructed using λ-Red recombination system. Phenotypic and biological analysis revealed that rfbG insertion or deletion mutants were similar to the wild-type strain in growth rate and biochemical properties, but the swimming motility was reduced. SE Slide Agglutination test and ELISA test showed that rfbG mutants do not stimulate animals to produce agglutinating antibody. In addition, the half-lethal dose (LD 50 ) of the rfbG deletion mutant strain was 10 6.6 -fold higher than that of the parent strain in a mouse model when injected intraperitoneally. These data indicate that the rfbG gene is involved in smooth-to-rough transition, swimming motility and virulence of S. Enteritidis. Furthermore, somatic O-antigen antibody-based approach to screen signature-tagged transposon mutants is feasible to clarify LPS biosynthesis and to find suitable markers in DIVA-vaccine research.

  18. Human Health Screening and Public Health Significance of ...

    Science.gov (United States)

    The source water and treated drinking water from twenty five drinking water treatment plants (DWTPs) across the United States were sampled in 2010 – 2012. Samples were analyzed for 247 contaminants using 15 chemical and microbiological methods. Most of these contaminants are not regulated currently either in drinking water or in discharges to ambient water by the United States Environmental Protection Agency (EPA) or other U.S. regulatory agencies. This analysis shows that there is little public health concern for most of the contaminants detected in treated water from the 25 DWTPs participating in this study. For vanadium, the calculated MOE was less than the screening MOE in two DWTPs. Additional study, for example a national survey may be needed to determine the number of people ingesting vanadium above a level of concern. In addition, the concentrations of lithium found in treated water from several DWTPs are within the range previous research has suggested to have a human health effect. Additional investigation of this issue may also be appropriate. Finally, new toxicological data suggests that exposure to manganese at levels in public water supplies may present a public health concern which may warrant a more robust assessment of this information. This paper provides a screening-level human health risk assessment using the margin of exposure of exposure approach, of contaminants of emerging concern detected in drinking water. As far as we are a

  19. Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion.

    Science.gov (United States)

    Schiebler, Mark; Brown, Karen; Hegyi, Krisztina; Newton, Sandra M; Renna, Maurizio; Hepburn, Lucy; Klapholz, Catherine; Coulter, Sarah; Obregón-Henao, Andres; Henao Tamayo, Marcela; Basaraba, Randall; Kampmann, Beate; Henry, Katherine M; Burgon, Joseph; Renshaw, Stephen A; Fleming, Angeleen; Kay, Robert R; Anderson, Karen E; Hawkins, Phillip T; Ordway, Diane J; Rubinsztein, David C; Floto, Rodrigo Andres

    2015-02-01

    Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell-based screening of FDA-approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug-resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR-independent pathway controlled by cellular depletion of myo-inositol. While strain-specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug-resistant mycobacterial infection. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  20. Elaborate cellulosome architecture of Acetivibrio cellulolyticus revealed by selective screening of cohesin–dockerin interactions

    Directory of Open Access Journals (Sweden)

    Yuval Hamberg

    2014-10-01

    cellulosome’s total cohesin and dockerin sequence diversity, emphasizing the testing of unusual and previously-unknown protein modules. The screen revealed several novel cell-bound cellulosome architectures, thus expanding on those previously known, as well as soluble cellulose systems that are not bound to the bacterial cell surface. This study sets the stage for screening the entire complement of cellulosomal components from A. cellulolyticus and other organisms with large cellulosome systems. The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.

  1. [Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

    Science.gov (United States)

    Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

    2012-06-01

    To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

  2. Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

    Science.gov (United States)

    Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

    2015-03-23

    Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

  3. Screens

    OpenAIRE

    2016-01-01

    This Sixth volume in the series The Key Debates. Mutations and Appropriations in European Film Studies investigates the question of screens in the context both of the dematerialization due to digitalization and the multiplication of media screens. Scholars offer various infomations and theories of topics such as the archeology of screen, film and media theories, contemporary art, pragmatics of new ways of screening (from home video to street screening).

  4. Zebrafish chemical screening reveals the impairment of dopaminergic neuronal survival by cardiac glycosides.

    Directory of Open Access Journals (Sweden)

    Yaping Sun

    Full Text Available Parkinson's disease is a neurodegenerative disorder characterized by the prominent degeneration of dopaminergic (DA neurons among other cell types. Here we report a first chemical screen of over 5,000 compounds in zebrafish, aimed at identifying small molecule modulators of DA neuron development or survival. We find that Neriifolin, a member of the cardiac glycoside family of compounds, impairs survival but not differentiation of both zebrafish and mammalian DA neurons. Cardiac glycosides are inhibitors of Na(+/K(+ ATPase activity and widely used for treating heart disorders. Our data suggest that Neriifolin impairs DA neuronal survival by targeting the neuronal enriched Na(+/K(+ ATPase α3 subunit (ATP1A3. Modulation of ionic homeostasis, knockdown of p53, or treatment with antioxidants protects DA neurons from Neriifolin-induced death. These results reveal a previously unknown effect of cardiac glycosides on DA neuronal survival and suggest that it is mediated through ATP1A3 inhibition, oxidative stress, and p53. They also elucidate potential approaches for counteracting the neurotoxicity of this valuable class of medications.

  5. In vivo RNAi screen reveals neddylation genes as novel regulators of Hedgehog signaling.

    Directory of Open Access Journals (Sweden)

    Juan Du

    Full Text Available Hedgehog (Hh signaling is highly conserved in all metazoan animals and plays critical roles in many developmental processes. Dysregulation of the Hh signaling cascade has been implicated in many diseases, including cancer. Although key components of the Hh pathway have been identified, significant gaps remain in our understanding of the regulation of individual Hh signaling molecules. Here, we report the identification of novel regulators of the Hh pathway, obtained from an in vivo RNA interference (RNAi screen in Drosophila. By selectively targeting critical genes functioning in post-translational modification systems utilizing ubiquitin (Ub and Ub-like proteins, we identify two novel genes (dUba3 and dUbc12 that negatively regulate Hh signaling activity. We provide in vivo and in vitro evidence illustrating that dUba3 and dUbc12 are essential components of the neddylation pathway; they function in an enzyme cascade to conjugate the ubiquitin-like NEDD8 modifier to Cullin proteins. Neddylation activates the Cullin-containing ubiquitin ligase complex, which in turn promotes the degradation of Cubitus interruptus (Ci, the downstream transcription factor of the Hh pathway. Our study reveals a conserved molecular mechanism of the neddylation pathway in Drosophila and sheds light on the complex post-translational regulations in Hh signaling.

  6. Screening of the Open Source Malaria Box Reveals an Early Lead Compound for the Treatment of Alveolar Echinococcosis.

    Directory of Open Access Journals (Sweden)

    Britta Stadelmann

    2016-03-01

    Full Text Available The metacestode (larval stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE, a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV, comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 μM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 μM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC50 value below 5 μM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load.

  7. An overexpression screen of Toxoplasma gondii Rab-GTPases reveals distinct transport routes to the micronemes.

    Directory of Open Access Journals (Sweden)

    Katrin Kremer

    2013-03-01

    Full Text Available The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.

  8. Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells

    Science.gov (United States)

    Kraehling, Jan R.; Chidlow, John H.; Rajagopal, Chitra; Sugiyama, Michael G.; Fowler, Joseph W.; Lee, Monica Y.; Zhang, Xinbo; Ramírez, Cristina M.; Park, Eon Joo; Tao, Bo; Chen, Keyang; Kuruvilla, Leena; Larriveé, Bruno; Folta-Stogniew, Ewa; Ola, Roxana; Rotllan, Noemi; Zhou, Wenping; Nagle, Michael W.; Herz, Joachim; Williams, Kevin Jon; Eichmann, Anne; Lee, Warren L.; Fernández-Hernando, Carlos; Sessa, William C.

    2016-01-01

    In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL. PMID:27869117

  9. A kinome-wide RNAi screen in Drosophila Glia reveals that the RIO kinases mediate cell proliferation and survival through TORC2-Akt signaling in glioblastoma.

    Directory of Open Access Journals (Sweden)

    Renee D Read

    Full Text Available Glioblastoma, the most common primary malignant brain tumor, is incurable with current therapies. Genetic and molecular analyses demonstrate that glioblastomas frequently display mutations that activate receptor tyrosine kinase (RTK and Pi-3 kinase (PI3K signaling pathways. In Drosophila melanogaster, activation of RTK and PI3K pathways in glial progenitor cells creates malignant neoplastic glial tumors that display many features of human glioblastoma. In both human and Drosophila, activation of the RTK and PI3K pathways stimulates Akt signaling along with other as-yet-unknown changes that drive oncogenesis. We used this Drosophila glioblastoma model to perform a kinome-wide genetic screen for new genes required for RTK- and PI3K-dependent neoplastic transformation. Human orthologs of novel kinases uncovered by these screens were functionally assessed in mammalian glioblastoma models and human tumors. Our results revealed that the atypical kinases RIOK1 and RIOK2 are overexpressed in glioblastoma cells in an Akt-dependent manner. Moreover, we found that overexpressed RIOK2 formed a complex with RIOK1, mTor, and mTor-complex-2 components, and that overexpressed RIOK2 upregulated Akt signaling and promoted tumorigenesis in murine astrocytes. Conversely, reduced expression of RIOK1 or RIOK2 disrupted Akt signaling and caused cell cycle exit, apoptosis, and chemosensitivity in glioblastoma cells by inducing p53 activity through the RpL11-dependent ribosomal stress checkpoint. These results imply that, in glioblastoma cells, constitutive Akt signaling drives RIO kinase overexpression, which creates a feedforward loop that promotes and maintains oncogenic Akt activity through stimulation of mTor signaling. Further study of the RIO kinases as well as other kinases identified in our Drosophila screen may reveal new insights into defects underlying glioblastoma and related cancers and may reveal new therapeutic opportunities for these cancers.

  10. Fine-scaled human genetic structure revealed by SNP microarrays.

    Science.gov (United States)

    Xing, Jinchuan; Watkins, W Scott; Witherspoon, David J; Zhang, Yuhua; Guthery, Stephen L; Thara, Rangaswamy; Mowry, Bryan J; Bulayeva, Kazima; Weiss, Robert B; Jorde, Lynn B

    2009-05-01

    We report an analysis of more than 240,000 loci genotyped using the Affymetrix SNP microarray in 554 individuals from 27 worldwide populations in Africa, Asia, and Europe. To provide a more extensive and complete sampling of human genetic variation, we have included caste and tribal samples from two states in South India, Daghestanis from eastern Europe, and the Iban from Malaysia. Consistent with observations made by Charles Darwin, our results highlight shared variation among human populations and demonstrate that much genetic variation is geographically continuous. At the same time, principal components analyses reveal discernible genetic differentiation among almost all identified populations in our sample, and in most cases, individuals can be clearly assigned to defined populations on the basis of SNP genotypes. All individuals are accurately classified into continental groups using a model-based clustering algorithm, but between closely related populations, genetic and self-classifications conflict for some individuals. The 250K data permitted high-level resolution of genetic variation among Indian caste and tribal populations and between highland and lowland Daghestani populations. In particular, upper-caste individuals from Tamil Nadu and Andhra Pradesh form one defined group, lower-caste individuals from these two states form another, and the tribal Irula samples form a third. Our results emphasize the correlation of genetic and geographic distances and highlight other elements, including social factors that have contributed to population structure.

  11. Identifying and annotating human bifunctional RNAs reveals their versatile functions.

    Science.gov (United States)

    Chen, Geng; Yang, Juan; Chen, Jiwei; Song, Yunjie; Cao, Ruifang; Shi, Tieliu; Shi, Leming

    2016-10-01

    Bifunctional RNAs that possess both protein-coding and noncoding functional properties were less explored and poorly understood. Here we systematically explored the characteristics and functions of such human bifunctional RNAs by integrating tandem mass spectrometry and RNA-seq data. We first constructed a pipeline to identify and annotate bifunctional RNAs, leading to the characterization of 132 high-confidence bifunctional RNAs. Our analyses indicate that bifunctional RNAs may be involved in human embryonic development and can be functional in diverse tissues. Moreover, bifunctional RNAs could interact with multiple miRNAs and RNA-binding proteins to exert their corresponding roles. Bifunctional RNAs may also function as competing endogenous RNAs to regulate the expression of many genes by competing for common targeting miRNAs. Finally, somatic mutations of diverse carcinomas may generate harmful effect on corresponding bifunctional RNAs. Collectively, our study not only provides the pipeline for identifying and annotating bifunctional RNAs but also reveals their important gene-regulatory functions.

  12. Duplex Alu Screening for Degraded DNA of Skeletal Human Remains

    Directory of Open Access Journals (Sweden)

    Fabian Haß

    2017-10-01

    Full Text Available The human-specific Alu elements, belonging to the class of Short INterspersed Elements (SINEs, have been shown to be a powerful tool for population genetic studies. An earlier study in this department showed that it was possible to analyze Alu presence/absence in 3000-year-old skeletal human remains from the Bronze Age Lichtenstein cave in Lower Saxony, Germany. We developed duplex Alu screening PCRs with flanking primers for two Alu elements, each combined with a single internal Alu primer. By adding an internal primer, the approximately 400–500 bp presence signals of Alu elements can be detected within a range of less than 200 bp. Thus, our PCR approach is suited for highly fragmented ancient DNA samples, whereas NGS analyses frequently are unable to handle repetitive elements. With this analysis system, we examined remains of 12 individuals from the Lichtenstein cave with different degrees of DNA degradation. The duplex PCRs showed fully informative amplification results for all of the chosen Alu loci in eight of the 12 samples. Our analysis system showed that Alu presence/absence analysis is possible in samples with different degrees of DNA degradation and it reduces the amount of valuable skeletal material needed by a factor of four, as compared with a singleplex approach.

  13. Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling.

    Directory of Open Access Journals (Sweden)

    Aruna D Balgi

    Full Text Available BACKGROUND: Mammalian target of rapamycin complex 1 (mTORC1 is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy, a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties. METHODOLOGY/PRINCIPAL FINDINGS: Using an automated cell-based assay, we screened a collection of >3,500 chemicals and identified three approved drugs (perhexiline, niclosamide, amiodarone and one pharmacological reagent (rottlerin capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline, niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2, a negative regulator of mTORC1, was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline, niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions, by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2, an upstream regulator of mTORC1. By contrast, transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition, sustained inhibition of cell growth and no selective cell killing in starvation. CONCLUSION/SIGNIFICANCE: The observation that drugs already

  14. RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell.

    Directory of Open Access Journals (Sweden)

    Ryan Chong

    Full Text Available Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin

  15. A complex regulatory network coordinating cell cycles during C. elegans development is revealed by a genome-wide RNAi screen.

    Science.gov (United States)

    Roy, Sarah H; Tobin, David V; Memar, Nadin; Beltz, Eleanor; Holmen, Jenna; Clayton, Joseph E; Chiu, Daniel J; Young, Laura D; Green, Travis H; Lubin, Isabella; Liu, Yuying; Conradt, Barbara; Saito, R Mako

    2014-02-28

    The development and homeostasis of multicellular animals requires precise coordination of cell division and differentiation. We performed a genome-wide RNA interference screen in Caenorhabditis elegans to reveal the components of a regulatory network that promotes developmentally programmed cell-cycle quiescence. The 107 identified genes are predicted to constitute regulatory networks that are conserved among higher animals because almost half of the genes are represented by clear human orthologs. Using a series of mutant backgrounds to assess their genetic activities, the RNA interference clones displaying similar properties were clustered to establish potential regulatory relationships within the network. This approach uncovered four distinct genetic pathways controlling cell-cycle entry during intestinal organogenesis. The enhanced phenotypes observed for animals carrying compound mutations attest to the collaboration between distinct mechanisms to ensure strict developmental regulation of cell cycles. Moreover, we characterized ubc-25, a gene encoding an E2 ubiquitin-conjugating enzyme whose human ortholog, UBE2Q2, is deregulated in several cancers. Our genetic analyses suggested that ubc-25 acts in a linear pathway with cul-1/Cul1, in parallel to pathways employing cki-1/p27 and lin-35/pRb to promote cell-cycle quiescence. Further investigation of the potential regulatory mechanism demonstrated that ubc-25 activity negatively regulates CYE-1/cyclin E protein abundance in vivo. Together, our results show that the ubc-25-mediated pathway acts within a complex network that integrates the actions of multiple molecular mechanisms to control cell cycles during development. Copyright © 2014 Roy et al.

  16. Statistical universals reveal the structures and functions of human music.

    Science.gov (United States)

    Savage, Patrick E; Brown, Steven; Sakai, Emi; Currie, Thomas E

    2015-07-21

    Music has been called "the universal language of mankind." Although contemporary theories of music evolution often invoke various musical universals, the existence of such universals has been disputed for decades and has never been empirically demonstrated. Here we combine a music-classification scheme with statistical analyses, including phylogenetic comparative methods, to examine a well-sampled global set of 304 music recordings. Our analyses reveal no absolute universals but strong support for many statistical universals that are consistent across all nine geographic regions sampled. These universals include 18 musical features that are common individually as well as a network of 10 features that are commonly associated with one another. They span not only features related to pitch and rhythm that are often cited as putative universals but also rarely cited domains including performance style and social context. These cross-cultural structural regularities of human music may relate to roles in facilitating group coordination and cohesion, as exemplified by the universal tendency to sing, play percussion instruments, and dance to simple, repetitive music in groups. Our findings highlight the need for scientists studying music evolution to expand the range of musical cultures and musical features under consideration. The statistical universals we identified represent important candidates for future investigation.

  17. Recombinant methods for screening human DNA excision repair proficiency

    International Nuclear Information System (INIS)

    Athas, W.F.

    1988-01-01

    A method for measuring DNA excision repair in response to ultraviolet radiation (UV)-induced DNA damage has been developed, validated, and field-tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiologic studies seeking to investigate associations between excision repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the belief that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum (XP) primarily is a result of the reduced capacity of patients cells to repair UV-induced DNA damage. For assay, UV-irradiated non-replicating recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (CAT) indicator gene is introduced into lymphocytes using DEAE-dextran short-term transfection conditions. Exposure to UV induces transcriptionally-inactivating DNA photoproducts in the plasmid DNA which inactivate CAT gene expression. Excision repair of the damaged CAT gene is monitored indirectly as a function of reactivated CAT enzyme activity following a 40 hour repair/expression incubation period

  18. Redesign of a computerized clinical reminder for colorectal cancer screening: a human-computer interaction evaluation

    Directory of Open Access Journals (Sweden)

    Saleem Jason J

    2011-11-01

    Full Text Available Abstract Background Based on barriers to the use of computerized clinical decision support (CDS learned in an earlier field study, we prototyped design enhancements to the Veterans Health Administration's (VHA's colorectal cancer (CRC screening clinical reminder to compare against the VHA's current CRC reminder. Methods In a controlled simulation experiment, 12 primary care providers (PCPs used prototypes of the current and redesigned CRC screening reminder in a within-subject comparison. Quantitative measurements were based on a usability survey, workload assessment instrument, and workflow integration survey. We also collected qualitative data on both designs. Results Design enhancements to the VHA's existing CRC screening clinical reminder positively impacted aspects of usability and workflow integration but not workload. The qualitative analysis revealed broad support across participants for the design enhancements with specific suggestions for improving the reminder further. Conclusions This study demonstrates the value of a human-computer interaction evaluation in informing the redesign of information tools to foster uptake, integration into workflow, and use in clinical practice.

  19. Phytochemicals Screening and In Vitro Antibacterial Activity of Elaeis guineensis Leaves Extracts Against Human Pathogenic Bacteria

    International Nuclear Information System (INIS)

    Noorshilawati Abdul Aziz; Umi Nadhirah Halim; Nur Suraya Abdullah

    2015-01-01

    Chloroform and methanol extracts of Elaeis guineensis leaves were investigated for in vitro antibacterial activity against the human pathogenic bacteria Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Four different concentrations of both extracts consists of 50, 100, 200 and 300 mg/ ml were prepared for antibacterial activity using disc diffusion method. The results revealed that chloroform and methonal extract showed high toxicity against all bacterial strain tested. However, both extracts is more effective and exhibit better inhibiting activity against gram positive bacteria, S. aureus compared to gram negative bacteria (E. coli and P. aeruginosa). Methanol extract of Elaeis guineensis leaves shows greater inhibition zone compared to chloroform extract as phyto chemical screening revealed that this extracts contain terpenoids, tannins and saponin. The highest antibacterial activity was exhibited by 300 mg/ ml methanolic extracts against S. aureus which inhibited 10.67 ± 0.33 mm of the diameter zone. Followed by 200 mg/ ml methanolic extracts and 300 mg/ ml chloroform extracts against S. aureus which inhibited 9.17 ± 0.17 mm and 8.33 ± 1.67 mm respectively. This result revealed the potentials of Elaeis guineensis as antibacterial agent in combating infections from human pathogenic bacteria. However, further studies, including identification and purification of the active compounds, will need to be pursued. (author)

  20. BMI and breast cancer prognosis benefit: mammography screening reveals differences between normal weight and overweight women.

    Science.gov (United States)

    Crispo, Anna; Grimaldi, Maria; D'Aiuto, Massimiliano; Rinaldo, Massimo; Capasso, Immacolata; Amore, Alfonso; D'Aiuto, Giuseppe; Giudice, Aldo; Ciliberto, Gennaro; Montella, Maurizio

    2015-02-01

    Few studies are available on the potential impact of body weight on breast cancer prognosis in screen-detected patients. Moreover, it is not known whether body mass index (BMI) could have a different prognostic impact in screen-detected versus symptomatic breast cancer patients. To investigate these unsolved issues, we carried out a retrospective study evaluating the effect of BMI on breast cancer prognosis in screen-detected vs symptomatic breast cancer patients. We conducted a follow-up study on 448 women diagnosed with incident, histologically-confirmed breast cancer. Patients were categorized according to their BMI as normal weight, overweight and obese. Disease free survival (DFS), overall survival (OS), and BMI curves were compared according to mode of cancer detection. Among screen-detected patients, higher BMI was associated with a significant lower DFS, whereas no significant difference was observed among symptomatic patients. OS showed similar results. In the multivariate analysis adjusting for age, education, tumor size, nodal status, estrogen receptor (ER), progesterone receptor (PR) and menopausal status, the risk for high level of BMI among screen-detected patients did not reach the statistical significance for either recurrence or survival. Our study highlights the potential impact of high bodyweight in breast cancer prognosis, the findings confirm that obesity plays a role in women breast cancer prognosis independently from diagnosis mode. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. A Tendon Cell Specific RNAi Screen Reveals Novel Candidates Essential for Muscle Tendon Interaction.

    Directory of Open Access Journals (Sweden)

    Prabhat Tiwari

    Full Text Available Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. In the present study, we used dorsal longitudinal muscles of Drosophila and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. We performed a genetic screen using RNAi-mediated knockdown in tendon cells to find out molecular players involved in the formation and maintenance of myotendinous junction and found 21 candidates out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating our screen. We have found candidates with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV and BM-40 from hemocytes and fat cells.

  2. A Genome-wide multidimensional RNAi screen reveals pathways controlling MHC class II antigen presentation

    NARCIS (Netherlands)

    Paul, Petra; van den Hoorn, Tineke; Jongsma, Marlieke L. M.; Bakker, Mark J.; Hengeveld, Rutger; Janssen, Lennert; Cresswell, Peter; Egan, David A.; van Ham, Marieke; ten Brinke, Anja; Ovaa, Huib; Beijersbergen, Roderick L.; Kuijl, Coenraad; Neefjes, Jacques

    2011-01-01

    MHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and

  3. Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer

    Science.gov (United States)

    Duffy, Supipi; Fam, Hok Khim; Wang, Yi Kan; Styles, Erin B.; Kim, Jung-Hyun; Ang, J. Sidney; Singh, Tejomayee; Larionov, Vladimir; Shah, Sohrab P.; Andrews, Brenda; Boerkoel, Cornelius F.; Hieter, Philip

    2016-01-01

    Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1. Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors. PMID:27551064

  4. A functional microRNA library screen reveals miR-410 as a novel anti-apoptotic regulator of cholangiocarcinoma

    International Nuclear Information System (INIS)

    Palumbo, Tiziana; Poultsides, George A.; Kouraklis, Grigorios; Liakakos, Theodore; Drakaki, Alexandra; Peros, George; Hatziapostolou, Maria; Iliopoulos, Dimitrios

    2016-01-01

    Cholangiocarcinoma is characterized by late diagnosis and a poor survival rate. MicroRNAs have been involved in the pathogenesis of different cancer types, including cholangiocarcinoma. Our aim was to identify novel microRNAs regulating cholangiocarcinoma cell growth in vitro and in vivo. A functional microRNA library screen was performed in human cholangiocarcinoma cells to identify microRNAs that regulate cholangiocarcinoma cell growth. Real-time PCR analysis evaluated miR-9 and XIAP mRNA levels in cholangiocarcinoma cells and tumors. The screen identified 21 microRNAs that regulated >50 % cholangiocarcinoma cell growth. MiR-410 was identified as the top suppressor of growth, while its overexpression significantly inhibited the invasion and colony formation ability of cholangiocarcinoma cells. Bioinformatics analysis revealed that microRNA-410 exerts its effects through the direct regulation of the X-linked inhibitor of apoptosis protein (XIAP). Furthermore, overexpression of miR-410 significantly reduced cholangiocarcinoma tumor growth in a xenograft mouse model through induction of apoptosis. In addition, we identified an inverse relationship between miR-410 and XIAP mRNA levels in human cholangiocarcinomas. Taken together, our study revealed a novel microRNA signaling pathway involved in cholangiocarcinoma and suggests that manipulation of the miR-410/XIAP pathway could have a therapeutic potential for cholangiocarcinoma. The online version of this article (doi:10.1186/s12885-016-2384-0) contains supplementary material, which is available to authorized users

  5. Scaling up cervical cancer screening in the midst of human papillomavirus vaccination advocacy in Thailand

    Directory of Open Access Journals (Sweden)

    Teerawattananon Yot

    2010-07-01

    Full Text Available Abstract Background Screening tests for cervical cancer are effective in reducing the disease burden. In Thailand, a Pap smear program has been implemented throughout the country for 40 years. In 2008 the Ministry of Public Health (MoPH unexpectedly decided to scale up the coverage of free cervical cancer screening services, to meet an ambitious target. This study analyzes the processes and factors that drove this policy innovation in the area of cervical cancer control in Thailand. Methods In-depth interviews with key policy actors and review of relevant documents were conducted in 2009. Data analysis was guided by a framework, developed on public policy models and existing literature on scaling-up health care interventions. Results Between 2006 and 2008 international organizations and the vaccine industry advocated the introduction of Human Papillomavirus (HPV vaccine for the primary prevention of cervical cancer. Meanwhile, a local study suggested that the vaccine was considerably less cost-effective than cervical cancer screening in the Thai context. Then, from August to December 2008, the MoPH carried out a campaign to expand the coverage of its cervical cancer screening program, targeting one million women. The study reveals that several factors were influential in focusing the attention of policymakers on strengthening the screening services. These included the high burden of cervical cancer in Thailand, the launch of the HPV vaccine onto the global and domestic markets, the country’s political instability, and the dissemination of scientific evidence regarding the appropriateness of different options for cervical cancer prevention. Influenced by the country’s political crisis, the MoPH’s campaign was devised in a very short time. In the view of the responsible health officials, the campaign was not successful and indeed, did not achieve its ambitious target. Conclusion The Thai case study suggests that the political crisis was a

  6. Scaling up cervical cancer screening in the midst of human papillomavirus vaccination advocacy in Thailand.

    Science.gov (United States)

    Yothasamut, Jomkwan; Putchong, Choenkwan; Sirisamutr, Teera; Teerawattananon, Yot; Tantivess, Sripen

    2010-07-02

    Screening tests for cervical cancer are effective in reducing the disease burden. In Thailand, a Pap smear program has been implemented throughout the country for 40 years. In 2008 the Ministry of Public Health (MoPH) unexpectedly decided to scale up the coverage of free cervical cancer screening services, to meet an ambitious target. This study analyzes the processes and factors that drove this policy innovation in the area of cervical cancer control in Thailand. In-depth interviews with key policy actors and review of relevant documents were conducted in 2009. Data analysis was guided by a framework, developed on public policy models and existing literature on scaling-up health care interventions. Between 2006 and 2008 international organizations and the vaccine industry advocated the introduction of Human Papillomavirus (HPV) vaccine for the primary prevention of cervical cancer. Meanwhile, a local study suggested that the vaccine was considerably less cost-effective than cervical cancer screening in the Thai context. Then, from August to December 2008, the MoPH carried out a campaign to expand the coverage of its cervical cancer screening program, targeting one million women. The study reveals that several factors were influential in focusing the attention of policymakers on strengthening the screening services. These included the high burden of cervical cancer in Thailand, the launch of the HPV vaccine onto the global and domestic markets, the country's political instability, and the dissemination of scientific evidence regarding the appropriateness of different options for cervical cancer prevention. Influenced by the country's political crisis, the MoPH's campaign was devised in a very short time. In the view of the responsible health officials, the campaign was not successful and indeed, did not achieve its ambitious target. The Thai case study suggests that the political crisis was a crucial factor that drew the attention of policymakers to the cervical

  7. Women's attitude towards routine human platelet antigen-screening in pregnancy.

    Science.gov (United States)

    Winkelhorst, Dian; Loeff, Rosanne M; van den Akker-Van Marle, M Elske; de Haas, Masja; Oepkes, Dick

    2017-08-01

    Fetal and neonatal alloimmune thrombocytopenia is a potentially life-threatening disease with excellent preventative treatment available for subsequent pregnancies. To prevent index cases, the effectiveness of a population-based screening program has been suggested repeatedly. Therefore, we aimed to evaluate women's attitude towards possible future human platelet antigen-screening in pregnancy. We performed a cross-sectional questionnaire study among healthy pregnant women receiving prenatal care in one of seven participating midwifery practices. Attitude was assessed using a questionnaire based on the validated Multidimensional Measurement of Informed Choice model, containing questions assessing knowledge, attitude and intention to participate. A total of 143 of the 220 women (65%) completed and returned the questionnaire. A positive attitude towards human platelet antigen-screening was expressed by 91% of participants, of which 94% was based on sufficient knowledge. Attitude was more likely to be negatively influenced by the opinion that screening can be frightening. Informed choices were made in 87% and occurred significantly less in women from non-European origin, 89% in European women vs. 60% in non-European women (p = 0.03). Pregnant women in the Netherlands expressed a positive attitude towards human platelet antigen-screening in pregnancy. We therefore expect a high rate of informed uptake when human platelet antigen-screening is implemented. In future counseling on human platelet antigen-screening, ethnicity and possible anxiety associated with a screening test need to be specifically addressed. © 2017 Nordic Federation of Societies of Obstetrics and Gynecology.

  8. Screening Bioactives Reveals Nanchangmycin as a Broad Spectrum Antiviral Active against Zika Virus

    OpenAIRE

    Rausch, Keiko; Hackett, Brent A.; Weinbren, Nathan L.; Reeder, Sophia M.; Sadovsky, Yoel; Hunter, Christopher A.; Schultz, David C.; Coyne, Carolyn B.; Cherry, Sara

    2017-01-01

    Zika virus is an emerging arthropod-borne flavivirus for which there are no vaccines or specific therapeutics. We screened a library of 2,000 bioactive compounds for their ability to block Zika virus infection in three distinct cell types with two different strains of Zika virus. Using a microscopy-based assay, we validated 38 drugs that inhibited Zika virus infection, including FDA-approved nucleoside analogs. Cells expressing high levels of the attachment factor AXL can be protected from in...

  9. Visual screening for localized RNAs in yeast revealed novel RNAs at the bud-tip

    International Nuclear Information System (INIS)

    Andoh, Tomoko; Oshiro, Yukiko; Hayashi, Sachiko; Takeo, Hideki; Tani, Tokio

    2006-01-01

    Several RNAs, including rRNAs, snRNAs, snoRNAs, and some mRNAs, are known to be localized at specific sites in a cell. Although methods have been established to visualize RNAs in a living cell, no large-scale visual screening of localized RNAs has been performed. In this study, we constructed a genomic library in which random genomic fragments were inserted downstream of U1A-tag sequences under a GAL1 promoter. In a living yeast cell, transcribed U1A-tagged RNAs were visualized by U1A-GFP that binds the RNA sequence of the U1A-tag. In this screening, many RNAs showed nuclear signals. Since the nuclear signals of some RNAs were not seen when the U1A-tag was connected to the 3' ends of the RNAs, it is suggested that their nuclear signals correspond to nascent transcripts on GAL1 promoter plasmids. Using this screening method, we successfully identified two novel localized mRNAs, CSR2 and DAL81, which showed bud-tip localization

  10. Structure of the human protein kinase MPSK1 reveals an atypical activation loop architecture.

    Science.gov (United States)

    Eswaran, Jeyanthy; Bernad, Antonio; Ligos, Jose M; Guinea, Barbara; Debreczeni, Judit E; Sobott, Frank; Parker, Sirlester A; Najmanovich, Rafael; Turk, Benjamin E; Knapp, Stefan

    2008-01-01

    The activation segment of protein kinases is structurally highly conserved and central to regulation of kinase activation. Here we report an atypical activation segment architecture in human MPSK1 comprising a beta sheet and a large alpha-helical insertion. Sequence comparisons suggested that similar activation segments exist in all members of the MPSK1 family and in MAST kinases. The consequence of this nonclassical activation segment on substrate recognition was studied using peptide library screens that revealed a preferred substrate sequence of X-X-P/V/I-phi-H/Y-T*-N/G-X-X-X (phi is an aliphatic residue). In addition, we identified the GTPase DRG1 as an MPSK1 interaction partner and specific substrate. The interaction domain in DRG1 was mapped to the N terminus, leading to recruitment and phosphorylation at Thr100 within the GTPase domain. The presented data reveal an atypical kinase structural motif and suggest a role of MPSK1 regulating DRG1, a GTPase involved in regulation of cellular growth.

  11. Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G + C-rich attenuating structure

    International Nuclear Information System (INIS)

    Soreq, H.; Ben-Aziz, R.; Prody, C.A.; Seidman, S.; Gnatt, A.; Neville, L.; Lieman-Hurwitz, J.; Lev-Lehman, E.; Ginzberg, D.; Lapidot-Lifson, Y.; Zakut, H.

    1990-01-01

    To study the primary structure of human acetylcholinesterase and its gene expression and amplification, cDNA libraries from human tissues expressing oocyte-translatable AcChoEase mRNA were constructed and screened with labeled oligodeoxynucleotide probes. Several cDNA clones were isolated that encoded a polypeptide with ≥50% identically aligned amino acids to Torpedo AcChoEase and human butyrylcholinesterase. However, these cDNA clones were all truncated within a 300-nucleotide-long G + C-rich region with a predicted pattern of secondary structure having a high Gibbs free energy downstream from the expected 5' end of the coding region. Screening of a genomic DNA library revealed the missing 5' domain. When ligated to the cDNA and constructed into a transcription vector, this sequence encoded a synthetic mRNA translated in microinjected oocytes into catalytically active AcChoEase with marked preference for acetylthiocholine over butyrylthiocholine as a substrate, susceptibility to inhibition by the AcChoEase inhibitor BW284C51, and resistance to the AcChoEase inhibitor tetraisopropylpyrophosphoramide. Blot hybridization of genomic DNA from different individuals carrying amplified AcChoEase genes revealed variable intensities and restriction patterns with probes from the regions upstream and downstream from the predicted G + C-rich structure. Thus, the human AcChoEase gene includes a putative G + C-rich attenuator domain and is subject to structural alterations in cases of AcChoEase gene amplification

  12. Self-Sampling for Human Papillomavirus Testing: Increased Cervical Cancer Screening Participation and Incorporation in International Screening Programs

    Science.gov (United States)

    Gupta, Sarah; Palmer, Christina; Bik, Elisabeth M.; Cardenas, Juan P.; Nuñez, Harold; Kraal, Laurens; Bird, Sara W.; Bowers, Jennie; Smith, Alison; Walton, Nathaniel A.; Goddard, Audrey D.; Almonacid, Daniel E.; Zneimer, Susan; Richman, Jessica; Apte, Zachary S.

    2018-01-01

    In most industrialized countries, screening programs for cervical cancer have shifted from cytology (Pap smear or ThinPrep) alone on clinician-obtained samples to the addition of screening for human papillomavirus (HPV), its main causative agent. For HPV testing, self-sampling instead of clinician-sampling has proven to be equally accurate, in particular for assays that use nucleic acid amplification techniques. In addition, HPV testing of self-collected samples in combination with a follow-up Pap smear in case of a positive result is more effective in detecting precancerous lesions than a Pap smear alone. Self-sampling for HPV testing has already been adopted by some countries, while others have started trials to evaluate its incorporation into national cervical cancer screening programs. Self-sampling may result in more individuals willing to participate in cervical cancer screening, because it removes many of the barriers that prevent women, especially those in low socioeconomic and minority populations, from participating in regular screening programs. Several studies have shown that the majority of women who have been underscreened but who tested HPV-positive in a self-obtained sample will visit a clinic for follow-up diagnosis and management. In addition, a self-collected sample can also be used for vaginal microbiome analysis, which can provide additional information about HPV infection persistence as well as vaginal health in general. PMID:29686981

  13. A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables.

    Science.gov (United States)

    Wang, Yuanze; van Oosterwijk, Niels; Ali, Ameena M; Adawy, Alaa; Anindya, Atsarina L; Dömling, Alexander S S; Groves, Matthew R

    2017-08-24

    Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which "helper" molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the "helper" molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.

  14. The human oral metaproteome reveals potential biomarkers for caries disease

    DEFF Research Database (Denmark)

    Belda-Ferre, Pedro; Williamson, James; Simón-Soro, Áurea

    2015-01-01

    metabolism and immune response. We applied multivariate analysis in order to find the minimum set of proteins that better allows discrimination of healthy and caries-affected dental plaque samples, detecting seven bacterial and five human protein functions that allow determining the health status......Tooth decay is considered the most prevalent human disease worldwide. We present the first metaproteomic study of the oral biofilm, using different mass spectrometry approaches that have allowed us to quantify individual peptides in healthy and caries-bearing individuals. A total of 7771 bacterial...... and 853 human proteins were identified in 17 individuals, which provide the first available protein repertoire of human dental plaque. Actinomyces and Coryneybacterium represent a large proportion of the protein activity followed by Rothia and Streptococcus. Those four genera account for 60-90% of total...

  15. Human papillomavirus self-sampling for screening nonattenders

    DEFF Research Database (Denmark)

    Lam, Janni Uyen Hoa; Rebolj, Matejka; Ejegod, Ditte Møller

    2017-01-01

    was well-accepted among nonattenders. Adopting modern technology-based platforms into the current organized screening program would serve as a convenient communication method between health authority and citizens, allowing easy access for the citizen and reducing the work load in administrating self-sampling......In organized cervical screening programs, typically 25% of the invited women do not attend. The Copenhagen Self-sampling Initiative (CSi) aimed to gain experiences on participation among screening nonattenders in the Capital Region of Denmark. Here, we report on the effectiveness of different...... communication platforms used in the pilot with suggestions for strategies prior to a full-implementation. Moreover, an innovative approach using self-sampling brushes with unique radio frequency identification chips allowed for unprecedented levels patient identification safety. Nonattenders from the capital...

  16. A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction

    DEFF Research Database (Denmark)

    Bowl, Michael R.; Simon, Michelle M.; Ingham, Neil J.

    2017-01-01

    The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. O...

  17. Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

    Science.gov (United States)

    Takayama, Kazuo; Mizuguchi, Hiroyuki

    2017-02-01

    Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  18. Screening the budding yeast genome reveals unique factors affecting K2 toxin susceptibility.

    Science.gov (United States)

    Servienė, Elena; Lukša, Juliana; Orentaitė, Irma; Lafontaine, Denis L J; Urbonavičius, Jaunius

    2012-01-01

    Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.

  19. Revealing molecular mechanisms by integrating high-dimensional functional screens with protein interaction data.

    Directory of Open Access Journals (Sweden)

    Angela Simeone

    2014-09-01

    Full Text Available Functional genomics screens using multi-parametric assays are powerful approaches for identifying genes involved in particular cellular processes. However, they suffer from problems like noise, and often provide little insight into molecular mechanisms. A bottleneck for addressing these issues is the lack of computational methods for the systematic integration of multi-parametric phenotypic datasets with molecular interactions. Here, we present Integrative Multi Profile Analysis of Cellular Traits (IMPACT. The main goal of IMPACT is to identify the most consistent phenotypic profile among interacting genes. This approach utilizes two types of external information: sets of related genes (IMPACT-sets and network information (IMPACT-modules. Based on the notion that interacting genes are more likely to be involved in similar functions than non-interacting genes, this data is used as a prior to inform the filtering of phenotypic profiles that are similar among interacting genes. IMPACT-sets selects the most frequent profile among a set of related genes. IMPACT-modules identifies sub-networks containing genes with similar phenotype profiles. The statistical significance of these selections is subsequently quantified via permutations of the data. IMPACT (1 handles multiple profiles per gene, (2 rescues genes with weak phenotypes and (3 accounts for multiple biases e.g. caused by the network topology. Application to a genome-wide RNAi screen on endocytosis showed that IMPACT improved the recovery of known endocytosis-related genes, decreased off-target effects, and detected consistent phenotypes. Those findings were confirmed by rescreening 468 genes. Additionally we validated an unexpected influence of the IGF-receptor on EGF-endocytosis. IMPACT facilitates the selection of high-quality phenotypic profiles using different types of independent information, thereby supporting the molecular interpretation of functional screens.

  20. Species Richness and Diversity Reveal that Human-Modified ...

    African Journals Online (AJOL)

    Family diversity and richness showed no significant differences across the sites. The spider species consisted of primarily three functional groups: ground wanderers, web builders and plant wanderers, and showed no within-group differences in abundance between sites. Similarity index between the study sites revealed a ...

  1. A genetic screen reveals a periplasmic copper chaperone required for nitrite reductase activity in pathogenic Neisseria.

    Science.gov (United States)

    Jen, Freda E-C; Djoko, Karrera Y; Bent, Stephen J; Day, Christopher J; McEwan, Alastair G; Jennings, Michael P

    2015-09-01

    Under conditions of low oxygen availability, Neisseria meningitidis and Neisseria gonorrhoeae are able to respire via a partial denitrification pathway in which nitrite is converted to nitrous oxide. In this process, nitrite reductase (AniA), a copper (Cu)-containing protein converts nitrite to NO, and this product is converted to nitrous oxide by nitric oxide reductase (NorB). NorB also confers protection against toxic NO, and so we devised a conditional lethal screen, using a norB mutant, to identify mutants that were resistant to nitrite-dependent killing. After random-deletion mutagenesis of N. meningitidis, this genetic screen identified a gene encoding a Cu chaperone that is essential for AniA function, AccA. Purified AccA binds one Cu (I) ion and also possesses a second binding site for Cu (II). This novel periplasmic Cu chaperone (AccA) appears to be essential for provision of Cu ions to AniA of pathogenic Neisseria to generate an active nitrite reductase. Apart from the Neisseria genus, AccA is distributed across a wide range of environmental Proteobacteria species. © FASEB.

  2. Screening in larval zebrafish reveals tissue-specific distribution of fifteen fluorescent compounds

    Directory of Open Access Journals (Sweden)

    Yuxiao Yao

    2017-09-01

    Full Text Available The zebrafish is a prominent vertebrate model for low-cost in vivo whole organism screening. In our recent screening of the distribution patterns of fluorescent compounds in live zebrafish larvae, fifteen compounds with tissue-specific distributions were identified. Several compounds were observed to accumulate in tissues where they were reported to induce side-effects, and compounds with similar structures tended to be enriched in the same tissues, with minor differences. In particular, we found three novel red fluorescent bone-staining dyes: purpurin, lucidin and 3-hydroxy-morindone; purpurin can effectively label bones in both larval and adult zebrafish, as well as in postnatal mice, without significantly affecting bone mass and density. Moreover, two structurally similar chemotherapeutic compounds, doxorubicin and epirubicin, were observed to have distinct distribution preferences in zebrafish. Epirubicin maintained a relatively higher concentration in the liver, and performed better in inhibiting hepatic hyperplasia caused by the over-expression of krasG12V. In total, our study suggests that the transparent zebrafish larvae serve as valuable tools for identifying tissue-specific distributions of fluorescent compounds.

  3. A Cell-Based Screen Reveals that the Albendazole Metabolite, Albendazole Sulfone, Targets Wolbachia

    Science.gov (United States)

    Bray, Walter M.; White, Pamela M.; Ruybal, Jordan; Lokey, R. Scott; Debec, Alain; Sullivan, William

    2012-01-01

    Wolbachia endosymbionts carried by filarial nematodes give rise to the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. Here we identify new Wolbachia-disrupting compounds by conducting high-throughput cell-based chemical screens using a Wolbachia-infected, fluorescently labeled Drosophila cell line. This screen yielded several Wolbachia-disrupting compounds including three that resembled Albendazole, a widely used anthelmintic drug that targets nematode microtubules. Follow-up studies demonstrate that a common Albendazole metabolite, Albendazole sulfone, reduces intracellular Wolbachia titer both in Drosophila melanogaster and Brugia malayi, the nematode responsible for lymphatic filariasis. Significantly, Albendazole sulfone does not disrupt Drosophila microtubule organization, suggesting that this compound reduces titer through direct targeting of Wolbachia. Accordingly, both DNA staining and FtsZ immunofluorescence demonstrates that Albendazole sulfone treatment induces Wolbachia elongation, a phenotype indicative of binary fission defects. This suggests that the efficacy of Albendazole in treating filarial nematode-based diseases is attributable to dual targeting of nematode microtubules and their Wolbachia endosymbionts. PMID:23028321

  4. Transcriptome sequencing from diverse human populations reveals differentiated regulatory architecture.

    Directory of Open Access Journals (Sweden)

    Alicia R Martin

    2014-08-01

    Full Text Available Large-scale sequencing efforts have documented extensive genetic variation within the human genome. However, our understanding of the origins, global distribution, and functional consequences of this variation is far from complete. While regulatory variation influencing gene expression has been studied within a handful of populations, the breadth of transcriptome differences across diverse human populations has not been systematically analyzed. To better understand the spectrum of gene expression variation, alternative splicing, and the population genetics of regulatory variation in humans, we have sequenced the genomes, exomes, and transcriptomes of EBV transformed lymphoblastoid cell lines derived from 45 individuals in the Human Genome Diversity Panel (HGDP. The populations sampled span the geographic breadth of human migration history and include Namibian San, Mbuti Pygmies of the Democratic Republic of Congo, Algerian Mozabites, Pathan of Pakistan, Cambodians of East Asia, Yakut of Siberia, and Mayans of Mexico. We discover that approximately 25.0% of the variation in gene expression found amongst individuals can be attributed to population differences. However, we find few genes that are systematically differentially expressed among populations. Of this population-specific variation, 75.5% is due to expression rather than splicing variability, and we find few genes with strong evidence for differential splicing across populations. Allelic expression analyses indicate that previously mapped common regulatory variants identified in eight populations from the International Haplotype Map Phase 3 project have similar effects in our seven sampled HGDP populations, suggesting that the cellular effects of common variants are shared across diverse populations. Together, these results provide a resource for studies analyzing functional differences across populations by estimating the degree of shared gene expression, alternative splicing, and

  5. High-throughput screening reveals alsterpaullone, 2-cyanoethyl as a potent p27Kip1 transcriptional inhibitor.

    Directory of Open Access Journals (Sweden)

    Brandon J Walters

    Full Text Available p27Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27Kip1 promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a "bioactive" library consisting of 8,904 (4,359 unique compounds, of which 830 are Food and Drug Administration (FDA approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of p27Kip1. These hits were further refined using a battery of secondary screens. Here we report four novel p27Kip1 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM Alsterpaullone 2-cyanoethyl, inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies.

  6. Protein crystallization with microseed matrix screening: application to human germline antibody Fabs

    International Nuclear Information System (INIS)

    Obmolova, Galina; Malia, Thomas J.; Teplyakov, Alexey; Sweet, Raymond W.; Gilliland, Gary L.

    2014-01-01

    The power of microseed matrix screening is demonstrated in the crystallization of a panel of antibody Fab fragments. The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization

  7. An Aboriginal Australian genome reveals separate human dispersals into Asia.

    Science.gov (United States)

    Rasmussen, Morten; Guo, Xiaosen; Wang, Yong; Lohmueller, Kirk E; Rasmussen, Simon; Albrechtsen, Anders; Skotte, Line; Lindgreen, Stinus; Metspalu, Mait; Jombart, Thibaut; Kivisild, Toomas; Zhai, Weiwei; Eriksson, Anders; Manica, Andrea; Orlando, Ludovic; De La Vega, Francisco M; Tridico, Silvana; Metspalu, Ene; Nielsen, Kasper; Ávila-Arcos, María C; Moreno-Mayar, J Víctor; Muller, Craig; Dortch, Joe; Gilbert, M Thomas P; Lund, Ole; Wesolowska, Agata; Karmin, Monika; Weinert, Lucy A; Wang, Bo; Li, Jun; Tai, Shuaishuai; Xiao, Fei; Hanihara, Tsunehiko; van Driem, George; Jha, Aashish R; Ricaut, François-Xavier; de Knijff, Peter; Migliano, Andrea B; Gallego Romero, Irene; Kristiansen, Karsten; Lambert, David M; Brunak, Søren; Forster, Peter; Brinkmann, Bernd; Nehlich, Olaf; Bunce, Michael; Richards, Michael; Gupta, Ramneek; Bustamante, Carlos D; Krogh, Anders; Foley, Robert A; Lahr, Marta M; Balloux, Francois; Sicheritz-Pontén, Thomas; Villems, Richard; Nielsen, Rasmus; Wang, Jun; Willerslev, Eske

    2011-10-07

    We present an Aboriginal Australian genomic sequence obtained from a 100-year-old lock of hair donated by an Aboriginal man from southern Western Australia in the early 20th century. We detect no evidence of European admixture and estimate contamination levels to be below 0.5%. We show that Aboriginal Australians are descendants of an early human dispersal into eastern Asia, possibly 62,000 to 75,000 years ago. This dispersal is separate from the one that gave rise to modern Asians 25,000 to 38,000 years ago. We also find evidence of gene flow between populations of the two dispersal waves prior to the divergence of Native Americans from modern Asian ancestors. Our findings support the hypothesis that present-day Aboriginal Australians descend from the earliest humans to occupy Australia, likely representing one of the oldest continuous populations outside Africa.

  8. Knowing beans: Human mirror mechanisms revealed through motor adaptation

    Directory of Open Access Journals (Sweden)

    Arthur M Glenberg

    2010-11-01

    Full Text Available Human mirror mechanisms (MMs respond during both performed and observed action and appear to underlie action goal recognition. We introduce a behavioral procedure for discovering and clarifying functional MM properties: Blindfolded participants repeatedly move beans either toward or away from themselves to induce motor adaptation. Then, the bias for perceiving direction of ambiguous visual movement in depth is measured. Bias is affected by a number of beans moved, b movement direction, and c similarity of the visual stimulus to the hand used to move beans. This cross-modal adaptation pattern supports both the validity of human MMs and functionality of our testing instrument. We also discuss related work that extends the motor adaptation paradigm to investigate contributions of MMs to speech perception and language comprehension.

  9. Sequencing of 50 human exomes reveals adaptation to high altitude

    DEFF Research Database (Denmark)

    Yi, Xin; Liang, Yu; Huerta-Sanchez, Emilia

    2010-01-01

    Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which repres...... in genetic adaptation to high altitude.......Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which...... represent strong candidates for altitude adaptation, were identified. The strongest signal of natural selection came from endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1), a transcription factor involved in response to hypoxia. One single-nucleotide polymorphism (SNP) at EPAS1 shows a 78% frequency...

  10. Screening natural libraries of human milk oligosaccharides against lectins using CaR-ESI-MS.

    Science.gov (United States)

    El-Hawiet, Amr; Chen, Yajie; Shams-Ud-Doha, Km; Kitova, Elena N; Kitov, Pavel I; Bode, Lars; Hage, Naim; Falcone, Franco H; Klassen, John S

    2018-01-15

    Human milk oligosaccharides (HMOs) afford many health benefits to breast-fed infants, such as protection against infection and regulation of the immune system, through the formation of non-covalent interactions with protein receptors. However, the molecular details of these interactions are poorly understood. Here, we describe the application of catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) for screening natural libraries of HMOs against lectins. The HMOs in the libraries were first identified based on molecular weights (MWs), ion mobility separation arrival times (IMS-ATs) and collision-induced dissociation (CID) fingerprints of their deprotonated anions. The libraries were then screened against lectins and the ligands identified from the MWs, IMS-ATs and CID fingerprints of HMOs released from the lectin in the gas phase. To demonstrate the assay, four fractions, extracted from pooled human milk and containing ≥35 different HMOs, were screened against a C-terminal fragment of human galectin-3 (hGal-3C), for which the HMOs specificities have been previously investigated, and a fragment of the blood group antigen-binding adhesin (BabA) from Helicobacter pylori, for which the HMO specificities have not been previously established. The structures of twenty-one ligands, corresponding to both neutral and acidic HMOs, of hGal-3C were identified; all twenty-one were previously shown to be ligands for this lectin. The presence of HMO ligands at six other MWs was also ascertained. Application of the assay to BabA revealed nineteen specific HMO structures that are recognized by the protein and HMO ligands at two other MWs. Notably, it was found that BabA exhibits broad specificity for HMOs, and recognizes both neutral HMOs, including non-fucosylated ones, and acidic HMOs. The results of competitive binding experiments indicate that HMOs can interact with BabA at previously unknown binding sites. The affinities of eight purified HMOs for BabA were

  11. Eye movements reveal epistemic curiosity in human observers.

    Science.gov (United States)

    Baranes, Adrien; Oudeyer, Pierre-Yves; Gottlieb, Jacqueline

    2015-12-01

    Saccadic (rapid) eye movements are primary means by which humans and non-human primates sample visual information. However, while saccadic decisions are intensively investigated in instrumental contexts where saccades guide subsequent actions, it is largely unknown how they may be influenced by curiosity - the intrinsic desire to learn. While saccades are sensitive to visual novelty and visual surprise, no study has examined their relation to epistemic curiosity - interest in symbolic, semantic information. To investigate this question, we tracked the eye movements of human observers while they read trivia questions and, after a brief delay, were visually given the answer. We show that higher curiosity was associated with earlier anticipatory orienting of gaze toward the answer location without changes in other metrics of saccades or fixations, and that these influences were distinct from those produced by variations in confidence and surprise. Across subjects, the enhancement of anticipatory gaze was correlated with measures of trait curiosity from personality questionnaires. Finally, a machine learning algorithm could predict curiosity in a cross-subject manner, relying primarily on statistical features of the gaze position before the answer onset and independently of covariations in confidence or surprise, suggesting potential practical applications for educational technologies, recommender systems and research in cognitive sciences. With this article, we provide full access to the annotated database allowing readers to reproduce the results. Epistemic curiosity produces specific effects on oculomotor anticipation that can be used to read out curiosity states. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Chromosomal clustering of a human transcriptome reveals regulatory background

    Directory of Open Access Journals (Sweden)

    Purmann Antje

    2005-09-01

    Full Text Available Abstract Background There has been much evidence recently for a link between transcriptional regulation and chromosomal gene order, but the relationship between genomic organization, regulation and gene function in higher eukaryotes remains to be precisely defined. Results Here, we present evidence for organization of a large proportion of a human transcriptome into gene clusters throughout the genome, which are partly regulated by the same transcription factors, share biological functions and are characterized by non-housekeeping genes. This analysis was based on the cardiac transcriptome identified by our genome-wide array analysis of 55 human heart samples. We found 37% of these genes to be arranged mainly in adjacent pairs or triplets. A significant number of pairs of adjacent genes are putatively regulated by common transcription factors (p = 0.02. Furthermore, these gene pairs share a significant number of GO functional classification terms. We show that the human cardiac transcriptome is organized into many small clusters across the whole genome, rather than being concentrated in a few larger clusters. Conclusion Our findings suggest that genes expressed in concert are organized in a linear arrangement for coordinated regulation. Determining the relationship between gene arrangement, regulation and nuclear organization as well as gene function will have broad biological implications.

  13. Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches.

    Science.gov (United States)

    Richter, Lilian H J; Kaminski, Yeda Rumi; Noor, Fozia; Meyer, Markus R; Maurer, Hans H

    2016-09-01

    Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans.

  14. High-Throughput Screening Enhances Kidney Organoid Differentiation from Human Pluripotent Stem Cells and Enables Automated Multidimensional Phenotyping.

    Science.gov (United States)

    Czerniecki, Stefan M; Cruz, Nelly M; Harder, Jennifer L; Menon, Rajasree; Annis, James; Otto, Edgar A; Gulieva, Ramila E; Islas, Laura V; Kim, Yong Kyun; Tran, Linh M; Martins, Timothy J; Pippin, Jeffrey W; Fu, Hongxia; Kretzler, Matthias; Shankland, Stuart J; Himmelfarb, Jonathan; Moon, Randall T; Paragas, Neal; Freedman, Benjamin S

    2018-05-15

    Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. Here, we present a fully automated, HTS-compatible platform for enhanced differentiation and phenotyping of human kidney organoids. The entire 21-day protocol, from plating to differentiation to analysis, can be performed automatically by liquid-handling robots, or alternatively by manual pipetting. High-content imaging analysis reveals both dose-dependent and threshold effects during organoid differentiation. Immunofluorescence and single-cell RNA sequencing identify previously undetected parietal, interstitial, and partially differentiated compartments within organoids and define conditions that greatly expand the vascular endothelium. Chemical modulation of toxicity and disease phenotypes can be quantified for safety and efficacy prediction. Screening in gene-edited organoids in this system reveals an unexpected role for myosin in polycystic kidney disease. Organoids in HTS formats thus establish an attractive platform for multidimensional phenotypic screening. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. An Aboriginal Australian Genome Reveals Separate Human Dispersals into Asia

    OpenAIRE

    Rasmussen, Morten; Guo, Xiaosen; Wang, Yong; Lohmueller, Kirk E.; Rasmussen, Simon; Albrechtsen, Anders; Skotte, Line; Lindgreen, Stinus; Metspalu, Mait; Jombart, Thibaut; Kivisild, Toomas; Zhai, Weiwei; Eriksson, Anders; Manica, Andrea; Orlando, Ludovic

    2011-01-01

    We present an Aboriginal Australian genomic sequence obtained from a 100-year-old lock of hair donated by an Aboriginal man from southern Western Australia in the early 20th century. We detect no evidence of European admixture and estimate contamination levels to be below 0.5%. We show that Aboriginal Australians are descendants of an early human dispersal into eastern Asia, possibly 62,000 to 75,000 years ago. This dispersal is separate from the one that gave rise to modern Asians 25,000 to ...

