Publications by authors named "David J Holmes"

37 Publications

Left Atrial Appendage Closure and Systemic Homeostasis: The LAA HOMEOSTASIS Study.

J Am Coll Cardiol 2018 01;71(2):135-144

Mayo Clinic, Rochester, Minnesota.

Background: The impact of left atrial appendage (LAA) exclusion, comparing an epicardial LAA or an endocardial LAA device, on systemic homeostasis remains unknown.

Objectives: This study compared the effects of epicardial or endocardial LAA devices on the neurohormonal profiles of patients, emphasizing the roles of the renin-angiotensin-aldosterone system and the autonomic nervous system.

Methods: This is a prospective, single-center, observational study including 77 patients who underwent LAA closure by an epicardial (n = 38) or endocardial (n = 39) device. Key hormones involved in the adrenergic system (adrenaline, noradrenaline), renin-angiotensin-aldosterone system (aldosterone, renin), metabolic system (adiponectin, free fatty acids, insulin, β-hydroxybutyrate, and free glycerols), and natriuresis (atrial and B-type natriuretic peptides) were assessed immediately before the procedure, immediately after device deployment, at 24 h, and at 3 months follow-up.

Results: In the endocardial LAA device group, when compared with baseline blood adrenaline, noradrenaline and aldosterone were significantly lower at 24 h and 3 months (p < 0.05). There was no significant change in levels post-endocardial LAA device implantation. After epicardial LAA device implantation, there were significant increases in adiponectin and insulin, with decreased free fatty acids at 3 months. There was no significant change in these levels post-endocardial LAA device. N-terminal pro-A-type natriuretic peptide and N-terminal pro-B-type natriuretic peptide were significantly decreased in the acute phase after epicardial LAA device implantation, which subsequently normalized at 3 months. Post endocardial LAA device implantation, the levels increased immediately and normalized after 24 h. Systemic blood pressure was also significantly lower at all time points after epicardial LAA device implantation, which was not seen post-endocardial LAA device implantation.

Conclusions: There are substantial differences in hemodynamics and neurohormonal effects of LAA exclusion with epicardial and endocardial devices. Further studies are required to elucidate the underlying mechanism of these physiological changes.
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http://dx.doi.org/10.1016/j.jacc.2017.10.092DOI Listing
January 2018

Macrolones Are a Novel Class of Macrolide Antibiotics Active against Key Resistant Respiratory Pathogens In Vitro and In Vivo.

Antimicrob Agents Chemother 2016 09 22;60(9):5337-48. Epub 2016 Aug 22.

GlaxoSmithKline Research Centre Zagreb, Zagreb, Croatia.

As we face an alarming increase in bacterial resistance to current antibacterial chemotherapeutics, expanding the available therapeutic arsenal in the fight against resistant bacterial pathogens causing respiratory tract infections is of high importance. The antibacterial potency of macrolones, a novel class of macrolide antibiotics, against key respiratory pathogens was evaluated in vitro and in vivo MIC values against Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, and Haemophilus influenzae strains sensitive to macrolide antibiotics and with defined macrolide resistance mechanisms were determined. The propensity of macrolones to induce the expression of inducible erm genes was tested by the triple-disk method and incubation in the presence of subinhibitory concentrations of compounds. In vivo efficacy was assessed in a murine model of S. pneumoniae-induced pneumonia, and pharmacokinetic (PK) profiles in mice were determined. The in vitro antibacterial profiles of macrolones were superior to those of marketed macrolide antibiotics, including the ketolide telithromycin, and the compounds did not induce the expression of inducible erm genes. They acted as typical protein synthesis inhibitors in an Escherichia coli transcription/translation assay. Macrolones were characterized by low to moderate systemic clearance, a large volume of distribution, a long half-life, and low oral bioavailability. They were highly efficacious in a murine model of pneumonia after intraperitoneal application even against an S. pneumoniae strain with constitutive resistance to macrolide-lincosamide-streptogramin B antibiotics. Macrolones are the class of macrolide antibiotics with an outstanding antibacterial profile and reasonable PK parameters resulting in good in vivo efficacy.
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http://dx.doi.org/10.1128/AAC.00524-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4997857PMC
September 2016

Selective Spectrum Antibiotic Modulation of the Gut Microbiome in Obesity and Diabetes Rodent Models.

PLoS One 2015 28;10(12):e0145499. Epub 2015 Dec 28.

Computational Biology, Target Sciences, Research and Development, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America.

