86 results on '"Phillip A. Yates"'
Search Results
2. Refinement of Leishmania donovani Genome Annotations in the Light of Ribosome-Protected mRNAs Fragments (Ribo-Seq Data)
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Alejandro Sánchez-Salvador, Sandra González-de la Fuente, Begoña Aguado, Phillip A. Yates, and Jose M. Requena
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Leishmania ,ribosome profiling ,Ribo-seq ,uORFs ,genome ,transcriptome ,Genetics ,QH426-470 - Abstract
Advances in next-generation sequencing methodologies have facilitated the assembly of an ever-increasing number of genomes. Gene annotations are typically conducted via specialized software, but the most accurate results require additional manual curation that incorporates insights derived from functional and bioinformatic analyses (e.g., transcriptomics, proteomics, and phylogenetics). In this study, we improved the annotation of the Leishmania donovani (strain HU3) genome using publicly available data from the deep sequencing of ribosome-protected mRNA fragments (Ribo-Seq). As a result of this analysis, we uncovered 70 previously non-annotated protein-coding genes and improved the annotation of around 600 genes. Additionally, we present evidence for small upstream open reading frames (uORFs) in a significant number of transcripts, indicating their potential role in the translational regulation of gene expression. The bioinformatics pipelines developed for these analyses can be used to improve the genome annotations of other organisms for which Ribo-Seq data are available. The improvements provided by these studies will bring us closer to the ultimate goal of a complete and accurately annotated L. donovani genome and will enhance future transcriptomics, proteomics, and genetics studies.
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- 2023
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3. Safety, tolerability and antiviral activity of the antisense oligonucleotide bepirovirsen in patients with chronic hepatitis B: a phase 2 randomized controlled trial
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Yvonne Tami, Shihyun You, Melanie Paff, Jeong-Won Jang, Robert Elston, Sung-Jae Park, Phillip J. Yates, Yu Tao, Jung Hwan Yoon, C. Frank Bennett, Jennifer Cremer, Jeong Heo, Dickens Theodore, T. Jesse Kwoh, Man-Fung Yuen, Young-Oh Kweon, and Fiona Campbell
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Adult ,Male ,medicine.medical_specialty ,HBsAg ,Hepatitis B virus ,Adolescent ,Diseases ,Placebo ,medicine.disease_cause ,Gastroenterology ,Antiviral Agents ,General Biochemistry, Genetics and Molecular Biology ,Article ,law.invention ,Polyethylene Glycols ,Placebos ,Young Adult ,Hepatitis B, Chronic ,Randomized controlled trial ,law ,Internal medicine ,Republic of Korea ,medicine ,Humans ,Spotlight ,Adverse effect ,Hepatitis B Surface Antigens ,business.industry ,General Medicine ,Hepatitis B ,Middle Aged ,Oligonucleotides, Antisense ,medicine.disease ,Tolerability ,Hepatocellular carcinoma ,Infectious diseases ,Drug Therapy, Combination ,Female ,business - Abstract
Chronic infection with hepatitis B virus (HBV) leads to an increased risk of death from cirrhosis and hepatocellular carcinoma. Functional cure rates are low with current treatment options (nucleos(t)ide analogs (NAs) and pegylated interferons). Bepirovirsen is an antisense oligonucleotide targeting all HBV messenger RNAs; in cell culture and animal models, bepirovirsen leads to reductions in HBV-derived RNAs, HBV DNA and viral proteins. This phase 2 double-blinded, randomized, placebo-controlled trial is the first evaluation of the safety and activity of an antisense oligonucleotide targeting HBV RNA in both treatment-naïve and virally suppressed individuals with chronic HBV infection. The primary objective was to assess the safety and tolerability of bepirovirsen in individuals with chronic hepatitis B (CHB) (NCT02981602). The secondary objective was to assess antiviral activity, including the change from baseline to day 29 in serum hepatitis B surface antigen (HBsAg) concentration. Participants with CHB infection ≥6 months and serum HBsAg ≥50 IU ml−1 were enrolled from seven centers across Hong Kong and the Republic of Korea and randomized (3:1 within each dose cohort) to receive bepirovirsen or placebo via subcutaneous injection twice weekly during weeks 1 and 2 (days 1, 4, 8 and 11) and once weekly during weeks 3 and 4 (days 15 and 22). Participants were then followed for 26 weeks. Twenty-four participants were treatment-naïve and seven were receiving stable NA therapy. Treatment-emergent adverse events were mostly mild/moderate (most commonly injection site reactions). Eleven (61.1%) and three (50.0%) treatment-naïve participants experienced one or more treatment-emergent adverse event in the bepirovirsen and placebo groups, respectively. In participants receiving NA therapy, the corresponding numbers were three (60.0%) and one (50.0%). Transient, self-resolving alanine aminotransferase flares (≥2× upper limit of normal) were observed in eight treatment-naïve participants and three participants on stable NA regimens in the bepirovirsen treatment arms. HBsAg reductions were observed and were significant versus placebo for treatment-naïve participants receiving bepirovirsen 300 mg (P = 0.001), but not for the bepirovirsen 150 mg group (P = 0.245) or participants receiving stable NA therapy (P = 0.762). Two participants in each of the 300 mg dose groups achieved HBsAg levels below the lower limit of quantitation by day 29 (n = 3) or day 36 (n = 1). Bepirovirsen had a favorable safety profile. These preliminary observations warrant further investigation of the safety and activity of bepirovirsen in a larger CHB patient population., A first-in-human study of an antisense oligonucleotide targeting hepatitis B virus (HBV) RNA provides initial insights into this potential new therapeutic modality for individuals with chronic HBV infection.
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- 2021
4. 304. Viral resistance analysis in the COMET-PEAK study: sotrovimab treatment in participants with mild-to-moderate COVID-19
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Phillip J Yates, Jennifer Moore, Jennifer Han, Andrew Skingsley, Gretja Schnell, Andrea L Cathcart, Melissa Aldinger, Amanda Peppercorn, and Jill Walker
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Infectious Diseases ,Oncology - Abstract
Background Sotrovimab is a human monoclonal antibody targeting a conserved region of the SARS-CoV-2 spike (S) protein. COMET-PEAK was a 3-part, phase 2 study that evaluated intravenous (500 mg) and intramuscular (500 mg and 250 mg) administration of sotrovimab in outpatients (n=353) with mild-to-moderate COVID-19. We assessed amino acid substitutions in the SARS-CoV-2 S protein and circulating variants of concern/interest (VOC/VOI) in COMET-PEAK participants (enrolled Feb–July 2021). Methods Mid-turbinate (Part A) or nasopharyngeal (Part B/C) samples were obtained from all participants at Baseline and Post-Baseline visits. Next generation sequencing of the SARS-CoV-2 S gene was conducted using Illumina MiSeq with a ≥5% allelic frequency cut-off for samples with a viral load above 3.0 log10 copies/mL. Baseline, post-baseline and treatment-emergent (TE) substitutions were assessed, and prevalence of VOC/VOI was evaluated. Phenotypic analyses of epitope substitutions were conducted using a pseudotyped virus assay. Results In total, 282/353 participants had sequencing results for ≥1 visit (253 baseline, 248 post-baseline), and 219 had paired baseline and post-baseline sequences; among these, 149 (68%) had TE substitutions in the S protein (26 [12%] in the epitope). E340K was the predominant TE substitution in the epitope (15 [7%]). Across all arms 92/245 (38%) experienced virologic rebound, 8 of whom (Part A: 2; Part B: 2; Part C: 4) had TE substitutions in the epitope; none had evidence of clinical progression to severe disease. Prevalence of VOC/VOI or single amino acid substitutions of concern was 94% (266/282); the most frequent were Alpha (Part A: 8/16 [50%]; Part B: 75/128 [59%]) and Delta (Part C: 99/122 [81%]). Of 7 participants with evidence of clinical progression, none had S protein substitutions in the epitope and all had VOC/VOI (3 Alpha, 3 Delta, 1 Gamma). Sotrovimab effectively neutralized most epitope substitutions tested in vitro; P337L and E340A/K/V conferred significantly reduced susceptibility. Conclusion There was no evidence that sotrovimab epitope substitutions were associated with clinical progression or virologic rebound. These data are consistent with those from the COMET-ICE study. Funding Vir Biotechnology/GSK (NCT04779879). Disclosures Phillip J Yates, PhD, GlaxoSmithKline: Employee Jennifer Moore, MD, GlaxoSmithKline: Employee Jennifer Han, MD, GlaxoSmithKline: Employee Andrew Skingsley, MD, GlaxoSmithKline: Employee|GlaxoSmithKline: Stocks/Bonds Gretja Schnell, PhD, Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Andrea L. Cathcart, PhD, Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Melissa Aldinger, PharmD, Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Amanda Peppercorn, MD, GlaxoSmithKline: Employee|GlaxoSmithKline: Stocks/Bonds Jill Walker, PhD, GlaxoSmithKline: Employee.
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- 2022
5. ASAR lncRNAs control DNA replication timing through interactions with multiple hnRNP/RNA binding proteins
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Mathew J. Thayer, Michael B. Heskett, Leslie G. Smith, Paul T. Spellman, and Phillip A. Yates.
- Abstract
ASARs are a family of very-long noncoding RNAs that control replication timing on individual human autosomes, and are essential for chromosome stability. The eight known ASAR genes express RNAs that remain closely associated with their parent chromosomes. Analysis of RNA-protein interaction data (from ENCODE) revealed numerous RBPs with significant interactions with multiple ASAR RNAs, with several hnRNPs as abundant interactors. An ∼7kb domain within the ASAR6-141 RNA shows a striking density of RBP interaction sites. Genetic deletion and ectopic integration assays indicate that this ∼7kb RNA binding protein domain contains functional sequences for controlling replication timing of entire chromosomes incis. shRNA-mediated depletion of HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, and HNRNPUL1 results in dissociation of ASAR RNAs from their chromosome territories, and disrupts the synchronous replication that occurs on all autosome pairs, recapitulating the effect of individual ASAR gene knockouts on a genome-wide scale. Our results further demonstrate the role that ASARs play during the temporal order of genome-wide replication, and that ASARs function as essential RNA scaffolds for the assembly of hnRNP complexes that help maintain the structural integrity of each mammalian chromosome.
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- 2022
6. Epigenetic Control of Hundreds of Chromosome-Associated lncRNA Genes Essential for Replication and Stability
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Michael B. Heskett, Athanasios E. Vouzas, Leslie G. Smith, Phillip A. Yates, Christopher Boniface, Eric E. Bouhassira, Paul Spellman, David M. Gilbert, and Mathew J. Thayer
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ASARs are long noncoding RNA genes that control replication timing of entire human chromosomes in cis. The three known ASAR genes are located on human chromosomes 6 and 15, and are essential for chromosome integrity. To identify ASARs on all human chromosomes we utilized a set of distinctive ASAR characteristics that allowed for the identification of hundreds of autosomal loci with epigenetically controlled, allele-restricted behavior in expression and replication timing of coding and noncoding genes, and is distinct from genomic imprinting. Disruption of noncoding RNA genes at five of five tested loci resulted in chromosome-wide delayed replication and chromosomal instability, validating their ASAR activity. In addition to the three known essential cis-acting chromosomal loci, origins, centromeres, and telomeres, we propose that all mammalian chromosomes also contain “Inactivation/Stability Centers” that display allele-restricted epigenetic regulation of protein coding and noncoding ASAR genes that are essential for replication and stability of each chromosome.
