Doggett, Robert G. (Texas Institute for Rehabilitation and Research, Houston), Gunyon M. Harrison, Richard N. Stillwell, and Everett S. Wallis. Enzymatic action on the capsular material produced by Pseudomonas aeruginosa of cystic fibrosis origin. J. Bacteriol. 89:476–480. 1965.—An enzymatic study of the action of α- and β-amylases on the slime envelope produced by Pseudomonas aeruginosa originating from patients with cystic fibrosis (CF) has been reported. It has been shown that these two enzymes in combination prevent the formation of the abnormal ethanol-benzene insoluble mucoid present in the slime layer of the above organism of CF origin. Evidence is also given which suggests that this organism can serve as a tool for studies of chemical abnormalities in the mucous metabolism of CF individuals.
Mitchell, Paul D. (West Virginia University Medical Center, Morgantown), and Robert G. Burrell. Serology of the Mima-Herellea group and the genus Moraxella. J. Bacteriol. 87:900–909. 1964.—The identity of organisms which have been assigned to the Mima-Herellea group of the tribe Mimeae and their taxonomic position are uncertain. In considering the possible relationship of the Mima-Herellea group to the genus Moraxella, the approach used was the isolation and identification of representative antigenic constituents of strains designated Mima polymorpha, M. polymorpha var. oxidans, Herellea vaginicola, Moraxella lwoffi, M. liquefaciens, M. non-liquefaciens, and Bacterium anitratum. Cell-free extracts of sonically disrupted cells were used in these studies, and the precipitinogens derived by this method were considered to be of capsular and somatic origin. Identity among the strains was established by immunodiffusion, with immune sera and antigenic extracts. Heterologous and homologous reactions were performed with adsorbed and unadsorbed sera to verify the cases of identity. In every instance of homologous reaction, at least five distinct antigen-antibody systems were discernible within the Mima-Herellea group. However, as evidenced by heterologous studies, the demonstrable antigenic composition varied among the species and, in some instances, among the strains. M. polymorpha var. oxidans appeared to be serologically distinct from H. vaginicola and M. polymorpha, whereas strains of the latter two organisms were theorized to be closely related serologically. Serological cross-reactions in heterologous studies and reciprocal adsorption tests revealed the existence of a serological relationship between members of the Mima-Herellea group and the genus Moraxella. A tentative scheme of identity is postulated on the basis of these crossreactions and cultural and biochemical reactions among the designated strains of Herellea, Mima, Moraxella, and Bacterium.
Tourtellotte, Mark E. (University of Connecticut, Storrs), Robert G. Jensen, George W. Gander, and Harold J. Morowitz. Lipid composition and synthesis in the pleuropneumonia-like organism Mycoplasma gallisepticum. J. Bacteriol. 86:370–379. 1963.—A simple method of extracting lipids from cells of Mycoplasma gallisepticum by use of silicic acid columns is described. Proteolipids (peptides) extracted with chloroform-methanol (2:1) by other methods were not extracted with the lipid by this method, nor were proteins and nucleic acids. Fractionation of lipids of M. gallisepticum demonstrated the presence of saturated hydrocarbons, free fatty acids, cholesterol, cholesterol esters, di- and triglycerides, phosphatidic acids, cephalins, inositides, phosphatidyl choline, and sphingomyelin. The fatty acid composition of the various fractions was also determined. The positive identification of cholesterol and cholesterol esters in this organism by chromatography, melting point, and infrared spectroscopy confirms reports by others that cholesterol is present in the pleuropneumonia group of microorganisms. The incorporation of P32 orthophosphate into four phospholipid fractions, of oleic acid-1-C14 into neutral and phospholipids, and cholesterol-4-C14 into cholesterol esters clearly demonstrated the ability of M. gallisepticum to synthesize these lipids from simpler compounds. Between 70 and 80% of the lipid of this organism was found in the membrane.
Doggett, Robert G. (Texas Institute for Rehabilitation and Research, Houston), Gunyon M. Garrison, and Everett S. Wallis. Comparison of some properties of Pseudomonas aeruginosa isolated from infections in persons with and without cystic fibrosis. J. Bacteriol. 87:427–431. 1964.—Pseudomonas aeruginosa, isolated from the respiratory tract of a group of patients diagnosed as having cystic fibrosis (CF) of the pancreas, attained the ability to produce in its capsule a material which was insoluble in certain organic solvents, such as ethanol. The capsule obtained from P. aeruginosa isolated from infected individuals who did not have CF was ethanol-soluble. This alcohol-insoluble mucoid from the CF P. aeruginosa could be demonstrated to persist after sequential subcultures of this organism. The relative viscosity and carbohydrate moiety of this insoluble mucoid fraction of P. aeruginosa from individuals with CF differed from that of the non-CF P. aeruginosa soluble mucoid fraction.
To develop a system for measuring the teaching effort of medical school faculty and to implement a payment system that is based on it.
An interventional study with outcomes measured before and after the intervention.
A department of internal medicine with a university hospital and an affiliated Veterans Administration hospital.
We assigned a value in teaching units to each teaching activity in proportion to the time expended by the faculty and the intensity of their effort. We then calculated total teaching units for each faculty member in the Division of General Internal Medicine and for combined faculty effort in each subspecialty division in the Department of Medicine. After determining the dollar value for a teaching unit, we distributed discretionary teaching dollars to each faculty member in the Division of General Internal Medicine and to each subspecialty division according to total teaching units.
