Publications by authors named "Reading N"

67 Publications

Molecular heterogeneity of glucose-6-phosphate dehydrogenase deficiency in Gaza Strip Palestinians.

Blood Cells Mol Dis 2012 Oct 15-Dec 15;49(3-4):152-8. Epub 2012 Jul 5.

ARUP Laboratories and University of Utah School of Medicine, Salt Lake City, UT 84132-2408, USA.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting more than 500 million people worldwide, is one of the most common of inherited disorders. There are 186 G6PD mutations published, with mutational clustering within defined ethnic/racial groups. However comprehensive molecular characterization of ethnically associated G6PD mutants and their clinical implications are lacking.

Design And Methods: Eighty unrelated Palestinian children hospitalized for hemolysis were studied. G6PD activity was determined by quantitative spectrophotometry and G6PD mutations were analyzed by sequencing of gDNA.

Results: 65 of 80 children (81%) had G6PD deficiency, accounting for most of the hemolytic disease in this age group. G6PD Mediterranean(c.563T), African G6PD A-(c.202A/c.376G), and G6PD Cairo(c.404C) were common with relative allele frequencies of 0.33 [1], 0.26, and 0.18 respectively. Two other variants were discovered, G6PD Beverly Hills(c.1160A) mutation, and a novel G6PD missense mutation c.536G>A (Ser179Asn), designated G6PD "Gaza". Three samples exhibited enzyme deficiency without detectable exonic or exon/intron boundary mutations.

Conclusion: G6PD deficiency accounts for the majority of diagnoses for hemolysis in Palestinian children (81%), providing support for newborn G6PD deficiency screening programs. We report unanticipated molecular heterogeneity of G6PD variants among Gaza Strip Palestinians greater than reported in neighboring Arab populations. We report a high proportion of affected children with G6PD Cairo, which was observed previously in only a single Egyptian, and a novel mutation G6PD "Gaza".
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http://dx.doi.org/10.1016/j.bcmd.2012.06.003DOI Listing
March 2013

Gut immune maturation depends on colonization with a host-specific microbiota.

Cell 2012 Jun;149(7):1578-93

Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Gut microbial induction of host immune maturation exemplifies host-microbe mutualism. We colonized germ-free (GF) mice with mouse microbiota (MMb) or human microbiota (HMb) to determine whether small intestinal immune maturation depends on a coevolved host-specific microbiota. Gut bacterial numbers and phylum abundance were similar in MMb and HMb mice, but bacterial species differed, especially the Firmicutes. HMb mouse intestines had low levels of CD4(+) and CD8(+) T cells, few proliferating T cells, few dendritic cells, and low antimicrobial peptide expression--all characteristics of GF mice. Rat microbiota also failed to fully expand intestinal T cell numbers in mice. Colonizing GF or HMb mice with mouse-segmented filamentous bacteria (SFB) partially restored T cell numbers, suggesting that SFB and other MMb organisms are required for full immune maturation in mice. Importantly, MMb conferred better protection against Salmonella infection than HMb. A host-specific microbiota appears to be critical for a healthy immune system.
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http://dx.doi.org/10.1016/j.cell.2012.04.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3442780PMC
June 2012

Hemolysis and Mediterranean G6PD mutation (c.563 C>T) and c.1311 C>T polymorphism among Palestinians at Gaza Strip.

Blood Cells Mol Dis 2012 Apr 22;48(4):203-8. Epub 2012 Feb 22.

Associated Regional and University Pathologists, Inc., ARUP Laboratories and University of Utah, Salt Lake City, UT 84132-2408, USA.

Background: The G6PD c.563 C>T deficient mutation is endemic among Mediterranean populations but its clinical significance is not well delineated. We set up to estimate the proportion of G6PD deficient children presenting with hemolytic anemia at Al Nasser Pediatric Hospital at Gaza Strip, Palestine. We then established the prevalence of c.563T Mediterranean mutation and its linkage to c.1311 C>T polymorphism in this population.

Design And Methods: G6PD deficiency was identified in children presenting with hemolytic anemia at Al Nasser Pediatric Hospital by spectrophotometric measurement of G6PD activity. G6PD exon 6 and exon 11 were amplified from genomic DNA and evaluated for c.563T mutation by sequencing and the c.1311T polymorphism by restriction fragment analysis. Seventy X-chromosomes (60 males and 5 females) from G6PD deficient patients and 40 X-chromosomes from a control group known to be not G6PD deficient were tested.