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- 2022
7. Lessons from resistance analysis in clinical trials of IV zanamivir
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Phillip J, Yates, Nalini, Mehta, Helen A, Watson, and Amanda F, Peppercorn
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Cancer Research ,Infectious Diseases ,Virology - Abstract
Influenza infection causes substantial morbidity and mortality during seasonal epidemics and pandemics. Antivirals, including neuraminidase inhibitors, play an important role in the treatment of severely ill patients infected with influenza. Resistance is a key factor that can affect the efficacy of neuraminidase inhibitors (NAIs). It is a recommendation by regulatory authorities to monitor for resistance during the development of anti-influenza medications. An additional requirement by regulators is to examine amino acid sequences for minority species harbouring resistance substitutions. In a Phase III study of intravenous (IV) zanamivir respiratory samples were analysed for the presence of resistant quasi species using Next Generation Sequencing (NGS). In this study ten resistance substitutions, two of which were treatment emergent, were detected by NGS that otherwise would not have been detectable by Sanger sequencing. None of the substitutions were present at any other timepoints analysed. The effect these mutations have on clinical response is difficult to characterize; in fact, all patients from which these variants were isolated had a successful clinical outcome and the effect on clinical response was therefore likely minimal. Although NGS is becoming a routine method for nucleic acid sequencing and will detect substitutions previously undetected by Sanger sequencing, the value of this technique in identifying minority species with resistance substitutions that are clinically meaningful remains to be demonstrated, particularly with acute infections such as influenza.
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- 2023
8. Nutrient sensing in Leishmania : Flagellum and cytosol
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Felice D. Kelly, Scott M. Landfear, and Phillip A. Yates
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Protozoan Proteins ,Nutrient sensing ,Flagellum ,Microbiology ,Article ,03 medical and health sciences ,Cytosol ,biology.animal ,Animals ,Humans ,Leishmaniasis ,Molecular Biology ,030304 developmental biology ,Leishmania ,0303 health sciences ,Parasitic mode ,biology ,030306 microbiology ,Host (biology) ,Vertebrate ,Nutrients ,biology.organism_classification ,Alimentary tract ,Cell biology ,Flagella - Abstract
Parasites are by definition organisms that utilize resources from a host to support their existence, thus, promoting their ability to establish long-term infections and disease. Hence, sensing and acquiring nutrients for which the parasite and host compete is central to the parasitic mode of existence. Leishmania are flagellated kinetoplastid parasites that parasitize phagocytic cells, principally macrophages, of vertebrate hosts and the alimentary tract of sand fly vectors. Because nutritional supplies vary over time within both these hosts and are often restricted in availability, these parasites must sense a plethora of nutrients and respond accordingly. The flagellum has been recognized as an "antenna" that plays a core role in sensing environmental conditions, and various flagellar proteins have been implicated in sensing roles. In addition, these parasites exhibit non-flagellar intracellular mechanisms of nutrient sensing, several of which have been explored. Nonetheless, mechanistic details of these sensory pathways are still sparse and represent a challenging frontier for further experimental exploration.
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- 2020
9. Considerations from the Innovation and Quality Induction Working Group in Response to Drug-Drug Interaction Guidance from Regulatory Agencies: Guidelines on Model Fitting and Recommendations on Time Course for In Vitro Cytochrome P450 Induction Studies Including Impact on Drug Interaction Risk Assessment
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Heidi J. Einolf, Barry Jones, Phillip D. Yates, Theunis C. Goosen, Diane Ramsden, Conrad Fung, Y. Amy Siu, Niresh Hariparsad, Simon Wong, Liangfu Chen, Jairam Palamanda, George Zhang, Donald J. Tweedie, and Shannon Dallas
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CYP2B6 ,Pharmaceutical Science ,Guidelines as Topic ,Pharmacology ,Models, Biological ,Risk Assessment ,030226 pharmacology & pharmacy ,03 medical and health sciences ,0302 clinical medicine ,Drug Development ,Cytochrome P-450 CYP1A2 ,Cytochrome P-450 CYP3A ,Humans ,Medicine ,Drug Interactions ,Pharmacokinetics ,EC50 ,ADME ,CYP3A4 ,business.industry ,CYP1A2 ,Reproducibility of Results ,Drug interaction ,Confidence interval ,Cytochrome P-450 CYP2B6 ,Enzyme Induction ,030220 oncology & carcinogenesis ,Hepatocytes ,Drug and Narcotic Control ,business ,Risk assessment - Abstract
Translational and ADME Sciences Leadership Group Induction Working Group (IWG) presents an analysis on the time course for cytochrome P450 induction in primary human hepatocytes. Induction of CYP1A2, CYP2B6, and CYP3A4 was evaluated by seven IWG laboratories after incubation with prototypical inducers (omeprazole, phenobarbital, rifampicin, or efavirenz) for 6-72 hours. The effect of incubation duration and model-fitting approaches on induction parameters (Emax and EC50) and drug-drug interaction (DDI) risk assessment was determined. Despite variability in induction response across hepatocyte donors, the following recommendations are proposed: 1) 48 hours should be the primary time point for in vitro assessment of induction based on mRNA level or activity, with no further benefit from 72 hours; 2) when using mRNA, 24-hour incubations provide reliable assessment of induction and DDI risk; 3) if validated using prototypical inducers (>10-fold induction), 12-hour incubations may provide an estimate of induction potential, including characterization as negative if
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- 2020
10. Population Pharmacokinetic/Pharmacodynamic Analysis of Intravenous Zanamivir in Healthy Adults and Hospitalized Adult and Pediatric Subjects With Influenza
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Phillip J. Yates, Malek Okour, Jon Collins, Grace Roberts, Peiying Zuo, Helen A. Watson, Denise Shortino, Aline Barth, Mohammad Hossain, and Amanda Peppercorn
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Male ,030213 general clinical medicine ,Time Factors ,Datasets as Topic ,Pharmacology ,030226 pharmacology & pharmacy ,0302 clinical medicine ,Multicenter Studies as Topic ,Zanamivir ,General Pharmacology, Toxicology and Pharmaceutics ,Child ,Host cell membrane ,Aged, 80 and over ,education.field_of_study ,Inhalation ,biology ,General Neuroscience ,lcsh:Public aspects of medicine ,Age Factors ,virus diseases ,General Medicine ,Articles ,Middle Aged ,Viral Load ,Healthy Volunteers ,Hospitalization ,Influenza A virus ,Child, Preschool ,Administration, Intravenous ,Female ,medicine.drug ,Glomerular Filtration Rate ,Adult ,Adolescent ,Population ,Neuraminidase ,Antiviral Agents ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,Article ,Drug Administration Schedule ,03 medical and health sciences ,Young Adult ,Clinical Trials, Phase II as Topic ,Pharmacokinetics ,Influenza, Human ,medicine ,Humans ,Dosing ,education ,Aged ,Dose-Response Relationship, Drug ,business.industry ,Research ,lcsh:RM1-950 ,Infant ,lcsh:RA1-1270 ,United States ,Renal Elimination ,lcsh:Therapeutics. Pharmacology ,Clinical Trials, Phase III as Topic ,Pharmacodynamics ,biology.protein ,business - Abstract
Zanamivir is a potent and highly selective inhibitor of influenza neuraminidase in which the inhibition of this enzyme prevents the virus from infecting other cells and specifically prevents release of the new virion from the host cell membrane. It is available as an oral powder for inhalation and intravenous formulations. The current population pharmacokinetic model based on data from eight studies of subjects treated with the intravenous formulation (125 healthy adults and 533 hospitalized adult and pediatric subjects with suspected or confirmed influenza) suggested a decreased zanamivir clearance in pediatric and renal impairment adult subjects. It also indicates that b.i.d. dosing is necessary to keep the exposure in influenza infected subjects above the 90% inhibitory concentration values of recently circulating viruses over the dosing interval. In the exposure-response analysis (phases II and III studies), no apparent relationship was found between zanamivir exposure and clinically relevant pharmacodynamic end points.
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- 2020
11. Metabolic reprogramming during purine stress in the protozoan pathogen Leishmania donovani.
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Jessica L Martin, Phillip A Yates, Radika Soysa, Joshua F Alfaro, Feng Yang, Kristin E Burnum-Johnson, Vladislav A Petyuk, Karl K Weitz, David G Camp, Richard D Smith, Phillip A Wilmarth, Larry L David, Gowthaman Ramasamy, Peter J Myler, and Nicola S Carter
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over three months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.
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- 2014
- Full Text
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12. The impact of low temperature on fraction unbound for plasma and tissue
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Jonathan J. Novak, Phillip D. Yates, Li Di, Roshan Patel, and Sangwoo Ryu
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0301 basic medicine ,Pharmacology ,Chemistry ,Pharmaceutical Science ,Fraction (chemistry) ,General Medicine ,Plasma ,Plasma protein binding ,Prodrug ,030226 pharmacology & pharmacy ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Tissue binding ,Biophysics ,Pharmacology (medical) ,Incubation ,Plasma stability - Abstract
The effect of low temperature (4 °C) on plasma protein binding and tissue binding was evaluated for the first time using a large set of structurally diverse compounds covering a wide range of physiochemical properties and fraction unbound values. These results show that temperature has little effect on plasma protein binding and tissue binding and that the measured binding values at 4 °C are equivalent, on average, to those at physiological temperature (37 °C). The exception is indomethacin, where binding component(s) changed during long incubation at 37 °C. These data suggest that binding experiments can be conducted at 4 °C for unstable compounds in early drug discovery. Furthermore, plasma protein binding and tissue binding are likely to have little change during hypothermia conditions.
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- 2018
13. Novel aspects of iron homeostasis in pathogenic bloodstream form Trypanosoma brucei
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Lucy J. Cornell, Youssef Madbouly, Michele Tinti, Zhihao Lai, Calvin Tiengwe, Phillip A. Yates, Carla Gilabert Carbajo, and Wellcome Trust
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Protozoan Proteins ,RNA-binding protein ,Artificial Gene Amplification and Extension ,Biochemistry ,Polymerase Chain Reaction ,Transcriptome ,RNA interference ,1108 Medical Microbiology ,Antibiotics ,Medicine and Health Sciences ,Homeostasis ,Biology (General) ,Cells, Cultured ,chemistry.chemical_classification ,Protozoans ,0303 health sciences ,Secretory Pathway ,biology ,Chemistry ,Antimicrobials ,Messenger RNA ,030302 biochemistry & molecular biology ,RNA-Binding Proteins ,Eukaryota ,Drugs ,Adaptation, Physiological ,Endocytosis ,Cell biology ,Nucleic acids ,RNA silencing ,Genetic interference ,1107 Immunology ,Tetracyclines ,Cell Processes ,Epigenetics ,0605 Microbiology ,Research Article ,Trypanosoma ,QH301-705.5 ,Iron ,Immunology ,Trypanosoma brucei brucei ,Transferrin receptor ,Trypanosoma brucei ,Research and Analysis Methods ,Microbiology ,03 medical and health sciences ,Downregulation and upregulation ,Virology ,Microbial Control ,Receptors, Transferrin ,Genetics ,Trypanosoma Brucei ,Molecular Biology Techniques ,Molecular Biology ,030304 developmental biology ,Pharmacology ,Organisms ,Biology and Life Sciences ,Cell Biology ,RC581-607 ,biology.organism_classification ,Parasitic Protozoans ,Trypanosomiasis, African ,Transferrin ,RNA ,Parasitology ,Gene expression ,Immunologic diseases. Allergy ,Trypanosoma Brucei Gambiense - Abstract
Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms by which T. brucei subsequently handles iron remains enigmatic. Here, we analyse the transcriptome of T. brucei cultured in iron-rich and iron-poor conditions. We show that adaptation to iron-deprivation induces upregulation of TfR, a cohort of parasite-specific genes (ESAG3, PAGS), genes involved in glucose uptake and glycolysis (THT1 and hexokinase), endocytosis (Phosphatidic Acid Phosphatase, PAP2), and most notably a divergent RNA binding protein RBP5, indicative of a non-canonical mechanism for regulating intracellular iron levels. We show that cells depleted of TfR by RNA silencing import free iron as a compensatory survival strategy. The TfR and RBP5 iron response are reversible by genetic complementation, the response kinetics are similar, but the regulatory mechanisms are distinct. Increased TfR protein is due to increased mRNA. Increased RBP5 expression, however, occurs by a post-transcriptional feedback mechanism whereby RBP5 interacts with its own, and with PAP2 mRNAs. Further observations suggest that increased RBP5 expression in iron-deprived cells has a maximum threshold as ectopic overexpression above this threshold disrupts normal cell cycle progression resulting in an accumulation of anucleate cells and cells in G2/M phase. This phenotype is not observed with overexpression of RPB5 containing a point mutation (F61A) in its single RNA Recognition Motif. Our experiments shed new light on how T. brucei BSFs reorganise their transcriptome to deal with iron stress revealing the first iron responsive RNA binding protein that is co-regulated with TfR, is important for cell viability and iron homeostasis; two essential processes for successful proliferation., Author summary African trypanosomes are single-celled extracellular parasites of humans and animals relying on essential host nutrients for survival. They satisfy their iron needs by capturing host transferrin-bound iron using a surface-localised transferrin receptor (TfR) that is structurally distinct from its host counterpart. Little is known about the trypanosome response to fluctuations in host iron availability, with the exception of modulated TfR expression. We show that unlike other eukaryotes, at the transcriptome level, trypanosomes do not regulate iron-dependent enzymes as a mechanism to cope with iron deprivation. Instead, we identify a group of novel iron responsive trypanosome-specific genes, particularly an RNA Binding Protein RBP5 that is responsive to iron levels, albeit mediated by a distinct mechanism from TfR. We show that although RBP5 expression is elevated at the mRNA and protein levels, increased abundance above a maximum threshold is toxic. The trypanosome TfR has been suggested as a therapeutic target, but whether it is essential for optimal host colonisation is unclear. Our data demonstrate that trypanosomes efficiently import free iron from their environment independent of TfR suggesting that alternative iron uptake pathways exist, and that any therapeutic interventions targeting TfR must be evaluated with caution.