The gastrointestinal tract microbiome has been suggested as a potential therapeutic target for metabolic diseases such as obesity and Type 2 diabetes mellitus (T2DM). However, the relationship between changes in microbial communities and metabolic disease-phenotypes are still poorly understood. In this study, we used antibiotics with markedly different antibacterial spectra to modulate the gut microbiome in a diet-induced obesity mouse model and then measured relevant biochemical, hormonal and phenotypic biomarkers of obesity and T2DM. Mice fed a high-fat diet were treated with either ceftazidime (a primarily anti-Gram negative bacteria antibiotic) or vancomycin (mainly anti-Gram positive bacteria activity) in an escalating three-dose regimen. We also dosed animals with a well-known prebiotic weight-loss supplement, 10% oligofructose saccharide (10% OFS). Vancomycin treated mice showed little weight change and no improvement in glycemic control while ceftazidime and 10% OFS treatments induced significant weight loss. However, only ceftazidime showed significant, dose dependent improvement in key metabolic variables including glucose, insulin, protein tyrosine tyrosine (PYY) and glucagon-like peptide-1 (GLP-1). Subsequently, we confirmed the positive hyperglycemic control effects of ceftazidime in the Zucker diabetic fatty (ZDF) rat model. Metagenomic DNA sequencing of bacterial 16S rRNA gene regions V1-V3 showed that the microbiomes of ceftazidime dosed mice and rats were enriched for the phylum Firmicutes while 10% OFS treated mice had a greater abundance of Bacteroidetes. We show that specific changes in microbial community composition are associated with obesity and glycemic control phenotypes. More broadly, our study suggests that in vivo modulation of the microbiome warrants further investigation as a potential therapeutic strategy for metabolic diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145499PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692534PMC
July 2016

Frequency of Spontaneous Resistance to Peptide Deformylase Inhibitor GSK1322322 in Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae.

Antimicrob Agents Chemother 2015 Aug 26;59(8):4644-52. Epub 2015 May 26.

Antibacterial Discovery Performance Unit, Infectious Disease Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania, USA.

The continuous emergence of multidrug-resistant pathogenic bacteria is compromising the successful treatment of serious microbial infections. GSK1322322, a novel peptide deformylase (PDF) inhibitor, shows good in vitro antibacterial activity and has demonstrated safety and efficacy in human proof-of-concept clinical studies. In vitro studies were performed to determine the frequency of resistance (FoR) to this antimicrobial agent in major pathogens that cause respiratory tract and skin infections. Resistance to GSK1322322 occurred at high frequency through loss-of-function mutations in the formyl-methionyl transferase (FMT) protein in Staphylococcus aureus (4/4 strains) and Streptococcus pyogenes (4/4 strains) and via missense mutations in Streptococcus pneumoniae (6/21 strains), but the mutations were associated with severe in vitro and/or in vivo fitness costs. The overall FoR to GSK1322322 was very low in Haemophilus influenzae, with only one PDF mutant being identified in one of four strains. No target-based mutants were identified from S. pyogenes, and only one or no PDF mutants were isolated in three of the four S. aureus strains studied. In S. pneumoniae, PDF mutants were isolated from only six of 21 strains tested; an additional 10 strains did not yield colonies on GSK1322322-containing plates. Most of the PDF mutants characterized from those three organisms (35/37 mutants) carried mutations in residues at or in close proximity to one of three highly conserved motifs that are part of the active site of the PDF protein, with 30 of the 35 mutations occurring at position V71 (using the S. pneumoniae numbering system).
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http://dx.doi.org/10.1128/AAC.00484-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505285PMC
August 2015

Microbiome changes in healthy volunteers treated with GSK1322322, a novel antibiotic targeting bacterial peptide deformylase.

Antimicrob Agents Chemother 2015 Feb 8;59(2):1182-92. Epub 2014 Dec 8.

Computational Biology, GSK R&D, Collegeville, Pennsylvania, USA

GSK1322322 is a novel antibacterial agent under development, and it has known antibacterial activities against multidrug-resistant respiratory and skin pathogens through its inhibition of the bacterial peptide deformylase. Here, we used next-generation sequencing (NGS) of the bacterial 16S rRNA genes from stool samples collected from 61 healthy volunteers at the predosing and end-of-study time points to determine the effects of GSK1322322 on the gastrointestinal (GI) microbiota in a phase I, randomized, double-blind, and placebo-controlled study. GSK1322322 was administered either intravenously (i.v.) only or in an oral-i.v. combination in single- and repeat-dose-escalation infusions. Analysis of the 16S rRNA sequence data found no significant changes in the relative abundances of GI operational taxonomic units (OTUs) between the prestudy and end-of-study samples for either the placebo- or i.v.-only-treated subjects. However, oral-i.v. treatment resulted in significant decreases in some bacterial taxa, the Firmicutes and Bacteroidales, and increases in others, the Betaproteobacteria, Gammaproteobacteria, and Bifidobacteriaceae. Microbiome diversity plots clearly differentiated the end-of-study oral-i.v.-dosed samples from all others collected. The changes in genome function as inferred from species composition suggest an increase in bacterial transporter and xenobiotic metabolism pathways in these samples. A phylogenetic analysis of the peptide deformylase protein sequences collected from the published genomes of clinical isolates previously tested for GSK1322322 in vitro susceptibility and GI bacterial reference genomes suggests that antibiotic target homology is one of several factors that influences the response of GI microbiota to this antibiotic. Our study shows that dosing regimen and target class are important factors when considering the impact of antibiotic usage on GI microbiota. (This clinical trial was registered at the GlaxoSmithKline Clinical Study Register under study identifier PDF 113376.).
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http://dx.doi.org/10.1128/AAC.04506-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4335841PMC
February 2015

Bacterial resistance to leucyl-tRNA synthetase inhibitor GSK2251052 develops during treatment of complicated urinary tract infections.