Results: Over 80% of these children presenting with hemolytic anemia were G6PD deficient and 34% of these had the Mediterranean G6PD deficient variant. The allelic frequencies of Mediterranean c.563T and c.1311T polymorphisms among G6PD deficient patients were 0.33 and 0.38 respectively. The c.1311T polymorphism was linked in 95.2% of patients with the Mediterranean mutation, an allele frequency of 0.87, compared to the control non-G6PD deficient group with an allele frequency of 0.18.

Conclusions: We conclude that G6PD deficiency accounts for majority of hemolytic anemia encountered in Gaza children treated at Al Nasser Pediatric Hospital Emergency department. The Mediterranean mutation c.563T, while not accounting for a majority of G6PD deficiency, is common among G6PD deficient Gaza Strip Palestinians and is frequently, but not always, linked to the c.1311T polymorphism. This work provides a foundation for the population screening of Palestinians for G6PD deficiency and for investigations of ancestral origin of the Mediterranean variant in world populations.
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http://dx.doi.org/10.1016/j.bcmd.2012.01.007DOI Listing
April 2012

The starting lineup: key microbial players in intestinal immunity and homeostasis.

Front Microbiol 2011 7;2:148. Epub 2011 Jul 7.

Channing Laboratory, Department of Medicine, Brigham and Women's Hospital Boston, MA, USA.

The complexity of microbiota inhabiting the intestine is increasingly apparent. Delicate balance of numerous bacterial species can affect development of the immune system, how susceptible a host is to pathogenic organisms, and the auto-inflammatory state of the host. In the last decade, with the increased use of germ-free mice, gnotobiotic mice, and animal models in which a germ-free animal has been colonized with a foreign microbiota such as humanized mice, it has been possible to delineate relationships that specific bacteria have with the host immune system and to show what role they may play in overall host health. These models have not only allowed us to tease out the roles of individual species, but have also allowed the discovery and characterization of functionally unknown organisms. For example, segmented filamentous bacteria (SFB) have been shown to play a vital role in expansion of IL-17 producing cells. Prior to linking their key role in immune system development, little was known about these organisms. Bacteroides fragilis can rescue some of the immune defects of gnotobiotic mice after mono-colonization and have anti-inflammatory properties that can alleviate colitis and experimental allergic encephalitis in murine models. Additionally, Clostridium species have most recently been shown to expand regulatory T-cell populations leading to anti-inflammatory conditions. This review will highlight and summarize some of the major findings within the last decade concerning the role of select groups of bacteria including SFB, Clostridium, Bacteroides, Bifidobacterium, and Lactobacillus, and their impact on host mucosal immune systems.
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http://dx.doi.org/10.3389/fmicb.2011.00148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133820PMC
November 2011

Association of G6PD with lower haemoglobin concentration but not increased haemolysis in patients with sickle cell anaemia.

Br J Haematol 2010 Jul 9;150(2):218-25. Epub 2010 May 9.

Center for Sickle Cell Disease, Howard University, Washington, DC 20060, USA.

The genetic bases of the highly variable degrees of anaemia and haemolysis in persons with Hb SS are not fully known, but several studies have indicated that G6PD deficiency is not a factor. The G6PD(202A) and G6PD(376G) alleles and alpha-thalassaemia were determined by molecular genetic testing in 261 children and adolescents with Hb SS in a multicentre study. G6PD(202A,376G) (G6PD A-) was defined as hemizygosity for both alleles in males and homozygosity in females. Among the participants 41% were receiving hydroxycarbamide. The prevalence of G6PD(202A,376G) was 13.6% in males and 3.3% in females with an overall prevalence of 8.7%. G6PD(202A,376G) was associated with a 10 g/l decrease in haemoglobin concentration (P = 0.008) but not with increased haemolysis as measured by lactate dehydrogenase, bilirubin, aspartate-aminotransferase, reticulocyte count or a haemolytic component derived from these markers (P > 0.09). Similar results were found within a sub-group of children who were not receiving hydroxycarbamide. By comparison, single and double alpha-globin deletions were associated with progressively higher haemoglobin concentrations (P = 0.005 for trend), progressively lower values for haemolytic component (P = 0.007), and increased severe pain episodes (P < 0.001). In conclusion, G6PD(202A,376G) may be associated with lower haemoglobin concentration in sickle cell anaemia by a mechanism other than increased haemolysis.
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http://dx.doi.org/10.1111/j.1365-2141.2010.08215.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2906678PMC
July 2010

A transcriptome study of the QseEF two-component system and the QseG membrane protein in enterohaemorrhagic Escherichia coli O157 : H7.