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- 2021
14. Purine-responsive expression of the
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M Haley, Licon and Phillip A, Yates
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Base Sequence ,Trypanosoma brucei brucei ,Protozoan Proteins ,Genomic Instability ,Culture Media ,Mutagenesis ,Purines ,parasitic diseases ,Nucleobase Transport Proteins ,Nucleic Acid Conformation ,Gene Regulation ,RNA, Messenger ,3' Untranslated Regions ,Leishmania donovani - Abstract
The ability to modulate gene expression in response to changes in the host environment is essential for survival of the kinetoplastid parasite Leishmania. Unlike most eukaryotes, gene expression in kinetoplastids is predominately regulated posttranscriptionally. Consequently, RNA-binding proteins and mRNA-encoded sequence elements serve as primary determinants of gene regulation in these organisms; however, few have defined roles in specific stress response pathways. Leishmania species cannot synthesize purines de novo and must scavenge these essential nutrients from the host. Leishmania have evolved a robust stress response to withstand sustained periods of purine scarcity during their life cycle. The purine nucleobase transporter LdNT3 is among the most substantially up-regulated proteins in purine-starved Leishmania donovani parasites. Here we report that the posttranslational stability of the LdNT3 protein is unchanged in response to purine starvation. Instead, LdNT3 up-regulation is primarily mediated by a 33-nucleotide-long sequence in the LdNT3 mRNA 3′ UTR that is predicted to adopt a stem–loop structure. Although this sequence is highly conserved within the mRNAs of orthologous transporters in multiple kinetoplastid species, putative stem–loops from L. donovani and Trypanosoma brucei nucleobase transporter mRNAs were not functionally interchangeable for purine-responsive regulation. Through mutational analysis of the element, we demonstrate that species specificity is attributable to just three variant bases within the predicted loop. Finally, we provide evidence that the abundance of the trans-acting factor that binds the LdNT3 stem–loop in vivo is substantially higher than required for regulation of LdNT3 alone, implying a potential role in regulating other purine-responsive genes.
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- 2020
15. 11β-Hydroxysteroid Dehydrogenase 1 Human Tissue Distribution, Selective Inhibitor, and Role in Doxorubicin Metabolism
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Xin Yang, Wenyi Hua, Hui Zhang, Cheng Chang, Sangwoo Ryu, Phillip D. Yates, and Li Di
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Pharmacology ,chemistry.chemical_classification ,medicine.medical_specialty ,Kidney ,Carbonyl Reductase ,biology ,Chemistry ,Pharmaceutical Science ,Kidney metabolism ,Cytochrome P450 ,Dehydrogenase ,Metabolism ,030226 pharmacology & pharmacy ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,Enzyme ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Internal medicine ,medicine ,Microsome ,biology.protein ,hormones, hormone substitutes, and hormone antagonists - Abstract
11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) is distributed mainly in the human liver, with no detectable levels in the intestine or kidney, based on a newly developed proteomic approach. 11β-HSD1 is mostly membrane-bound and retained in the liver microsomal fraction. Interindividual variability of 11β-HSD1 is relatively low, with about a 3-fold difference. A significant correlation was not observed between various demographic variables (ethnicity, gender, age, weight, smoking, and alcohol use) and 11β-HSD1 protein expression or activity based on data from 31 donors. PF-915275 has been identified as a selective 11β-HSD1 inhibitor with minimal effects on carbonyl reductase 1 and major cytochrome P450 enzymes. 11β-HSD1 has been shown, for the first time, to be involved in doxorubicin metabolism, accounting for approximately 30% of doxorubicinol formation in human hepatocytes.
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- 2018
16. Comparison of Species and Cell-Type Differences in Fraction Unbound of Liver Tissues, Hepatocytes, and Cell Lines
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Brian R. Holder, Dhirender Singh, Sangwoo Ryu, Keith Riccardi, Jian Lin, Rui Li, Phillip D. Yates, Brendon Kapinos, David A. Tess, George Chang, and Li Di
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Cell type ,Metabolic Clearance Rate ,Cell ,Pharmaceutical Science ,030226 pharmacology & pharmacy ,Mice ,03 medical and health sciences ,Dogs ,0302 clinical medicine ,Drug Discovery ,medicine ,Animals ,Humans ,Rats, Wistar ,Pharmacology ,Kidney ,Chemistry ,HEK 293 cells ,Embryonic stem cell ,Rats ,Macaca fascicularis ,HEK293 Cells ,medicine.anatomical_structure ,Liver ,Pharmaceutical Preparations ,Biochemistry ,Cell culture ,030220 oncology & carcinogenesis ,Hepatocyte ,Hepatocytes ,Intracellular - Abstract
Fraction unbound (fu) of liver tissue, hepatocytes, and other cell types is an essential parameter used to estimate unbound liver drug concentration and intracellular free drug concentration. fu,liver and fu,cell are frequently measured in multiple species and cell types in drug discovery and development for various applications. A comparison study of 12 matrices for fu,liver and fu,cell of hepatocytes in five different species (mouse, rat, dog, monkey, and human), as well as fu,cell of Huh7 and human embryonic kidney 293 cell lines, was conducted for 22 structurally diverse compounds with the equilibrium dialysis method. Using an average bioequivalence approach, our results show that the average difference in binding to liver tissue, hepatocytes, or different cell types was within 2-fold of that of the rat fu,liver Therefore, we recommend using rat fu,liver as a surrogate for liver binding in other species and cell types in drug discovery. This strategy offers the potential to simplify binding studies and reduce cost, thereby enabling a more effective and practical determination of fu for liver tissues, hepatocytes, and other cell types. In addition, fu under hepatocyte stability incubation conditions should not be confused with fu,cell, as one is a diluted fu and the other is an undiluted fu Cell density also plays a critical role in the accurate measurement of fu,cell.
- Published
- 2018
17. Comparing lamin proteins post-translational relative stability using a 2A peptide-based system reveals elevated resistance of progerin to cellular degradation
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Di Wu, Phillip A. Yates, Kan Cao, and Haoyue Zhang
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0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,Biology ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,Amino Acid Sequence ,Nuclear membrane ,lamin B1 ,Original Research ,lamin A ,Progeria ,integumentary system ,Protein Stability ,protein relative stability ,Wild type ,DNA replication ,Mesenchymal Stem Cells ,Cell Biology ,Fibroblasts ,Lamin Type A ,medicine.disease ,Progerin ,Molecular biology ,protein relative abundance ,Cell biology ,Chromatin ,030104 developmental biology ,medicine.anatomical_structure ,progerin ,Protein Biosynthesis ,FTI ,Proteolysis ,embryonic structures ,Nuclear lamina ,Peptides ,030217 neurology & neurosurgery ,Lamin - Abstract
Nuclear lamins are the major components of the nuclear lamina at the periphery of the nucleus, supporting the nuclear envelope and participating in many nuclear processes, including DNA replication, transcription and chromatin organization. A group of diseases, the laminopathies, is associated with mutations in lamin genes. One of the most striking cases is Hutchinson-Gilford progeria syndrome (HGPS) which is the consequence of a lamin A dominant negative mutant named progerin. Due to the abnormal presence of a permanent C-terminal farnesyl tail, progerin gradually accumulates on the nuclear membrane, perturbing a diversity of signalings and transcriptional events. The accumulation of progerin has led to the speculation that progerin possesses higher stability than the wild type lamin A protein. However, the low solubility of lamin proteins renders traditional immunoprecipitation-dependent methods such as pulse-chase analysis ineffective for comparing the relative stabilities of mutant and wild type lamins. Here, we employ a novel platform for inferring differences in lamin stability, which is based on normalization to a co-translated reporter protein following porcine teschovirus-1 2A peptide-mediated co-translational cleavage. The results obtained using this method support the notion that progerin is more stable than lamin A. Moreover, treatment of FTI reduces progerin relative stability to the level of wild type lamin A.
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- 2016
18. Vaccinia Virus Vectors Targeting Peptides for MHC Class II Presentation to CD4
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Diana Ortiz, Phillip A. Yates, Scott M. Landfear, Samuel J. Hobbs, Jake C. Harbour, and Jeffrey C. Nolz
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CD4-Positive T-Lymphocytes ,T cell ,Immunology ,Epitopes, T-Lymphocyte ,Vaccinia virus ,Adaptive Immunity ,CD8-Positive T-Lymphocytes ,Epitope ,Article ,Mice ,Antigen ,medicine ,Immunology and Allergy ,Cytotoxic T cell ,Animals ,Antigens ,MHC class II ,biology ,Immunodominant Epitopes ,Histocompatibility Antigens Class II ,General Medicine ,T lymphocyte ,Acquired immune system ,Virology ,Mice, Inbred C57BL ,medicine.anatomical_structure ,biology.protein ,Peptides ,CD8 - Abstract
CD4+ helper T cells play important roles in providing help to B cells, macrophages, and cytotoxic CD8+ T cells, but also exhibit direct effector functions against a variety of different pathogens. In contrast to CD8+ T cells, CD4+ T cells typically exhibit broader specificities and undergo less clonal expansion during many types of viral infections, which often makes the identification of virus-specific CD4+ T cells technically challenging. In this study, we have generated recombinant vaccinia virus (VacV) vectors that target I-Ab–restricted peptides for MHC class II (MHC-II) presentation to activate CD4+ T cells in mice. Conjugating the lymphocytic choriomeningitis virus immunodominant epitope GP61–80 to either LAMP1 to facilitate lysosomal targeting or to the MHC-II invariant chain (Ii) significantly increased the activation of Ag-specific CD4+ T cells in vivo. Immunization with VacV-Ii-GP61–80 activated endogenous Ag-specific CD4+ T cells that formed memory and rapidly re-expanded following heterologous challenge. Notably, immunization of mice with VacV expressing an MHC-II–restricted peptide from Leishmania species (PEPCK335–351) conjugated to either LAMP1 or Ii also generated Ag-specific memory CD4+ T cells that underwent robust secondary expansion following a visceral leishmaniasis infection, suggesting this approach could be used to generate Ag-specific memory CD4+ T cells against a variety of different pathogens. Overall, our data show that VacV vectors targeting peptides for MHC-II presentation is an effective strategy to activate Ag-specific CD4+ T cells in vivo and could be used to study Ag-specific effector and memory CD4+ T cell responses against a variety of viral, bacterial, or parasitic infections.
