Publications by authors named "Danielle Johnson"

30 Publications

  • Page 1 of 1

Targeting DNA Repair and Chromatin Crosstalk in Cancer Therapy.

Cancers (Basel) 2021 Jan 20;13(3). Epub 2021 Jan 20.

Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 84112, USA.

Aberrant DNA repair pathways that underlie developmental diseases and cancers are potential targets for therapeutic intervention. Targeting DNA repair signal effectors, modulators and checkpoint proteins, and utilizing the synthetic lethality phenomena has led to seminal discoveries. Efforts to efficiently translate the basic findings to the clinic are currently underway. Chromatin modulation is an integral part of DNA repair cascades and an emerging field of investigation. Here, we discuss some of the key advancements made in DNA repair-based therapeutics and what is known regarding crosstalk between chromatin and repair pathways during various cellular processes, with an emphasis on cancer.
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http://dx.doi.org/10.3390/cancers13030381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864178PMC
January 2021

Prevalence and predictors of weight loss during induction therapy for childhood acute lymphoblastic leukemia.

Nutrition 2021 01 3;81:110937. Epub 2020 Jul 3.

Department of Pediatric Hematology/Oncology, Cook Children's Health Care System, Fort Worth, Texas. Electronic address:

Objective: Children with acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma often experience significant weight gain during induction therapy. However, a subgroup of patients may experience weight loss, which can impact outcomes; thus, identifying and understanding this underrecognized concern is important. Our aim was to identify the prevalence and predictors for weight loss during ALL induction therapy.

Methods: This was a single-institution retrospective study of 187 patients, ages 2 to 20 y, diagnosed with ALL or lymphoblastic lymphoma. We analyzed weight trends during induction therapy and predictors of weight loss.

Results: Significant weight loss (≥5%) occurred in 17% of patients. Having high-risk disease, trisomy 21, overweight/obese status at the time of diagnosis, and/or hyperglycemia were positively associated with weight loss and negatively associated with weight gain during induction therapy.

Conclusion: Future studies should aim to better understand the etiology and importance of weight loss during induction therapy.
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http://dx.doi.org/10.1016/j.nut.2020.110937DOI Listing
January 2021

Adrenaline autoinjectors.

Clin Exp Allergy 2020 06;50(6):652-653

The Anaphylaxis Campaign, Farnborough, UK.

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http://dx.doi.org/10.1111/cea.13615DOI Listing
June 2020

Effects of anionic supplement source in prepartum negative dietary cation-anion difference diets on serum calcium, feed intake, and lactational performance of multiparous dairy cows.

J Dairy Sci 2020 May 26;103(5):4302-4314. Epub 2020 Feb 26.

Department of Animal Science, University of Minnesota, Saint Paul 55108. Electronic address:

Incidence of subclinical hypocalcemia in early postpartum dairy cows continues to be an animal welfare concern and an economic burden for producers. Feeding prepartum negative dietary cation-anion difference (DCAD) diets produces metabolic acidosis, which supports mobilization of bone calcium and reduces the incidence of hypocalcemia. Achieving a sufficient degree of metabolic acidosis without reducing dry matter intake (DMI) can be difficult. This study compared the ability of MegAnion (MA; Origination O2D Inc., Maplewood, MN), a new DCAD supplement designed to be more palatable than typical anionic salt sources, and another palatable commercial DCAD product, SoyChlor (SC; Landus Cooperative, Ralston, IA), to reduce urine pH (a surrogate for metabolic acidosis) without reducing prepartum DMI. A secondary objective was to assess the effect of these anionic supplements on postpartum serum calcium concentrations and DMI. Prepartum multiparous Holstein (HO) and crossbred (XX) cows were blocked by breed and expected calving date and randomly assigned within breed to total mixed rations (TMR) with MA or SC and DCAD values of -215 mEq/kg of DM. Cows (n = 56; 15 MA-HO, 12 SC-HO, 15 MA-XX, 14 SC-XX) consumed the treatment TMR for at least 19 d and completed the 28 d in milk (DIM) phase of the study. Urine and blood samples were collected weekly and at 1, 2, and 3 DIM. Data were analyzed as a randomized block design by repeated measures with week or DIM as the repeated effect. Prepartum urine pH decreased from 8.15 ± 0.27 before treatment to 6.12 ± 0.14 during treatment, was not affected by anionic supplement, and increased immediately after calving when all cows consumed the same early-lactation TMR. Prepartum serum calcium concentrations were not affected (2.34 vs. 2.33 ± 0.02 mmol/L) by treatment, whereas nonesterified fatty acids were lower (86 vs. 120 ± 10 mmol/L) and insulin was greater (215 vs. 174 ± 10 pmol/L) in cows fed MA than in cows fed SC. These differences are supported by the numerically greater prepartum DMI (1.2 kg/d) and energy balance (1.8 Mcal/d) of cows fed MA. However, pre- and postpartum DMI and other production variables, including body weight, body condition score, milk yield, and energy balance, were not affected by treatment. This lack of difference indicates that MA provides another effective source of anionic salts for diets designed to reduce urine pH and induce metabolic acidosis in prepartum dairy cows.
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http://dx.doi.org/10.3168/jds.2019-16991DOI Listing
May 2020

Evidence to Support Inclusion of Pharmacogenetic Biomarkers in Randomised Controlled Trials.