  16. Screening Bioactives Reveals Nanchangmycin as a Broad Spectrum Antiviral Active against Zika Virus

    Directory of Open Access Journals (Sweden)

    Keiko Rausch

    2017-01-01

    Full Text Available Zika virus is an emerging arthropod-borne flavivirus for which there are no vaccines or specific therapeutics. We screened a library of 2,000 bioactive compounds for their ability to block Zika virus infection in three distinct cell types with two different strains of Zika virus. Using a microscopy-based assay, we validated 38 drugs that inhibited Zika virus infection, including FDA-approved nucleoside analogs. Cells expressing high levels of the attachment factor AXL can be protected from infection with receptor tyrosine kinase inhibitors, while placental-derived cells that lack AXL expression are insensitive to this inhibition. Importantly, we identified nanchangmycin as a potent inhibitor of Zika virus entry across all cell types tested, including physiologically relevant primary cells. Nanchangmycin also was active against other medically relevant viruses, including West Nile, dengue, and chikungunya viruses that use a similar route of entry. This study provides a resource of small molecules to study Zika virus pathogenesis.

  17. Population Screening Using Sewage Reveals Pan-Resistant Bacteria in Hospital and Community Samples.

    Science.gov (United States)

    Meir-Gruber, Lital; Manor, Yossi; Gefen-Halevi, Shiraz; Hindiyeh, Musa Y; Mileguir, Fernando; Azar, Roberto; Smollan, Gill; Belausov, Natasha; Rahav, Galia; Shamiss, Ari; Mendelson, Ella; Keller, Nathan

    2016-01-01

    The presence of pan-resistant bacteria worldwide possesses a threat to global health. It is difficult to evaluate the extent of carriage of resistant bacteria in the population. Sewage sampling is a possible way to monitor populations. We evaluated the presence of pan-resistant bacteria in Israeli sewage collected from all over Israel, by modifying the pour plate method for heterotrophic plate count technique using commercial selective agar plates. This method enables convenient and fast sewage sampling and detection. We found that sewage in Israel contains multiple pan-resistant bacteria including carbapenemase resistant Enterobacteriacae carrying blaKPC and blaNDM-1, MRSA and VRE. blaKPC carrying Klebsiella pneumonia and Enterobacter cloacae were the most common Enterobacteriacae drug resistant bacteria found in the sewage locations we sampled. Klebsiella pneumonia, Enterobacter spp., Escherichia coli and Citrobacter spp. were the 4 main CRE isolated from Israeli sewage and also from clinical samples in our clinical microbiology laboratory. Hospitals and Community sewage had similar percentage of positive samplings for blaKPC and blaNDM-1. VRE was found to be more abundant in sewage in Israel than MRSA but there were more locations positive for MRSA and VRE bacteria in Hospital sewage than in the Community. Therefore, our upgrade of the pour plate method for heterotrophic plate count technique using commercial selective agar plates can be a useful tool for routine screening and monitoring of the population for pan-resistant bacteria using sewage.

  18. Lipidomics reveals a remarkable diversity of lipids in human plasma.

    Science.gov (United States)

    Quehenberger, Oswald; Armando, Aaron M; Brown, Alex H; Milne, Stephen B; Myers, David S; Merrill, Alfred H; Bandyopadhyay, Sibali; Jones, Kristin N; Kelly, Samuel; Shaner, Rebecca L; Sullards, Cameron M; Wang, Elaine; Murphy, Robert C; Barkley, Robert M; Leiker, Thomas J; Raetz, Christian R H; Guan, Ziqiang; Laird, Gregory M; Six, David A; Russell, David W; McDonald, Jeffrey G; Subramaniam, Shankar; Fahy, Eoin; Dennis, Edward A

    2010-11-01

    The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patient's blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules.

  19. An Aboriginal Australian Genome Reveals Separate Human Dispersals into Asia

    DEFF Research Database (Denmark)

    Rasmussen, Morten; Guo, Xiaosen; Wang, Yong

    2011-01-01

    We present an Aboriginal Australian genomic sequence obtained from a 100-year-old lock of hair donated by an Aboriginal man from southern Western Australia in the early 20th century. We detect no evidence of European admixture and estimate contamination levels to be below 0.5%. We show that Abori......We present an Aboriginal Australian genomic sequence obtained from a 100-year-old lock of hair donated by an Aboriginal man from southern Western Australia in the early 20th century. We detect no evidence of European admixture and estimate contamination levels to be below 0.5%. We show...... that Aboriginal Australians are descendants of an early human dispersal into eastern Asia, possibly 62,000 to 75,000 years ago. This dispersal is separate from the one that gave rise to modern Asians 25,000 to 38,000 years ago. We also find evidence of gene flow between populations of the two dispersal waves...... prior to the divergence of Native Americans from modern Asian ancestors. Our findings support the hypothesis that present-day Aboriginal Australians descend from the earliest humans to occupy Australia, likely representing one of the oldest continuous populations outside Africa....

  20. Three-cohort targeted gene screening reveals a non-synonymous TRKA polymorphism associated with schizophrenia

    DEFF Research Database (Denmark)

    van Schijndel, Jessica E; van Loo, Karen M J; van Zweeden, Martine

    2009-01-01

    selected non-synonymous single-nucleotide polymorphisms (SNPs) in three independent Caucasian schizophrenia case-control cohorts (USA, Denmark and Norway). A meta-analysis revealed ten non-synonymous SNPs that were nominally associated with schizophrenia, nine of which have not been previously linked...... attractive candidate for further study concerns SNP rs6336 (q=0.12) that causes the substitution of an evolutionarily highly conserved amino acid residue in the kinase domain of the neurodevelopmentally important receptor TRKA. Thus, TRKA signaling may represent a novel susceptibility pathway...

  1. Functional genomic screening reveals asparagine dependence as a metabolic vulnerability in sarcoma

    OpenAIRE

    Hettmer, Simone; Schinzel, Anna C; Tchessalova, Daria; Schneider, Michaela; Parker, Christina L; Bronson, Roderick T; Richards, Nigel GJ; Hahn, William C; Wagers, Amy J

    2015-01-01

    eLife digest Sarcoma is a type of cancer that forms in the connective tissues of the body, such as bone, cartilage, muscle and fat. Usually, treatment involves surgical removal of the tumor and/or radiation to kill the tumor cells. However, if sarcomas spread to other parts of the body, the treatment options are limited. Genetic studies have revealed several genetic changes that contribute to the formation of sarcomas. Many sarcomas have a mutation in a gene that encodes a protein called Ras....

  2. Spatial interactions reveal inhibitory cortical networks in human amblyopia.

    Science.gov (United States)

    Wong, Erwin H; Levi, Dennis M; McGraw, Paul V

    2005-10-01

    Humans with amblyopia have a well-documented loss of sensitivity for first-order, or luminance defined, visual information. Recent studies show that they also display a specific loss of sensitivity for second-order, or contrast defined, visual information; a type of image structure encoded by neurons found predominantly in visual area A18/V2. In the present study, we investigate whether amblyopia disrupts the normal architecture of spatial interactions in V2 by determining the contrast detection threshold of a second-order target in the presence of second-order flanking stimuli. Adjacent flanks facilitated second-order detectability in normal observers. However, in marked contrast, they suppressed detection in each eye of the majority of amblyopic observers. Furthermore, strabismic observers with no loss of visual acuity show a similar pattern of detection suppression. We speculate that amblyopia results in predominantly inhibitory cortical interactions between second-order neurons.

  3. Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells

    Directory of Open Access Journals (Sweden)

    Chad M. Toledo

    2015-12-01

    Full Text Available To identify therapeutic targets for glioblastoma (GBM, we performed genome-wide CRISPR-Cas9 knockout (KO screens in patient-derived GBM stem-like cells (GSCs and human neural stem/progenitors (NSCs, non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers. In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.

  4. Screening of a long-term sample set reveals two Ranavirus lineages in British herpetofauna.

    Directory of Open Access Journals (Sweden)

    Stephen J Price

    Full Text Available Reports of severe disease outbreaks in amphibian communities in mainland Europe due to strains of the common midwife toad virus (CMTV-like clade of Ranavirus are increasing and have created concern due to their considerable population impacts. In Great Britain, viruses in another clade of Ranavirus-frog virus 3 (FV3-like-have caused marked declines of common frog (Rana temporaria populations following likely recent virus introductions. The British public has been reporting mortality incidents to a citizen science project since 1992, with carcasses submitted for post-mortem examination, resulting in a long-term tissue archive spanning 25 years. We screened this archive for ranavirus (458 individuals from 228 incidents using molecular methods and undertook preliminary genotyping of the ranaviruses detected. In total, ranavirus was detected in 90 individuals from 41 incidents focused in the north and south of England. The majority of detections involved common frogs (90% but also another anuran, a caudate and a reptile. Most incidents were associated with FV3-like viruses but two, separated by 300 km and 16 years, involved CMTV-like viruses. These British CMTV-like viruses were more closely related to ranaviruses from mainland Europe than to each other and were estimated to have diverged at least 458 years ago. This evidence of a CMTV-like virus in Great Britain in 1995 represents the earliest confirmed case of a CMTV associated with amphibians and raises important questions about the history of ranavirus in Great Britain and the epidemiology of CMTV-like viruses. Despite biases present in the opportunistic sample used, this study also demonstrates the role of citizen science projects in generating resources for research and the value of maintaining long-term wildlife tissue archives.

  5. Human Vascular Microphysiological System for in vitro Drug Screening.

    Science.gov (United States)

    Fernandez, C E; Yen, R W; Perez, S M; Bedell, H W; Povsic, T J; Reichert, W M; Truskey, G A

    2016-02-18

    In vitro human tissue engineered human blood vessels (TEBV) that exhibit vasoactivity can be used to test human toxicity of pharmaceutical drug candidates prior to pre-clinical animal studies. TEBVs with 400-800 μM diameters were made by embedding human neonatal dermal fibroblasts or human bone marrow-derived mesenchymal stem cells in dense collagen gel. TEBVs were mechanically strong enough to allow endothelialization and perfusion at physiological shear stresses within 3 hours after fabrication. After 1 week of perfusion, TEBVs exhibited endothelial release of nitric oxide, phenylephrine-induced vasoconstriction, and acetylcholine-induced vasodilation, all of which were maintained up to 5 weeks in culture. Vasodilation was blocked with the addition of the nitric oxide synthase inhibitor L-N(G)-Nitroarginine methyl ester (L-NAME). TEBVs elicited reversible activation to acute inflammatory stimulation by TNF-α which had a transient effect upon acetylcholine-induced relaxation, and exhibited dose-dependent vasodilation in response to caffeine and theophylline. Treatment of TEBVs with 1 μM lovastatin for three days prior to addition of Tumor necrosis factor - α (TNF-α) blocked the injury response and maintained vasodilation. These results indicate the potential to develop a rapidly-producible, endothelialized TEBV for microphysiological systems capable of producing physiological responses to both pharmaceutical and immunological stimuli.

  6. The problem of false-positive human papillomavirus DNA tests in cervical screening

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Pribac, Igor; Frederiksen, Maria Eiholm

    2013-01-01

    Human Papillomavirus (HPV) testing has been extensively studied in randomized controlled trials of primary cervical screening. Based on encouraging results concerning its high detection rates and a high negative predictive value for high-grade cervical intraepithelial neoplasia (CIN), HPV testing...... will probably replace cytology in future primary cervical screening. However, HPV testing is associated with more frequent false-positive tests compared to cytology. False-positive tests are defined as positive screening tests which are not subsequently confirmed with high-grade CIN. Several authors have...

  7. Cervical human papilloma virus (HPV) DNA primary screening test: Results of a population-based screening programme in central Italy.

    Science.gov (United States)

    Passamonti, Basilio; Gustinucci, Daniela; Giorgi Rossi, Paolo; Cesarini, Elena; Bulletti, Simonetta; Carlani, Angela; Martinelli, Nadia; Broccolini, Massimo; D'Angelo, Valentina; D'Amico, Maria Rosaria; Di Dato, Eugenio; Galeazzi, Paola; Malaspina, Morena; Spita, Nicoletta; Tintori, Beatrice; Giaimo, Maria Donata

    2017-09-01

    Objective To present the results of the first and second round human papilloma virus (HPV)-based screening programme in the Umbria region after three years. Methods From August 2010 to November 2011, the entire female population aged 35-64 in a local health district was invited for HPV testing (HPV-DNA cobas4800 on a liquid-based cytology sample). HPV-negative women were re-invited after three years. For HPV-positive women, a slide was prepared and interpreted. Positive cytologies were referred to colposcopy; negatives were referred to repeat HPV after one year. If HPV was persistently positive, women were referred to colposcopy; if negative, to normal screening. Indicators of the first and second round are compared with those of cytology screening in the same area in the preceding three years. Results Participation was 56.5%, the same as cytology (56.6%). HPV-positivity was 6.4% (396/6272), cytology triage positivity was 35.6%; 251 cytology negative women were referred to one-year HPV retesting, 84.1% complied, and 55.5% were positive. Total colposcopy referral was 4.1%, and for cytology 1%. The detection rate for cervical intraepithelial neoplasia grade 2 or more severe was 10‰, compared with 3.7‰ using cytology. After three years, HPV-positivity was 3.4% (129/3831), overall colposcopy referral was 2.3% (most at one-year follow-up), and detection rate was 0.5/1000. Conclusions The first round detection rate was more than twice that of cytology screening, while colposcopy referral increased fourfold. At the second round, the detection rate decreased dramatically, showing that longer interval and more conservative protocols are needed.

  8. Dosage and dose schedule screening of drug combinations in agent-based models reveals hidden synergies

    Directory of Open Access Journals (Sweden)

    Lisa Corina Barros de Andrade e Sousa1

    2016-01-01

    Full Text Available The fungus Candida albicans is the most common causative agent of human fungal infections and better drugs or drug combination strategies are urgently needed. Here, we present an agent-based model of the interplay of C. albicans with the host immune system and with the microflora of the host. We took into account the morphological change of C. albicans from the yeast to hyphae form and its dynamics during infection. The model allowed us to follow the dynamics of fungal growth and morphology, of the immune cells and of microflora in different perturbing situations. We specifically focused on the consequences of microflora reduction following antibiotic treatment. Using the agent-based model, different drug types have been tested for their effectiveness, namely drugs that inhibit cell division and drugs that constrain the yeast-to-hyphae transition. Applied individually, the division drug turned out to successfully decrease hyphae while the transition drug leads to a burst in hyphae after the end of the treatment. To evaluate the effect of different drug combinations, doses, and schedules, we introduced a measure for the return to a healthy state, the infection score. Using this measure, we found that the addition of a transition drug to a division drug treatment can improve the treatment reliability while minimizing treatment duration and drug dosage. In this work we present a theoretical study. Although our model has not been calibrated to quantitative experimental data, the technique of computationally identifying synergistic treatment combinations in an agent based model exemplifies the importance of computational techniques in translational research.

  9. Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer.

    LENUS (Irish Health Repository)

    Perry, Antoinette S

    2013-04-15

    Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2\\/20) (SFRP1), 64.86% (48\\/74) (SFRP2), 0% (0\\/20) (SFRP4) and 60% (12\\/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6\\/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7\\/69), p < 0.0001) and BPH (11.43% (4\\/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

  10. Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Mohan Babu

    2014-02-01

    Full Text Available Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI screens can provide insights into the biological role(s of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.

  11. Systematic screen for mutants resistant to TORC1 inhibition in fission yeast reveals genes involved in cellular ageing and growth

    Directory of Open Access Journals (Sweden)

    Charalampos Rallis

    2014-01-01

    Target of rapamycin complex 1 (TORC1, which controls growth in response to nutrients, promotes ageing in multiple organisms. The fission yeast Schizosaccharomyces pombe emerges as a valuable genetic model system to study TORC1 function and cellular ageing. Here we exploited the combinatorial action of rapamycin and caffeine, which inhibit fission yeast growth in a TORC1-dependent manner. We screened a deletion library, comprising ∼84% of all non-essential fission yeast genes, for drug-resistant mutants. This screen identified 33 genes encoding functions such as transcription, kinases, mitochondrial respiration, biosynthesis, intra-cellular trafficking, and stress response. Among the corresponding mutants, 5 showed shortened and 21 showed increased maximal chronological lifespans; 15 of the latter mutants showed no further lifespan increase with rapamycin and might thus represent key targets downstream of TORC1. We pursued the long-lived sck2 mutant with additional functional analyses, revealing that the Sck2p kinase functions within the TORC1 network and is required for normal cell growth, global protein translation, and ribosomal S6 protein phosphorylation in a nutrient-dependent manner. Notably, slow cell growth was associated with all long-lived mutants while oxidative-stress resistance was not.

  12. In Vivo Screening Using Transgenic Zebrafish Embryos Reveals New Effects of HDAC Inhibitors Trichostatin A and Valproic Acid on Organogenesis.

    Directory of Open Access Journals (Sweden)

    Ling Li

    Full Text Available The effects of endocrine disrupting chemicals (EDCs on reproduction are well known, whereas their developmental effects are much less characterized. However, exposure to endocrine disruptors during organogenesis may lead to deleterious and permanent problems later in life. Zebrafish (Danio rerio transgenic lines expressing the green fluorescent protein (GFP in specific organs and tissues are powerful tools to uncover developmental defects elicited by EDCs. Here, we used seven transgenic lines to visualize in vivo whether a series of EDCs and other pharmaceutical compounds can alter organogenesis in zebrafish. We used transgenic lines expressing GFP in pancreas, liver, blood vessels, inner ear, nervous system, pharyngeal tooth and pectoral fins. This screen revealed that four of the tested chemicals have detectable effects on different organs, which shows that the range of effects elicited by EDCs is wider than anticipated. The endocrine disruptor tetrabromobisphenol-A (TBBPA, as well as the three drugs diclofenac, trichostatin A (TSA and valproic acid (VPA induced abnormalities in the embryonic vascular system of zebrafish. Moreover, TSA and VPA induced specific alterations during the development of pancreas, an observation that was confirmed by in situ hybridization with specific markers. Developmental delays were also induced by TSA and VPA in the liver and in pharyngeal teeth, resulting in smaller organ size. Our results show that EDCs can induce a large range of developmental alterations during embryogenesis of zebrafish and establish GFP transgenic lines as powerful tools to screen for EDCs effects in vivo.

  13. Fragment Screening of Human Kynurenine Aminotransferase-II.

    Science.gov (United States)

    Jayawickrama, Gayan S; Nematollahi, Alireza; Sun, Guanchen; Church, W Bret

    2018-03-01

    Kynurenine aminotransferase-II (KAT-II) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that acts in the tryptophan metabolic pathway by catalyzing the transamination of kynurenine into kynurenic acid (KYNA). It is one of four isoforms in the KAT family, of which it is the primary homologue responsible for KYNA production in the mammalian brain. KAT-II is targeted for inhibition as KYNA is implicated in diseases such as schizophrenia, where it is found in elevated concentrations. Previously, many different approaches have been taken to develop KAT-II inhibitors, and herein fragment-based drug design (FBDD) approaches have been exploited to provide further lead compounds that can be designed into novel inhibitors. Surface plasmon resonance (SPR) was used to screen a fragment library containing 1000 compounds, of which 41 hits were identified. These hits were further evaluated with SPR, and 18 were selected for inhibition studies. From these hits, two fragments, F6037-0164 and F0037-7280, were pursued and determined to have an IC 50 of 524.5 (± 25.6) μM and 115.2 (± 4.5) μM, respectively. This strategy shows the viability of using FBDD in gleaning knowledge about KAT-II inhibition and generating leads for the production of KAT-II inhibitors.

  14. Developmental neurotoxicity screening using human embryonic stem cells.

    Science.gov (United States)

    Bosnjak, Zeljko J

    2012-09-01

    Research in the area of stem cell biology and regenerative medicine, along with neuroscience, will further our understanding of drug-induced death of neurons during their development. With the development of an in vitro model of stem cell-derived human neural cell lines investigators can, under control conditions and during intense neuronal growth, examine molecular mechanisms of various drugs and conditions on early developmental neuroapoptosis in humans. If the use of this model will lead to fewer risks, or identification of drugs and anesthetics that are less likely to cause the death of neurons, this approach will be a major stride toward assuring the safety of drugs during the brain development. The ultimate goal would be not only to find the trigger for the catastrophic chain of events, but also to prevent neuronal cell death itself. Copyright © 2012. Published by Elsevier Inc.

  15. RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation

    Science.gov (United States)

    van Nierop, Pim; Vormer, Tinke L.; Foijer, Floris; Verheij, Joanne; Lodder, Johannes C.; Andersen, Jesper B.; Mansvelder, Huibert D.; te Riele, Hein

    2018-01-01

    To identify coding and non-coding suppressor genes of anchorage-independent proliferation by efficient loss-of-function screening, we have developed a method for enzymatic production of low complexity shRNA libraries from subtracted transcriptomes. We produced and screened two LEGO (Low-complexity by Enrichment for Genes shut Off) shRNA libraries that were enriched for shRNA vectors targeting coding and non-coding polyadenylated transcripts that were reduced in transformed Mouse Embryonic Fibroblasts (MEFs). The LEGO shRNA libraries included ~25 shRNA vectors per transcript which limited off-target artifacts. Our method identified 79 coding and non-coding suppressor transcripts. We found that taurine-responsive GABAA receptor subunits, including GABRA5 and GABRB3, were induced during the arrest of non-transformed anchor-deprived MEFs and prevented anchorless proliferation. We show that taurine activates chloride currents through GABAA receptors on MEFs, causing seclusion of cell volume in large membrane protrusions. Volume seclusion from cells by taurine correlated with reduced proliferation and, conversely, suppression of this pathway allowed anchorage-independent proliferation. In human cholangiocarcinomas, we found that several proteins involved in taurine signaling via GABAA receptors were repressed. Low GABRA5 expression typified hyperproliferative tumors, and loss of taurine signaling correlated with reduced patient survival, suggesting this tumor suppressive mechanism operates in vivo. PMID:29787571

  16. Comparison of RAW264.7, human whole blood and PBMC assays to screen for immunomodulators.

    Science.gov (United States)

    Elisia, Ingrid; Pae, Han Bee; Lam, Vivian; Cederberg, Rachel; Hofs, Elyse; Krystal, Gerald

    2018-01-01

    The RAW264.7 mouse macrophage cell line is used extensively to carry out in vitro screens for immunomodulators. Compounds that are effective at reducing the expression of pro-inflammatory cytokines or nitric oxide (NO) from lipopolysaccharide (LPS)-stimulated RAW264.7 cells are often considered candidate anti-inflammatory agents for humans. There is, however, very little data on the reliability of this screen to identify bona fide human immunomodulators. We compared the efficacy of 37 purported immunomodulators to modulate LPS or E. coli-induced inflammatory responses in RAW264.7 cell, whole human blood and human peripheral blood mononuclear cell (PBMC) assays. Interestingly, there was no significant correlation (R=0.315) between the responses obtained with RAW264.7 cells and the whole blood assay (WBA), suggesting that compounds demonstrating efficacy in RAW264.7 cells may be ineffective in humans, and, more importantly, compounds that are effective in humans may be missed with a RAW264.7 screen. Interestingly, there was also no significant correlation between the WBA and human PBMCs when the latter were cultured with 10% FCS, but a moderate correlation was seen when the PBMCs were cultured with 25% autologous plasma. The presence of plasma thus contributes to the overall inflammatory response observed in the WBA. We then asked if RAW264.7 cells, given that they are mouse macrophage-like cells, respond in a manner similar to primary murine derived macrophages. Intriguingly, there was no significant correlation (R=0.012) with the 37 putative immunomodulators, pointing to distinct inflammatory response mechanisms in the two model systems. We conclude that the use of a WBA to confirm potential immunomodulators obtained from high throughput screening with RAW264.7 cells is advisable and that future screens be carried out using a WBA. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Complete genome-wide screening and subtractive genomic approach revealed new virulence factors, potential drug targets against bio-war pathogen Brucella melitensis 16M

    Directory of Open Access Journals (Sweden)

    Pradeepkiran JA

    2015-03-01

    Full Text Available Jangampalli Adi Pradeepkiran,1* Sri Bhashyam Sainath,2,3* Konidala Kranthi Kumar,1 Matcha Bhaskar1 1Division of Animal Biotechnology, Department of Zoology, Sri Venkateswara University, Tirupati, India; 2CIMAR/CIIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental, Universidade do Porto, Rua dos Bragas, Porto, Portugal, 3Department of Biotechnology, Vikrama Simhapuri University, Nellore, Andhra Pradesh, India *These authors contributed equally to this work Abstract: Brucella melitensis 16M is a Gram-negative coccobacillus that infects both animals and humans. It causes a disease known as brucellosis, which is characterized by acute febrile illness in humans and causes abortions in livestock. To prevent and control brucellosis, identification of putative drug targets is crucial. The present study aimed to identify drug targets in B. melitensis 16M by using a subtractive genomic approach. We used available database repositories (Database of Essential Genes, Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server, and Kyoto Encyclopedia of Genes and Genomes to identify putative genes that are nonhomologous to humans and essential for pathogen B. melitensis 16M. The results revealed that among 3 Mb genome size of pathogen, 53 putative characterized and 13 uncharacterized hypothetical genes were identified; further, from Basic Local Alignment Search Tool protein analysis, one hypothetical protein showed a close resemblance (50% to Silicibacter pomeroyi DUF1285 family protein (2RE3. A further homology model of the target was constructed using MODELLER 9.12 and optimized through variable target function method by molecular dynamics optimization with simulating annealing. The stereochemical quality of the restrained model was evaluated by PROCHECK, VERIFY-3D, ERRAT, and WHATIF servers. Furthermore, structure-based virtual screening was carried out against the predicted active site of the respective protein using the

  18. Screening for tuberculosis and testing for human immunodeficiency virus in Zambian prisons

    Science.gov (United States)

    Maggard, Katie R; Hatwiinda, Sisa; Harris, Jennifer B; Phiri, Winifreda; Krüüner, Annika; Kaunda, Kaunda; Topp, Stephanie M; Kapata, Nathan; Ayles, Helen; Chileshe, Chisela; Henostroza, German

    2015-01-01

    Abstract Objective To improve the Zambia Prisons Service’s implementation of tuberculosis screening and human immunodeficiency virus (HIV) testing. Methods For both tuberculosis and HIV, we implemented mass screening of inmates and community-based screening of those residing in encampments adjacent to prisons. We also established routine systems – with inmates as peer educators – for the screening of newly entered or symptomatic inmates. We improved infection control measures, increased diagnostic capacity and promoted awareness of tuberculosis in Zambia’s prisons. Findings In a period of 9 months, we screened 7638 individuals and diagnosed 409 new patients with tuberculosis. We tested 4879 individuals for HIV and diagnosed 564 cases of infection. An additional 625 individuals had previously been found to be HIV-positive. Including those already on tuberculosis treatment at the time of screening, the prevalence of tuberculosis recorded in the prisons and adjacent encampments – 6.4% (6428/100 000) – is 18 times the national prevalence estimate of 0.35%. Overall, 22.9% of the inmates and 13.8% of the encampment residents were HIV-positive. Conclusion Both tuberculosis and HIV infection are common within Zambian prisons. We enhanced tuberculosis screening and improved the detection of tuberculosis and HIV in this setting. Our observations should be useful in the development of prison-based programmes for tuberculosis and HIV elsewhere. PMID:25883402

  19. Listen to them draw: screening children in primary care through the use of human figure drawings.

    Science.gov (United States)

    Tielsch, Anna H; Allen, Patricia Jackson

    2005-01-01

    This literature review focuses on the Human Figure Drawing (HFD) methods put forth by Elizabeth Koppitz as a screening instrument. Children's drawings have potential as a mental health screening aide for health care practitioners in the primary care setting. This paper focuses on self-portrait drawings as a screening technique for emotional well-being, anxiety, and depression in school-aged children (6-12 years old). Using Koppitz's emotional indicators checklist for mental health, practitioners can use the child's HFD as a quick screening tool. Although the HFD is not diagnostic and can not be used as the sole indicator for anxiety or depression, two or more emotional indicators may signal to the clinician that further psychiatric assessment and referral is needed.

  20. A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development.

    Science.gov (United States)

    Covassin, L D; Siekmann, A F; Kacergis, M C; Laver, E; Moore, J C; Villefranc, J A; Weinstein, B M; Lawson, N D

    2009-05-15

    In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.

  1. Complete genome sequence analysis of novel human bocavirus reveals genetic recombination between human bocavirus 2 and human bocavirus 4.

    Science.gov (United States)

    Khamrin, Pattara; Okitsu, Shoko; Ushijima, Hiroshi; Maneekarn, Niwat

    2013-07-01

    Epidemiological surveillance of human bocavirus (HBoV) was conducted on fecal specimens collected from hospitalized children with diarrhea in Chiang Mai, Thailand in 2011. By partial sequence analysis of VP1 gene, an unusual strain of HBoV (CMH-S011-11), was initially identified as HBoV4. The complete genome sequence of CMH-S011-11 was performed and analyzed further to clarify whether it was a recombinant strain or a new HBoV variant. Analysis of complete genome sequence revealed that the coding sequence starting from NS1, NP1 to VP1/VP2 was 4795 nucleotides long. Interestingly, the nucleotide sequence of NS1 gene of CMH-S011-11 was most closely related to the HBoV2 reference strains detected in Pakistan, which contradicted to the initial genotyping result of the partial VP1 region in the previous study. In addition, comparison of NP1 nucleotide sequence of CMH-S011-11 with those of other HBoV1-4 reference strains also revealed a high level of sequence identity with HBoV2. On the other hand, nucleotide sequence of VP1/VP2 gene of CMH-S011-11 was most closely related to those of HBoV4 reference strains detected in Nigeria. The overall full-length sequence analysis revealed that this CMH-S011-11 was grouped within HBoV4 species, but located in a separate branch from other HBoV4 prototype strains. Recombination analysis revealed that CMH-S011-11 was the result of recombination between HBoV2 and HBoV4 strains with the break point located near the start codon of VP2. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Restriction of human papillomavirus DNA testing in primary cervical screening to women above age 30

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Njor, Sisse H; Lynge, Elsebeth

    2012-01-01

    Cervical screening with human papillomavirus (HPV) testing is less specific for high-grade cervical intraepithelial neoplasia (=CIN3) than cytology. The aim of this systematic review was to determine whether a restriction of HPV testing to women aged at least 30 years would eliminate the problem...

  3. Concordant testing results between various Human Papillomavirus assays in primary cervical cancer screening

    DEFF Research Database (Denmark)

    de Thurah, Lena; Bonde, Jesper; Hoa Lam, Janni Uyen

    2018-01-01

    OBJECTIVES: Human Papillomavirus (HPV) assays are increasingly used for primary cervical screening and HPV vaccination effect monitoring. We undertook a systematic literature review to determine the concordance in positive test results (i.e., detection of HPV infections) between Hybrid Capture 2 ...

  4. Self-Sampling for Human Papillomavirus Testing among Non-Attenders Increases Attendance to the Norwegian Cervical Cancer Screening Programme

    DEFF Research Database (Denmark)

    Enerly, Espen; Bonde, Jesper; Schee, Kristina

    2016-01-01

    Increasing attendance to screening offers the best potential for improving the effectiveness of well-established cervical cancer screening programs. Self-sampling at home for human papillomavirus (HPV) testing as an alternative to a clinical sampling can be a useful policy to increase attendance....... To determine whether self-sampling improves screening attendance for women who do not regularly attend the Norwegian Cervical Cancer Screening Programme (NCCSP), 800 women aged 25-69 years in the Oslo area who were due to receive a 2nd reminder to attend regular screening were randomly selected and invited...... alternative for increasing cervical cancer screening coverage in Norway....

  5. A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila.

    Directory of Open Access Journals (Sweden)

    Dariel Ashton-Beaucage

    2014-03-01

    Full Text Available The small GTPase RAS is among the most prevalent oncogenes. The evolutionarily conserved RAF-MEK-MAPK module that lies downstream of RAS is one of the main conduits through which RAS transmits proliferative signals in normal and cancer cells. Genetic and biochemical studies conducted over the last two decades uncovered a small set of factors regulating RAS/MAPK signaling. Interestingly, most of these were found to control RAF activation, thus suggesting a central regulatory role for this event. Whether additional factors are required at this level or further downstream remains an open question. To obtain a comprehensive view of the elements functionally linked to the RAS/MAPK cascade, we used a quantitative assay in Drosophila S2 cells to conduct a genome-wide RNAi screen for factors impacting RAS-mediated MAPK activation. The screen led to the identification of 101 validated hits, including most of the previously known factors associated to this pathway. Epistasis experiments were then carried out on individual candidates to determine their position relative to core pathway components. While this revealed several new factors acting at different steps along the pathway--including a new protein complex modulating RAF activation--we found that most hits unexpectedly work downstream of MEK and specifically influence MAPK expression. These hits mainly consist of constitutive splicing factors and thereby suggest that splicing plays a specific role in establishing MAPK levels. We further characterized two representative members of this group and surprisingly found that they act by regulating mapk alternative splicing. This study provides an unprecedented assessment of the factors modulating RAS/MAPK signaling in Drosophila. In addition, it suggests that pathway output does not solely rely on classical signaling events, such as those controlling RAF activation, but also on the regulation of MAPK levels. Finally, it indicates that core splicing

  6. Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d

    Directory of Open Access Journals (Sweden)

    Moffatt Barbara A

    2010-08-01

    Full Text Available Abstract Background Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB for coplanar aromatic motifs similar to those found in known glycan-binding proteins. Results The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192 in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Conclusions Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

  7. Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d.

    Science.gov (United States)

    Doxey, Andrew C; Cheng, Zhenyu; Moffatt, Barbara A; McConkey, Brendan J

    2010-08-03

    Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB) for coplanar aromatic motifs similar to those found in known glycan-binding proteins. The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO) enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192) in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

  8. A joint model of persistent human papillomavirus infection and cervical cancer risk: Implications for cervical cancer screening.

    Science.gov (United States)

    Katki, Hormuzd A; Cheung, Li C; Fetterman, Barbara; Castle, Philip E; Sundaram, Rajeshwari

    2015-10-01

    New cervical cancer screening guidelines in the US and many European countries recommend that women get tested for human papillomavirus (HPV). To inform decisions about screening intervals, we calculate the increase in precancer/cancer risk per year of continued HPV infection. However, both time to onset of precancer/cancer and time to HPV clearance are interval-censored, and onset of precancer/cancer strongly informatively censors HPV clearance. We analyze this bivariate informatively interval-censored data by developing a novel joint model for time to clearance of HPV and time to precancer/cancer using shared random-effects, where the estimated mean duration of each woman's HPV infection is a covariate in the submodel for time to precancer/cancer. The model was fit to data on 9,553 HPV-positive/Pap-negative women undergoing cervical cancer screening at Kaiser Permanente Northern California, data that were pivotal to the development of US screening guidelines. We compare the implications for screening intervals of this joint model to those from population-average marginal models of precancer/cancer risk. In particular, after 2 years the marginal population-average precancer/cancer risk was 5%, suggesting a 2-year interval to control population-average risk at 5%. In contrast, the joint model reveals that almost all women exceeding 5% individual risk in 2 years also exceeded 5% in 1 year, suggesting that a 1-year interval is better to control individual risk at 5%. The example suggests that sophisticated risk models capable of predicting individual risk may have different implications than population-average risk models that are currently used for informing medical guideline development.

  9. Developing a Bacteroides System for Function-Based Screening of DNA from the Human Gut Microbiome.

    Science.gov (United States)

    Lam, Kathy N; Martens, Eric C; Charles, Trevor C

    2018-01-01

    Functional metagenomics is a powerful method that allows the isolation of genes whose role may not have been predicted from DNA sequence. In this approach, first, environmental DNA is cloned to generate metagenomic libraries that are maintained in Escherichia coli, and second, the cloned DNA is screened for activities of interest. Typically, functional screens are carried out using E. coli as a surrogate host, although there likely exist barriers to gene expression, such as lack of recognition of native promoters. Here, we describe efforts to develop Bacteroides thetaiotaomicron as a surrogate host for screening metagenomic DNA from the human gut. We construct a B. thetaiotaomicron-compatible fosmid cloning vector, generate a fosmid clone library using DNA from the human gut, and show successful functional complementation of a B. thetaiotaomicron glycan utilization mutant. Though we were unable to retrieve the physical fosmid after complementation, we used genome sequencing to identify the complementing genes derived from the human gut microbiome. Our results demonstrate that the use of B. thetaiotaomicron to express metagenomic DNA is promising, but they also exemplify the challenges that can be encountered in the development of new surrogate hosts for functional screening. IMPORTANCE Human gut microbiome research has been supported by advances in DNA sequencing that make it possible to obtain gigabases of sequence data from metagenomes but is limited by a lack of knowledge of gene function that leads to incomplete annotation of these data sets. There is a need for the development of methods that can provide experimental data regarding microbial gene function. Functional metagenomics is one such method, but functional screens are often carried out using hosts that may not be able to express the bulk of the environmental DNA being screened. We expand the range of current screening hosts and demonstrate that human gut-derived metagenomic libraries can be

  10. A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1.

    Science.gov (United States)

    Wang, Yuru; Havel, Jocelyn; Beal, Peter A

    2015-11-20

    Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. ADAR1 and ADAR2 are two members of the family that have been shown to be catalytically active. Earlier, we reported a phenotypic screen for the study of human ADAR2 using budding yeast S. cerevisiae as the host system. While this screen has been successfully applied to the study of ADAR2, it failed with ADAR1. Here, we report a new reporter that uses a novel editing substrate and is suitable for the study of ADAR1. We screened plasmid libraries with randomized codons for two important residues in human ADAR1 (G1007 and E1008). The screening results combined with in vitro deamination assays led to the identification of mutants that are more active than the wild type protein. Furthermore, a screen of the ADAR1 E1008X library with a reporter construct bearing an A•G mismatch at the editing site suggests one role for the residue at position 1008 is to sense the identity of the base pairing partner for the editing site adenosine. This work has provided a starting point for future in vitro evolution studies of ADAR1 and led to new insight into ADAR's editing site selectivity.

  11. Adherence patterns to extended cervical screening intervals in women undergoing human papillomavirus (HPV) and cytology cotesting.

    Science.gov (United States)

    Rendle, Katharine A; Schiffman, Mark; Cheung, Li C; Kinney, Walter K; Fetterman, Barbara; Poitras, Nancy E; Lorey, Thomas; Castle, Philip E

    2018-04-01

    Although guidelines have recommended extended interval cervical screening using concurrent human papillomavirus (HPV) and cytology ("cotesting") for over a decade, little is known about its adoption into routine care. Using longitudinal medical record data (2003-2015) from Kaiser Permanente Northern California (KPNC), which adopted triennial cotesting in 2003, we examined adherence to extended interval screening. We analyzed predictors of screening intervals among 491,588 women undergoing routine screening, categorizing interval length into early (<2.5years), adherent (2.5<3.5years), or late (3.5<6.0years). We also examined repeated early screening in a subgroup of 50,691 women. Predictors examined included: cohort year (defined by baseline cotest, 2003-2009), race/ethnicity, and baseline age. Compared to the 2003 cohort, women in the 2009 cohort were significantly less likely to screen early (aOR=0.22, 95% CI=0.21, 0.23) or late (aOR=0.47, 95% CI=0.45, 0.49). African American (AA) and Hispanic women were less adherent overall than Non-Hispanic White women, with increased early [(AA: aOR=1.21, 95%CI=1.17, 1.25) (Hispanic: aOR=1.08, 95%CI=1.06, 1.11)] and late screening [(AA: aOR=1.23, 95%CI=1.19, 1.27) (Hispanic: aOR=1.06, 95%CI=1.03, 1.08)]. Asian women were slightly more likely to screen early (aOR=1.03, 95%CI=1.01, 1.05), and less likely to screen late (aOR=0.92, 95% CI=0.90, 0.94). Women aged 60-64years were most likely to screen early for two consecutive intervals (aOR=2.09, 95%CI=1.91, 2.29). Our study found that widespread and rapid adoption of extended interval cervical cancer screening is possible, at least in this managed care setting. Further research examining multilevel drivers promoting or restricting extended interval screening across diverse healthcare settings is needed. Copyright © 2018 Elsevier Inc. All rights reserved.

  12. Economic evaluation, human immunodeficiency virus infection and screening: a review and critical appraisal of economic studies.

    Science.gov (United States)

    Dibosa-Osadolor, Onome; Roberts, Tracy

    2010-07-01

    The aim of this study was to review, systematically and critically, evidence used to derive estimates of cost-effectiveness of human immunodeficiency virus (HIV) screening. A systematic review was conducted. Searched were three main electronic bibliographic databases from 1993 to 2008 using key words including HIV, mass screening, HAART, economic evaluation, cost-effectiveness analysis, modeling. We included studies of sexually transmitted HIV infection in both sexes, including studies comparing diagnostic testing protocols and partner notification. Outcomes included were cases of HIV infection detected, deterioration to the AIDS state, secondary transmission of HIV, the quality-adjusted life-years/survival, costs, and cost-effectiveness of HIV screening. Eighty-four papers were identified; ten of which were formal economic evaluations, one cost study, three effectiveness studies, and three systematic reviews of HIV prevention programs. The predominant assertion was that HIV screening is cost-effective; methodological problems, such as the preponderance of static models which are inappropriate for infectious diseases, varying perspectives from which the studies were analyzed, and arbitrary threshold incremental cost-effectiveness ratio levels, limited the validity of these findings, and their usefulness in informing health policy decisions. The majority of published economic evaluations are based on inappropriate static models. This flaw renders the results of these studies as inconclusive and the purported cost-effectiveness of HIV screening debatable. The results of this review could form a basis for consideration of further research and analysis by health economists into the cost-effectiveness of HIV screening.

  13. High-content screening of small compounds on human embryonic stem cells.

    Science.gov (United States)

    Barbaric, Ivana; Gokhale, Paul J; Andrews, Peter W

    2010-08-01

    Human ES (embryonic stem) cells and iPS (induced pluripotent stem) cells have been heralded as a source of differentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson's disease or diabetes. Despite the great potential for their use in regenerative therapy, the challenge remains to understand the basic biology of these remarkable cells, in order to differentiate them into any functional cell type. Given the scale of the task, high-throughput screening of agents and culture conditions offers one way to accelerate these studies. The screening of small-compound libraries is particularly amenable to such high-throughput methods. Coupled with high-content screening technology that enables simultaneous assessment of multiple cellular features in an automated and quantitative way, this approach is proving powerful in identifying both small molecules as tools for manipulating stem cell fates and novel mechanisms of differentiation not previously associated with stem cell biology. Such screens performed on human ES cells also demonstrate the usefulness of human ES/iPS cells as cellular models for pharmacological testing of drug efficacy and toxicity, possibly a more imminent use of these cells than in regenerative medicine.

  14. Prevalence of Human Papillomavirus in Self-Taken Samples from Screening Nonattenders

    DEFF Research Database (Denmark)

    Lam, J U H; Rebolj, M; Ejegod, D M

    2017-01-01

    The Copenhagen Self-Sampling Initiative (CSi) has shown how human papillomavirus (HPV)-based self-sampling can be used to increase screening participation among 23,632 nonattenders in the Capital Region of Denmark. In this study, we describe HPV prevalence and genotype frequency in 4,824 self...... study, which had been undertaken in the same screening laboratory. Nonattenders had an HPV prevalence of 11.3% as determined by the CLART assay, which was lower than that for women from the Horizon study (18.5%). One-third of the CSi women who tested HPV positive by self-sampling tested HPV negative...

  15. A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle.

    Science.gov (United States)

    Modrzynska, Katarzyna; Pfander, Claudia; Chappell, Lia; Yu, Lu; Suarez, Catherine; Dundas, Kirsten; Gomes, Ana Rita; Goulding, David; Rayner, Julian C; Choudhary, Jyoti; Billker, Oliver

    2017-01-11

    A family of apicomplexa-specific proteins containing AP2 DNA-binding domains (ApiAP2s) was identified in malaria parasites. This family includes sequence-specific transcription factors that are key regulators of development. However, functions for the majority of ApiAP2 genes remain unknown. Here, a systematic knockout screen in Plasmodium berghei identified ten ApiAP2 genes that were essential for mosquito transmission: four were critical for the formation of infectious ookinetes, and three were required for sporogony. We describe non-essential functions for AP2-O and AP2-SP proteins in blood stages, and identify AP2-G2 as a repressor active in both asexual and sexual stages. Comparative transcriptomics across mutants and developmental stages revealed clusters of co-regulated genes with shared cis promoter elements, whose expression can be controlled positively or negatively by different ApiAP2 factors. We propose that stage-specific interactions between ApiAP2 proteins on partly overlapping sets of target genes generate the complex transcriptional network that controls the Plasmodium life cycle. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    Science.gov (United States)

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

    1992-09-01

    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  17. Functional microRNA high throughput screening reveals miR-9 as a central regulator of liver oncogenesis by affecting the PPARA-CDH1 pathway

    International Nuclear Information System (INIS)

    Drakaki, Alexandra; Hatziapostolou, Maria; Polytarchou, Christos; Vorvis, Christina; Poultsides, George A.; Souglakos, John; Georgoulias, Vassilis; Iliopoulos, Dimitrios

    2015-01-01

    Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths, reflecting the aggressiveness of this type of cancer and the absence of effective therapeutic regimens. MicroRNAs have been involved in the pathogenesis of different types of cancers, including liver cancer. Our aim was to identify microRNAs that have both functional and clinical relevance in HCC and examine their downstream signaling effectors. MicroRNA and gene expression levels were measured by quantitative real-time PCR in HCC tumors and controls. A TargetScan algorithm was used to identify miR-9 downstream direct targets. A high-throughput screen of the human microRNAome revealed 28 microRNAs as regulators of liver cancer cell invasiveness. MiR-9, miR-21 and miR-224 were the top inducers of HCC invasiveness and also their expression was increased in HCC relative to control liver tissues. Integration of the microRNA screen and expression data revealed miR-9 as the top microRNA, having both functional and clinical significance. MiR-9 levels correlated with HCC tumor stage and miR-9 overexpression induced SNU-449 and HepG2 cell growth, invasiveness and their ability to form colonies in soft agar. Bioinformatics and 3′UTR luciferase analyses identified E-cadherin (CDH1) and peroxisome proliferator-activated receptor alpha (PPARA) as direct downstream effectors of miR-9 activity. Inhibition of PPARA suppressed CDH1 mRNA levels, suggesting that miR-9 regulates CDH1 expression directly through binding in its 3′UTR and indirectly through PPARA. On the other hand, miR-9 inhibition of overexpression suppressed HCC tumorigenicity and invasiveness. PPARA and CDH1 mRNA levels were decreased in HCC relative to controls and were inversely correlated with miR-9 levels. Taken together, this study revealed the involvement of the miR-9/PPARA/CDH1 signaling pathway in HCC oncogenesis. The online version of this article (doi:10.1186/s12885-015-1562-9) contains supplementary material, which is

  18. Human glial chimeric mice reveal astrocytic dependence of JC virus infection

    DEFF Research Database (Denmark)

    Kondo, Yoichi; Windrem, Martha S; Zou, Lisa

    2014-01-01

    with humanized white matter by engrafting human glial progenitor cells (GPCs) into neonatal immunodeficient and myelin-deficient mice. Intracerebral delivery of JCV resulted in infection and subsequent demyelination of these chimeric mice. Human GPCs and astrocytes were infected more readily than...... that was chimeric for human astrocytes and GPCs. JCV effectively propagated in these mice, which indicates that astroglial infection is sufficient for JCV spread. Sequencing revealed progressive mutation of the JCV capsid protein VP1 after infection, suggesting that PML may evolve with active infection...

  19. Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection.

    Directory of Open Access Journals (Sweden)

    Celine Deffrasnes

    2016-03-01

    Full Text Available Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.

  20. Screening vaccine formulations for biological activity using fresh human whole blood.

    Science.gov (United States)

    Brookes, Roger H; Hakimi, Jalil; Ha, Yukyung; Aboutorabian, Sepideh; Ausar, Salvador F; Hasija, Manvi; Smith, Steven G; Todryk, Stephen M; Dockrell, Hazel M; Rahman, Nausheen

    2014-01-01

    Understanding the relevant biological activity of any pharmaceutical formulation destined for human use is crucial. For vaccine-based formulations, activity must reflect the expected immune response, while for non-vaccine therapeutic agents, such as monoclonal antibodies, a lack of immune response to the formulation is desired. During early formulation development, various biochemical and biophysical characteristics can be monitored in a high-throughput screening (HTS) format. However, it remains impractical and arguably unethical to screen samples in this way for immunological functionality in animal models. Furthermore, data for immunological functionality lag formulation design by months, making it cumbersome to relate back to formulations in real-time. It is also likely that animal testing may not accurately reflect the response in humans. For a more effective formulation screen, a human whole blood (hWB) approach can be used to assess immunological functionality. The functional activity relates directly to the human immune response to a complete formulation (adjuvant/antigen) and includes adjuvant response, antigen response, adjuvant-modulated antigen response, stability, and potentially safety. The following commentary discusses the hWB approach as a valuable new tool to de-risk manufacture, formulation design, and clinical progression.

  1. Human scenarios for the screening assessment. Columbia River Comprehensive Impact Assessment

    International Nuclear Information System (INIS)

    Napier, B.A.; Harper, B.L.; Lane, N.K.; Strenge, D.L.; Spivey, R.B.

    1996-03-01

    Because of past nuclear production operations along the Columbia River, there is intense public and tribal interest in assessing any residual Hanford Site related contamination along the river from the Hanford Reach to the Pacific Ocean. The Columbia River Impact Assessment (CRCIA) was proposed to address these concerns. The assessment of the Columbia River is being conducted in phases. The initial phase is a screening assessment of risk, which addresses current environmental conditions for a range of potential uses. One component of the screening assessment estimates the risk from contaminants in the Columbia River to humans. Because humans affected by the Columbia river are involved in a wide range of activities, various scenarios have been developed on which to base the risk assessments. The scenarios illustrate the range of activities possible by members of the public coming in contact with the Columbia River so that the impact of contaminants in the river on human health can be assessed. Each scenario illustrates particular activity patterns by a specific group. Risk will be assessed at the screening level for each scenario. This report defines the scenarios and the exposure factors that will be the basis for estimating the potential range of risk to human health from Hanford-derived radioactive as well as non-radioactive contaminants associated with the Columbia River

  2. Human scenarios for the screening assessment. Columbia River Comprehensive Impact Assessment

    Energy Technology Data Exchange (ETDEWEB)

    Napier, B.A.; Harper, B.L.; Lane, N.K.; Strenge, D.L.; Spivey, R.B.

    1996-03-01

    Because of past nuclear production operations along the Columbia River, there is intense public and tribal interest in assessing any residual Hanford Site related contamination along the river from the Hanford Reach to the Pacific Ocean. The Columbia River Impact Assessment (CRCIA) was proposed to address these concerns. The assessment of the Columbia River is being conducted in phases. The initial phase is a screening assessment of risk, which addresses current environmental conditions for a range of potential uses. One component of the screening assessment estimates the risk from contaminants in the Columbia River to humans. Because humans affected by the Columbia river are involved in a wide range of activities, various scenarios have been developed on which to base the risk assessments. The scenarios illustrate the range of activities possible by members of the public coming in contact with the Columbia River so that the impact of contaminants in the river on human health can be assessed. Each scenario illustrates particular activity patterns by a specific group. Risk will be assessed at the screening level for each scenario. This report defines the scenarios and the exposure factors that will be the basis for estimating the potential range of risk to human health from Hanford-derived radioactive as well as non-radioactive contaminants associated with the Columbia River.

  3. A review of the use of human papilloma virus (HPV) in cervical screening.

    Science.gov (United States)

    Crossley, B; Crossley, J

    2017-07-01

    Using key words online databases were searched to identify relevant publications to review the use of Human papilloma virus (HPV) in cervical screening. The mode of cervical screening in the UK has been decided but implementation plans have yet to be announced. The protracted uncertainty surrounding the initial announcement to move to HPV primary screening together with the lack of a national steer has resulted in a flight of staff which threatens the provision of the current and future service. The transition will be a challenging time but analysis of data from more than 176,000 women has shown clear evidence of a reduction in the incidence of cancer where HPV testing is used. There will however, be a population of women who are cytologically negative but high-risk HPV positive and the management of these women will be key to maximising the benefits of HPV primary screening. As cervical cytology becomes increasingly rare its effectiveness and role in cervical screening will come under scrutiny and we must ensure the specificity of reporting is maintained in order for it to survive.

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

    Science.gov (United States)

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

    2011-03-01

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

  5. STRP Screening Sets for the human genome at 5 cM density

    Directory of Open Access Journals (Sweden)

    Marth Gabor

    2003-02-01

    Full Text Available Abstract Background Short tandem repeat polymorphisms (STRPs are powerful tools for gene mapping and other applications. A STRP genome scan of 10 cM is usually adequate for mapping single gene disorders. However mapping studies involving genetically complex disorders and especially association (linkage disequilibrium often require higher STRP density. Results We report the development of two separate 10 cM human STRP Screening Sets (Sets 12 and 52 which span all chromosomes. When combined, the two Sets contain a total of 782 STRPs, with average STRP spacing of 4.8 cM, average heterozygosity of 0.72, and total sex-average coverage of 3535 cM. The current Sets are comprised almost entirely of STRPs based on tri- and tetranucleotide repeats. We also report correction of primer sequences for many STRPs used in previous Screening Sets. Detailed information for the new Screening Sets is available from our web site: http://research.marshfieldclinic.org/genetics. Conclusion Our new human STRP Screening Sets will improve the quality and cost effectiveness of genotyping for gene mapping and other applications.

  6. Human thrombomodulin knock-in mice reveal differential effects of human thrombomodulin on thrombosis and atherosclerosis.

    Science.gov (United States)

    Raife, Thomas J; Dwyre, Denis M; Stevens, Jeff W; Erger, Rochelle A; Leo, Lorie; Wilson, Katina M; Fernández, Jose A; Wilder, Jennifer; Kim, Hyung-Suk; Griffin, John H; Maeda, Nobuyo; Lentz, Steven R

    2011-11-01

    We sought to develop a murine model to examine the antithrombotic and antiinflammatory functions of human thrombomodulin in vivo. Knock-in mice that express human thrombomodulin from the murine thrombomodulin gene locus were generated. Compared with wild-type mice, human thrombomodulin knock-in mice exhibited decreased protein C activation in the aorta (Pknock-in mice compared with wild-type mice (Pknock-in mice (12±3 minutes) than in wild-type mice (31±6 minutes; Pknock-in and wild-type mice after injection of endotoxin. When crossed with apolipoprotein E-deficient mice and fed a Western diet, knock-in mice had a further decrease in protein C activation but did not exhibit increased atherosclerosis. Expression of human thrombomodulin in place of murine thrombomodulin produces viable mice with a prothrombotic phenotype but unaltered responses to systemic inflammatory or atherogenic stimuli. This humanized animal model will be useful for investigating the function of human thrombomodulin under pathophysiological conditions in vivo.

  7. Classification of human pathogen bacteria for early screening using electronic nose

    Science.gov (United States)

    Zulkifli, Syahida Amani; Mohamad, Che Wan Syarifah Robiah; Abdullah, Abu Hassan

    2017-10-01

    This paper present human pathogen bacteria for early screening using electronic nose. Electronic nose (E-nose) known as gas sensor array is a device that analyze the odor measurement give the fast response and less time consuming for clinical diagnosis. Many bacterial pathogens could lead to life threatening infections. Accurate and rapid diagnosis is crucial for the successful management of these infections disease. The conventional method need more time to detect the growth of bacterial. Alternatively, the bacteria are Pseudomonas aeruginosa and Shigella cultured on different media agar can be detected and classifies according to the volatile compound in shorter time using electronic nose (E-nose). Then, the data from electronic nose (E-nose) is processed using statistical method which is principal component analysis (PCA). The study shows the capability of electronic nose (E-nose) for early screening for bacterial infection in human stomach.

  8. Pathway-based analysis of genome-wide siRNA screens reveals the regulatory landscape of APP processing.

    Directory of Open Access Journals (Sweden)

    Luiz Miguel Camargo

    Full Text Available The progressive aggregation of Amyloid-β (Aβ in the brain is a major trait of Alzheimer's Disease (AD. Aβ is produced as a result of proteolytic processing of the β-amyloid precursor protein (APP. Processing of APP is mediated by multiple enzymes, resulting in the production of distinct peptide products: the non-amyloidogenic peptide sAPPα and the amyloidogenic peptides sAPPβ, Aβ40, and Aβ42. Using a pathway-based approach, we analyzed a large-scale siRNA screen that measured the production of different APP proteolytic products. Our analysis identified many of the biological processes/pathways that are known to regulate APP processing and have been implicated in AD pathogenesis, as well as revealing novel regulatory mechanisms. Furthermore, we also demonstrate that some of these processes differentially regulate APP processing, with some mechanisms favouring production of certain peptide species over others. For example, synaptic transmission having a bias towards regulating Aβ40 production over Aβ42 as well as processes involved in insulin and pancreatic biology having a bias for sAPPβ production over sAPPα. In addition, some of the pathways identified as regulators of APP processing contain genes (CLU, BIN1, CR1, PICALM, TREM2, SORL1, MEF2C, DSG2, EPH1A recently implicated with AD through genome wide association studies (GWAS and associated meta-analysis. In addition, we provide supporting evidence and a deeper mechanistic understanding of the role of diabetes in AD. The identification of these processes/pathways, their differential impact on APP processing, and their relationships to each other, provide a comprehensive systems biology view of the "regulatory landscape" of APP.

  9. High-risk human papillomavirus (HPV screening and detection in healthy patient saliva samples: a pilot study

    Directory of Open Access Journals (Sweden)

    Wang Robert C

    2011-10-01

    Full Text Available Abstract Background The human papillomaviruses (HPV are a large family of non-enveloped DNA viruses, mainly associated with cervical cancers. Recent epidemiologic evidence has suggested that HPV may be an independent risk factor for oropharyngeal cancers. Evidence now suggests HPV may modulate the malignancy process in some tobacco- and alcohol-induced oropharynx tumors, but might also be the primary oncogenic factor for inducing carcinogenesis among some non-smokers. More evidence, however, is needed regarding oral HPV prevalence among healthy adults to estimate risk. The goal of this study was to perform an HPV screening of normal healthy adults to assess oral HPV prevalence. Methods Healthy adult patients at a US dental school were selected to participate in this pilot study. DNA was isolated from saliva samples and screened for high-risk HPV strains HPV16 and HPV18 and further processed using qPCR for quantification and to confirm analytical sensitivity and specificity. Results Chi-square analysis revealed the patient sample was representative of the general clinic population with respect to gender, race and age (p Conclusions The successful recruitment and screening of healthy adult patients revealed HPV16, but not HPV18, was present in a small subset. These results provide new information about oral HPV status, which may help to contextualize results from other studies that demonstrate oral cancer rates have risen in the US among both females and minorities and in some geographic areas that are not solely explained by rates of tobacco and alcohol use. The results of this study may be of significant value to further our understanding of oral health and disease risk, as well as to help design future studies exploring the role of other factors that influence oral HPV exposure, as well as the short- and long-term consequences of oral HPV infection.