Microbiology (Reading) 2010 Apr 7;156(Pt 4):1167-1175. Epub 2010 Jan 7.

Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9048, USA.

QseE is a sensor kinase that responds to epinephrine, sulfate and phosphate. QseE constitutes a two-component signalling system together with the QseF sigma(54)-dependent response regulator. Encoded within the same operon as qseEF is the qseG gene, which encodes a membrane protein involved in the translocation of a type III secretion effector protein of enterohaemorrhagic Escherichia coli (EHEC) into epithelial cells. The qseEGF genes also form an operon with the glnB gene, which encodes the E. coli nitrogen sensor PII protein. Here we report a transcriptome analysis comparing qseE, qseF andqseG single mutants with the wild-type strain. This study revealed that the proteins encoded by these genes play a modest but significant role in iron uptake. Although QseEFG regulate genes involved in nitrogen utilization, these proteins do not play a notable role in nitrogen metabolism. In addition, QseEFG regulate transcription of the rcsBC and phoPQ two-component systems, linking several signal transduction pathways. The similarity of the microarray profiles of these mutants also indicates that these proteins work together. These data indicate that QseEFG are involved in the regulation of virulence and metabolism in EHEC.
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http://dx.doi.org/10.1099/mic.0.033027-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889445PMC
April 2010

Evaluation of diagnostic tests for Clostridium difficile infection.

J Clin Microbiol 2010 Feb 23;48(2):606-8. Epub 2009 Dec 23.

Department of Microbiology, City Hospital, Dudley Road, Birmingham, United Kingdom.

We evaluated toxigenic Clostridium difficile detection by a lateral flow assay for antigen and toxin, an enzyme immunoassay, and two commercial PCR methods. Compared to the cell cytotoxicity neutralization assay and toxigenic culture, both toxin detection methods lacked sensitivity. PCR following combined antigen and toxin detection provided the most useful diagnostic information.
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http://dx.doi.org/10.1128/JCM.01579-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2815642PMC
February 2010

Life-threatening carotid haemorrhage following blunt trauma.

J Laryngol Otol 2010 Sep 22;124(9):1030-2. Epub 2009 Dec 22.

ENT Department, Whipps Cross University Hospital NHS Trust, London, UK.

Introduction: We report a case of internal carotid arterial damage following blunt neck trauma. This rare mechanism of injury demands a high index of suspicion to enable prompt specialist management.

Case Report: A 22-year-old man presented to hospital after sustaining blunt neck trauma. Rapid onset of stridor necessitated an emergency tracheostomy. Computed tomography angiography demonstrated a tear of the right internal carotid artery, which was repaired surgically.

Discussion: Blunt carotid vessel injury, although rare, has a high mortality rate. Mechanisms of injury include hyperextension and contralateral neck rotation, a direct blow to the vessel, and laceration by adjacent bony structures. The 'gold standard' investigation for suspected blunt carotid vessel injury is catheter angiography, although this carries a small risk of stroke. Computed tomography angiography is a less invasive, alternative investigation which has almost equivalent accuracy. The extent of damage to the vessel wall will dictate treatment. In our literature review, we discuss the presentation, investigation and different treatment modalities available.

Conclusion: This case highlights an unusual mechanism of carotid artery injury, with a delayed, potentially fatal presentation. Such injury demands a high index of suspicion, and confirmation with specific investigations. Management is hazardous and requires experienced personnel in all aspects of care.
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http://dx.doi.org/10.1017/S0022215109992465DOI Listing
September 2010

Angiogenic and inflammatory markers of cardiopulmonary changes in children and adolescents with sickle cell disease.

PLoS One 2009 Nov 23;4(11):e7956. Epub 2009 Nov 23.

Center for Sickle Cell Disease, Howard University, Washington, DC, United States of America.