- Published
- 2019
19. Efficacy and Safety of Danirixin (GSK1325756) Co-administered With Standard-of-Care Antiviral (Oseltamivir): A Phase 2b, Global, Randomized Study of Adults Hospitalized With Influenza
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Phillip J. Yates, Sumita Roy-Ghanta, G B Roberts, Michael L Washburn, Andrew J. Peat, Michael F. Parry, Yu Tao, Anuradha Madan, Shuguang Chen, Otis Barnum, and Micah T. McClain
- Subjects
0301 basic medicine ,Oseltamivir ,medicine.medical_specialty ,030106 microbiology ,Placebo ,law.invention ,Major Articles ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Randomized controlled trial ,neutrophils ,law ,Internal medicine ,medicine ,Clinical endpoint ,030212 general & internal medicine ,Adverse effect ,business.industry ,Confidence interval ,Clinical trial ,Infectious Diseases ,Oncology ,chemistry ,Tolerability ,CXCR2 antagonist ,danirixin ,business ,influenza ,hospitalization - Abstract
Background Excessive neutrophil migration has been correlated with influenza symptom severity. Danirixin (GSK1325756), a selective and reversible antagonist of C-X-C chemokine receptor 2, decreases neutrophil activation and transmigration to areas of inflammation. This study evaluated the efficacy and safety of intravenous (IV) danirixin co-administered with oseltamivir for the treatment of adults hospitalized with influenza. Methods In this phase 2b, double-blind, 3-arm study (NCT02927431), influenza-positive participants were randomized 2:2:1 to receive danirixin 15mg intravenously (IV) twice daily (bid) + oral oseltamivir 75mg bid (OSV), danirixin 50mg IV bid + OSV, or placebo IV bid + OSV, for up to 5 days. The primary endpoint was time to clinical response (TTCR). Results In total, 10 participants received study treatment (danirixin 15mg + OSV, n = 4; danirixin 50mg + OSV, n = 4; placebo + OSV, n = 2) before the study was terminated early due to low enrollment. All participants achieved a clinical response. Median (95% confidence interval) TTCR was 4.53 days (2.95, 5.71) for danirixin 15mg + OSV, 4.76 days (2.71, 5.25) for danirixin 50mg + OSV, and 1.33 days (0.71, 1.95) for placebo + OSV. Adverse events (AEs) were generally of mild or moderate intensity; no serious AEs were considered treatment-related. Interleukin-8 levels increased in nasal samples (using synthetic absorptive matrix strips) and decreased serum neutrophil-elastase–mediated degradation of elastin decreased in danirixin-treated participants, suggesting effective target engagement. Conclusions Interpretation of efficacy results is restricted by the low participant numbers. The safety and tolerability profile of danirixin was consistent with previous studies. Clinical trial information The registration data for the trial are in the ClinicalTrials.gov database, number NCT02927431, and in the EU Clinical Trials Register (https://www.clinicaltrialsregister.eu/) as GSK study 201023, EudraCT 2016-002512-40. Anonymized individual participant data and study documents can be requested for further research from www.clinicalstudydatarequest.com., In this phase 2b, randomized, placebo-controlled study of intravenous danirixin in hospitalized influenza patients, all participants achieved a clinical response; danirixin had an acceptable safety profile. Early termination due to low recruitment and small sample size limits interpretation.
- Published
- 2019
20. Myeloperoxidase inhibition in mice alters atherosclerotic lesion composition
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Andrew Robertson, Amit S. Kalgutkar, Eliza Bollinger, Leonard Buckbinder, Timothy M. Coskran, Rachel J. Roth Flach, Edmund J. Keliher, Christian Cortes, Chunyan Su, Albert M. Kim, Kevin P. Maresca, Alan C. Opsahl, and Phillip D. Yates
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0301 basic medicine ,Pathology ,030204 cardiovascular system & hematology ,Pathology and Laboratory Medicine ,Biochemistry ,Vascular Medicine ,White Blood Cells ,Mice ,0302 clinical medicine ,Animal Cells ,Acetamides ,Medicine and Health Sciences ,Myocardial infarction ,Receptor ,Immune Response ,Aorta ,Mice, Knockout ,Multidisciplinary ,biology ,Animal Models ,Lipids ,Plaque, Atherosclerotic ,Experimental Organism Systems ,Myeloperoxidase ,Medicine ,medicine.symptom ,Anatomy ,Cellular Types ,Research Article ,medicine.medical_specialty ,Imaging Techniques ,Immune Cells ,Science ,Immunology ,Inflammation ,Mouse Models ,Pyrimidinones ,Research and Analysis Methods ,Lesion ,03 medical and health sciences ,Model Organisms ,Signs and Symptoms ,Diagnostic Medicine ,medicine.artery ,Fluorescence Imaging ,medicine ,Animals ,Lipoprotein oxidation ,Nutrition ,Peroxidase ,Blood Cells ,business.industry ,Macrophages ,Biology and Life Sciences ,Cell Biology ,medicine.disease ,Atherosclerosis ,Diet ,030104 developmental biology ,Receptors, LDL ,Heart failure ,biology.protein ,Cardiovascular Anatomy ,Animal Studies ,Blood Vessels ,business ,Oils - Abstract
Myeloperoxidase (MPO) is a highly abundant protein within the neutrophil that is associated with lipoprotein oxidation, and increased plasma MPO levels are correlated with poor prognosis after myocardial infarct. Thus, MPO inhibitors have been developed for the treatment of heart failure and acute coronary syndrome in humans. 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide PF-06282999 is a recently described selective small molecule mechanism-based inactivator of MPO. Here, utilizing PF-06282999, we investigated the role of MPO to regulate atherosclerotic lesion formation and composition in the Ldlr-/- mouse model of atherosclerosis. Though MPO inhibition did not affect lesion area in Ldlr-/- mice fed a Western diet, reduced necrotic core area was observed in aortic root sections after MPO inhibitor treatment. MPO inhibition did not alter macrophage content in and leukocyte homing to atherosclerotic plaques. To assess non-invasive monitoring of plaque inflammation, [18F]-Fluoro-deoxy-glucose (FDG) was administered to Ldlr-/- mice with established atherosclerosis that had been treated with clinically relevant doses of PF-06282999, and reduced FDG signal was observed in animals treated with a dose of PF-06282999 that corresponded with reduced necrotic core area. These data suggest that MPO inhibition does not alter atherosclerotic plaque area or leukocyte homing, but rather alters the inflammatory tone of atherosclerotic lesions; thus, MPO inhibition could have utility to promote atherosclerotic lesion stabilization and prevent atherosclerotic plaque rupture.
- Published
- 2019
21. Randomized, Double-Blind, Placebo-Controlled Study of the Safety, Tolerability, and Clinical Effect of Danirixin in Adults With Acute, Uncomplicated Influenza
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Jill Walker, Michael L Washburn, Sumita Roy-Ghanta, Phillip J. Yates, Andrew J. Peat, Edward Kerwin, Gary Soucie, G B Roberts, Anuradha Madan, and Shuguang Chen
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0301 basic medicine ,safety ,medicine.medical_specialty ,Oseltamivir ,030106 microbiology ,Placebo-controlled study ,Placebo ,Major Articles ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,danirixin (DNX) ,Internal medicine ,Medicine ,CXC chemokine receptor 2 (CXCR2) antagonist ,030212 general & internal medicine ,Adverse effect ,business.industry ,Surrogate endpoint ,Incidence (epidemiology) ,Infectious Diseases ,Oncology ,Tolerability ,chemistry ,outpatient ,business ,influenza ,Viral load - Abstract
Background Danirixin (DNX), a selective and reversible CXC chemokine receptor 2 antagonist, inhibits neutrophil transmigration and activation. This study assessed the safety, tolerability, and clinical effect of DNX with and without oseltamivir (OSV) in adults with acute, uncomplicated influenza. Methods This was a placebo-controlled, double-blind, Phase IIa study. Participants (18–64 years) with influenza-like symptoms (onset ≤48 hours) and positive influenza rapid antigen test were randomized 2:1:2:1 to DNX, placebo, DNX+OSV, or OSV (75 mg each, administered twice daily for 5 days) and followed for 28 days. Primary endpoints included frequency of adverse events (AEs) and serious AEs (SAEs). The effect of DNX on virologic response and clinical effect on influenza symptoms were secondary endpoints. Results A total of 45 participants were enrolled, 35 of whom were confirmed influenza positive by polymerase chain reaction analysis. The highest incidence of AEs was in the placebo group (4 of 7, 57%), followed by the DNX+OSV (7 of 16, 44%), DNX (3 of 15, 20%), and OSV (0 of 7, 0%) groups. One SAE (T-wave abnormality) was reported in the DNX group (unrelated to treatment). No differences in viral load assessments were observed among treatment groups. Conclusions Danirixin treatment was well tolerated and did not impede viral clearance.
- Published
- 2018
22. An inferential framework for biological network hypothesis tests.
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Phillip D. Yates and Nitai D. Mukhopadhyay
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- 2013
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23. Leishmania-infected macrophages release extracellular vesicles that can promote lesion development
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Anna Gioseffi, Naixin Zhang, Phillip A. Yates, Kha Van, Timothy Hamerly, Rhoel R. Dinglasan, and Peter E. Kima
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Resource ,0301 basic medicine ,Angiogenesis ,Health, Toxicology and Mutagenesis ,Transgene ,Leishmania donovani ,Plant Science ,Biology ,Epithelial cell migration ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Lesion ,03 medical and health sciences ,0302 clinical medicine ,parasitic diseases ,medicine ,skin and connective tissue diseases ,Tube formation ,Ecology ,biology.organism_classification ,Leishmania ,Cell biology ,Blot ,030104 developmental biology ,030220 oncology & carcinogenesis ,sense organs ,medicine.symptom - Abstract
Macrophages infected with Leishmania donovani release extracellular vesicles that are composed of parasite and host-derived molecules that have the potential to induce vascular changes in tissues., Leishmania donovani infection of macrophages results in quantitative and qualitative changes in the protein profile of extracellular vesicles (EVs) released by the infected host cells. We confirmed mass spectrometry results orthogonally by performing Western blots for several Leishmania-infected macrophage-enriched EVs (LieEVs) molecules. Several host cell proteins in LieEVs have been implicated in promoting vascular changes in other systems. We also identified 59 parasite-derived proteins in LieEVs, including a putative L. donovani homolog of mammalian vasohibins (LdVash), which in mammals promotes angiogenesis. We developed a transgenic parasite that expressed an endogenously tagged LdVash/mNeonGreen (mNG) and confirmed that LdVash/mNG is indeed expressed in infected macrophages and in LieEVs. We further observed that LieEVs induce endothelial cells to release angiogenesis promoting mediators including IL-8, G-CSF/CSF-3, and VEGF-A. In addition, LieEVs induce epithelial cell migration and tube formation by endothelial cells in surrogate angiogenesis assays. Taken together, these studies show that Leishmania infection alters the composition of EVs from infected cells and suggest that LieEVs may play a role in the promotion of vascularization of Leishmania infections.
- Published
- 2020
24. A role for adenine nucleotides in the sensing mechanism to purine starvation inLeishmania donovani
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Jessica L. Martin, Nicola S. Carter, Audrey L. Fulwiler, Buddy Ullman, Maria B. Cassera, Dennis R. Koop, Jan M. Boitz, and Phillip A. Yates
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0301 basic medicine ,Purine ,chemistry.chemical_classification ,Guanine ,030106 microbiology ,Purine riboswitch ,Purine nucleoside phosphorylase ,Biology ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,Biochemistry ,Adenine nucleotide ,Nucleotide ,Purine metabolism ,Molecular Biology ,Nucleotide salvage - Abstract
Purine salvage by Leishmania is an obligatory nutritional process that impacts both cell viability and growth. Previously, we have demonstrated that the removal of purines in culture provokes significant metabolic changes that enable Leishmania to survive prolonged periods of purine starvation. In order to understand how Leishmania sense and respond to changes in their purine environment, we have exploited several purine pathway mutants, some in which adenine and guanine nucleotide metabolism is uncoupled. While wild type parasites grow in any one of a variety of naturally occurring purines, the proliferation of these purine pathway mutants requires specific types or combinations of exogenous purines. By culturing purine pathway mutants in high levels of extracellular purines that are either permissive or non-permissive for growth and monitoring for previously defined markers of the adaptive response to purine starvation, we determined that adaptation arises from a surveillance of intracellular purine nucleotide pools rather than from a direct sensing of the extracellular purine content of the environment. Specifically, our data suggest that perturbation of intracellular adenine-containing nucleotide pools provides a crucial signal for inducing the metabolic changes necessary for the long-term survival of Leishmania in a purine-scarce environment.