  10. Screening vaccine formulations for biological activity using fresh human whole blood.

    OpenAIRE

    Brookes, RH; Hakimi, J; Ha, Y; Aboutorabian, S; Ausar, SF; Hasija, M; Smith, SG; Todryk, SM; Dockrell, HM; Rahman, N

    2014-01-01

    Understanding the relevant biological activity of any pharmaceutical formulation destined for human use is crucial. For vaccine-based formulations, activity must reflect the expected immune response, while for non-vaccine therapeutic agents, such as monoclonal antibodies, a lack of immune response to the formulation is desired. During early formulation development, various biochemical and biophysical characteristics can be monitored in a high-throughput screening (HTS) format. However, it rem...

  11. Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function

    Directory of Open Access Journals (Sweden)

    Matthew J. Birket

    2015-10-01

    Full Text Available Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC model of hypertrophic cardiomyopathy (HCM. A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

  12. Screening Out Controversy: Human Genetics, Emerging Techniques of Diagnosis, and the Origins of the Social Issues Committee of the American Society of Human Genetics, 1964-1973.

    Science.gov (United States)

    Mitchell, M X

    2017-05-01

    In the years following World War II, and increasingly during the 1960s and 1970s, professional scientific societies developed internal sub-committees to address the social implications of their scientific expertise (Moore, Disrupting Science: Social Movements, American Scientists, and the Politics of the Military, 1945-1975. Princeton: Princeton University Press, 2008). This article explores the early years of one such committee, the American Society of Human Genetics' "Social Issues Committee," founded in 1967. Although the committee's name might suggest it was founded to increase the ASHG's public and policy engagement, exploration of the committee's early years reveals a more complicated reality. Affronted by legislators' recent unwillingness to seek the expert advice of human geneticists before adopting widespread neonatal screening programs for phenylketonuria (PKU), and feeling pressed to establish their relevance in an increasingly resource-scarce funding environment, committee members sought to increase the discipline's expert authority. Painfully aware of controversy over abortion rights and haunted by the taint of the discipline's eugenic past, however, the committee proceeded with great caution. Seeking to harness interest in and assert professional control over emerging techniques of genetic diagnosis, the committee strove to protect the society's image by relegating ethical and policy questions about their use to the individual consciences of member scientists. It was not until 1973, after the committee's modest success in organizing support for a retrospective public health study of PKU screening and following the legalization of abortion on demand, that the committee decided to take a more publicly engaged stance.

  13. Cost-effectiveness of human papillomavirus vaccination and cervical cancer screening in Thailand.

    Science.gov (United States)

    Sharma, M; Ortendahl, J; van der Ham, E; Sy, S; Kim, J J

    2012-01-01

    To assess the health and economic outcomes of various screening and vaccination strategies for cervical cancer prevention. Cost-effectiveness analysis from a societal perspective. Thailand. Females aged 9 years and older. Using a mathematical model of human papillomavirus (HPV) infection and cervical cancer, calibrated to epidemiological data from Thailand, we estimated the cost-effectiveness of pre-adolescent HPV vaccination, screening [visual inspection with acetic acid (VIA), HPV DNA testing, and cytology] between one and five times per lifetime in adulthood, and combined pre-adolescent vaccination and screening. Vaccine efficacy, coverage, cost, and screening frequency were varied in sensitivity analyses. Incremental cost-effectiveness ratios, expressed as cost per year of life saved (YLS). Assuming lifelong efficacy and 80% coverage, pre-adolescent HPV vaccination alone was projected to reduce the lifetime risk of cervical cancer by 55%, which was greater than any strategy of screening alone. When cost per vaccinated girl was I$10 (approximately $2 per dose) or less, HPV vaccination alone was cost saving. Pre-adolescent vaccination and HPV DNA testing five times per lifetime, starting at age 35 years, reduced the lifetime cervical cancer risk by 70%, and had a cost-effectiveness ratio less than Thailand's GDP per capita (I$8100), provided the cost per vaccinated girl was I$200 or less. Low cost pre-adolescent HPV vaccination followed by HPV screening five times per lifetime is an efficient strategy for Thailand. Costs may need to be lower, however, for this strategy to be affordable. If vaccination is not feasible, HPV DNA testing five times per lifetime is efficient. © 2011 The Authors BJOG An International Journal of Obstetrics and Gynaecology © 2011 RCOG.

  14. Annual Papanicolaou screening for 5 years among human papillomavirus-negative women

    International Nuclear Information System (INIS)

    Petry, Karl Ulrich; Rinnau, Franziska; Böhmer, Gerd; Hollwitz, Bettina; Luyten, Alexander; Buttmann, Nina; Brünger, Martin; Iftner, Thomas

    2013-01-01

    Primary human papilloma virus (HPV) screening is more effective than cytology in reducing the risk of cervical cancer, but screening intervals should be extended in HPV-negative women. However, some Markov models predicted that long intervals are associated with an excess risk of cervical cancer. The aim of this analysis was to estimate the real-life risks and benefits of annual Papanicolaou (Pap) screening in HPV-negative women with normal cytology. Women with negative Hybrid Capture 2 (HC2) results and normal cytology at the time of inclusion in the Hannover HPV screening trial underwent annual Pap smears for 5 years. A subgroup was randomly selected for retesting with cytology, HC2, and colposcopy 60–68 months after recruitment. Of 4236 women included, 3406 had at least one Pap smear, but only 1185 attended all five annual screening visits. The proportion of women with at least one abnormal smear was 14.4% in 60 months. The probability of abnormal smears increased continuously over time. No case of ≥ CIN2+ was observed during 5 years. Of 605 women selected for subgroup analysis, 292 agreed to be retested (48.3%). The rate of high-risk HPV at 60–68 months was 3.0% (9/296). The long-term risk of high-grade neoplasia after an initial negative HC2 test and normal cytology result was low, while the rate of false-positive abnormal Pap smears was significant and increased constantly over time. Pap smear screening of HPV-negative women more frequently than every 5 years could be potentially harmful and seems to be of little clinical value

  15. [Human papillomavirus testing in cervical cancer screening at a public health service of Santiago, Chile].

    Science.gov (United States)

    Terrazas, Solana; Ibáñez, Carolina; Lagos, Marcela; Poggi, Helena; Brañes, Jorge; Barriga, María Isabel; Cartagena, Jaime; Núñez, Felipe; González, Francisca; Cook, Paz; Van De Wyngard, Vanessa; Ferreccio, Catterina

    2015-01-01

    Molecular techniques for human papillomavirus (HPV) detection have a good performance as screening tests and could be included in cervical cancer early detection programs. We conducted a population-based trial comparing HPV detection and Papanicolaou as primary screening tests, in a public health service in Santiago, Chile. To describe the experience of implementing this new molecular test and present the main results of the study. Women aged 25 to 64 enrolled in three public health centers were invited to participate. In all women, samples were collected for Papanicolaou and HPV DNA testing, and naked-eye visual inspection of the cervix with acetic acid was performed. Women with any positive screening test were referred to the local area hospital for diagnostic confirmation with colposcopy and biopsy of suspicious lesions. Screening results were obtained for 8265 women, of whom 931 (11.3%) were positive to any test. The prevalence of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was 1.1%; nine women had invasive cervical cancer. Sensitivities for the detection of CIN2+ were 22.1% (95% confidence interval (CI) 16.4-29.2) for Papanicolaou and 92.7% (95% CI 84.4-96.8) for HPV testing; specificities were 98.9% (95% CI 98.7-99.0) and 92.0% (95% CI 91.4-92.6) respectively. This experience showed that the implementation of a molecular test for cervical cancer screening is not a major challenge in Chile: it was well accepted by both the health team and the participants, and it may improve the effectiveness of the screening program.

  16. Integration of human papillomavirus vaccination and cervical cancer screening in Latin America and the Caribbean.

    Science.gov (United States)

    Franco, Eduardo L; Tsu, Vivien; Herrero, Rolando; Lazcano-Ponce, Eduardo; Hildesheim, Allan; Muñoz, Nubia; Murillo, Raul; Sánchez, Gloria Ines; Andrus, Jon Kim

    2008-08-19

    Despite substantial efforts to control cervical cancer by screening, most Latin American and Caribbean countries continue to experience incidence rates of this disease that are much higher than those of other Western countries. The implementation of universal human papillomavirus (HPV) vaccination for young adolescent women is the best prospect for changing this situation. Even though there are financial challenges to overcome to implement such a policy, there is broad political support in the region for adopting universal HPV vaccination. The costs of implementing this policy could be largely alleviated by changing cervical cancer control practices that rely on inefficient use of resources presently allocated to cytology screening. In view of the strong evidence base concerning cervical cancer prevention technologies in the region and the expected impact of vaccination on the performance of cytology, we propose a reformulation of cervical cancer screening policies to be based on HPV testing using validated methods followed by cytologic triage. This approach would serve as the central component of a system that plays the dual role of providing screening and surveillance as integrated and complementary activities sharing centralized resources and coordination.

  17. Study on a noninvasive method for rapid screening Human Serum albumin injectables by Raman spectroscopy

    Directory of Open Access Journals (Sweden)

    Yu Zhao

    2017-01-01

    Full Text Available Human serum albumin (HSA injectable product is a severely afflicted area on drug safety due to its high price and restricted supply. Raman spectroscopy performances high specificity on HSA detection and it is even possible to determine HSA injectable products noninvasively. In this study, we developed a noninvasive rapid screening method for of HSA injectable products by using portable Raman spectrometer. Qualitative models were established by using principal component analysis combined with classical least squares (PCA-CLS algorithm, while quantitative model was established by using partial least squares (PLS algorithm. Model transfer in different instruments of both the same and different apparatus modules was further discussed in this paper. A total of 34 HSA injectable samples collected from markets were used for verification. The identification results showed 100% accuracy and the predicted concentrations of those identified as true HSA were consistent with their labeled concentrations. The quantitative results also indicated that model transfer was excellent in the same apparatus modules of Raman spectrometer at all concentration levels, and still good enough in the different apparatus modules although the relative standard deviation (RSD value showed a little increasing trend at low HSA concentration level. In conclusion, the method was proved to be feasible and efficient for screening HSA injections, especially on its screening speed and the consideration of glass containers. Moreover, with inspiring results on the model transfer, the method could be used as a universal screening mean to different Raman instruments.

  18. Nucleic acid amplification technology screening for hepatitis C virus and human immunodeficiency virus for blood donations

    International Nuclear Information System (INIS)

    Bamaga, Mohammad S.; Bokhari, Fawzi F.; Aboud, Abdulrehman M.; Al-Malki, M.; Alenzi, Faris Q.

    2006-01-01

    To investigate the performance of the commercial Roche COBAS AmpliScreen assay, and demonstrate whether the COBAS AmpliScreen human immunodeficiency virus-1 (HIV-1) test, v1.5, and COBAS AmpliScreen hepatitis C virus (HCV) v 2.0 for screening for HIV-1 and HCV RNA in the donated blood units from which plasma mini pools were collected, by nucleic acid amplification technology (NAT), could detect the positive pools and reduce the risk of transmission of infections for those routinely tested by serological assays. The study was performed on 3288 plasma samples collected from blood donors in a period of 13 months, from August 2004 to August 2005, at Al-Hada Armed Forces Hospital, Molecular Pathology Laboratory, Taif, Kingdom of Saudi Arabia. The samples were tested by the reverse transcriptase polymerase chain reaction (RT-PCR) after RNA extraction (this represents the major method in NAT assays), in parallel with the routine serological testing to detect qualitatively for HIV-1 and HCV. The NAT assays that include an automated COBAS AmpliPrep system for RNA extraction and COBAS Amplicor Analyzer using AmpliScreen kits for RT-PCR assays, and the routine serological screening assays for the detection of the HIV-1 and HCV RNA in the plasma samples from the blood donors have shown to be a reliable combination that would meet our requirements. The collected data further confirms the results from the serological assays and enables us to decrease the residual risk of transmission to a minimum with the finding of no seronegative window period donation. The results demonstrate that out of 3288 samples, the percentages of RT-PCR (NAT) negative blood donations that were also confirmed as seronegative were 99% for HCV, and 99.1% for HIV-1. The modified combined systems (automated COBAS AmpliPrep system for RNA extraction and COBAS Amplicor Analyzer using AmpliScreen kits for RT-PCR assays) for NAT screening assays has allowed the release of all blood donations supplied in the

  19. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay.

    Science.gov (United States)

    Alley, M C; Scudiero, D A; Monks, A; Hursey, M L; Czerwinski, M J; Fine, D L; Abbott, B J; Mayo, J G; Shoemaker, R H; Boyd, M R

    1988-02-01

    For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

  20. Ghost-in-the-Machine reveals human social signals for human-robot interaction.

    Science.gov (United States)

    Loth, Sebastian; Jettka, Katharina; Giuliani, Manuel; de Ruiter, Jan P

    2015-01-01

    We used a new method called "Ghost-in-the-Machine" (GiM) to investigate social interactions with a robotic bartender taking orders for drinks and serving them. Using the GiM paradigm allowed us to identify how human participants recognize the intentions of customers on the basis of the output of the robotic recognizers. Specifically, we measured which recognizer modalities (e.g., speech, the distance to the bar) were relevant at different stages of the interaction. This provided insights into human social behavior necessary for the development of socially competent robots. When initiating the drink-order interaction, the most important recognizers were those based on computer vision. When drink orders were being placed, however, the most important information source was the speech recognition. Interestingly, the participants used only a subset of the available information, focussing only on a few relevant recognizers while ignoring others. This reduced the risk of acting on erroneous sensor data and enabled them to complete service interactions more swiftly than a robot using all available sensor data. We also investigated socially appropriate response strategies. In their responses, the participants preferred to use the same modality as the customer's requests, e.g., they tended to respond verbally to verbal requests. Also, they added redundancy to their responses, for instance by using echo questions. We argue that incorporating the social strategies discovered with the GiM paradigm in multimodal grammars of human-robot interactions improves the robustness and the ease-of-use of these interactions, and therefore provides a smoother user experience.

  1. Novel PI3K/Akt Inhibitors Screened by the Cytoprotective Function of Human Immunodeficiency Virus Type 1 Tat

    Science.gov (United States)

    Kim, Dong-Hyun; Kim, Baek

    2011-01-01

    The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors. PMID:21765914

  2. Novel PI3K/Akt inhibitors screened by the cytoprotective function of human immunodeficiency virus type 1 Tat.

    Directory of Open Access Journals (Sweden)

    Yuri Kim

    Full Text Available The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors.

  3. A random PCR screening system for the identification of type 1 human herpes simplex virus.

    Science.gov (United States)

    Yu, Xuelian; Shi, Bisheng; Gong, Yan; Zhang, Xiaonan; Shen, Silan; Qian, Fangxing; Gu, Shimin; Hu, Yunwen; Yuan, Zhenghong

    2009-10-01

    Several viral diseases exhibit measles-like symptoms. Differentiation of suspected cases of measles with molecular epidemiological techniques in the laboratory is useful for measles surveillance. In this study, a random PCR screening system was undertaken for the identification of isolates from patients with measles-like symptoms who exhibited cytopathic effects, but who had negative results for measles virus-specific reverse transcription (RT)-PCR and indirect immunofluorescence assays. Sequence analysis of random amplified PCR products showed that they were highly homologous to type 1 human herpes simplex virus (HSV-1). The results were further confirmed by an HSV-1-specific TaqMan real-time PCR assay. The random PCR screening system described in this study provides an efficient procedure for the identification of unknown viral pathogens. Measles-like symptoms can also be caused by HSV-1, suggesting the need to include HSV-1 in differential diagnoses of measles-like diseases.

  4. Human Papillomavirus Assays and Cytology in Primary Cervical Screening of Women Aged 30 Years and Above

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Bonde, Jesper; Preisler, Sarah

    2016-01-01

    In women aged ≥30 years, Human Papillomavirus testing will replace cytology for primary cervical screening. We compared Hybrid Capture 2 (HC2), cobas, CLART, and APTIMA HPV assays with cytology on 2869 SurePath samples from women undergoing routine screening at 30-65 years in Copenhagen, Denmark....... Women with cytological abnormalities were managed according to routine recommendations, with 92% completeness. Those with cytology-normal/HPV-positive samples (on any of the four assays) were invited for repeated cytology and HPV testing in 1.5 year, and 58% had additional testing. HPV testing detected...... more ≥CIN3 than cytology (HC2: 35, cobas, CLART: 37, APTIMA: 34, cytology: 31), although statistically the differences were not significant. Cobas and CLART detected significantly more ≥CIN2 than cytology (cobas, CLART: 49, cytology: 39). The proportion of women with false-positive test results...

  5. Screening of Compounds Toxicity against Human Monocytic cell line-THP-1 by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Pick Neora

    2004-01-01

    Full Text Available The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound's toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound's toxicity associated with cell death.

  6. A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs.

    Science.gov (United States)

    Karlas, Alexander; Berre, Stefano; Couderc, Thérèse; Varjak, Margus; Braun, Peter; Meyer, Michael; Gangneux, Nicolas; Karo-Astover, Liis; Weege, Friderike; Raftery, Martin; Schönrich, Günther; Klemm, Uwe; Wurzlbauer, Anne; Bracher, Franz; Merits, Andres; Meyer, Thomas F; Lecuit, Marc

    2016-05-12

    Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents.

  7. Translational analysis of mouse and human placental protein and mRNA reveals distinct molecular pathologies in human preeclampsia.

    Science.gov (United States)

    Cox, Brian; Sharma, Parveen; Evangelou, Andreas I; Whiteley, Kathie; Ignatchenko, Vladimir; Ignatchenko, Alex; Baczyk, Dora; Czikk, Marie; Kingdom, John; Rossant, Janet; Gramolini, Anthony O; Adamson, S Lee; Kislinger, Thomas

    2011-12-01

    Preeclampsia (PE) adversely impacts ~5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent

  8. Applying a gender lens on human papillomavirus infection: cervical cancer screening, HPV DNA testing, and HPV vaccination.

    Science.gov (United States)

    Branković, Ivan; Verdonk, Petra; Klinge, Ineke

    2013-02-08

    Our aim is to provide a state-of-the-art overview of knowledge on sex (biological) and gender (sociocultural) aspects of Human papillomavirus (HPV) and cervical cancer for educational purposes. Considerable disparities exist in cervical cancer incidences between different subgroups of women. We provide an outline on the crucial issues and debates based on the recent literature published in leading gender medicine journals. Intersectionality was applied in order to help categorise the knowledge. Key terms (HPV, cervical cancer) were screened in Gender Medicine, Journal of Women's Health and Women & Health from January 2005-June 2012. Additional searches were conducted for topics insufficiently mentioned, such as HPV vaccination of boys. In total, 71 publications were included (56 original papers, four reviews, six reports, three commentaries, one editorial and one policy statement). Research reveals complexity in the way various subgroups of women adhere to cervical screening. Less educated women, older women, uninsured women, homeless women, migrant women facing language barriers, women who have sex with women and obese women participate in Pap smears less frequently. A series of barriers can act to impede decisions to vaccinate against HPV. Both male and female controlled preventive methods and treatment measures should be developed in order to tackle HPV infection and different strategies are needed for different subgroups. A substantial discussion and research on alternative methods of prevention was and is lacking. In future research, sex and gender aspects of HPV-related diseases of boys and men as well as subgroup differences in HPV risk need to be addressed.

  9. Screening organic chemicals in commerce for emissions in the context of environmental and human exposure.

    Science.gov (United States)

    Breivik, Knut; Arnot, Jon A; Brown, Trevor N; McLachlan, Michael S; Wania, Frank

    2012-08-01

    Quantitative knowledge of organic chemical release into the environment is essential to understand and predict human exposure as well as to develop rational control strategies for any substances of concern. While significant efforts have been invested to characterize and screen organic chemicals for hazardous properties, relatively less effort has been directed toward estimating emissions and hence also risks. Here, a rapid throughput method to estimate emissions of discrete organic chemicals in commerce has been developed, applied and evaluated to support screening studies aimed at ranking and identifying chemicals of potential concern. The method builds upon information in the European Union Technical Guidance Document and utilizes information on quantities in commerce (production and/or import rates), chemical function (use patterns) and physical-chemical properties to estimate emissions to air, soil and water within the OECD for five stages of the chemical life-cycle. The method is applied to 16,029 discrete substances (identified by CAS numbers) from five national and international high production volume lists. As access to consistent input data remains fragmented or even impossible, particular attention is given to estimating, evaluating and discussing uncertainties in the resulting emission scenarios. The uncertainty for individual substances typically spans 3 to 4 orders of magnitude for this initial tier screening method. Information on uncertainties in emissions is useful as any screening or categorization methods which solely rely on threshold values are at risk of leading to a significant number of either false positives or false negatives. A limited evaluation of the screening method's estimates for a sub-set of about 100 substances, compared against independent and more detailed emission scenarios presented in various European Risk Assessment Reports, highlights that up-to-date and accurate information on quantities in commerce as well as a detailed

  10. Individual decision making in relation to participation in cardiovascular screening: a study of revealed and stated preferences.

    Science.gov (United States)

    Søgaard, Rikke; Lindholt, Jes; Gyrd-Hansen, Dorte

    2013-02-01

    The (cost-)effectiveness of a screening programme may be strongly influenced by the participation rate. The objective of this study was to compare participants' and non-participants' motives for the attendance decision as well as their overall preferences for participation in cardiovascular disease screening. This study sampled 1053 participants and 1006 non-participants from a screening trial and randomly allocated the participants to receive different levels of additional information about the screening programme. An ad hoc survey questionnaire about doubt and arguments in relation to the participation decision was given to participants and non-participants along with a contingent valuation task. Among participants, 5% had doubt about participation and the most frequent argument was that they did not want the test result. Among non-participants, 40% would reconsider their non-participation decision after having received additional information while the remainder 60% stood by their decision and provided explicit arguments for it. After having received additional information the participants still valued the programme significantly higher than non-participants, but the difference was relatively small. Participants and non-participants in cardiovascular screening programmes seem to have different strengths of preferences, which signals that their behavioural choice is founded in rational thinking. Furthermore, it appears that additional information and a second reflection about the participation decision may affect a substantial proportion of non-participants to reverse their decision, a finding that should receive policy interest.

  11. In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen

    Science.gov (United States)

    Plouffe, David; Brinker, Achim; McNamara, Case; Henson, Kerstin; Kato, Nobutaka; Kuhen, Kelli; Nagle, Advait; Adrián, Francisco; Matzen, Jason T.; Anderson, Paul; Nam, Tae-gyu; Gray, Nathanael S.; Chatterjee, Arnab; Janes, Jeff; Yan, S. Frank; Trager, Richard; Caldwell, Jeremy S.; Schultz, Peter G.; Zhou, Yingyao; Winzeler, Elizabeth A.

    2008-01-01

    The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of ≈1.7 million compounds, we identified a diverse collection of ≈6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities. PMID:18579783

  12. A synthetic interaction screen identifies factors selectively required for proliferation and TERT transcription in p53-deficient human cancer cells.

    Directory of Open Access Journals (Sweden)

    Li Xie

    Full Text Available Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi-based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53- human cancer cells. We find that compared to p53-competent (p53+ human cancer cell lines, diverse p53- human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53- cells, RNAi-mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53- but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53- cancer cells.

  13. Human Engineered Cardiac Tissues Created Using Induced Pluripotent Stem Cells Reveal Functional Characteristics of BRAF-Mediated Hypertrophic Cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Timothy J Cashman

    Full Text Available Hypertrophic cardiomyopathy (HCM is a leading cause of sudden cardiac death that often goes undetected in the general population. HCM is also prevalent in patients with cardio-facio-cutaneous syndrome (CFCS, which is a genetic disorder characterized by aberrant signaling in the RAS/MAPK signaling cascade. Understanding the mechanisms of HCM development in such RASopathies may lead to novel therapeutic strategies, but relevant experimental models of the human condition are lacking. Therefore, the objective of this study was to develop the first 3D human engineered cardiac tissue (hECT model of HCM. The hECTs were created using human cardiomyocytes obtained by directed differentiation of induced pluripotent stem cells derived from a patient with CFCS due to an activating BRAF mutation. The mutant myocytes were directly conjugated at a 3:1 ratio with a stromal cell population to create a tissue of defined composition. Compared to healthy patient control hECTs, BRAF-hECTs displayed a hypertrophic phenotype by culture day 6, with significantly increased tissue size, twitch force, and atrial natriuretic peptide (ANP gene expression. Twitch characteristics reflected increased contraction and relaxation rates and shorter twitch duration in BRAF-hECTs, which also had a significantly higher maximum capture rate and lower excitation threshold during electrical pacing, consistent with a more arrhythmogenic substrate. By culture day 11, twitch force was no longer different between BRAF and wild-type hECTs, revealing a temporal aspect of disease modeling with tissue engineering. Principal component analysis identified diastolic force as a key factor that changed from day 6 to day 11, supported by a higher passive stiffness in day 11 BRAF-hECTs. In summary, human engineered cardiac tissues created from BRAF mutant cells recapitulated, for the first time, key aspects of the HCM phenotype, offering a new in vitro model for studying intrinsic mechanisms and

  14. Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy

    Directory of Open Access Journals (Sweden)

    Johanna L. Höög

    2015-11-01

    Full Text Available Human ejaculates contain extracellular vesicles (EVs, that to a large extent are considered to originate from the prostate gland, and are often denominated “prostasomes.” These EVs are important for human fertility, for example by promoting sperm motility and by inducing immune tolerance of the female immune system to the spermatozoa. So far, the EVs present in human ejaculate have not been studied in their native state, inside the seminal fluid without prior purification and isolation procedures. Using cryo-electron microscopy and tomography, we performed a comprehensive inventory of human ejaculate EVs. The sample was neither centrifuged, fixed, filtered or sectioned, nor were heavy metals added. Approximately 1,500 extracellular structures were imaged and categorized. The extracellular environment of human ejaculate was found to be diverse, with 5 major subcategories of EVs and 6 subcategories of extracellular membrane compartments, including lamellar bodies. Furthermore, 3 morphological features, including electron density, double membrane bilayers and coated surface, are described in all subcategories. This study reveals that the extracellular environment in human ejaculate is multifaceted. Several novel morphological EV subcategories are identified and clues to their cellular origin may be found in their morphology. This inventory is therefore important for developing future experimental approaches, and to interpret previously published data to understand the role of EVs for human male fertility.

  15. Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A*

    Science.gov (United States)

    Han, Gil-Soo; Sreenivas, Avula; Choi, Mal-Gi; Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

    2005-01-01

    CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. PMID:16179339

  16. Screening for fecal carriage of MCR-producing Enterobacteriaceae in healthy humans and primary care patients

    Directory of Open Access Journals (Sweden)

    Katrin Zurfluh

    2017-03-01

    Full Text Available Abstract Background The extent of the occurrence of the plasmid-encoded colistin resistance genes mcr-1 and mcr-2 among humans is currently sparsely studied in Western Europe. Objectives To determine the occurrence of MCR-producing Enterobacteriaceae in fecal samples of healthy humans with high occupational exposure to food and primary care patients in Switzerland. Methods Stool samples from 1091 healthy individuals and fecal swabs from 53 primary care patients were screened for polymyxin-resistant Enterobacteriaceae using LB agar containing 4 mg/L colistin. Minimal inhibitory concentrations (MICs of colistin were determined for non-intrinsic colistin-resistant isolates. Isolates were screened by PCR for the presence of mcr-1 and mcr-2 genes. Results The fecal carriage rate of colistin resistant (MIC value >2 mg/l Enterobacteriaceae was 1.5% for healthy people and 3.8% for primary care patients. Isolates included Hafnia alvei (n = 9, Escherichia coli (n = 3, Enterobacter cloacae (n = 4, Klebsiella pneumoniae (n = 1 and Raoultella ornithinolytica (n = 1. None of the isolates harbored the mcr-1 or mcr-2 genes. Conclusions There is no evidence for the presence of MCR-producers in the fecal flora of healthy people or primary care patients. Therefore, the risk of transfer of mcr genes from animals, food or the environment to humans is likely to be low in Switzerland.

  17. Three-dimensional multispectral optoacoustic mesoscopy reveals melanin and blood oxygenation in human skin in vivo.

    Science.gov (United States)

    Schwarz, Mathias; Buehler, Andreas; Aguirre, Juan; Ntziachristos, Vasilis

    2016-01-01

    Optical imaging plays a major role in disease detection in dermatology. However, current optical methods are limited by lack of three-dimensional detection of pathophysiological parameters within skin. It was recently shown that single-wavelength optoacoustic (photoacoustic) mesoscopy resolves skin morphology, i.e. melanin and blood vessels within epidermis and dermis. In this work we employed illumination at multiple wavelengths for enabling three-dimensional multispectral optoacoustic mesoscopy (MSOM) of natural chromophores in human skin in vivo operating at 15-125 MHz. We employ a per-pulse tunable laser to inherently co-register spectral datasets, and reveal previously undisclosed insights of melanin, and blood oxygenation in human skin. We further reveal broadband absorption spectra of specific skin compartments. We discuss the potential of MSOM for label-free visualization of physiological biomarkers in skin in vivo. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Inter-species activity correlations reveal functional correspondences between monkey and human brain areas

    Science.gov (United States)

    Mantini, Dante; Hasson, Uri; Betti, Viviana; Perrucci, Mauro G.; Romani, Gian Luca; Corbetta, Maurizio; Orban, Guy A.; Vanduffel, Wim

    2012-01-01

    Evolution-driven functional changes in the primate brain are typically assessed by aligning monkey and human activation maps using cortical surface expansion models. These models use putative homologous areas as registration landmarks, assuming they are functionally correspondent. In cases where functional changes have occurred in an area, this assumption prohibits to reveal whether other areas may have assumed lost functions. Here we describe a method to examine functional correspondences across species. Without making spatial assumptions, we assess similarities in sensory-driven functional magnetic resonance imaging responses between monkey (Macaca mulatta) and human brain areas by means of temporal correlation. Using natural vision data, we reveal regions for which functional processing has shifted to topologically divergent locations during evolution. We conclude that substantial evolution-driven functional reorganizations have occurred, not always consistent with cortical expansion processes. This novel framework for evaluating changes in functional architecture is crucial to building more accurate evolutionary models. PMID:22306809

  19. High-grade cervical intraepithelial neoplasia in human papillomavirus self-sampling of screening non-attenders

    DEFF Research Database (Denmark)

    Lam, J U H; Elfström, K M; Ejegod, D. M.

    2018-01-01

    precancer lesions. Here, we compare the cervical intraepithelial neoplasia grade 2 or worse (⩾CIN2) detection rate between non-attenders who participated in self-sampling and women attending routine screening. METHODS: A total of 23 632 women who were qualified as non-attenders in the Copenhagen Region were......BACKGROUND: Self-sampling for human papillomavirus (HPV) offered to women who do not participate in cervical cancer screening is an increasingly popular method to increase screening coverage. The rationale behind self-sampling is that unscreened women harbour a high proportion of undetected...... higher detection rates for ⩾CIN2 than routine cytology-based screening, and similar detection rates as HPV and cytology co-testing. This reinforces the importance of self-sampling for screening non-attenders in organised cervical cancer screening.British Journal of Cancer advance online publication, 14...

  20. Correlates of women’s intentions to be screened for human papillomavirus for cervical cancer screening with an extended interval

    Directory of Open Access Journals (Sweden)

    Gina S. Ogilvie

    2016-03-01

    Full Text Available Abstract Background High-risk HPV DNA testing has been proposed as a primary tool for cervical cancer screening (HPV-CCS as an alternative to the Papanicolaou cytology- method. This study describes factors associated with women’s intentions to attend cervical cancer screening if high-risk HPV DNA testing (HPV-CCS was implemented as a primary screening tool, and if screening were conducted every 4 years starting after age 25. Methods This online survey was designed using the Theory of Planned Behaviour to assess factors that impact women’s intentions to attend HPV-CCS among women aged 25–69 upon exit of the HPV FOCAL trial. Univariate and regression analyses were performed to compare the demographic, sexual history, and smoking characteristics between women willing and unwilling to screen, and scales for intention to attend HPV-CCS. A qualitative analysis was performed by compiling and coding the comments section of the survey. Results Of the 981 women who completed the survey in full, only 51.4 % responded that they intended to attend HPV-CCS with a delayed start age and extended screening interval. Women who intended to screen were more likely to have higher education (AOR 0.59, 95 % CI [0.37, 0.93], while both positive attitudes (AOR 1.26, 95 % CI [1.23, 1.30] and perceived behavior control (AOR 1.06, 95 % CI [1.02, 1.10] were significant predictors of intention to screen. Among women who provided comments in the survey, a large number of women expressed fears about not being checked more than every 4 years, but 12 % stated that these fears may be alleviated by having more information. Conclusions Acceptability of increased screening intervals and starting age could be improved through enhanced education of benefits. Program planners should consider measures to assess and improve women’s knowledge, attitudes and beliefs prior to the implementation of new screening programs to avoid unintended consequences.

  1. Dried blood spots, valid screening for viral hepatitis and human immunodeficiency virus in real-life

    DEFF Research Database (Denmark)

    Mössner, Belinda K; Staugaard, Benjamin; Jensen, Janne

    2016-01-01

    AIM: To detect chronic hepatitis B (CHB), chronic hepatitis C (CHC) and human immunodeficiency virus (HIV) infections in dried blood spot (DBS) and compare these samples to venous blood sampling in real-life. METHODS: We included prospective patients with known viral infections from drug treatment......, but correctly classified 95% of the anti-HCV-positive patients with chronic and past infections. Anti-HBc and anti-HBS showed low sensitivity in DBS (68% and 42%). CONCLUSION: DBS sampling, combined with an automated analysis system, is a feasible screening method to diagnose chronic viral hepatitis and HIV...

  2. Identification of genes important for cutaneous function revealed by a large scale reverse genetic screen in the mouse.

    Directory of Open Access Journals (Sweden)

    Tia DiTommaso

    2014-10-01

    Full Text Available The skin is a highly regenerative organ which plays critical roles in protecting the body and sensing its environment. Consequently, morbidity and mortality associated with skin defects represent a significant health issue. To identify genes important in skin development and homeostasis, we have applied a high throughput, multi-parameter phenotype screen to the conditional targeted mutant mice generated by the Wellcome Trust Sanger Institute's Mouse Genetics Project (Sanger-MGP. A total of 562 different mouse lines were subjected to a variety of tests assessing cutaneous expression, macroscopic clinical disease, histological change, hair follicle cycling, and aberrant marker expression. Cutaneous lesions were associated with mutations in 23 different genes. Many of these were not previously associated with skin disease in the organ (Mysm1, Vangl1, Trpc4ap, Nom1, Sparc, Farp2, and Prkab1, while others were ascribed new cutaneous functions on the basis of the screening approach (Krt76, Lrig1, Myo5a, Nsun2, and Nf1. The integration of these skin specific screening protocols into the Sanger-MGP primary phenotyping pipelines marks the largest reported reverse genetic screen undertaken in any organ and defines approaches to maximise the productivity of future projects of this nature, while flagging genes for further characterisation.

  3. A Genomewide Screen for Tolerance to Cationic Drugs Reveals Genes Important for Potassium Homeostasis in Saccharomyces cerevisiae

    Czech Academy of Sciences Publication Activity Database

    Barreto, L.; Canadell, D.; Petrezsélyová, Silvia; Navarrete, C.; Marešová, Lydie; Peréz-Valle, J.; Herrera, R.; Olier, I.; Giraldo, J.; Sychrová, Hana; Yenush, L.; Ramos, J.; Ariňo, J.

    2011-01-01

    Roč. 10, č. 9 (2011), s. 1241-1250 ISSN 1535-9778 R&D Projects: GA MŠk(CZ) LC531; GA AV ČR(CZ) IAA500110801 Institutional research plan: CEZ:AV0Z50110509 Keywords : Potassium homeostasis * Saccharomyces cerevisiae * genomewide screen Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.604, year: 2011

  4. K -shell decomposition reveals hierarchical cortical organization of the human brain

    International Nuclear Information System (INIS)

    Lahav, Nir; Ksherim, Baruch; Havlin, Shlomo; Ben-Simon, Eti; Maron-Katz, Adi; Cohen, Reuven

    2016-01-01

    In recent years numerous attempts to understand the human brain were undertaken from a network point of view. A network framework takes into account the relationships between the different parts of the system and enables to examine how global and complex functions might emerge from network topology. Previous work revealed that the human brain features ‘small world’ characteristics and that cortical hubs tend to interconnect among themselves. However, in order to fully understand the topological structure of hubs, and how their profile reflect the brain’s global functional organization, one needs to go beyond the properties of a specific hub and examine the various structural layers that make up the network. To address this topic further, we applied an analysis known in statistical physics and network theory as k-shell decomposition analysis. The analysis was applied on a human cortical network, derived from MRI/DSI data of six participants. Such analysis enables us to portray a detailed account of cortical connectivity focusing on different neighborhoods of inter-connected layers across the cortex. Our findings reveal that the human cortex is highly connected and efficient, and unlike the internet network contains no isolated nodes. The cortical network is comprised of a nucleus alongside shells of increasing connectivity that formed one connected giant component, revealing the human brain’s global functional organization. All these components were further categorized into three hierarchies in accordance with their connectivity profile, with each hierarchy reflecting different functional roles. Such a model may explain an efficient flow of information from the lowest hierarchy to the highest one, with each step enabling increased data integration. At the top, the highest hierarchy (the nucleus) serves as a global interconnected collective and demonstrates high correlation with consciousness related regions, suggesting that the nucleus might serve as a

  5. RNAi Reveals Phase-Specific Global Regulators of Human Somatic Cell Reprogramming

    Directory of Open Access Journals (Sweden)

    Cheng-Xu Delon Toh

    2016-06-01

    Full Text Available Incomplete knowledge of the mechanisms at work continues to hamper efforts to maximize reprogramming efficiency. Here, we present a systematic genome-wide RNAi screen to determine the global regulators during the early stages of human reprogramming. Our screen identifies functional repressors and effectors that act to impede or promote the reprogramming process. Repressors and effectors form close interacting networks in pathways, including RNA processing, G protein signaling, protein ubiquitination, and chromatin modification. Combinatorial knockdown of five repressors (SMAD3, ZMYM2, SFRS11, SAE1, and ESET synergistically resulted in ∼85% TRA-1-60-positive cells. Removal of the novel splicing factor SFRS11 during reprogramming is accompanied by rapid acquisition of pluripotency-specific spliced forms. Mechanistically, SFRS11 regulates exon skipping and mutually exclusive splicing of transcripts in genes involved in cell differentiation, mRNA splicing, and chromatin modification. Our study provides insights into the reprogramming process, which comprises comprehensive and multi-layered transcriptional, splicing, and epigenetic machineries.

  6. Analysis of human reticulocyte genes reveals altered erythropoiesis: potential use to detect recombinant human erythropoietin doping.

    Science.gov (United States)

    Varlet-Marie, Emmanuelle; Audran, Michel; Lejeune, Mireille; Bonafoux, Béatrice; Sicart, Marie-Therese; Marti, Jacques; Piquemal, David; Commes, Thérèse

    2004-08-01

    Enhancement of oxygen delivery to tissues is associated with improved sporting performance. One way of enhancing oxygen delivery is to take recombinant human erythropoietin (rHuEpo), which is an unethical and potentially dangerous practice. However, detection of the use of rHuEpo remains difficult in situations such as: i) several days after the end of treatment ii) when a treatment with low doses is conducted iii) if the rHuEpo effect is increased by other substances. In an attempt to detect rHuEpo abuse, we selected erythroid gene markers from a SAGE library and analyzed the effects of rHuEpo administration on expression of the HBB, FTL and OAZ genes. Ten athletes were assigned to the rHuEpo or placebo group. The rHuEpo group received subcutaneous injections of rHuEpo (50 UI/kg three times a week, 4 weeks; 20 UI/kg three times a week, 2 weeks). HBB, FTL and OAZ gene profiles were monitored by real time-polymerase chain reaction (PCR) quantification during and for 3 weeks after drug administration. The global analysis of these targeted genes detected in whole blood samples showed a characteristic profile of subjects misusing rHuEpo with a increase above the threshold levels. The individual analysis of OAZ mRNA seemed indicative of rHuEpo treatment. The performance-enhancing effect of rHuEpo treatment is greater than the duration of hematologic changes associated with rHuEpo misuse. Although direct electrophoretic methods to detect rHuEpo have been developed, recombinant isoforms of rHuEpo are not detectable some days after the last subcutaneous injection. To overcome these limitations indirect OFF models have been developed. Our data suggest that, in the near future, it will be possible to consolidate results achievable with the OFF models by analyzing selected erythroid gene markers as a supplement to indirect methods.

  7. Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening.

    Science.gov (United States)

    Smith, Alec S T; Macadangdang, Jesse; Leung, Winnie; Laflamme, Michael A; Kim, Deok-Ho

    Improved methodologies for modeling cardiac disease phenotypes and accurately screening the efficacy and toxicity of potential therapeutic compounds are actively being sought to advance drug development and improve disease modeling capabilities. To that end, much recent effort has been devoted to the development of novel engineered biomimetic cardiac tissue platforms that accurately recapitulate the structure and function of the human myocardium. Within the field of cardiac engineering, induced pluripotent stem cells (iPSCs) are an exciting tool that offer the potential to advance the current state of the art, as they are derived from somatic cells, enabling the development of personalized medical strategies and patient specific disease models. Here we review different aspects of iPSC-based cardiac engineering technologies. We highlight methods for producing iPSC-derived cardiomyocytes (iPSC-CMs) and discuss their application to compound efficacy/toxicity screening and in vitro modeling of prevalent cardiac diseases. Special attention is paid to the application of micro- and nano-engineering techniques for the development of novel iPSC-CM based platforms and their potential to advance current preclinical screening modalities. Published by Elsevier Inc.

  8. Recent adaptive events in human brain revealed by meta-analysis of positively selected genes.

    Directory of Open Access Journals (Sweden)

    Yue Huang

    Full Text Available BACKGROUND AND OBJECTIVES: Analysis of positively-selected genes can help us understand how human evolved, especially the evolution of highly developed cognitive functions. However, previous works have reached conflicting conclusions regarding whether human neuronal genes are over-represented among genes under positive selection. METHODS AND RESULTS: We divided positively-selected genes into four groups according to the identification approaches, compiling a comprehensive list from 27 previous studies. We showed that genes that are highly expressed in the central nervous system are enriched in recent positive selection events in human history identified by intra-species genomic scan, especially in brain regions related to cognitive functions. This pattern holds when different datasets, parameters and analysis pipelines were used. Functional category enrichment analysis supported these findings, showing that synapse-related functions are enriched in genes under recent positive selection. In contrast, immune-related functions, for instance, are enriched in genes under ancient positive selection revealed by inter-species coding region comparison. We further demonstrated that most of these patterns still hold even after controlling for genomic characteristics that might bias genome-wide identification of positively-selected genes including gene length, gene density, GC composition, and intensity of negative selection. CONCLUSION: Our rigorous analysis resolved previous conflicting conclusions and revealed recent adaptation of human brain functions.

  9. Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis

    Science.gov (United States)

    Bos, Kirsten I.; Harkins, Kelly M.; Herbig, Alexander; Coscolla, Mireia; Weber, Nico; Comas, Iñaki; Forrest, Stephen A.; Bryant, Josephine M.; Harris, Simon R.; Schuenemann, Verena J.; Campbell, Tessa J.; Majander, Kerrtu; Wilbur, Alicia K.; Guichon, Ricardo A.; Wolfe Steadman, Dawnie L.; Cook, Della Collins; Niemann, Stefan; Behr, Marcel A.; Zumarraga, Martin; Bastida, Ricardo; Huson, Daniel; Nieselt, Kay; Young, Douglas; Parkhill, Julian; Buikstra, Jane E.; Gagneux, Sebastien; Stone, Anne C.; Krause, Johannes

    2015-01-01

    Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact1. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World2. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch3, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-old mycobacterial genomes from Peruvian human skeletons, revealing that a member of the M. tuberculosis complex caused human disease before contact. The ancient strains are distinct from known human-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean. PMID:25141181

  10. The tomography of human mobility - what do shortest-path trees reveal?

    Energy Technology Data Exchange (ETDEWEB)

    Thiemann, Christian [Eng. Sci. and Appl. Math, Northwestern University, Evanston, IL (United States); Max-Planck-Institute for Dynamics and Self-Organization, Goettingen (Germany); Grady, Daniel; Brockmann, Dirk [Eng. Sci. and Appl. Math, Northwestern University, Evanston, IL (United States)

    2010-07-01

    Similar to illustrating the anatomy of organs using pictures of tissue slices taken at various depths, we construct shortest-path trees of different nodes to create tomograms of large-scale human mobility networks. This tomography allows us to measure global properties of the system conditioned on a reference location in the network to gain a fuller characterization of a node. It also suggests a canonical coordinate system for representing complex networks and dynamical processes thereon in a simplified way, revealing a new symmetry in the human mobility networks we investigated. Furthermore, introducing the notion of tree similarity, we devised a new technique for clustering nodes with similar topological footprint, yielding a unique and efficient method for community identification and topological backbone extraction. We applied these methods to a multi-scale human mobility network obtained from the dollar-bill-tracking site wheresgoerge.com and to the U.S. and world-wide air transportation network.

  11. Human papilloma virus (HPV) prophylactic vaccination: challenges for public health and implications for screening.

    Science.gov (United States)

    Adams, M; Jasani, B; Fiander, A

    2007-04-20

    Prophylactic vaccination against high risk human papilloma virus (HPV) 16 and 18 represents an exciting means of protection against HPV related malignancy. However, this strategy alone, even if there is a level of cross protection against other oncogenic viruses, cannot completely prevent cervical cancer. In some developed countries cervical screening programmes have reduced the incidence of invasive cervical cancer by up to 80% although this decline has now reached a plateau with current cancers occurring in patients who have failed to attend for screening or where the sensitivity of the tests have proved inadequate. Cervical screening is inevitably associated with significant anxiety for the many women who require investigation and treatment following abnormal cervical cytology. However, it is vitally important to stress the need for continued cervical screening to complement vaccination in order to optimise prevention in vaccinees and prevent cervical cancer in older women where the value of vaccination is currently unclear. It is likely that vaccination will ultimately change the natural history of HPV disease by reducing the influence of the highly oncogenic types HPV 16 and 18. In the long term this is likely to lead to an increase in recommended screening intervals. HPV vaccination may also reduce the positive predictive value of cervical cytology by reducing the number of truly positive abnormal smears. Careful consideration is required to ensure vaccination occurs at an age when the vaccine is most effective immunologically and when uptake is likely to be high. Antibody titres following vaccination in girls 12-16 years have been shown to be significantly higher than in older women, favouring vaccination in early adolescence prior contact with the virus. Highest prevalence rates for HPV infection are seen following the onset of sexual activity and therefore vaccination would need to be given prior to sexual debut. Since 20% of adolescents are sexually

  12. Knowledge and Intention to Participate in Cervical Cancer Screening after the Human Papillomavirus Vaccine

    Science.gov (United States)

    Price, Rebecca Anhang; Koshiol, Jill; Kobrin, Sarah; Tiro, Jasmin A.

    2011-01-01

    Background If women who receive the human papillomavirus (HPV) vaccine are unduly reassured about the cancer prevention benefits of vaccination, they may choose not to participate in screening, thereby increasing their risk for cervical cancer. This study assesses adult women’s knowledge of the need to continue cervical cancer screening after HPV vaccination, describes Pap test intentions of vaccinated young adult women, and evaluates whether knowledge and intentions differ across groups at greatest risk for cervical cancer. Methods Data were from the 2008 Health Information National Trends Survey (HINTS) and the 2008 National Health Interview Survey (NHIS), which initiated data collection approximately 18 months after the first FDA approval of an HPV vaccine. We calculated associations between independent variables and the outcomes using chi-square tests. Results Of 1,586 female HINTS respondents ages 18 through 74, 95.6% knew that HPV-vaccinated women should continue to receive Pap tests. This knowledge did not vary significantly by race/ethnicity, education, income, or healthcare access. Among 1,101 female NHIS respondents ages 18 to 26 who had ever received a Pap test, the proportion (12.7%; n = 139) who reported receipt of the HPV vaccine were more likely than those not vaccinated to plan to receive a Pap test within three years (98.1% vs. 92.5%, pknowledge and intention to participate in Pap testing after HPV vaccination. The vast majority of young adult women who received the HPV vaccine within its first two years on the market intend to participate in cervical cancer screening in the near future. Future studies are needed to examine whether those vaccinated in adolescence will become aware of, and adhere to, screening guidelines as they become eligible. PMID:21473953

  13. Identification by virtual screening and in vitro testing of human DOPA decarboxylase inhibitors.

    Directory of Open Access Journals (Sweden)

    Frederick Daidone

    Full Text Available Dopa decarboxylase (DDC, a pyridoxal 5'-phosphate (PLP enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD. PD is a neurodegenerative disease mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a to use virtual screening to identify potential human DDC inhibitors and (b to evaluate the reliability of our virtual-screening (VS protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.

  14. Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening.

    Science.gov (United States)

    Tong, Zhi-Bin; Hogberg, Helena; Kuo, David; Sakamuru, Srilatha; Xia, Menghang; Smirnova, Lena; Hartung, Thomas; Gerhold, David

    2017-02-01

    More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Genome editing reveals a role for OCT4 in human embryogenesis.

    Science.gov (United States)

    Fogarty, Norah M E; McCarthy, Afshan; Snijders, Kirsten E; Powell, Benjamin E; Kubikova, Nada; Blakeley, Paul; Lea, Rebecca; Elder, Kay; Wamaitha, Sissy E; Kim, Daesik; Maciulyte, Valdone; Kleinjung, Jens; Kim, Jin-Soo; Wells, Dagan; Vallier, Ludovic; Bertero, Alessandro; Turner, James M A; Niakan, Kathy K

    2017-10-05

    Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

  16. New Details of the Human Corneal Limbus Revealed With Second Harmonic Generation Imaging.

    Science.gov (United States)

    Park, Choul Yong; Lee, Jimmy K; Zhang, Cheng; Chuck, Roy S

    2015-09-01

    To report novel findings of the human corneal limbus by using second harmonic generation (SHG) imaging. Corneal limbus was imaged by using an inverted two-photon excitation fluorescence microscope. Laser (Ti:Sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of SHG and autofluorescence (AF) were collected through a 425/30-nm emission filter and a 525/45-emission filter, respectively. Multiple, consecutive, and overlapping image stacks (z-stack) were acquired for the corneal limbal area. Two novel collagen structures were revealed by SHG imaging at the limbus: an anterior limbal cribriform layer and presumed anchoring fibers. Anterior limbal cribriform layer is an intertwined reticular collagen architecture just beneath the limbal epithelial niche and is located between the peripheral cornea and Tenon's/scleral tissue. Autofluorescence imaging revealed high vascularity in this structure. Central to the anterior limbal cribriform layer, radial strands of collagen were found to connect the peripheral cornea to the limbus. These presumed anchoring fibers have both collagen and elastin and were found more extensively in the superficial layers than deep layer and were absent in very deep limbus near Schlemm's canal. By using SHG imaging, new details of the collagen architecture of human corneal limbal area were elucidated. High resolution images with volumetric analysis revealed two novel collagen structures.

  17. Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family

    International Nuclear Information System (INIS)

    Vanderslice, P.; Ballinger, S.M.; Tam, E.K.; Goldstein, S.M.; Craik, C.S.; Caughey, G.H.

    1990-01-01

    Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the ∼1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family

  18. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells

    DEFF Research Database (Denmark)

    Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas

    2016-01-01

    to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially...... kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified...... as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human...

  19. Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

    Directory of Open Access Journals (Sweden)

    Ellen C Kammula

    Full Text Available HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

  20. Human iPSC Glial Mouse Chimeras Reveal Glial Contributions to Schizophrenia

    DEFF Research Database (Denmark)

    Windrem, Martha S.; Osipovitch, Mikhail; Liu, Zhengshan

    2017-01-01

    with childhood-onset SCZ. After neonatal implantation into myelin-deficient shiverer mice, SCZ GPCs showed premature migration into the cortex, leading to reduced white matter expansion and hypomyelination relative to controls. The SCZ glial chimeras also showed delayed astrocytic differentiation and abnormal...... astrocytic morphologies. When established in myelin wild-type hosts, SCZ glial mice showed reduced prepulse inhibition and abnormal behavior, including excessive anxiety, antisocial traits, and disturbed sleep. RNA-seq of cultured SCZ human glial progenitor cells (hGPCs) revealed disrupted glial...

  1. Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier

    DEFF Research Database (Denmark)

    Dreier, Jes; Sørensen, Jens A; Brewer, Jonathan R

    2016-01-01

    In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human...... skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm...... liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers...

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

    Science.gov (United States)

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

    2015-11-01

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

  3. Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

    Science.gov (United States)

    Džunková, Mária; D'Auria, Giuseppe; Pérez-Villarroya, David; Moya, Andrés

    2012-01-01

    Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.

  4. Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

    Directory of Open Access Journals (Sweden)

    Mária Džunková

    Full Text Available Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.

  5. A joint model of persistent human papillomavirus infection and cervical cancer risk: Implications for cervical cancer screening

    OpenAIRE

    Katki, Hormuzd A.; Cheung, Li C.; Fetterman, Barbara; Castle, Philip E.; Sundaram, Rajeshwari

    2015-01-01

    New cervical cancer screening guidelines in the US and many European countries recommend that women get tested for human papillomavirus (HPV). To inform decisions about screening intervals, we calculate the increase in precancer/cancer risk per year of continued HPV infection. However, both time to onset of precancer/cancer and time to HPV clearance are interval-censored, and onset of precancer/cancer strongly informatively censors HPV clearance. We analyze this bivariate informatively interv...

  6. Revealing the cerebello-ponto-hypothalamic pathway in the human brain.

    Science.gov (United States)

    Kamali, Arash; Karbasian, Niloofar; Rabiei, Pejman; Cano, Andres; Riascos, Roy F; Tandon, Nitin; Arevalo, Octavio; Ocasio, Laura; Younes, Kyan; Khayat-Khoei, Mahsa; Mirbagheri, Saeedeh; Hasan, Khader M

    2018-04-16

    The cerebellum is shown to be involved in some limbic functions of the human brain such as emotion and affect. The major connection of the cerebellum with the limbic system is known to be through the cerebello-hypothalamic pathways. The consensus is that the projections from the cerebellar nuclei to the limbic system, and particularly the hypothalamus, or from the hypothalamus to the cerebellar nuclei, are through multisynaptic pathways in the bulbar reticular formation. The detailed anatomy of the pathways responsible for mediating these responses, however, is yet to be determined. Diffusion tensor imaging may be helpful in better visualizing the surgical anatomy of the cerebello-ponto-hypothalamic (CPH) pathway. This study aimed to investigate the utility of high-spatial-resolution diffusion tensor tractography for mapping the trajectory of the CPH tract in the human brain. Fifteen healthy adults were studied. We delineated, for the first time, the detailed trajectory of the CPH tract of the human brain in fifteen normal adult subjects using high-spatial-resolution diffusion tensor tractography. We further revealed the close relationship of the CPH tract with the optic tract, temporo-pontine tract, amygdalofugal tract and the fornix in the human brain. Copyright © 2018. Published by Elsevier B.V.

  7. A directed RNAi screen based on larval growth arrest reveals new modifiers of C. elegans insulin signaling.

    Directory of Open Access Journals (Sweden)

    Ola Billing

    Full Text Available Genes regulating Caenorhabditis elegans insulin/IGF signaling (IIS have largely been identified on the basis of their involvement in dauer development or longevity. A third IIS phenotype is the first larval stage (L1 diapause, which is also influenced by asna-1, a regulator of DAF-28/insulin secretion. We reasoned that new regulators of IIS strength might be identified in screens based on the L1 diapause and the asna-1 phenotype. Eighty- six genes were selected for analysis by virtue of their predicted interaction with ASNA-1 and screened for asna-1-like larval arrest. ykt-6, mrps-2, mrps-10 and mrpl-43 were identified as genes which, when inactivated, caused larval arrest without any associated feeding defects. Several tests indicated that IIS strength was weaker and that insulin secretion was defective in these animals. This study highlights the role of the Golgi network and the mitochondria in insulin secretion and provides a new list of genes that modulate IIS in C. elegans.

  8. A direct pre-screen for marine bacteria producing compounds inhibiting quorum sensing reveals diverse planktonic bacteria that are bioactive.

    Science.gov (United States)

    Linthorne, Jamie S; Chang, Barbara J; Flematti, Gavin R; Ghisalberti, Emilio L; Sutton, David C

    2015-02-01

    A promising new strategy in antibacterial research is inhibition of the bacterial communication system termed quorum sensing. In this study, a novel and rapid pre-screening method was developed to detect the production of chemical inhibitors of this system (quorum-quenching compounds) by bacteria isolated from marine and estuarine waters. This method involves direct screening of mixed populations on an agar plate, facilitating specific isolation of bioactive colonies. The assay showed that between 4 and 46 % of culturable bacteria from various samples were bioactive, and of the 95 selectively isolated bacteria, 93.7 % inhibited Vibrio harveyi bioluminescence without inhibiting growth, indicating potential production of quorum-quenching compounds. Of the active isolates, 21 % showed further activity against quorum-sensing-regulated pigment production by Serratia marcescens. The majority of bioactive isolates were identified by 16S ribosomal DNA (rDNA) amplification and sequencing as belonging to the genera Vibrio and Pseudoalteromonas. Extracts of two strongly bioactive Pseudoalteromonas isolates (K1 and B2) were quantitatively assessed for inhibition of growth and quorum-sensing-regulated processes in V. harveyi, S. marcescens and Chromobacterium violaceum. Extracts of the isolates reduced V. harveyi bioluminescence by as much as 98 % and C. violaceum pigment production by 36 % at concentrations which had no adverse effect on growth. The activity found in the extracts indicated that the isolates may produce quorum-quenching compounds. This study further supports the suggestion that quorum quenching may be a common attribute among culturable planktonic marine and estuarine bacteria.