J Pers Med 2019 09 1;9(3). Epub 2019 Sep 1.

Institute of Translational Medicine, Department of Biostatistics, University of Liverpool, Waterhouse Building, 1-5 Brownlow Street, Liverpool L69 3GL, UK.

Pharmacogenetics and biomarkers are becoming normalised as important technologies to improve drug efficacy rates, reduce the incidence of adverse drug reactions, and make informed choices for targeted therapies. However, their wider clinical implementation has been limited by a lack of robust evidence. Suitable evidence is required before a biomarker's clinical use, and also before its use in a clinical trial. We have undertaken a review of five pharmacogenetic biomarker-guided randomised controlled trials (RCTs) and evaluated the evidence used by these trials to justify biomarker inclusion. We assessed and quantified the evidence cited in published rationale papers, or where these were not available, obtained protocols from trial authors. Very different levels of evidence were provided by the trials. We used these observations to write recommendations for future justifications of biomarker use in RCTs and encourage regulatory authorities to write clear guidelines.
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http://dx.doi.org/10.3390/jpm9030042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789450PMC
September 2019

Assessment of epigenetic mechanisms and DNA double-strand break repair using laser micro-irradiation technique developed for hematological cells.

EBioMedicine 2019 May 16;43:138-149. Epub 2019 Apr 16.

Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA; Department of Radiation Oncology, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA. Electronic address:

Background: Certain tumors rely heavily on their DNA repair capability to survive the DNA damage induced by chemotherapeutic agents. Therefore, it is important to monitor the dynamics of DNA repair in patient samples during the course of their treatment, in order to determine whether a particular drug regimen perturbs the DNA repair networks in cancer cells and provides therapeutic benefits. Quantitative measurement of proteins and/or their posttranslational modification(s) at DNA double strand breaks (DSBs) induced by laser microirradiation provides an applicable diagnostic approach to examine DNA repair and its dynamics. However, its use is restricted to adherent cell lines and not employed in suspension tumor cells that include the many hematological malignancies.

Methods: Here, we report the development of an assay to laser micro-irradiate and quantitatively measure DNA repair transactions at DSB sites in normal mononuclear cells and a variety of suspension leukemia and lymphoma cells including primary patient samples.

Findings: We show that global changes in the H3K27me3-ac switch modulated by inhibitors of Class I HDACs, EZH2 methyltransferase and (or) H3K27me3 demethylases do not reflect the dynamic changes in H3K27me3 that occur at double-strand break sites during DNA repair.

Interpretation: Results from our mechanistic studies and proof-of-principle data with patient samples together show the effectiveness of using the modified micro-laser-based assay to examine DNA repair directly in suspension cancer cells, and has important clinical implications by serving as a valuable tool to assess drug efficacies in hematological cancer cells that grow in suspension.
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http://dx.doi.org/10.1016/j.ebiom.2019.03.083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562062PMC
May 2019

Engaging Undergraduate Kinesiology Students in Clinically-Based Research.

Quest 2018 22;70(3):292-303. Epub 2017 Nov 22.

University of Utah.

Many undergraduate students in kinesiology are interested in clinical careers and seek research opportunities for advanced study and unique learning experiences. This paper describes a process of engaging undergraduate students in a multi-disciplinary, NIH-funded Program Project investigating factors that may affect pelvic floor support and symptoms in primiparous women during the first year postpartum. Students complete general and protocol-specific training prior to engagement, have specific tasks that reinforce skill development and require independence, and are invited to participate in additional opportunities with the investigative team. The topic of pelvic floor health is novel to most students and participation in this research expands their knowledge beyond a mainstream kinesiology curriculum. Institutionalizing this type of program could formalize undergraduate student research experiences and facilitate ongoing clinical research efforts with a kinesiology focus.
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http://dx.doi.org/10.1080/00336297.2017.1380054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6133246PMC
November 2017

Late Endosomal/Lysosomal Cholesterol Accumulation Is a Host Cell-Protective Mechanism Inhibiting Endosomal Escape of Influenza A Virus.

mBio 2018 07 24;9(4). Epub 2018 Jul 24.

Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, University of Muenster, Muenster, Germany

To transfer the viral genome into the host cell cytoplasm, internalized influenza A virus (IAV) particles depend on the fusion of the IAV envelope with host endosomal membranes. The antiviral host interferon (IFN) response includes the upregulation of interferon-induced transmembrane protein 3 (IFITM3), which inhibits the release of the viral content into the cytosol. Although IFITM3 induction occurs concomitantly with late endosomal/lysosomal (LE/L) cholesterol accumulation, the functional significance of this process is not well understood. Here we report that LE/L cholesterol accumulation itself plays a pivotal role in the early antiviral defense. We demonstrate that inducing LE/L cholesterol accumulation is antiviral in non-IFN-primed cells, restricting incoming IAV particles and impairing mixing of IAV/endosomal membrane lipids. Our results establish a protective function of LE/L cholesterol accumulation and suggest endosomal cholesterol balance as a possible antiviral target. With annual epidemics occurring in all parts of the world and the risk of global outbreaks, influenza A virus (IAV) infections remain a major threat to public health. Infected host cells detect viral components and mount an interferon (IFN)-mediated response to restrict virus propagation and spread of infection. Identification of cellular factors and underlying mechanisms that establish such an antiviral state can provide novel strategies for the development of antiviral drugs. The contribution of LE/L cholesterol levels, especially in the context of the IFN-induced antiviral response, has remained controversial so far. Here, we report that accumulation of cholesterol in the LE/L compartment contributes to the IFN-induced host cell defense against incoming IAV. Our results establish cholesterol accumulation in LE/L as a novel antiviral barrier and suggest the endosomal cholesterol balance as a putative druggable host cell factor in IAV infection.
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http://dx.doi.org/10.1128/mBio.01345-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058292PMC
July 2018