- Published
- 2016
25. RCMAT: a regularized covariance matrix approach to testing gene sets.
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Phillip D. Yates and Mark A. Reimers
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- 2009
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26. Contralateral HeRO Graft Insertion to Treat Severe Venous Hypertension and Preserve Arteriovenous Fistula Patency
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Jonathan P. O'Doherty, Pat Cain, Abigail Johnson, Phillip J. Yates, James Harding, and Karl A. Holden
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medicine.medical_specialty ,education.field_of_study ,business.industry ,Fistula ,Population ,Central venous pressure ,Arteriovenous fistula ,General Medicine ,030204 cardiovascular system & hematology ,medicine.disease ,030218 nuclear medicine & medical imaging ,Surgery ,03 medical and health sciences ,Stenosis ,0302 clinical medicine ,Superior vena cava ,Blood vessel prosthesis ,medicine ,Cardiology and Cardiovascular Medicine ,business ,education ,Internal jugular vein - Abstract
Background Central venous stenosis and occlusion (CVO) is an increasing problem in the growing hemodialysis population. Sequelae include loss of access, loss of sites suitable for future venous access, and venous hypertension. Endoluminal techniques are often unsuitable to treat long-standing stenoses, and open surgery confers higher morbidity and is not appropriate in many patients. Case We present a case of long-standing central venous stenosis with an ipsilateral functioning fistula but with significant symptoms and signs of venous hypertension. The stenosis was not considered appropriate for endoluminal treatment, and the patient was considered to be at too high risk for open surgery. The Hemodialysis Reliable Outflow (HeRO) (Merit Medical Systems, UT) device was used to bypass the fistula to the superior vena cava via the contralateral internal jugular vein. Conclusions This case demonstrates the utility of the HeRO device in cases of long-standing CVO necessitating contralateral bypass. This technique confers the benefits of open surgery while minimizing the associated risks.
- Published
- 2020
27. Multi-system bleeding risk with a cutaneous angiosarcoma at an arteriovenous fistula site
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Phillip J Yates, Abheesh R. Prasad, Dominic Heining, Andrew Bentall, Jonathan Senior, and Michael Thomas
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medicine.medical_specialty ,Poor prognosis ,business.industry ,030232 urology & nephrology ,Subdural haematoma ,Arteriovenous fistula ,Soft tissue ,030204 cardiovascular system & hematology ,medicine.disease ,Microbiology ,Bleeding diathesis ,03 medical and health sciences ,0302 clinical medicine ,Infectious Diseases ,Lung malignancy ,medicine ,Parasitology ,Angiosarcoma ,Radiology ,Differential diagnosis ,business - Abstract
We present a case of angiosarcoma at an arteriovenous fistula site in a non-immunocompromised patient presenting as a soft tissue swelling with associated findings suggestive of lung malignancy, metastases and bleeding diathesis. This patient died of an otherwise unexplained subdural haematoma. Given the ability of this tumour to metastasise early and the poor prognosis of angiosarcoma without adequate resection, this needs to be considered early in any differential diagnosis of soft tissue masses near an arteriovenous fistula.
- Published
- 2018
28. Regulation and biological function of a flagellar glucose transporter inLeishmania mexicana:a potential glucose sensor
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Scott M. Landfear, Shaden Kamhawi, Khoa D. Tran, Phillip A. Yates, Dayana Rodriguez-Contreras, Hamide Aslan, and Xiuhong Feng
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Recombinant Fusion Proteins ,Genes, Protozoan ,Leishmania mexicana ,Glucose Transport Proteins, Facilitative ,Protozoan Proteins ,Leishmaniasis, Cutaneous ,Biochemistry ,Cell Line ,Mice ,Genetics ,Animals ,Humans ,Amastigote ,Receptor ,Molecular Biology ,Ciliary membrane ,Mice, Inbred BALB C ,Autocommentaries ,biology ,Permease ,Glucose transporter ,biology.organism_classification ,Leishmania ,Cell biology ,Glucose ,Gene Expression Regulation ,Flagella ,Mutation ,Female ,Psychodidae ,Intracellular ,Biotechnology - Abstract
In Leishmania mexicana parasites, a unique glucose transporter, LmxGT1, is selectively targeted to the flagellar membrane, suggesting a possible sensory role that is often associated with ciliary membrane proteins. Expression of LmxGT1 is down-regulated ∼20-fold by increasing cell density but is up-regulated ∼50-fold by depleting glucose from the medium, and the permease is strongly down-regulated when flagellated insect-stage promastigotes invade mammalian macrophages and transform into intracellular amastigotes. Regulation of LmxGT1 expression by glucose and during the lifecycle operates at the level of protein stability. Significantly, a ∆lmxgt1 null mutant, grown in abundant glucose, undergoes catastrophic loss of viability when parasites deplete glucose from the medium, a property not exhibited by wild-type or add-back lines. These results suggest that LmxGT1 may function as a glucose sensor that allows parasites to enter the stationary phase when they deplete glucose and that in the absence of this sensor, parasites do not maintain viability when they run out of glucose. However, alternate roles for LmxGT1 in monitoring glucose availability are considered. The absence of known sensory receptors with defined ligands and biologic functions in Leishmania and related kinetoplastid parasites underscores the potential significance of these observations.
- Published
- 2014
29. Downstream effects of striatal-enriched protein tyrosine phosphatase reduction on RNA expression in vivo and in vitro
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Thomas A. Lanz, R. Gerwien, V.L. Reinhart, Phillip D. Yates, T. Nguyen, and Max Kuhn
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Male ,MAP Kinase Signaling System ,PTPN5 ,Gene Expression ,Protein tyrosine phosphatase ,Biology ,Hippocampus ,Small hairpin RNA ,Mice ,Gene expression ,Animals ,Humans ,Nerve Growth Factors ,Mice, Knockout ,Neurons ,Gene knockdown ,General Neuroscience ,Molecular biology ,Corpus Striatum ,Rats ,HEK293 Cells ,nervous system ,RNA ,NMDA receptor ,Phosphorylation ,Protein Tyrosine Phosphatases ,Signal transduction ,Signal Transduction - Abstract
Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific tyrosine phosphatase that has been shown to de-phosphorylate several key neuronal signaling proteins, including kinases (extracellular signal-regulated kinase (ERK1/2), FYN, PYK2) and glutamate receptor subunits (N-methyl-d-aspartate receptor subtype 2B (NR2B), glutamate receptor 2 (GLUR2)). Step knock-out mice have increased phosphorylation of these substrates in the brain, with potential functional consequences in synaptic plasticity and cognitive tasks. It is therefore of interest to identify the molecular pathways and downstream transcriptional targets that are impacted by Step knockdown. In the present study, striatal RNA samples from Step wild-type, knock-out and heterozygous mice were hybridized to Affymetrix microarray chips and evaluated for transcriptional changes between genotypes. Pathway analysis highlighted Erk signaling and multiple pathways related to neurotrophin signaling, neuronal development and synaptic transmission. Potential genes of interest identified by microarray were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) in the cortex and hippocampus, which shared several transcriptional alterations with the striatum. In order to evaluate Step knockdown in an in vitro system, a panel of genes were evaluated using qRT-PCR in rat cortical neurons that were transduced with lentivirus expressing short hairpin RNA against Step or a non-targeting control. Our data suggest that Step has a role in the expression of immediate early genes relevant to synaptic plasticity, in both in vitro and in vivo systems.
- Published
- 2014
30. Intravenous Zanamivir in Hospitalized Patients With Influenza
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Jose R. Romero, John S. Bradley, Bryan E. Adams, Roberta L. DeBiasi, Jeffrey L. Blumer, Denise Shortino, Phillip J. Yates, Marian G. Michaels, Go Yamamoto, Amanda Peppercorn, David W. Kimberlin, Barbara A. Pahud, Flor M. Munoz, G B Roberts, and Mohammad Hossain
- Subjects
0301 basic medicine ,Male ,medicine.medical_specialty ,Neutropenia ,Adolescent ,030106 microbiology ,Vital signs ,Antiviral Agents ,03 medical and health sciences ,Zanamivir ,Pharmacokinetics ,Internal medicine ,Influenza, Human ,medicine ,Humans ,Adverse effect ,Child ,Infusions, Intravenous ,business.industry ,Mortality rate ,Infant ,Viral Load ,medicine.disease ,Surgery ,Clinical trial ,Hospitalization ,Child, Preschool ,Pediatrics, Perinatology and Child Health ,Female ,business ,Viral load ,medicine.drug - Abstract
BACKGROUND: Children with severe influenza infection may require parenteral therapy if oral or inhaled therapies are ineffective or cannot be administered. Results from a study investigating intravenous (IV) zanamivir for the treatment of hospitalized infants and children with influenza are presented. METHODS: This phase II, open-label, multicenter, single-arm study assessed the safety of investigational IV zanamivir in hospitalized children with influenza. Safety outcomes included treatment-emergent adverse events (TEAEs), clinical laboratory measurements, and vital signs. Clinical outcomes, pharmacokinetics, and virologic efficacy data were collected as key secondary outcomes. RESULTS: In total, 71 children received treatment with investigational IV zanamivir (exposure comparable to 600 mg twice daily in adults). TEAEs and serious TEAEs (STEAEs) were reported in 51 (72%) and 15 (21%) patients, respectively. The mortality rate was 7%, and median durations of hospital and ICU stays were 6 and 7.5 days, respectively. No STEAEs or deaths were considered related to IV zanamivir treatment, and no patterns of TEAEs, laboratory abnormalities, or vital signs were observed. The mean zanamivir exposures from 34 patients with normal renal function who received 12 mg/kg, 14 mg/kg, or 600 mg of IV zanamivir ranged from 64.5 to 110 hour·µg/mL. The median change from baseline in the viral load was −1.81 log10 copies per mL after 2 days of treatment. CONCLUSIONS: The safety profile of IV zanamivir was favorable, with no drug-related STEAEs reported. The majority of children experienced virologic response and clinical improvement during the treatment course. Systemic zanamivir exposures in children were consistent with adults.