  9. Docking-based Screening of Ficus religiosa Phytochemicals as Inhibitors of Human Histamine H2 Receptor.

    Science.gov (United States)

    Chaudhary, Amit; Yadav, Birendra Singh; Singh, Swati; Maurya, Pramod Kumar; Mishra, Alok; Srivastva, Shweta; Varadwaj, Pritish Kumar; Singh, Nand Kumar; Mani, Ashutosh

    2017-10-01

    Ficus religiosa L. is generally known as Peepal and belongs to family Moraceae . The tree is a source of many compounds having high medicinal value. In gastrointestinal tract, histamine H2 receptors have key role in histamine-stimulated gastric acid secretion. Their over stimulation causes its excessive production which is responsible for gastric ulcer. This study aims to screen the range of phytochemicals present in F. religiosa for binding with human histamine H2 and identify therapeutics for a gastric ulcer from the plant. In this work, a 3D-structure of human histamine H2 receptor was modeled by using homology modeling and the predicted model was validated using PROCHECK. Docking studies were also performed to assess binding affinities between modeled receptor and 34 compounds. Molecular dynamics simulations were done to identify most stable receptor-ligand complexes. Absorption, distribution, metabolism, excretion, and screening was done to evaluate pharmacokinetic properties of compounds. The results suggest that seven ligands, namely, germacrene, bergaptol, lanosterol, Ergost-5-en-3beta-ol, α-amyrin acetate, bergapten, and γ-cadinene showed better binding affinities. Among seven phytochemicals, lanosterol and α-amyrin acetate were found to have greater stability during simulation studies. These two compounds may be a suitable therapeutic agent against histamine H2 receptor. This study was performed to screen antiulcer compounds from F. religiosa . Molecular modeling, molecular docking and MD simulation studies were performed with selected phytochemicals from F. religiosa . The analysis suggests that Lanosterol and α-amyrin may be a suitable therapeutic agent against histamine H2 receptor. This study facilitates initiation of the herbal drug discovery process for the antiulcer activity. Abbreviations used: ADMET: Absorption, distribution, metabolism, excretion and toxicity, DOPE: Discrete Optimized Potential Energy, OPLS: Optimized potential for liquid

  10. Screening, sensitivity, and uncertainty for the CREAM method of Human Reliability Analysis

    International Nuclear Information System (INIS)

    Bedford, Tim; Bayley, Clare; Revie, Matthew

    2013-01-01

    This paper reports a sensitivity analysis of the Cognitive Reliability and Error Analysis Method for Human Reliability Analysis. We consider three different aspects: the difference between the outputs of the Basic and Extended methods, on the same HRA scenario; the variability in outputs through the choices made for common performance conditions (CPCs); and the variability in outputs through the assignment of choices for cognitive function failures (CFFs). We discuss the problem of interpreting categories when applying the method, compare its quantitative structure to that of first generation methods and discuss also how dependence is modelled with the approach. We show that the control mode intervals used in the Basic method are too narrow to be consistent with the Extended method. This motivates a new screening method that gives improved accuracy with respect to the Basic method, in the sense that (on average) halves the uncertainty associated with the Basic method. We make some observations on the design of a screening method that are generally applicable in Risk Analysis. Finally, we propose a new method of combining CPC weights with nominal probabilities so that the calculated probabilities are always in range (i.e. between 0 and 1), while satisfying sensible properties that are consistent with the overall CREAM method

  11. [Usefulness of human papillomavirus testing in anal intraepithelial neoplasia screening in a risk behaviour population].

    Science.gov (United States)

    Padilla-España, Laura; Repiso-Jiménez, Bosco; Fernández-Sánchez, Fernando; Frieyro-Elicegui, Marta; Fernández-Morano, Teresa; Pereda, Teresa; Rivas-Ruiz, Francisco; Redondo, Maximino; de-Troya Martín, Magdalena

    2014-11-01

    The incidence of intraepithelial anal neoplasia is increasing in certain risk behaviour groups, and human papillomavirus (HPV) infection is involved in its pathogenesis. The systematic use of anal cytology, and more recently HPV detection by hybrid capture and genotyping, have been introduced into screening programs in recent decades. A retrospective cohort study was carried out on individuals with risk behaviours of developing intraepithelial anal neoplasia and who attended Sexually Transmitted Infections clinics in the Dermatology area of the Hospital Costa del Sol from January 2010 to December 2012. The intraepithelial anal neoplasia screening was performed using anal cytology and HPV genotyping. Half (50%) of the study population were HIV positive. A high frequency of anal dysplasia and presence of HPV in cytology (82.1%) and genotype (79%) was found. A statistically significant association (P<.005) was obtained between the presence of high-risk HPV genotypes and the presence of high-grade dysplasia in the second directed cytology. HPV genotyping enabled 17 cases (22%) of severe dysplasia to be identified that were under-diagnosed in the first cytology. Cases of high-grade dysplasia can be under-diagnosed by a first anal cytology. Detection of HPV can supplement this procedure, leading to the identification of those patients most at risk of developing high-grade anal dysplasia. Copyright © 2013 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  12. Patterns of cervical coinfection with multiple human papilloma virus types in a screening population in Denmark

    DEFF Research Database (Denmark)

    Goldman, Bryan; Rebolj, Matejka; Rygaard, Carsten

    2013-01-01

    Patterns of cervical human papillomavirus (HPV) infection suggest that HPV genotypes are not independent of each other. This may be explained by risk factors common to all HPV infections, but type-specific biological factors may also play a role. This raises the question of whether widespread use...... of the quadrivalent vaccine (covering HPV6, 11, 16, 18) may indirectly affect the prevalence of any non-vaccine types. Routine screening samples from 5014 Danish women were tested for 35 HPV genotypes (including 13 high-risk) using the Genomica CLART(®) HPV2 kit, which is a low-density microarray based on PCR...... amplification. Simulation studies were performed both under independence between genotypes and under a common dependence structure as would arise from common risk factors, and simulation results were compared to observed coinfection patterns. Overall HPV prevalence was 37.4%, with multiple infections in 17...

  13. Phenotypic Screening Identifies Modulators of Amyloid Precursor Protein Processing in Human Stem Cell Models of Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Philip W. Brownjohn

    2017-04-01

    Full Text Available Summary: Human stem cell models have the potential to provide platforms for phenotypic screens to identify candidate treatments and cellular pathways involved in the pathogenesis of neurodegenerative disorders. Amyloid precursor protein (APP processing and the accumulation of APP-derived amyloid β (Aβ peptides are key processes in Alzheimer's disease (AD. We designed a phenotypic small-molecule screen to identify modulators of APP processing in trisomy 21/Down syndrome neurons, a complex genetic model of AD. We identified the avermectins, commonly used as anthelmintics, as compounds that increase the relative production of short Aβ peptides at the expense of longer, potentially more toxic peptides. Further studies demonstrated that this effect is not due to an interaction with the core γ-secretase responsible for Aβ production. This study demonstrates the feasibility of phenotypic drug screening in human stem cell models of Alzheimer-type dementia, and points to possibilities for indirectly modulating APP processing, independently of γ-secretase modulation. : In this article, Livesey and colleagues perform a phenotypic drug screen in a human stem cell model of Alzheimer's disease. The anthelminthic avermectins are identified as a family of compounds that increase the production of short Aβ peptides over longer more toxic Aβ forms. The effect is analogous to existing γ-secretase modulators, but is independent of the core γ-secretase complex. Keywords: neural stem cells, Alzheimer's disease, phenotypic screening, iPSCs, human neurons, dementia, Down syndrome, amyloid beta, ivermectin, selamectin

  14. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

    Directory of Open Access Journals (Sweden)

    Ingrid R. Cordeiro

    2015-09-01

    Full Text Available Human adipose-derived stromal cells (hADSC are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1 regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

  15. Revealing topological organization of human brain functional networks with resting-state functional near infrared spectroscopy.

    Science.gov (United States)

    Niu, Haijing; Wang, Jinhui; Zhao, Tengda; Shu, Ni; He, Yong

    2012-01-01

    The human brain is a highly complex system that can be represented as a structurally interconnected and functionally synchronized network, which assures both the segregation and integration of information processing. Recent studies have demonstrated that a variety of neuroimaging and neurophysiological techniques such as functional magnetic resonance imaging (MRI), diffusion MRI and electroencephalography/magnetoencephalography can be employed to explore the topological organization of human brain networks. However, little is known about whether functional near infrared spectroscopy (fNIRS), a relatively new optical imaging technology, can be used to map functional connectome of the human brain and reveal meaningful and reproducible topological characteristics. We utilized resting-state fNIRS (R-fNIRS) to investigate the topological organization of human brain functional networks in 15 healthy adults. Brain networks were constructed by thresholding the temporal correlation matrices of 46 channels and analyzed using graph-theory approaches. We found that the functional brain network derived from R-fNIRS data had efficient small-world properties, significant hierarchical modular structure and highly connected hubs. These results were highly reproducible both across participants and over time and were consistent with previous findings based on other functional imaging techniques. Our results confirmed the feasibility and validity of using graph-theory approaches in conjunction with optical imaging techniques to explore the topological organization of human brain networks. These results may expand a methodological framework for utilizing fNIRS to study functional network changes that occur in association with development, aging and neurological and psychiatric disorders.

  16. Transcriptomic analysis of human retinal detachment reveals both inflammatory response and photoreceptor death.

    Directory of Open Access Journals (Sweden)

    Marie-Noëlle Delyfer

    Full Text Available BACKGROUND: Retinal detachment often leads to a severe and permanent loss of vision and its therapeutic management remains to this day exclusively surgical. We have used surgical specimens to perform a differential analysis of the transcriptome of human retinal tissues following detachment in order to identify new potential pharmacological targets that could be used in combination with surgery to further improve final outcome. METHODOLOGY/PRINCIPAL FINDINGS: Statistical analysis reveals major involvement of the immune response in the disease. Interestingly, using a novel approach relying on coordinated expression, the interindividual variation was monitored to unravel a second crucial aspect of the pathological process: the death of photoreceptor cells. Within the genes identified, the expression of the major histocompatibility complex I gene HLA-C enables diagnosis of the disease, while PKD2L1 and SLCO4A1 -which are both down-regulated- act synergistically to provide an estimate of the duration of the retinal detachment process. Our analysis thus reveals the two complementary cellular and molecular aspects linked to retinal detachment: an immune response and the degeneration of photoreceptor cells. We also reveal that the human specimens have a higher clinical value as compared to artificial models that point to IL6 and oxidative stress, not implicated in the surgical specimens studied here. CONCLUSIONS/SIGNIFICANCE: This systematic analysis confirmed the occurrence of both neurodegeneration and inflammation during retinal detachment, and further identifies precisely the modification of expression of the different genes implicated in these two phenomena. Our data henceforth give a new insight into the disease process and provide a rationale for therapeutic strategies aimed at limiting inflammation and photoreceptor damage associated with retinal detachment and, in turn, improving visual prognosis after retinal surgery.

  17. Improving virtual screening predictive accuracy of Human kallikrein 5 inhibitors using machine learning models.

    Science.gov (United States)

    Fang, Xingang; Bagui, Sikha; Bagui, Subhash

    2017-08-01

    The readily available high throughput screening (HTS) data from the PubChem database provides an opportunity for mining of small molecules in a variety of biological systems using machine learning techniques. From the thousands of available molecular descriptors developed to encode useful chemical information representing the characteristics of molecules, descriptor selection is an essential step in building an optimal quantitative structural-activity relationship (QSAR) model. For the development of a systematic descriptor selection strategy, we need the understanding of the relationship between: (i) the descriptor selection; (ii) the choice of the machine learning model; and (iii) the characteristics of the target bio-molecule. In this work, we employed the Signature descriptor to generate a dataset on the Human kallikrein 5 (hK 5) inhibition confirmatory assay data and compared multiple classification models including logistic regression, support vector machine, random forest and k-nearest neighbor. Under optimal conditions, the logistic regression model provided extremely high overall accuracy (98%) and precision (90%), with good sensitivity (65%) in the cross validation test. In testing the primary HTS screening data with more than 200K molecular structures, the logistic regression model exhibited the capability of eliminating more than 99.9% of the inactive structures. As part of our exploration of the descriptor-model-target relationship, the excellent predictive performance of the combination of the Signature descriptor and the logistic regression model on the assay data of the Human kallikrein 5 (hK 5) target suggested a feasible descriptor/model selection strategy on similar targets. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Mitochondrial damage and cytoskeleton reorganization in human dermal fibroblasts exposed to artificial visible light similar to screen-emitted light.

    Science.gov (United States)

    Rascalou, Adeline; Lamartine, Jérôme; Poydenot, Pauline; Demarne, Frédéric; Bechetoille, Nicolas

    2018-05-05

    Artificial visible light is everywhere in modern life. Social communication confronts us with screens of all kinds, and their use is on the rise. We are therefore increasingly exposed to artificial visible light, the effects of which on skin are poorly known. The purpose of this study was to model the artificial visible light emitted by electronic devices and assess its effect on normal human fibroblasts. The spectral irradiance emitted by electronic devices was optically measured and equipment was developed to accurately reproduce such artificial visible light. Effects on normal human fibroblasts were analyzed on human genome microarray-based gene expression analysis. At cellular level, visualization and image analysis were performed on the mitochondrial network and F-actin cytoskeleton. Cell proliferation, ATP release and type I procollagen secretion were also measured. We developed a device consisting of 36 LEDs simultaneously emitting blue, green and red light at distinct wavelengths (450 nm, 525 nm and 625 nm) with narrow spectra and equivalent radiant power for the three colors. A dose of 99 J/cm 2 artificial visible light was selected so as not to induce cell mortality following exposure. Microarray analysis revealed 2984 light-modulated transcripts. Functional annotation of light-responsive genes revealed several enriched functions including, amongst others, the "mitochondria" and "integrin signaling" categories. Selected results were confirmed by real-time quantitative PCR, analyzing 24 genes representing these two categories. Analysis of micro-patterned culture plates showed marked fragmentation of the mitochondrial network and disorganization of the F-actin cytoskeleton following exposure. Functionally, there was considerable impairment of cell growth and spread, ATP release and type I procollagen secretion in exposed fibroblasts. Artificial visible light induces drastic molecular and cellular changes in normal human fibroblasts. This may impede

  19. Modeling human papillomavirus and cervical cancer in the United States for analyses of screening and vaccination

    Directory of Open Access Journals (Sweden)

    Ortendahl Jesse

    2007-10-01

    Full Text Available Abstract Background To provide quantitative insight into current U.S. policy choices for cervical cancer prevention, we developed a model of human papillomavirus (HPV and cervical cancer, explicitly incorporating uncertainty about the natural history of disease. Methods We developed a stochastic microsimulation of cervical cancer that distinguishes different HPV types by their incidence, clearance, persistence, and progression. Input parameter sets were sampled randomly from uniform distributions, and simulations undertaken with each set. Through systematic reviews and formal data synthesis, we established multiple epidemiologic targets for model calibration, including age-specific prevalence of HPV by type, age-specific prevalence of cervical intraepithelial neoplasia (CIN, HPV type distribution within CIN and cancer, and age-specific cancer incidence. For each set of sampled input parameters, likelihood-based goodness-of-fit (GOF scores were computed based on comparisons between model-predicted outcomes and calibration targets. Using 50 randomly resampled, good-fitting parameter sets, we assessed the external consistency and face validity of the model, comparing predicted screening outcomes to independent data. To illustrate the advantage of this approach in reflecting parameter uncertainty, we used the 50 sets to project the distribution of health outcomes in U.S. women under different cervical cancer prevention strategies. Results Approximately 200 good-fitting parameter sets were identified from 1,000,000 simulated sets. Modeled screening outcomes were externally consistent with results from multiple independent data sources. Based on 50 good-fitting parameter sets, the expected reductions in lifetime risk of cancer with annual or biennial screening were 76% (range across 50 sets: 69–82% and 69% (60–77%, respectively. The reduction from vaccination alone was 75%, although it ranged from 60% to 88%, reflecting considerable parameter

  20. Environmental Toxin Screening Using Human-Derived 3D Bioengineered Liver and Cardiac Organoids

    Directory of Open Access Journals (Sweden)

    Steven D. Forsythe

    2018-04-01

    Full Text Available IntroductionEnvironmental toxins, such as lead and other heavy metals, pesticides, and other compounds, represent a significant health concern within the USA and around the world. Even in the twenty-first century, a plethora of cities and towns in the U.S. have suffered from exposures to lead in drinking water or other heavy metals in food or the earth, while there is a high possibility of further places to suffer such exposures in the near future.MethodsWe employed bioengineered 3D human liver and cardiac organoids to screen a panel of environmental toxins (lead, mercury, thallium, and glyphosate, and charted the response of the organoids to these compounds. Liver and cardiac organoids were exposed to lead (10 µM–10 mM, mercury (200 nM–200 µM, thallium (10 nM–10 µM, or glyphosate (25 µM–25 mM for a duration of 48 h. The impacts of toxin exposure were then assessed by LIVE/DEAD viability and cytotoxicity staining, measuring ATP activity and determining IC50 values, and determining changes in cardiac organoid beating activity.ResultsAs expected, all of the toxins induced toxicity in the organoids. Both ATP and LIVE/DEAD assays showed toxicity in both liver and cardiac organoids. In particular, thallium was the most toxic, with IC50 values of 13.5 and 1.35 µM in liver and cardiac organoids, respectively. Conversely, glyphosate was the least toxic of the four compounds, with IC50 values of 10.53 and 10.85 mM in liver and cardiac organoids, respectively. Additionally, toxins had a negative influence on cardiac organoid beating activity as well. Thallium resulting in the most significant decreases in beating rate, followed by mercury, then glyphosate, and finally, lead. These results suggest that the 3D organoids have significant utility to be deployed in additional toxicity screening applications, and future development of treatments to mitigate exposures.Conclusion3D organoids have significant utility to be

  1. Efficacy of human papillomavirus-based screen-and-treat for cervical cancer prevention among HIV-infected women.

    Science.gov (United States)

    Kuhn, Louise; Wang, Chunhui; Tsai, Wei-Yann; Wright, Thomas C; Denny, Lynette

    2010-10-23

    Cervical cancer prevention should be provided as part of primary healthcare services for HIV-infected women but conventional screening programs are difficult to implement in low-resource settings. Here, we evaluate the efficacy among HIV-infected women of a simpler, screen-and-treat strategy in which all women with a positive screening test are treated with cryotherapy. We conducted a randomized clinical trial of two screen-and-treat strategies among 6555 women in Cape Town, South Africa, among whom 956 were HIV-positive. Women were randomized to screen-and-treat utilizing either human papillomavirus DNA testing or visual inspection with acetic acid as the screening method or to a control group. Women were followed for up to 36 months after randomization with colposcopy and biopsy to determine the study endpoint of cervical intraepithelial neoplasia grade 2 or higher. In the control group, HIV-positive women had higher rates of cervical intraepithelial neoplasia grade 2 or higher detected by 36 months (14.9%) than HIV-negative women (4.6%) (P = 0.0006). Screen-and-treat utilizing human papillomavirus DNA testing significantly reduced cervical intraepithelial neoplasia grade 2 or higher through 36 months in both HIV-positive (relative risk = 0.20, 95% confidence interval 0.06-0.69) and HIV-negative women (relative risk = 0.31, 95% confidence interval 0.20-0.50). Reductions in the visual inspection with acetic acid-and-treat group were less marked. Complications of cryotherapy were mostly minor and did not differ in frequency between HIV-positive and HIV-negative women. Screen-and-treat using human papillomavirus testing is a simple and effective method to reduce high-grade cervical cancer precursors in HIV-infected women.

  2. Screening Test for Shed Skin Cells by Measuring the Ratio of Human DNA to Staphylococcus epidermidis DNA.

    Science.gov (United States)

    Nakanishi, Hiroaki; Ohmori, Takeshi; Hara, Masaaki; Takahashi, Shirushi; Kurosu, Akira; Takada, Aya; Saito, Kazuyuki

    2016-05-01

    A novel screening method for shed skin cells by detecting Staphylococcus epidermidis (S. epidermidis), which is a resident bacterium on skin, was developed. Staphylococcus epidermidis was detected using real-time PCR. Staphylococcus epidermidis was detected in all 20 human skin surface samples. Although not present in blood and urine samples, S. epidermidis was detected in 6 of 20 saliva samples, and 5 of 18 semen samples. The ratio of human DNA to S. epidermidisDNA was significantly smaller in human skin surface samples than in saliva and semen samples in which S. epidermidis was detected. Therefore, although skin cells could not be identified by detecting only S. epidermidis, they could be distinguished by measuring the S. epidermidis to human DNA ratio. This method could be applied to casework touch samples, which suggests that it is useful for screening whether skin cells and human DNA are present on potential evidentiary touch samples. © 2016 American Academy of Forensic Sciences.

  3. Hemispheric Asymmetry of Human Brain Anatomical Network Revealed by Diffusion Tensor Tractography

    Directory of Open Access Journals (Sweden)

    Ni Shu

    2015-01-01

    Full Text Available The topological architecture of the cerebral anatomical network reflects the structural organization of the human brain. Recently, topological measures based on graph theory have provided new approaches for quantifying large-scale anatomical networks. However, few studies have investigated the hemispheric asymmetries of the human brain from the perspective of the network model, and little is known about the asymmetries of the connection patterns of brain regions, which may reflect the functional integration and interaction between different regions. Here, we utilized diffusion tensor imaging to construct binary anatomical networks for 72 right-handed healthy adult subjects. We established the existence of structural connections between any pair of the 90 cortical and subcortical regions using deterministic tractography. To investigate the hemispheric asymmetries of the brain, statistical analyses were performed to reveal the brain regions with significant differences between bilateral topological properties, such as degree of connectivity, characteristic path length, and betweenness centrality. Furthermore, local structural connections were also investigated to examine the local asymmetries of some specific white matter tracts. From the perspective of both the global and local connection patterns, we identified the brain regions with hemispheric asymmetries. Combined with the previous studies, we suggested that the topological asymmetries in the anatomical network may reflect the functional lateralization of the human brain.

  4. Human colorectal mucosal microbiota correlates with its host niche physiology revealed by endomicroscopy.

    Science.gov (United States)

    Wang, Ai-Hua; Li, Ming; Li, Chang-Qing; Kou, Guan-Jun; Zuo, Xiu-Li; Li, Yan-Qing

    2016-02-26

    The human gut microbiota plays a pivotal role in the maintenance of health, but how the microbiota interacts with the host at the colorectal mucosa is poorly understood. We proposed that confocal laser endomicroscopy (CLE) might help to untangle this relationship by providing in vivo physiological information of the mucosa. We used CLE to evaluate the in vivo physiology of human colorectal mucosa, and the mucosal microbiota was quantified using 16 s rDNA pyrosequencing. The human mucosal microbiota agglomerated to three major clusters dominated by Prevotella, Bacteroides and Lactococcus. The mucosal microbiota clusters did not significantly correlate with the disease status or biopsy sites but closely correlated with the mucosal niche physiology, which was non-invasively revealed by CLE. Inflammation tilted two subnetworks within the mucosal microbiota. Infiltration of inflammatory cells significantly correlated with multiple components in the predicted metagenome, such as the VirD2 component of the type IV secretory pathway. Our data suggest that a close correlation exists between the mucosal microbiota and the colorectal mucosal physiology, and CLE is a clinically available tool that can be used to facilitate the study of the in vivo correlation between colorectal mucosal physiology and the mucosal microbiota.

  5. Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Melo, Rossana C.N., E-mail: rossana.melo@ufjf.edu.br [Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, UFJF, Rua José Lourenço Kelmer, Juiz de Fora, MG 36036-900 (Brazil); Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS 943, Boston, MA 02215 (United States); Weller, Peter F. [Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS 943, Boston, MA 02215 (United States)

    2016-10-01

    Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombrero Vesicles – EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses. - Highlights: • Application of EM to understand the complex secretory pathway in human eosinophils. • EM techniques reveal an active vesicular system associated with secretory granules. • Tubular vesicles are involved in the transport of granule-derived immune mediators.

  6. Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy

    International Nuclear Information System (INIS)

    Melo, Rossana C.N.; Weller, Peter F.

    2016-01-01

    Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombrero Vesicles – EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses. - Highlights: • Application of EM to understand the complex secretory pathway in human eosinophils. • EM techniques reveal an active vesicular system associated with secretory granules. • Tubular vesicles are involved in the transport of granule-derived immune mediators.

  7. Comparison of the IKr blockers moxifloxacin, dofetilide and E-4031 in five screening models of pro-arrhythmia reveals lack of specificity of isolated cardiomyocytes

    DEFF Research Database (Denmark)

    Nalos, L; Varkevisser, R; Jonsson, Mkb

    2012-01-01

    Background and purpose Drug discovery and development require testing of new chemical entities for possible adverse effects. For cardiac safety screening, improved assays are urgently needed and isolated adult cardiomyocytes (CM) and human embryonic stem cell-derived cardiomyocytes (hESC-CM) may......-4031 (unsafe compounds). Experimental approach The assays included: 1. The anesthetized remodeled chronic complete AV-block (CAVB) dog, 2. The anesthetized methoxamine sensitized unremodeled rabbit, 3. Multi-cellular hESC-CM clusters, 4. Isolated CM obtained from the CAVB dog and 5. Isolated CM...

  8. A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling.

    Directory of Open Access Journals (Sweden)

    Neil Dani

    Full Text Available A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS 6-O-sulfotransferase (hs6st and sulfatase (sulf1, which bidirectionally control HS proteoglycan (HSPG sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st and increased (sulf1 neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg and BMP (Glass Bottom Boat; Gbb ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects.

  9. A genome-wide RNAi screen reveals MAP kinase phosphatases as key ERK pathway regulators during embryonic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Shen-Hsi Yang

    Full Text Available Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Complex interlinked transcriptional and signaling networks control the fate of these cells towards maintenance of pluripotency or differentiation. In this study we have focused on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase and GSK3 signaling pathways play in this process. Through a genome-wide siRNA screen we have identified more than 400 genes involved in loss of pluripotency and promoting the onset of differentiation. These genes were functionally associated with the ERK and/or GSK3 pathways, providing an important resource for studying the roles of these pathways in controlling escape from the pluripotent ground state. More detailed analysis identified MAP kinase phosphatases as a focal point of regulation and demonstrated an important role for these enzymes in controlling ERK activation kinetics and subsequently determining early embryonic stem cell fate decisions.

  10. Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics

    Science.gov (United States)

    van der Ploeg, René; Goudelis, Spyridon Theodoros; den Blaauwen, Tanneke

    2015-01-01

    The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s) simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes) that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET) assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl) isothiourea (A22) or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors. PMID:26263980

  11. Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics

    Directory of Open Access Journals (Sweden)

    René van der Ploeg

    2015-07-01

    Full Text Available The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl isothiourea (A22 or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors.

  12. Mutational screening of the USH2A gene in Spanish USH patients reveals 23 novel pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Diaz-Llopis Manuel

    2011-10-01

    Full Text Available Abstract Background Usher Syndrome type II (USH2 is an autosomal recessive disorder, characterized by moderate to severe hearing impairment and retinitis pigmentosa (RP. Among the three genes implicated, mutations in the USH2A gene account for 74-90% of the USH2 cases. Methods To identify the genetic cause of the disease and determine the frequency of USH2A mutations in a cohort of 88 unrelated USH Spanish patients, we carried out a mutation screening of the 72 coding exons of this gene by direct sequencing. Moreover, we performed functional minigene studies for those changes that were predicted to affect splicing. Results As a result, a total of 144 DNA sequence variants were identified. Based upon previous studies, allele frequencies, segregation analysis, bioinformatics' predictions and in vitro experiments, 37 variants (23 of them novel were classified as pathogenic mutations. Conclusions This report provide a wide spectrum of USH2A mutations and clinical features, including atypical Usher syndrome phenotypes resembling Usher syndrome type I. Considering only the patients clearly diagnosed with Usher syndrome type II, and results obtained in this and previous studies, we can state that mutations in USH2A are responsible for 76.1% of USH2 disease in patients of Spanish origin.

  13. A screen for constituents of motor control and decision making in Drosophila reveals visual distance-estimation neurons

    Science.gov (United States)

    Triphan, Tilman; Nern, Aljoscha; Roberts, Sonia F.; Korff, Wyatt; Naiman, Daniel Q.; Strauss, Roland

    2016-01-01

    Climbing over chasms larger than step size is vital to fruit flies, since foraging and mating are achieved while walking. Flies avoid futile climbing attempts by processing parallax-motion vision to estimate gap width. To identify neuronal substrates of climbing control, we screened a large collection of fly lines with temporarily inactivated neuronal populations in a novel high-throughput assay described here. The observed climbing phenotypes were classified; lines in each group are reported. Selected lines were further analysed by high-resolution video cinematography. One striking class of flies attempts to climb chasms of unsurmountable width; expression analysis guided us to C2 optic-lobe interneurons. Inactivation of C2 or the closely related C3 neurons with highly specific intersectional driver lines consistently reproduced hyperactive climbing whereas strong or weak artificial depolarization of C2/C3 neurons strongly or mildly decreased climbing frequency. Contrast-manipulation experiments support our conclusion that C2/C3 neurons are part of the distance-evaluation system. PMID:27255169

  14. A forward-genetic screen and dynamic analysis of lambda phage host-dependencies reveals an extensive interaction network and a new anti-viral strategy.

    Directory of Open Access Journals (Sweden)

    Nathaniel D Maynard

    2010-07-01

    Full Text Available Latently infecting viruses are an important class of virus that plays a key role in viral evolution and human health. Here we report a genome-scale forward-genetics screen for host-dependencies of the latently-infecting bacteriophage lambda. This screen identified 57 Escherichia coli (E. coli genes--over half of which have not been previously associated with infection--that when knocked out inhibited lambda phage's ability to replicate. Our results demonstrate a highly integrated network between lambda and its host, in striking contrast to the results from a similar screen using the lytic-only infecting T7 virus. We then measured the growth of E. coli under normal and infected conditions, using wild-type and knockout strains deficient in one of the identified host genes, and found that genes from the same pathway often exhibited similar growth dynamics. This observation, combined with further computational and experimental analysis, led us to identify a previously unannotated gene, yneJ, as a novel regulator of lamB gene expression. A surprising result of this work was the identification of two highly conserved pathways involved in tRNA thiolation-one pathway is required for efficient lambda replication, while the other has anti-viral properties inhibiting lambda replication. Based on our data, it appears that 2-thiouridine modification of tRNAGlu, tRNAGln, and tRNALys is particularly important for the efficient production of infectious lambda phage particles.

  15. Dual Transcriptome Profiling of Leishmania-Infected Human Macrophages Reveals Distinct Reprogramming Signatures.

    Science.gov (United States)

    Fernandes, Maria Cecilia; Dillon, Laura A L; Belew, Ashton Trey; Bravo, Hector Corrada; Mosser, David M; El-Sayed, Najib M

    2016-05-10

    Macrophages are mononuclear phagocytes that constitute a first line of defense against pathogens. While lethal to many microbes, they are the primary host cells of Leishmania spp. parasites, the obligate intracellular pathogens that cause leishmaniasis. We conducted transcriptomic profiling of two Leishmania species and the human macrophage over the course of intracellular infection by using high-throughput RNA sequencing to characterize the global gene expression changes and reprogramming events that underlie the interactions between the pathogen and its host. A systematic exclusion of the generic effects of large-particle phagocytosis revealed a vigorous, parasite-specific response of the human macrophage early in the infection that was greatly tempered at later time points. An analogous temporal expression pattern was observed with the parasite, suggesting that much of the reprogramming that occurs as parasites transform into intracellular forms generally stabilizes shortly after entry. Following that, the parasite establishes an intracellular niche within macrophages, with minimal communication between the parasite and the host cell later during the infection. No significant difference was observed between parasite species transcriptomes or in the transcriptional response of macrophages infected with each species. Our comparative analysis of gene expression changes that occur as mouse and human macrophages are infected by Leishmania spp. points toward a general signature of the Leishmania-macrophage infectome. Little is known about the transcriptional changes that occur within mammalian cells harboring intracellular pathogens. This study characterizes the gene expression signatures of Leishmania spp. parasites and the coordinated response of infected human macrophages as the pathogen enters and persists within them. After accounting for the generic effects of large-particle phagocytosis, we observed a parasite-specific response of the human macrophages early in

  16. A human reliability assessment screening method for the NRU upgrade project

    International Nuclear Information System (INIS)

    Bremner, F.M.; Alsop, C.J.

    1997-01-01

    The National Research Universal (NRU) reactor is a 130MW, low pressure, heavy water cooled and moderated research reactor. The reactor is used for research, both in support of Canada's CANDU development program, and for a wide variety of other research applications. In addition, NRU plays an important part in the production of medical isotopes, e.g., generating 80% of worldwide supplies of Molybdenum-99. NRU is owned and operated by Atomic Energy of Canada Ltd. (AECL), and is currently undergoing upgrading as part of AECL's continuing commitment to operate their facilities in a safe manner. As part of these upgrades both deterministic and probabilistic safety assessments are being carried out. It was recognized that the assignment of Human Error Probabilities (HEPs) is an important part of the Probabilistic Safety Assessment (PSA) studies, particularly for a facility whose design predates modern ergonomic practices, and which will undergo a series of backfitted modifications whilst continuing to operate. A simple Human Reliability Assessment (HRA) screening method, looking at both pre- and post-accident errors, was used in the initial safety studies. However, following review of this method within AECL and externally by the regulator, it was judged that benefits could be gained for future error reduction by including additional features, as later described in this document. The HRA development project consisted of several stages; needs analysis, literature review, development of method (including testing and evaluation), and implementation. This paper discusses each of these stages in further detail. (author)

  17. MALDI Q-TOF CID MS for Diagnostic Ion Screening of Human Milk Oligosaccharide Samples

    Directory of Open Access Journals (Sweden)

    Marko Jovanović

    2014-04-01

    Full Text Available Human milk oligosaccharides (HMO represent the bioactive components of human milk, influencing the infant’s gastrointestinal microflora and immune system. Structurally, they represent a highly complex class of analyte, where the main core oligosaccharide structures are built from galactose and N-acetylglucosamine, linked by 1-3 or 1-4 glycosidic linkages and potentially modified with fucose and sialic acid residues. The core structures can be linear or branched. Additional structural complexity in samples can be induced by endogenous exoglycosidase activity or chemical procedures during the sample preparation. Here, we show that using matrix-assisted laser desorption/ionization (MALDI quadrupole-time-of-flight (Q-TOF collision-induced dissociation (CID as a fast screening method, diagnostic structural information about single oligosaccharide components present in a complex mixture can be obtained. According to sequencing data on 14 out of 22 parent ions detected in a single high molecular weight oligosaccharide chromatographic fraction, 20 different oligosaccharide structure types, corresponding to over 30 isomeric oligosaccharide structures and over 100 possible HMO isomers when biosynthetic linkage variations were taken into account, were postulated. For MS/MS data analysis, we used the de novo sequencing approach using diagnostic ion analysis on reduced oligosaccharides by following known biosynthetic rules. Using this approach, de novo characterization has been achieved also for the structures, which could not have been predicted.

  18. High throughput Screening to Identify Natural Human Monoamine Oxidase B Inhibitors

    Science.gov (United States)

    Mazzio, E; Deiab, S; Park, K; Soliman, KFA

    2012-01-01

    Age-related increase in monoamine oxidase B (MAO-B) may contribute to CNS neurodegenerative diseases. Moreover, MAO-B inhibitors are used in the treatment of idiopathic Parkinson disease as preliminary monotherapy or adjunct therapy with L-dopa. To date, meager natural sources of MAO-B inhibitors have been identified, and the relative strength, potency and rank of many plants relative to standard drugs such as Selegiline (L-deprenyl, Eldepryl) are not known. In this work, we developed and utilized a high throughput enzyme microarray format to screen and evaluate 905 natural product extracts (0.025–.7 mg/ml) to inhibit human MAO-B derived from BTI-TN-5B1-4 cells infected with recombinant baculovirus. The protein sequence of purified enzyme was confirmed using 1D gel electrophoresis-matrix assisted laser desorption ionization-time-of-flight-tandem mass spectroscopy, and enzyme activity was confirmed by [1] substrate conversion (3-mM benzylamine) to H202 and [2] benzaldehyde. Of the 905 natural extracts tested, the lowest IC50s [Comfrey, Bringraj, Skullcap, Kava-kava, Wild Indigo, Gentian and Green Tea. In conclusion, the data reflect relative potency information by rank of commonly used herbs and plants that contain human MAO-B inhibitory properties in their natural form. PMID:22887993

  19. Integrated Metagenomics/Metaproteomics Reveals Human Host-Microbiota Signatures of Crohn's Disease

    Science.gov (United States)

    Darzi, Youssef; Mongodin, Emmanuel F.; Pan, Chongle; Shah, Manesh; Halfvarson, Jonas; Tysk, Curt; Henrissat, Bernard; Raes, Jeroen; Verberkmoes, Nathan C.; Jansson, Janet K.

    2012-01-01

    Crohn's disease (CD) is an inflammatory bowel disease of complex etiology, although dysbiosis of the gut microbiota has been implicated in chronic immune-mediated inflammation associated with CD. Here we combined shotgun metagenomic and metaproteomic approaches to identify potential functional signatures of CD in stool samples from six twin pairs that were either healthy, or that had CD in the ileum (ICD) or colon (CCD). Integration of these omics approaches revealed several genes, proteins, and pathways that primarily differentiated ICD from healthy subjects, including depletion of many proteins in ICD. In addition, the ICD phenotype was associated with alterations in bacterial carbohydrate metabolism, bacterial-host interactions, as well as human host-secreted enzymes. This eco-systems biology approach underscores the link between the gut microbiota and functional alterations in the pathophysiology of Crohn's disease and aids in identification of novel diagnostic targets and disease specific biomarkers. PMID:23209564

  20. Integrated metagenomics/metaproteomics reveals human host-microbiota signatures of Crohn's disease.

    Directory of Open Access Journals (Sweden)

    Alison R Erickson

    Full Text Available Crohn's disease (CD is an inflammatory bowel disease of complex etiology, although dysbiosis of the gut microbiota has been implicated in chronic immune-mediated inflammation associated with CD. Here we combined shotgun metagenomic and metaproteomic approaches to identify potential functional signatures of CD in stool samples from six twin pairs that were either healthy, or that had CD in the ileum (ICD or colon (CCD. Integration of these omics approaches revealed several genes, proteins, and pathways that primarily differentiated ICD from healthy subjects, including depletion of many proteins in ICD. In addition, the ICD phenotype was associated with alterations in bacterial carbohydrate metabolism, bacterial-host interactions, as well as human host-secreted enzymes. This eco-systems biology approach underscores the link between the gut microbiota and functional alterations in the pathophysiology of Crohn's disease and aids in identification of novel diagnostic targets and disease specific biomarkers.

  1. RNAi screen reveals a role of SPHK2 in dengue virus-mediated apoptosis in hepatic cell lines.

    Directory of Open Access Journals (Sweden)

    Atthapan Morchang

    Full Text Available Hepatic dysfunction is a feature of dengue virus (DENV infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show apoptosis, which relates to the pathogenesis of DENV infection. However, how DENV induced liver injury is not fully understood. In this study, we aim to identify the factors that influence cell death by employing an apoptosis-related siRNA library screening. Our results show the effect of 558 gene silencing on caspase 3-mediated apoptosis in DENV-infected Huh7 cells. The majority of genes that contributed to apoptosis were the apoptosis-related kinase enzymes. Tumor necrosis factor superfamily member 12 (TNFSF12, and sphingosine kinase 2 (SPHK2, were selected as the candidate genes to further validate their influences on DENV-induced apoptosis. Transfection of siRNA targeting SPHK2 but not TNFSF12 genes reduced apoptosis determined by Annexin V/PI staining. Knockdown of SPHK2 did not reduce caspase 8 activity; however, did significantly reduce caspase 9 activity, suggesting its involvement of SPHK2 in the intrinsic pathway of apoptosis. Treatment of ABC294649, an inhibitor of SPHK2, reduced the caspase 3 activity, suggesting the involvement of its kinase activity in apoptosis. Knockdown of SPHK2 significantly reduced caspase 3 activity not only in DENV-infected Huh7 cells but also in DENV-infected HepG2 cells. Our results were consistent across all of the four serotypes of DENV infection, which supports the pro-apoptotic role of SPHK2 in DENV-infected liver cells.

  2. Systematic analysis of off-target effects in an RNAi screen reveals microRNAs affecting sensitivity to TRAIL-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Enright Anton J

    2010-03-01

    Full Text Available Abstract Background RNA inhibition by siRNAs is a frequently used approach to identify genes required for specific biological processes. However RNAi screening using siRNAs is hampered by non-specific or off target effects of the siRNAs, making it difficult to separate genuine hits from false positives. It is thought that many of the off-target effects seen in RNAi experiments are due to siRNAs acting as microRNAs (miRNAs, causing a reduction in gene expression of unintended targets via matches to the 6 or 7 nt 'seed' sequence. We have conducted a careful examination of off-target effects during an siRNA screen for novel regulators of the TRAIL apoptosis induction pathway(s. Results We identified 3 hexamers and 3 heptamer seed sequences that appeared multiple times in the top twenty siRNAs in the TRAIL apoptosis screen. Using a novel statistical enrichment approach, we systematically identified a further 17 hexamer and 13 heptamer seed sequences enriched in high scoring siRNAs. The presence of one of these seeds sequences (which could explain 6 of 8 confirmed off-target effects is sufficient to elicit a phenotype. Three of these seed sequences appear in the human miRNAs miR-26a, miR-145 and miR-384. Transfection of mimics of these miRNAs protects several cell types from TRAIL-induced cell death. Conclusions We have demonstrated a role for miR-26a, miR-145 and miR-26a in TRAIL-induced apoptosis. Further these results show that RNAi screening enriches for siRNAs with relevant off-target effects. Some of these effects can be identified by the over-representation of certain seed sequences in high-scoring siRNAs and we demonstrate the usefulness of such systematic analysis of enriched seed sequences.

  3. Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells

    Science.gov (United States)

    Ali, Dalia; Hamam, Rimi; Alfayez, Musaed; Kassem, Moustapha; Aldahmash, Abdullah

    2016-01-01

    The epigenetic mechanisms promoting lineage-specific commitment of human skeletal (mesenchymal or stromal) stem cells (hMSCs) into adipocytes or osteoblasts are still not fully understood. Herein, we performed an epigenetic library functional screen and identified several novel compounds, including abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell proliferation and differentiation that were targeted by abexinostat. Concordantly, ChIP-quantitative polymerase chain reaction revealed marked increase in H3K9Ac epigenetic mark on the promoter region of AdipoQ, FABP4, PPARγ, KLF15, CEBPA, SP7, and ALPL in abexinostat-treated hMSCs. Pharmacological inhibition of focal adhesion kinase (PF-573228) or insulin-like growth factor-1R/insulin receptor (NVP-AEW51) signaling exhibited significant inhibition of abexinostat-mediated adipocytic differentiation, whereas inhibition of WNT (XAV939) or transforming growth factor-β (SB505124) signaling abrogated abexinostat-mediated osteogenic differentiation of hMSCs. Our findings provide insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways governing adipocyte and osteoblast differentiation. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies and tissue engineering. Significance This unbiased epigenetic library functional screen identified several novel compounds, including abexinostat, that promoted adipocytic and osteoblastic differentiation of human skeletal (mesenchymal or stromal) stem cells (hMSCs). These data provide new insight into the understanding of the relationship between the epigenetic effect of histone deacetylase

  4. Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hui; Klein, Michael G.; Snell, Gyorgy; Lane, Weston; Zou, Hua; Levin, Irena; Li, Ke; Sang, Bi-Ching (Takeda Cali)

    2016-07-01

    Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

  5. MR diffusion histology and micro-tractography reveal mesoscale features of the human cerebellum.

    Science.gov (United States)

    Dell'Acqua, Flavio; Bodi, Istvan; Slater, David; Catani, Marco; Modo, Michel

    2013-12-01

    After 140 years from the discovery of Golgi's black reaction, the study of connectivity of the cerebellum remains a fascinating yet challenging task. Current histological techniques provide powerful methods for unravelling local axonal architecture, but the relatively low volume of data that can be acquired in a reasonable amount of time limits their application to small samples. State-of-the-art in vivo magnetic resonance imaging (MRI) methods, such as diffusion tractography techniques, can reveal trajectories of the major white matter pathways, but their correspondence with underlying anatomy is yet to be established. Hence, a significant gap exists between these two approaches as neither of them can adequately describe the three-dimensional complexity of fibre architecture at the level of the mesoscale (from a few millimetres to micrometres). In this study, we report the application of MR diffusion histology and micro-tractography methods to reveal the combined cytoarchitectural organisation and connectivity of the human cerebellum at a resolution of 100-μm (2 nl/voxel volume). Results show that the diffusion characteristics for each layer of the cerebellar cortex correctly reflect the known cellular composition and its architectural pattern. Micro-tractography also reveals details of the axonal connectivity of individual cerebellar folia and the intra-cortical organisation of the different cerebellar layers. The direct correspondence between MR diffusion histology and micro-tractography with immunohistochemistry indicates that these approaches have the potential to complement traditional histology techniques by providing a non-destructive, quantitative and three-dimensional description of the microstructural organisation of the healthy and pathological tissue.

  6. The clinical effectiveness and cost-effectiveness of primary human papillomavirus cervical screening in England: extended follow-up of the ARTISTIC randomised trial cohort through three screening rounds.

    Science.gov (United States)

    C Kitchener, Henry; Canfell, Karen; Gilham, Clare; Sargent, Alexandra; Roberts, Chris; Desai, Mina; Peto, Julian

    2014-04-01

    The ARTISTIC (A Randomised Trial In Screening To Improve Cytology) trial originally reported after two rounds of primary cervical screening with human papillomavirus (HPV). Extended follow-up of the randomised trial cohort through a third round could provide valuable insight into the duration of protection of a negative HPV test, which could allow extended screening intervals. If HPV primary screening is to be considered in the national programme, then determining its cost-effectiveness is key, and a detailed economic analysis using ARTISTIC data is needed. (1) To determine the round 3 and cumulative rates of cervical intraepithelial neoplasia (CIN) grade 2 or worse (2+) and CIN grade 3 or worse (CIN3+) between the revealed and concealed arms of ARTISTIC after three screening rounds over 6 years. (2) To compare the cumulative incidence of CIN2+ over three screening rounds following negative screening cytology with that following negative baseline HPV. (3) To determine whether or not HPV screening could safely extend the screening interval from 3 to 6 years. (4) To study the potential clinical utility of an increased cut-off of 2 relative light unit/mean control (RLU/Co) for Hybrid Capture 2 (HC2) and HPV genotyping in primary cervical screening. (5) To determine the potential impact of HPV vaccination with Cervarix™ in terms of preventing abnormal cytology and CIN2+. (6) To determine the cost-effectiveness of HPV primary screening compared with current practice using cervical cytology in England. The ARTISTIC study cohort was recalled for a third round of screening 3 years after round 2 and 6 years following their enrolment to the study. Both arms of the original trial used a single protocol during round 3. ARTISTIC study cohort undergoing cervical screening in primary care in Greater Manchester, UK. Between July 2007 and September 2009, 8873 women participated in round 3; 6337 had been screened in round 2 and 2536 had not been screened since round 1. All women

  7. Indeterminate human immunodeficiency virus western blot results in Iranian patients with discordant screening assay results

    International Nuclear Information System (INIS)

    Ravanshad, M.; Sabahi, F.; Mahboudi, F.; Sabahi, F.

    2006-01-01

    The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection and confirmation of antibodies to human immunodeficiency virus type 1 (HIV-1) and 2 (HIV-2). However, indeterminate WB reactivity to HIV-1 and HIV-2 proteins may occur in individuals who do not appear to be infected with HIV. In this study, we describe the results of indeterminate WB reactivity in Iranian patients with discordant screening assays. The samples were obtained from Iranian Blood Transfusion Center, Tehran, Iran and evaluated in the Biotechnology Process Development Center, Pasteur Institute of Iran, Tehran, Iran between 2003 and 2004. A total of 4707 were tested for the presence of HIV-1 antibodies. Six hundred and four (12.8%) patients tested for HIV were positive for HIV-1 antibody. Nine (1.49%) have discordant results among screening assays and indeterminate WB results as interpreted by Centers for Disease Control and Prevention (CDC) criteria. Most (66.7%) of these indeterminate WB results were due to p24 reactivity. However, 2(22.2%) display reactivity to both gp41 and gp120 proteins [Positive by World Health Organization (WHO) criteria]. Of 9 WB assays initially indeterminate by the CDC criteria and with follow-up samples 8(88.8%) became negative when retested subsequently while one (11.1%) remained indeterminate for more than a year and were thus considered negative. In addition all the indeterminate samples were negative when assessed by polymerase chain reaction assay. In general, there were was an 88.8% concordance between the CDC and WHO criteria for an indeterminate WB result. The CDC II criteria for an indeterminate WB result. The CDC II criteria best met the specified objectives for diagnosis in our setting. (author)

  8. Screening for the synthetic cannabinoid JWH-018 and its major metabolites in human doping controls.

    Science.gov (United States)

    Möller, Ines; Wintermeyer, Annette; Bender, Katja; Jübner, Martin; Thomas, Andreas; Krug, Oliver; Schänzer, Wilhelm; Thevis, Mario

    2011-09-01

    Referred to as 'spice', several new drugs, advertised as herbal blends, have appeared on the market in the last few years, in which the synthetic cannabinoids JWH-018 and a C(8) homologue of CP 47,497 were identified as major active ingredients. Due to their reported cannabis-like effects, many European countries have banned these substances. The World Anti-Doping Agency has also explicitly prohibited synthetic cannabinoids in elite sport in-competition. Since urine specimens have been the preferred doping control samples, the elucidation of the metabolic pathways of these substances is of particular importance to implement them in sports drug testing programmes. In a recent report, an in vitro phase-I metabolism study of JWH-018 was presented yielding mainly hydroxylated and N-dealkylated metabolites. Due to these findings, a urine sample of a healthy man declaring to have smoked a 'spice' product was screened for potential phase-I and -II metabolites by high-resolution/high-accuracy mass spectrometry in the present report. The majority of the phase-I metabolites observed in earlier in vitro studies of JWH-018 were detected in this urine specimen and furthermore most of their respective monoglucuronides. As no intact JWH-018 was detectable, the monohydroxylated metabolite being the most abundant one was chosen as a target analyte for sports drug testing purposes; a detection method was subsequently developed and validated in accordance to conventional screening protocols based on enzymatic hydrolysis, liquid-liquid extraction, and liquid chromatography/electrospray tandem mass spectrometry analysis. The method was applied to approximately 7500 urine doping control samples yielding two JWH-018 findings and demonstrated its capability for a sensitive and selective identification of JWH-018 and its metabolites in human urine. Copyright © 2010 John Wiley & Sons, Ltd.

  9. Human papillomavirus-based cervical cancer prevention: long-term results of a randomized screening trial.

    Science.gov (United States)

    Denny, Lynette; Kuhn, Louise; Hu, Chih-Chi; Tsai, Wei-Yann; Wright, Thomas C

    2010-10-20

    Screen-and-treat approaches to cervical cancer prevention are an attractive option for low-resource settings, but data on their long-term efficacy are lacking. We evaluated the efficacy of two screen-and-treat approaches through 36 months of follow-up in a randomized trial. A total of 6637 unscreened South African women aged 35-65 years who were tested for the presence of high-risk human papillomavirus (HPV) DNA in cervical samples underwent visual inspection of the cervix using acetic acid staining and HIV serotesting. Of these, 6555 were randomly assigned to three study arms: 1) HPV-and-treat, in which all women with a positive HPV DNA test result underwent cryotherapy; 2) visual inspection-and-treat, in which all women with a positive visual inspection test result underwent cryotherapy; or 3) control, in which further evaluation or treatment was delayed for 6 months. All women underwent colposcopy with biopsy at 6 months. All women who were HPV DNA- or visual inspection-positive at enrollment, and a subset of all other women had extended follow-up to 36 months (n = 3639) with yearly colposcopy. The endpoint-cervical intraepithelial neoplasia grade 2 or worse (CIN2+)-was analyzed using actuarial life-table methods. All statistical tests were two-sided. After 36 months, there was a sustained statistically significant decrease in the cumulative detection of CIN2+ in the HPV-and-treat arm compared with the control arm (1.5% vs 5.6%, difference = 4.1%, 95% confidence interval [CI] = 2.8% to 5.3%, P cryotherapy.

  10. Prevent cervical cancer by screening with reliable human papillomavirus detection and genotyping

    International Nuclear Information System (INIS)

    Ge, Shichao; Gong, Bo; Cai, Xushan; Yang, Xiaoer; Gan, Xiaowei; Tong, Xinghai; Li, Haichuan; Zhu, Meijuan; Yang, Fengyun; Zhou, Hongrong; Hong, Guofan

    2012-01-01

    The incidence of cervical cancer is expected to rise sharply in China. A reliable routine human papillomavirus (HPV) detection and genotyping test to be supplemented by the limited Papanicolaou cytology facilities is urgently needed to help identify the patients with cervical precancer for preventive interventions. To this end, we evaluated a nested polymerase chain reaction (PCR) protocol for detection of HPV L1 gene DNA in cervicovaginal cells. The PCR amplicons were genotyped by direct DNA sequencing. In parallel, split samples were subjected to a Digene HC2 HPV test which has been widely used for “cervical cancer risk” screen. Of the 1826 specimens, 1655 contained sufficient materials for analysis and 657 were truly negative. PCR/DNA sequencing showed 674 infected by a single high-risk HPV, 188 by a single low-risk HPV, and 136 by multiple HPV genotypes with up to five HPV genotypes in one specimen. In comparison, the HC2 test classified 713 specimens as infected by high-risk HPV, and 942 as negative for HPV infections. The high-risk HC2 test correctly detected 388 (57.6%) of the 674 high-risk HPV isolates in clinical specimens, mislabeled 88 (46.8%) of the 188 low-risk HPV isolates as high-risk genotypes, and classified 180 (27.4%) of the 657 “true-negative” samples as being infected by high-risk HPV. It was found to cross-react with 20 low-risk HPV genotypes. We conclude that nested PCR detection of HPV followed by short target DNA sequencing can be used for screening and genotyping to formulate a paradigm in clinical management of HPV-related disorders in a rapidly developing economy

  11. 3-Dimensional culture systems for anti-cancer compound profiling and high-throughput screening reveal increases in EGFR inhibitor-mediated cytotoxicity compared to monolayer culture systems.

    Science.gov (United States)

    Howes, Amy L; Richardson, Robyn D; Finlay, Darren; Vuori, Kristiina

    2014-01-01

    3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.

  12. Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier.

    Directory of Open Access Journals (Sweden)

    Jes Dreier

    Full Text Available In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer lipid layers of the stratum corneum. It was also found that the flexible liposomes showed a greater delivery of the fluorophore into the stratum corneum, indicating that they functioned as chemical permeability enhancers.

  13. Computational dissection of human episodic memory reveals mental process-specific genetic profiles

    Science.gov (United States)

    Luksys, Gediminas; Fastenrath, Matthias; Coynel, David; Freytag, Virginie; Gschwind, Leo; Heck, Angela; Jessen, Frank; Maier, Wolfgang; Milnik, Annette; Riedel-Heller, Steffi G.; Scherer, Martin; Spalek, Klara; Vogler, Christian; Wagner, Michael; Wolfsgruber, Steffen; Papassotiropoulos, Andreas; de Quervain, Dominique J.-F.

    2015-01-01

    Episodic memory performance is the result of distinct mental processes, such as learning, memory maintenance, and emotional modulation of memory strength. Such processes can be effectively dissociated using computational models. Here we performed gene set enrichment analyses of model parameters estimated from the episodic memory performance of 1,765 healthy young adults. We report robust and replicated associations of the amine compound SLC (solute-carrier) transporters gene set with the learning rate, of the collagen formation and transmembrane receptor protein tyrosine kinase activity gene sets with the modulation of memory strength by negative emotional arousal, and of the L1 cell adhesion molecule (L1CAM) interactions gene set with the repetition-based memory improvement. Furthermore, in a large functional MRI sample of 795 subjects we found that the association between L1CAM interactions and memory maintenance revealed large clusters of differences in brain activity in frontal cortical areas. Our findings provide converging evidence that distinct genetic profiles underlie specific mental processes of human episodic memory. They also provide empirical support to previous theoretical and neurobiological studies linking specific neuromodulators to the learning rate and linking neural cell adhesion molecules to memory maintenance. Furthermore, our study suggests additional memory-related genetic pathways, which may contribute to a better understanding of the neurobiology of human memory. PMID:26261317

  14. Human subcortical brain asymmetries in 15,847 people worldwide reveal effects of age and sex.

    Science.gov (United States)

    Guadalupe, Tulio; Mathias, Samuel R; vanErp, Theo G M; Whelan, Christopher D; Zwiers, Marcel P; Abe, Yoshinari; Abramovic, Lucija; Agartz, Ingrid; Andreassen, Ole A; Arias-Vásquez, Alejandro; Aribisala, Benjamin S; Armstrong, Nicola J; Arolt, Volker; Artiges, Eric; Ayesa-Arriola, Rosa; Baboyan, Vatche G; Banaschewski, Tobias; Barker, Gareth; Bastin, Mark E; Baune, Bernhard T; Blangero, John; Bokde, Arun L W; Boedhoe, Premika S W; Bose, Anushree; Brem, Silvia; Brodaty, Henry; Bromberg, Uli; Brooks, Samantha; Büchel, Christian; Buitelaar, Jan; Calhoun, Vince D; Cannon, Dara M; Cattrell, Anna; Cheng, Yuqi; Conrod, Patricia J; Conzelmann, Annette; Corvin, Aiden; Crespo-Facorro, Benedicto; Crivello, Fabrice; Dannlowski, Udo; de Zubicaray, Greig I; de Zwarte, Sonja M C; Deary, Ian J; Desrivières, Sylvane; Doan, Nhat Trung; Donohoe, Gary; Dørum, Erlend S; Ehrlich, Stefan; Espeseth, Thomas; Fernández, Guillén; Flor, Herta; Fouche, Jean-Paul; Frouin, Vincent; Fukunaga, Masaki; Gallinat, Jürgen; Garavan, Hugh; Gill, Michael; Suarez, Andrea Gonzalez; Gowland, Penny; Grabe, Hans J; Grotegerd, Dominik; Gruber, Oliver; Hagenaars, Saskia; Hashimoto, Ryota; Hauser, Tobias U; Heinz, Andreas; Hibar, Derrek P; Hoekstra, Pieter J; Hoogman, Martine; Howells, Fleur M; Hu, Hao; Hulshoff Pol, Hilleke E; Huyser, Chaim; Ittermann, Bernd; Jahanshad, Neda; Jönsson, Erik G; Jurk, Sarah; Kahn, Rene S; Kelly, Sinead; Kraemer, Bernd; Kugel, Harald; Kwon, Jun Soo; Lemaitre, Herve; Lesch, Klaus-Peter; Lochner, Christine; Luciano, Michelle; Marquand, Andre F; Martin, Nicholas G; Martínez-Zalacaín, Ignacio; Martinot, Jean-Luc; Mataix-Cols, David; Mather, Karen; McDonald, Colm; McMahon, Katie L; Medland, Sarah E; Menchón, José M; Morris, Derek W; Mothersill, Omar; Maniega, Susana Munoz; Mwangi, Benson; Nakamae, Takashi; Nakao, Tomohiro; Narayanaswaamy, Janardhanan C; Nees, Frauke; Nordvik, Jan E; Onnink, A Marten H; Opel, Nils; Ophoff, Roel; Paillère Martinot, Marie-Laure; Papadopoulos Orfanos, Dimitri; Pauli, Paul; Paus, Tomáš; Poustka, Luise; Reddy, Janardhan Yc; Renteria, Miguel E; Roiz-Santiáñez, Roberto; Roos, Annerine; Royle, Natalie A; Sachdev, Perminder; Sánchez-Juan, Pascual; Schmaal, Lianne; Schumann, Gunter; Shumskaya, Elena; Smolka, Michael N; Soares, Jair C; Soriano-Mas, Carles; Stein, Dan J; Strike, Lachlan T; Toro, Roberto; Turner, Jessica A; Tzourio-Mazoyer, Nathalie; Uhlmann, Anne; Hernández, Maria Valdés; van den Heuvel, Odile A; van der Meer, Dennis; van Haren, Neeltje E M; Veltman, Dick J; Venkatasubramanian, Ganesan; Vetter, Nora C; Vuletic, Daniella; Walitza, Susanne; Walter, Henrik; Walton, Esther; Wang, Zhen; Wardlaw, Joanna; Wen, Wei; Westlye, Lars T; Whelan, Robert; Wittfeld, Katharina; Wolfers, Thomas; Wright, Margaret J; Xu, Jian; Xu, Xiufeng; Yun, Je-Yeon; Zhao, JingJing; Franke, Barbara; Thompson, Paul M; Glahn, David C; Mazoyer, Bernard; Fisher, Simon E; Francks, Clyde

    2017-10-01

    The two hemispheres of the human brain differ functionally and structurally. Despite over a century of research, the extent to which brain asymmetry is influenced by sex, handedness, age, and genetic factors is still controversial. Here we present the largest ever analysis of subcortical brain asymmetries, in a harmonized multi-site study using meta-analysis methods. Volumetric asymmetry of seven subcortical structures was assessed in 15,847 MRI scans from 52 datasets worldwide. There were sex differences in the asymmetry of the globus pallidus and putamen. Heritability estimates, derived from 1170 subjects belonging to 71 extended pedigrees, revealed that additive genetic factors influenced the asymmetry of these two structures and that of the hippocampus and thalamus. Handedness had no detectable effect on subcortical asymmetries, even in this unprecedented sample size, but the asymmetry of the putamen varied with age. Genetic drivers of asymmetry in the hippocampus, thalamus and basal ganglia may affect variability in human cognition, including susceptibility to psychiatric disorders.