Pharmacologic Characterization of AMG8379, a Potent and Selective Small Molecule Sulfonamide Antagonist of the Voltage-Gated Sodium Channel Na1.7.

J Pharmacol Exp Ther 2017 07 4;362(1):146-160. Epub 2017 May 4.

Department of Neuroscience (T.J.K., R.Y., S.A, C.P.I., M.J., D.J., J.H.L., S.G.L., J.Li., D.L., J.Lu., D.M., D.O., K.T., J.W., V.Y., D.X.D.Z., R.T.F., B.D.M.), Department of Medicinal Chemistry (M.M.W.), and Department of Pharmacokinetics and Drug Metabolism (X.B., V.B., J.R.), Amgen Inc., Cambridge, Massachusetts and Thousand Oaks, California

Potent and selective antagonists of the voltage-gated sodium channel Na1.7 represent a promising avenue for the development of new chronic pain therapies. We generated a small molecule atropisomer quinolone sulfonamide antagonist AMG8379 and a less active enantiomer AMG8380. Here we show that AMG8379 potently blocks human Na1.7 channels with an IC of 8.5 nM and endogenous tetrodotoxin (TTX)-sensitive sodium channels in dorsal root ganglion (DRG) neurons with an IC of 3.1 nM in whole-cell patch clamp electrophysiology assays using a voltage protocol that interrogates channels in a partially inactivated state. AMG8379 was 100- to 1000-fold selective over other Na family members, including Na1.4 expressed in muscle and Na1.5 expressed in the heart, as well as TTX-resistant Na channels in DRG neurons. Using an ex vivo mouse skin-nerve preparation, AMG8379 blocked mechanically induced action potential firing in C-fibers in both a time-dependent and dose-dependent manner. AMG8379 similarly reduced the frequency of thermally induced C-fiber spiking, whereas AMG8380 affected neither mechanical nor thermal responses. In vivo target engagement of AMG8379 in mice was evaluated in multiple Na1.7-dependent behavioral endpoints. AMG8379 dose-dependently inhibited intradermal histamine-induced scratching and intraplantar capsaicin-induced licking, and reversed UVB radiation skin burn-induced thermal hyperalgesia; notably, behavioral effects were not observed with AMG8380 at similar plasma exposure levels. AMG8379 is a potent and selective Na1.7 inhibitor that blocks sodium current in heterologous cells as well as DRG neurons, inhibits action potential firing in peripheral nerve fibers, and exhibits pharmacodynamic effects in translatable models of both itch and pain.
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http://dx.doi.org/10.1124/jpet.116.239590DOI Listing
July 2017

V-ATPases Containing a3 Subunit Play a Direct Role in Enamel Development in Mice.

J Cell Biochem 2017 10 3;118(10):3328-3340. Epub 2017 May 3.

Faculty of Dentistry, Dental Research Institute, University of Toronto, Toronto, Ontario, Canada.

Vacuolar H -ATPases (V-ATPases) are ubiquitous multisubunit proton pumps responsible for organellar pH maintenance. Mutations in the a3 subunit of V-ATPases cause autosomal recessive osteopetrosis, a rare disease due to impaired bone resorption. Patients with osteopetrosis also display dental anomalies, such as enamel defects; however, it is not clear whether these enamel abnormalities are a direct consequence of the a3 mutations. We investigated enamel mineralization, spatiotemporal expression of enamel matrix proteins and the a3 protein during tooth development using an osteopetrotic mouse model with a R740S point mutation in the V-ATPase a3 subunit. Histology revealed aberrations in both crown and root development, whereas SEM analysis demonstrated delayed enamel mineralization in homozygous animals. Enamel thickness and mineralization were significantly decreased in homozygous mice as determined by μCT analysis. The expression patterns of the enamel matrix proteins amelogenin, amelotin, and odontogenic ameloblast-associated protein (ODAM) suggested a delay in transition to the maturation stage in homozygous animals. Protein expression of the a3 subunit was detected in ameloblasts in all three genotypes, suggesting that a3-containing V-ATPases play a direct role in amelogenesis, and mutations in a3 delay transition from the secretory to the maturation stage, resulting in hypomineralized and hypoplastic enamel. J. Cell. Biochem. 118: 3328-3340, 2017. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jcb.25986DOI Listing
October 2017

Cresyl violet: a superior fluorescent lysosomal marker.

Traffic 2016 12 17;17(12):1313-1321. Epub 2016 Oct 17.

Program in Cell Biology, Hospital for Sick Children, Toronto, Canada.