- Published
- 2017
31. Intravenous zanamivir or oral oseltamivir for hospitalised patients with influenza: an international, randomised, double-blind, double-dummy, phase 3 trial
- Author
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Helen A. Watson, Francisco M. Marty, Denise D Shortino, Sandeep Kumar Gupta, Joan Vidal-Puigserver, Petr Husa, Frédérique Jacobs, Esperanza Merino, Phillip J. Yates, Eduardo Rodríguez-Noriega, Marianne J. Chapman, Carol L. Clark, Amanda Peppercorn, and Denis Garot
- Subjects
0301 basic medicine ,Pulmonary and Respiratory Medicine ,Adult ,Male ,Oseltamivir ,medicine.medical_specialty ,Adolescent ,030106 microbiology ,Population ,Administration, Oral ,Pharmacology ,Antiviral Agents ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,Young Adult ,0302 clinical medicine ,Zanamivir ,Randomized controlled trial ,Double-Blind Method ,law ,Internal medicine ,Influenza, Human ,Clinical endpoint ,Medicine ,Humans ,030212 general & internal medicine ,Adverse effect ,education ,Aged ,Aged, 80 and over ,education.field_of_study ,biology ,business.industry ,virus diseases ,Middle Aged ,3. Good health ,Hospitalization ,Treatment Outcome ,Respiratory failure ,chemistry ,biology.protein ,Administration, Intravenous ,Female ,business ,Neuraminidase ,medicine.drug - Abstract
Background Neuraminidase inhibitors are effective for the treatment of acute uncomplicated influenza. However, there is an unmet need for intravenous treatment for patients admitted to hospital with severe influenza. We studied whether intravenous zanamivir was a suitable treatment in this setting. Methods In this international, randomised, double-blind, double-dummy, phase 3 trial, we recruited patients aged 16 years or older with severe influenza admitted to 97 hospitals from 26 countries. We randomly assigned patients (1:1:1 stratified by symptom onset
- Published
- 2017
32. A 'middle-out' approach to human pharmacokinetic predictions for OATP substrates using physiologically-based pharmacokinetic modeling
- Author
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Angela Wolford, Hugh A. Barton, Avijit Ghosh, Phillip D. Yates, Tristan S. Maurer, Keith Riccardi, and Rui Li
- Subjects
Pharmacology ,Physiologically based pharmacokinetic modelling ,Models, Statistical ,Computer science ,Estimation theory ,Pharmacology toxicology ,Pharmacokinetic modeling ,Organic Anion Transporters ,Context (language use) ,computer.software_genre ,Resampling ,Confidence Intervals ,Hepatocytes ,Humans ,Identifiability ,Pharmacokinetics ,Data mining ,Biological system ,computer ,Algorithms ,Cells, Cultured ,Middle out - Abstract
Physiologically based pharmacokinetic (PBPK) models provide a framework useful for generating credible human pharmacokinetic predictions from data available at the earliest, preclinical stages of pharmaceutical research. With this approach, the pharmacokinetic implications of in vitro data are contextualized via scaling according to independent physiological information. However, in many cases these models also require model-based estimation of additional empirical scaling factors (SFs) in order to accurately recapitulate known human pharmacokinetic behavior. While this practice clearly improves data characterization, the introduction of empirically derived SFs may belie the extrapolative power commonly attributed to PBPK. This is particularly true when such SFs are compound dependent and/or when there are issues with regard to identifiability. As such, when empirically-derived SFs are necessary, a critical evaluation of parameter estimation and model structure are prudent. In this study, we applied a global optimization method to support model-based estimation of a single set of empirical SFs from intravenous clinical data on seven OATP substrates within the context of a previously published PBPK model as well as a revised PBPK model. The revised model with experimentally measured unbound fraction in liver, permeability between liver compartments, and permeability limited distribution to selected tissues improved data characterization. We utilized large-sample approximation and resampling approaches to estimate confidence intervals for the revised model in support of forward predictions that reflect the derived uncertainty. This work illustrates an objective approach to estimating empirically-derived SFs, systematically refining PBPK model performance and conveying the associated confidence in subsequent forward predictions.
- Published
- 2014
33. Evaluation of the time-course of CYP Induction and impact on drug interaction risk assessment from the IQ Induction working group
- Author
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Kelly Nulick, Niresh Hariparsad, Jane R. Kenny, Barry Jones, Donald J. Tweedie, Theunis C. Goosen, Heidi J. Einolf, Shannon Dallas, David B. Buckley, Diane Ramsden, Josh Dekeyser, George Zhang, Jairam Palamanda, Phillip D. Yates, Mike Mohutsky, Amy Siu, and Simon Wong
- Subjects
Pharmacology ,medicine.medical_specialty ,business.industry ,Group (mathematics) ,Internal medicine ,Time course ,Pharmaceutical Science ,Medicine ,Pharmacology (medical) ,Drug interaction ,business ,Risk assessment - Published
- 2019
34. Substrate Inhibition of Uracil Phosphoribosyltransferase by Uracil Can Account for the Uracil Growth Sensitivity of Leishmania donovani Pyrimidine Auxotrophs
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Buddy Ullman, Phillip A. Yates, Isaac P. Forquer, Zachary N. Wilson, Johannes Elferich, Ujwal Shinde, Radika Soysa, and Michael K. Riscoe
- Subjects
Purine ,Pyrimidine ,Cations, Divalent ,Protozoan Proteins ,Phosphoribosyl Pyrophosphate ,Biology ,Biochemistry ,Thiouracil ,Substrate Specificity ,chemistry.chemical_compound ,Pyrimidine analogue ,parasitic diseases ,Enzyme Stability ,heterocyclic compounds ,Nucleotide ,Pentosyltransferases ,Uracil ,Molecular Biology ,Feedback, Physiological ,chemistry.chemical_classification ,Uracil phosphoribosyltransferase ,Temperature ,Cell Biology ,Hydrogen-Ion Concentration ,Recombinant Proteins ,Kinetics ,Metabolism ,Pyrimidines ,chemistry ,Spectrophotometry ,Mutation ,Pyrimidine metabolism ,Chromatography, Gel ,Fluorouracil ,Protein Multimerization ,Leishmania donovani - Abstract
The pathogenic protozoan parasite Leishmania donovani is capable of both de novo pyrimidine biosynthesis and salvage of pyrimidines from the host milieu. Genetic analysis has authenticated L. donovani uracil phosphoribosyltransferase (LdUPRT), an enzyme not found in mammalian cells, as the focal enzyme of pyrimidine salvage because all exogenous pyrimidines that can satisfy the requirement of the parasite for pyrimidine nucleotides are funneled to uracil and then phosphoribosylated to UMP in the parasite by LdUPRT. To characterize this unique parasite enzyme, LdUPRT was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Kinetic analysis revealed apparent Km values of 20 and 99 μM for the natural substrates uracil and phosphoribosylpyrophosphate, respectively, as well as apparent Km values 6 and 7 μM for the pyrimidine analogs 5-fluorouracil and 4-thiouracil, respectively. Size exclusion chromatography revealed the native LdUPRT to be tetrameric and retained partial structure and activity in high concentrations of urea. L. donovani mutants deficient in de novo pyrimidine biosynthesis, which require functional LdUPRT for growth, are hypersensitive to high concentrations of uracil, 5-fluorouracil, and 4-thiouracil in the growth medium. This hypersensitivity can be explained by the observation that LdUPRT is substrate-inhibited by uracil and 4-thiouracil, but 5-fluorouracil toxicity transpires via an alternative mechanism. This substrate inhibition of LdUPRT provides a protective mechanism for the parasite by facilitating purine and pyrimidine nucleotide pool balance and by sparing phosphoribosylpyrophosphate for consumption by the nutritionally indispensable purine salvage process.
- Published
- 2013
35. KHARON1 Mediates Flagellar Targeting of a Glucose Transporter in Leishmania mexicana and Is Critical for Viability of Infectious Intracellular Amastigotes
- Author
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Danielle P. Vieira, Johannes Elferich, Phillip A. Yates, Dayana Rodriguez-Contreras, Khoa D. Tran, Larry L. David, Scott M. Landfear, and Wandy L. Beatty
- Subjects
Monosaccharide Transport Proteins ,Leishmania mexicana ,Molecular Sequence Data ,Mutant ,Protozoan Proteins ,Flagellum ,medicine.disease_cause ,Biochemistry ,Chromatography, Affinity ,Animals, Genetically Modified ,parasitic diseases ,Protein targeting ,medicine ,Animals ,Amino Acid Sequence ,Molecular Biology ,Integral membrane protein ,DNA Primers ,Base Sequence ,Sequence Homology, Amino Acid ,biology ,musculoskeletal, neural, and ocular physiology ,fungi ,Kinetoplastida ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Transport protein ,Cell biology ,Protein Transport ,Glucose ,Membrane protein ,Flagella ,bacteria - Abstract
The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.
- Published
- 2013
36. Virus Susceptibility Analyses from a Phase IV Clinical Trial of Inhaled Zanamivir Treatment in Children Infected with Influenza
- Author
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Nalini Mehta, Phillip J. Yates, Margaret Tisdale, and Joseph Horton
- Subjects
Sequence analysis ,viruses ,Molecular Sequence Data ,Population ,Neuraminidase ,Hemagglutinin (influenza) ,Polymerase Chain Reaction ,Virus ,Microbiology ,Viral Proteins ,Zanamivir ,In vivo ,Drug Resistance, Viral ,Influenza, Human ,medicine ,Humans ,Pharmacology (medical) ,education ,Pharmacology ,chemistry.chemical_classification ,education.field_of_study ,biology ,Virology ,Amino acid ,Infectious Diseases ,Amino Acid Substitution ,chemistry ,Susceptibility ,Mutation ,biology.protein ,medicine.drug - Abstract
A zanamivir postapproval efficacy study was conducted in children ( n = 279) in Japan during three influenza seasons. Pharyngeal swab specimens ( n = 714) were obtained for detailed resistance analysis. From 371 cultured viruses, 3 viruses (A/H1N1) from two subjects showed reduced susceptibility to zanamivir at day 1 (before treatment), 1 had an N74S amino acid substitution (fold shift, 46), and 2 (day 1 and day 2) had a Q136K amino acid substitution (fold shifts, 292 and 301). Q136K was detected only in cultured virus and not in the swab. From the remaining 118 cultured viruses obtained during or after treatment with zanamivir, no shifts in virus susceptibility were detected. Neuraminidase (NA) population sequencing showed that viruses from 12 subjects had emergent amino acid substitutions, but 3 with susceptibility data were not zanamivir resistant. The remainder may be natural variants. Further analysis is planned. Hemagglutinin (HA) sequencing showed that viruses from 20 subjects had 9 HA amino acid substitutions that were previously implicated in resistance to neuraminidase inhibitors in in vitro assays or that were close to the receptor binding site. Their role in in vivo resistance appears to be less important but is not well understood. NA clonal sequence analysis was undertaken to determine if minority species of resistant viruses were present. A total of 1,682 clones from 90 subjects were analyzed. Single clones from 12 subjects contained amino acid substitutions close to the NA active site. It is unclear whether these single amino acid substitutions could have been amplified after drug pressure or are just chance mutations introduced during PCR.
- Published
- 2013
37. Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani
- Author
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Armando Jardim, Phillip A. Yates, Rona Strasser, Buddy Ullman, and Jan M. Boitz
- Subjects
Purine ,Purine-Pyrimidine Metabolism, Inborn Errors ,Leishmania donovani ,Biochemistry ,Microbiology ,Adenylosuccinate Synthase ,Mice ,Open Reading Frames ,chemistry.chemical_compound ,parasitic diseases ,medicine ,Animals ,RNA, Messenger ,Autistic Disorder ,Cloning, Molecular ,Adenylosuccinate lyase ,Purine metabolism ,Molecular Biology ,Adenylosuccinate lyase deficiency ,Mice, Inbred BALB C ,biology ,Macrophages ,Genetic Complementation Test ,Adenylosuccinate Lyase ,Adenylosuccinate synthase ,Cell Biology ,Purine/pyrimidine metabolism ,medicine.disease ,biology.organism_classification ,Kinetics ,Phenotype ,Metabolism ,Liver ,chemistry ,Purines ,Drug Design ,Mutation ,Adenylosuccinate ,biology.protein ,Leishmaniasis, Visceral ,Female ,Subcellular Fractions - Abstract
Leishmania are auxotrophic for purines, and consequently purine acquisition from the host is a requisite nutritional function for the parasite. Both adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) have been identified as vital components of purine salvage in Leishmania donovani, and therefore Δadss and Δasl null mutants were constructed to test this hypothesis. Unlike wild type L. donovani, Δadss and Δasl parasites in culture exhibited a profoundly restricted growth phenotype in which the only permissive growth conditions were a 6-aminopurine source in the presence of 2'-deoxycoformycin, an inhibitor of adenine aminohydrolase activity. Although both knock-outs showed a diminished capacity to infect murine peritoneal macrophages, only the Δasl null mutant was profoundly incapacitated in its ability to infect mice. The enormous discrepancy in parasite loads observed in livers and spleens from mice infected with either Δadss or Δasl parasites can be explained by selective accumulation of adenylosuccinate in the Δasl knock-out and consequent starvation for guanylate nucleotides. Genetic complementation of a Δasl lesion in Escherichia coli implied that the L. donovani ASL could also recognize 5-aminoimidazole-(N-succinylocarboxamide) ribotide as a substrate, and purified recombinant ASL displayed an apparent Km of ∼24 μm for adenylosuccinate. Unlike many components of the purine salvage pathway of L. donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania.