  15. The gating mechanism of the human aquaporin 5 revealed by molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Lorant Janosi

    Full Text Available Aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins. The high-resolution X-ray structure of the human aquaporin 5 (HsAQP5 shows that HsAQP5, as all the other known aquaporins, exhibits tetrameric structure. By means of molecular dynamics simulations we analyzed the role of spontaneous fluctuations on the structural behavior of the human AQP5. We found that different conformations within the tetramer lead to a distribution of monomeric channel structures, which can be characterized as open or closed. The switch between the two states of a channel is a tap-like mechanism at the cytoplasmic end which regulates the water passage through the pore. The channel is closed by a translation of the His67 residue inside the pore. Moreover, water permeation rate calculations revealed that the selectivity filter, located at the other end of the channel, regulates the flow rate of water molecules when the channel is open, by locally modifying the orientation of His173. Furthermore, the calculated permeation rates of a fully open channel are in good agreement with the reported experimental value.

  16. Computational dissection of human episodic memory reveals mental process-specific genetic profiles.

    Science.gov (United States)

    Luksys, Gediminas; Fastenrath, Matthias; Coynel, David; Freytag, Virginie; Gschwind, Leo; Heck, Angela; Jessen, Frank; Maier, Wolfgang; Milnik, Annette; Riedel-Heller, Steffi G; Scherer, Martin; Spalek, Klara; Vogler, Christian; Wagner, Michael; Wolfsgruber, Steffen; Papassotiropoulos, Andreas; de Quervain, Dominique J-F

    2015-09-01

    Episodic memory performance is the result of distinct mental processes, such as learning, memory maintenance, and emotional modulation of memory strength. Such processes can be effectively dissociated using computational models. Here we performed gene set enrichment analyses of model parameters estimated from the episodic memory performance of 1,765 healthy young adults. We report robust and replicated associations of the amine compound SLC (solute-carrier) transporters gene set with the learning rate, of the collagen formation and transmembrane receptor protein tyrosine kinase activity gene sets with the modulation of memory strength by negative emotional arousal, and of the L1 cell adhesion molecule (L1CAM) interactions gene set with the repetition-based memory improvement. Furthermore, in a large functional MRI sample of 795 subjects we found that the association between L1CAM interactions and memory maintenance revealed large clusters of differences in brain activity in frontal cortical areas. Our findings provide converging evidence that distinct genetic profiles underlie specific mental processes of human episodic memory. They also provide empirical support to previous theoretical and neurobiological studies linking specific neuromodulators to the learning rate and linking neural cell adhesion molecules to memory maintenance. Furthermore, our study suggests additional memory-related genetic pathways, which may contribute to a better understanding of the neurobiology of human memory.

  17. Repeated Structural Imaging Reveals Nonlinear Progression of Experience-Dependent Volume Changes in Human Motor Cortex.

    Science.gov (United States)

    Wenger, Elisabeth; Kühn, Simone; Verrel, Julius; Mårtensson, Johan; Bodammer, Nils Christian; Lindenberger, Ulman; Lövdén, Martin

    2017-05-01

    Evidence for experience-dependent structural brain change in adult humans is accumulating. However, its time course is not well understood, as intervention studies typically consist of only 2 imaging sessions (before vs. after training). We acquired up to 18 structural magnetic resonance images over a 7-week period while 15 right-handed participants practiced left-hand writing and drawing. After 4 weeks, we observed increases in gray matter of both left and right primary motor cortices relative to a control group; 3 weeks later, these differences were no longer reliable. Time-series analyses revealed that gray matter in the primary motor cortices expanded during the first 4 weeks and then partially renormalized, in particular in the right hemisphere, despite continued practice and increasing task proficiency. Similar patterns of expansion followed by partial renormalization are also found in synaptogenesis, cortical map plasticity, and maturation, and may qualify as a general principle of structural plasticity. Research on human brain plasticity needs to encompass more than 2 measurement occasions to capture expansion and potential renormalization processes over time. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. Latent physiological factors of complex human diseases revealed by independent component analysis of clinarrays

    Directory of Open Access Journals (Sweden)

    Chen David P

    2010-10-01

    Full Text Available Abstract Background Diagnosis and treatment of patients in the clinical setting is often driven by known symptomatic factors that distinguish one particular condition from another. Treatment based on noticeable symptoms, however, is limited to the types of clinical biomarkers collected, and is prone to overlooking dysfunctions in physiological factors not easily evident to medical practitioners. We used a vector-based representation of patient clinical biomarkers, or clinarrays, to search for latent physiological factors that underlie human diseases directly from clinical laboratory data. Knowledge of these factors could be used to improve assessment of disease severity and help to refine strategies for diagnosis and monitoring disease progression. Results Applying Independent Component Analysis on clinarrays built from patient laboratory measurements revealed both known and novel concomitant physiological factors for asthma, types 1 and 2 diabetes, cystic fibrosis, and Duchenne muscular dystrophy. Serum sodium was found to be the most significant factor for both type 1 and type 2 diabetes, and was also significant in asthma. TSH3, a measure of thyroid function, and blood urea nitrogen, indicative of kidney function, were factors unique to type 1 diabetes respective to type 2 diabetes. Platelet count was significant across all the diseases analyzed. Conclusions The results demonstrate that large-scale analyses of clinical biomarkers using unsupervised methods can offer novel insights into the pathophysiological basis of human disease, and suggest novel clinical utility of established laboratory measurements.

  19. Lack of tissue renewal in human adult Achilles tendon is revealed by nuclear bomb 14C

    Science.gov (United States)

    Heinemeier, Katja Maria; Schjerling, Peter; Heinemeier, Jan; Magnusson, Stig Peter; Kjaer, Michael

    2013-01-01

    Tendons are often injured and heal poorly. Whether this is caused by a slow tissue turnover is unknown, since existing data provide diverging estimates of tendon protein half-life that range from 2 mo to 200 yr. With the purpose of determining life-long turnover of human tendon tissue, we used the 14C bomb-pulse method. This method takes advantage of the dramatic increase in atmospheric levels of 14C, produced by nuclear bomb tests in 1955–1963, which is reflected in all living organisms. Levels of 14C were measured in 28 forensic samples of Achilles tendon core and 4 skeletal muscle samples (donor birth years 1945–1983) with accelerator mass spectrometry (AMS) and compared to known atmospheric levels to estimate tissue turnover. We found that Achilles tendon tissue retained levels of 14C corresponding to atmospheric levels several decades before tissue sampling, demonstrating a very limited tissue turnover. The tendon concentrations of 14C approximately reflected the atmospheric levels present during the first 17 yr of life, indicating that the tendon core is formed during height growth and is essentially not renewed thereafter. In contrast, 14C levels in muscle indicated continuous turnover. Our observation provides a fundamental premise for understanding tendon function and pathology, and likely explains the poor regenerative capacity of tendon tissue.—Heinemeier, K. M., Schjerling, P., Heinemeier, J., Magnusson, S. P., Kjaer, M. Lack of tissue renewal in human adult Achilles tendon is revealed by nuclear bomb 14C. PMID:23401563

  20. GAPscreener: An automatic tool for screening human genetic association literature in PubMed using the support vector machine technique

    Directory of Open Access Journals (Sweden)

    Khoury Muin J

    2008-04-01

    Full Text Available Abstract Background Synthesis of data from published human genetic association studies is a critical step in the translation of human genome discoveries into health applications. Although genetic association studies account for a substantial proportion of the abstracts in PubMed, identifying them with standard queries is not always accurate or efficient. Further automating the literature-screening process can reduce the burden of a labor-intensive and time-consuming traditional literature search. The Support Vector Machine (SVM, a well-established machine learning technique, has been successful in classifying text, including biomedical literature. The GAPscreener, a free SVM-based software tool, can be used to assist in screening PubMed abstracts for human genetic association studies. Results The data source for this research was the HuGE Navigator, formerly known as the HuGE Pub Lit database. Weighted SVM feature selection based on a keyword list obtained by the two-way z score method demonstrated the best screening performance, achieving 97.5% recall, 98.3% specificity and 31.9% precision in performance testing. Compared with the traditional screening process based on a complex PubMed query, the SVM tool reduced by about 90% the number of abstracts requiring individual review by the database curator. The tool also ascertained 47 articles that were missed by the traditional literature screening process during the 4-week test period. We examined the literature on genetic associations with preterm birth as an example. Compared with the traditional, manual process, the GAPscreener both reduced effort and improved accuracy. Conclusion GAPscreener is the first free SVM-based application available for screening the human genetic association literature in PubMed with high recall and specificity. The user-friendly graphical user interface makes this a practical, stand-alone application. The software can be downloaded at no charge.

  1. RODENT AND HUMAN NEUROPROGENITOR CELLS FOR HIGH-CONTENT SCREENS OF CHEMICAL EFFECTS ON PROLIFERATION AND APOPTOSIS

    Science.gov (United States)

    The objective of these experiments is to develop high-throughput screens for proliferation and apoptosis in order to compare rodent and human neuroprogenitor cell responses to potential developmental neurotoxicants. Effects of 4 chemicals on proliferation and apoptosis in mouse c...

  2. Gas chromatographic quadrupole time-of-flight full scan high resolution mass spectrometric screening of human urine in antidoping analysis

    NARCIS (Netherlands)

    Abushareeda, Wadha; Lyris, Emmanouil; Kraiem, Suhail; Wahaibi, Aisha Al; Alyazidi, Sameera; Dbes, Najib; Lommen, Arjen; Nielen, Michel; Horvatovich, Peter L.; Alsayrafi, Mohammed; Georgakopoulos, Costas

    2017-01-01

    This paper presents the development and validation of a high-resolution full scan (FS) electron impact ionization (EI) gas chromatography coupled to quadrupole Time-of-Flight mass spectrometry (GC/QTOF) platform for screening anabolic androgenic steroids (AAS) in human urine samples. The World

  3. Gas chromatographic quadrupole time-of-flight full scan high resolution mass spectrometric screening of human urine in antidoping analysis

    NARCIS (Netherlands)

    Abushareeda, Wadha; Lyris, Emmanouil; Kraiem, Suhail; Wahaibi, Aisha Al; Alyazidi, Sameera; Dbes, Najib; Lommen, Arjen; Nielen, Michel; Horvatovich, Peter L.; Alsayrafi, Mohammed; Georgakopoulos, Costas

    2017-01-01

    This paper presents the development and validation of a high-resolution full scan (FS) electron impact ionization (EI) gas chromatography coupled to quadrupole Time-of-Flight mass spectrometry (GC/QTOF) platform for screening anabolic androgenic steroids (AAS) in human urine samples. The World

  4. Improvement of Gynecological Screening of Female Renal Transplant Recipients by Self-Sampling for Human Papillomavirus Detection

    NARCIS (Netherlands)

    Hinten, F.; Hilbrands, L.B.; Meeuwis, K.A.P.; Bergen-Verkuyten, M.C. van; Slangen, B.F.; Rossum, M.M. van; Rahamat-Langendoen, J.C.; Massuger, L.F.A.G.; Hullu, J.A. de; Melchers, W.J.G.

    2017-01-01

    OBJECTIVES: Female renal transplant recipients (RTRs) have increased risk for developing human papillomavirus (HPV)-related (pre)malignancies of the lower genital tract. Annual cervical screening is advised for RTRs, but the participation rate is low. The aim of this study is to investigate whether

  5. A genetic screen reveals Arabidopsis stomatal and/or apoplastic defenses against Pseudomonas syringae pv. tomato DC3000.

    Directory of Open Access Journals (Sweden)

    Weiqing Zeng

    2011-10-01

    Full Text Available Bacterial infection of plants often begins with colonization of the plant surface, followed by entry into the plant through wounds and natural openings (such as stomata, multiplication in the intercellular space (apoplast of the infected tissues, and dissemination of bacteria to other plants. Historically, most studies assess bacterial infection based on final outcomes of disease and/or pathogen growth using whole infected tissues; few studies have genetically distinguished the contribution of different host cell types in response to an infection. The phytotoxin coronatine (COR is produced by several pathovars of Pseudomonas syringae. COR-deficient mutants of P. s. tomato (Pst DC3000 are severely compromised in virulence, especially when inoculated onto the plant surface. We report here a genetic screen to identify Arabidopsis mutants that could rescue the virulence of COR-deficient mutant bacteria. Among the susceptible to coronatine-deficient Pst DC3000 (scord mutants were two that were defective in stomatal closure response, two that were defective in apoplast defense, and four that were defective in both stomatal and apoplast defense. Isolation of these three classes of mutants suggests that stomatal and apoplastic defenses are integrated in plants, but are genetically separable, and that COR is important for Pst DC3000 to overcome both stomatal guard cell- and apoplastic mesophyll cell-based defenses. Of the six mutants defective in bacterium-triggered stomatal closure, three are defective in salicylic acid (SA-induced stomatal closure, but exhibit normal stomatal closure in response to abscisic acid (ABA, and scord7 is compromised in both SA- and ABA-induced stomatal closure. We have cloned SCORD3, which is required for salicylic acid (SA biosynthesis, and SCORD5, which encodes an ATP-binding cassette (ABC protein, AtGCN20/AtABCF3, predicted to be involved in stress-associated protein translation control. Identification of SCORD5 begins to

  6. An EST screen from the annelid Pomatoceros lamarckii reveals patterns of gene loss and gain in animals

    Directory of Open Access Journals (Sweden)

    Chen Wei-Chung

    2009-09-01

    Full Text Available Abstract Background Since the drastic reorganisation of the phylogeny of the animal kingdom into three major clades of bilaterians; Ecdysozoa, Lophotrochozoa and Deuterostomia, it became glaringly obvious that the selection of model systems with extensive molecular resources was heavily biased towards only two of these three clades, namely the Ecdysozoa and Deuterostomia. Increasing efforts have been put towards redressing this imbalance in recent years, and one of the principal phyla in the vanguard of this endeavour is the Annelida. Results In the context of this effort we here report our characterisation of an Expressed Sequence Tag (EST screen in the serpulid annelid, Pomatoceros lamarckii. We have sequenced over 5,000 ESTs which consolidate into over 2,000 sequences (clusters and singletons. These sequences are used to build phylogenetic trees to estimate relative branch lengths amongst different taxa and, by comparison to genomic data from other animals, patterns of gene retention and loss are deduced. Conclusion The molecular phylogenetic trees including the P. lamarckii sequences extend early observations that polychaetes tend to have relatively short branches in such trees, and hence are useful taxa with which to reconstruct gene family evolution. Also, with the availability of lophotrochozoan data such as that of P. lamarckii, it is now possible to make much more accurate reconstructions of the gene complement of the ancestor of the bilaterians than was previously possible from comparisons of ecdysozoan and deuterostome genomes to non-bilaterian outgroups. It is clear that the traditional molecular model systems for protostomes (e.g. Drosophila melanogaster and Caenorhabditis elegans, which are restricted to the Ecdysozoa, have undergone extensive gene loss during evolution. These ecdysozoan systems, in terms of gene content, are thus more derived from the bilaterian ancestral condition than lophotrochozoan systems like the polychaetes

  7. Targeted Genetic Screen in Amyotrophic Lateral Sclerosis Reveals Novel Genetic Variants with Synergistic Effect on Clinical Phenotype

    Directory of Open Access Journals (Sweden)

    Johnathan Cooper-Knock

    2017-11-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is underpinned by an oligogenic rare variant architecture. Identified genetic variants of ALS include RNA-binding proteins containing prion-like domains (PrLDs. We hypothesized that screening genes encoding additional similar proteins will yield novel genetic causes of ALS. The most common genetic variant of ALS patients is a G4C2-repeat expansion within C9ORF72. We have shown that G4C2-repeat RNA sequesters RNA-binding proteins. A logical consequence of this is that loss-of-function mutations in G4C2-binding partners might contribute to ALS pathogenesis independently of and/or synergistically with C9ORF72 expansions. Targeted sequencing of genomic DNA encoding either RNA-binding proteins or known ALS genes (n = 274 genes was performed in ALS patients to identify rare deleterious genetic variants and explore genotype-phenotype relationships. Genomic DNA was extracted from 103 ALS patients including 42 familial ALS patients and 61 young-onset (average age of onset 41 years sporadic ALS patients; patients were chosen to maximize the probability of identifying genetic causes of ALS. Thirteen patients carried a G4C2-repeat expansion of C9ORF72. We identified 42 patients with rare deleterious variants; 6 patients carried more than one variant. Twelve mutations were discovered in known ALS genes which served as a validation of our strategy. Rare deleterious variants in RNA-binding proteins were significantly enriched in ALS patients compared to control frequencies (p = 5.31E-18. Nineteen patients featured at least one variant in a RNA-binding protein containing a PrLD. The number of variants per patient correlated with rate of disease progression (t-test, p = 0.033. We identified eighteen patients with a single variant in a G4C2-repeat binding protein. Patients with a G4C2-binding protein variant in combination with a C9ORF72 expansion had a significantly faster disease course (t-test, p = 0.025. Our data are

  8. Impact of enrofloxacin on the human intestinal microbiota revealed by comparative molecular analysis.

    Science.gov (United States)

    Kim, Bong-Soo; Kim, Jong Nam; Yoon, Seok-Hwan; Chun, Jongsik; Cerniglia, Carl E

    2012-06-01

    The indigenous human intestinal microbiota could be disrupted by residues of antibiotics in foods as well as therapeutically administered antibiotics to humans. These disruptions may lead to adverse health outcomes. To observe the possible impact of residues of antibiotics at concentrations below therapeutic levels on human intestinal microbiota, we performed studies using in vitro cultures of fecal suspensions from three individuals with 10 different concentrations (0, 0.1, 0.5, 1, 5, 10, 15, 25, 50 and 150 μg/ml) of the fluoroquinolone, enrofloxacin. The bacterial communities of the control and enrofloxacin dosed fecal samples were analyzed by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. In addition, changes of functional gene expression were analyzed by a pyrosequencing-based random whole-community mRNA sequencing method. Although each individual had a unique microbial composition, the communities of all individuals were affected by enrofloxacin. The proportions of two phyla, namely, Bacteroidetes and Proteobacteria, were significantly reduced with increasing concentrations of enrofloxacin exposure, while the proportion of Firmicutes increased. Principal Coordinate Analysis (PCoA) using the Fast UniFrac indicated that the community structures of intestinal microbiota were shifted by enrofloxacin. Most of the mRNA transcripts and the anti-microbial drug resistance genes increased with increasing concentrations of enrofloxacin. 16S rRNA gene pyrosequencing of control and enrofloxacin treated fecal suspensions provided valuable information of affected bacterial taxa down to the species level, and the community transcriptomic analyses using mRNA revealed the functional gene expression responses of the changed bacterial communities by enrofloxacin. Published by Elsevier Ltd.

  9. Mechanistic Insights on Human Phosphoglucomutase Revealed by Transition Path Sampling and Molecular Dynamics Calculations.

    Science.gov (United States)

    Brás, Natércia F; Fernandes, Pedro A; Ramos, Maria J; Schwartz, Steven D

    2018-02-06

    Human α-phosphoglucomutase 1 (α-PGM) catalyzes the isomerization of glucose-1-phosphate into glucose-6-phosphate (G6P) through two sequential phosphoryl transfer steps with a glucose-1,6-bisphosphate (G16P) intermediate. Given that the release of G6P in the gluconeogenesis raises the glucose output levels, α-PGM represents a tempting pharmacological target for type 2 diabetes. Here, we provide the first theoretical study of the catalytic mechanism of human α-PGM. We performed transition-path sampling simulations to unveil the atomic details of the two catalytic chemical steps, which could be key for developing transition state (TS) analogue molecules with inhibitory properties. Our calculations revealed that both steps proceed through a concerted S N 2-like mechanism, with a loose metaphosphate-like TS. Even though experimental data suggests that the two steps are identical, we observed noticeable differences: 1) the transition state ensemble has a well-defined TS region and a late TS for the second step, and 2) larger coordinated protein motions are required to reach the TS of the second step. We have identified key residues (Arg23, Ser117, His118, Lys389), and the Mg 2+ ion that contribute in different ways to the reaction coordinate. Accelerated molecular dynamics simulations suggest that the G16P intermediate may reorient without leaving the enzymatic binding pocket, through significant conformational rearrangements of the G16P and of specific loop regions of the human α-PGM. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Lifespan Development of the Human Brain Revealed by Large-Scale Network Eigen-Entropy

    Directory of Open Access Journals (Sweden)

    Yiming Fan

    2017-09-01

    Full Text Available Imaging connectomics based on graph theory has become an effective and unique methodological framework for studying functional connectivity patterns of the developing and aging brain. Normal brain development is characterized by continuous and significant network evolution through infancy, childhood, and adolescence, following specific maturational patterns. Normal aging is related to some resting state brain networks disruption, which are associated with certain cognitive decline. It is a big challenge to design an integral metric to track connectome evolution patterns across the lifespan, which is to understand the principles of network organization in the human brain. In this study, we first defined a brain network eigen-entropy (NEE based on the energy probability (EP of each brain node. Next, we used the NEE to characterize the lifespan orderness trajectory of the whole-brain functional connectivity of 173 healthy individuals ranging in age from 7 to 85 years. The results revealed that during the lifespan, the whole-brain NEE exhibited a significant non-linear decrease and that the EP distribution shifted from concentration to wide dispersion, implying orderness enhancement of functional connectome over age. Furthermore, brain regions with significant EP changes from the flourishing (7–20 years to the youth period (23–38 years were mainly located in the right prefrontal cortex and basal ganglia, and were involved in emotion regulation and executive function in coordination with the action of the sensory system, implying that self-awareness and voluntary control performance significantly changed during neurodevelopment. However, the changes from the youth period to middle age (40–59 years were located in the mesial temporal lobe and caudate, which are associated with long-term memory, implying that the memory of the human brain begins to decline with age during this period. Overall, the findings suggested that the human connectome

  11. A Human Open Field Test Reveals Thigmotaxis Related to Agoraphobic Fear.

    Science.gov (United States)

    Walz, Nora; Mühlberger, Andreas; Pauli, Paul

    2016-09-01

    Thigmotaxis refers to a specific behavior of animals (i.e., to stay close to walls when exploring an open space). Such behavior can be assessed with the open field test (OFT), which is a well-established indicator of animal fear. The detection of similar open field behavior in humans may verify the translational validity of this paradigm. Enhanced thigmotaxis related to anxiety may suggest the relevance of such behavior for anxiety disorders, especially agoraphobia. A global positioning system was used to analyze the behavior of 16 patients with agoraphobia and 18 healthy individuals with a risk for agoraphobia (i.e., high anxiety sensitivity) during a human OFT and compare it with appropriate control groups (n = 16 and n = 19). We also tracked 17 patients with agoraphobia and 17 control participants during a city walk that involved walking through an open market square. Our human OFT triggered thigmotaxis in participants; patients with agoraphobia and participants with high anxiety sensitivity exhibited enhanced thigmotaxis. This behavior was evident in increased movement lengths along the wall of the natural open field and fewer entries into the center of the field despite normal movement speed and length. Furthermore, participants avoided passing through the market square during the city walk, indicating again that thigmotaxis is related to agoraphobia. This study is the first to our knowledge to verify the translational validity of the OFT and to reveal that thigmotaxis, an evolutionarily adaptive behavior shown by most species, is related to agoraphobia, a pathologic fear of open spaces, and anxiety sensitivity, a risk factor for agoraphobia. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  12. Barcoded pyrosequencing reveals that consumption of galactooligosaccharides results in a highly specific bifidogenic response in humans.

    Directory of Open Access Journals (Sweden)

    Lauren M G Davis

    Full Text Available Prebiotics are selectively fermented ingredients that allow specific changes in the gastrointestinal microbiota that confer health benefits to the host. However, the effects of prebiotics on the human gut microbiota are incomplete as most studies have relied on methods that fail to cover the breadth of the bacterial community. The goal of this research was to use high throughput multiplex community sequencing of 16S rDNA tags to gain a community wide perspective of the impact of prebiotic galactooligosaccharide (GOS on the fecal microbiota of healthy human subjects. Fecal samples from eighteen healthy adults were previously obtained during a feeding trial in which each subject consumed a GOS-containing product for twelve weeks, with four increasing dosages (0, 2.5, 5, and 10 gram of GOS. Multiplex sequencing of the 16S rDNA tags revealed that GOS induced significant compositional alterations in the fecal microbiota, principally by increasing the abundance of organisms within the Actinobacteria. Specifically, several distinct lineages of Bifidobacterium were enriched. Consumption of GOS led to five- to ten-fold increases in bifidobacteria in half of the subjects. Increases in Firmicutes were also observed, however, these changes were detectable in only a few individuals. The enrichment of bifidobacteria was generally at the expense of one group of bacteria, the Bacteroides. The responses to GOS and the magnitude of the response varied between individuals, were reversible, and were in accordance with dosage. The bifidobacteria were the only bacteria that were consistently and significantly enriched by GOS, although this substrate supported the growth of diverse colonic bacteria in mono-culture experiments. These results suggest that GOS can be used to enrich bifidobacteria in the human gastrointestinal tract with remarkable specificity, and that the bifidogenic properties of GOS that occur in vivo are caused by selective fermentation as well as by

  13. Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying; Takagi, Akira [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kayano, Hidekazu [Department of Pathology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Koyama, Isamu [Department of Digestive and General Surgery, Saitama International Medical Center, Faculty of Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    In a previous study, we developed five kinds of monoclonal antibodies against different portions of human mEH: three, anti-N-terminal; one, anti-C-terminal; one, anti-conformational epitope. Using them, we stained the intact and the permeabilized human cells of various kinds and performed flow cytometric analysis. Primary hepatocytes and peripheral blood mononuclear cells (PBMC) showed remarkable differences. On the surface, hepatocytes exhibited 4 out of 5 epitopes whereas PBMC did not show any of the epitopes. mEH was detected inside both cell types, but the most prominent expression was observed for the conformational epitope in the hepatocytes and the two N-terminal epitopes in PBMC. These differences were also observed between hepatocyte-derived cell lines and mononuclear cell-derived cell lines. In addition, among each group, there were several differences which may be related to the cultivation, the degree of differentiation, or the original cell subsets. We also noted that two glioblastoma cell lines reveal marked expression of the conformational epitope on the surface which seemed to correlate with the brain tumor-associated antigen reported elsewhere. Several cell lines also underwent selective permeabilization before flow cytometric analysis, and we noticed that the topological orientation of mEH on the ER membrane in those cells was in accordance with the previous report. However, the orientation on the cell surface was inconsistent with the report and had a great variation between the cells. These findings show the multiple mode of expression of mEH which may be possibly related to the multiple roles that mEH plays in different cells. -- Highlights: ► We examine expression of five mEH epitopes in human cells. ► Remarkable differences exist between hepatocytes and PBMC. ► mEH expression in cell lines differs depending on several factors. ► Some glioblastoma cell lines reveal marked surface expression of mEH. ► Topology of mEH on the cell

  14. Muscle biopsies from human muscle diseases with myopathic pathology reveal common alterations in mitochondrial function.

    Science.gov (United States)

    Sunitha, Balaraju; Gayathri, Narayanappa; Kumar, Manish; Keshava Prasad, Thottethodi Subrahmanya; Nalini, Atchayaram; Padmanabhan, Balasundaram; Srinivas Bharath, Muchukunte Mukunda

    2016-07-01

    Muscle diseases are clinically and genetically heterogeneous and manifest as dystrophic, inflammatory and myopathic pathologies, among others. Our previous study on the cardiotoxin mouse model of myodegeneration and inflammation linked muscle pathology with mitochondrial damage and oxidative stress. In this study, we investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies from muscle disease patients, represented by dysferlinopathy (dysfy) (dystrophic pathology; n = 43), polymyositis (PM) (inflammatory pathology; n = 24), and distal myopathy with rimmed vacuoles (DMRV) (distal myopathy; n = 31) were analyzed. Mitochondrial damage (ragged blue and COX-deficient fibers) was revealed in dysfy, PM, and DMRV cases by enzyme histochemistry (SDH and COX-SDH), electron microscopy (vacuolation and altered cristae) and biochemical assays (significantly increased ADP/ATP ratio). Proteomic analysis of muscle mitochondria from all three muscle diseases by isobaric tag for relative and absolute quantitation labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated down-regulation of electron transport chain (ETC) complex subunits, assembly factors and Krebs cycle enzymes. Interestingly, 80 of the under-expressed proteins were common among the three pathologies. Assay of ETC and Krebs cycle enzyme activities validated the MS data. Mitochondrial proteins from muscle pathologies also displayed higher tryptophan (Trp) oxidation and the same was corroborated in the cardiotoxin model. Molecular modeling predicted Trp oxidation to alter the local structure of mitochondrial proteins. Our data highlight mitochondrial alterations in muscle pathologies, represented by morphological changes, altered mitochondrial proteome and protein oxidation, thereby establishing the role of mitochondrial damage in human muscle diseases. We investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies

  15. Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity

    Science.gov (United States)

    Marjanovic, Jasmina; Chalupska, Dominika; Patenode, Caroline; Coster, Adam; Arnold, Evan; Ye, Alice; Anesi, George; Lu, Ying; Okun, Ilya; Tkachenko, Sergey; Haselkorn, Robert; Gornicki, Piotr

    2010-01-01

    Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism with multiple isozymes often expressed in different eukaryotic cellular compartments. ACC-made malonyl-CoA serves as a precursor for fatty acids; it also regulates fatty acid oxidation and feeding behavior in animals. ACC provides an important target for new drugs to treat human diseases. We have developed an inexpensive nonradioactive high-throughput screening system to identify new ACC inhibitors. The screen uses yeast gene-replacement strains depending for growth on cloned human ACC1 and ACC2. In “proof of concept” experiments, growth of such strains was inhibited by compounds known to target human ACCs. The screen is sensitive and robust. Medium-size chemical libraries yielded new specific inhibitors of human ACC2. The target of the best of these inhibitors was confirmed with in vitro enzymatic assays. This compound is a new drug chemotype inhibiting human ACC2 with 2.8 μM IC50 and having no effect on human ACC1 at 100 μM. PMID:20439761

  16. Methylation screening of the TGFBI promoter in human lung and prostate cancer by methylation-specific PCR

    International Nuclear Information System (INIS)

    Shah, Jinesh N; Shao, Genze; Hei, Tom K; Zhao, Yongliang

    2008-01-01

    Hypermethylation of the TGFBI promoter has been shown to correlate with decreased expression of this gene in human tumor cell lines. In this study, we optimized a methylation-specific polymerase chain reaction (MSP) method and investigated the methylation status of the TGFBI promoter in human lung and prostate cancer specimens. Methylation-specific primers were designed based on the methylation profiles of the TGFBI promoter in human tumor cell lines, and MSP conditions were optimized for accurate and efficient amplification. Genomic DNA was isolated from lung tumors and prostatectomy tissues of prostate cancer patients, bisulfite-converted, and analyzed by MSP. Among 50 lung cancer samples, 44.0% (22/50) harbored methylated CpG sites in the TGFBI promoter. An analysis correlating gene methylation status with clinicopathological cancer features revealed that dense methylation of the TGFBI promoter was associated with a metastatic phenotype, with 42.9% (6/14) of metastatic lung cancer samples demonstrating dense methylation vs. only 5.6% (2/36) of primary lung cancer samples (p < 0.05). Similar to these lung cancer results, 82.0% (41/50) of prostate cancer samples harbored methylated CpG sites in the TGFBI promoter, and dense methylation of the promoter was present in 38.9% (7/18) of prostate cancer samples with the feature of locoregional invasiveness vs. only 19.4% (6/31) of prostate cancer samples without locoregional invasiveness (p < 0.05). Furthermore, promoter hypermethylation correlated with highly reduced expression of the TGFBI gene in human lung and prostate tumor cell lines. We successfully optimized a MSP method for the precise and efficient screening of TGFBI promoter methylation status. Dense methylation of the TGFBI promoter correlated with the extent of TGFBI gene silencing in tumor cell lines and was related to invasiveness of prostate tumors and metastatic status of lung cancer tumors. Thus, TGFBI promoter methylation can be used as a potential

  17. Impact of variations in triage cytology interpretation on human papillomavirus-based cervical screening and implications for screening algorithms.

    Science.gov (United States)

    Ronco, Guglielmo; Zappa, Marco; Franceschi, Silvia; Tunesi, Sara; Caprioglio, Adele; Confortini, Massimo; Del Mistro, Annarosa; Carozzi, Francesca; Segnan, Nereo; Zorzi, Manuel; Giorgi-Rossi, Paolo

    2016-11-01

    Women positive to human papillomavirus (HPV+) testing at cervical screening need triage, typically cytology and immediate colposcopy in case of atypical squamous cells of undetermined significance (ASCUS) or worse (ASCUS+) or, in cytology-normal HPV+ women, HPV test repeat after 1 year and colposcopy referral if still HPV+. Our hypothesis was that substantial variations in triage positivity and sensitivity may produce little variation in overall referral to colposcopy and on sensitivity of the entire screening process. Centre- and age-aggregated data from 72,869 women aged 35-64 years were derived from 10 organised screening programmes which have piloted HPV screening in Italy since 2012. Overall colposcopy referral was evaluated as a function of immediate colposcopy referral and overall CIN2+ detection as a function of the proportion of all CIN2+ detected by immediate referral (a proxy of cytology's sensitivity). We fitted additive regression models, adjusted for centre, age, compliance to HPV retesting and to colposcopy, by generalised estimation equations. The proportion of HPV+ women directly referred to colposcopy varied across programmes (20-57%; average 37%) and so did CIN2+ detection (49-94%; average 77%). Overall, 63% (range 41-75%) of HPV+ were referred to colposcopy either immediately or at HPV repeat. An absolute 10% increase in immediate colposcopy referral resulted in 4.2% (95% CI: 3.3-5.1%) increase in overall referral. An absolute 10% increase in cytology's sensitivity resulted in a 1.1% (95% CI: 0.1-2.0%) increase in overall CIN2+ detection. Repeat HPV testing limits the effect of subjectivity of cytology interpretation on overall referral and sensitivity. These will change only slightly when replacing cytology with another test if the interval to HPV repeat remains unchanged. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. Transcriptomic profiling-based mutant screen reveals three new transcription factors mediating menadione resistance in Neurospora crassa.

    Science.gov (United States)

    Zhu, Jufen; Yu, Xinxu; Xie, Baogui; Gu, Xiaokui; Zhang, Zhenying; Li, Shaojie

    2013-06-01

    To gain insight into the regulatory mechanisms of oxidative stress responses in filamentous fungi, the genome-wide transcriptional response of Neurospora crassa to menadione was analysed by digital gene expression (DGE) profiling, which identified 779 upregulated genes and 576 downregulated genes. Knockout mutants affecting 130 highly-upregulated genes were tested for menadione sensitivity, which revealed that loss of the transcription factor siderophore regulation (SRE) (a transcriptional repressor for siderophore biosynthesis), catatase-3, cytochrome c peroxidase or superoxide dismutase 1 copper chaperone causes hypersensitivity to menadione. Deletion of sre dramatically increased transcription of the siderophore biosynthesis gene ono and the siderophore iron transporter gene sit during menadione stress, suggesting that SRE is required for repression of iron uptake under oxidative stress conditions. Contrary to its phenotype, the sre deletion mutant showed higher transcriptional levels of genes encoding reactive oxygen species (ROS) scavengers than wild type during menadione stress, which implies that the mutant suffers a higher level of oxidative stress than wild type. Uncontrolled iron uptake in the sre mutant might exacerbate cellular oxidative stress. This is the first report of a negative regulator of iron assimilation participating in the fungal oxidative stress response. In addition to SRE, eight other transcription factor genes were also menadione-responsive but their single gene knockout mutants showed wild-type menadione sensitivity. Two of them, named as mit-2 (menadione induced transcription factor-2) and mit-4 (menadione induced transcription factor-4), were selected for double mutant analysis. The double mutant was hypersensitive to menadione. Similarly, the double mutation of mit-2 and sre also had additive effects on menadione sensitivity, suggesting multiple transcription factors mediate oxidative stress resistance in an additive manner

  19. A Genetic Screen Reveals that Synthesis of 1,4-Dihydroxy-2-Naphthoate (DHNA), but Not Full-Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival.

    Science.gov (United States)

    Chen, Grischa Y; McDougal, Courtney E; D'Antonio, Marc A; Portman, Jonathan L; Sauer, John-Demian

    2017-03-21

    Through unknown mechanisms, the host cytosol restricts bacterial colonization; therefore, only professional cytosolic pathogens are adapted to colonize this host environment. Listeria monocytogenes is a Gram-positive intracellular pathogen that is highly adapted to colonize the cytosol of both phagocytic and nonphagocytic cells. To identify L. monocytogenes determinants of cytosolic survival, we designed and executed a novel screen to isolate L. monocytogenes mutants with cytosolic survival defects. Multiple mutants identified in the screen were defective for synthesis of menaquinone (MK), an essential molecule in the electron transport chain. Analysis of an extensive set of MK biosynthesis and respiratory chain mutants revealed that cellular respiration was not required for cytosolic survival of L. monocytogenes but that, instead, synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an MK biosynthesis intermediate, was essential. Recent discoveries showed that modulation of the central metabolism of both host and pathogen can influence the outcome of host-pathogen interactions. Our results identify a potentially novel function of the MK biosynthetic intermediate DHNA and specifically highlight how L. monocytogenes metabolic adaptations promote cytosolic survival and evasion of host immunity. IMPORTANCE Cytosolic bacterial pathogens, such as Listeria monocytogenes and Francisella tularensis , are exquisitely evolved to colonize the host cytosol in a variety of cell types. Establishing an intracellular niche shields these pathogens from effectors of humoral immunity, grants access to host nutrients, and is essential for pathogenesis. Through yet-to-be-defined mechanisms, the host cytosol restricts replication of non-cytosol-adapted bacteria, likely through a combination of cell autonomous defenses (CADs) and nutritional immunity. Utilizing a novel genetic screen, we identified determinants of L. monocytogenes cytosolic survival and virulence and identified a role

  20. A high content screening assay to predict human drug-induced liver injury during drug discovery.

    Science.gov (United States)

    Persson, Mikael; Løye, Anni F; Mow, Tomas; Hornberg, Jorrit J

    2013-01-01

    Adverse drug reactions are a major cause for failures of drug development programs, drug withdrawals and use restrictions. Early hazard identification and diligent risk avoidance strategies are therefore essential. For drug-induced liver injury (DILI), this is difficult using conventional safety testing. To reduce the risk for DILI, drug candidates with a high risk need to be identified and deselected. And, to produce drug candidates without that risk associated, risk factors need to be assessed early during drug discovery, such that lead series can be optimized on safety parameters. This requires methods that allow for medium-to-high throughput compound profiling and that generate quantitative results suitable to establish structure-activity-relationships during lead optimization programs. We present the validation of such a method, a novel high content screening assay based on six parameters (nuclei counts, nuclear area, plasma membrane integrity, lysosomal activity, mitochondrial membrane potential (MMP), and mitochondrial area) using ~100 drugs of which the clinical hepatotoxicity profile is known. We find that a 100-fold TI between the lowest toxic concentration and the therapeutic Cmax is optimal to classify compounds as hepatotoxic or non-hepatotoxic, based on the individual parameters. Most parameters have ~50% sensitivity and ~90% specificity. Drugs hitting ≥2 parameters at a concentration below 100-fold their Cmax are typically hepatotoxic, whereas non-hepatotoxic drugs typically hit based on nuclei count, MMP and human Cmax, we identified an area without a single false positive, while maintaining 45% sensitivity. Hierarchical clustering using the multi-parametric dataset roughly separates toxic from non-toxic compounds. We employ the assay in discovery projects to prioritize novel compound series during hit-to-lead, to steer away from a DILI risk during lead optimization, for risk assessment towards candidate selection and to provide guidance of safe

  1. A novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells.

    Science.gov (United States)

    Koban, Robert; Neumann, Markus; Daugs, Aila; Bloch, Oliver; Nitsche, Andreas; Langhammer, Stefan; Ellerbrok, Heinz

    2018-02-01

    Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) and FDA approved for treatment of non-small cell lung cancer. In a previous study we could show the in vitro efficacy of gefitinib for treatment of poxvirus infections in monolayer (2D) cultivated cell lines. Permanent cell lines and 2D cultures, however, are known to be rather unphysiological; therefore it is difficult to predict whether determined effective concentrations or the drug efficacy per se are transferable to the in vivo situation. 3D cell cultures, which meanwhile are widely distributed across all fields of research, are a promising tool for more predictive in vitro investigations of antiviral compounds. In this study the spreading of cowpox virus and the antiviral efficacy of gefitinib were analyzed in primary human keratinocytes (NHEK) grown in a novel 3D extracellular matrix-based cell culture model and compared to the respective monolayer culture. 3D-cultivated NHEK grew in a polarized and thus a more physiological manner with altered morphology and close cell-cell contact. Infected cultures showed a strongly elevated sensitivity towards gefitinib. EGFR phosphorylation, cell proliferation, and virus replication were significantly reduced in 3D cultures at gefitinib concentrations which were at least 100-fold lower than those in monolayer cultures and well below the level of cytotoxicity. Our newly established 3D cell culture model with primary human cells is an easy-to-handle alternative to conventional monolayer cell cultures and previously described more complex 3D cell culture systems. It can easily be adapted to other cell types and a broad spectrum of viruses for antiviral drug screening and many other aspects of virus research under more in vivo-like conditions. In consequence, it may contribute to a more targeted realization of necessary in vivo experiments. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Cross-sensory facilitation reveals neural interactions between visual and tactile motion in humans

    Directory of Open Access Journals (Sweden)

    Monica eGori

    2011-04-01

    Full Text Available Many recent studies show that the human brain integrates information across the different senses and that stimuli of one sensory modality can enhance the perception of other modalities. Here we study the processes that mediate cross-modal facilitation and summation between visual and tactile motion. We find that while summation produced a generic, non-specific improvement of thresholds, probably reflecting higher-order interaction of decision signals, facilitation reveals a strong, direction-specific interaction, which we believe reflects sensory interactions. We measured visual and tactile velocity discrimination thresholds over a wide range of base velocities and conditions. Thresholds for both visual and tactile stimuli showed the characteristic dipper function, with the minimum thresholds occurring at a given pedestal speed. When visual and tactile coherent stimuli were combined (summation condition the thresholds for these multi-sensory stimuli also showed a dipper function with the minimum thresholds occurring in a similar range to that for unisensory signals. However, the improvement of multisensory thresholds was weak and not directionally specific, well predicted by the maximum likelihood estimation model (agreeing with previous research. A different technique (facilitation did, however, reveal direction-specific enhancement. Adding a non-informative pedestal motion stimulus in one sensory modality (vision or touch selectively lowered thresholds in the other, by the same amount as pedestals in the same modality. Facilitation did not occur for neutral stimuli like sounds (that would also have reduced temporal uncertainty, nor for motion in opposite direction, even in blocked trials where the subjects knew that the motion was in the opposite direction showing that the facilitation was not under subject control. Cross-sensory facilitation is strong evidence for functionally relevant cross-sensory integration at early levels of sensory

  3. Self-Sampling for Human Papillomavirus Testing among Non-Attenders Increases Attendance to the Norwegian Cervical Cancer Screening Programme.

    Directory of Open Access Journals (Sweden)

    Espen Enerly

    Full Text Available Increasing attendance to screening offers the best potential for improving the effectiveness of well-established cervical cancer screening programs. Self-sampling at home for human papillomavirus (HPV testing as an alternative to a clinical sampling can be a useful policy to increase attendance. To determine whether self-sampling improves screening attendance for women who do not regularly attend the Norwegian Cervical Cancer Screening Programme (NCCSP, 800 women aged 25-69 years in the Oslo area who were due to receive a 2nd reminder to attend regular screening were randomly selected and invited to be part of the intervention group. Women in this group received one of two self-sampling devices, Evalyn Brush or Delphi Screener. To attend screening, women in the intervention group had the option of using the self-sampling device (self-sampling subgroup or visiting their physician for a cervical smear. Self-sampled specimens were split and analyzed for the presence of high-risk (hr HPV by the CLART® HPV2 test and the digene® Hybrid Capture (HC2 test. The control group consisted of 2593 women who received a 2nd reminder letter according to the current guidelines of the NCCSP. The attendance rates were 33.4% in the intervention group and 23.2% in the control group, with similar attendance rates for both self-sampling devices. Women in the self-sampling subgroup responded favorably to both self-sampling devices and cited not remembering receiving a call for screening as the most dominant reason for previous non-attendance. Thirty-two of 34 (94.1% hrHPV-positive women in the self-sampling subgroup attended follow-up. In conclusion, self-sampling increased attendance rates and was feasible and well received. This study lends further support to the proposal that self-sampling may be a valuable alternative for increasing cervical cancer screening coverage in Norway.

  4. Self-Sampling for Human Papillomavirus Testing among Non-Attenders Increases Attendance to the Norwegian Cervical Cancer Screening Programme.

    Science.gov (United States)

    Enerly, Espen; Bonde, Jesper; Schee, Kristina; Pedersen, Helle; Lönnberg, Stefan; Nygård, Mari

    2016-01-01

    Increasing attendance to screening offers the best potential for improving the effectiveness of well-established cervical cancer screening programs. Self-sampling at home for human papillomavirus (HPV) testing as an alternative to a clinical sampling can be a useful policy to increase attendance. To determine whether self-sampling improves screening attendance for women who do not regularly attend the Norwegian Cervical Cancer Screening Programme (NCCSP), 800 women aged 25-69 years in the Oslo area who were due to receive a 2nd reminder to attend regular screening were randomly selected and invited to be part of the intervention group. Women in this group received one of two self-sampling devices, Evalyn Brush or Delphi Screener. To attend screening, women in the intervention group had the option of using the self-sampling device (self-sampling subgroup) or visiting their physician for a cervical smear. Self-sampled specimens were split and analyzed for the presence of high-risk (hr) HPV by the CLART® HPV2 test and the digene® Hybrid Capture (HC)2 test. The control group consisted of 2593 women who received a 2nd reminder letter according to the current guidelines of the NCCSP. The attendance rates were 33.4% in the intervention group and 23.2% in the control group, with similar attendance rates for both self-sampling devices. Women in the self-sampling subgroup responded favorably to both self-sampling devices and cited not remembering receiving a call for screening as the most dominant reason for previous non-attendance. Thirty-two of 34 (94.1%) hrHPV-positive women in the self-sampling subgroup attended follow-up. In conclusion, self-sampling increased attendance rates and was feasible and well received. This study lends further support to the proposal that self-sampling may be a valuable alternative for increasing cervical cancer screening coverage in Norway.

  5. Disease modeling and phenotypic drug screening for diabetic cardiomyopathy using human induced pluripotent stem cells.

    Science.gov (United States)

    Drawnel, Faye M; Boccardo, Stefano; Prummer, Michael; Delobel, Frédéric; Graff, Alexandra; Weber, Michael; Gérard, Régine; Badi, Laura; Kam-Thong, Tony; Bu, Lei; Jiang, Xin; Hoflack, Jean-Christophe; Kiialainen, Anna; Jeworutzki, Elena; Aoyama, Natsuyo; Carlson, Coby; Burcin, Mark; Gromo, Gianni; Boehringer, Markus; Stahlberg, Henning; Hall, Benjamin J; Magnone, Maria Chiara; Kolaja, Kyle; Chien, Kenneth R; Bailly, Jacques; Iacone, Roberto

    2014-11-06

    Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Screening human populations for chromosome damage. Progress report, March 1982-November 1982

    International Nuclear Information System (INIS)

    Norman, A.

    1982-01-01

    The micronuclear counts in 73 relatively young and healthy patients obtained in previous studies were examined. The natural logarithm of the micronuclear counts (LMNC) was approximately normally distributed so we have tested the effects of age, sex, and medical x-ray exposure on the counts. The results show a clear dependence of micronuclear counts on age, and demonstrate that studies of chromosome damage in radiation workers or in other populations exposed to radiation may be misinterpreted if the effects of age and medical x-ray examinations are not controlled. The results also show that the variability in LNMC among the individuals examined cannot be accounted for totally by the factors of age, sex, or medical x-rays. There are at least two other important sources of variation: counting statistics and degree of lymphocyte proliferation. A single set of harlequin stained cells may be sufficient for estimating micronuclear yields, the degree of lymphocyte proliferation, and possibly the frequency of chromosome aberrations. These results point to the usefulness of the micronucleus assay for screening human populations for chromosome damage

  7. Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen

    Directory of Open Access Journals (Sweden)

    Arnaud Carpentier

    2016-05-01

    Full Text Available The establishment of protocols to differentiate human pluripotent stem cells (hPSCs including embryonic (ESC and induced pluripotent (iPSC stem cells into functional hepatocyte-like cells (HLCs creates new opportunities to study liver metabolism, genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses in the context of specific genetic background. While supporting efficient differentiation to HLCs, the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells, which remain differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation, metabolism, genetic network, and response to infection or other external stimuli.

  8. Disease Modeling and Phenotypic Drug Screening for Diabetic Cardiomyopathy using Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Faye M. Drawnel

    2014-11-01

    Full Text Available Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

  9. Plasma metabolomics reveal the correlation of metabolic pathways and Prakritis of humans

    Directory of Open Access Journals (Sweden)

    Amey Shirolkar

    2018-04-01

    Full Text Available Background: Ayurveda, an ancient Indian medicinal system, has categorized human body constitutions in three broad constitutional types (prakritis i.e. Vata, Pitta and Kapha. Objectives: Analysis of plasma metabolites and related pathways to classify Prakriti specific dominant marker metabolites and metabolic pathways. Materials and methods: 38 healthy male individuals were assessed for dominant Prakritis and their fasting blood samples were collected. The processed plasma samples were subjected to rapid resolution liquid chromatography–electrospray ionization–quadrupole time of flight mass spectrometry (RRLC–ESI–QTOFMS. Mass profiles were aligned and subjected to multivariate analysis. Results: Partial least square discriminant analysis (PLS-DA model showed 97.87% recognition capability. List of PLS-DA metabolites was subjected to permutative Benjamini–Hochberg false discovery rate (FDR correction and final list of 76 metabolites with p  2.0 was identified. Pathway analysis using metascape and JEPETTO plugins in Cytoscape revealed that steroidal hormone biosynthesis, amino acid, and arachidonic acid metabolism are major pathways varying with different constitution. Biological Go processes analysis showed that aromatic amino acids, sphingolipids, and pyrimidine nucleotides metabolic processes were dominant in kapha type of body constitution. Fat soluble vitamins, cellular amino acid, and androgen biosynthesis process along with branched chain amino acid and glycerolipid catabolic processes were dominant in pitta type individuals. Vata Prakriti was found to have dominant catecholamine, arachidonic acid and hydrogen peroxide metabolomics processes. Conclusion: The neurotransmission and oxidative stress in vata, BCAA catabolic, androgen, xenobiotics metabolic processes in pitta, and aromatic amino acids, sphingolipid, and pyrimidine metabolic process in kapha Prakriti were the dominant marker pathways. Keywords: Ayurveda, Prakriti, Human

  10. Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease.

    Science.gov (United States)

    Lewis, Wesley R; Malarkey, Erik B; Tritschler, Douglas; Bower, Raqual; Pasek, Raymond C; Porath, Jonathan D; Birket, Susan E; Saunier, Sophie; Antignac, Corinne; Knowles, Michael R; Leigh, Margaret W; Zariwala, Maimoona A; Challa, Anil K; Kesterson, Robert A; Rowe, Steven M; Drummond, Iain A; Parant, John M; Hildebrandt, Friedhelm; Porter, Mary E; Yoder, Bradley K; Berbari, Nicolas F

    2016-07-01

    Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in

  11. Comparative study of human mitochondrial proteome reveals extensive protein subcellular relocalization after gene duplications

    Directory of Open Access Journals (Sweden)

    Huang Yong

    2009-11-01

    Full Text Available Abstract Background Gene and genome duplication is the principle creative force in evolution. Recently, protein subcellular relocalization, or neolocalization was proposed as one of the mechanisms responsible for the retention of duplicated genes. This hypothesis received support from the analysis of yeast genomes, but has not been tested thoroughly on animal genomes. In order to evaluate the importance of subcellular relocalizations for retention of duplicated genes in animal genomes, we systematically analyzed nuclear encoded mitochondrial proteins in the human genome by reconstructing phylogenies of mitochondrial multigene families. Results The 456 human mitochondrial proteins selected for this study were clustered into 305 gene families including 92 multigene families. Among the multigene families, 59 (64% consisted of both mitochondrial and cytosolic (non-mitochondrial proteins (mt-cy families while the remaining 33 (36% were composed of mitochondrial proteins (mt-mt families. Phylogenetic analyses of mt-cy families revealed three different scenarios of their neolocalization following gene duplication: 1 relocalization from mitochondria to cytosol, 2 from cytosol to mitochondria and 3 multiple subcellular relocalizations. The neolocalizations were most commonly enabled by the gain or loss of N-terminal mitochondrial targeting signals. The majority of detected subcellular relocalization events occurred early in animal evolution, preceding the evolution of tetrapods. Mt-mt protein families showed a somewhat different pattern, where gene duplication occurred more evenly in time. However, for both types of protein families, most duplication events appear to roughly coincide with two rounds of genome duplications early in vertebrate evolution. Finally, we evaluated the effects of inaccurate and incomplete annotation of mitochondrial proteins and found that our conclusion of the importance of subcellular relocalization after gene duplication on

  12. Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease.

    Directory of Open Access Journals (Sweden)

    Wesley R Lewis

    2016-07-01

    Full Text Available Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400. While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8. GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC protein 4 (DRC4 where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR to generate one of these human missense

  13. Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals.

    Directory of Open Access Journals (Sweden)

    Yeon Sook Cho

    Full Text Available Interleukin-7 (IL-7 is a cytokine essential for T cell homeostasis, and is clinically important. However, the regulatory mechanism of IL-7 gene expression is not well known, and a systematic approach to screen chemicals that regulate IL-7 expression has not yet been developed. In this study, we attempted to develop human reporter cell lines using CRISPR/Cas9-mediated genome editing technology. For this purpose, we designed donor DNA that contains an enhanced green fluorescent protein (eGFP gene, drug selection cassette, and modified homologous arms which are considered to enhance the translation of the eGFP reporter transcript, and also a highly efficient single-guide RNA with a minimal off-target effect to target the IL-7 start codon region. By applying this system, we established IL-7 eGFP reporter cell lines that could report IL-7 gene transcription based on the eGFP protein signal. Furthermore, we utilized the cells to run a pilot screen campaign for IL-7-upregulating chemicals in a high-throughput format, and identified a chemical that can up-regulate IL-7 gene transcription. Collectively, these results suggest that our IL-7 reporter system can be utilized in large-scale chemical library screening to reveal novel IL-7 regulatory pathways and to identify potential drugs for development of new treatments in immunodeficiency disease.

  14. A buoyancy-based screen of Drosophila larvae for fat-storage mutants reveals a role for Sir2 in coupling fat storage to nutrient availability.

    Directory of Open Access Journals (Sweden)

    Tânia Reis

    2010-11-01

    Full Text Available Obesity has a strong genetic component, but few of the genes that predispose to obesity are known. Genetic screens in invertebrates have the potential to identify genes and pathways that regulate the levels of stored fat, many of which are likely to be conserved in humans. To facilitate such screens, we have developed a simple buoyancy-based screening method for identifying mutant Drosophila larvae with increased levels of stored fat. Using this approach, we have identified 66 genes that when mutated increase organismal fat levels. Among these was a sirtuin family member, Sir2. Sirtuins regulate the storage and metabolism of carbohydrates and lipids by deacetylating key regulatory proteins. However, since mammalian sirtuins function in many tissues in different ways, it has been difficult to define their role in energy homeostasis accurately under normal feeding conditions. We show that knockdown of Sir2 in the larval fat body results in increased fat levels. Moreover, using genetic mosaics, we demonstrate that Sir2 restricts fat accumulation in individual cells of the fat body in a cell-autonomous manner. Consistent with this function, changes in the expression of metabolic enzymes in Sir2 mutants point to a shift away from catabolism. Surprisingly, although Sir2 is typically upregulated under conditions of starvation, Sir2 mutant larvae survive better than wild type under conditions of amino-acid starvation as long as sugars are provided. Our findings point to a Sir2-mediated pathway that activates a catabolic response to amino-acid starvation irrespective of the sugar content of the diet.