Antimicrob Agents Chemother 2015 Jan 27;59(1):289-98. Epub 2014 Oct 27.

Computational Biology, Quantitative Sciences, GlaxoSmithKline R&D, Collegeville, Pennsylvania, USA

GSK2251052, a novel leucyl-tRNA synthetase (LeuRS) inhibitor, was in development for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. In a phase II study (study LRS114688) evaluating the efficacy of GSK2251052 in complicated urinary tract infections, resistance developed very rapidly in 3 of 14 subjects enrolled, with ≥32-fold increases in the GSK2251052 MIC of the infecting pathogen being detected. A fourth subject did not exhibit the development of resistance in the baseline pathogen but posttherapy did present with a different pathogen resistant to GSK2251052. Whole-genome DNA sequencing of Escherichia coli isolates collected longitudinally from two study LRS114688 subjects confirmed that GSK2251052 resistance was due to specific mutations, selected on the first day of therapy, in the LeuRS editing domain. Phylogenetic analysis strongly suggested that resistant Escherichia coli isolates resulted from clonal expansion of baseline susceptible strains. This resistance development likely resulted from the confluence of multiple factors, of which only some can be assessed preclinically. Our study shows the challenges of developing antibiotics and the importance of clinical studies to evaluate their effect on disease pathogenesis. (These studies have been registered at ClinicalTrials.gov under registration no. NCT01381549 for the study of complicated urinary tract infections and registration no. NCT01381562 for the study of complicated intra-abdominal infections.).
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http://dx.doi.org/10.1128/AAC.03774-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291364PMC
January 2015

Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

ACS Med Chem Lett 2013 Dec 18;4(12):1208-12. Epub 2013 Oct 18.

Astex Pharmaceuticals Inc., 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, United Kingdom.

Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase.
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http://dx.doi.org/10.1021/ml4003277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027552PMC
December 2013

Synthesis of macrolones with central piperazine ring in the linker and its influence on antibacterial activity.

Bioorg Med Chem 2011 Dec 21;19(23):7281-98. Epub 2011 Jul 21.

GlaxoSmithKline Research Centre Zagreb, Croatia.

Three macrolides, clarithromycin, azithromycin and 11-O-Me-azithromycin have been selected for the construction of a series of new macrolone derivatives. Quinolone-linker intermediates are prepared by Sonogashira-type C(6)-alkynylation of 6-iodoquinolone precursors. The final macrolones, differing by macrolide moiety and substituents at the position N-1 of the quinolone or by the presence of an ethyl ester or free acid on the quinolone unit attached via a linker. The linker comprises of a central piperazine ring bonded to the 4″-O position of cladinose by 3-carbon ester or ether functionality. Modifications of the linker did not improve antibacterial properties compared to the previously reported macrolone compounds. Linker flexibility seems to play an important role for potency against macrolide resistant respiratory pathogens.
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http://dx.doi.org/10.1016/j.bmc.2011.07.010DOI Listing
December 2011

Synthesis and activity of new macrolones: conjugates between 6(7)-(2'-aminoethyl)-amino-1-cyclopropyl-3-carboxylic acid (2'-hydroxyethyl) amides and 4″-propenoyl-azithromycin.

Bioorg Med Chem 2011 Dec 21;19(23):7270-80. Epub 2011 Jul 21.

GlaxoSmithKline Research Centre Zagreb Ltd, Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia.

A set of novel macrolones containing the flexible C8 basic linker and quinolone 3-(2'-hydroxyethyl)carboxamido group has been prepared and structurally characterized by NMR and IR spectroscopy, mass spectrometry and molecular modeling. The new compounds were evaluated in vitro against a panel of erythromycin-susceptible and erythromycin-resistant Gram-positive and Gram-negative bacterial strains. Compared to azithromycin, most of the compounds exhibited improved in vitro potency against the key respiratory pathogens.
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http://dx.doi.org/10.1016/j.bmc.2011.07.011DOI Listing
December 2011

Orientation masking and cross-orientation suppression (XOS): implications for estimates of filter bandwidth.

J Vis 2010 Oct 1;10(12). Epub 2010 Oct 1.

School of Life and Health Sciences, Aston University, Birmingham, UK.