Background: Pulmonary hypertension and left ventricular diastolic dysfunction are complications of sickle cell disease. Pulmonary hypertension is associated with hemolysis and hypoxia, but other unidentified factors are likely involved in pathogenesis as well.

Design And Methods: Plasma concentrations of three angiogenic markers (fibroblast growth factor, platelet derived growth factor-BB [PDGF-BB], vascular endothelial growth factor [VEGF]) and seven inflammatory markers implicated in pulmonary hypertension in other settings were determined by Bio-Plex suspension array in 237 children and adolescents with sickle cell disease at steady state and 43 controls. Tricuspid regurgitation velocity (which reflects systolic pulmonary artery pressure), mitral valve E/Edti ratio (which reflects left ventricular diastolic dysfunction), and a hemolytic component derived from four markers of hemolysis and hemoglobin oxygen saturation were also determined.

Results: Plasma concentrations of interleukin-8, interleukin-10 and VEGF were elevated in the patients with sickle cell disease compared to controls (P
Conclusions: Circulating concentrations of angiogenic and pro-Inflammatory markers are altered in sickle cell disease children and adolescents with elevated tricuspid regurgitation velocity, a subgroup that may be at risk for developing worsening pulmonary hypertension. Further studies to understand the molecular changes in these children are indicated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0007956PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2776981PMC
November 2009

The two-component system QseEF and the membrane protein QseG link adrenergic and stress sensing to bacterial pathogenesis.

Proc Natl Acad Sci U S A 2009 Apr 16;106(14):5889-94. Epub 2009 Mar 16.

Department of Microbiology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9048, USA.

Bacterial pathogens sense host cues to activate expression of virulence genes. Most of these signals are sensed through histidine kinases (HKs), which comprise the main sensory mechanism in bacteria. The host stress hormones epinephrine (Epi) and norepinephrine are sensed through the QseC HK, which initiates a complex signaling cascade to regulate virulence gene expression in enterohemorrhagic Escherichia coli (EHEC). Epi signaling through QseC activates expression of the genes encoding the QseEF 2-component system. QseE is an HK, and QseF is a response regulator. Here, we show that QseE is a second bacterial adrenergic receptor that gauges the stress signals Epi, sulfate, and phosphate. The qseEF genes are organized within an unusual operonic structure, in that a gene is encoded between qseE and qseF. This gene was renamed qseG, and it was shown to encode an outer membrane (OM) protein. EHEC uses a type III secretion system (TTSS) to translocate effector proteins to the epithelial cells that rearrange the host cytoskeleton to form pedestal-like structures that cup the bacterium. QseE, QseG, and QseF are necessary for pedestal formation. Although QseE and QseF are involved in the transcriptional control of genes necessary for pedestal formation, QseG is necessary for translocation of effectors into epithelial cells. QseG is an OM protein necessary for translocation of TTSS effectors that also works in conjunction with a 2-component signaling system that senses host stress signals.
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http://dx.doi.org/10.1073/pnas.0811409106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2667056PMC
April 2009

Concordance of assays designed for the quantification of JAK2V617F: a multicenter study.

Haematologica 2009 Jan 10;94(1):38-45. Epub 2008 Nov 10.

Laboratoire d'Hématologie, Centre Hospitalier Universitaire, Institut de Biologie, Bordeaux, France.

Background: Many different techniques have been designed for the quantification of JAK2V617F allelic burden, sometimes producing discrepant results.

Design And Methods: JAK2V617F quantification techniques were compared among 16 centers using 11 assays based on quantitative polymerase chain reaction (with mutation-specific primers or probes, or fluorescent resonance energy transfer/melting curve analysis), allele-specific polymerase chain reaction, conventional sequencing or pyrosequencing.

Results: A first series of blinded samples (granulocyte DNA, n=29) was analyzed. Seven assays (12 centers) reported values inside the mean +/- 2SD; the mean coefficient of variation was 31%. Sequencing techniques lacked sensitivity, and strong discrepancies were observed with four techniques, which could be attributed to inadequate standards or to different modes of expression of results. Indeed, quantification of JAK2V617F in relation to another control gene produced higher than expected values, suggesting the possibility of more than two JAK2 copies/cell. After calibration of assays with common 1% to 100% JAK2V617F standards (dilutions of UKE-1 cells in normal leukocytes), 14 centers tested ten new samples. JAK2V617F allelic burdens greater or equal than 1% were then reliably quantified by five techniques -- one allele specific-polymerase chain reaction and four TaqMan allele-specific quantitative polymerase chain reaction assays, including one previously giving results outside the mean +/- 2SD -- with a lower mean coefficient of variation (21%). Of these, only the two TaqMan allele-specific quantitative polymerase chain reaction assays with primer-based specificity could detect 0.2% JAK2V617F.