  15. Biomarker screening of oral cancer cell lines revealed sub-populations of CD133-, CD44-, CD24- and ALDH1- positive cancer stem cells

    Directory of Open Access Journals (Sweden)

    Kendall K

    2013-05-01

    Full Text Available Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for cancer-related mortality. For the past several decades the mainstay of treatment for HNSCC has been surgery and external beam radiation, although more recent trials combining chemotherapy and radiation have demonstrated improvements. However, cancer recurrence and treatment failures continue to occur in a significant percentage of patients. Recent advances in tumor biology have led to the discovery that many cancers, including HNSCC, may contain subpopulations of cells with stem cell-like properties that may explain relapse and recurrence. The objective of this study was to screen existing oral cancer cell lines for biomarkers specific for cells with stem cell-like properties. RNA was isolated for RT-PCR screening using primers for specific mRNA of the biomarkers: CD44, CD24, CD133, NANOG, Nestin, ALDH1, and ABCG2 in CAL27, SCC25 and SCC15 cells. This analysis revealed that some oral cancer cell lines (CAL27 and SCC25 may contain small subpopulations of adhesion- and contact-independent cells (AiDC that also express tumor stem cell markers, including CD44, CD133, and CD24. In addition, CAL27 cells also expressed the intracellular tumor stem cell markers, ALDH1 and ABCG2. Isolation and culture of the adhesion- and contact-independent cells from CAL27 and SCC25 populations revealed differential proliferation rates and more robust inhibition by the MEK inhibitor PD98059, as well as the chemotherapeutic agents Cisplatin and Paclitaxel, within the AiDC CAL27 cells. At least one oral cancer cell line (CAL27 contained subpopulations of cells that express specific biomarkers associated with tumor stem cells which were morphologically and phenotypically distinct from other cells within this cell line.

  16. Activity in human visual and parietal cortex reveals object-based attention in working memory.

    Science.gov (United States)

    Peters, Benjamin; Kaiser, Jochen; Rahm, Benjamin; Bledowski, Christoph

    2015-02-25

    Visual attention enables observers to select behaviorally relevant information based on spatial locations, features, or objects. Attentional selection is not limited to physically present visual information, but can also operate on internal representations maintained in working memory (WM) in service of higher-order cognition. However, only little is known about whether attention to WM contents follows the same principles as attention to sensory stimuli. To address this question, we investigated in humans whether the typically observed effects of object-based attention in perception are also evident for object-based attentional selection of internal object representations in WM. In full accordance with effects in visual perception, the key behavioral and neuronal characteristics of object-based attention were observed in WM. Specifically, we found that reaction times were shorter when shifting attention to memory positions located on the currently attended object compared with equidistant positions on a different object. Furthermore, functional magnetic resonance imaging and multivariate pattern analysis of visuotopic activity in visual (areas V1-V4) and parietal cortex revealed that directing attention to one position of an object held in WM also enhanced brain activation for other positions on the same object, suggesting that attentional selection in WM activates the entire object. This study demonstrated that all characteristic features of object-based attention are present in WM and thus follows the same principles as in perception. Copyright © 2015 the authors 0270-6474/15/353360-10$15.00/0.

  17. Metabolic connectivity mapping reveals effective connectivity in the resting human brain.

    Science.gov (United States)

    Riedl, Valentin; Utz, Lukas; Castrillón, Gabriel; Grimmer, Timo; Rauschecker, Josef P; Ploner, Markus; Friston, Karl J; Drzezga, Alexander; Sorg, Christian

    2016-01-12

    Directionality of signaling among brain regions provides essential information about human cognition and disease states. Assessing such effective connectivity (EC) across brain states using functional magnetic resonance imaging (fMRI) alone has proven difficult, however. We propose a novel measure of EC, termed metabolic connectivity mapping (MCM), that integrates undirected functional connectivity (FC) with local energy metabolism from fMRI and positron emission tomography (PET) data acquired simultaneously. This method is based on the concept that most energy required for neuronal communication is consumed postsynaptically, i.e., at the target neurons. We investigated MCM and possible changes in EC within the physiological range using "eyes open" versus "eyes closed" conditions in healthy subjects. Independent of condition, MCM reliably detected stable and bidirectional communication between early and higher visual regions. Moreover, we found stable top-down signaling from a frontoparietal network including frontal eye fields. In contrast, we found additional top-down signaling from all major clusters of the salience network to early visual cortex only in the eyes open condition. MCM revealed consistent bidirectional and unidirectional signaling across the entire cortex, along with prominent changes in network interactions across two simple brain states. We propose MCM as a novel approach for inferring EC from neuronal energy metabolism that is ideally suited to study signaling hierarchies in the brain and their defects in brain disorders.

  18. Pathophysiological Significance of Dermatan Sulfate Proteoglycans Revealed by Human Genetic Disorders

    Directory of Open Access Journals (Sweden)

    Shuji Mizumoto

    2017-03-01

    Full Text Available The indispensable roles of dermatan sulfate-proteoglycans (DS-PGs have been demonstrated in various biological events including construction of the extracellular matrix and cell signaling through interactions with collagen and transforming growth factor-β, respectively. Defects in the core proteins of DS-PGs such as decorin and biglycan cause congenital stromal dystrophy of the cornea, spondyloepimetaphyseal dysplasia, and Meester-Loeys syndrome. Furthermore, mutations in human genes encoding the glycosyltransferases, epimerases, and sulfotransferases responsible for the biosynthesis of DS chains cause connective tissue disorders including Ehlers-Danlos syndrome and spondyloepimetaphyseal dysplasia with joint laxity characterized by skin hyperextensibility, joint hypermobility, and tissue fragility, and by severe skeletal disorders such as kyphoscoliosis, short trunk, dislocation, and joint laxity. Glycobiological approaches revealed that mutations in DS-biosynthetic enzymes cause reductions in enzymatic activities and in the amount of synthesized DS and also disrupt the formation of collagen bundles. This review focused on the growing number of glycobiological studies on recently reported genetic diseases caused by defects in the biosynthesis of DS and DS-PGs.

  19. Mutation analysis of the human CYP3A4 gene 5' regulatory region: population screening using non-radioactive SSCP.

    Science.gov (United States)

    Hamzeiy, Hossein; Vahdati-Mashhadian, Nasser; Edwards, Helen J; Goldfarb, Peter S

    2002-03-20

    Human CYP3A4 is the major cytochrome P450 isoenzyme in adult human liver and is known to metabolise many xenobiotic and endogenous compounds. There is substantial inter-individual variation in the hepatic levels of CYP3A4. Although, polymorphic mutations have been reported in the 5' regulatory region of the CYP3A4 gene, those that have been investigated so far do not appear to have any effect on gene expression. To determine whether other mutations exist in this region of the gene, we have performed a new population screen on a panel of 101 human DNA samples. A 1140 bp section of the 5' proximal regulatory region of the CYP3A4 gene, containing numerous regulatory motifs, was amplified from genomic DNA as three overlapping segments. The 300 bp distal enhancer region at -7.9kb containing additional regulatory motifs was also amplified. Mutation analysis of the resulting PCR products was carried out using non-radioactive single strand conformation polymorphism (SSCP) and confirmatory sequencing of both DNA strands in those samples showing extra SSCP bands. In addition to detection of the previously reported CYP3A4*1B allele in nine subjects, three novel alleles were found: CYP3A4*1E (having a T-->A transversion at -369 in one subject), CYP3A4*1F (having a C-->G tranversion at -747 in 17 subjects) and CYP3A4*15B containing a nine-nucleotide insertion between -845 and -844 linked to an A-->G transition at -392 and a G-->A transition in exon 6 (position 485 in the cDNA) in one subject. All the novel alleles were heterozygous. No mutations were found in the upstream distal enhancer region. Our results clearly indicate that this rapid and simple SSCP approach can reveal mutant alleles in drug metabolising enzyme genes. Detection and determination of the frequency of novel alleles in CYP3A4 will assist investigation of the relationship between genotype, xenobiotic metabolism and toxicity in the CYP3A family of isoenzymes.

  20. Dual microRNA Screens Reveal That the Immune-Responsive miR-181 Promotes Henipavirus Entry and Cell-Cell Fusion.

    Directory of Open Access Journals (Sweden)

    Chwan Hong Foo

    2016-10-01

    Full Text Available Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus are bat-borne viruses that cause fatal disease in humans and a range of other mammalian species. Gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new anti-viral therapies. Here we have performed genome-wide high-throughput agonist and antagonist screens at biosafety level 4 to identify host-encoded microRNAs (miRNAs impacting henipavirus infection in human cells. Members of the miR-181 and miR-17~93 families strongly promoted Hendra virus infection. miR-181 also promoted Nipah virus infection, but did not affect infection by paramyxoviruses from other genera, indicating specificity in the virus-host interaction. Infection promotion was primarily mediated via the ability of miR-181 to significantly enhance henipavirus-induced membrane fusion. Cell signalling receptors of ephrins, namely EphA5 and EphA7, were identified as novel negative regulators of henipavirus fusion. The expression of these receptors, as well as EphB4, were suppressed by miR-181 overexpression, suggesting that simultaneous inhibition of several Ephs by the miRNA contributes to enhanced infection and fusion. Immune-responsive miR-181 levels was also up-regulated in the biofluids of ferrets and horses infected with Hendra virus, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. This study is the first genome-wide screen of miRNAs influencing infection by a clinically significant mononegavirus and nominates select miRNAs as targets for future anti-viral therapy development.

  1. Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

    Science.gov (United States)

    Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

    2009-12-15

    Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

  2. Pluripotent stem cells reveal the developmental biology of human megakaryocytes and provide a source of platelets for clinical application.

    Science.gov (United States)

    Takayama, Naoya; Eto, Koji

    2012-10-01

    Human pluripotent stem cells [PSCs; including human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] can infinitely proliferate in vitro and are easily accessible for gene manipulation. Megakaryocytes (MKs) and platelets can be created from human ESCs and iPSCs in vitro and represent a potential source of blood cells for transfusion and a promising tool for studying the human thrombopoiesis. Moreover, disease-specific iPSCs are a powerful tool for elucidating the pathogenesis of hematological diseases and for drug screening. In that context, we and other groups have developed in vitro MK and platelet differentiation systems from human pluripotent stem cells (PSCs). Combining this co-culture system with a drug-inducible gene expression system enabled us to clarify the novel role played by c-MYC during human thrombopoiesis. In the next decade, technical advances (e.g., high-throughput genomic sequencing) will likely enable the identification of numerous gene mutations associated with abnormal thrombopoiesis. Combined with such technology, an in vitro system for differentiating human PSCs into MKs and platelets could provide a novel platform for studying human gene function associated with thrombopoiesis.

  3. Microbioreactor arrays for full factorial screening of exogenous and paracrine factors in human embryonic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Drew M Titmarsh

    Full Text Available Timed exposure of pluripotent stem cell cultures to exogenous molecules is widely used to drive differentiation towards desired cell lineages. However, screening differentiation conditions in conventional static cultures can become impractical in large parameter spaces, and is intrinsically limited by poor spatiotemporal control of the microenvironment that also makes it impossible to determine whether exogenous factors act directly or through paracrine-dependent mechanisms. We detail here the development of a continuous flow microbioreactor array platform that combines full-factorial multiplexing of input factors with progressive accumulation of paracrine factors through serially-connected culture chambers, and further, the use of this system to explore the combinatorial parameter space of both exogenous and paracrine factors involved in human embryonic stem cell (hESC differentiation to a MIXL1-GFP(+ primitive streak-like population. We show that well known inducers of primitive streak (BMP, Activin and Wnt signals do not simply act directly on hESC to induce MIXL1 expression, but that this requires accumulation of surplus, endogenous factors; and, that conditioned medium or FGF-2 supplementation is able to offset this. Our approach further reveals the presence of a paracrine, negative feedback loop to the MIXL1-GFP(+ population, which can be overcome with GSK-3β inhibitors (BIO or CHIR99021, implicating secreted Wnt inhibitory signals such as DKKs and sFRPs as candidate effectors. Importantly, modulating paracrine effects identified in microbioreactor arrays by supplementing FGF-2 and CHIR in conventional static culture vessels resulted in improved differentiation outcomes. We therefore demonstrate that this microbioreactor array platform uniquely enables the identification and decoding of complex soluble factor signalling hierarchies, and that this not only challenges prevailing strategies for extrinsic control of hESC differentiation, but

  4. Model for screening-level assessment of near-field human exposure to neutral organic chemicals released indoors.

    Science.gov (United States)

    Zhang, Xianming; Arnot, Jon A; Wania, Frank

    2014-10-21

    Screening organic chemicals for hazard and risk to human health requires near-field human exposure models that can be readily parametrized with available data. The integration of a model of human exposure, uptake, and bioaccumulation into an indoor mass balance model provides a quantitative framework linking emissions in indoor environments with human intake rates (iRs), intake fractions (iFs) and steady-state concentrations in humans (C) through consideration of dermal permeation, inhalation, and nondietary ingestion exposure pathways. Parameterized based on representative indoor and adult human characteristics, the model is applied here to 40 chemicals of relevance in the context of human exposure assessment. Intake fractions and human concentrations (C(U)) calculated with the model based on a unit emission rate to air for these 40 chemicals span 2 and 5 orders of magnitude, respectively. Differences in priority ranking based on either iF or C(U) can be attributed to the absorption, biotransformation and elimination processes within the human body. The model is further applied to a large data set of hypothetical chemicals representative of many in-use chemicals to show how the dominant exposure pathways, iF and C(U) change as a function of chemical properties and to illustrate the capacity of the model for high-throughput screening. These simulations provide hypotheses for the combination of chemical properties that may result in high exposure and internal dose. The model is further exploited to highlight the role human contaminant uptake plays in the overall fate of certain chemicals indoors and consequently human exposure.

  5. Functional Toxicogenomic Assessment of Triclosan in Human HepG2 Cells Using Genome-Wide CRISPR-Cas9 Screening.

    Science.gov (United States)

    Xia, Pu; Zhang, Xiaowei; Xie, Yuwei; Guan, Miao; Villeneuve, Daniel L; Yu, Hongxia

    2016-10-04

    There are thousands of chemicals used by humans and detected in the environment for which limited or no toxicological data are available. Rapid and cost-effective approaches for assessing the toxicological properties of chemicals are needed. We used CRISPR-Cas9 functional genomic screening to identify the potential molecular mechanism of a widely used antimicrobial triclosan (TCS) in HepG2 cells. Resistant genes at IC50 (the concentration causing a 50% reduction in cell viability) were significantly enriched in the adherens junction pathway, MAPK signaling pathway, and PPAR signaling pathway, suggesting a potential role in the molecular mechanism of TCS-induced cytotoxicity. Evaluation of the top-ranked resistant genes, FTO (encoding an mRNA demethylase) and MAP2K3 (a MAP kinase kinase family gene), revealed that their loss conferred resistance to TCS. In contrast, sensitive genes at IC10 and IC20 were specifically enriched in pathways involved with immune responses, which was concordant with transcriptomic profiling of TCS at concentrations of CRISPR-Cas9 fingerprint may reveal the patterns of TCS toxicity at low concentration levels. Moreover, we retrieved the potential connection between CRISPR-Cas9 fingerprint and disease terms, obesity, and breast cancer from an existing chemical-gene-disease database. Overall, CRISPR-Cas9 functional genomic screening offers an alternative approach for chemical toxicity testing.

  6. UFLC-Q-TOF-MS/MS-Based Screening and Identification of Flavonoids and Derived Metabolites in Human Urine after Oral Administration of Exocarpium Citri Grandis Extract

    Directory of Open Access Journals (Sweden)

    Xuan Zeng

    2018-04-01

    Full Text Available Exocarpium Citri grandis (ECG is an important Traditional Chinese Medicine (TCM for the treatment of cough and phlegm, and the flavonoids contained were considered the main effective components. To date, the systematic chemical profiling of these flavonoids and derived in vivo metabolites in human have not been well investigated. ECG was extracted using boiling water and then provided to volunteers for oral administration. Following the ingestion, urine samples were collected from volunteers over 48 h. The extract and urine samples were analyzed using ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UFLC-Q-TOF-MS/MS system to screen and identify flavonoids and derived in vivo metabolites. A total of 18 flavonoids were identified in the ECG extract, and 20 metabolites, mainly glucuronide and sulfate conjugates, were screened in urine samples collected post consumption. The overall excretion of naringenin metabolites corresponded to 5.45% of intake and occurred mainly within 4–12 h after the ingestion. Meanwhile, another 29 phenolic catabolites were detected in urine. Obtained data revealed that flavonoids were abundant in the ECG extract, and these components underwent extensive phase II metabolism in humans. These results provided valuable information for further study of the pharmacology and mechanism of action of ECG.

  7. Cervical cancer screening through human papillomavirus testing in community health campaigns versus health facilities in rural western Kenya.

    Science.gov (United States)

    Huchko, Megan J; Ibrahim, Saduma; Blat, Cinthia; Cohen, Craig R; Smith, Jennifer S; Hiatt, Robert A; Bukusi, Elizabeth

    2018-04-01

    To determine the effectiveness of community health campaigns (CHCs) as a strategy for human papillomavirus (HPV)-based cervical cancer screening in rural western Kenya. Between January and November 2016, a cluster-randomized trial was carried out in 12 communities in western Kenya to investigate high-risk HPV testing offered via self-collection to women aged 25-65 years in CHCs versus government health facilities. Outcome measures were the total number of women accessing cervical cancer screening and the proportion of HPV-positive women accessing treatment. In total, 4944 women underwent HPV-based cervical cancer screening in CHCs (n=2898) or health facilities (n=2046). Screening uptake as a proportion of total eligible women in the population was greater in communities assigned to CHCs (60.0% vs 37.0%, P<0.001). Rates of treatment acquisition were low in both arms (CHCs 39.2%; health facilities 31.5%; P=0.408). Cervical cancer screening using HPV testing of self-collected samples reached a larger proportion of women when offered through periodic CHCs compared with health facilities. The community-based model is a promising strategy for cervical cancer prevention. Lessons learned from this trial can be used to identify ways of maximizing the impact of such strategies through greater community participation and improved linkage to treatment. ClinicalTrials.gov registration: NCT02124252. © 2017 International Federation of Gynecology and Obstetrics.

  8. Isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism

    NARCIS (Netherlands)

    Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker; van der Hoek, Lia

    2013-01-01

    The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in

  9. Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

    Directory of Open Access Journals (Sweden)

    Jorge S.B.

    2003-01-01

    Full Text Available Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns, the alpha-globin genes are duplicate (alpha2 and alpha1 and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP. Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem(TM and GenePhor(TM, Amersham Biosciences, different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem(TM and 20% gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.

  10. Validation of a Human Papillomavirus (HPV) DNA Cervical Screening Test That Provides Expanded HPV Typing.

    Science.gov (United States)

    Demarco, Maria; Carter-Pokras, Olivia; Hyun, Noorie; Castle, Philip E; He, Xin; Dallal, Cher M; Chen, Jie; Gage, Julia C; Befano, Brian; Fetterman, Barbara; Lorey, Thomas; Poitras, Nancy; Raine-Bennett, Tina R; Wentzensen, Nicolas; Schiffman, Mark

    2018-05-01

    As cervical cancer screening shifts from cytology to human papillomavirus (HPV) testing, a major question is the clinical value of identifying individual HPV types. We aimed to validate Onclarity (Becton Dickinson Diagnostics, Sparks, MD), a nine-channel HPV test recently approved by the FDA, by assessing (i) the association of Onclarity types/channels with precancer/cancer; (ii) HPV type/channel agreement between the results of Onclarity and cobas (Roche Molecular Systems, Pleasanton, CA), another FDA-approved test; and (iii) Onclarity typing for all types/channels compared to typing results from a research assay (linear array [LA]; Roche). We compared Onclarity to histopathology, cobas, and LA. We tested a stratified random sample ( n = 9,701) of discarded routine clinical specimens that had tested positive by Hybrid Capture 2 (HC2; Qiagen, Germantown, MD). A subset had already been tested by cobas and LA ( n = 1,965). Cervical histopathology was ascertained from electronic health records. Hierarchical Onclarity channels showed a significant linear association with histological severity. Onclarity and cobas had excellent agreement on partial typing of HPV16, HPV18, and the other 12 types as a pool (sample-weighted kappa value of 0.83); cobas was slightly more sensitive for HPV18 and slightly less sensitive for the pooled high-risk types. Typing by Onclarity showed excellent agreement with types and groups of types identified by LA (kappa values from 0.80 for HPV39/68/35 to 0.97 for HPV16). Onclarity typing results corresponded well to histopathology and to an already validated HPV DNA test and could provide additional clinical typing if such discrimination is determined to be clinically desirable. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

  11. The psychosocial burden of human papillomavirus related disease and screening interventions.

    Science.gov (United States)

    Pirotta, M; Ung, L; Stein, A; Conway, E L; Mast, T C; Fairley, C K; Garland, S

    2009-12-01

    (i) To assess the psychosocial burden of testing for human papillomavirus (HPV) related genital disease or of a HPV-related diagnosis; (ii) to compare an instrument specifically designed to measure HPV-related psychosocial burden with other generic quality of life (QoL) instruments. A cross-sectional design. Researchers recruited women from outpatient clinics at a major tertiary women's hospital and a sexual health centre who completed surveys within 3 months of receiving 331 women, 18-45 years, who had experienced a normal cervical Papanicolaou (Pap) result, an abnormal Pap result, biopsy confirmed cervical intraepithelial neoplasia (CIN) or external genital warts (EGW). The HPV impact profile (HIP) designed to assess the psychosocial impact of HPV; two general health-related QoL surveys-the EuroQoL VAS and the Sheehan disability scale; and a HPV knowledge survey. Response rate was 78%. Significant psychosocial impacts were found for women screened for, or having a diagnosis of, HPV-related genital disease. The largest impact was in women with CIN 2/3 and EGW. This HPV-related psychosocial impact was most sensitively detected with the HIP. Relative to generic measures of QoL, the HIP provided insight into the full range of psychosocial impacts of HPV testing and diagnoses. Clinicians need to be aware of the potential psychosocial impact of testing for or diagnosing HPV-related genital disease, in particular CIN 2/3 and EGW. The HIP survey is a more sensitive measure of the psychosocial impact of HPV-related genital disease than generic QoL surveys.

  12. Rapid molecular evolution of human bocavirus revealed by Bayesian coalescent inference.

    Science.gov (United States)

    Zehender, Gianguglielmo; De Maddalena, Chiara; Canuti, Marta; Zappa, Alessandra; Amendola, Antonella; Lai, Alessia; Galli, Massimo; Tanzi, Elisabetta

    2010-03-01

    Human bocavirus (HBoV) is a linear single-stranded DNA virus belonging to the Parvoviridae family that has recently been isolated from the upper respiratory tract of children with acute respiratory infection. All of the strains observed so far segregate into two genotypes (1 and 2) with a low level of polymorphism. Given the recent description of the infection and the lack of epidemiological and molecular data, we estimated the virus's rates of molecular evolution and population dynamics. A dataset of forty-nine dated VP2 sequences, including also eight new isolates obtained from pharyngeal swabs of Italian patients with acute respiratory tract infections, was submitted to phylogenetic analysis. The model parameters, evolutionary rates and population dynamics were co-estimated using a Bayesian Markov Chain Monte Carlo approach, and site-specific positive and negative selection was also investigated. Recombination was investigated by seven different methods and one suspected recombinant strain was excluded from further analysis. The estimated mean evolutionary rate of HBoV was 8.6x10(-4)subs/site/year, and that of the 1st+2nd codon positions was more than 15 times less than that of the 3rd codon position. Viral population dynamics analysis revealed that the two known genotypes diverged recently (mean tMRCA: 24 years), and that the epidemic due to HBoV genotype 2 grew exponentially at a rate of 1.01year(-1). Selection analysis of the partial VP2 showed that 8.5% of sites were under significant negative pressure and the absence of positive selection. Our results show that, like other parvoviruses, HBoV is characterised by a rapid evolution. The low level of polymorphism is probably due to a relatively recent divergence between the circulating genotypes and strong purifying selection acting on viral antigens.

  13. Unbiased plasma metabolomics reveal the correlation of metabolic pathways and Prakritis of humans.

    Science.gov (United States)

    Shirolkar, Amey; Chakraborty, Sutapa; Mandal, Tusharkanti; Dabur, Rajesh

    2017-11-25

    Ayurveda, an ancient Indian medicinal system, has categorized human body constitutions in three broad constitutional types (prakritis) i.e. Vata, Pitta and Kapha. Analysis of plasma metabolites and related pathways to classify Prakriti specific dominant marker metabolites and metabolic pathways. 38 healthy male individuals were assessed for dominant Prakritis and their fasting blood samples were collected. The processed plasma samples were subjected to rapid resolution liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry (RRLC-ESI-QTOFMS). Mass profiles were aligned and subjected to multivariate analysis. Partial least square discriminant analysis (PLS-DA) model showed 97.87% recognition capability. List of PLS-DA metabolites was subjected to permutative Benjamini-Hochberg false discovery rate (FDR) correction and final list of 76 metabolites with p  2.0 was identified. Pathway analysis using metascape and JEPETTO plugins in Cytoscape revealed that steroidal hormone biosynthesis, amino acid, and arachidonic acid metabolism are major pathways varying with different constitution. Biological Go processes analysis showed that aromatic amino acids, sphingolipids, and pyrimidine nucleotides metabolic processes were dominant in kapha type of body constitution. Fat soluble vitamins, cellular amino acid, and androgen biosynthesis process along with branched chain amino acid and glycerolipid catabolic processes were dominant in pitta type individuals. Vata Prakriti was found to have dominant catecholamine, arachidonic acid and hydrogen peroxide metabolomics processes. The neurotransmission and oxidative stress in vata, BCAA catabolic, androgen, xenobiotics metabolic processes in pitta, and aromatic amino acids, sphingolipid, and pyrimidine metabolic process in kaphaPrakriti were the dominant marker pathways. Copyright © 2017 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Published by Elsevier B.V. All rights

  14. Brain-to-brain hyperclassification reveals action-specific motor mapping of observed actions in humans.

    Science.gov (United States)

    Smirnov, Dmitry; Lachat, Fanny; Peltola, Tomi; Lahnakoski, Juha M; Koistinen, Olli-Pekka; Glerean, Enrico; Vehtari, Aki; Hari, Riitta; Sams, Mikko; Nummenmaa, Lauri

    2017-01-01

    Seeing an action may activate the corresponding action motor code in the observer. It remains unresolved whether seeing and performing an action activates similar action-specific motor codes in the observer and the actor. We used novel hyperclassification approach to reveal shared brain activation signatures of action execution and observation in interacting human subjects. In the first experiment, two "actors" performed four types of hand actions while their haemodynamic brain activations were measured with 3-T functional magnetic resonance imaging (fMRI). The actions were videotaped and shown to 15 "observers" during a second fMRI experiment. Eleven observers saw the videos of one actor, and the remaining four observers saw the videos of the other actor. In a control fMRI experiment, one of the actors performed actions with closed eyes, and five new observers viewed these actions. Bayesian canonical correlation analysis was applied to functionally realign observers' and actors' fMRI data. Hyperclassification of the seen actions was performed with Bayesian logistic regression trained on actors' data and tested with observers' data. Without the functional realignment, between-subjects accuracy was at chance level. With the realignment, the accuracy increased on average by 15 percentage points, exceeding both the chance level and the accuracy without functional realignment. The highest accuracies were observed in occipital, parietal and premotor cortices. Hyperclassification exceeded chance level also when the actor did not see her own actions. We conclude that the functional brain activation signatures underlying action execution and observation are partly shared, yet these activation signatures may be anatomically misaligned across individuals.

  15. Human processing of short temporal intervals as revealed by an ERP waveform analysis

    Directory of Open Access Journals (Sweden)

    Yoshitaka eNakajima

    2011-12-01

    Full Text Available To clarify the time course over which the human brain processes information about durations up to ~300 ms, we reanalyzed the data that were previously reported by Mitsudo et al. (2009 using a multivariate analysis method. Event-related potentials were recorded from 19 scalp electrodes on 11 (9 original and 2 additional participants while they judged whether two neighboring empty time intervals—called t1 and t2 and marked by three tone bursts—had equal durations. There was also a control condition in which the participants were presented the same temporal patterns but without a judgment task. In the present reanalysis, we sought to visualize how the temporal patterns were represented in the brain over time. A correlation matrix across channels was calculated for each temporal pattern. Geometric separations between the correlation matrices were calculated, and subjected to multidimensional scaling. We performed such analyses for a moving 100-ms time window after the t1 presentations. In the windows centered at < 100 ms after the t2 presentation, the analyses revealed the local maxima of categorical separation between temporal patterns of perceptually equal durations versus perceptually unequal durations, both in the judgment condition and in the control condition. Such categorization of the temporal patterns was prominent only in narrow temporal regions. The analysis indicated that the participants determined whether the two neighboring time intervals were of equal duration mostly within 100 ms after the presentation of the temporal patterns. A very fast brain activity was related to the perception of elementary temporal patterns without explicit judgments. This is consistent with the findings of Mitsudo et al., and it is in line with the processing time hypothesis proposed by Nakajima et al. (2004. The validity of the correlation matrix analyses turned out to be an effective tool to grasp the overall responses of the brain to temporal

  16. Quantitative membrane proteomics reveals a role for tetraspanin enriched microdomains during entry of human cytomegalovirus.

    Directory of Open Access Journals (Sweden)

    Kasinath Viswanathan

    Full Text Available Human cytomegalovirus (HCMV depends on and modulates multiple host cell membrane proteins during each stage of the viral life cycle. To gain a global view of the impact of HCMV-infection on membrane proteins, we analyzed HCMV-induced changes in the abundance of membrane proteins in fibroblasts using stable isotope labeling with amino acids (SILAC, membrane fractionation and protein identification by two-dimensional liquid chromatography and tandem mass spectrometry. This systematic approach revealed that CD81, CD44, CD98, caveolin-1 and catenin delta-1 were down-regulated during infection whereas GRP-78 was up-regulated. Since CD81 downregulation was also observed during infection with UV-inactivated virus we hypothesized that this tetraspanin is part of the viral entry process. Interestingly, additional members of the tetraspanin family, CD9 and CD151, were also downregulated during HCMV-entry. Since tetraspanin-enriched microdomains (TEM cluster host cell membrane proteins including known CMV receptors such as integrins, we studied whether TEMs are required for viral entry. When TEMs were disrupted with the cholesterol chelator methyl-β-cylcodextrin, viral entry was inhibited and this inhibition correlated with reduced surface levels of CD81, CD9 and CD151, whereas integrin levels remained unchanged. Furthermore, simultaneous siRNA-mediated knockdown of multiple tetraspanins inhibited viral entry whereas individual knockdown had little effect suggesting essential, but redundant roles for individual tetraspanins during entry. Taken together, our data suggest that TEM act as platforms for receptors utilized by HCMV for entry into cells.

  17. Knowledge and Attitudes About Human Papilloma Virus (HPV) Vaccination and Cervical Cancer Screening Among Women in Rural Uganda

    Science.gov (United States)

    2016-06-15

    1- Knowledge and attitudes about Human Papilloma Virus (HPV) vaccination and cervical cancer screening among women in rural Uganda Authors...vaccination among parents/guardians of the vaccinated girls and to assess the attitudes to HPV vaccination among parents/guardians of the vaccinated girls...general attitude towards HPV vaccination was positive among mothers though there is still need for the populations to appreciate HPV and cervical

  18. Cytology and Human Papillomavirus Testing 6 to 12 Months after ASCUS or LSIL Cytology in Organized Screening To Predict High-Grade Cervical Neoplasia between Screening Rounds

    Science.gov (United States)

    Sjøborg, Katrine D.; Nygård, Mari; Røysland, Kjetil; Campbell, Suzanne; Alfsen, G. Cecilie; Jonassen, Christine M.

    2012-01-01

    We carried out a prospective study comparing the performance of human papillomavirus (HPV) E6/E7 mRNA (PreTect HPV-Proofer; NorChip, Klokkarstua, Norway) and DNA (Amplicor HPV test; Roche Diagnostics, Basel, Switzerland) triage testing of women 6 to 12 months after atypical-squamous-cells-of-undetermined-significance (ASCUS) or low-grade-squamous-intraepithelial-lesion (LSIL) cytology in organized screening to predict high-grade cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) between screening rounds. Between January 2005 and April 2008, 692 study women with screening-detected ASCUS/LSIL cytology 6 to 12 months earlier returned for HPV mRNA and DNA testing and repeat cytology. The median follow-up time was 3 years, using existing health care facilities. Follow-up test results were available for 625 women. Of the 145 CIN2+ cases detected during the study period, 95 (65.5%) were HPV mRNA positive 6 to 12 months after screening-detected ASCUS/LSIL, 44 (30.4%) were HPV mRNA negative, and 6 (4.1%) were invalid. The corresponding HPV DNA results were 139 (95.9%), 5 (3.4%), and 1 (0.7%), respectively. The cumulative incidences of CIN2+ 3 years after a negative HPV mRNA and DNA test were 10.3% (95% confidence interval [CI], 7.2 to 13.3%) and 1.8% (95% CI, 0.0 to 3.6%), respectively. The cumulative incidences of CIN2+ 3 years after positive HPV mRNA and DNA tests were 52.8% (95% CI, 40.1 to 60.1%) and 41.3% (95% CI, 35.5 to 46.6%), respectively. In conclusion, both positive HPV mRNA and DNA test results have a high enough long-term prediction of CIN2+ risk to consider referral to colposcopy as good practice when performed in delayed triage of women with ASCUS/LSIL cytology. In addition, the low CIN2+ risk among women with a negative Amplicor HPV test in our study confirms its safe use in a clinical setting. PMID:22518869

  19. Design, development, and validation of a high-throughput drug-screening assay for targeting of human leukemia

    Science.gov (United States)

    Karjalainen, Katja; Pasqualini, Renata; Cortes, Jorge E.; Kornblau, Steven M.; Lichtiger, Benjamin; O'Brien, Susan; Kantarjian, Hagop M.; Sidman, Richard L.; Arap, Wadih; Koivunen, Erkki

    2015-01-01

    Background We introduce an ex vivo methodology to perform drug library screening against human leukemia. Method Our strategy relies on human blood or bone marrow cultures under hypoxia; under these conditions, leukemia cells deplete oxygen faster than normal cells, causing a hemoglobin oxygenation shift. We demonstrate several advantages: (I) partial recapitulation of the leukemia microenvironment, (ii) use of native hemoglobin oxygenation as real-time sensor/reporter, (iii) cost-effectiveness, (iv) species-specificity, and (v) format that enables high-throughput screening. Results As a proof-of-concept, we screened a chemical library (size ∼20,000) against human leukemia cells. We identified 70 compounds (“hit” rate=0.35%; Z-factor=0.71) with activity; we examined 20 to find 18 true-positives (90%). Finally, we show that carbonohydraxonic diamide group-containing compounds are potent anti-leukemia agents that induce cell death in leukemia cells and patient-derived samples. Conclusions This unique functional assay can identify novel drug candidates as well as find future applications in personalized drug selection for leukemia patients. PMID:24496871

  20. Cost-Effectiveness of Cervical Cancer Screening With Human Papillomavirus DNA Testing and HPV-16,18 Vaccination

    Science.gov (United States)

    Goldhaber-Fiebert, Jeremy D.; Stout, Natasha K.; Salomon, Joshua A.; Kuntz, Karen M.; Goldie, Sue J.

    2011-01-01

    Background The availability of human papillomavirus (HPV) DNA testing and vaccination against HPV types 16 and 18 (HPV-16,18) motivates questions about the cost-effectiveness of cervical cancer prevention in the United States for unvaccinated older women and for girls eligible for vaccination. Methods An empirically calibrated model was used to assess the quality-adjusted life years (QALYs), lifetime costs, and incremental cost-effectiveness ratios (2004 US dollars per QALY) of screening, vaccination of preadolescent girls, and vaccination combined with screening. Screening varied by initiation age (18, 21, or 25 years), interval (every 1, 2, 3, or 5 years), and test (HPV DNA testing of cervical specimens or cytologic evaluation of cervical cells with a Pap test). Testing strategies included: 1) cytology followed by HPV DNA testing for equivocal cytologic results (cytology with HPV test triage); 2) HPV DNA testing followed by cytology for positive HPV DNA results (HPV test with cytology triage); and 3) combined HPV DNA testing and cytology. Strategies were permitted to switch once at age 25, 30, or 35 years. Results For unvaccinated women, triennial cytology with HPV test triage, beginning by age 21 years and switching to HPV testing with cytology triage at age 30 years, cost $78 000 per QALY compared with the next best strategy. For girls vaccinated before age 12 years, this same strategy, beginning at age 25 years and switching at age 35 years, cost $41 000 per QALY with screening every 5 years and $188 000 per QALY screening triennially, each compared with the next best strategy. These strategies were more effective and cost-effective than screening women of all ages with cytology alone or cytology with HPV triage annually or biennially. Conclusions For both vaccinated and unvaccinated women, age-based screening by use of HPV DNA testing as a triage test for equivocal results in younger women and as a primary screening test in older women is expected to be more

  1. Comparison of independent screens on differentially vulnerable motor neurons reveals alpha-synuclein as a common modifier in motor neuron diseases.

    Science.gov (United States)

    Kline, Rachel A; Kaifer, Kevin A; Osman, Erkan Y; Carella, Francesco; Tiberi, Ariana; Ross, Jolill; Pennetta, Giuseppa; Lorson, Christian L; Murray, Lyndsay M

    2017-03-01

    The term "motor neuron disease" encompasses a spectrum of disorders in which motor neurons are the primary pathological target. However, in both patients and animal models of these diseases, not all motor neurons are equally vulnerable, in that while some motor neurons are lost very early in disease, others remain comparatively intact, even at late stages. This creates a valuable system to investigate the factors that regulate motor neuron vulnerability. In this study, we aim to use this experimental paradigm to identify potential transcriptional modifiers. We have compared the transcriptome of motor neurons from healthy wild-type mice, which are differentially vulnerable in the childhood motor neuron disease Spinal Muscular Atrophy (SMA), and have identified 910 transcriptional changes. We have compared this data set with published microarray data sets on other differentially vulnerable motor neurons. These neurons were differentially vulnerable in the adult onset motor neuron disease Amyotrophic Lateral Sclerosis (ALS), but the screen was performed on the equivalent population of neurons from neurologically normal human, rat and mouse. This cross species comparison has generated a refined list of differentially expressed genes, including CELF5, Col5a2, PGEMN1, SNCA, Stmn1 and HOXa5, alongside a further enrichment for synaptic and axonal transcripts. As an in vivo validation, we demonstrate that the manipulation of a significant number of these transcripts can modify the neurodegenerative phenotype observed in a Drosophila line carrying an ALS causing mutation. Finally, we demonstrate that vector-mediated expression of alpha-synuclein (SNCA), a transcript decreased in selectively vulnerable motor neurons in all four screens, can extend life span, increase weight and decrease neuromuscular junction pathology in a mouse model of SMA. In summary, we have combined multiple data sets to identify transcripts, which are strong candidates for being phenotypic modifiers

  2. High content image-based screening of a protease inhibitor library reveals compounds broadly active against Rift Valley fever virus and other highly pathogenic RNA viruses.

    Directory of Open Access Journals (Sweden)

    Rajini Mudhasani

    2014-08-01

    Full Text Available High content image-based screening was developed as an approach to test a protease inhibitor small molecule library for antiviral activity against Rift Valley fever virus (RVFV and to determine their mechanism of action. RVFV is the causative agent of severe disease of humans and animals throughout Africa and the Arabian Peninsula. Of the 849 compounds screened, 34 compounds exhibited ≥ 50% inhibition against RVFV. All of the hit compounds could be classified into 4 distinct groups based on their unique chemical backbone. Some of the compounds also showed broad antiviral activity against several highly pathogenic RNA viruses including Ebola, Marburg, Venezuela equine encephalitis, and Lassa viruses. Four hit compounds (C795-0925, D011-2120, F694-1532 and G202-0362, which were most active against RVFV and showed broad-spectrum antiviral activity, were selected for further evaluation for their cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the trans Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their

  3. Within-Host Variations of Human Papillomavirus Reveal APOBEC-Signature Mutagenesis in the Viral Genome.

    Science.gov (United States)

    Hirose, Yusuke; Onuki, Mamiko; Tenjimbayashi, Yuri; Mori, Seiichiro; Ishii, Yoshiyuki; Takeuchi, Takamasa; Tasaka, Nobutaka; Satoh, Toyomi; Morisada, Tohru; Iwata, Takashi; Miyamoto, Shingo; Matsumoto, Koji; Sekizawa, Akihiko; Kukimoto, Iwao

    2018-03-28

    Persistent infection with oncogenic human papillomaviruses (HPVs) causes cervical cancer, accompanied with the accumulation of somatic mutations into the host genome. There are concomitant genetic changes in the HPV genome during viral infection; however, their relevance to cervical carcinogenesis is poorly understood. Here we explored within-host genetic diversity of HPV by performing deep sequencing analyses of viral whole-genome sequences in clinical specimens. The whole genomes of HPV types 16, 52 and 58 were amplified by type-specific PCR from total cellular DNA of cervical exfoliated cells collected from patients with cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (ICC), and were deep-sequenced. After constructing a reference vial genome sequence for each specimen, nucleotide positions showing changes with > 0.5% frequencies compared to the reference sequence were determined for individual samples. In total, 1,052 positions of nucleotide variations were detected in HPV genomes from 151 samples (CIN1, n = 56; CIN2/3, n = 68; ICC, n = 27), with varying numbers per sample. Overall, C-to-T and C-to-A substitutions were the dominant changes observed across all histological grades. While C-to-T transitions were predominantly detected in CIN1, their prevalence was decreased in CIN2/3 and fell below that of C-to-A transversions in ICC. Analysis of the tri-nucleotides context encompassing substituted bases revealed that Tp C pN, a preferred target sequence for cellular APOBEC cytosine deaminases, was a primary site for C-to-T substitutions in the HPV genome. These results strongly imply that the APOBEC proteins are drivers of HPV genome mutation, particularly in CIN1 lesions. IMPORTANCE HPVs exhibit surprisingly high levels of genetic diversity, including a large repertoire of minor genomic variants in each viral genotype. Here, by conducting deep sequencing analyses, we show for the first time a comprehensive snapshot of the "within

  4. The gene order on Human Chromosome 15 and Chicken Chromosome 10 reveal multiple inter- and intrachromosomal rearrangements

    NARCIS (Netherlands)

    Crooijmans, R.P.M.A.; Dijkhof, R.J.M.; Veenendaal, T.; Poel, van der J.J.; Groenen, M.A.M.

    2001-01-01

    Comparative mapping between the human and chicken genomes has revealed a striking conservation of synteny between the genomes of these two species, but the results have been based on low-resolution comparative maps. To address this conserved synteny in much more detail, a high-resolution

  5. mtDNA variation predicts population size in humans and reveals a major Southern Asian chapter in human prehistory.

    Science.gov (United States)

    Atkinson, Quentin D; Gray, Russell D; Drummond, Alexei J

    2008-02-01

    The relative timing and size of regional human population growth following our expansion from Africa remain unknown. Human mitochondrial DNA (mtDNA) diversity carries a legacy of our population history. Given a set of sequences, we can use coalescent theory to estimate past population size through time and draw inferences about human population history. However, recent work has challenged the validity of using mtDNA diversity to infer species population sizes. Here we use Bayesian coalescent inference methods, together with a global data set of 357 human mtDNA coding-region sequences, to infer human population sizes through time across 8 major geographic regions. Our estimates of relative population sizes show remarkable concordance with the contemporary regional distribution of humans across Africa, Eurasia, and the Americas, indicating that mtDNA diversity is a good predictor of population size in humans. Plots of population size through time show slow growth in sub-Saharan Africa beginning 143-193 kya, followed by a rapid expansion into Eurasia after the emergence of the first non-African mtDNA lineages 50-70 kya. Outside Africa, the earliest and fastest growth is inferred in Southern Asia approximately 52 kya, followed by a succession of growth phases in Northern and Central Asia (approximately 49 kya), Australia (approximately 48 kya), Europe (approximately 42 kya), the Middle East and North Africa (approximately 40 kya), New Guinea (approximately 39 kya), the Americas (approximately 18 kya), and a second expansion in Europe (approximately 10-15 kya). Comparisons of relative regional population sizes through time suggest that between approximately 45 and 20 kya most of humanity lived in Southern Asia. These findings not only support the use of mtDNA data for estimating human population size but also provide a unique picture of human prehistory and demonstrate the importance of Southern Asia to our recent evolutionary past.

  6. Human papillomavirus type-specific prevalence in the cervical cancer screening population of Czech women.

    Directory of Open Access Journals (Sweden)

    Ruth Tachezy

    Full Text Available BACKGROUND: Infection with high-risk human papillomavirus (HPVtypes has been recognized as a causal factor for the development of cervical cancer and a number of other malignancies. Today, vaccines against HPV, highly effective in the prevention of persistent infection and precancerous lesions, are available for the routine clinical practice. OBJECTIVES: The data on the prevalence and type-specific HPV distribution in the population of each country are crucial for the surveillance of HPV type-specific prevalence at the onset of vaccination against HPV. METHODS: Women attending a preventive gynecological examination who had no history of abnormal cytological finding and/or surgery for cervical lesions were enrolled. All samples were tested for the presence of HPV by High-Risk Hybrid Capture 2 (HR HC2 and by a modified PCR-reverse line blot assay with broad spectrum primers (BS-RLB. RESULTS: Cervical smears of 1393 women were analyzed. In 6.5% of women, atypical cytological findings were detected. Altogether, 28.3% (394/1393 of women were positive for any HPV type by BS-RLB, 18.2% (254/1393 by HR HC2, and 22.3% (310/1393 by BS-RLB for HR HPV types. In women with atypical findings the prevalence for HR and any HPV types were significantly higher than in women with normal cytological findings. Overall, 36 different HPV types were detected, with HPV 16 being the most prevalent (4.8%. HPV positivity decreased with age; the highest prevalence was 31.5% in the age group 21-25 years. CONCLUSIONS: Our study subjects represent the real screening population. HPV prevalence in this population in the Czech Republic is higher than in other countries of Eastern Europe. Also the spectrum of the most prevalent HPV types differs from those reported by others but HPV 16 is, concordantly, the most prevalent type. Country-specific HPV type-specific prevalences provide baseline information which will enable to measure the impact of HPV vaccination in the future.

  7. Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse

    OpenAIRE

    Zhang, Ye; Sloan, Steven A.; Clarke, Laura E.; Caneda, Christine; Plaza, Colton A.; Blumenthal, Paul D.; Vogel, Hannes; Steinberg, Gary K.; Edwards, Michael S. B.; Li, Gordon; Duncan, John A.; Cheshier, Samuel H.; Shuer, Lawrence M.; Chang, Edward F.; Grant, Gerald A.

    2015-01-01

    The functional and molecular similarities and distinctions between human and murine astrocytes are poorly understood. Here we report the development of an immunopanning method to acutely purify astrocytes from fetal, juvenile, and adult human brains, and to maintain these cells in serum-free cultures. We found that human astrocytes have similar abilities to murine astrocytes in promoting neuronal survival, inducing functional synapse formation, and engulfing synaptosomes. In contrast to exist...

  8. Neuropeptidomics Mass Spectrometry Reveals Signaling Networks Generated by Distinct Protease Pathways in Human Systems

    Science.gov (United States)

    Hook, Vivian; Bandeira, Nuno

    2015-12-01

    Neuropeptides regulate intercellular signaling as neurotransmitters of the central and peripheral nervous systems, and as peptide hormones in the endocrine system. Diverse neuropeptides of distinct primary sequences of various lengths, often with post-translational modifications, coordinate and integrate regulation of physiological functions. Mass spectrometry-based analysis of the diverse neuropeptide structures in neuropeptidomics research is necessary to define the full complement of neuropeptide signaling molecules. Human neuropeptidomics has notable importance in defining normal and dysfunctional neuropeptide signaling in human health and disease. Neuropeptidomics has great potential for expansion in translational research opportunities for defining neuropeptide mechanisms of human diseases, providing novel neuropeptide drug targets for drug discovery, and monitoring neuropeptides as biomarkers of drug responses. In consideration of the high impact of human neuropeptidomics for health, an observed gap in this discipline is the few published articles in human neuropeptidomics compared with, for example, human proteomics and related mass spectrometry disciplines. Focus on human neuropeptidomics will advance new knowledge of the complex neuropeptide signaling networks participating in the fine control of neuroendocrine systems. This commentary review article discusses several human neuropeptidomics accomplishments that illustrate the rapidly expanding diversity of neuropeptides generated by protease processing of pro-neuropeptide precursors occurring within the secretory vesicle proteome. Of particular interest is the finding that human-specific cathepsin V participates in producing enkephalin and likely other neuropeptides, indicating unique proteolytic mechanisms for generating human neuropeptides. The field of human neuropeptidomics has great promise to solve new mechanisms in disease conditions, leading to new drug targets and therapeutic agents for human

  9. A trans-Amazonian screening of mtDNA reveals deep intraspecific divergence in forest birds and suggests a vast underestimation of species diversity.

    Directory of Open Access Journals (Sweden)

    Borja Milá

    Full Text Available The Amazonian avifauna remains severely understudied relative to that of the temperate zone, and its species richness is thought to be underestimated by current taxonomy. Recent molecular systematic studies using mtDNA sequence reveal that traditionally accepted species-level taxa often conceal genetically divergent subspecific lineages found to represent new species upon close taxonomic scrutiny, suggesting that intraspecific mtDNA variation could be useful in species discovery. Surveys of mtDNA variation in Holarctic species have revealed patterns of variation that are largely congruent with species boundaries. However, little information exists on intraspecific divergence in most Amazonian species. Here we screen intraspecific mtDNA genetic variation in 41 Amazonian forest understory species belonging to 36 genera and 17 families in 6 orders, using 758 individual samples from Ecuador and French Guiana. For 13 of these species, we also analyzed trans-Andean populations from the Ecuadorian Chocó. A consistent pattern of deep intraspecific divergence among trans-Amazonian haplogroups was found for 33 of the 41 taxa, and genetic differentiation and genetic diversity among them was highly variable, suggesting a complex range of evolutionary histories. Mean sequence divergence within families was the same as that found in North American birds (13%, yet mean intraspecific divergence in Neotropical species was an order of magnitude larger (2.13% vs. 0.23%, with mean distance between intraspecific lineages reaching 3.56%. We found no clear relationship between genetic distances and differentiation in plumage color. Our results identify numerous genetically and phenotypically divergent lineages which may result in new species-level designations upon closer taxonomic scrutiny and thorough sampling, although lineages in the tropical region could be older than those in the temperate zone without necessarily representing separate species. In

  10. LC-ESI/MS/MS method for rapid screening and confirmation of 44 exogenous anabolic steroids in human urine.

    Science.gov (United States)

    Jeon, Byoung Wook; Yoo, Hye Hyun; Jeong, Eun Sook; Kim, Ho Jun; Jin, Changbae; Kim, Dong Hyun; Lee, Jaeick

    2011-09-01

    A sensitive and rapid method based on liquid chromatography-triple-quadrupole tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) has been developed and validated for the screening and confirmation of 44 exogenous anabolic steroids (29 parent steroids and 15 metabolites) in human urine. The method involves an enzymatic hydrolysis, liquid-liquid extraction, and detection by LC-MS/MS. A triple-quadrupole mass spectrometer was operated in positive ESI mode with selected reaction monitoring (SRM) mode for the screening and product ion scan mode for the confirmation. The protonated molecular ions were used as precursor ions for the SRM analysis and product ion scan. The intraday and interday precisions of the target analytes at concentrations of the minimum required performance levels for the screening were 2-14% and 2-15%, respectively. The limits of detection for the screening and confirmation method were 0.1-10 ng/mL and 0.2-10 ng/mL, respectively, for 44 steroids. This method was successfully applied to analysis of urine samples from suspected anabolic steroid abusers.

  11. Human recombinant beta-secretase immobilized enzyme reactor for fast hits' selection and characterization from a virtual screening library.

    Science.gov (United States)

    De Simone, Angela; Mancini, Francesca; Cosconati, Sandro; Marinelli, Luciana; La Pietra, Valeria; Novellino, Ettore; Andrisano, Vincenza

    2013-01-25

    In the present work, a human recombinant BACE1 immobilized enzyme reactor (hrBACE1-IMER) has been applied for the sensitive fast screening of 38 compounds selected through a virtual screening approach. HrBACE1-IMER was inserted into a liquid chromatograph coupled with a fluorescent detector. A fluorogenic peptide substrate (M-2420), containing the β-secretase site of the Swedish mutation of APP, was injected and cleaved in the on-line HPLC-hrBACE1-IMER system, giving rise to the fluorescent product. The compounds of the library were tested for their ability to inhibit BACE1 in the immobilized format and to reduce the area related to the chromatographic peak of the fluorescent enzymatic product. The results were validated in solution by using two different FRET methods. Due to the efficient virtual screening methodology, more than fifty percent of the selected compounds showed a measurable inhibitory activity. One of the most active compound (a bis-indanone derivative) was characterized in terms of IC(50) and K(i) determination on the hrBACE1-IMER. Thus, the hrBACE1-IMER has been confirmed as a valid tool for the throughput screening of different chemical entities with potency lower than 30μM for the fast hits' selection and for mode of action determination. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. The potential usefulness of human leukocyte antigen typing for celiac disease screening: a systematic review and meta-analysis

    Directory of Open Access Journals (Sweden)

    Alicia Díaz-Redondo

    2015-07-01

    Full Text Available Background and aim: The presence of specific human leukocyte antigen-DQ2 and DQ8 seems to be necessary for celiac disease development, but the real contribution of its typing for screening is still uncertain. We aim to conduct a systematic review and meta-analysis of the diagnostic performance of human leukocyte antigen typing tests for celiac disease screening. Methods: Systematic review of published studies assessing accuracy of human leukocyte antigen DQ2 and DQ8 typing for the detection of celiac disease were selected. MEDLINE and EMBASE were searched from 1st January 2004 until 31st December 2013. Two independent researchers carried out selection and classification of studies, data extraction and analysis. Meta-analysis combining sensitivities, specificities and likelihood ratios of HLA-DQ2 and DQ8 for the diagnosis of celiac disease were carried out. Results: 6 studies including 1303 individuals were finally evaluated. Pooled sensitivity was 98% (95% confidence interval: 97-99. Overall specificity was 45% (95% confidence interval: 41-48. Regarding specificity, studies were heterogeneous and a subgroup analysis was done according to the type of population included. Overall negative likelihood ratio was 0.05 (0.03-0.09. Conclusions: Due to its great sensitivity and low negative likelihood ratio, human leukocyte antigen-DQ2/DQ8 typing would be an appropriate test for ruling out celiac disease in the general population suffering related symptoms, and even more in at risk population.

  13. Large Scale Immune Profiling of Infected Humans and Goats Reveals Differential Recognition of Brucella melitensis Antigens

    Science.gov (United States)

    Liang, Li; Leng, Diana; Burk, Chad; Nakajima-Sasaki, Rie; Kayala, Matthew A.; Atluri, Vidya L.; Pablo, Jozelyn; Unal, Berkay; Ficht, Thomas A.; Gotuzzo, Eduardo; Saito, Mayuko; Morrow, W. John W.; Liang, Xiaowu; Baldi, Pierre; Gilman, Robert H.; Vinetz, Joseph M.; Tsolis, Renée M.; Felgner, Philip L.

    2010-01-01

    Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host. PMID:20454614

  14. Long livestock farming history and human landscape shaping revealed by lake sediment DNA.

    Science.gov (United States)

    Giguet-Covex, Charline; Pansu, Johan; Arnaud, Fabien; Rey, Pierre-Jérôme; Griggo, Christophe; Gielly, Ludovic; Domaizon, Isabelle; Coissac, Eric; David, Fernand; Choler, Philippe; Poulenard, Jérôme; Taberlet, Pierre

    2014-01-01

    The reconstruction of human-driven, Earth-shaping dynamics is important for understanding past human/environment interactions and for helping human societies that currently face global changes. However, it is often challenging to distinguish the effects of the climate from human activities on environmental changes. Here we evaluate an approach based on DNA metabarcoding used on lake sediments to provide the first high-resolution reconstruction of plant cover and livestock farming history since the Neolithic Period. By comparing these data with a previous reconstruction of erosive event frequency, we show that the most intense erosion period was caused by deforestation and overgrazing by sheep and cowherds during the Late Iron Age and Roman Period. Tracking plants and domestic mammals using lake sediment DNA (lake sedDNA) is a new, promising method for tracing past human practices, and it provides a new outlook of the effects of anthropogenic factors on landscape-scale changes.

  15. Evaluation of dried blood spot samples for screening of hepatitis C and human immunodeficiency virus in a real-world setting.

    Science.gov (United States)

    Vázquez-Morón, Sonia; Ryan, Pablo; Ardizone-Jiménez, Beatriz; Martín, Dolores; Troya, Jesus; Cuevas, Guillermo; Valencia, Jorge; Jimenez-Sousa, María A; Avellón, Ana; Resino, Salvador

    2018-01-30

    Both hepatitis C virus (HCV) infection and human immunodeficiency virus (HIV) infection are underdiagnosed, particularly in low-income countries and in difficult-to-access populations. Our aim was to develop and evaluate a methodology for the detection of HCV and HIV infection based on capillary dry blood spot (DBS) samples taken under real-world conditions. We carried out a cross-sectional study of 139 individuals (31 healthy controls, 68 HCV-monoinfected patients, and 40 HCV/HIV-coinfected patients). ELISA was used for anti-HCV and anti-HIV antibody detection; and SYBR Green RT-PCR was used for HCV-RNA detection. The HIV serological analysis revealed 100% sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The HCV serological analysis revealed a sensitivity of 92.6%, specificity of 100%, PPV of 100%, and NPV of 79.5%. Finally, the HCV-RNA detection test revealed a detection limit of 5 copies/µl with an efficiency of 100% and sensitivity of 99.1%, specificity of 100%, PPV of 100%, and NPV of 96.9%. In conclusion, our methodology was able to detect both HCV infection and HIV infection from the same DBS sample with good diagnostic performance. Screening for HCV and HIV using DBS might be a key strategy in the implementation of national programs for the control of both infections.

  16. Screening of random peptide library of hemagglutinin from pandemic 2009 A(H1N1 influenza virus reveals unexpected antigenically important regions.

    Directory of Open Access Journals (Sweden)

    Wanghui Xu

    Full Text Available The antigenic structure of the membrane protein hemagglutinin (HA from the 2009 A(H1N1 influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify "hot spots" or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins.

  17. Screening Assessment of Potential Human-Health Risk from Future Natural-Gas Drilling Near Project Rulison in Western Colorado

    International Nuclear Information System (INIS)

    Daniels, Jeffrey I.; Chapman, Jenny B.