Most contemporary models of spatial vision include a cross-oriented route to suppression (masking from a broadly tuned inhibitory pool), which is most potent at low spatial and high temporal frequencies (T. S. Meese & D. J. Holmes, 2007). The influence of this pathway can elevate orientation-masking functions without exciting the target mechanism, and because early psychophysical estimates of filter bandwidth did not accommodate this, it is likely that they have been overestimated for this corner of stimulus space. Here we show that a transient 40% contrast mask causes substantial binocular threshold elevation for a transient vertical target, and this declines from a mask orientation of 0° to about 40° (indicating tuning), and then more gently to 90°, where it remains at a factor of ∼4. We also confirm that cross-orientation masking is diminished or abolished at high spatial frequencies and for sustained temporal modulation. We fitted a simple model of pedestal masking and cross-orientation suppression (XOS) to our data and those of G. C. Phillips and H. R. Wilson (1984) and found the dependency of orientation bandwidth on spatial frequency to be much less than previously supposed. An extension of our linear spatial pooling model of contrast gain control and dilution masking (T. S. Meese & R. J. Summers, 2007) is also shown to be consistent with our results using filter bandwidths of ±20°. Both models include tightly and broadly tuned components of divisive suppression. More generally, because XOS and/or dilution masking can affect the shape of orientation-masking curves, we caution that variations in bandwidth estimates might reflect variations in processes that have nothing to do with filter bandwidth.
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http://dx.doi.org/10.1167/10.12.9DOI Listing
October 2010

Synthesis and properties of macrolones characterized by two ether bonds in the linker.

Bioorg Med Chem 2010 Sep 14;18(17):6578-88. Epub 2010 Jul 14.

GlaxoSmithKline Research Centre Zagreb Ltd, Prilaz baruna Filipovića 29, HR-10000 Zagreb, Croatia.

In this paper synthesis of macrolones 1-18 starting from azithromycin is reported. Two key steps in the construction of the linker between macrolide and quinolone moiety, are formation of central ether bond by alkylation of unactivated OH group, and formation of terminal C-C bond at 6-position of the quinolone unit. Due to the difficulty in formation of these two bonds the study of alternative synthetic methodologies and optimization of the conditions for the selected routes was required. Formation of C-4''-O-ether bond was completed by modified Michael addition, whereas O-alkylation via diazonium cation proved to be the most effective in formation of the central allylic or propargylic ether bond. Comparison of Heck and Sonogashira reaction revealed the former as preferred route to the C-C bond formation at C(6) position of the quinolone unit. Most of the target compounds exhibited highly favorable antibacterial activity against common respiratory pathogens, without significant cytotoxicity profile when tested in vitro on eukaryotic cell lines.
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http://dx.doi.org/10.1016/j.bmc.2010.07.007DOI Listing
September 2010

6-Alkylquinolone-3-carboxylic acid tethered to macrolides synthesis and antimicrobial profile.

Bioorg Med Chem 2010 Sep 3;18(17):6569-77. Epub 2010 Jul 3.

GlaxoSmithKline Research Centre Zagreb Ltd, Prilaz baruna Filipovića 29, HR-10000 Zagreb, Croatia.

Two series of clarithromycin and azithromycin derivatives with terminal 6-alkylquinolone-3-carboxylic unit with central ether bond in the linker were prepared and tested for antimicrobial activity. Quinolone-linker intermediates were prepared by Sonogashira-type C(6)-alkynylation of 6-iodo-quinolone precursors. In the last step, 4'' site-selective acylation of 2'-protected macrolides was completed with the EDC reagent, which selectively activated a terminal, aliphatic carboxylic group in dicarboxylic intermediates. Antimicrobial activity of the new series of macrolones is discussed. The most potent compound, 4''-O-{6-[3-(3-carboxy-1-ethyl-4-oxo-1,4-dihydroquinolin-6-yl)-propoxy]-hexanoyl}-azithromycin (10), is highly active against bacterial respiratory pathogens resistant to macrolide antibiotics and represents a promising lead for further investigation.
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http://dx.doi.org/10.1016/j.bmc.2010.06.048DOI Listing
September 2010

Synthesis and biological activity of 4''-O-acyl derivatives of 14- and 15-membered macrolides linked to omega-quinolone-carboxylic unit.

Bioorg Med Chem 2010 Sep 22;18(17):6547-58. Epub 2010 Jun 22.

GlaxoSmithKline Research Centre Zagreb Ltd, Prilaz baruna Filipovića 29, HR-10000 Zagreb, Croatia.

The synthesis and antimicrobial activity of a new class of macrolide antibiotics which consist of a macrolide scaffold and a quinolone unit covalently connected by an appropriate linker are described. Optimization of several synthetic steps and structural properties of lead compound 26 are discussed. Promising antibacterial properties of this compound and some of its analogues are reported.
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http://dx.doi.org/10.1016/j.bmc.2010.06.050DOI Listing
September 2010

Genetic characterization of Vga ABC proteins conferring reduced susceptibility to pleuromutilins in Staphylococcus aureus.

Antimicrob Agents Chemother 2008 Dec 6;52(12):4507-9. Epub 2008 Oct 6.

Department of Microbiology, ID-CEDD, GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA.

Retapamulin MICs of > or =2 microg/ml were noted for 6 of 5,676 S. aureus recent clinical isolates evaluated. The ABC proteins VgaAv and VgaA were found to be responsible for the reduced susceptibility to pleuromutilins exhibited by these six isolates.
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http://dx.doi.org/10.1128/AAC.00915-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2592886PMC
December 2008

Clinical and research issues regarding chronic advanced coronary artery disease: part I: Contemporary and emerging therapies.