Conclusions: Techniques expressing the allelic burden as JAK2V617F/total JAK2 and using a common set of standards produced similar quantification results but with variable sensitivity. Calibration to a reference standard improved reproducibility.
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http://dx.doi.org/10.3324/haematol.13486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2625411PMC
January 2009

Targeting QseC signaling and virulence for antibiotic development.

Science 2008 Aug;321(5892):1078-80

Department of Microbiology, University of Texas (UT) Southwestern Medical Center, Dallas, TX 75390, USA.

Many bacterial pathogens rely on a conserved membrane histidine sensor kinase, QseC, to respond to host adrenergic signaling molecules and bacterial signals in order to promote the expression of virulence factors. Using a high-throughput screen, we identified a small molecule, LED209, that inhibits the binding of signals to QseC, preventing its autophosphorylation and consequently inhibiting QseC-mediated activation of virulence gene expression. LED209 is not toxic and does not inhibit pathogen growth; however, this compound markedly inhibits the virulence of several pathogens in vitro and in vivo in animals. Inhibition of signaling offers a strategy for the development of broad-spectrum antimicrobial drugs.
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http://dx.doi.org/10.1126/science.1160354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2605406PMC
August 2008

Missense mutation of the last nucleotide of exon 1 (G->C) of beta globin gene not only leads to undetectable mutant peptide and transcript but also interferes with the expression of wild allele.

Haematologica 2007 Dec;92(12):1715-6

Hematology Division, University of Utah School of Medicine, Salt Lake City, UT, USA.

Hemoglobin Monroe (beta globin G->C, codon 30) is a missense mutation. We could not detect either the mutant peptide or transcript in reticulocyte-enriched preparation and in expanded erythroid progenitor cells. By quantitative gene expression assay beta globin mRNA was found to be reduced by more than 70% in all heterozygous subjects with different haplotypes. We conclude that this mutation also interferes with expression of wild type allele.
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http://dx.doi.org/10.3324/haematol.11543DOI Listing
December 2007

A novel two-component signaling system that activates transcription of an enterohemorrhagic Escherichia coli effector involved in remodeling of host actin.

J Bacteriol 2007 Mar 12;189(6):2468-76. Epub 2007 Jan 12.

Dept. of Microbiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9048, USA.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for worldwide outbreaks of bloody diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. After colonizing the large intestine, EHEC forms attaching and effacing (AE) lesions on intestinal epithelial cells. These lesions cause destruction of the microvilli and elicit actin rearrangement to form pedestals that cup each bacterium individually. EHEC responds to a signal produced by the intestinal microbial flora, autoinducer-3 (AI-3), and the host hormones epinephrine and norepinephrine to activate transcription of the genes involved in AE lesion formation. These three signals, involved in interkingdom communication, are sensed by bacterial sensor kinases. Here we describe a novel two-component system, QseEF (quorum-sensing E. coli regulators E and F), which is part of the AI-3/epinephrine/norepinephrine signaling system. QseE is the sensor kinase and QseF the response regulator. The qseEF genes are cotranscribed, and transcription of qseEF is activated by epinephrine through the QseC sensor. A qseF mutant does not form AE lesions. QseF activates transcription of the gene encoding EspFu, an effector protein translocated to the host cell by the EHEC, which mimics a eukaryotic SH2/SH3 adapter protein to engender actin polymerization during pedestal formation. Expression of the espFu gene from a plasmid restored AE lesion formation to the qseF mutant, suggesting that lack of espFu expression in this mutant was responsible for the loss of pedestal formation. These findings suggest the QseEF is a two-component system involved in the regulation of AE lesion formation by EHEC.
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http://dx.doi.org/10.1128/JB.01848-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1899401PMC
March 2007

Detection of acquired Janus kinase 2 V617F mutation in myeloproliferative disorders by fluorescence melting curve analysis.