    2012-01-01

    The Project Rulison underground nuclear test was conducted in 1969 at a depth of 8,400 ft in the Williams Fork Formation of the Piceance Basin, west-central Colorado (Figure 1). The U.S. Department of Energy Office of Legacy Management (LM) is the steward of the site. Their management is guided by data collected from past site investigations and current monitoring, and by the results of calculations of expected behavior of contaminants remaining in the deep subsurface. The purpose of this screening risk assessment is to evaluate possible health risks from current and future exposure to Rulison contaminants so the information can be factored into LM's stewardship decisions. For example, these risk assessment results can inform decisions regarding institutional controls at the site and appropriate monitoring of nearby natural-gas extraction activities. Specifically, the screening risk analysis can provide guidance for setting appropriate action levels for contaminant monitoring to ensure protection of human health.

  18. Screening Assessment of Potential Human-Health Risk from Future Natural-Gas Drilling Near Project Rulison in Western Colorado

    Energy Technology Data Exchange (ETDEWEB)

    Daniels Jeffrey I.,Chapman Jenny B.

    2012-01-01

    The Project Rulison underground nuclear test was conducted in 1969 at a depth of 8,400 ft in the Williams Fork Formation of the Piceance Basin, west-central Colorado (Figure 1). The U.S. Department of Energy Office of Legacy Management (LM) is the steward of the site. Their management is guided by data collected from past site investigations and current monitoring, and by the results of calculations of expected behavior of contaminants remaining in the deep subsurface. The purpose of this screening risk assessment is to evaluate possible health risks from current and future exposure to Rulison contaminants so the information can be factored into LM's stewardship decisions. For example, these risk assessment results can inform decisions regarding institutional controls at the site and appropriate monitoring of nearby natural-gas extraction activities. Specifically, the screening risk analysis can provide guidance for setting appropriate action levels for contaminant monitoring to ensure protection of human health.

  19. Small molecule inhibitors of the LEDGF site of human immunodeficiency virus integrase identified by fragment screening and structure based design.

    Directory of Open Access Journals (Sweden)

    Thomas S Peat

    Full Text Available A fragment-based screen against human immunodeficiency virus type 1 (HIV integrase led to a number of compounds that bound to the lens epithelium derived growth factor (LEDGF binding site of the integrase catalytic core domain. We determined the crystallographic structures of complexes of the HIV integrase catalytic core domain for 10 of these compounds and quantitated the binding by surface plasmon resonance. We demonstrate that the compounds inhibit the interaction of LEDGF with HIV integrase in a proximity AlphaScreen assay, an assay for the LEDGF enhancement of HIV integrase strand transfer and in a cell based assay. The compounds identified represent a potential framework for the development of a new series of HIV integrase inhibitors that do not bind to the catalytic site of the enzyme.

  20. High throughput screening of human subtelomeric DNA for copy number changes using multiplex amplifiable probe hybridisation (MAPH).

    Science.gov (United States)

    Hollox, E J; Atia, T; Cross, G; Parkin, T; Armour, J A L

    2002-11-01

    Subtelomeric regions of the human genome are gene rich, with a high level of sequence polymorphism. A number of clinical conditions, including learning disability, have been attributed to subtelomeric deletions or duplications, but screening for deletion in these regions using conventional cytogenetic methods and fluorescence in situ hybridisation (FISH) is laborious. Here we report that a new method, multiplex amplifiable probe hybridisation (MAPH), can be used to screen for copy number at subtelomeric regions. We have constructed a set of MAPH probes with each subtelomeric region represented at least once, so that one gel lane can assay copy number at all chromosome ends in one person. Each probe has been sequenced and, where possible, its position relative to the telomere determined by comparison with mapped clones. The sensitivity of the probes has been characterised on a series of cytogenetically verified positive controls and 83 normal controls were used to assess the frequency of polymorphic copy number with no apparent phenotypic effect. We have also used MAPH to test a cohort of 37 people selected from males referred for fragile X syndrome testing and found six changes that were confirmed by dosage PCR. MAPH can be used to screen subtelomeric regions of chromosomes for deletions and duplications before confirmation by FISH or dosage PCR. The high throughput nature of this technique allows it to be used for large scale screening of subtelomeric copy number, before confirmation by FISH. In practice, the availability of a rapid and efficient screen may allow subtelomeric analysis to be applied to a wider selection of patients than is currently possible using FISH alone.

  1. Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells.

    Science.gov (United States)

    Sherman, Sean P; Pyle, April D

    2013-01-01

    Differentiated cells from human embryonic stem cells (hESCs) provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs) provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

  2. Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Sean P Sherman

    Full Text Available Differentiated cells from human embryonic stem cells (hESCs provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

  3. Integrative analyses of human reprogramming reveal dynamic nature of induced pluripotency

    Science.gov (United States)

    Cacchiarelli, Davide; Trapnell, Cole; Ziller, Michael J.; Soumillon, Magali; Cesana, Marcella; Karnik, Rahul; Donaghey, Julie; Smith, Zachary D.; Ratanasirintrawoot, Sutheera; Zhang, Xiaolan; Ho Sui, Shannan J.; Wu, Zhaoting; Akopian, Veronika; Gifford, Casey A.; Doench, John; Rinn, John L.; Daley, George Q.; Meissner, Alexander; Lander, Eric S.; Mikkelsen, Tarjei S.

    2015-01-01

    Summary Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered reactivation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PMID:26186193

  4. Lachnospiraceae and Bacteroidales Alternative Fecal Indicators Reveal Chronic Human Sewage Contamination in an Urban Harbor▿†

    Science.gov (United States)

    Newton, Ryan J.; VandeWalle, Jessica L.; Borchardt, Mark A.; Gorelick, Marc H.; McLellan, Sandra L.

    2011-01-01

    The complexity of fecal microbial communities and overlap among human and other animal sources have made it difficult to identify source-specific fecal indicator bacteria. However, the advent of next-generation sequencing technologies now provides increased sequencing power to resolve microbial community composition within and among environments. These data can be mined for information on source-specific phylotypes and/or assemblages of phylotypes (i.e., microbial signatures). We report the development of a new genetic marker for human fecal contamination identified through microbial pyrotag sequence analysis of the V6 region of the 16S rRNA gene. Sequence analysis of 37 sewage samples and comparison with database sequences revealed a human-associated phylotype within the Lachnospiraceae family, which was closely related to the genus Blautia. This phylotype, termed Lachno2, was on average the second most abundant fecal bacterial phylotype in sewage influent samples from Milwaukee, WI. We developed a quantitative PCR (qPCR) assay for Lachno2 and used it along with the qPCR-based assays for human Bacteroidales (based on the HF183 genetic marker), total Bacteroidales spp., and enterococci and the conventional Escherichia coli and enterococci plate count assays to examine the prevalence of fecal and human fecal pollution in Milwaukee's harbor. Both the conventional fecal indicators and the human-associated indicators revealed chronic fecal pollution in the harbor, with significant increases following heavy rain events and combined sewer overflows. The two human-associated genetic marker abundances were tightly correlated in the harbor, a strong indication they target the same source (i.e., human sewage). Human adenoviruses were routinely detected under all conditions in the harbor, and the probability of their occurrence increased by 154% for every 10-fold increase in the human indicator concentration. Both Lachno2 and human Bacteroidales increased specificity to

  5. Interaction studies reveal specific recognition of an anti-inflammatory polyphosphorhydrazone dendrimer by human monocytes.

    Science.gov (United States)

    Ledall, Jérémy; Fruchon, Séverine; Garzoni, Matteo; Pavan, Giovanni M; Caminade, Anne-Marie; Turrin, Cédric-Olivier; Blanzat, Muriel; Poupot, Rémy

    2015-11-14

    Dendrimers are nano-materials with perfectly defined structure and size, and multivalency properties that confer substantial advantages for biomedical applications. Previous work has shown that phosphorus-based polyphosphorhydrazone (PPH) dendrimers capped with azabisphosphonate (ABP) end groups have immuno-modulatory and anti-inflammatory properties leading to efficient therapeutic control of inflammatory diseases in animal models. These properties are mainly prompted through activation of monocytes. Here, we disclose new insights into the molecular mechanisms underlying the anti-inflammatory activation of human monocytes by ABP-capped PPH dendrimers. Following an interdisciplinary approach, we have characterized the physicochemical and biological behavior of the lead ABP dendrimer with model and cell membranes, and compared this experimental set of data to predictive computational modelling studies. The behavior of the ABP dendrimer was compared to the one of an isosteric analog dendrimer capped with twelve azabiscarboxylate (ABC) end groups instead of twelve ABP end groups. The ABC dendrimer displayed no biological activity on human monocytes, therefore it was considered as a negative control. In detail, we show that the ABP dendrimer can bind both non-specifically and specifically to the membrane of human monocytes. The specific binding leads to the internalization of the ABP dendrimer by human monocytes. On the contrary, the ABC dendrimer only interacts non-specifically with human monocytes and is not internalized. These data indicate that the bioactive ABP dendrimer is recognized by specific receptor(s) at the surface of human monocytes.

  6. Comparing immediate-type food allergy in humans and companion animals-revealing unmet needs.

    Science.gov (United States)

    Pali-Schöll, I; De Lucia, M; Jackson, H; Janda, J; Mueller, R S; Jensen-Jarolim, E

    2017-11-01

    Adverse food reactions occur in human as well as veterinary patients. Systematic comparison may lead to improved recommendations for prevention and treatment in both. In this position paper, we summarize the current knowledge on immediate-type food allergy vs other food adverse reactions in companion animals, and compare this to the human situation. While the prevalence of food allergy in humans has been well studied for some allergens, this remains to be investigated for animal patients, where owner-reported as well as veterinarian-diagnosed food adverse reactions are on the increase. The characteristics of the disease in humans vs dogs, cats, and horses are most often caused by similar, but sometimes species-dependent different pathophysiological mechanisms, prompting the specific clinical symptoms, diagnoses, and treatments. Furthermore, little is known about the allergen molecules causative for type I food allergy in animals, which, like in human patients, could represent predictive biomarkers for risk evaluation. The definite diagnosis of food allergy relies-as in humans-on elimination diet and provocation tests. Besides allergen avoidance in daily practice, novel treatment options and tolerization strategies are underway. Taken together, numerous knowledge gaps were identified in veterinary food allergy, which need to be filled by systematic comparative studies. © 2017 The Authors. Allergy Published by John Wiley & Sons Ltd.

  7. Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Leon, Ileana R; Bak, Steffen

    2011-01-01

    . In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomic study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins...... in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates....... Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes....

  8. A Genome-Wide Screen for Interactions Reveals a New Locus on 4p15 Modifying the Effect of Waist-to-Hip Ratio on Total Cholesterol

    NARCIS (Netherlands)

    Surakka, Ida; Isaacs, Aaron; Karssen, Lennart C.; Laurila, Pirkka-Pekka P.; Middelberg, Rita P. S.; Tikkanen, Emmi; Ried, Janina S.; Lamina, Claudia; Mangino, Massimo; Igl, Wilmar; Hottenga, Jouke-Jan; Lagou, Vasiliki; van der Harst, Pim; Mateo Leach, Irene; Esko, Tonu; Kutalik, Zoltan; Wainwright, Nicholas W.; Struchalin, Maksim V.; Sarin, Antti-Pekka; Kangas, Antti J.; Viikari, Jorma S.; Perola, Markus; Rantanen, Taina; Petersen, Ann-Kristin; Soininen, Pasi; Johansson, Asa; Soranzo, Nicole; Heath, Andrew C.; Papamarkou, Theodore; Prokopenko, Inga; Toenjes, Anke; Kronenberg, Florian; Doering, Angela; Rivadeneira, Fernando; Montgomery, Grant W.; Whitfield, John B.; Kahonen, Mika; Lehtimaki, Terho; Freimer, Nelson B.; Willemsen, Gonneke; de Geus, Eco J. C.; Palotie, Aarno; Sandhu, Manj S.; Waterworth, Dawn M.; Metspalu, Andres; Stumvoll, Michael; Uitterlinden, Andre G.; Navis, Gerjan; Wijmenga, Cisca; Wolffenbuttel, Bruce H. R.

    2011-01-01

    Recent genome-wide association (GWA) studies described 95 loci controlling serum lipid levels. These common variants explain similar to 25% of the heritability of the phenotypes. To date, no unbiased screen for gene-environment interactions for circulating lipids has been reported. We screened for

  9. A Genome-Wide Screen for Interactions Reveals a New Locus on 4p15 Modifying the Effect of Waist-to-Hip Ratio on Total Cholesterol

    NARCIS (Netherlands)

    Surakka, I.; Isaacs, A.; Karssen, L.C.; Laurila, P.P.P.; Middelberg, R.P.S.; Tikkanen, E.; Ried, J.S.; Lamina, C.; Mangino, M.; Igl, W.; Hottenga, J.J.; Lagou, V.; van der Harst, P.; Mateo Leach, I.; Esko, T.; Kutalik, Z.; Wainwright, N.W.; Struchalin, M.V.; Sarin, A.P.; Kangas, A.J.; Viikari, J.S.; Perola, M.; Rantanen, T.; Petersen, A.K.; Soininen, P.; Johansson, Å.; Soranzo, N.; Heath, A.C.; Papamarkou, T.; Prokopenko, I.; Tönjes, A.; Kronenberg, F.; Döring, A.; Rivadeneira, F.; Montgomery, GW; Whitfield, J.B.; Kähönen, M.; Lehtimäki, T.; Freimer, N.B.; Willemsen, G.; de Geus, E.J.C.; Palotie, A.; Sandhu, M.S.; Waterworth, D.; Metspalu, A.; Stumvoll, M.; Uitterlinden, A.G.; Navis, G.; Wijmenga, C.; Wolffenbuttel, B.H.R.; Taskinen, M.R.; Ala-Korpela, M.; Kaprio, J.; Kyvik, K.O.; Boomsma, D.I.; Pedersen, N.L.; Gyllensten, U.; Wilson, J.F.; Rudan, I.; Campbell, H.; Pramstaller, P.P.; Spector, T.D.; Witteman, J.C.M.; Eriksson, J.G.; Salomaa, V.; Oostra, B.A.; Raitakari, O.T.; Wichmann, H.E.; Gieger, C.; Järvelin, M.J.; Martin, N.G.; Hofman, A.; McCarthy, M.I.; Peltonen, L.; van Duijn, C.M.; Aulchenko, Y.S.; Ripatti, S.

    2011-01-01

    Recent genome-wide association (GWA) studies described 95 loci controlling serum lipid levels. These common variants explain ~25% of the heritability of the phenotypes. To date, no unbiased screen for gene-environment interactions for circulating lipids has been reported. We screened for variants

  10. A genome-wide screen for interactions reveals a new locus on 4p15 modifying the effect of waist-to-hip ratio on total cholesterol

    NARCIS (Netherlands)

    I. Surakka (Ida); A.J. Isaacs (Aaron); L.C. Karssen (Lennart); P.-P.P. Laurila; R.P.S. Middelberg (Rita); E. Tikkanen (Emmi); J.S. Ried (Janina); C. Lamina (Claudia); M. Mangino (Massimo); W. Igl (Wilmar); J.J. Hottenga (Jouke Jan); V. Lagou (Vasiliki); P. van der Harst (Pim); I.M. Leach (Irene Mateo); T. Esko (Tõnu); Z. Kutalik (Zoltán); N.W. Wainwright (Nicholas); M.V. Struchalin (Maksim); A.-P. Sarin; A.J. Kangas (Antti); J. Viikari (Jorma); M. Perola (Markus); T. Rantanen (Taina); A.K. Petersen; P. Soininen (Pasi); A. Johansson (Åsa); N. Soranzo (Nicole); A.C. Heath (Andrew); T. Papamarkou (Theodore); I. Prokopenko (Inga); A. Tönjes (Anke); F. Kronenberg (Florian); A. Döring (Angela); F. Rivadeneira Ramirez (Fernando); G.W. Montgomery (Grant); J.B. Whitfield (John); M. Kähönen (Mika); T. Lehtimäki (Terho); N.B. Freimer (Nelson); G.A.H.M. Willemsen (Gonneke); E.J.C. de Geus (Eco); A. Palotie (Aarno); M.S. Sandhu (Manj); D. Waterworth (Dawn); A. Metspalu (Andres); M. Stumvoll (Michael); A.G. Uitterlinden (André); A. Jula (Antti); G. Navis (Gerjan); C. Wijmenga (Cisca); B.H.R. Wolffenbuttel (Bruce); M.-R. Taskinen; M. Ala-Korpela (Mika); J. Kaprio (Jaakko); K.O. Kyvik (Kirsten Ohm); D.I. Boomsma (Dorret); N.L. Pedersen (Nancy); U. Gyllensten (Ulf); J.F. Wilson (James); I. Rudan (Igor); H. Campbell (Harry); P.P. Pramstaller (Peter Paul); T.D. Spector (Timothy); J.C.M. Witteman (Jacqueline); J.G. Eriksson (Johan); V. Salomaa (Veikko); B.A. Oostra (Ben); O. Raitakari (Olli); H.E. Wichmann (Heinz Erich); C. Gieger (Christian); M.R. Järvelin; N.G. Martin (Nicholas); A. Hofman (Albert); M.I. McCarthy (Mark); Y.S. Aulchenko (Yurii); L. Peltonen (Leena Johanna); P. Tikka-Kleemola (Päivi); S. Ripatti (Samuli)

    2011-01-01

    textabstractRecent genome-wide association (GWA) studies described 95 loci controlling serum lipid levels. These common variants explain ~25% of the heritability of the phenotypes. To date, no unbiased screen for gene-environment interactions for circulating lipids has been reported. We screened for

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

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2017-01-01

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

  12. The largest human cognitive performance dataset reveals insights into the effects of lifestyle factors and aging

    Directory of Open Access Journals (Sweden)

    Daniel A Sternberg

    2013-06-01

    Full Text Available Making new breakthroughs in understanding the processes underlying human cognition may depend on the availability of very large datasets that have not historically existed in psychology and neuroscience. Lumosity is a web-based cognitive training platform that has grown to include over 600 million cognitive training task results from over 35 million individuals, comprising the largest existing dataset of human cognitive performance. As part of the Human Cognition Project, Lumosity’s collaborative research program to understand the human mind, Lumos Labs researchers and external research collaborators have begun to explore this dataset in order uncover novel insights about the correlates of cognitive performance. This paper presents two preliminary demonstrations of some of the kinds of questions that can be examined with the dataset. The first example focuses on replicating known findings relating lifestyle factors to baseline cognitive performance in a demographically diverse, healthy population at a much larger scale than has previously been available. The second example examines a question that would likely be very difficult to study in laboratory-based and existing online experimental research approaches: specifically, how learning ability for different types of cognitive tasks changes with age. We hope that these examples will provoke the imagination of researchers who are interested in collaborating to answer fundamental questions about human cognitive performance.

  13. Genetics of the pig tapeworm in madagascar reveal a history of human dispersal and colonization.

    Science.gov (United States)

    Yanagida, Tetsuya; Carod, Jean-François; Sako, Yasuhito; Nakao, Minoru; Hoberg, Eric P; Ito, Akira

    2014-01-01

    An intricate history of human dispersal and geographic colonization has strongly affected the distribution of human pathogens. The pig tapeworm Taenia solium occurs throughout the world as the causative agent of cysticercosis, one of the most serious neglected tropical diseases. Discrete genetic lineages of T. solium in Asia and Africa/Latin America are geographically disjunct; only in Madagascar are they sympatric. Linguistic, archaeological and genetic evidence has indicated that the people in Madagascar have mixed ancestry from Island Southeast Asia and East Africa. Hence, anthropogenic introduction of the tapeworm from Southeast Asia and Africa had been postulated. This study shows that the major mitochondrial haplotype of T. solium in Madagascar is closely related to those from the Indian Subcontinent. Parasitological evidence presented here, and human genetics previously reported, support the hypothesis of an Indian influence on Malagasy culture coinciding with periods of early human migration onto the island. We also found evidence of nuclear-mitochondrial discordance in single tapeworms, indicating unexpected cross-fertilization between the two lineages of T. solium. Analyses of genetic and geographic populations of T. solium in Madagascar will shed light on apparently rapid evolution of this organism driven by recent (<2,000 yr) human migrations, following tens of thousands of years of geographic isolation.

  14. Environmentally sensitive molecular probes reveal mutations and epigenetic 5-methyl cytosine in human oncogenes

    DEFF Research Database (Denmark)

    Taskova, M.; Barducci, M. C.; Astakhova, K.

    2017-01-01

    There is currently an unmet need for reliable tools that allow for direct detection and quantification of modifications in genomic DNA. For example, in cancer research and clinical diagnostics, target DNA has to be amplified and sequenced in order to reveal mutations. For 5-methylcytosine detecti...

  15. Lack of tissue renewal in human adult Achilles tendon is revealed by nuclear bomb C

    DEFF Research Database (Denmark)

    Heinemeier, Katja Maria; Schjerling, Peter; Heinemeier, J.

    2013-01-01

    the 14C bomb-pulse method. This method takes advantage of the dramatic increase in atmospheric levels of 14C, produced by nuclear bomb tests in 1955-1963, which is reflected in all living organisms. Levels of 14C were measured in 28 forensic samples of Achilles tendon core and 4 skeletal muscle samples...... is revealed by nuclear bomb 14C....

  16. Sequence-based Screening for Rare Enzymes: New Insights into the World of AMDases Reveal a Conserved Motif and 58 Novel Enzymes Clustering in Eight Distinct Families.

    Directory of Open Access Journals (Sweden)

    Janine Maimanakos

    2016-08-01

    Full Text Available Arylmalonate-Decarboxylases (AMDases, EC 4.1.1.76 are very rare and mostly underexplored enzymes. Currently only four known and biochemically characterized representatives exist. However, their ability to decarboxylate α-disubstituted malonic acid derivatives to optically pure products without cofactors makes them attractive and promising candidates for the use as biocatalysts in industrial processes. Until now, AMDases could not be separated from other members of the aspartate/glutamate racemase superfamily based on their gene sequences. Within this work, a search algorithm was developed that enables a reliable prediction of AMDase activity for potential candidates. Based on specific sequence patterns and screening methods 58 novel AMDase candidate genes could be identified in this work. Thereby, AMDases with the conserved sequence pattern of Bordetella bronchiseptica’s prototype appeared to be limited to the classes of Alpha-, Beta- and Gammaproteobacteria. Amino acid homologies and comparison of gene surrounding sequences enabled the classification of eight enzyme clusters. Particularly striking is the accumulation of genes coding for different transporters of the TTT family, TRAP transporters and ABC transporters as well as genes coding for mandelate racemases/muconate lactonizing enzymes that might be involved in substrate uptake or degradation of AMDase products. Further, three novel AMDases were characterized which showed a high enantiomeric excess (>99% of the (R-enantiomer of flurbiprofen. These are the recombinant AmdA and AmdV from Variovorax sp. strains HH01 and HH02, originated from soil, and AmdP from Polymorphum gilvum found by a data base search. Altogether our findings give new insights into the class of AMDases and reveal many previously unknown enzyme candidates with high potential for bioindustrial processes.

  17. Clustering patterns of LOD scores for asthma-related phenotypes revealed by a genome-wide screen in 295 French EGEA families.

    Science.gov (United States)

    Bouzigon, Emmanuelle; Dizier, Marie-Hélène; Krähenbühl, Christine; Lemainque, Arnaud; Annesi-Maesano, Isabella; Betard, Christine; Bousquet, Jean; Charpin, Denis; Gormand, Frédéric; Guilloud-Bataille, Michel; Just, Jocelyne; Le Moual, Nicole; Maccario, Jean; Matran, Régis; Neukirch, Françoise; Oryszczyn, Marie-Pierre; Paty, Evelyne; Pin, Isabelle; Rosenberg-Bourgin, Myriam; Vervloet, Daniel; Kauffmann, Francine; Lathrop, Mark; Demenais, Florence

    2004-12-15

    A genome-wide scan for asthma phenotypes was conducted in the whole sample of 295 EGEA families selected through at least one asthmatic subject. In addition to asthma, seven phenotypes involved in the main asthma physiopathological pathways were considered: SPT (positive skin prick test response to at least one of 11 allergens), SPTQ score being the number of positive skin test responses to 11 allergens, Phadiatop (positive specific IgE response to a mixture of allergens), total IgE levels, eosinophils, bronchial responsiveness (BR) to methacholine challenge and %predicted FEV(1). Four regions showed evidence for linkage (Pscreens, 6q14 appears to be a new region potentially linked to %FEV(1). To determine which of these various asthma phenotypes are more likely to share common genetic determinants, a principal component analysis was applied to the genome-wide LOD scores. This analysis revealed clustering of LODs for asthma, SPT and Phadiatop on one axis and clustering of LODs for %FEV(1), BR and SPTQ on the other, while LODs for IgE and eosinophils appeared to be independent from all other LODs. These results provide new insights into the potential sharing of genetic determinants by asthma-related phenotypes.

  18. High-throughput and automatic typing via human papillomavirus identification map for cervical cancer screening and prognosis.

    Science.gov (United States)

    Yi, Linglu; Xu, Xueqin; Lin, Xuexia; Li, Haifang; Ma, Yuan; Lin, Jin-Ming

    2014-07-07

    A novel human papillomavirus (HPV) typing assay for cervical cancer screening and prognosis was developed by the combination of restriction fragment length polymorphism (RFLP) and microchip electrophoresis (MCE) to achieve higher levels of sensitivity and throughput. The detection limit of 2 × 10(2) copies, high sensitivity and typing accuracy on the account of PCR-RFLP-MCE method guarantee the successful diagnosis results of 4-fold higher infection rate over cytologic tests. From clinical samples, eleven kinds of HPV types were identified with a good compatibility degree of over 90%. The described method showed good reliability in clinical samples and provided a promising alternative for pathological studies at the molecular level.

  19. Development of an Intracellular Screen for New Compounds Able To Inhibit Mycobacterium tuberculosis Growth in Human Macrophages.

    Science.gov (United States)

    Sorrentino, Flavia; Gonzalez del Rio, Ruben; Zheng, Xingji; Presa Matilla, Jesus; Torres Gomez, Pedro; Martinez Hoyos, Maria; Perez Herran, Maria Esther; Mendoza Losana, Alfonso; Av-Gay, Yossef

    2016-01-01

    Here we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  20. Within- and across-trial dynamics of human EEG reveal cooperative interplay between reinforcement learning and working memory.

    Science.gov (United States)

    Collins, Anne G E; Frank, Michael J

    2018-03-06

    Learning from rewards and punishments is essential to survival and facilitates flexible human behavior. It is widely appreciated that multiple cognitive and reinforcement learning systems contribute to decision-making, but the nature of their interactions is elusive. Here, we leverage methods for extracting trial-by-trial indices of reinforcement learning (RL) and working memory (WM) in human electro-encephalography to reveal single-trial computations beyond that afforded by behavior alone. Neural dynamics confirmed that increases in neural expectation were predictive of reduced neural surprise in the following feedback period, supporting central tenets of RL models. Within- and cross-trial dynamics revealed a cooperative interplay between systems for learning, in which WM contributes expectations to guide RL, despite competition between systems during choice. Together, these results provide a deeper understanding of how multiple neural systems interact for learning and decision-making and facilitate analysis of their disruption in clinical populations.

  1. Intergenic DNA sequences from the human X chromosome reveal high rates of global gene flow

    Directory of Open Access Journals (Sweden)

    Wall Jeffrey D

    2008-11-01

    Full Text Available Abstract Background Despite intensive efforts devoted to collecting human polymorphism data, little is known about the role of gene flow in the ancestry of human populations. This is partly because most analyses have applied one of two simple models of population structure, the island model or the splitting model, which make unrealistic biological assumptions. Results Here, we analyze 98-kb of DNA sequence from 20 independently evolving intergenic regions on the X chromosome in a sample of 90 humans from six globally diverse populations. We employ an isolation-with-migration (IM model, which assumes that populations split and subsequently exchange migrants, to independently estimate effective population sizes and migration rates. While the maximum effective size of modern humans is estimated at ~10,000, individual populations vary substantially in size, with African populations tending to be larger (2,300–9,000 than non-African populations (300–3,300. We estimate mean rates of bidirectional gene flow at 4.8 × 10-4/generation. Bidirectional migration rates are ~5-fold higher among non-African populations (1.5 × 10-3 than among African populations (2.7 × 10-4. Interestingly, because effective sizes and migration rates are inversely related in African and non-African populations, population migration rates are similar within Africa and Eurasia (e.g., global mean Nm = 2.4. Conclusion We conclude that gene flow has played an important role in structuring global human populations and that migration rates should be incorporated as critical parameters in models of human demography.

  2. In silico assessment of genetic variation in KCNA5 reveals multiple mechanisms of human atrial arrhythmogenesis

    DEFF Research Database (Denmark)

    Colman, Michael A; Ni, Haibo; Liang, Bo

    2017-01-01

    and quantify the functional impact of these KCNA5 mutations on atrial electrical activity. A multi-scale model of the human atria was updated to incorporate detailed experimental data on IKur from both wild-type and mutants. The effects of the mutations on human atrial action potential and rate dependence were...... provides new insights into understanding the mechanisms by which mutant IKur contributes to atrial arrhythmias. In addition, as IKur is an atrial-specific channel and a number of IKur-selective blockers have been developed as anti-AF agents, this study also helps to understand some contradictory results...

  3. A highly variable segment of human subterminal 16p reveals a history of population growth for modern humans outside Africa

    Science.gov (United States)

    Alonso, Santos; Armour, John A. L.

    2001-01-01

    We have sequenced a highly polymorphic subterminal noncoding region from human chromosome 16p13.3, flanking the 5′ end of the hypervariable minisatellite MS205, in 100 chromosomes sampled from different African and Euroasiatic populations. Coalescence analysis indicates that the time to the most recent common ancestor (approximately 1 million years) predates the appearance of anatomically modern human forms. The root of the network describing this variability lies in Africa. African populations show a greater level of diversity and deeper branches. Most Euroasiatic variability seems to have been generated after a recent out-of-Africa range expansion. A history of population growth is the most likely scenario for the Euroasiatic populations. This pattern of nuclear variability can be reconciled with inferences based on mitochondrial DNA. PMID:11158547

  4. Cryo-EM structure of human adenovirus D26 reveals the conservation of structural organization among human adenoviruses.

    Science.gov (United States)

    Yu, Xiaodi; Veesler, David; Campbell, Melody G; Barry, Mary E; Asturias, Francisco J; Barry, Michael A; Reddy, Vijay S

    2017-05-01

    Human adenoviruses (HAdVs) cause acute respiratory, ocular, and gastroenteric diseases and are also frequently used as gene and vaccine delivery vectors. Unlike the archetype human adenovirus C5 (HAdV-C5), human adenovirus D26 (HAdV-D26) belongs to species-D HAdVs, which target different cellular receptors, and is differentially recognized by immune surveillance mechanisms. HAdV-D26 is being championed as a lower seroprevalent vaccine and oncolytic vector in preclinical and human clinical studies. To understand the molecular basis for their distinct biological properties and independently validate the structures of minor proteins, we determined the first structure of species-D HAdV at 3.7 Å resolution by cryo-electron microscopy. All the hexon hypervariable regions (HVRs), including HVR1, have been identified and exhibit a distinct organization compared to those of HAdV-C5. Despite the differences in the arrangement of helices in the coiled-coil structures, protein IX molecules form a continuous hexagonal network on the capsid exterior. In addition to the structurally conserved region (3 to 300) of IIIa, we identified an extra helical domain comprising residues 314 to 390 that further stabilizes the vertex region. Multiple (two to three) copies of the cleaved amino-terminal fragment of protein VI (pVIn) are observed in each hexon cavity, suggesting that there could be ≥480 copies of VI present in HAdV-D26. In addition, a localized asymmetric reconstruction of the vertex region provides new details of the three-pronged "claw hold" of the trimeric fiber and its interactions with the penton base. These observations resolve the previous conflicting assignments of the minor proteins and suggest the likely conservation of their organization across different HAdVs.

  5. Isolation and Characterization of Current Human Coronavirus Strains in Primary Human Epithelial Cell Cultures Reveal Differences in Target Cell Tropism

    Science.gov (United States)

    Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker

    2013-01-01

    The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in nonhospitalized and hospitalized children. Studies of the fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism, are seriously hampered by the lack of a universal culture system or suitable animal models. To expand the knowledge on fundamental virus-host interactions for all four HCoVs at the site of primary infection, we used pseudostratified HAE cell cultures to isolate and characterize representative clinical HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained, representing HCoV-229E (n = 1), HCoV-NL63 (n = 1), HCoV-HKU1 (n = 4), and HCoV-OC43 (n = 4). For each strain, we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection by confocal microscopy, we observed that HCoV-229E employs a target cell tropism for nonciliated cells, whereas HCoV-OC43, HCoV-HKU1, and HCoV-NL63 all infect ciliated cells. Collectively, the data demonstrate that HAE cell cultures, which morphologically and functionally resemble human airways in vivo, represent a robust universal culture system for isolating and comparing all contemporary HCoV strains. PMID:23427150

  6. Identification of human flap endonuclease 1 (FEN1) inhibitors using a machine learning based consensus virtual screening.

    Science.gov (United States)

    Deshmukh, Amit Laxmikant; Chandra, Sharat; Singh, Deependra Kumar; Siddiqi, Mohammad Imran; Banerjee, Dibyendu

    2017-07-25

    Human Flap endonuclease1 (FEN1) is an enzyme that is indispensable for DNA replication and repair processes and inhibition of its Flap cleavage activity results in increased cellular sensitivity to DNA damaging agents (cisplatin, temozolomide, MMS, etc.), with the potential to improve cancer prognosis. Reports of the high expression levels of FEN1 in several cancer cells support the idea that FEN1 inhibitors may target cancer cells with minimum side effects to normal cells. In this study, we used large publicly available, high-throughput screening data of small molecule compounds targeted against FEN1. Two machine learning algorithms, Support Vector Machine (SVM) and Random Forest (RF), were utilized to generate four classification models from huge PubChem bioassay data containing probable FEN1 inhibitors and non-inhibitors. We also investigated the influence of randomly selected Zinc-database compounds as negative data on the outcome of classification modelling. The results show that the SVM model with inactive compounds was superior to RF with Matthews's correlation coefficient (MCC) of 0.67 for the test set. A Maybridge database containing approximately 53 000 compounds was screened and top ranking 5 compounds were selected for enzyme and cell-based in vitro screening. The compound JFD00950 was identified as a novel FEN1 inhibitor with in vitro inhibition of flap cleavage activity as well as cytotoxic activity against a colon cancer cell line, DLD-1.

  7. An association between Trichomonas vaginalis and high-risk human papillomavirus in rural Tanzanian women undergoing cervical cancer screening.

    Science.gov (United States)

    Lazenby, Gweneth B; Taylor, Peyton T; Badman, Barbara S; McHaki, Emil; Korte, Jeffrey E; Soper, David E; Young Pierce, Jennifer

    2014-01-01

    The goal of this study was to determine the prevalence of vaginitis and its association with high-risk human papillomavirus (HR HPV) in women undergoing cervical cancer screening in rural Tanzania. For the purpose of cervical cancer screening, cytology and HR HPV polymerase chain reaction data were collected from 324 women aged between 30 and 60 years. Microscopy and gram stains were used to detect yeast and bacterial vaginosis. Cervical nucleic acid amplification test specimens were collected for the detection of Trichomonas vaginalis (TV), Chlamydia trachomatis, and Neisseria gonorrhoeae. The majority of women were married (320 of 324) and reported having a single sexual partner (270 of 324); the median age of participants was 41 years. HR HPV was detected in 42 participants. Forty-seven percent of women had vaginitis. Bacterial vaginosis was the most common infection (32.4%), followed by TV (10.4%), and yeast (6.8%). In multivariable logistic regression analysis, TV was associated with an increased risk of HR HPV (odds ratio, 4.2 [95% CI, 1.7-10.3]). Patients with TV were 6.5 times more likely to have HPV type 16 than patients negative for TV (50% vs 13.3%) (odds ratio, 6.5 [95% CI, 1.1-37]). Among rural Tanzanian women who presented for cervical cancer screening, Trichomonas vaginitis was significantly associated with HR HPV infection (specifically type 16). © 2014 Published by Elsevier HS Journals, Inc.

  8. CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome.

    Science.gov (United States)

    Klann, Tyler S; Black, Joshua B; Chellappan, Malathi; Safi, Alexias; Song, Lingyun; Hilton, Isaac B; Crawford, Gregory E; Reddy, Timothy E; Gersbach, Charles A

    2017-06-01

    Large genome-mapping consortia and thousands of genome-wide association studies have identified non-protein-coding elements in the genome as having a central role in various biological processes. However, decoding the functions of the millions of putative regulatory elements discovered in these studies remains challenging. CRISPR-Cas9-based epigenome editing technologies have enabled precise perturbation of the activity of specific regulatory elements. Here we describe CRISPR-Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. Using dCas9 KRAB repressor and dCas9 p300 activator constructs and lentiviral single guide RNA libraries to target DNase I hypersensitive sites surrounding a gene of interest, we carried out both loss- and gain-of-function screens to identify regulatory elements for the β-globin and HER2 loci in human cells. CERES readily identified known and previously unidentified regulatory elements, some of which were dependent on cell type or direction of perturbation. This technology allows the high-throughput functional annotation of putative regulatory elements in their native chromosomal context.

  9. Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening.

    Science.gov (United States)

    Lin, Shih-Hung; Huang, Kao-Jean; Weng, Ching-Feng; Shiuan, David

    2015-01-01

    Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR). The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank) database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity) properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration) values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening.

  10. A review of human pluripotent stem cell-derived cardiomyocytes for high-throughput drug discovery, cardiotoxicity screening, and publication standards.

    Science.gov (United States)

    Mordwinkin, Nicholas M; Burridge, Paul W; Wu, Joseph C

    2013-02-01

    Drug attrition rates have increased in past years, resulting in growing costs for the pharmaceutical industry and consumers. The reasons for this include the lack of in vitro models that correlate with clinical results and poor preclinical toxicity screening assays. The in vitro production of human cardiac progenitor cells and cardiomyocytes from human pluripotent stem cells provides an amenable source of cells for applications in drug discovery, disease modeling, regenerative medicine, and cardiotoxicity screening. In addition, the ability to derive human-induced pluripotent stem cells from somatic tissues, combined with current high-throughput screening and pharmacogenomics, may help realize the use of these cells to fulfill the potential of personalized medicine. In this review, we discuss the use of pluripotent stem cell-derived cardiomyocytes for drug discovery and cardiotoxicity screening, as well as current hurdles that must be overcome for wider clinical applications of this promising approach.

  11. Diet-microbe co-metabolic interactions in wild primates reveal clues on human evolution

    Czech Academy of Sciences Publication Activity Database

    Gomez, A. M.; Rothman, J. M.; Petrželková, Klára Judita; Yeoman, C. J.; Vlčková, K.; Umana, J. D.; Carr, M.; Modrý, D.; Tod, A.; Nelson, K.; Stumpf, R. M.; Wilson, B. A.; White, B. A.; Leigh, S. R.

    2015-01-01

    Roč. 156, č. 60 (2015), s. 149 ISSN 0002-9483. [Annual Meeting of the American Association of Physical Anthropologists /84./. 25.03.2015-28.03.2015, St Louis] Institutional support: RVO:68081766 Keywords : wild primates * human evolution Subject RIV: EE - Microbiology, Virology

  12. Concordance of gene expression in human protein complexes reveals tissue specificity and pathology

    DEFF Research Database (Denmark)

    Börnigen, Daniela; Pers, Tune Hannes; Thorrez, Lieven

    2013-01-01

    Disease-causing variants in human genes usually lead to phenotypes specific to only a few tissues. Here, we present a method for predicting tissue specificity based on quantitative deregulation of protein complexes. The underlying assumption is that the degree of coordinated expression among prot...

  13. Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life

    DEFF Research Database (Denmark)

    Lynnerup, Niels; Kjeldsen, Henrik; Heegaard, Steffen

    2008-01-01

    , there is no subsequent remodelling of these fibers, nor removal of degraded lens fibers. Human tissue ultimately derives its (14)C content from the atmospheric carbon dioxide. The (14)C content of the lens proteins thus reflects the atmospheric content of (14)C when the lens crystallines were formed. Precise radiocarbon...

  14. Two ancient human genomes reveal Polynesian ancestry among the indigenous Botocudos of Brazil

    DEFF Research Database (Denmark)

    Malaspinas, Anna-Sapfo; Lao, Oscar; Schroeder, Hannes

    2014-01-01

    Understanding the peopling of the Americas remains an important and challenging question. Here, we present 14C dates, and morphological, isotopic and genomic sequence data from two human skulls from the state of Minas Gerais, Brazil, part of one of the indigenous groups known as ‘Botocudos’. We...

  15. Zebrafish syntenic relationship to human/mouse genomes revealed by radiation hybrid mapping

    International Nuclear Information System (INIS)

    Samonte, Irene E.

    2007-01-01

    Zebrafish (Danio rerio) is an excellent model system for vertebrate developmental analysis and a new model for human disorders. In this study, however, zebrafish was used to determine its syntenic relationship to human/mouse genomes using the zebrafish-hamster radiation hybrid panel. The focus was on genes residing on chromosomes 6 and 17 of human and mouse, respectively, and some other genes of either immunologic or evolutionary importance. Gene sequences of interest and zebrafish expressed sequence tags deposited in the GenBank were used in identifying zebrafish homologs. Polymerase chain reaction (PCR) amplification, cloning and subcloning, sequencing, and phylogenetic analysis were done to confirm the homology of the candidate genes in zebrafish. The promising markers were then tested in the 94 zebrafish-hamster radiation hybrid panel cell lines and submitted for logarithm of the odds (LOD) score analysis to position genes on the zebrafish map. A total of 19 loci were successfully mapped to zebrafish linkage groups 1, 14, 15, 19, and 20. Four of these loci were positioned in linkage group 20, whereas, 3 more loci were added in linkage group 19, thus increasing to 34 loci the number of human genes syntenic to the group. With the sequencing of the zebrafish genome, about 20 more MHC genes were reported linked on the same group. (Author)

  16. Structure of Human Tyrosinase Related Protein 1 Reveals a Binuclear Zinc Active Site Important for Melanogenesis

    NARCIS (Netherlands)

    Lai, Xuelei; Wichers, Harry J.; Soler-Lopez, Montserrat; Dijkstra, Bauke W.

    2017-01-01

    Tyrosinase-related protein 1 (TYRP1) is one of three tyrosinase-like glycoenzymes in human melanocytes that are key to the production of melanin, the compound responsible for the pigmentation of skin, eye, and hair. Difficulties with producing these enzymes in pure form have hampered the

  17. Associations between common intestinal parasites and bacteria in humans as revealed by qPCR

    DEFF Research Database (Denmark)

    O'Brien Andersen, L.; Karim, A. B.; Roager, Henrik Munch

    2016-01-01

    Several studies have shown associations between groups of intestinal bacterial or specific ratios between bacterial groups and various disease traits. Meanwhile, little is known about interactions and associations between eukaryotic and prokaryotic microorganisms in the human gut. In this work, we...

  18. Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway

    NARCIS (Netherlands)

    A.M. Brum (Andrea M.); J. van de Peppel (Jeroen); C.S. Van Der Leije (Cindy S.); M. Schreuders-Koedam (M.); H.J.M. Eijken (Marco); B.C.J. van der Eerden (Bram); J.P.T.M. van Leeuwen (Hans)

    2015-01-01

    textabstractOsteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. In this study, we have identified pathways that stimulate differentiation of bone forming osteoblasts from human mesenchymal stromal cells (hMSCs).

  19. Xenobiotics and the Human Gut Microbiome: Metatranscriptomics Reveal the Active Players

    OpenAIRE

    Ursell, Luke K.; Knight, Rob

    2013-01-01

    The human gut microbiome plays an important role in the metabolism of xenobiotics. In a recent issue of Cell, Maurice et al. identify the active members of the gut microbiome and show how gene expression profiles change within the gut microbial community in response to antibiotics and host-targeted xenobiotics.

  20. Human subcortical brain asymmetries in 15,847 people worldwide reveal effects of age and sex

    NARCIS (Netherlands)

    Guadalupe, Tulio; Mathias, Samuel R.; vanErp, Theo G.M.; Whelan, Christopher D.; Zwiers, Marcel P.; Abe, Yoshinari; Abramovic, Lucija; Agartz, Ingrid; Andreassen, Ole A.; Arias-Vásquez, Alejandro; Aribisala, Benjamin S.; Armstrong, Nicola J.; Arolt, Volker; Artiges, Eric; Ayesa-Arriola, Rosa; Baboyan, Vatche G.; Banaschewski, Tobias; Barker, Gareth; Bastin, Mark E.; Baune, Bernhard T.; Blangero, John; Bokde, Arun L.W.; Boedhoe, Premika S.W.; Bose, Anushree; Brem, Silvia; Brodaty, Henry; Bromberg, Uli; Brooks, Samantha; Büchel, Christian; Buitelaar, Jan; Calhoun, Vince D.; Cannon, Dara M.; Cattrell, Anna; Cheng, Yuqi; Conrod, Patricia J.; Conzelmann, Annette; Corvin, Aiden; Crespo-Facorro, Benedicto; Crivello, Fabrice; Dannlowski, Udo; de Zubicaray, Greig I.; de Zwarte, Sonja M.C.; Deary, Ian J.; Desrivières, Sylvane; Doan, Nhat Trung; Donohoe, Gary; Dørum, Erlend S.; Ehrlich, Stefan; Espeseth, Thomas; Fernández, Guillén; Flor, Herta; Fouche, Jean Paul; Frouin, Vincent; Fukunaga, Masaki; Gallinat, Jürgen; Garavan, Hugh; Gill, Michael; Suarez, Andrea Gonzalez; Gowland, Penny; Grabe, Hans J.; Grotegerd, Dominik; Gruber, Oliver; Hagenaars, Saskia; Hashimoto, Ryota; Hauser, Tobias U.; Heinz, Andreas; Hibar, Derrek P.; Hoekstra, Pieter J.; Hoogman, Martine; Howells, Fleur M.; Hu, Hao; Hulshoff Pol, Hilleke E.; Huyser, Chaim; Ittermann, Bernd; Jahanshad, Neda; Jönsson, Erik G.; Jurk, Sarah; Kahn, Rene S.; Kelly, Sinead; Kraemer, Bernd; Kugel, Harald; Kwon, Jun Soo; Lemaitre, Herve; Lesch, Klaus Peter; Lochner, Christine; Luciano, Michelle; Marquand, Andre F.; Martin, Nicholas G.; Martínez-Zalacaín, Ignacio; Martinot, Jean Luc; Mataix-Cols, David; Mather, Karen; McDonald, Colm; McMahon, Katie L.; Medland, Sarah E.; Menchón, José M.; Morris, Derek W.; Mothersill, Omar; Maniega, Susana Munoz; Mwangi, Benson; Nakamae, Takashi; Nakao, Tomohiro; Narayanaswaamy, Janardhanan C.; Nees, Frauke; Nordvik, Jan E.; Onnink, A. Marten H.; Opel, Nils; Ophoff, Roel; Paillère Martinot, Marie Laure; Papadopoulos Orfanos, Dimitri; Pauli, Paul; Paus, Tomáš; Poustka, Luise; Reddy, Janardhan Yc; Renteria, Miguel E.; Roiz-Santiáñez, Roberto; Roos, Annerine; Royle, Natalie A.; Sachdev, Perminder; Sánchez-Juan, Pascual; Schmaal, Lianne; Schumann, Gunter; Shumskaya, Elena; Smolka, Michael N.; Soares, Jair C.; Soriano-Mas, Carles; Stein, Dan J.; Strike, Lachlan T.; Toro, Roberto; Turner, Jessica A.; Tzourio-Mazoyer, Nathalie; Uhlmann, Anne; Hernández, Maria Valdés; van den Heuvel, Odile A.; van der Meer, Dennis; van Haren, Neeltje E.M.; Veltman, Dick J.; Venkatasubramanian, Ganesan; Vetter, Nora C.; Vuletic, Daniella; Walitza, Susanne; Walter, Henrik; Walton, Esther; Wang, Zhen; Wardlaw, Joanna; Wen, Wei; Westlye, Lars T.; Whelan, Robert; Wittfeld, Katharina; Wolfers, Thomas; Wright, Margaret J.; Xu, Jian; Xu, Xiufeng; Yun, Je Yeon; Zhao, Jing Jing; Franke, Barbara; Thompson, Paul M.; Glahn, David C.; Mazoyer, Bernard; Fisher, Simon E.; Francks, Clyde

    2017-01-01

    The two hemispheres of the human brain differ functionally and structurally. Despite over a century of research, the extent to which brain asymmetry is influenced by sex, handedness, age, and genetic factors is still controversial. Here we present the largest ever analysis of subcortical brain

  1. Transcriptome profiling reveals bisphenol A alternatives activate estrogen receptor alpha in human breast cancer cells

    Science.gov (United States)

    Plasticizers with estrogenic activity, such as bisphenol A (BPA), have potential adverse health effects in humans. Due to mounting evidence of these health effects, BPA is being phased out and replaced by other bisphenol variants in “BPA-free” products. We have compared estrogeni...

  2. A high-quality human reference panel reveals the complexity and distribution of genomic structural variants

    NARCIS (Netherlands)

    Hehir-Kwa, J.Y.; Marschall, T.; Kloosterman, W.P.; Francioli, L.C.; Baaijens, J.A.; Dijkstra, L.J.; Abdellaoui, A.; Koval, V.; Thung, D.T.; Wardenaar, R.; Renkens, I.; Coe, B.P.; Deelen, P.; de Ligt, J.; Lameijer, E.W.; Dijk, F.; Hormozdiari, F.; Uitterlinden, A.G.; van Duijn, C.M.; Eichler, E.E.; Bakker, P.I.W.; Swertz, M.A.; Wijmenga, C.; van Ommen, G.J.B; Slagboom, P.E.; Boomsma, D.I.; Schönhuth, A.; Ye, K.; Guryev, V.

    2016-01-01

    Structural variation (SV) represents a major source of differences between individual human genomes and has been linked to disease phenotypes. However, the majority of studies provide neither a global view of the full spectrum of these variants nor integrate them into reference panels of genetic

  3. The bacteriological screening of donated human milk: laboratory experience of British Paediatric Association's published guidelines.

    Science.gov (United States)

    Wright, K C; Feeney, A M

    1998-01-01

    This study was undertaken to assess the application of the British Paediatric Association's (BPA) published guidelines to the bacteriological screening of breast milk donated to a District General Hospital milk bank. Samples of donated milk were subjected to bacterial counts and provisional identification after both 24 and 48 h incubation on cysteine lactose electrolyte-deficient (CLED) and Columbia blood agar. 21.8% (76 out of 348) donations of milk failed to reach the BPA acceptable criteria. The organisms responsible for the rejection of these samples were all evident within 24 h incubation, and were not significantly confined to one medium. A large percentage of rejected samples originated from a small number of donor mothers; 63.2% came from one donor. In applying BPA guidelines, both CLED and Columbia blood agar were found to be equally effective in screening for unacceptable organisms in prepasteurization donated breast milk. The 24 h period allowed for bacteriological screening, prior to pasteurization of milk samples, was sufficient to allow the growth of all potentially pathogenic bacteria in this study. To prevent the donation of consistently contaminated milk, more active communication between the milk bank staff and the donor is recommended.

  4. Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism.

    Science.gov (United States)

    Mohammed, Akram; Guda, Chittibabu

    2015-01-01

    Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids, cofactors and

  5. Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism

    Science.gov (United States)

    2015-01-01

    Background Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. Results ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids

  6. Genome-wide comparative analysis reveals human-mouse regulatory landscape and evolution.

    Science.gov (United States)

    Denas, Olgert; Sandstrom, Richard; Cheng, Yong; Beal, Kathryn; Herrero, Javier; Hardison, Ross C; Taylor, James

    2015-02-14

    Because species-specific gene expression is driven by species-specific regulation, understanding the relationship between sequence and function of the regulatory regions in different species will help elucidate how differences among species arise. Despite active experimental and computational research, relationships among sequence, conservation, and function are still poorly understood. We compared transcription factor occupied segments (TFos) for 116 human and 35 mouse TFs in 546 human and 125 mouse cell types and tissues from the Human and the Mouse ENCODE projects. We based the map between human and mouse TFos on a one-to-one nucleotide cross-species mapper, bnMapper, that utilizes whole genome alignments (WGA). Our analysis shows that TFos are under evolutionary constraint, but a substantial portion (25.1% of mouse and 25.85% of human on average) of the TFos does not have a homologous sequence on the other species; this portion varies among cell types and TFs. Furthermore, 47.67% and 57.01% of the homologous TFos sequence shows binding activity on the other species for human and mouse respectively. However, 79.87% and 69.22% is repurposed such that it binds the same TF in different cells or different TFs in the same cells. Remarkably, within the set of repurposed TFos, the corresponding genome regions in the other species are preferred locations of novel TFos. These events suggest exaptation of some functional regulatory sequences into new function. Despite TFos repurposing, we did not find substantial changes in their predicted target genes, suggesting that CRMs buffer evolutionary events allowing little or no change in the TFos - target gene associations. Thus, the small portion of TFos with strictly conserved occupancy underestimates the degree of conservation of regulatory interactions. We mapped regulatory sequences from an extensive number of TFs and cell types between human and mouse using WGA. A comparative analysis of this correspondence unveiled the

  7. Pattern Analyses Reveal Separate Experience-Based Fear Memories in the Human Right Amygdala.

    Science.gov (United States)

    Braem, Senne; De Houwer, Jan; Demanet, Jelle; Yuen, Kenneth S L; Kalisch, Raffael; Brass, Marcel

    2017-08-23

    Learning fear via the experience of contingencies between a conditioned stimulus (CS) and an aversive unconditioned stimulus (US) is often assumed to be fundamentally different from learning fear via instructions. An open question is whether fear-related brain areas respond differently to experienced CS-US contingencies than to merely instructed CS-US contingencies. Here, we contrasted two experimental conditions where subjects were instructed to expect the same CS-US contingencies while only one condition was characterized by prior experience with the CS-US contingency. Using multivoxel pattern analysis of fMRI data, we found CS-related neural activation patterns in the right amygdala (but not in other fear-related regions) that dissociated between whether a CS-US contingency had been instructed and experienced versus merely instructed. A second experiment further corroborated this finding by showing a category-independent neural response to instructed and experienced, but not merely instructed, CS presentations in the human right amygdala. Together, these findings are in line with previous studies showing that verbal fear instructions have a strong impact on both brain and behavior. However, even in the face of fear instructions, the human right amygdala still shows a separable neural pattern response to experience-based fear contingencies. SIGNIFICANCE STATEMENT In our study, we addressed a fundamental problem of the science of human fear learning and memory, namely whether fear learning via experience in humans relies on a neural pathway that can be separated from fear learning via verbal information. Using two new procedures and recent advances in the analysis of brain imaging data, we localized purely experience-based fear processing and memory in the right amygdala, thereby making a direct link between human and animal research. Copyright © 2017 the authors 0270-6474/17/378116-15$15.00/0.

  8. Future perspective of induced pluripotent stem cells for diagnosis, drug screening and treatment of human diseases.

    Science.gov (United States)

    Lian, Qizhou; Chow, Yenyen; Esteban, Miguel Angel; Pei, Duanqing; Tse, Hung-Fat

    2010-07-01

    Recent advances in stem cell biology have transformed the understanding of cell physiology and developmental biology such that it can now play a more prominent role in the clinical application of stem cell and regenerative medicine. Success in the generation of human induced pluripotent stem cells (iPS) as well as related emerging technology on the iPS platform provide great promise in the development of regenerative medicine. Human iPS cells show almost identical properties to human embryonic stem cells (ESC) in pluripotency, but avoid many of their limitations of use. In addition, investigations into reprogramming of somatic cells to pluripotent stem cells facilitate a deeper understanding of human stem cell biology. The iPS cell technology has offered a unique platform for studying the pathogenesis of human disease, pharmacological and toxicological testing, and cell-based therapy. Nevertheless, significant challenges remain to be overcome before the promise of human iPS cell technology can be realised.

  9. Multistructure index in revealing complexity of regulatory mechanisms of human cardiovascular system at rest and orthostatic stress in healthy humans

    Science.gov (United States)

    Makowiec, Danuta; Graff, Beata; Struzik, Zbigniew R.

    2017-02-01

    Biological regulation is sufficiently complex to pose an enduring challenge for characterization of both its equilibrium and transient non-equilibrium dynamics. Two univariate but coupled observables, heart rate and systolic blood pressure, are commonly characterized in the benchmark example of the human cardiovascular regulatory system. Asymmetric distributions of accelerations and decelerations of heart rate, as well as rises and falls in systolic blood pressure, recorded in humans during a head-up tilt test provide insights into the dynamics of cardiovascular response to a rapid, controlled deregulation of the system's homeostasis. The baroreflex feedback loop is assumed to be the fundamental physiological mechanism for ensuring homeostatic blood supply to distant organs at rest and during orthostatic stress, captured in a classical beat-to-beat autoregressive model of baroreflex by de Boer et al. (1987). For model corroboration, a multistructure index statistic is proposed, seamlessly evaluating the size spectrum of magnitudes of neural reflexes such as baroreflex, responsible for maintaining the homeostatic dynamics. The multistructure index exposes a distinctly different dynamics of multiscale asymmetry between results obtained from real-life signals recorded from healthy subjects and those simulated using both the classical and perturbed versions of the model. Nonlinear effects observed suggest the pronounced presence of complex mechanisms resulting from baroreflex regulation when a human is at rest, which is aggravated in the system's response to orthostatic stress. Using our methodology of multistructure index, we therefore show a marked difference between model and real-life scenarios, which we attribute to multiscale asymmetry of non-linear origin in real-life signals, which we are not reproducible by the classical model.

  10. Screening for exogenous androgen anabolic steroids in human hair by liquid chromatography/orbitrap-high resolution mass spectrometry

    International Nuclear Information System (INIS)

    Strano-Rossi, Sabina; Castrignanò, Erika; Anzillotti, Luca; Odoardi, Sara; De-Giorgio, Fabio; Bermejo, Ana; Pascali, Vincenzo L.