Am Heart J 2008 Mar 31;155(3):418-34. Epub 2008 Jan 31.

Duke University, Durham, NC, USA.

The following report is based on a working group meeting about advanced coronary artery disease for patients with refractory ischemia who cannot receive revascularization. The aims were to review currently available treatment strategies, define unmet clinical needs, explore clinical trial design issues, and identify promising novel therapeutic targets and approaches for patients with chronic ischemia. The Working Group brought together medical experts in the management of refractory angina with representatives from regulatory agencies, Centers for Medicare and Medicaid Services, and industry. The meeting began with presentations reviewing the limitations of the current medical therapies and revascularization strategies and focused on lessons learned from past therapeutic attempts to optimize outcomes and on what are considered to be the most promising new approaches. Perspectives from clinical experts and from regulatory agencies were juxtaposed against needs and concerns of industry regarding development of new therapeutic strategies. This report presents the considerations and conclusions of the meeting on December 4-5, 2006. This document has been developed as a 2-part article, with contemporary and emerging therapies for advanced coronary artery disease reviewed first. Trial design, end points, and regulatory issues will be discussed in the second part of the article.
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http://dx.doi.org/10.1016/j.ahj.2007.12.004DOI Listing
March 2008

Selection for high-level telithromycin resistance in Staphylococcus aureus yields mutants resulting from an rplB-to-rplV gene conversion-like event.

Antimicrob Agents Chemother 2008 Mar 14;52(3):1156-8. Epub 2008 Jan 14.

Department of Microbiology, ID-CEDD, GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA.

While most Staphylococcus aureus telithromycin-resistant mutants isolated in this study possessed duplications within rplV (encoding ribosomal protein L22), four isolates possessed insertions within rplV that were identical to a portion of the gene rplB (encoding ribosomal protein L2). This novel type of mutation is the result of an apparent gene conversion-like event.
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http://dx.doi.org/10.1128/AAC.00923-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2258494PMC
March 2008

Contextual modulation involves suppression and facilitation from the center and the surround.

J Vis 2007 Mar 22;7(4). Epub 2007 Mar 22.

Neurosciences Research Institute, Aston University, Aston Triangle, Birmingham, UK.

In psychophysics, cross-orientation suppression (XOS) and cross-orientation facilitation (XOF) have been measured by investigating mask configuration on the detection threshold of a centrally placed patch of sine-wave grating. Much of the evidence for XOS and XOF comes from studies using low and high spatial frequencies, respectively, where the interactions are thought to arise from within (XOS) and outside (XOF) the footprint of the classical receptive field. We address the relation between these processes here by measuring the effects of various sizes of superimposed and annular cross-oriented masks on detection thresholds at two spatial scales (1 and 7 c/deg) and on contrast increment thresholds at 7 c/deg. A functional model of our results indicates the following (1) XOS and XOF both occur for superimposed and annular masks. (2) XOS declines with spatial frequency but XOF does not. (3) The spatial extent of the interactions does not scale with spatial frequency, meaning that surround-effects are seen primarily at high spatial frequencies. (4) There are two distinct processes involved in XOS: direct divisive suppression and modulation of self-suppression. (5) Whether XOS or XOF wins out depends upon their relative weights and mask contrast. These results prompt enquiry into the effect of spatial frequency at the single-cell level and place new constraints on image-processing models of early visual processing.
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http://dx.doi.org/10.1167/7.4.7DOI Listing
March 2007

Stepwise exposure of Staphylococcus aureus to pleuromutilins is associated with stepwise acquisition of mutations in rplC and minimally affects susceptibility to retapamulin.

Antimicrob Agents Chemother 2007 Jun 2;51(6):2048-52. Epub 2007 Apr 2.

UP1345, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, USA.

To assess their effects on susceptibility to retapamulin in Staphylococcus aureus, first-, second-, and third-step mutants with elevated MICs to tiamulin and other investigational pleuromutilin compounds were isolated and characterized through exposure to high drug concentrations. All first- and second-step mutations were in rplC, encoding ribosomal protein L3. Most third-step mutants acquired a third mutation in rplC. While first- and second-step mutations did cause an elevation in tiamulin and retapamulin MICs, a significant decrease in activity was not seen until a third mutation was acquired. All third-step mutants exhibited severe growth defects, and faster-growing variants arose at a high frequency from most isolates. These faster-growing variants were found to be more susceptible to pleuromutilins. In the case of a mutant with three alterations in rplC, the fast-growing variants acquired an additional mutation in rplC. In the case of fast-growing variants of isolates with two mutations in rplC and at least one mutation at an unmapped locus, one of the two rplC mutations reverted to wild type. These data indicate that mutations in rplC that lead to pleuromutilin resistance have a direct, negative effect on fitness. While reduction in activity of retapamulin against S. aureus can be seen through mutations in rplC, it is likely that target-specific resistance to retapamulin will be slow to emerge due to the need for three mutations for a significant effect on activity and the fitness cost of each mutational step.
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http://dx.doi.org/10.1128/AAC.01066-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891380PMC
June 2007

Remote facilitation in the Fourier domain.