Mol Diagn Ther 2006 ;10(5):311-7

Associated Regional and University Pathologists (ARUP) Laboratories, Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA.

Background: The genetic lesion underlying the pathogenesis of chronic myeloproliferative disorders (MPDs) has been identified in the Janus kinase 2 (JAK2) gene. A point mutation in codon 617 causes a valine to phenylalanine substitution (V617F) in the JH2 autoinhibitory region of the protein, resulting in constitutive activation of the tyrosine kinase. The high prevalence of this conserved mutation in MPD makes it an excellent candidate as a diagnostic molecular marker.

Methods And Results: We report here the development and validation of a single oligonucleotide probe-based PCR approach using fluorescence melting curve analysis for point mutation detection in DNA derived from unfractionated peripheral blood samples. Using this assay and serial dilutions of an erythroleukemia cell line harboring the homozygous JAK2 V617F mutation, we successfully detected the mutation within a background of wild type sequences at a sensitivity of 2.5%. Our novel fluorescence probe-based assay was compared with allele-specific PCR-gel assay and sequencing techniques. Using the single probe assay, we examined 70 cases with a presumptive diagnosis of MPD, of which 38 (54%) yielded positive results for the presence of the JAK2 V617F mutation, and 92 follicular lymphoma cases, which were negative for the JAK2 V617F mutation. Additionally, the probe-based assay detected a previously unreported T>C base substitution at nucleotide 2342 (JAK2, codon 616), which was not detected by an allele-specific PCR assay.

Conclusion: The single fluorescent probe-based assay described here is a rapid, homogeneous, and robust method for the detection of the JAK2 V617F mutation with favorable performance characteristics that make it advantageous for clinical diagnosis.
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http://dx.doi.org/10.1007/BF03256206DOI Listing
December 2006

Quorum sensing: the many languages of bacteria.

FEMS Microbiol Lett 2006 Jan;254(1):1-11

Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9048, USA.

In the conventional view of prokaryotic existence, bacteria live unicellularly, with responses to external stimuli limited to the detection of chemical and physical signals of environmental origin. This view of bacteriology is now recognized to be overly simplistic, because bacteria communicate with each other through small 'hormone-like' organic compounds referred to as autoinducers. These bacterial cell-to-cell signaling systems were initially described as mechanisms through which bacteria regulate gene expression via cell density and, therefore, they have been collectively termed quorum sensing. The functions controlled by quorum sensing are varied and reflect the needs of a particular species of bacteria to inhabit a given niche. Three major quorum-sensing circuits have been described: one used primarily by Gram-negative bacteria, one used primarily by Gram-positive bacteria, and one that has been proposed to be universal.
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http://dx.doi.org/10.1111/j.1574-6968.2005.00001.xDOI Listing
January 2006

Expression of the Rho-family GTPase gene RHOF in lymphocyte subsets and malignant lymphomas.

Br J Haematol 2005 May;129(4):531-3

Department of Hematology, University of Utah, 50 North Medical Drive, Salt Lake City, UT 84132, USA.

We have studied the expression of RHOF, a member of the Rho-GTPase family, in an array of lymphoid cells and tissues. Previous microarray studies demonstrated RHOF upregulation in a subset of transformed follicular lymphomas. Real-time quantitative polymerase chain reaction evaluated RHOF expression in lymphocyte subpopulations, and normal and malignant lymphoid tissue. Cells and tissues of B-cell origin expressed higher RHOF levels than their T-cell counterparts. Neoplastic cells and tissues of B-cell origin expressed higher levels of RHOF than their benign cellular counterparts. Relatively elevated levels of RHOF were seen in lymphomas derived from germinal centre origin.
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http://dx.doi.org/10.1111/j.1365-2141.2005.05481.xDOI Listing
May 2005

Identification and mapping of self-assembling protein domains encoded by the Escherichia coli K-12 genome by use of lambda repressor fusions.

J Bacteriol 2004 Mar;186(5):1311-9

Department of Biochemistry and Biophysics and Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843-2128, USA.