We have characterized cresyl violet as a membrane-permeant fluorophore that localizes to lysosomes and acidic vacuoles of budding yeast, Drosophila, human, murine and canine cells. An acidotropic weak base, cresyl violet is shown to be virtually insensitive to physiological alkali and divalent cations. Because of its unique spectral properties, it can be used in combination with green, red and far-red fluorophores, is less susceptible to photobleaching than alternative acidotropic probes, and does not undergo photoconversion. At concentrations that yield bright labeling of acidic compartments, cresyl violet does not alter the organellar pH nor does it affect the buffering capacity. Its affordability, together with its chemical and spectral properties, make cresyl violet a superior lysosomal marker devoid of many of the negative characteristics associated with other lysosomal probes.
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http://dx.doi.org/10.1111/tra.12447DOI Listing
December 2016

The position of lysosomes within the cell determines their luminal pH.

J Cell Biol 2016 Mar;212(6):677-92

Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada

We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H(+)-adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility.
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http://dx.doi.org/10.1083/jcb.201507112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792074PMC
March 2016

Inhibition of Inactive States of Tetrodotoxin-Sensitive Sodium Channels Reduces Spontaneous Firing of C-Fiber Nociceptors and Produces Analgesia in Formalin and Complete Freund's Adjuvant Models of Pain.

PLoS One 2015 17;10(9):e0138140. Epub 2015 Sep 17.

Department of Diagnostics and Biological Sciences, University of Minnesota School of Dentistry, Minneapolis, Minnesota, United States of America.

While genetic evidence shows that the Nav1.7 voltage-gated sodium ion channel is a key regulator of pain, it is unclear exactly how Nav1.7 governs neuronal firing and what biophysical, physiological, and distribution properties of a pharmacological Nav1.7 inhibitor are required to produce analgesia. Here we characterize a series of aminotriazine inhibitors of Nav1.7 in vitro and in rodent models of pain and test the effects of the previously reported "compound 52" aminotriazine inhibitor on the spiking properties of nociceptors in vivo. Multiple aminotriazines, including some with low terminal brain to plasma concentration ratios, showed analgesic efficacy in the formalin model of pain. Effective concentrations were consistent with the in vitro potency as measured on partially-inactivated Nav1.7 but were far below concentrations required to inhibit non-inactivated Nav1.7. Compound 52 also reversed thermal hyperalgesia in the complete Freund's adjuvant (CFA) model of pain. To study neuronal mechanisms, electrophysiological recordings were made in vivo from single nociceptive fibers from the rat tibial nerve one day after CFA injection. Compound 52 reduced the spontaneous firing of C-fiber nociceptors from approximately 0.7 Hz to 0.2 Hz and decreased the number of action potentials evoked by suprathreshold tactile and heat stimuli. It did not, however, appreciably alter the C-fiber thresholds for response to tactile or thermal stimuli. Surprisingly, compound 52 did not affect spontaneous activity or evoked responses of Aδ-fiber nociceptors. Results suggest that inhibition of inactivated states of TTX-S channels, mostly likely Nav1.7, in the peripheral nervous system produces analgesia by regulating the spontaneous discharge of C-fiber nociceptors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0138140PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4575030PMC
June 2016

The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

J Biol Chem 2015 Apr 20;290(15):9919-28. Epub 2015 Feb 20.

From the Department of Chemistry and Biology and the Molecular Science Program, Ryerson University, Toronto, Ontario M5B2K3, Canada and the Program in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G1X8, Canada

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes.
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http://dx.doi.org/10.1074/jbc.M114.613984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392288PMC
April 2015

HDAC1,2 inhibition impairs EZH2- and BBAP-mediated DNA repair to overcome chemoresistance in EZH2 gain-of-function mutant diffuse large B-cell lymphoma.

Oncotarget 2015 Mar;6(7):4863-87

Department of Radiation Oncology, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT, USA.

Gain-of-function mutations in the catalytic site of EZH2 (Enhancer of Zeste Homologue 2), is observed in about 22% of diffuse large B-cell lymphoma (DLBCL) cases. Here we show that selective inhibition of histone deacetylase 1,2 (HDAC1,2) activity using a small molecule inhibitor causes cytotoxic or cytostatic effects in EZH2 gain-of-function mutant (EZH2GOF) DLBCL cells. Our results show that blocking the activity of HDAC1,2 increases global H3K27ac without causing a concomitant global decrease in H3K27me3 levels. Our data shows that inhibition of HDAC1,2 is sufficient to decrease H3K27me3 present at DSBs, decrease DSB repair and activate the DNA damage response in these cells. In addition to increased H3K27me3, we found that the EZH2GOF DLBCL cells overexpress another chemotherapy resistance factor - B-lymphoma and BAL-associated protein (BBAP). BBAP monoubiquitinates histone H4K91, a residue that is also subjected to acetylation. Our results show that selective inhibition of HDAC1,2 increases H4K91ac, decreases BBAP-mediated H4K91 monoubiquitination, impairs BBAP-dependent DSB repair and sensitizes the refractory EZH2GOF DLBCL cells to treatment with doxorubicin, a chemotherapy agent. Hence, selective HDAC1,2 inhibition provides a novel DNA repair mechanism-based therapeutic approach as it can overcome both EZH2- and BBAP-mediated DSB repair in the EZH2GOF DLBCL cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467121PMC
http://dx.doi.org/10.18632/oncotarget.3120DOI Listing
March 2015

Suppression of mTORC1 activation in acid-α-glucosidase-deficient cells and mice is ameliorated by leucine supplementation.