    2013-01-01

    Graphical abstract: -- Highlights: •LC–HRMS screening method for the detection of a variety of anabolics in hair. •Detection of unmetabolized anabolic steroids and their esters in hair matrix by simple keratin pretreatment. •Identification of target compounds by retention time, accurate mass and isotopic cluster. •Quantitative determination of detected compounds. •Possibility to a retrospective re-analysis of the acquired datafile in case a further analyte is to be screened. -- Abstract: A method for the screening of various anabolic steroids and their esters in human hair, based on liquid-chromatography–high resolution mass spectrometry using an Exactive benchtop Orbitrap mass spectrometer, has been set up and validated. This method involved methanolic incubation of 30 mg of hair and analysis of the relevant extract in HPLC using a C18 column. The mass detector, with nominal resolving power of 100,000, operated in full scan mode in APCI under positive ionization mode. Analytes were identified by exact mass, correspondence of isotopic cluster and retention times. The limits of detection obtained varied from 10 to 50 pg mg −1 , and limits of quantitation were 0.5 ng mg −1 for all compounds. The method was linear for all analytes in the ranges from the LOQ to 6 ng mg −1 , giving correlation coefficients >0.99 for all analytes. Also accuracy (intended as %E) and repeatability (%CV) were always lower than 15%. Specificity was assessed by analysing ten blank samples and fifteen samples from polidrug abusers. This method was applied to a real-life case, resulting in the identification of testosterone undecanoate in the hair of a suspect. The analyte identity was confirmed by the analysis of its in-source fragmentation and comparison to a certified standard. Thanks to the scan acquisition, this method also enables retrospective re-analysis of the acquired datafile in case a further analyte needs to be screened

  11. Prospective assessment of early fetal loss using an immunoenzymometric screening assay for detection of urinary human chorionic gonadotropin.

    Science.gov (United States)

    Taylor, C A; Overstreet, J W; Samuels, S J; Boyers, S P; Canfield, R E; O'Connor, J F; Hanson, F W; Lasley, B L

    1992-06-01

    To develop an economical, nonradiometric immunoenzymometric assay (IEMA) for the detection of urinary human chorionic gonadotropin (hCG) in studies of early fetal loss. To be effective, the IEMA must have a sensitivity equal to the standard immunoradiometric assay (IRMA) and sufficient specificity to eliminate the need for screening most nonconceptive cycles with the expensive and labor-intensive IRMA. Two different assays were used to measure hCG in daily early morning urine samples from potential conceptive cycles. Women undergoing donor artificial insemination (AI) were evaluated in a prospective study. Ninety-two women volunteers were selected on the basis of apparent normal reproductive health. Artificial insemination with nonfrozen donor semen was performed by cervical cup twice each menstrual cycle at 48-hour intervals, and daily urine samples were self-collected throughout the menstrual cycle. An IEMA was developed to detect urinary hCG using the same antibodies as in the standard IRMA; a study was designed to determine whether this nonradiometric assay could successfully detect the early fetal loss that was detected by the IRMA. Of 224 menstrual cycles analyzed by both assays, a total of six early fetal losses were detected by the IRMA. When the tentative screening rule was set to allow all six of these losses and 95% of future losses to be detected by the IEMA, an additional 34 false-positive results were detected by the IEMA. The specificity of the IEMA with this rule was calculated to be 84%. An IEMA based on the same antibodies used for the standard IRMA can serve as an efficient screening assay for the detection of early fetal loss. When the IEMA is used in this manner, nearly 80% of screened menstrual cycles can be eliminated without further testing by the IRMA.

  12. High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells.

    Science.gov (United States)

    Karmaus, Agnes L; Toole, Colleen M; Filer, Dayne L; Lewis, Kenneth C; Martin, Matthew T

    2016-04-01

    Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology.

  13. Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening

    Directory of Open Access Journals (Sweden)

    Lin SH

    2015-06-01

    Full Text Available Shih-Hung Lin,1 Kao-Jean Huang,1,2 Ching-Feng Weng,1 David Shiuan1 1Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, Republic of China; 2Development Center of Biotechnology, Taipei, Taiwan, Republic of China Abstract: Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR. The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening. Keywords: HMG-CoA reductase, virtual screening, curcumin, salvianolic acid C

  14. Screening for exogenous androgen anabolic steroids in human hair by liquid chromatography/orbitrap-high resolution mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Strano-Rossi, Sabina, E-mail: sabina.stranorossi@rm.unicatt.it [Institute of Legal Medicine, Catholic University of Sacred Heart, L.go F. Vito, 1, 00168 Rome (Italy); Castrignanò, Erika; Anzillotti, Luca; Odoardi, Sara; De-Giorgio, Fabio [Institute of Legal Medicine, Catholic University of Sacred Heart, L.go F. Vito, 1, 00168 Rome (Italy); Bermejo, Ana [Institute of Legal Medicine, University of Santiago de Compostela, Av S. Francisco s/n, Santiago de Compostela (Spain); Pascali, Vincenzo L. [Institute of Legal Medicine, Catholic University of Sacred Heart, L.go F. Vito, 1, 00168 Rome (Italy)

    2013-09-02

    Graphical abstract: -- Highlights: •LC–HRMS screening method for the detection of a variety of anabolics in hair. •Detection of unmetabolized anabolic steroids and their esters in hair matrix by simple keratin pretreatment. •Identification of target compounds by retention time, accurate mass and isotopic cluster. •Quantitative determination of detected compounds. •Possibility to a retrospective re-analysis of the acquired datafile in case a further analyte is to be screened. -- Abstract: A method for the screening of various anabolic steroids and their esters in human hair, based on liquid-chromatography–high resolution mass spectrometry using an Exactive benchtop Orbitrap mass spectrometer, has been set up and validated. This method involved methanolic incubation of 30 mg of hair and analysis of the relevant extract in HPLC using a C18 column. The mass detector, with nominal resolving power of 100,000, operated in full scan mode in APCI under positive ionization mode. Analytes were identified by exact mass, correspondence of isotopic cluster and retention times. The limits of detection obtained varied from 10 to 50 pg mg{sup −1}, and limits of quantitation were 0.5 ng mg{sup −1} for all compounds. The method was linear for all analytes in the ranges from the LOQ to 6 ng mg{sup −1}, giving correlation coefficients >0.99 for all analytes. Also accuracy (intended as %E) and repeatability (%CV) were always lower than 15%. Specificity was assessed by analysing ten blank samples and fifteen samples from polidrug abusers. This method was applied to a real-life case, resulting in the identification of testosterone undecanoate in the hair of a suspect. The analyte identity was confirmed by the analysis of its in-source fragmentation and comparison to a certified standard. Thanks to the scan acquisition, this method also enables retrospective re-analysis of the acquired datafile in case a further analyte needs to be screened.

  15. Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma.

    Directory of Open Access Journals (Sweden)

    Ting-Lei Gu

    Full Text Available Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23 of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.

  16. Human figure drawing distinguishes Alzheimer's patients: a cognitive screening test study.

    Science.gov (United States)

    Stanzani Maserati, Michelangelo; D'Onofrio, Renato; Matacena, Corrado; Sambati, Luisa; Oppi, Federico; Poda, Roberto; De Matteis, Maddalena; Naldi, Ilaria; Liguori, Rocco; Capellari, Sabina

    2018-05-01

    To study human figure drawing in a group of Alzheimer's disease (AD) patients and compare it with a group of patients with mild cognitive impairment (MCI) and controls. We evaluated consecutive outpatients over a one-year period. Patients were classified as affected by AD or by MCI. All patients and controls underwent a simplified version of the human-figure drawing test and MMSE. A qualitative and quantitative analysis of all human figures was obtained. 112 AD, 100 MCI patients and 104 controls were enrolled. AD patients drew human figures poor in details and globally smaller than MCI patients and controls. Human figures drawn by MCI patients are intermediate in body height between those of the AD patients and the healthy subjects. The head-to-body ratio of human figures drawn by AD patients is greater than controls and MCI patients, while the human figure size-relative-to-page space index is significantly smaller. Body height is an independent predictor of cognitive impairment correlating with its severity and with the number of the figure's details. Human figures drawn by AD patients are different from those drawn by healthy subjects and MCI patients. Human figure drawing test is a useful tool for orienting cognitive impairment's diagnosis.

  17. The L1TD1 Protein Interactome Reveals the Importance of Post-transcriptional Regulation in Human Pluripotency

    Directory of Open Access Journals (Sweden)

    Maheswara Reddy Emani

    2015-03-01

    Full Text Available The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.

  18. Genome sequencing of chimpanzee malaria parasites reveals possible pathways of adaptation to human hosts

    KAUST Repository

    Otto, Thomas D.

    2014-09-09

    Plasmodium falciparum causes most human malaria deaths, having prehistorically evolved from parasites of African Great Apes. Here we explore the genomic basis of P. falciparum adaptation to human hosts by fully sequencing the genome of the closely related chimpanzee parasite species P. reichenowi, and obtaining partial sequence data from a more distantly related chimpanzee parasite (P. gaboni). The close relationship between P. reichenowi and P. falciparum is emphasized by almost complete conservation of genomic synteny, but against this strikingly conserved background we observe major differences at loci involved in erythrocyte invasion. The organization of most virulence-associated multigene families, including the hypervariable var genes, is broadly conserved, but P. falciparum has a smaller subset of rif and stevor genes whose products are expressed on the infected erythrocyte surface. Genome-wide analysis identifies other loci under recent positive selection, but a limited number of changes at the host–parasite interface may have mediated host switching.

  19. The human auditory brainstem response to running speech reveals a subcortical mechanism for selective attention.

    Science.gov (United States)

    Forte, Antonio Elia; Etard, Octave; Reichenbach, Tobias

    2017-10-10

    Humans excel at selectively listening to a target speaker in background noise such as competing voices. While the encoding of speech in the auditory cortex is modulated by selective attention, it remains debated whether such modulation occurs already in subcortical auditory structures. Investigating the contribution of the human brainstem to attention has, in particular, been hindered by the tiny amplitude of the brainstem response. Its measurement normally requires a large number of repetitions of the same short sound stimuli, which may lead to a loss of attention and to neural adaptation. Here we develop a mathematical method to measure the auditory brainstem response to running speech, an acoustic stimulus that does not repeat and that has a high ecological validity. We employ this method to assess the brainstem's activity when a subject listens to one of two competing speakers, and show that the brainstem response is consistently modulated by attention.

  20. Parallel or convergent evolution in human population genomic data revealed by genotype networks

    OpenAIRE

    Vahdati, Ali R; Wagner, Andreas

    2016-01-01

    Background Genotype networks are representations of genetic variation data that are complementary to phylogenetic trees. A genotype network is a graph whose nodes are genotypes (DNA sequences) with the same broadly defined phenotype. Two nodes are connected if they differ in some minimal way, e.g., in a single nucleotide. Results We analyze human genome variation data from the 1,000 genomes project, and construct haploid genotype (haplotype) networks for 12,235 protein coding genes. The struc...

  1. Genome-wide and paternal diversity reveal a recent origin of human populations in North Africa

    OpenAIRE

    Fadhlaoui-Zid, Karima; Haber, Marc, 1980-; Martínez Cruz, Begoña; Zalloua, Pierre A; Elgaaied, Amel Benammar; Comas, David, 1969-

    2013-01-01

    The geostrategic location of North Africa as a crossroad between three continents and as a stepping-stone outside Africa has evoked anthropological and genetic interest in this region. Numerous studies have described the genetic landscape of the human population in North Africa employing paternal, maternal, and biparental molecular markers. However, information from these markers which have different inheritance patterns has been mostly assessed independently, resulting in an incomplete descr...

  2. Human fMRI Reveals That Delayed Action Re-Recruits Visual Perception

    OpenAIRE

    Singhal, Anthony; Monaco, Simona; Kaufman, Liam D.; Culham, Jody C.

    2013-01-01

    Behavioral and neuropsychological research suggests that delayed actions rely on different neural substrates than immediate actions; however, the specific brain areas implicated in the two types of actions remain unknown. We used functional magnetic resonance imaging (fMRI) to measure human brain activation during delayed grasping and reaching. Specifically, we examined activation during visual stimulation and action execution separated by a 18-s delay interval in which subjects had to rememb...

  3. The genome of a Mongolian individual reveals the genetic imprints of Mongolians on modern human populations.

    Science.gov (United States)

    Bai, Haihua; Guo, Xiaosen; Zhang, Dong; Narisu, Narisu; Bu, Junjie; Jirimutu, Jirimutu; Liang, Fan; Zhao, Xiang; Xing, Yanping; Wang, Dingzhu; Li, Tongda; Zhang, Yanru; Guan, Baozhu; Yang, Xukui; Yang, Zili; Shuangshan, Shuangshan; Su, Zhe; Wu, Huiguang; Li, Wenjing; Chen, Ming; Zhu, Shilin; Bayinnamula, Bayinnamula; Chang, Yuqi; Gao, Ying; Lan, Tianming; Suyalatu, Suyalatu; Huang, Hui; Su, Yan; Chen, Yujie; Li, Wenqi; Yang, Xu; Feng, Qiang; Wang, Jian; Yang, Huanming; Wang, Jun; Wu, Qizhu; Yin, Ye; Zhou, Huanmin

    2014-11-05

    Mongolians have played a significant role in modern human evolution, especially after the rise of Genghis Khan (1162[?]-1227). Although the social cultural impacts of Genghis Khan and the Mongolian population have been well documented, explorations of their genome structure and genetic imprints on other human populations have been lacking. We here present the genome of a Mongolian male individual. The genome was de novo assembled using a total of 130.8-fold genomic data produced from massively parallel whole-genome sequencing. We identified high-confidence variation sets, including 3.7 million single nucleotide polymorphisms (SNPs) and 756,234 short insertions and deletions. Functional SNP analysis predicted that the individual has a pathogenic risk for carnitine deficiency. We located the patrilineal inheritance of the Mongolian genome to the lineage D3a through Y haplogroup analysis and inferred that the individual has a common patrilineal ancestor with Tibeto-Burman populations and is likely to be the progeny of the earliest settlers in East Asia. We finally investigated the genetic imprints of Mongolians on other human populations using different approaches. We found varying degrees of gene flows between Mongolians and populations living in Europe, South/Central Asia, and the Indian subcontinent. The analyses demonstrate that the genetic impacts of Mongolians likely resulted from the expansion of the Mongolian Empire in the 13th century. The genome will be of great help in further explorations of modern human evolution and genetic causes of diseases/traits specific to Mongolians. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  4. Deep RNA sequencing reveals dynamic regulation of myocardial noncoding RNAs in failing human heart and remodeling with mechanical circulatory support.

    Science.gov (United States)

    Yang, Kai-Chien; Yamada, Kathryn A; Patel, Akshar Y; Topkara, Veli K; George, Isaac; Cheema, Faisal H; Ewald, Gregory A; Mann, Douglas L; Nerbonne, Jeanne M

    2014-03-04

    Microarrays have been used extensively to profile transcriptome remodeling in failing human heart, although the genomic coverage provided is limited and fails to provide a detailed picture of the myocardial transcriptome landscape. Here, we describe sequencing-based transcriptome profiling, providing comprehensive analysis of myocardial mRNA, microRNA (miRNA), and long noncoding RNA (lncRNA) expression in failing human heart before and after mechanical support with a left ventricular (LV) assist device (LVAD). Deep sequencing of RNA isolated from paired nonischemic (NICM; n=8) and ischemic (ICM; n=8) human failing LV samples collected before and after LVAD and from nonfailing human LV (n=8) was conducted. These analyses revealed high abundance of mRNA (37%) and lncRNA (71%) of mitochondrial origin. miRNASeq revealed 160 and 147 differentially expressed miRNAs in ICM and NICM, respectively, compared with nonfailing LV. Among these, only 2 (ICM) and 5 (NICM) miRNAs are normalized with LVAD. RNASeq detected 18 480, including 113 novel, lncRNAs in human LV. Among the 679 (ICM) and 570 (NICM) lncRNAs differentially expressed with heart failure, ≈10% are improved or normalized with LVAD. In addition, the expression signature of lncRNAs, but not miRNAs or mRNAs, distinguishes ICM from NICM. Further analysis suggests that cis-gene regulation represents a major mechanism of action of human cardiac lncRNAs. The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

  5. Comparative genome analysis of Megasphaera sp. reveals niche specialization and its potential role in the human gut.

    Directory of Open Access Journals (Sweden)

    Sudarshan Anand Shetty

    Full Text Available With increasing number of novel bacteria being isolated from the human gut ecosystem, there is a greater need to study their role in the gut ecosystem and their effect on the host health. In the present study, we carried out in silico genome-wide analysis of two novel Megasphaera sp. isolates NM10 (DSM25563 and BL7 (DSM25562, isolated from feces of two healthy individuals and validated the key features by in vitro studies. The analysis revealed the general metabolic potential, adaptive features and the potential effects of these isolates on the host. The comparative genome analysis of the two human gut isolates NM10 and BL7 with ruminal isolate Megasphaera elsdenii (DSM20460 highlighted the differential adaptive features for their survival in human gut. The key findings include features like bile resistance, presence of various sensory and regulatory systems, stress response systems, membrane transporters and resistance to antibiotics. Comparison of the "glycobiome" based on the genomes of the ruminal isolate with the human gut isolates NM10 and BL revealed the presence of diverse and unique sets of Carbohydrate-Active enzymes (CAZymes amongst these isolates, with a higher collection of CAZymes in the human gut isolates. This could be attributed to the difference in host diet and thereby the environment, consequently suggesting host specific adaptation in these isolates. In silico analysis of metabolic potential predicted the ability of these isolates to produce important metabolites like short chain fatty acids (butyrate, acetate, formate, and caproate, vitamins and essential amino acids, which was further validated by in vitro experiments. The ability of these isolates to produce important metabolites advocates for a potential healthy influence on the host. Further in vivo studies including transcriptomic and proteomic analysis will be required for better understanding the role and impact of these Megasphaera sp. isolates NM10 and BL7 on the

  6. New patterns in human biogeography revealed by networks of contacts between linguistic groups.

    Science.gov (United States)

    Capitán, José A; Bock Axelsen, Jacob; Manrubia, Susanna

    2015-03-07

    Human languages differ broadly in abundance and are distributed highly unevenly on the Earth. In many qualitative and quantitative aspects, they strongly resemble biodiversity distributions. An intriguing and previously unexplored issue is the architecture of the neighbouring relationships between human linguistic groups. Here we construct and characterize these networks of contacts and show that they represent a new kind of spatial network with uncommon structural properties. Remarkably, language networks share a meaningful property with food webs: both are quasi-interval graphs. In food webs, intervality is linked to the existence of a niche space of low dimensionality; in language networks, we show that the unique relevant variable is the area occupied by the speakers of a language. By means of a range model analogous to niche models in ecology, we show that a geometric restriction of perimeter covering by neighbouring linguistic domains explains the structural patterns observed. Our findings may be of interest in the development of models for language dynamics or regarding the propagation of cultural innovations. In relation to species distribution, they pose the question of whether the spatial features of species ranges share architecture, and eventually generating mechanism, with the distribution of human linguistic groups. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  7. Human Brain Organoids on a Chip Reveal the Physics of Folding.

    Science.gov (United States)

    Karzbrun, Eyal; Kshirsagar, Aditya; Cohen, Sidney R; Hanna, Jacob H; Reiner, Orly

    2018-05-01

    Human brain wrinkling has been implicated in neurodevelopmental disorders and yet its origins remain unknown. Polymer gel models suggest that wrinkling emerges spontaneously due to compression forces arising during differential swelling, but these ideas have not been tested in a living system. Here, we report the appearance of surface wrinkles during the in vitro development and self-organization of human brain organoids in a micro-fabricated compartment that supports in situ imaging over a timescale of weeks. We observe the emergence of convolutions at a critical cell density and maximal nuclear strain, which are indicative of a mechanical instability. We identify two opposing forces contributing to differential growth: cytoskeletal contraction at the organoid core and cell-cycle-dependent nuclear expansion at the organoid perimeter. The wrinkling wavelength exhibits linear scaling with tissue thickness, consistent with balanced bending and stretching energies. Lissencephalic (smooth brain) organoids display reduced convolutions, modified scaling and a reduced elastic modulus. Although the mechanism here does not include the neuronal migration seen in in vivo , it models the physics of the folding brain remarkably well. Our on-chip approach offers a means for studying the emergent properties of organoid development, with implications for the embryonic human brain.

  8. Single-Cell Analysis of Human Pancreas Reveals Transcriptional Signatures of Aging and Somatic Mutation Patterns.

    Science.gov (United States)

    Enge, Martin; Arda, H Efsun; Mignardi, Marco; Beausang, John; Bottino, Rita; Kim, Seung K; Quake, Stephen R

    2017-10-05

    As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Boolean analysis reveals systematic interactions among low-abundance species in the human gut microbiome.

    Directory of Open Access Journals (Sweden)

    Jens Christian Claussen

    2017-06-01

    Full Text Available The analysis of microbiome compositions in the human gut has gained increasing interest due to the broader availability of data and functional databases and substantial progress in data analysis methods, but also due to the high relevance of the microbiome in human health and disease. While most analyses infer interactions among highly abundant species, the large number of low-abundance species has received less attention. Here we present a novel analysis method based on Boolean operations applied to microbial co-occurrence patterns. We calibrate our approach with simulated data based on a dynamical Boolean network model from which we interpret the statistics of attractor states as a theoretical proxy for microbiome composition. We show that for given fractions of synergistic and competitive interactions in the model our Boolean abundance analysis can reliably detect these interactions. Analyzing a novel data set of 822 microbiome compositions of the human gut, we find a large number of highly significant synergistic interactions among these low-abundance species, forming a connected network, and a few isolated competitive interactions.

  10. Dynamics of scene representations in the human brain revealed by magnetoencephalography and deep neural networks

    Science.gov (United States)

    Cichy, Radoslaw Martin; Khosla, Aditya; Pantazis, Dimitrios; Oliva, Aude

    2017-01-01

    Human scene recognition is a rapid multistep process evolving over time from single scene image to spatial layout processing. We used multivariate pattern analyses on magnetoencephalography (MEG) data to unravel the time course of this cortical process. Following an early signal for lower-level visual analysis of single scenes at ~100 ms, we found a marker of real-world scene size, i.e. spatial layout processing, at ~250 ms indexing neural representations robust to changes in unrelated scene properties and viewing conditions. For a quantitative model of how scene size representations may arise in the brain, we compared MEG data to a deep neural network model trained on scene classification. Representations of scene size emerged intrinsically in the model, and resolved emerging neural scene size representation. Together our data provide a first description of an electrophysiological signal for layout processing in humans, and suggest that deep neural networks are a promising framework to investigate how spatial layout representations emerge in the human brain. PMID:27039703

  11. Raman spectroscopy imaging reveals interplay between atherosclerosis and medial calcification in the human aorta

    Science.gov (United States)

    You, Amanda Y. F.; Bergholt, Mads S.; St-Pierre, Jean-Philippe; Kit-Anan, Worrapong; Pence, Isaac J.; Chester, Adrian H.; Yacoub, Magdi H.; Bertazzo, Sergio; Stevens, Molly M.

    2017-01-01

    Medial calcification in the human aorta accumulates during aging and is known to be aggravated in several diseases. Atherosclerosis, another major cause of cardiovascular calcification, shares some common aggravators. However, the mechanisms of cardiovascular calcification remain poorly understood. To elucidate the relationship between medial aortic calcification and atherosclerosis, we characterized the cross-sectional distributions of the predominant minerals in aortic tissue, apatite and whitlockite, and the associated extracellular matrix. We also compared the cellular changes between atherosclerotic and nonatherosclerotic human aortic tissues. This was achieved through the development of Raman spectroscopy imaging methods that adapted algorithms to distinguish between the major biomolecules present within these tissues. We present a relationship between apatite, cholesterol, and triglyceride in atherosclerosis, with the relative amount of all molecules concurrently increased in the atherosclerotic plaque. Further, the increase in apatite was disproportionately large in relation to whitlockite in the aortic media directly underlying a plaque, indicating that apatite is more pathologically significant in atherosclerosis-aggravated medial calcification. We also discovered a reduction of β-carotene in the whole aortic intima, including a plaque in atherosclerotic aortic tissues compared to nonatherosclerotic tissues. This unprecedented biomolecular characterization of the aortic tissue furthers our understanding of pathological and physiological cardiovascular calcification events in humans. PMID:29226241

  12. Human brain organoids on a chip reveal the physics of folding

    Science.gov (United States)

    Karzbrun, Eyal; Kshirsagar, Aditya; Cohen, Sidney R.; Hanna, Jacob H.; Reiner, Orly

    2018-05-01

    Human brain wrinkling has been implicated in neurodevelopmental disorders and yet its origins remain unknown. Polymer gel models suggest that wrinkling emerges spontaneously due to compression forces arising during differential swelling, but these ideas have not been tested in a living system. Here, we report the appearance of surface wrinkles during the in vitro development and self-organization of human brain organoids in a microfabricated compartment that supports in situ imaging over a timescale of weeks. We observe the emergence of convolutions at a critical cell density and maximal nuclear strain, which are indicative of a mechanical instability. We identify two opposing forces contributing to differential growth: cytoskeletal contraction at the organoid core and cell-cycle-dependent nuclear expansion at the organoid perimeter. The wrinkling wavelength exhibits linear scaling with tissue thickness, consistent with balanced bending and stretching energies. Lissencephalic (smooth brain) organoids display reduced convolutions, modified scaling and a reduced elastic modulus. Although the mechanism here does not include the neuronal migration seen in vivo, it models the physics of the folding brain remarkably well. Our on-chip approach offers a means for studying the emergent properties of organoid development, with implications for the embryonic human brain.

  13. Neural prediction errors reveal a risk-sensitive reinforcement-learning process in the human brain.

    Science.gov (United States)

    Niv, Yael; Edlund, Jeffrey A; Dayan, Peter; O'Doherty, John P

    2012-01-11

    Humans and animals are exquisitely, though idiosyncratically, sensitive to risk or variance in the outcomes of their actions. Economic, psychological, and neural aspects of this are well studied when information about risk is provided explicitly. However, we must normally learn about outcomes from experience, through trial and error. Traditional models of such reinforcement learning focus on learning about the mean reward value of cues and ignore higher order moments such as variance. We used fMRI to test whether the neural correlates of human reinforcement learning are sensitive to experienced risk. Our analysis focused on anatomically delineated regions of a priori interest in the nucleus accumbens, where blood oxygenation level-dependent (BOLD) signals have been suggested as correlating with quantities derived from reinforcement learning. We first provide unbiased evidence that the raw BOLD signal in these regions corresponds closely to a reward prediction error. We then derive from this signal the learned values of cues that predict rewards of equal mean but different variance and show that these values are indeed modulated by experienced risk. Moreover, a close neurometric-psychometric coupling exists between the fluctuations of the experience-based evaluations of risky options that we measured neurally and the fluctuations in behavioral risk aversion. This suggests that risk sensitivity is integral to human learning, illuminating economic models of choice, neuroscientific models of affective learning, and the workings of the underlying neural mechanisms.

  14. Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cell response to extracellular matrix

    International Nuclear Information System (INIS)

    Ma, Yufei; Ji, Yuan; Huang, Guoyou; Zhang, Xiaohui; Xu, Feng; Ling, Kai

    2015-01-01

    Periodontitis is an inflammatory disease negatively affecting up to 15% of adults worldwide. Periodontal ligament stem cells (PDLSCs) hold great promises for periodontal tissue regeneration, where it is necessary to find proper extracellular matrix (ECM) materials (e.g., composition, concentration). In this study, we proposed a bioprinting-based approach to generate nano-liter sized three-dimensional (3D) cell-laden hydrogel array with gradient of ECM components, through controlling the volume ratio of two hydrogels, such as gelatin methacrylate (GelMA) and poly(ethylene glycol) (PEG) dimethacrylate. The resulting cell-laden array with a gradient of GelMA/PEG composition was used to screen human PDLSC response to ECM. The behavior (e.g., cell viability, spreading) of human PDLSCs in GelMA/PEG array were found to be depended on the volume ratios of GelMA/PEG, with cell viability and spreading area decreased along with increasing the ratio of PEG. The developed approach would be useful for screening cell-biomaterial interaction in 3D and promoting regeneration of functional tissue. (paper)

  15. Genomic analysis of influenza A virus from captive wild boars in Brazil reveals a human-like H1N2 influenza virus.

    Science.gov (United States)

    Biondo, Natalha; Schaefer, Rejane; Gava, Danielle; Cantão, Mauricio E; Silveira, Simone; Mores, Marcos A Z; Ciacci-Zanella, Janice R; Barcellos, David E S N

    2014-01-10

    Influenza is a viral disease that affects human and several animal species. In Brazil, H1N1, H3N2 and 2009 pandemic H1N1 A(H1N1)pdm09 influenza A viruses (IAV) circulate in domestic swine herds. Wild boars are also susceptible to IAV infection but in Brazil until this moment there are no reports of IAV infection in wild boars or in captive wild boars populations. Herein the occurrence of IAV in captive wild boars with the presence of lung consolidation lesions during slaughter was investigated. Lung samples were screened by RT-PCR for IAV detection. IAV positive samples were further analyzed by quantitative real-time PCR (qRRT-PCR), virus isolation, genomic sequencing, histopathology and immunohistochemistry (IHC). Eleven out of 60 lungs (18.3%) were positive for IAV by RT-PCR and seven out of the eleven were also positive for A(H1N1)pdm09 by qRRT-PCR. Chronic diffuse bronchopneumonia was observed in all samples and IHC analysis was negative for influenza A antigen. Full genes segments of H1N2 IAV were sequenced using Illumina's genome analyzer platform (MiSeq). The genomic analysis revealed that the HA and NA genes clustered with IAVs of the human lineage and the six internal genes were derived from the H1N1pdm09 IAV. This is the first report of a reassortant human-like H1N2 influenza virus infection in captive wild boars in Brazil and indicates the need to monitor IAV evolution in Suidae populations. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Proteomics reveals the effects of sustained weight loss on the human plasma proteome

    DEFF Research Database (Denmark)

    Geyer, Philipp E; Wewer Albrechtsen, Nicolai J; Tyanova, Stefka

    2016-01-01

    Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed...... by a year of weight maintenance. Using mass spectrometry-based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual-specific protein levels with wide-ranging effects of losing weight on the plasma proteome reflected in 93 significantly...

  17. Pre-screening method for somatic cell contamination in human sperm epigenetic studies.

    Science.gov (United States)

    Jenkins, Timothy G; Liu, Lihua; Aston, Kenneth I; Carrell, Douglas T

    2018-04-01

    Sperm epigenetic profiles are frequently studied and are of great interest in many fields. One major technical concern when assessing these marks is the potential for somatic cell contamination. Because somatic cells have dramatically different epigenetic signatures, even small levels of contamination can result in significant problems in analysis and interpretation of data. In this study we evaluate an assay, which we designed to offer a reliable 'pre-screen' for somatic cell contamination that directly assesses the DNA being used in the study to determine tissue purity. In brief, we designed an inexpensive and simple assay that utilizes the strong differential methylation between sperm and somatic cells at four genomic loci to assess the general purity of samples prior to performing expensive and time intensive assays. The assay is able to reliably detect contamination qualitatively by running the sample on an agarose gel, or quantitatively with the use of a bioanalyzer. With this technique we have found that we can detect potentially contaminating signals in samples of many different types, including those from patients with poor sperm phenotypes (oligozoospermia, asthenozoospermia, and teratozoospermia). We also have found that the use of multiple sites to determine potential contamination is key, as some conditions (asthenozoospermia specifically) appear at one site to reflect a somatic-like profile, while at all other sites it appears to have very typical sperm DNA methylation signatures. Taken together, the use of the assay described herein was effective at identifying contamination and could be implemented in many labs to quickly and inexpensively pre-screen samples prior to performing far more expensive and labor intensive procedures. Additionally, the principles applied to the development of this assay could be easily adapted for the development of other assays to pre-screen different tissue/cell types or model organisms.

  18. Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human.

    Science.gov (United States)

    Zanotto-Filho, Alfeu; Dashnamoorthy, Ravi; Loranc, Eva; de Souza, Luis H T; Moreira, José C F; Suresh, Uthra; Chen, Yidong; Bishop, Alexander J R

    2016-01-01

    Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair), DNA-mRNA-protein metabolism (transcription/translation) and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.

  19. Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human.

    Directory of Open Access Journals (Sweden)

    Alfeu Zanotto-Filho

    Full Text Available Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair, DNA-mRNA-protein metabolism (transcription/translation and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress/Unfolded Protein Responses (UPR in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.

  20. Large scale aggregate microarray analysis reveals three distinct molecular subclasses of human preeclampsia.

    Science.gov (United States)

    Leavey, Katherine; Bainbridge, Shannon A; Cox, Brian J

    2015-01-01

    Preeclampsia (PE) is a life-threatening hypertensive pathology of pregnancy affecting 3-5% of all pregnancies. To date, PE has no cure, early detection markers, or effective treatments short of the removal of what is thought to be the causative organ, the placenta, which may necessitate a preterm delivery. Additionally, numerous small placental microarray studies attempting to identify "PE-specific" genes have yielded inconsistent results. We therefore hypothesize that preeclampsia is a multifactorial disease encompassing several pathology subclasses, and that large cohort placental gene expression analysis will reveal these groups. To address our hypothesis, we utilized known bioinformatic methods to aggregate 7 microarray data sets across multiple platforms in order to generate a large data set of 173 patient samples, including 77 with preeclampsia. Unsupervised clustering of these patient samples revealed three distinct molecular subclasses of PE. This included a "canonical" PE subclass demonstrating elevated expression of known PE markers and genes associated with poor oxygenation and increased secretion, as well as two other subclasses potentially representing a poor maternal response to pregnancy and an immunological presentation of preeclampsia. Our analysis sheds new light on the heterogeneity of PE patients, and offers up additional avenues for future investigation. Hopefully, our subclassification of preeclampsia based on molecular diversity will finally lead to the development of robust diagnostics and patient-based treatments for this disorder.

  1. Sensitivity of human auditory cortex to rapid frequency modulation revealed by multivariate representational similarity analysis.

    Science.gov (United States)

    Joanisse, Marc F; DeSouza, Diedre D

    2014-01-01

    Functional Magnetic Resonance Imaging (fMRI) was used to investigate the extent, magnitude, and pattern of brain activity in response to rapid frequency-modulated sounds. We examined this by manipulating the direction (rise vs. fall) and the rate (fast vs. slow) of the apparent pitch of iterated rippled noise (IRN) bursts. Acoustic parameters were selected to capture features used in phoneme contrasts, however the stimuli themselves were not perceived as speech per se. Participants were scanned as they passively listened to sounds in an event-related paradigm. Univariate analyses revealed a greater level and extent of activation in bilateral auditory cortex in response to frequency-modulated sweeps compared to steady-state sounds. This effect was stronger in the left hemisphere. However, no regions showed selectivity for either rate or direction of frequency modulation. In contrast, multivoxel pattern analysis (MVPA) revealed feature-specific encoding for direction of modulation in auditory cortex bilaterally. Moreover, this effect was strongest when analyses were restricted to anatomical regions lying outside Heschl's gyrus. We found no support for feature-specific encoding of frequency modulation rate. Differential findings of modulation rate and direction of modulation are discussed with respect to their relevance to phonetic discrimination.

  2. A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.

    Directory of Open Access Journals (Sweden)

    Jakob D Wikstrom

    Full Text Available The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets.The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets.The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

  3. Meta-analysis reveals a lack of sexual dimorphism in human amygdala volume.

    Science.gov (United States)

    Marwha, Dhruv; Halari, Meha; Eliot, Lise

    2017-02-15

    The amygdala plays a key role in many affective behaviors and psychiatric disorders that differ between men and women. To test whether human amygdala volume (AV) differs reliably between the sexes, we performed a systematic review and meta-analysis of AVs reported in MRI studies of age-matched healthy male and female groups. Using four search strategies, we identified 46 total studies (58 matched samples) from which we extracted effect sizes for the sex difference in AV. All data were converted to Hedges g values and pooled effect sizes were calculated using a random-effects model. Each dataset was further meta-regressed against study year and average participant age. We found that uncorrected amygdala volume is about 10% larger in males, with pooled sex difference effect sizes of g=0.581 for right amygdala (κ=28, n=2022), 0.666 for left amygdala (κ=28, n=2006), and 0.876 for bilateral amygdala (κ=16, n=1585) volumes (all p values brain volume (TBV; g=1.278, pbrain size in males. Among studies reporting AVs normalized for ICV or TBV, sex difference effect sizes were small and not statistically significant: g=0.171 for the right amygdala (p=0.206, κ=13, n=1560); 0.233 for the left amygdala (p=0.092, κ=12, n=1512); and 0.257 for bilateral volume (p=0.131, κ=5, n=1629). These values correspond to less than 0.1% larger corrected right AV and 2.5% larger corrected left AV in males compared to females. In summary, AV is not selectively enhanced in human males, as often claimed. Although we cannot rule out subtle male-female group differences, it is not accurate to refer to the human amygdala as "sexually dimorphic." Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Multispacer typing of Rickettsia isolates from humans and ticks in Tunisia revealing new genotypes.

    Science.gov (United States)

    Znazen, Abir; Khrouf, Fatma; Elleuch, Nihel; Lahiani, Dorra; Marrekchi, Chakib; M'Ghirbi, Youmna; Ben Jemaa, Mounir; Bouattour, Ali; Hammami, Adnene

    2013-12-31

    Rickettsioses are important remerging vector born infections. In Tunisia, many species have been described in humans and vectors. Genotyping is important for tracking pathogen movement between hosts and vectors. In this study, we characterized Rickettsia species detected in patients and vectors using multispacer typing (MST), proposed by Founier et al. and based on three intergenic spacers (dksA-xerC, rmpE- tRNA(fMet), mppA-pruC) sequencing. Our study included 25 patients hospitalized during 2009. Ticks and fleas were collected in the vicinity of confirmed cases. Serology was performed on serum samples by microimmunofluorescence using Rickettsia conorii and Rickettsia typhi antigens. To detect and identify Rickettsia species, PCR targeting ompA, ompB and gltA genes followed by sequencing was performed on 18 obtained skin biopsies and on all collected vectors. Rickettsia positive samples were further characterized using primers targeting three intergenic spacers (dksA-xerC, rmpE- tRNA(fMet) and mppA-purC). A rickettsial infection was confirmed in 15 cases (60%). Serology was positive in 13 cases (52%). PCR detected Rickettsia DNA in four biopsies (16%) allowing the identification of R. conorii subsp israelensis in three cases and R. conorii subsp conorii in one case. Among 380 collected ticks, nine presented positive PCR (2.4%) allowing the identification of six R. conorii subsp israelensis, two R. massiliae and one R. conorii subsp conorii. Among 322 collected fleas, only one was positive for R. felis. R. conorii subsp israelensis strains detected in humans and vectors clustered together and showed a new MST genotype. Similarly, R. conorii subsp conorii strains detected in a skin biopsy and a tick were genetically related and presented a new MST genotype. New Rickettsia spotted fever strain genotypes were found in Tunisia. Isolates detected in humans and vectors were genetically homogenous despite location differences in their original isolation suggesting

  5. What Lies Within: Using Radiopharmaceuticals to Reveal and Target Diseases Hidden Inside the Human Body

    International Nuclear Information System (INIS)

    Jawerth, Nicole

    2014-01-01

    The ability to pinpoint the location and size of a cancerous mass hidden inside of a patient’s body was unthinkable less than 100 years ago. Today, with the help of special scanning machines, doctors are able to use radioactive drugs known as radiopharmaceuticals to get a glimpse inside the human body, and these pharmaceuticals can even be used in treating many health conditions. In nuclear medicine, radiopharmaceuticals play an essential role for minimally invasive diagnostic, treatment and care management procedures for many diseases, especially cancer, as well as for relieving pain associated with certain cancers

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

    Directory of Open Access Journals (Sweden)

    Thomas Birkballe Hansen

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

  7. Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition.

    Science.gov (United States)

    Wibowo, Ardian S; Singh, Mirage; Reeder, Kristen M; Carter, Joshua J; Kovach, Alexander R; Meng, Wuyi; Ratnam, Manohar; Zhang, Faming; Dann, Charles E

    2013-09-17

    Antifolates, folate analogs that inhibit vitamin B9 (folic acid)-using cellular enzymes, have been used over several decades for the treatment of cancer and inflammatory diseases. Cellular uptake of the antifolates in clinical use occurs primarily via widely expressed facilitative membrane transporters. More recently, human folate receptors (FRs), high affinity receptors that transport folate via endocytosis, have been proposed as targets for the specific delivery of new classes of antifolates or folate conjugates to tumors or sites of inflammation. The development of specific, FR-targeted antifolates would be accelerated if additional biophysical data, particularly structural models of the receptors, were available. Here we describe six distinct crystallographic models that provide insight into biological trafficking of FRs and distinct binding modes of folate and antifolates to these receptors. From comparison of the structures, we delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes. Additionally, we describe the molecular details of human FR in complex with three clinically prevalent antifolates, pemetrexed (also Alimta), aminopterin, and methotrexate. On the whole, our data form the basis for rapid design and implementation of unique, FR-targeted, folate-based drugs for the treatment of cancer and inflammatory diseases.

  8. Potential human pathogenic bacteria in a mixed urban watershed as revealed by pyrosequencing.

    Directory of Open Access Journals (Sweden)

    A Mark Ibekwe

    Full Text Available Current microbial source tracking (MST methods for water depend on testing for fecal indicator bacterial counts or specific marker gene sequences to identify fecal contamination where potential human pathogenic bacteria could be present. In this study, we applied 454 high-throughput pyrosequencing to identify bacterial pathogen DNA sequences, including those not traditionally monitored by MST and correlated their abundances to specific sources of contamination such as urban runoff and agricultural runoff from concentrated animal feeding operations (CAFOs, recreation park area, waste-water treatment plants, and natural sites with little or no human activities. Samples for pyrosequencing were surface water, and sediment collected from 19 sites. A total of 12,959 16S rRNA gene sequences with average length of ≤400 bp were obtained, and were assigned to corresponding taxonomic ranks using ribosomal database project (RDP, Classifier and Greengenes databases. The percent of total potential pathogens were highest in urban runoff water (7.94%, agricultural runoff sediment (6.52%, and Prado Park sediment (6.00%, respectively. Although the numbers of DNA sequence tags from pyrosequencing were very high for the natural site, corresponding percent potential pathogens were very low (3.78-4.08%. Most of the potential pathogenic bacterial sequences identified were from three major phyla, namely, Proteobacteria, Bacteroidetes, and Firmicutes. The use of deep sequencing may provide improved and faster methods for the identification of pathogen sources in most watersheds so that better risk assessment methods may be developed to enhance public health.

  9. Altered hematopoiesis in trisomy 21 as revealed through in vitro differentiation of isogenic human pluripotent cells

    Science.gov (United States)

    MacLean, Glenn A.; Menne, Tobias F.; Guo, Guoji; Sanchez, Danielle J.; Park, In-Hyun; Daley, George Q.; Orkin, Stuart H.

    2012-01-01

    Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver, a preleukemic condition termed transient myeloproliferative disorder, and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins, human embryionic stem (hES), and induced pluripotent stem (iPS) cells, were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation, we isolated disomic and trisomic subclones from the same parental iPS line, thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like, γ-globin expressing, definitive hematopoiesis, we found that trisomic cells of hES, iPS, or isogenic origins exhibited a two- to fivefold increase in a population of CD43+(Leukosialin)/CD235+(Glycophorin A) hematopoietic cells, accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage. PMID:23045682

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

    Directory of Open Access Journals (Sweden)

    Dingxiao Zhang

    2016-03-01

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

  11. Human Thalamic-Prefrontal Peduncle Connectivity Revealed by Diffusion Spectrum Imaging Fiber Tracking

    Directory of Open Access Journals (Sweden)

    Chuanqi Sun

    2018-04-01

    Full Text Available The thalamic-prefrontal peduncle (TPP is a large bundle connecting the thalamus and prefrontal cortex. The definitive structure and function of the TPP are still controversial. To investigate the connectivity and segmentation patterns of the TPP, we employed diffusion spectrum imaging with generalized q-sampling reconstruction to perform both subject-specific and template-based analyses. Our results confirmed the trajectory and spatial relationship of the TPP in the human brain and identified the connection areas in the prefrontal cortex. The TPP-connecting areas identified based on Brodmann areas (BAs were BAs 8–11 and 45–47. Based on the automated anatomical atlas, these areas were the medial superior frontal gyrus, superior frontal gyrus, middle frontal gyrus, pars triangularis, pars orbitalis, anterior orbital gyrus, and lateral orbital gyrus. In addition, we identified the TPP connection areas in the thalamus, including the anterior and medial nuclei, and the lateral dorsal/lateral posterior nuclei. TPP fibers connected the thalamus with the ipsilateral prefrontal BAs 11, 47, 10, 46, 45, 9, and 8 seriatim from medial to lateral, layer by layer. Our results provide further details of the thalamic-prefrontal peduncle structure, and may aid future studies and a better understanding of the functional roles of the TPP in the human brain.

  12. Chromatic illumination discrimination ability reveals that human colour constancy is optimised for blue daylight illuminations.

    Directory of Open Access Journals (Sweden)

    Bradley Pearce

    Full Text Available The phenomenon of colour constancy in human visual perception keeps surface colours constant, despite changes in their reflected light due to changing illumination. Although colour constancy has evolved under a constrained subset of illuminations, it is unknown whether its underlying mechanisms, thought to involve multiple components from retina to cortex, are optimised for particular environmental variations. Here we demonstrate a new method for investigating colour constancy using illumination matching in real scenes which, unlike previous methods using surface matching and simulated scenes, allows testing of multiple, real illuminations. We use real scenes consisting of solid familiar or unfamiliar objects against uniform or variegated backgrounds and compare discrimination performance for typical illuminations from the daylight chromaticity locus (approximately blue-yellow and atypical spectra from an orthogonal locus (approximately red-green, at correlated colour temperature 6700 K, all produced in real time by a 10-channel LED illuminator. We find that discrimination of illumination changes is poorer along the daylight locus than the atypical locus, and is poorest particularly for bluer illumination changes, demonstrating conversely that surface colour constancy is best for blue daylight illuminations. Illumination discrimination is also enhanced, and therefore colour constancy diminished, for uniform backgrounds, irrespective of the object type. These results are not explained by statistical properties of the scene signal changes at the retinal level. We conclude that high-level mechanisms of colour constancy are biased for the blue daylight illuminations and variegated backgrounds to which the human visual system has typically been exposed.

  13. Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease

    Science.gov (United States)

    Cruz, Nelly M.; Song, Xuewen; Czerniecki, Stefan M.; Gulieva, Ramila E.; Churchill, Angela J.; Kim, Yong Kyun; Winston, Kosuke; Tran, Linh M.; Diaz, Marco A.; Fu, Hongxia; Finn, Laura S.; Pei, York; Himmelfarb, Jonathan; Freedman, Benjamin S.

    2017-11-01

    Polycystic kidney disease (PKD) is a life-threatening disorder, commonly caused by defects in polycystin-1 (PC1) or polycystin-2 (PC2), in which tubular epithelia form fluid-filled cysts. A major barrier to understanding PKD is the absence of human cellular models that accurately and efficiently recapitulate cystogenesis. Previously, we have generated a genetic model of PKD using human pluripotent stem cells and derived kidney organoids. Here we show that systematic substitution of physical components can dramatically increase or decrease cyst formation, unveiling a critical role for microenvironment in PKD. Removal of adherent cues increases cystogenesis 10-fold, producing cysts phenotypically resembling PKD that expand massively to 1-centimetre diameters. Removal of stroma enables outgrowth of PKD cell lines, which exhibit defects in PC1 expression and collagen compaction. Cyclic adenosine monophosphate (cAMP), when added, induces cysts in both PKD organoids and controls. These biomaterials establish a highly efficient model of PKD cystogenesis that directly implicates the microenvironment at the earliest stages of the disease.

  14. Human listening studies reveal insights into object features extracted by echolocating dolphins

    Science.gov (United States)

    Delong, Caroline M.; Au, Whitlow W. L.; Roitblat, Herbert L.

    2004-05-01

    Echolocating dolphins extract object feature information from the acoustic parameters of object echoes. However, little is known about which object features are salient to dolphins or how they extract those features. To gain insight into how dolphins might be extracting feature information, human listeners were presented with echoes from objects used in a dolphin echoic-visual cross-modal matching task. Human participants performed a task similar to the one the dolphin had performed; however, echoic samples consisting of 23-echo trains were presented via headphones. The participants listened to the echoic sample and then visually selected the correct object from among three alternatives. The participants performed as well as or better than the dolphin (M=88.0% correct), and reported using a combination of acoustic cues to extract object features (e.g., loudness, pitch, timbre). Participants frequently reported using the pattern of aural changes in the echoes across the echo train to identify the shape and structure of the objects (e.g., peaks in loudness or pitch). It is likely that dolphins also attend to the pattern of changes across echoes as objects are echolocated from different angles.

  15. Screening of Probiotic Candidates in Human Oral Bacteria for the Prevention of Dental Disease.

    Directory of Open Access Journals (Sweden)

    Tomohiko Terai

    Full Text Available The oral cavity in healthy subjects has a well-balanced microbiota that consists of more than 700 species. However, a disturbance of this balance, with an increase of harmful microbes and a decrease of beneficial microbes, causes oral disorders such as periodontal disease or dental caries. Nowadays, probiotics are expected to confer oral health benefits by modulating the oral microbiota. This study screened new probiotic candidates with potential oral health benefits and no harmful effects on the oral cavity. We screened 14 lactobacillus strains and 36 streptococcus strains out of 896 oral isolates derived from healthy subjects. These bacteria did not produce volatile sulfur compounds or water-insoluble glucan, had higher antibacterial activity against periodontal bacteria, and had higher adherence activity to oral epithelial cells or salivary-coated hydroxyapatite in vitro. We then evaluated the risk of primary cariogenicity and infective endocarditis of the selected oral isolates. As a result, Lactobacillus crispatus YIT 12319, Lactobacillus fermentum YIT 12320, Lactobacillus gasseri YIT 12321, and Streptococcus mitis YIT 12322 were selected because they showed no cariogenic potential in an artificial mouth system and a lower risk of experimental infective endocarditis in a rat model. These candidates are expected as new probiotics with potential oral health benefits and no adverse effects on general health.

  16. Screening of Probiotic Candidates in Human Oral Bacteria for the Prevention of Dental Disease.

    Science.gov (United States)

    Terai, Tomohiko; Okumura, Takekazu; Imai, Susumu; Nakao, Masumi; Yamaji, Kazuaki; Ito, Masahiko; Nagata, Tsuyoshi; Kaneko, Kimiyuki; Miyazaki, Kouji; Okada, Ayako; Nomura, Yoshiaki; Hanada, Nobuhiro

    2015-01-01

    The oral cavity in healthy subjects has a well-balanced microbiota that consists of more than 700 species. However, a disturbance of this balance, with an increase of harmful microbes and a decrease of beneficial microbes, causes oral disorders such as periodontal disease or dental caries. Nowadays, probiotics are expected to confer oral health benefits by modulating the oral microbiota. This study screened new probiotic candidates with potential oral health benefits and no harmful effects on the oral cavity. We screened 14 lactobacillus strains and 36 streptococcus strains out of 896 oral isolates derived from healthy subjects. These bacteria did not produce volatile sulfur compounds or water-insoluble glucan, had higher antibacterial activity against periodontal bacteria, and had higher adherence activity to oral epithelial cells or salivary-coated hydroxyapatite in vitro. We then evaluated the risk of primary cariogenicity and infective endocarditis of the selected oral isolates. As a result, Lactobacillus crispatus YIT 12319, Lactobacillus fermentum YIT 12320, Lactobacillus gasseri YIT 12321, and Streptococcus mitis YIT 12322 were selected because they showed no cariogenic potential in an artificial mouth system and a lower risk of experimental infective endocarditis in a rat model. These candidates are expected as new probiotics with potential oral health benefits and no adverse effects on general health.

  17. Cervical cancer screening, human papillomavirus vaccination practices and current infrastructure in Israel.

    Science.gov (United States)

    Schejter, Eduardo; Bornstein, Jacob; Siegler, Efraim

    2013-11-22

    The incidence rates for premalignant lesions in Jewish women in Israel are similar to those observed in Western countries, but the incidence of cervical cancer in Israel is low; this discrepancy is not yet clearly understood. Because of the low incidence of cervical cancer in Israel, it was decided to base cervical cancer prevention on opportunistic screening: every woman from the ages of 35-54 years can have a Pap test smear free of charge every 3 years. Over the last decade 12.2% of the women population had an annual Pap test. From 36 to 50% of women who attended the Clalit Health Maintenance Organization (HMO) and the Maccabi HMO, the two largest HMOs in Israel, did so. There were also discrepancies between women of different socio-economic status (SES): Israel Society of Obstetrics and Gynecology recommends continuing cytologic screening in vaccinated women as recommended for the general population. This article forms part of a regional report entitled "Comprehensive Control of HPV Infections and Related Diseases in Israel" Vaccine Volume 31, Supplement 8, 2013. Updates of the progress in the field are presented in a separate monograph entitled "Comprehensive Control of HPV Infections and Related Diseases" Vaccine Volume 30, Supplement 5, 2012. Copyright © 2013. Published by Elsevier Ltd.

  18. The musical brain: brain waves reveal the neurophysiological basis of musicality in human subjects.

    Science.gov (United States)

    Tervaniemi, M; Ilvonen, T; Karma, K; Alho, K; Näätänen, R

    1997-04-18

    To reveal neurophysiological prerequisites of musicality, auditory event-related potentials (ERPs) were recorded from musical and non-musical subjects, musicality being here defined as the ability to temporally structure auditory information. Instructed to read a book and to ignore sounds, subjects were presented with a repetitive sound pattern with occasional changes in its temporal structure. The mismatch negativity (MMN) component of ERPs, indexing the cortical preattentive detection of change in these stimulus patterns, was larger in amplitude in musical than non-musical subjects. This amplitude enhancement, indicating more accurate sensory memory function in musical subjects, suggests that even the cognitive component of musicality, traditionally regarded as depending on attention-related brain processes, in fact, is based on neural mechanisms present already at the preattentive level.

  19. Limitations of widely used high-risk human papillomavirus laboratory-developed testing in cervical cancer screening

    Directory of Open Access Journals (Sweden)

    Naryshkin S

    2012-11-01

    Full Text Available Sonya Naryshkin,1 R Marshall Austin21Department of Pathology, Mercy Health System, Janesville, WI; 2Department of Pathology, Magee-Womens Hospital of University of Pittsburgh Medical Center, Pittsburgh, PA, USAObjective: To increase awareness of the limitations of high-risk human papillomavirus (hrHPV laboratory-developed testing (LDT widely used in US cervical cancer screening.Methods and results: A young woman in her 30s was diagnosed and treated for stage 1B1 cervical squamous cell carcinoma in which HPV 16 DNA was detected using polymerase chain reaction testing. Both 1 month before and 42 months before cervical cancer diagnosis, the patient had highly abnormal cytology findings; however, residual SurePath™ (Becton, Dickson and Company, Franklin Lakes, NJ vial fluid yielded negative Hybrid Capture 2 (HC2; Qiagen NV, Hilden, Germany hrHPV LDT results from each of the two specimens. This prompted questions to be asked concerning the performance characteristics of hrHPV LDT. A review of the available data indicates that (1 purification of DNA from SurePath specimens requires complex sample preparation due to formaldehyde crosslinking of proteins and nucleic acids, (2 HC2–SurePath hrHPV testing had not been Food and Drug Administration-approved after multiple premarket approval submissions, (3 detectible hrHPV DNA in the SurePath vial decreases over time, and (4 US laboratories performing HC2–SurePath hrHPV LDT testing are not using a standardized manufacturer-endorsed procedure.Conclusion: Recently updated cervical screening guidelines in the US recommend against the use of hrHPV LDT in cervical screening, including widely used HC2 testing from the SurePath vial. The manufacturer recently issued a technical bulletin specifically warning that use of SurePath samples with the HC2 hrHPV test may provide false negative results and potentially compromise patient safety. Co-collection using a Food and Drug Administration-approved hrHPV test

  20. Computational challenges and human factors influencing the design and use of clinical research participant eligibility pre-screening tools

    Directory of Open Access Journals (Sweden)

    Pressler Taylor R

    2012-05-01

    Full Text Available Abstract Background Clinical trials are the primary mechanism for advancing clinical care and evidenced-based practice, yet challenges with the recruitment of participants for such trials are widely recognized as a major barrier to these types of studies. Data warehouses (DW store large amounts of heterogenous clinical data that can be used to enhance recruitment practices, but multiple challenges exist when using a data warehouse for such activities, due to the manner of collection, management, integration, analysis, and dissemination of the data. A critical step in leveraging the DW for recruitment purposes is being able to match trial eligibility criteria to discrete and semi-structured data types in the data warehouse, though trial eligibility criteria tend to be written without concern for their computability. We present the multi-modal evaluation of a web-based tool that can be used for pre-screening patients for clinical trial eligibility and assess the ability of this tool to be practically used for clinical research pre-screening and recruitment. Methods The study used a validation study, usability testing, and a heuristic evaluation to evaluate and characterize the operational characteristics of the software as well as human factors affecting its use. Results Clinical trials from the Division of Cardiology and the Department of Family Medicine were used for this multi-modal evaluation, which included a validation study, usability study, and a heuristic evaluation. From the results of the validation study, the software demonstrated a positive predictive value (PPV of 54.12% and 0.7%, respectively, and a negative predictive value (NPV of 73.3% and 87.5%, respectively, for two types of clinical trials. Heuristic principles concerning error prevention and documentation were characterized as the major usability issues during the heuristic evaluation. Conclusions This software is intended to provide an initial list of eligible patients to a

  1. Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Sirenko, Oksana, E-mail: oksana.sirenko@moldev.com [Molecular Devices LLC, Sunnyvale, CA 94089 (United States); Cromwell, Evan F., E-mail: evan.cromwell@moldev.com [Molecular Devices LLC, Sunnyvale, CA 94089 (United States); Crittenden, Carole [Molecular Devices LLC, Sunnyvale, CA 94089 (United States); Wignall, Jessica A. [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599 (United States); Wright, Fred A. [Department of Biostatistics, University of North Carolina, Chapel Hill, NC 27599 (United States); Rusyn, Ivan [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599 (United States)

    2013-12-15

    Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes show promise for screening during early drug development. Here, we tested a hypothesis that in vitro assessment of multiple cardiomyocyte physiological parameters enables predictive and mechanistically-interpretable evaluation of cardiotoxicity in a high-throughput format. Human iPSC-derived cardiomyocytes were exposed for 30 min or 24 h to 131 drugs, positive (107) and negative (24) for in vivo cardiotoxicity, in up to 6 concentrations (3 nM to 30 uM) in 384-well plates. Fast kinetic imaging was used to monitor changes in cardiomyocyte function using intracellular Ca{sup 2+} flux readouts synchronous with beating, and cell viability. A number of physiological parameters of cardiomyocyte beating, such as beat rate, peak shape (amplitude, width, raise, decay, etc.) and regularity were collected using automated data analysis. Concentration–response profiles were evaluated using logistic modeling to derive a benchmark concentration (BMC) point-of-departure value, based on one standard deviation departure from the estimated baseline in vehicle (0.3% dimethyl sulfoxide)-