Self-assembling proteins and protein fragments encoded by the Escherichia coli genome were identified from E. coli K-12 strain MG1655. Libraries of random DNA fragments cloned into a series of lambda repressor fusion vectors were subjected to selection for immunity to infection by phage lambda. Survivors were identified by sequencing the ends of the inserts, and the fused protein sequence was inferred from the known genomic sequence. Four hundred sixty-three nonredundant open reading frame-encoded interacting sequence tags (ISTs) were recovered from sequencing 2,089 candidates. These ISTs, which range from 16 to 794 amino acids in length, were clustered into families of overlapping fragments, identifying potential homotypic interactions encoded by 232 E. coli genes. Repressor fusions identified ISTs from genes in every protein-based functional category, but membrane proteins were underrepresented. The IST-containing genes were enriched for regulatory proteins and for proteins that form higher-order oligomers. Forty-eight (20.7%) homotypic proteins identified by ISTs are predicted to contain coiled coils. Although most of the IST-containing genes are identifiably related to proteins in other bacterial genomes, more than half of the ISTs do not have identifiable homologs in the Protein Data Bank, suggesting that they may include many novel structures. The data are available online at http://oligomers.tamu.edu/.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC344411PMC
http://dx.doi.org/10.1128/JB.186.5.1311-1319.2004DOI Listing
March 2004

5'-(RACE) identification of rare ALK fusion partner in anaplastic large cell lymphoma.

J Mol Diagn 2003 May;5(2):136-40

Institute for Clinical and Experimental Pathology, Associated Regional and University Pathologists Laboratories, Salt Lake City, Utah, USA.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1907320PMC
http://dx.doi.org/10.1016/S1525-1578(10)60463-1DOI Listing
May 2003

Using modifier -25.

Authors:
Nancy L Reading

Adv Nurse Pract 2002 May;10(5):28-9

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May 2002

Coding strategies for NPs.

Authors:
Nancy L Reading

Adv Nurse Pract 2002 Feb;10(2):22-3

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February 2002

Role of disulfide bonds in the stability of recombinant manganese peroxidase.

Biochemistry 2001 Jul;40(27):8161-8

Biotechnology Center, Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-4705, USA.

Phanerochaete chrysosporium manganese peroxidase (MnP) [isoenzyme H4] was engineered with additional disulfide bonds to provide structural reinforcement to the proximal and distal calcium-binding sites. This rational protein engineering investigated the effects of multiple disulfide bonds on the stabilization of the enzyme heme environment and oxidase activity. Stabilization of the heme environment was monitored by UV-visible spectroscopy based on the electronic state of the alkaline transition species of ferric and ferrous enzyme. The optical spectral data confirm an alkaline transition to hexacoordinate, low-spin heme species for native and wild-type MnP and show that the location of the engineered disulfide bonds in the protein can have significant effects on the electronic state of the enzyme. The addition of a single disulfide bond in the distal region of MnP resulted in an enzyme that maintained a pentacoordinate, high-spin heme at pH 9.0, whereas MnP with multiple engineered disulfide bonds did not exhibit an increase in stability of the pentacoordinate, high-spin state of the enzyme at alkaline pH. The mutant enzymes were assessed for increased stability by incubation at high pH. In comparison to wild-type MnP, enzymes containing engineered disulfide bonds in the distal and proximal regions of the protein retained greater levels of activity when restored to physiological pH. Additionally, when assayed for oxidase activity at pH 9.0, proteins containing engineered disulfide bonds exhibited slower rates of inactivation than wild-type MnP.
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http://dx.doi.org/10.1021/bi010440iDOI Listing
July 2001

Setting practice fees.

Authors:
N L Reading

J Med Pract Manage 2000 Jan-Feb;15(4):200-2

Reading Reimbursement Consultants, Salt Lake City, UT 84106, USA.

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June 2000

Engineering a disulfide bond in recombinant manganese peroxidase results in increased thermostability.

Biotechnol Prog 2000 May-Jun;16(3):326-33

Biotechnology Center, Utah State University, Logan, Utah 84322-4705, USA.