Vision Res 2007 Apr 26;47(8):1112-9. Epub 2006 Dec 26.

School of Life and Health Sciences, Aston University, Birmingham B47ET, UK.

To explore spatial interactions between visual mechanisms in the Fourier domain we measured detection thresholds for vertical and horizontal sine-wave gratings (4.4 deg diameter) over a range of spatial frequencies (0.5-23 c/deg) in the presence of grating and plaid masks with component contrasts of 8%, orientations of +/-45 degrees and a spatial frequency of 1c/deg. The mask suppressed the target grating over a range of +/-1 octave, and the plaid produced more suppression than the grating, consistent with summation of mask components in a broadly tuned contrast gain pool. At greater differences in spatial frequency ( approximately 3 octaves), the plaid and grating masks both produced about 3 dB of facilitation (they reduced detection thresholds by a factor of about square root 2). At yet further distances ( approximately 4 octaves) the masks had no effect. The facilitation cannot be attributed to a reduction of uncertainty by the mask because (a) it occurs for mask components that have very different spatial frequencies and orientations from the test and (b) the large stimulus size and central fixation point mean there was no spatial uncertainty that could be reduced. We suggest the results are due to long-range sensory interactions (in the Fourier domain) between mask and test-channels. The effects could be due to either direct facilitation or disinhibition.
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http://dx.doi.org/10.1016/j.visres.2006.11.010DOI Listing
April 2007

Drugs for bad bugs: confronting the challenges of antibacterial discovery.

Nat Rev Drug Discov 2007 Jan 8;6(1):29-40. Epub 2006 Dec 8.

Infectious Diseases Centre of Excellence for Drug Discovery, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, USA.

The sequencing of the first complete bacterial genome in 1995 heralded a new era of hope for antibacterial drug discoverers, who now had the tools to search entire genomes for new antibacterial targets. Several companies, including GlaxoSmithKline, moved back into the antibacterials area and embraced a genomics-derived, target-based approach to screen for new classes of drugs with novel modes of action. Here, we share our experience of evaluating more than 300 genes and 70 high-throughput screening campaigns over a period of 7 years, and look at what we learned and how that has influenced GlaxoSmithKline's antibacterials strategy going forward.
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http://dx.doi.org/10.1038/nrd2201DOI Listing
January 2007

Spatial and temporal dependencies of cross-orientation suppression in human vision.

Proc Biol Sci 2007 Jan;274(1606):127-36

School of Life and Health Sciences, Aston University, Birmingham B47ET, UK.

A well-known property of orientation-tuned neurons in the visual cortex is that they are suppressed by the superposition of an orthogonal mask. This phenomenon has been explained in terms of physiological constraints (synaptic depression), engineering solutions for components with poor dynamic range (contrast normalization) and fundamental coding strategies for natural images (redundancy reduction). A common but often tacit assumption is that the suppressive process is equally potent at different spatial and temporal scales of analysis. To determine whether it is so, we measured psychophysical cross-orientation masking (XOM) functions for flickering horizontal Gabor stimuli over wide ranges of spatio-temporal frequency and contrast. We found that orthogonal masks raised contrast detection thresholds substantially at low spatial frequencies and high temporal frequencies (high speeds), and that small and unexpected levels of facilitation were evident elsewhere. The data were well fit by a functional model of contrast gain control, where (i) the weight of suppression increased with the ratio of temporal to spatial frequency and (ii) the weight of facilitatory modulation was the same for all conditions, but outcompeted by suppression at higher contrasts. These results (i) provide new constraints for models of primary visual cortex, (ii) associate XOM and facilitation with the transient magno- and sustained parvostreams, respectively, and (iii) reconcile earlier conflicting psychophysical reports on XOM.
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http://dx.doi.org/10.1098/rspb.2006.3697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1679878PMC
January 2007

Independent binocular integration for form and colour.

Vision Res 2006 Mar 14;46(5):665-77. Epub 2005 Jul 14.

Department of Psychology, Wolfson Research Institute, University of Durham, UK.

Although different features of an object are processed in anatomically distinct regions of the cerebral cortex, they often appear bound together in perception. Here, using binocular rivalry, we reveal that the awareness of form can occur independently from the awareness of colour. First, we report that, if both eyes briefly view a grating stimulus prior to the presentation of the same grating in one eye and an orthogonal grating in the other, subjects tend to report perceptual dominance of the non-primed grating. The primer was most effective when it was similar in orientation, spatial frequency and spatial phase to one of the rival images. Next, we showed that the process underlying the binocular integration of chromatic information was selectively influenced by the colour of a previously presented stimulus. We then combined these paradigms by using a primer that had the same colour as one rival stimulus, but the same form as the other stimulus. In this situation, we found that rival stimuli differing in form and colour can sometimes achieve states of dominance in which the chromatic information from one eye's image combines with the form of the other eye's image temporarily creating a binocular impression that corresponds with neither monocular component. Finally, we demonstrated that during continuous viewing of rival stimuli differing in form and colour, chromatic integration could occur independently of form rivalry. Paradoxically, however, we found that changes to the form of the stimulus had more of an influence on chromatic integration than on form rivalry. Together these phenomena show that the neural processes involved in integrating information from the two eyes can operate selectively on different stimulus features.
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http://dx.doi.org/10.1016/j.visres.2005.05.023DOI Listing
March 2006

Grating and plaid masks indicate linear summation in a contrast gain pool.