Am J Physiol Regul Integr Comp Physiol 2014 Nov 17;307(10):R1251-9. Epub 2014 Sep 17.

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York; Department of Medicine, Albert Einstein College of Medicine, Bronx, New York

Pompe disease is due to a deficiency in acid-α-glucosidase (GAA) and results in debilitating skeletal muscle wasting, characterized by the accumulation of glycogen and autophagic vesicles. Given the role of lysosomes as a platform for mTORC1 activation, we examined mTORC1 activity in models of Pompe disease. GAA-knockdown C2C12 myoblasts and GAA-deficient human skin fibroblasts of infantile Pompe patients were found to have decreased mTORC1 activation. Treatment with the cell-permeable leucine analog L-leucyl-L-leucine methyl ester restored mTORC1 activation. In vivo, Pompe mice also displayed reduced basal and leucine-stimulated mTORC1 activation in skeletal muscle, whereas treatment with a combination of insulin and leucine normalized mTORC1 activation. Chronic leucine feeding restored basal and leucine-stimulated mTORC1 activation, while partially protecting Pompe mice from developing kyphosis and the decline in muscle mass. Leucine-treated Pompe mice showed increased spontaneous activity and running capacity, with reduced muscle protein breakdown and glycogen accumulation. Together, these data demonstrate that GAA deficiency results in reduced mTORC1 activation that is partly responsible for the skeletal muscle wasting phenotype. Moreover, mTORC1 stimulation by dietary leucine supplementation prevented some of the detrimental skeletal muscle dysfunction that occurs in the Pompe disease mouse model.
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http://dx.doi.org/10.1152/ajpregu.00212.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233288PMC
November 2014

Global Nav1.7 knockout mice recapitulate the phenotype of human congenital indifference to pain.

PLoS One 2014 4;9(9):e105895. Epub 2014 Sep 4.

Department of Neuroscience, Amgen Inc., Cambridge, Massachusetts, United States of America.

Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund's adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105895PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4154897PMC
April 2015

Identification of a potent, state-dependent inhibitor of Nav1.7 with oral efficacy in the formalin model of persistent pain.

J Med Chem 2011 Jul 2;54(13):4427-45. Epub 2011 Jun 2.

Department of Chemistry Research and Discovery, Amgen Inc., Cambridge, Massachusetts 02142, United States.

Clinical human genetic studies have recently identified the tetrodotoxin (TTX) sensitive neuronal voltage gated sodium channel Nav1.7 (SCN9A) as a critical mediator of pain sensitization. Herein, we report structure-activity relationships for a novel series of 2,4-diaminotriazines that inhibit hNav1.7. Optimization efforts culminated in compound 52, which demonstrated pharmacokinetic properties appropriate for in vivo testing in rats. The binding site of compound 52 on Nav1.7 was determined to be distinct from that of local anesthetics. Compound 52 inhibited tetrodotoxin-sensitive sodium channels recorded from rat sensory neurons and exhibited modest selectivity against the hERG potassium channel and against cloned and native tetrodotoxin-resistant sodium channels. Upon oral administration to rats, compound 52 produced dose- and exposure-dependent efficacy in the formalin model of pain.
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http://dx.doi.org/10.1021/jm200018kDOI Listing
July 2011

Cytosolic H+ microdomain developed around AE1 during AE1-mediated Cl-/HCO3- exchange.

J Physiol 2011 Apr 7;589(Pt 7):1551-69. Epub 2011 Feb 7.

Membrane Protein Research Group, Department of Physiology, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

Microdomains, regions of discontinuous cytosolic solute concentration enhanced by rapid solute transport and slow diffusion rates, have many cellular roles. pH-regulatory membrane transporters, like the Cl−/HCO3− exchanger AE1, could develop H+ microdomains since AE1 has a rapid transport rate and cytosolic H+ diffusion is slow. We examined whether the pH environment surrounding AE1 differs from other cellular locations. As AE1 drives Cl−/HCO3− exchange, differences in pH, near and remote from AE1, were monitored by confocal microscopy using two pH-sensitive fluorescent proteins: deGFP4 (GFP) and mNectarine (mNect). Plasma membrane (PM) pH (defined as ∼1 μm region around the cell periphery) was monitored by GFP fused to AE1 (GFP.AE1), and mNect fused to an inactive mutant of the Na+-coupled nucleoside co-transporter, hCNT3 (mNect.hCNT3). GFP.AE1 to mNect.hCNT3 distance was varied by co-expression of different amounts of the two proteins in HEK293 cells. As the GFP.AE1–mNect.hCNT3 distance increased, mNect.hCNT3 detected the Cl−/HCO3− exchange-associated cytosolic pH change with a time delay and reduced rate of pH change compared to GFP.AE1. We found that a H+ microdomain 0.3 μm in diameter forms around GFP.AE1 during physiological HCO3− transport. Carbonic anhydrase isoform II inhibition prevented H+ microdomain formation. We also measured the rate of H+ movement from PM GFP.AE1 to endoplasmic reticulum (ER), using mNect fused to the cytosolic face of ER-resident calnexin (CNX.mNect). The rate of H+ diffusion through cytosol was 60-fold faster than along the cytosolic surface of the plasma membrane. The pH environment surrounding pH regulatory transport proteins may differ as a result of H+ microdomain formation, which will affect nearby pH-sensitive processes.
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http://dx.doi.org/10.1113/jphysiol.2010.201483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3099015PMC
April 2011

Characterization of an epilepsy-associated variant of the human Cl-/HCO3(-) exchanger AE3.