Manganese peroxidase (MnP) produced by Phanerochaete chrysosporium, which catalyzes the oxidation of Mn(2+) to Mn(3+) by hydrogen peroxide, was shown to be susceptible to thermal inactivation due to the loss of calcium [Sutherland, G. R. J.; Aust, S. D. Arch. Biochem. Biophys. 1996, 332, 128-134]. The recombinant enzyme, lacking glycosylation, was found to be more susceptible [Nie, G.; Reading, N. S.; Aust, S. D. Arch. Biochem. Biophys. 1999, 365, 328-334]. On the basis of the properties and structure of peanut peroxidase, we have engineered a disulfide bond near the distal calcium binding site of MnP by means of the double mutation A48C and A63C. The mutant enzyme had activity and spectral properties similar to those of native, glycosylated MnP. The thermostabilities of native, recombinant, and mutant MnP were studied as a function of temperature and pH. MnPA48C/A63C exhibited kinetics of inactivation similar to that of native MnP. The addition of calcium decreased the rate of thermal inactivation of the enzymes, while EGTA increased the rate of inactivation. Thermally treated MnPA48C/A63C mutant was shown to contain one calcium, and it retained a percentage of its original manganese oxidase activity; native and recombinant MnP were inactivated by the removal of calcium from the protein.
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http://dx.doi.org/10.1021/bp0000151DOI Listing
August 2000

A pilot Internet teaching project to support specialist medical training.

Hosp Med 1999 Dec;60(12):904-7

Department of Education, Anglia Polytechnic University, Chelmsford, Essex.

Regional training programmes involving specialist medical trainees at geographically separate sites lend themselves to distance learning methods. This paper describes the setting up and early evaluation of an internet-based project designed to support regional study days across North East Thames for respiratory medicine.
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http://dx.doi.org/10.12968/hosp.1999.60.12.1258DOI Listing
December 1999

Substrate specificity of lignin peroxidase and a S168W variant of manganese peroxidase.

Arch Biochem Biophys 2000 Jan;373(1):147-53

Biotechnology Center, Utah State University, Logan, Utah, 84322-4705, USA.

Lignin peroxidase (LiP) and manganese peroxidase (MnP) are structurally similar heme-containing enzymes secreted by white-rot fungi. Unlike MnP, which is only specific for Mn(2+), LiP has broad substrate specificity, but it is not known if this versatility is due to multiple substrate-binding sites. We report here that a S168W variant of MnP from Phanerochaete chrysosporium not only retained full Mn(2+) oxidase activity, but also, unlike native or recombinant MnP, oxidized a multitude of LiP substrates, including small molecule and polymeric substrates. The kinetics of oxidation of most nonpolymeric substrates by the MnP variant and LiP were similar. The stoichiometries for veratryl alcohol oxidation by these two enzymes were identical. Some readily oxidizable substrates, such as guaiacol and ferrocyanide, were oxidized by MnP S168W and LiP both specifically and nonspecifically while recombinant MnP oxidized these substrates only nonspecifically. The functional similarities between this MnP variant and LiP provide evidence for the broad substrate specificity of a single oxidation site near the surface tryptophan.
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http://dx.doi.org/10.1006/abbi.1999.1562DOI Listing
January 2000

Relative stability of recombinant versus native peroxidases from Phanerochaete chrysosporium.

Arch Biochem Biophys 1999 May;365(2):328-34

Biotechnology Center, Utah State University, Logan, Utah, 84322-4705, USA.

Two types of glycosylated peroxidases are secreted by the white-rot fungus Phanerochaete chrysosporium, lignin peroxidase (LiP) and manganese peroxidase (MnP). The thermal stabilities of recombinant LiPH2, LiPH8, and MnPH4, which were expressed without glycosylation in Escherichia coli, were lower than those of corresponding native peroxidases isolated from P. chrysosporium. Recovery of thermally inactivated recombinant enzyme activities was higher than with that of the thermally inactivated native peroxidases. Removal of N-linked glycans from native LiPH8 and MnPH4 did not affect enzyme activities or thermal stabilities of the enzymes. Although LiPH2, LiPH8, and MnPH4 contained O-linked glycans, only the O-linked glycans from MnPH4 could be removed by O-glycosidase, and the glycan-depleted MnPH4 exhibited essentially the same activity as nondeglycosylated MnPH4, but thermal stability decreased. Periodate-treated MnPH4 exhibited even lower thermal stability than O-glycosidase treated MnPH4. The role of O-linked glycans in protein stability was also evidenced with LiPH2 and LiPH8. Based on these data, we propose that neither N- nor O-linked glycans are likely to have a direct role in enzyme activity of native LiPH2, LiPH8, and MnPH4 and that only O-linked glycans may play a crucial role in protein stability of native peroxidases.
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http://dx.doi.org/10.1006/abbi.1999.1180DOI Listing
May 1999
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