- Published
- 2013
38. Arginase Is Essential for Survival of Leishmania donovani Promastigotes but Not Intracellular Amastigotes
- Author
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Buddy Ullman, Tamara Olenyik, Sigrid C. Roberts, Kristie Dearman, Yuexin Li, Caslin Gilroy, Dustin K Paradis, Phillip A. Yates, Michael K. Riscoe, Jan M. Boitz, Jasmine Perdeh, and Madison Davis
- Subjects
0301 basic medicine ,polyamines ,Immunology ,Leishmania donovani ,Biology ,Ornithine Decarboxylase ,Microbiology ,Microbodies ,Ornithine decarboxylase ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Cytosol ,parasitic diseases ,Putrescine ,Animals ,Leishmania infantum ,Cells, Cultured ,Leishmania ,Mice, Inbred BALB C ,Cellular Microbiology: Pathogen-Host Cell Molecular Interactions ,Arginase ,Ornithine ,biology.organism_classification ,Spermidine ,030104 developmental biology ,Infectious Diseases ,chemistry ,Biochemistry ,biology.protein ,Leishmaniasis, Visceral ,Parasitology ,Female ,Spermidine synthase ,Polyamine - Abstract
Studies of Leishmania donovani have shown that both ornithine decarboxylase and spermidine synthase, two enzymes of the polyamine biosynthetic pathway, are critical for promastigote proliferation and required for maximum infection in mice. However, the importance of arginase (ARG), the first enzyme of the polyamine pathway in Leishmania , has not been analyzed in L. donovani . To test ARG function in intact parasites, we generated Δ arg null mutants in L. donovani and evaluated their ability to proliferate in vitro and trigger infections in mice. The Δ arg knockout was incapable of growth in the absence of polyamine supplementation, but the auxotrophic phenotype could be bypassed by addition of either millimolar concentrations of ornithine or micromolar concentrations of putrescine or by complementation with either glycosomal or cytosolic versions of ARG. Spermidine supplementation of the medium did not circumvent the polyamine auxotrophy of the Δ arg line. Although ARG was found to be essential for ornithine and polyamine synthesis, ornithine decarboxylase appeared to be the rate-limiting enzyme for polyamine production. Mouse infectivity studies revealed that the Δ arg lesion reduced parasite burdens in livers by an order of magnitude but had little impact on the numbers of parasites recovered from spleens. Thus, ARG is essential for proliferation of promastigotes but not intracellular amastigotes. Coupled with previous studies, these data support a model in which L. donovani amastigotes readily salvage ornithine and have some access to host spermidine pools, while host putrescine appears to be unavailable for salvage by the parasite.
- Published
- 2016
39. Feasibility of Singlet Analysis for Ligand Binding Assays: a Retrospective Examination of Data Generated Using the Gyrolab Platform
- Author
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Chad Ray, Aidong Wu, Kathleen Pelletier, Lindsay King, Yiqun Zhang, Allison Given Chunyk, Tracey Clark, Alison Joyce, Jo-Ann Wentland, Petar Pop-Damkov, Elizabeth A. Dreher, Laurie Tylaska, and Phillip D. Yates
- Subjects
Databases, Factual ,Statistics as Topic ,Cmax ,Pharmaceutical Science ,Ligands ,030226 pharmacology & pharmacy ,01 natural sciences ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Statistics ,Animals ,Singlet state ,Retrospective Studies ,Chemistry ,Ligand binding assay ,010401 analytical chemistry ,Replicate ,PK Parameters ,Haplorhini ,0104 chemical sciences ,Rats ,Standard curve ,Concordance correlation coefficient ,Pharmaceutical Preparations ,Feasibility Studies ,Analysis of variance ,Protein Binding - Abstract
There are many sources of analytical variability in ligand binding assays (LBA). One strategy to reduce variability has been duplicate analyses. With recent advances in LBA technologies, it is conceivable that singlet analysis is possible. We retrospectively evaluated singlet analysis using Gyrolab data. Relative precision of duplicates compared to singlets was evaluated using 60 datasets from toxicokinetic (TK) or pharmacokinetic (PK) studies which contained over 23,000 replicate pairs composed of standards, quality control (QC), and animal samples measured with 23 different bioanalytical assays. The comparison was first done with standard curve and QCs followed by PK parameters (i.e., Cmax and AUC). Statistical analyses were performed on combined duplicate versus singlets using a concordance correlation coefficient (CCC), a measurement used to assess agreement. Variance component analyses were conducted on PK estimates to assess the relative analytical and biological variability. Overall, 97.5% of replicate pairs had a %CV of11% and 50% of the results had a %CV of ≤1.38%. There was no observable bias in concentration comparing the first replicate with the second (CCC of 0.99746 and accuracy value of 1). The comparison of AUC and Cmax showed no observable difference between singlet and duplicate (CCC for AUC and Cmax0.99999). Analysis of variance indicated an AUC inter-subject variability 35.3-fold greater than replicate variability and 8.5-fold greater for Cmax. Running replicates from the same sample will not significantly reduce variation or change PK parameters. These analyses indicated the majority of variance was inter-subject and supported the use of a singlet strategy.
- Published
- 2016
40. A role for adenine nucleotides in the sensing mechanism to purine starvation in Leishmania donovani
- Author
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Jessica L, Martin, Phillip A, Yates, Jan M, Boitz, Dennis R, Koop, Audrey L, Fulwiler, Maria Belen, Cassera, Buddy, Ullman, and Nicola S, Carter
- Subjects
Guanine ,Adenine Nucleotides ,Purines ,Starvation ,Adenine ,Purine Nucleotides ,Guanine Nucleotides ,Leishmania donovani - Abstract
Purine salvage by Leishmania is an obligatory nutritional process that impacts both cell viability and growth. Previously, we have demonstrated that the removal of purines in culture provokes significant metabolic changes that enable Leishmania to survive prolonged periods of purine starvation. In order to understand how Leishmania sense and respond to changes in their purine environment, we have exploited several purine pathway mutants, some in which adenine and guanine nucleotide metabolism is uncoupled. While wild type parasites grow in any one of a variety of naturally occurring purines, the proliferation of these purine pathway mutants requires specific types or combinations of exogenous purines. By culturing purine pathway mutants in high levels of extracellular purines that are either permissive or non-permissive for growth and monitoring for previously defined markers of the adaptive response to purine starvation, we determined that adaptation arises from a surveillance of intracellular purine nucleotide pools rather than from a direct sensing of the extracellular purine content of the environment. Specifically, our data suggest that perturbation of intracellular adenine-containing nucleotide pools provides a crucial signal for inducing the metabolic changes necessary for the long-term survival of Leishmania in a purine-scarce environment.
- Published
- 2016
41. Phenotypic and genotypic analysis of influenza viruses isolated from adult subjects during a phase II study of intravenous zanamivir in hospitalised subjects
- Author
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Phillip J. Yates, Nalini Mehta, Amanda Peppercorn, Dawn S. Raimonde, Choy Y. Man, Helen M. Steel, and Henry H. Zhao
- Subjects
0301 basic medicine ,Adult ,Genotype ,030106 microbiology ,Virulence ,Phases of clinical research ,Neuraminidase ,Virus ,Microbiology ,03 medical and health sciences ,Inhibitory Concentration 50 ,Zanamivir ,Influenza A Virus, H1N1 Subtype ,Virology ,Drug Resistance, Viral ,Influenza, Human ,medicine ,Humans ,Pharmacology ,biology ,Influenza A Virus, H3N2 Subtype ,Sequence Analysis, DNA ,Phenotype ,Hospitalization ,030104 developmental biology ,Hemagglutinins ,biology.protein ,Administration, Intravenous ,Post treatment ,medicine.drug - Abstract
Intravenous zanamivir (IVZ) is a neuraminidase (NA) inhibitor (NAI) under investigation for the treatment of subjects hospitalised with influenza. The study included 130 symptomatic, hospitalised adults with influenza. Subjects received IVZ for 5-10 days. Viruses were cultured and analysed for susceptibility to zanamivir. Mean IC50s (n = 50) (±SD) for influenza A/H1N1pdm09, A/H3N2 and influenza B were 0.20 ± 0.06, 0.26 ± 0.07 and 1.61 ± 0.35 nM, respectively, and are comparable to data observed for sensitive isolates. A total of 185 NA and 180 haemagglutinin (HA) sequences were obtained from 123 subjects; the majority did not contain resistance substitutions. Four influenza A/H1N1pdm09 viruses from four subjects harboured NA resistance substitutions: three, Y155H, D199G and S247N, were present at Day 1 before IVZ exposure and the fourth, E119D/E, was detected at Post Treatment +5 Days but was not present at 5 other timepoints. Five subjects harboured virus with treatment-emergent NA substitutions not associated with resistance; N63D, V83A, W190C, M269K (A/H1N1pdm09) and R210K (A/H3N2). Viruses from fifteen subjects harboured HA resistance substitutions, (A/H1N1pdm09) one emerged during treatment: S162N (Day 5). Five viruses harboured treatment-emergent HA substitutions (A/H1N1pdm09) not associated with resistance: E81K, V108L, S164D, D168N and S185N. 10/92 subjects with A/H1N1pdm09 harboured a D222 HA substitution, which has been associated with increased virulence. The emergent substitutions are not associated with resistance but may have arisen due to selection pressure during IVZ treatment or by chance. In this study, there was evidence for resistance selection in a post treatment sample but the resistant variant did not persist in later visit samples.
- Published
- 2016
42. Statistical Methods for Drug Discovery
- Author
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Craig L. Hyde, Max Kuhn, and Phillip D. Yates
- Subjects
Computer science ,Drug discovery ,education ,Data science ,Statistician - Abstract
This chapter is a broad overview of the drug discovery process and areas where statistical input can have a key impact. The focus is primarily in a few key areas: target discovery, compound screening/optimization, and the characterization of important properties. Special attention is paid to working with assay data and phenotypic screens. A discussion of important skills for a nonclinical statistician supporting drug discovery concludes the chapter.
- Published
- 2016
43. Safety, Tolerability and Pharmacokinetics (PK) of Intravenous Zanamivir (IVZ) Treatment in Hospitalized Pediatric and Adolescent Patients with Influenza: A Phase II Open-Label, Multicenter, Single- Arm Study
- Author
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G B Roberts, David W. Kimberlin, Jeffrey L. Blumer, Amanda Peppercorn, Marian G. Michaels, Phillip J. Yates, Denise Shortino, Jose R. Romero, John S. Bradley, Go Yamamoto, Bryan Adams, and Mohammad Hossain
- Subjects
Pediatrics ,medicine.medical_specialty ,business.industry ,Safety tolerability ,Influenza a ,Infectious Diseases ,Zanamivir ,Oncology ,Pharmacokinetics ,medicine ,Open label ,business ,Single Arm Study ,medicine.drug - Published
- 2016
44. Nonconvergence/Improper Solution Problems With the Correlated-Trait Correlated-Method Parameterization of a Multitrait–Multimethod Matrix
- Author
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Phillip D. Yates and Levent Dumenci
- Subjects
Applied Mathematics ,Multitrait-multimethod matrix ,Sampling (statistics) ,Parameter space ,Education ,Correlation ,Matrix (mathematics) ,Goodness of fit ,Convergence (routing) ,Statistics ,Developmental and Educational Psychology ,Trait ,Applied Psychology ,Mathematics - Abstract
Estimation problems associated with the correlated-trait correlated-method (CTCM) parameterization of a multitrait–multimethod (MTMM) matrix are widely documented: the model often fails to converge; even when convergence is achieved, one or more of the parameter estimates are outside the admissible parameter space. In this study, the authors explore the potential contributions of two factors to the occurrence of nonconvergence and inadmissible solutions: (a) the minimum number of indicators per factor and (b) the deviation of the MTMM matrix from the CTCM model beyond sampling variability. Analyses of two published occupational preference data sets involving six traits and five methods and a simulation study show that the probability of obtaining problematic solutions diminishes as the minimum number of indicators per factor increases. Also, isolating factors contributing to model misfit beyond sampling variability reduces the probability of obtaining problematic solutions by one half. The implications of these findings for choosing a measurement design that improves the probability of achieving a proper solution in the analysis of an MTMM matrix using the CTCM parameterization are discussed.