Am J Physiol Cell Physiol 2009 Sep 15;297(3):C526-36. Epub 2009 Jul 15.

Dept. of Physiology, Univ. of Alberta, Edmonton, Alberta T6G 2H7, Canada.

Anion exchanger 3 (AE3), expressed in the brain, heart, and retina, extrudes intracellular HCO(3)(-) in exchange for extracellular Cl(-). The SLC4A3 gene encodes two variants of AE3, brain or full-length AE3 (AE3(fl)) and cardiac AE3 (cAE3). Epilepsy is a heterogeneous group of disorders characterized by recurrent unprovoked seizures that affect about 50 million people worldwide. The AE3-A867D allele in humans has been associated with the development of IGE (IGE), which accounts for approximately 30% of all epilepsies. To examine the molecular basis for the association of the A867D allele with IGE, we characterized wild-type (WT) and AE3(fl)-A867D in transfected human embryonic kidney (HEK)-293 cells. AE3(fl)-A867D had significantly reduced transport activity relative to WT (54 +/- 4%, P < 0.01). Differences in expression levels or the degree of protein trafficking to the plasma membrane did not account for the defect of AE3(fl)-A867D. Treatment with 8-bromo-cAMP (8-Br-cAMP) increased Cl(-)/HCO(3)(-) exchange activity of WT and AE3(fl)-A867D to a similar degree, which was abolished by preincubation with the protein kinase A (PKA)-specific inhibitor H89. This indicates that PKA regulates WT and AE3(fl)-A867D Cl(-)/HCO(3)(-) exchange activity. No difference in Cl(-)/HCO(3)(-) exchange activity was found between cultures of mixed populations of neonatal hippocampal cells from WT and slc4a3(-/-) mice. We conclude that the A867D allele is a functional (catalytic) mutant of AE3 and that the decreased activity of AE3(fl)-A867D may cause changes in cell volume and abnormal intracellular pH. In the brain, these alterations may promote neuron hyperexcitability and the generation of seizures.
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http://dx.doi.org/10.1152/ajpcell.00572.2008DOI Listing
September 2009

Red fluorescent protein pH biosensor to detect concentrative nucleoside transport.

J Biol Chem 2009 Jul 3;284(31):20499-511. Epub 2009 Jun 3.

Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

Human concentrative nucleoside transporter, hCNT3, mediates Na+/nucleoside and H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in HEK293 cells expressing mNect.hCNT3 or mNect.hCNT3-F563C, an inactive hCNT3 mutant. Free cytosolic mNect, mNect.hCNT3, and the traditional pH-sensitive dye, BCECF, reported cytosolic pH similarly in pH-clamped HEK293 cells. Cells were incubated at the permissive pH for H(+)-coupled nucleoside transport, pH 5.5, under both Na(+)-free and Na(+)-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mm uridine, providing the first direct evidence for H(+)-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H+ flux) in the presence or absence of Na+ (1.09 +/- 0.11 or 1.18 +/- 0.32 mm min(-1), respectively). This suggests that in acidic Na(+)-containing conditions, 1 Na+ and 1 H+ are transported per uridine molecule, while in acidic Na(+)-free conditions, 1 H+ alone is transported/uridine. In acid environments, including renal proximal tubule, H+/nucleoside co-transport may drive nucleoside accumulation by hCNT3. Fusion of mNect to hCNT3 provided a simple, self-referencing, and effective way to monitor nucleoside transport, suggesting an approach that may have applications in assays of transport activity of other H(+)-coupled transport proteins.
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http://dx.doi.org/10.1074/jbc.M109.019042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742814PMC
July 2009

Sentence comprehension in agrammatic aphasia: history and variability to clinical implications.

Clin Linguist Phon 2009 Jan;23(1):15-37

Fletcher Allen Health Care, Burlington, VT 05405, USA.

Individuals with Broca's aphasia often present with deficits in their ability to comprehend non-canonical sentences. This has been contrastingly characterized as a systematic loss of specific grammatical abilities or as individual variability in the dynamics between processing load and resource availability. The present study investigated sentence level comprehension in participants with Broca's aphasia in an attempt to integrate these contrasting views into a clinically useful process. Two participants diagnosed with Broca's aphasia were assessed using a sentence-to-picture matching paradigm and a truth-value judgement task, across sentence constructions thought to be problematic for this population. The data demonstrate markedly different patterns of performance between participants, as well as variability within participants (e.g. by sentence type). These findings support the notion of individual performance variability in persons with aphasia. Syntactic theory was instructive for assessing sentence level comprehension, leading to a clinically relevant process of identifying treatment targets considering both performance variability and syntactic complexity for this population.
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http://dx.doi.org/10.1080/02699200802394880DOI Listing
January 2009

Creating a "Code STEMI" improves care for cardiac patients.