J Vis 2004 Dec 22;4(12):1080-9. Epub 2004 Dec 22.

Neurosciences Research Institute, Aston University, Birmingham, UK.

In human vision, the response to luminance contrast at each small region in the image is controlled by a more global process where suppressive signals are pooled over spatial frequency and orientation bands. But what rules govern summation among stimulus components within the suppressive pool? We addressed this question by extending a pedestal plus pattern mask paradigm to use a stimulus with up to three mask components: a vertical 1 c/deg pedestal, plus pattern masks made from either a grating (orientation = -45 degrees ) or a plaid (orientation = +/-45 degrees ), with component spatial frequency of 3 c/deg. The overall contrast of both types of pattern mask was fixed at 20% (i.e., plaid component contrasts were 10%). We found that both of these masks transformed conventional dipper functions (threshold vs. pedestal contrast with no pattern mask) in exactly the same way: The dipper region was raised and shifted to the right, but the dipper handles superimposed. This equivalence of the two pattern masks indicates that contrast summation between the plaid components was perfectly linear prior to the masking stage. Furthermore, the pattern masks did not drive the detecting mechanism above its detection threshold because they did not abolish facilitation by the pedestal (Foley, 1994). Therefore, the pattern masking could not be attributed to within-channel masking, suggesting that linear summation of contrast signals takes place within a suppressive contrast gain pool. We present a quantitative model of the effects and discuss the implications for neurophysiological models of the process.
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http://dx.doi.org/10.1167/4.12.7DOI Listing
December 2004

Genomic approaches to antibacterial discovery.

Methods Mol Biol 2004 ;266:231-59

Department of Microbiology, Microbial, Musculoskeletal, Proliferative Diseases Centr for Excellence and Drug Discovery, GlaxoSmithKline, Collegeville, PA, USA.

This chapter describes two key strategies for the discovery of new antibacterial agents and illustrates the critical role played by genomics in each. The first approach is genomic target-based screening. Comparative genomics and bioinformatics are used to identify novel, selective antibacterial targets of the appropriate antibacterial spectrum. Genetic technologies integral for the success of this approach, such as essentiality testing, are also described. An unprecedented number of novel targets have been discovered via this approach, and a plethora of examples are discussed. This section concludes with the case history of a target successfully progressed from identification by genomics, to high-throughput screening, and onto proof of concept in curing experimental infections. The second approach is based on screening for compounds with antibacterial activity and then employing a broad variety of newer technologies to identify the molecular target of the antibacterial agent. The advantage of this approach is that compounds already possess antibacterial activity, which is a property often challenging to engineer into molecules obtained from enzyme-based screening approaches. The recent development of novel biochemical and genomic technologies that facilitate identification and characterization of the mode of action of these agents has made this approach as attractive as the genomic target-based screening strategy.
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http://dx.doi.org/10.1385/1-59259-763-7:231DOI Listing
August 2004

Characterization of Streptococcus pneumoniae TrmD, a tRNA methyltransferase essential for growth.

J Bacteriol 2004 Apr;186(8):2346-54

Microbial, Musculoskeletal and Proliferative Diseases CEDD, GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA.

Down-regulation of expression of trmD, encoding the enzyme tRNA (guanosine-1)-methyltransferase, has shown that this gene is essential for growth of Streptococcus pneumoniae. The S. pneumoniae trmD gene has been isolated and expressed in Escherichia coli by using a His-tagged T7 expression vector. Recombinant protein has been purified, and its catalytic and physical properties have been characterized. The native enzyme displays a molecular mass of approximately 65,000 Da, suggesting that streptococcal TrmD is a dimer of two identical subunits. In fact, this characteristic can be extended to several other TrmD orthologs, including E. coli TrmD. Kinetic studies show that the streptococcal enzyme utilizes a sequential mechanism. Binding of tRNA by gel mobility shift assays gives a dissociation constant of 22 nM for one of its substrates, tRNA(Leu)(CAG). Other heterologous nonsubstrate tRNA species, like, tRNA (Thr)(GGT), tRNA(Phe), and tRNA (Ala)(TGC), bind the enzyme with similar affinities, suggesting that tRNA specificity is achieved via a postbinding event(s).
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC412112PMC
http://dx.doi.org/10.1128/jb.186.8.2346-2354.2004DOI Listing
April 2004

A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function.

J Mol Microbiol Biotechnol 2003 ;6(2):109-26

Microbial, Musculoskeletal and Proliferative Diseases CEDD, Collegeville, PA 17426, USA.

Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.
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http://dx.doi.org/10.1159/000076741DOI Listing
June 2004

Validation of antibacterial mechanism of action using regulated antisense RNA expression in Staphylococcus aureus.

FEMS Microbiol Lett 2004 Feb;231(2):177-84

Veterinary and Biomedical Sciences Department, University of Minnesota, 1971 Commonwealth Avenue, St. Paul, MN 55108, USA.

Validation of antibiotic mode of action in whole bacterial cells is a key step for antibiotic drug discovery. In this study, one potential drug target, enoyl-acyl carrier protein reductase (FabI), an essential enzyme in the fatty acid biosynthesis pathway, was used to evaluate the feasibility of using a regulated antisense RNA interference approach to determine antibiotic mode of action. Antisense isogenic strains expressing antisense RNA to fabI were created using a tetracycline-regulated vector in Staphylococcus aureus. We demonstrated that down-regulation of FabI expression by induction of fabI antisense RNA induces a conditional lethal phenotype. In contrast, partial down-regulation gives a viable cell with a significant increase in sensitivity to FabI-specific inhibitors (i.e., a sensitized phenotype). More importantly, the mode of action for novel FabI inhibitors has been confirmed using this genetic approach in whole cell assay. These results indicate that controlled antisense technology provides a robust tool for defining and tracking the mode of action of novel antibacterial agents.
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http://dx.doi.org/10.1016/S0378-1097(03)00931-5DOI Listing
February 2004

Identification of antimicrobial targets using a comprehensive genomic approach.

Pharmacogenomics 2004 Jan;5(1):101-13

Department of Microbiology, GlaxoSmithKline Pharmaceuticals Research and Development, Collegeville, PA 19462, USA.

Regulated antisense RNA enables the construction of a defined set of conditional growth-defective/lethal strains. In this study, we expanded the regulated antisense RNA interference technology and developed a high-throughput screening strategy to identify the potential drug targets of novel antimicrobials. To prove this concept, the specific antisense sublibrary of different essential open reading frames were pooled in the presence of an inducer, and treated with or without sublethal levels of mupirocin, triclosan, or gentamicin. Antisense RNA-expressing strains that were sensitized for increased susceptibility to the antibiotics were selectively detected via DNA subtractive hybridization, microarray, and whole-cell analyses. No strain was identified as supersensitive to gentamicin because there was no target-specific antisense strain in this sublibrary. In contrast, strains expressing antisense to isoleucine tRNA synthetase (ileS) and enoyl-[acyl-carrier-protein] reductase (fabI) were specifically identified as having increased susceptibility to mupirocin and triclosan, respectively. These results demonstrated that ileS and fabI antisense strains showed significant increases of susceptibility only to their specific inhibitors. This data demonstrates that a regulated antisense RNA expression library provides an effective tool to assist in the identification of potential targets for novel antibacterial agents.
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http://dx.doi.org/10.1517/phgs.5.1.101.25679DOI Listing
January 2004

New benzylidenethiazolidinediones as antibacterial agents.

Bioorg Med Chem Lett 2003 Nov;13(21):3771-3

Medicinal Chemistry Department, Microbial, Musculoskeletal and Proliferative Diseases, GlaxoSmithKline Pharmaceuticals, 1250 S. Collegeville Road, Collegeville, PA 19426, USA.

A novel benzylidenethiazolidinedione has been discovered with antimicrobial activity. Here, we present the results of a structure-activity study on this compound with respect to its antimicrobial activity.
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http://dx.doi.org/10.1016/j.bmcl.2003.07.010DOI Listing
November 2003

Horizontal transfer of drug-resistant aminoacyl-transfer-RNA synthetases of anthrax and Gram-positive pathogens.

EMBO Rep 2003 Jul;4(7):692-8

Bioinformatics Division, GlaxoSmithKline, 1250 South Collegeville Road, UP1345, Collegeville, Pennsylvania 19426, USA.

The screening of new antibiotics against several bacterial strains often reveals unexpected occurrences of natural drug resistance. Two examples of this involve specific inhibitors of Staphylococcus aureus isoleucyl-transfer-RNA synthetase 1 (IleRS1) and, more recently, Streptococcus pneumoniae methionyl-tRNA synthetase 1 (MetRS1). In both cases, resistance is due to the presence of a second gene that encodes another synthetase (IleRS2 or MetRS2). Here, we show that both S. pneumoniae MetRS2 and S. aureus IleRS2 have closely related homologues in the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax. Furthermore, similar to drug-resistant pathogens, strains of B. anthracis and its closest relative, B. cereus, also have wild-type ileS1 and metS1 genes. Clostridium perfringens, the causative agent of gangrene, also has two metS genes, whereas Oceanobacillus iheyensis isolated from deep-sea sediments has a single ileS2-type gene. This study shows the importance of understanding complex evolutionary networks of ancient horizontal gene transfer for the development of novel antibiotics.
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http://dx.doi.org/10.1038/sj.embor.embor881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1326320PMC
July 2003