- Published
- 2012
45. Genetic Dissection of Pyrimidine Biosynthesis and Salvage in Leishmania donovani
- Author
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Zachary N. Wilson, Jan M. Boitz, Caslin Gilroy, Buddy Ullman, and Phillip A. Yates
- Subjects
Pyrimidine ,Auxotrophy ,Molecular Sequence Data ,Carbamoyl-Phosphate Synthase (Ammonia) ,Biology ,Biochemistry ,Mice ,chemistry.chemical_compound ,parasitic diseases ,Animals ,Homeostasis ,Pentosyltransferases ,Phosphorylation ,Uracil ,Amastigote ,Leishmaniasis Vaccines ,Uridine ,Molecular Biology ,Mice, Inbred BALB C ,Uracil phosphoribosyltransferase ,Cell Biology ,Salvage enzyme ,Pyrimidines ,Metabolism ,chemistry ,Pyrimidine metabolism ,Macrophages, Peritoneal ,Leishmaniasis, Visceral ,Female ,Pyrimidine nucleoside salvage ,Leishmania donovani - Abstract
Protozoan parasites of the Leishmania genus express the metabolic machinery to synthesize pyrimidine nucleotides via both de novo and salvage pathways. To evaluate the relative contributions of pyrimidine biosynthesis and salvage to pyrimidine homeostasis in both life cycle stages of Leishmania donovani, individual mutant lines deficient in either carbamoyl phosphate synthetase (CPS), the first enzyme in pyrimidine biosynthesis, uracil phosphoribosyltransferase (UPRT), a salvage enzyme, or both CPS and UPRT were constructed. The Δcps lesion conferred pyrimidine auxotrophy and a growth requirement for medium supplementation with one of a plethora of pyrimidine nucleosides or nucleobases, although only dihydroorotate or orotate could circumvent the pyrimidine auxotrophy of the Δcps/Δuprt double knockout. The Δuprt null mutant was prototrophic for pyrimidines but could not salvage uracil or any pyrimidine nucleoside. The capability of the Δcps parasites to infect mice was somewhat diminished but still robust, indicating active pyrimidine salvage by the amastigote form of the parasite, but the Δcps/Δuprt mutant was completely attenuated with no persistent parasites detected after a 4-week infection. Complementation of the Δcps/Δuprt clone with either CPS or UPRT restored infectivity. These data establish that an intact pyrimidine biosynthesis pathway is essential for the growth of the promastigote form of L. donovani in culture, that all uracil and pyrimidine nucleoside salvage in the parasite is mediated by UPRT, and that both the biosynthetic and salvage pathways contribute to a robust infection of the mammalian host by the amastigote. These findings impact potential therapeutic design and vaccine strategies for visceral leishmaniasis.
- Published
- 2012
46. Ischemic preconditioning, retinal neuroprotection and histone deacetylase activities
- Author
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C. James Chou, Jie Fan, Dennis S. Rice, Phillip W. Yates, Nicole Cathleen Goodwin, Craig E. Crosson, and Oday Alsarraf
- Subjects
0301 basic medicine ,Male ,Blotting, Western ,Ischemia ,Pharmacology ,Biology ,Neuroprotection ,Histone Deacetylases ,Retina ,Article ,Histones ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,medicine ,Electroretinography ,Animals ,Ischemic Preconditioning ,Analysis of Variance ,medicine.diagnostic_test ,Caspase 3 ,Retinal ,HDAC8 ,Acetylation ,medicine.disease ,Immunohistochemistry ,Sensory Systems ,Rats ,Ophthalmology ,Disease Models, Animal ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Biochemistry ,Ischemic preconditioning ,Histone deacetylase - Abstract
Increased histone deacetylase (HDAC) activity and the resulting dysregulation of protein acetylation is an integral event in retinal degenerations associated with ischemia and ocular hypertension. This study investigates the role of preconditioning on the process of acetylation in ischemic retinal injury. Rat eyes were unilaterally subjected to retinal injury by 45 minutes of acute ischemia, and retinal neuroprotection induced by 5 minutes of an ischemic preconditioning (IPC) event. HDAC activity was evaluated by a fluorometric enzymatic assay with selective isoform inhibitors. Retinal localization of acetylated histone-H3 was determined by immunohistochemistry on retina cross sections. Cleaved caspase-3 level was evaluated by Western blots. Electroretinogram (ERG) analyses were used to assess differences in retinal function seven days following ischemic injury. In control eyes, analysis of HDAC isoforms demonstrated that HDAC1/2 accounted for 28.4 ± 1.6%, HDAC3 for 42.4 ± 1.5% and HDAC6 activity 27.3 ± 3.5% of total activity. Following ischemia, total Class-I HDAC activity increased by 21.2 ± 6.2%, and this increase resulted solely from a rise in HDAC1/2 activity. No change in HDAC3 activity was measured. Activity of Class-II HDACs and HDAC8 was negligible. IPC stimulus prior to ischemic injury also suppressed the rise in Class-I HDAC activity, cleaved caspase-3 levels, and increased acetylated histone-H3 in the retina. In control animals 7 days post ischemia, ERG a- and b-wave amplitudes were significantly reduced by 34.9 ± 3.1% and 42.4 ± 6.3%, respectively. In rats receiving an IPC stimulus, the ischemia-induced decline in ERG a- and b-wave amplitudes was blocked. Although multiple HDACs were detected in the retina, these studies provide evidence that hypoacetylation associated with ischemic injury results from the selective rise in HDAC1/2 activity and that neuroprotection induced by IPC is mediated in part by suppressing HDAC activity.
- Published
- 2015
47. The Leishmania donovani UMP Synthase Is Essential for Promastigote Viability and Has an Unusual Tetrameric Structure That Exhibits Substrate-controlled Oligomerization
- Author
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D. Radika Soysa, Steven E. Ealick, Buddy Ullman, Nicola S. Carter, Phillip A. Yates, Jarrod B. French, Jan M. Boitz, and Bailey Chang
- Subjects
Models, Molecular ,Orotate Phosphoribosyltransferase ,Stereochemistry ,Orotidine-5'-Phosphate Decarboxylase ,Protozoan Proteins ,Biochemistry ,Glycosome ,chemistry.chemical_compound ,Multienzyme Complexes ,Orotidine ,Transferase ,Phosphofructokinase 2 ,Protein Structure, Quaternary ,Molecular Biology ,biology ,Active site ,Cell Biology ,Lyase ,Enzyme structure ,Protein Structure, Tertiary ,chemistry ,Protein Structure and Folding ,biology.protein ,Orotate phosphoribosyltransferase ,Protein Multimerization ,Uridine Monophosphate ,Leishmania donovani - Abstract
The final two steps of de novo uridine 5'-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is an essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.
- Published
- 2011
48. Aberrantly silenced promoters retain a persistent memory of the silenced state after long-term reactivation
- Author
-
Jon A. Oyer, Mitchell S. Turker, Sarah Godsey, and Phillip A. Yates
- Subjects
Chromatin Immunoprecipitation ,Time Factors ,Health, Toxicology and Mutagenesis ,Adenine Phosphoribosyltransferase ,Decitabine ,Hydroxamic Acids ,Methylation ,Article ,Histones ,Mice ,Cell Line, Tumor ,Genetics ,Animals ,Gene silencing ,Promoter Regions, Genetic ,DNA Modification Methylases ,Molecular Biology ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Lysine ,Promoter ,DNA Methylation ,Molecular biology ,Chromatin ,Gene Expression Regulation, Neoplastic ,Histone Deacetylase Inhibitors ,Histone ,CpG site ,DNA methylation ,Azacitidine ,biology.protein ,CpG Islands ,Chromatin immunoprecipitation - Abstract
A hallmark of aberrant DNA methylation-associated silencing is reversibility. However, long-term stability of reactivated promoters has not been explored. To examine this issue, spontaneous reactivant clones were isolated from mouse embryonal carcinoma cells bearing aberrantly silenced Aprt alleles and re-silencing frequencies were determined as long as three months after reactivation occurred. Despite continuous selection for expression of the reactivated Aprt alleles, exceptionally high spontaneous re-silencing frequencies were observed. A DNA methylation analysis demonstrated retention of sporadic methylation of CpG sites in a protected region of the Aprt promoter in many reactivant alleles suggesting a role for these methylated sites in the re-silencing process. In contrast, a chromatin immunoprecipitation (ChIP) analysis for methyl-H3K4, acetyl-H3K9, and dimethyl-H3K9 levels failed to reveal a specific histone modification that could explain high frequency re-silencing. These results demonstrate that aberrantly silenced and reactivated promoters retain a persistent memory of having undergone the silencing process and suggest the failure to eliminate all CpG methylation as a potential contributing mechanism.
- Published
- 2011
49. Leishmania donovani Ornithine Decarboxylase Is Indispensable for Parasite Survival in the Mammalian Host
- Author
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Phillip A. Yates, Jan M. Boitz, Mary E. Wilson, Chelsey D. Kline, Sigrid C. Roberts, Upasna Gaur, and Buddy Ullman
- Subjects
Auxotrophy ,Immunology ,Leishmania donovani ,Ornithine Decarboxylase ,Microbiology ,Gene Expression Regulation, Enzymologic ,Host-Parasite Interactions ,Ornithine decarboxylase ,Mice ,chemistry.chemical_compound ,Putrescine ,medicine ,Animals ,Amastigote ,Gene knockout ,Infectivity ,Mice, Inbred BALB C ,biology ,Macrophages ,biology.organism_classification ,medicine.disease ,Infectious Diseases ,Visceral leishmaniasis ,chemistry ,Biochemistry ,Leishmaniasis, Visceral ,Female ,Parasitology ,Fungal and Parasitic Infections ,Polyamine ,Gene Deletion - Abstract
Mutations within the polyamine biosynthetic pathway of Leishmania donovani , the etiological agent of visceral leishmaniasis, confer polyamine auxotrophy to the insect vector or promastigote form of the parasite. However, whether the infectious or amastigote form of the parasite requires an intact polyamine pathway has remained an open question. To address this issue, conditionally lethal Δ odc mutants lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, were created by double targeted gene replacement within a virulent strain of L. donovani . ODC-deficient promastigotes and axenic amastigotes were auxotrophic for polyamines and capable of robust growth only when exogenous putrescine was supplied in the culture medium, confirming that polyamine biosynthesis is an essential nutritional pathway for L. donovani promastigotes. To assess whether the Δ odc lesion also affected the ability of amastigotes to sustain a robust infection, macrophage and mouse infectivity experiments were performed. Parasite loads in murine macrophages infected with each of two independent Δ odc knockout lines were decreased ∼80% compared to their wild-type counterpart. Furthermore, α-difluoromethylornithine, a suicide inhibitor of ODC, inhibited growth of wild-type L. donovani amastigotes and effectively cured macrophages of parasites, thereby preventing host cell destruction. Strikingly, however, parasitemias of both Δ odc null mutants were reduced by 6 and 3 orders of magnitude, respectively, in livers and spleens of BALB/c mice. The compromised infectivity phenotypes of the Δ odc knockouts in both macrophages and mice were rescued by episomal complementation of the genetic lesion. These genetic and pharmacological studies strongly implicate ODC as an essential cellular determinant that is necessary for the viability and growth of both L. donovani promastigotes and amastigotes and intimate that pharmacological inhibition of ODC is a promising therapeutic paradigm for the treatment of visceral and perhaps other forms of leishmaniasis.
- Published
- 2009
50. Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis
- Author
-
Buddy Ullman, Keirei Ri, Phillip A. Yates, and Cassandra S. Arendt
- Subjects
Protozoan Proteins ,Biophysics ,Mutagenesis (molecular biology technique) ,Crithidia fasciculata ,Biology ,medicine.disease_cause ,Biochemistry ,Article ,Saccharomyces ,Gene Expression Regulation, Fungal ,Escherichia coli ,medicine ,Animals ,Cysteine ,Selection, Genetic ,Codon ,Saturated mutagenesis ,Molecular Biology ,Alanine ,chemistry.chemical_classification ,Mutation ,Guanosine ,Permease ,Cell Membrane ,Membrane Transport Proteins ,Cell Biology ,biology.organism_classification ,Inosine ,Amino acid ,chemistry ,Mutagenesis - Abstract
We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2 to facilitate biochemical studies using thiol-specific modifying reagents. Of 10 endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site saturation mutagenesis scheme based on the Stratagene Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in Saccharomyces cerevisiae cells auxotrophic for purines, several highly functional noncysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position.
- Published
- 2007
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