Nursing 2008 Jun;38(6):17-8

Florida Hospital Institute of Translational Research, USA.

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http://dx.doi.org/10.1097/01.NURSE.0000320336.43639.64DOI Listing
June 2008

Interaction of integrin-linked kinase with the kidney chloride/bicarbonate exchanger, kAE1.

J Biol Chem 2007 Aug 6;282(32):23205-18. Epub 2007 Jun 6.

Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

Kidney anion exchanger 1 (kAE1) mediates chloride/bicarbonate exchange at the basolateral membrane of kidney alpha-intercalated cells, thereby facilitating bicarbonate reabsorption into the blood. Human kAE1 lacks the N-terminal 65 residues of the erythroid form (AE1, band 3), which are essential for binding of cytoskeletal and cytosolic proteins. Yeast two-hybrid screening identified integrin-linked kinase (ILK), a serine/threonine kinase, and an actin-binding protein as an interacting partner with the N-terminal domain of kAE1. Interaction between kAE1 and ILK was confirmed in co-expression experiments in HEK 293 cells and is mediated by a previously unidentified calponin homology domain in the kAE1 N-terminal region. The calponin homology domain of kAE1 binds the C-terminal catalytic domain of ILK to enhance association of kAE1 with the actin cytoskeleton. Overexpression of ILK increased kAE1 levels at the cell surface as shown by flow cytometry, cell surface biotinylation, and anion transport activity assays. Pulse-chase experiments revealed that ILK associates with kAE1 early in biosynthesis, likely in the endoplasmic reticulum. ILK co-localized with kAE1 at the basolateral membrane of polarized Madin-Darby canine kidney cells and in alpha-intercalated cells of human kidneys. Taken together these results suggest that ILK and kAE1 traffic together from the endoplasmic reticulum to the basolateral membrane. ILK may provide a linkage between kAE1 and the underlying actin cytoskeleton to stabilize kAE1 at the basolateral membrane, resulting in higher levels of cell surface expression.
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http://dx.doi.org/10.1074/jbc.M702139200DOI Listing
August 2007

Carbonic anhydrase inhibition prevents and reverts cardiomyocyte hypertrophy.

J Physiol 2007 Feb 23;579(Pt 1):127-45. Epub 2006 Nov 23.

Department of Physiology, Membrane Protein Research Group, University of Alberta, Edmonton, Canada T6G2H7.

Hypertrophic cardiomyocyte growth contributes substantially to the progression of heart failure. Activation of the plasma membrane Na+-H+ exchanger (NHE1) and Cl- -HCO3- exchanger (AE3) has emerged as a central point in the hypertrophic cascade. Both NHE1 and AE3 bind carbonic anhydrase (CA), which activates their transport flux, by providing H+ and HCO3-, their respective transport substrates. We examined the contribution of CA activity to the hypertrophic response of cultured neonatal and adult rodent cardiomyocytes. Phenylephrine (PE) increased cell size by 37 +/- 2% and increased expression of the hypertrophic marker, atrial natriuretic factor mRNA, twofold in cultured neonatal rat cardiomyocytes. Cell size was also increased in adult cardiomyocytes subjected to angiotensin II or PE treatment. These effects were associated with increased expression of cytosolic CAII protein and the membrane-anchored isoform, CAIV. The membrane-permeant CA inhibitor, 6-ethoxyzolamide (ETZ), both prevented and reversed PE-induced hypertrophy in a concentration-dependent manner in neonate cardiomyocytes (IC50=18 microm). ETZ and the related CA inhibitor methazolamide prevented hypertrophy in adult cardiomyocytes. In addition, ETZ inhibited transport activity of NHE1 and the AE isoform, AE3, with respective EC50 values of 1.2 +/- 0.3 microm and 2.7 +/- 0.3 microm. PE significantly increased neonatal cardiomyocyte Ca2+ transient frequency from 0.33 +/- 0.4 Hz to 0.77 +/- 0.04 Hz following 24 h treatment; these Ca2+ -handling abnormalities were completely prevented by ETZ (0.28 +/- 0.07 Hz). Our study demonstrates a novel role for CA in mediating the hypertrophic response of cardiac myocytes to PE and suggests that CA inhibition represents an effective therapeutic approach towards mitigation of the hypertrophic phenotype.
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http://dx.doi.org/10.1113/jphysiol.2006.123638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2075384PMC
February 2007

The bicarbonate transport metabolon.

J Enzyme Inhib Med Chem 2004 Jun;19(3):231-6

Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

To allow cells to control their pH and bicarbonate levels, cells express bicarbonate transport proteins that rapidly and selectively move bicarbonate across the plasma membrane. Physical interactions have been identified between the carbonic anhydrase isoform, CAII, and the erythrocyte membrane Cl- /HCO3(-) anion exchanger, AE1, mediated by an acidic motif in the AE1 C-terminus. We have found that the presence of CAII attached to AE1 accelerates AE1 HCO3(-) transport activity, as AE1 moves bicarbonate either into or out of the cell. In efflux mode the presence of CAII attached to AE1 will increase the local concentration of bicarbonate at the AE1 transport site. As bicarbonate is transported into the cell by AE1, the presence of CAII on the cytosolic surface accelerates transport by consumption of bicarbonate, thereby maximizing the transmembrane bicarbonate concentration gradient experienced by the AE1 molecule. Functional and physical interactions also occur between CAII and Na+/HCO3(-) co-transporter isoforms NBC1 and NBC3. All examined bicarbonate transport proteins, except the DRA (SLC26A3) Cl-/HCO3(-) exchange protein, have a consensus CAII binding site in their cytoplasmic C-terminus. Interestingly, CAII does not bind DRA. CAIV is anchored to the extracellular surface of cells via a glycosylphosphatidyl inositol linkage. We have identified extracellular regions of AE1 and NBC1 that directly interact with CAIV, to form a physical complex between the proteins. In summary, bicarbonate transporters directly interact with the CAII and CAIV carbonic anhydrases to increase the transmembrane bicarbonate flux. The complex of a bicarbonate transporter with carbonic anhydrase forms a "Bicarbonate Transport Metabolon."
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http://dx.doi.org/10.1080/14756360410001704443DOI Listing
June 2004

A fully human monoclonal antibody to the insulin-like growth factor I receptor blocks ligand-dependent signaling and inhibits human tumor growth in vivo.

Cancer Res 2003 Dec;63(24):8912-21

ImClone Systems Incorporated, New York, New York 10014, USA.

The insulin-like growth factor I receptor (IGF-IR) is overexpressed in many diverse tumor types and is a critical signaling molecule for tumor cell proliferation and survival. Therapeutic strategies targeting the IGF-IR may therefore be effective broad-spectrum anticancer agents. Through screening of a Fab phage display library, we have generated a fully human antibody (A12) that binds to the IGF-IR with high affinity (4.11 x 10(-11) M) and inhibits ligand binding with an IC(50) of 0.6-1 nM. Antibody-mediated blockade of ligand binding to the IGF-IR inhibited downstream signaling of the two major insulin-like growth factor (IGF) pathways, mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/Akt, in MCF7 human breast cancer cells. As a result, the mitogenic and proliferative potential of IGF-I and IGF-II were significantly reduced. A12 did not block insulin binding to the insulin receptor but could block binding to atypical IGF-IR in MCF7 cells. In addition, A12 was shown to induce IGF-IR internalization and degradation on specific binding to tumor cells, resulting in a significant reduction in cell surface receptor density. In xenograft tumor models in vivo, IGF-IR blockade by A12 was shown to occur rapidly, resulting in significant growth inhibition of breast, renal, and pancreatic tumors. Histological analysis of tumor sections demonstrated a marked increase in apoptotic tumor cells in antibody-treated animals. These results demonstrate that A12 possesses strong antitumor activity in vitro and in vivo and may therefore be an effective therapeutic candidate for the treatment of cancers that are dependent on IGF-IR signaling for growth and survival.
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December 2003

Chronic fatigue syndrome: symptom subtypes in a community based sample.

Women Health 2003 ;37(1):1-13

Center for Community Research, DePaul University, Chicago, IL 60614, USA.

Most studies of Chronic Fatigue Syndrome (CFS) have been based on patients recruited from primary or tertiary care settings. Patients from such settings might not be typical of patients in the general population. The present investigation involved examining individuals with CFS from a community-based study. A random sample of 18,675 respondents in Chicago were first interviewed by telephone. A group of individuals with chronic fatigue accompanied by at least four Fukuda et al. (1994) symptoms associated with CFS were given medical and psychiatric examinations. From this sample, a physician review group diagnosed individuals with CFS. Those diagnosed with CFS were subclassified based on frequency of symptoms. Important differences emerged on measures of sociodemographics and disability. The implications of these findings and others are discussed.
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http://dx.doi.org/10.1300/J013v37n01_01DOI Listing
March 2003

A factor analysis of chronic fatigue symptoms in a community-based sample.

Soc Psychiatry Psychiatr Epidemiol 2002 Apr;37(4):183-9

Department of Psychology, DePaul University, Chicago, IL 60614, USA.

Background: This study examined characteristics of fatigue in individuals with chronic fatigue from a community-based study. Most studies of chronic fatigue have been based on patients recruited from primary or tertiary care settings. Samples such as these might not be representative of patients within the general population. The purpose of this study was to determine the factor structure of participants' symptoms in a random community sample of individuals with chronic fatigue.

Method: A random sample of 18,675 respondents in Chicago received a brief telephone questionnaire designed to identify individuals with chronic fatigue. A group of 780 (4.2%) with chronic fatigue received further interview via telephone questionnaire involving characteristics of their fatigue. The analyses for this study were based on those people identified with having chronic fatigue. A factor analysis was conducted on responses to questionnaire items, and a four-factor solution emerged. Mean factor scores were derived and analyzed in relation to sociodemographic characteristics and sample subgroups.

Results: The four factors were labeled: Lack of Energy, Physical Exertion, Cognitive Functioning, and Fatigue and Rest.

Conclusions: Results indicated that individuals with chronic fatigue have symptoms that can be differentiated into theoretically distinct factors.
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http://dx.doi.org/10.1007/s001270200013DOI Listing
April 2002