MEASUREMENTS AND MAIN RESULTS
The distribution of discretionary teaching dollars was determined. In the year after the intervention, there was a substantial redistribution of discretionary teaching dollars among divisions. Compared with an increase in total discretionary dollars of 11.4%, the change in allocation for individual divisions ranged from an increase of 78.2% to a decrease of −28.5%. Further changes in the second year after the intervention were modest. The distribution of teaching units among divisions was similar to the distribution of questions across subspecialties on the American College of Physicians In-Training Examination (r = .67) and the American Board of Internal Medicine Certifying Examination (r = .88).
It is possible to measure the value of teaching effort by medical school faculty and to distribute discretionary teaching funds among divisions according to the value of teaching effort. When this intervention was used at our institution, there were substantial changes in the amounts received by some divisions. We believe that the new distribution more closely approximates the desired distribution because it reflects the desired emphasis on knowledge as measured by two of the most experienced professional groups in internal medicine. We also believe that our method is flexible and adaptable to the needs of most clinical teaching
teaching, reimbursement; teaching, recognition
The effect of autonomic denervation on the metabolic and hormonal responses during intracellular glucopenia in man was investigated. 2-Deoxy-d-glucose (2 DG), a competitive inhibitor of glucose metabolism, was administered intravenously to nine normal volunteers and to five patients, three with complete cervical cord transection (C-6) and two with idiopathic orthostatic hypotension. Before, during, and after a 20 min infusion of 2 DG (50 mg/kg) plasma concentrations of glucose, lactate, FFA, total catecholamines, immunoreactive insulin (IRI), human growth hormone (HGH), and cortisol were determined for periods up to 150 min. In control subjects, the initial elevation of FFA, glucose. HGH, and cortisol corresponded with the rise in total catecholamines, with maximal levels attained at 60 min, lactate rose at a slower rate, reaching peak levels at 150 min: although no change in IRI was noted. In contrast, 2 DG-induced glucopenic stress in the autonomic denervated subjects was characterized by no detectable catecholamine release or significant rise in glucose, FFA, lactate, or IRI. However, HGH and cortisol responses in four of the five patients were of a similar or greater magnitude than controls.
These studies demonstrate that the integrity of the sympathoadrenomedullary axis is essential for the counter-regulatory response to intracellular glucopenia in man. Cortisol and HGH have no apparent role in these events.
Antimicrobial treatment in critically ill patients can either be started as soon as infection is suspected or after objective data confirm an infection. We postulated that delaying antimicrobial treatment of patients with suspected infections in the surgical intensive care unit (SICU) until objective evidence of infection had been obtained would not worsen patient mortality.
We did a 2-year, quasi-experimental, before and after observational cohort study of patients aged 18 years or older who were admitted to the SICU of the University of Virginia (Charlottesville, VA, USA). From Sept 1, 2008, to Aug 31, 2009, aggressive treatment was used: patients suspected of having an infection on the basis of clinical grounds had blood cultures sent and antimicrobial treatment started. From Sept 1, 2009, to Aug 31, 2010, a conservative strategy was used, with antimicrobial treatment started only after objective findings confirmed an infection. Our primary outcome was in-hospital mortality. Analyses were by intention to treat.
Admissions to the SICU for the first and second years were 762 and 721, respectively, with 101 patients with SICU-acquired infections during the aggressive year and 100 patients during the conservative year. Compared with the aggressive approach, the conservative approach was associated with lower all-cause mortality (13/100 [13%] vs 27/101 [27%]; p=0.015), more initially appropriate therapy (158/214 [74%] vs 144/231 [62%]; p=0.0095), and a shorter mean duration of therapy (12.5 days [SD 10.7] vs 17.7 [28.1]; p=0.0080). After adjusting for age, sex, trauma involvement, acute physiology and chronic health evaluation (APACHE) II score, and site of infection, the odds ratio for the risk of mortality in the aggressive therapy group compared with the conservative therapy group was 2.5 (95% CI 1.5−4.0).
Waiting for objective data to diagnose infection before treatment with antimicrobial drugs for suspected SICU-acquired infections does not worsen mortality and might be associated with better outcomes and use of antimicrobial drugs.
Free radical-induced oxidation of membrane phospholipids generates complex mixtures of oxidized phospholipids (oxPLs). The combinatorial operation of a few dozen reaction types upon a few dozen phospholipid structures results in the production of a dauntingly vast diversity of oxPL molecular species. Structural identification of the individual oxPL in these mixtures is a redoubtable challenge that is absolutely essential to allow determination of the biological activities of individual species. With an emphasis on cardiovascular consequences, this review focuses on biological activities of oxPLs whose molecular structures are known and highlights two diametric opposite approaches that were used to determine those structures, i.e. (1) the classical approach from bioactivity of a complex mixture to isolation and structural characterization of the active molecule followed by confirmation of the structure by unambiguous chemical synthesis, and (2) hypothesis of products likely to be generated by lipid oxidation, followed by synthesis, and then detection in vivo guided by the availability of authentic standards, and lastly, characterization of biological activities. Especially important for the application of the second paradigm is the capability of LC-MS/MS and derivatizations to selectively detect and quantify specific oxPL in complex mixtures, without the need for their isolation or complete separation. This technology can provide strong evidence for identity by comparisons with pure, well characterized samples available by chemical syntheses. Those pure samples are critical for determining the biological activities attributable to specific molecular species of oxPLs in the complex mixtures generated in vivo as a consequence of oxidative stress.
Cardiovascular Disease; Oxidized Phospholipids; Liquid Chromatography-Mass Spectrometry
Many of the pathological effects of lipid peroxidation are mediated by aldehydes generated through fragmentation of lipid peroxides. Among these aldehydes, the γ-hydroxy- and γ-oxo-α,β-alkenals, e.g., 4-hydroxy-2-nonenal (HNE), and 4-oxo-2-nonenal (ONE), are especially prone to modify proteins and DNA through covalent adduction. In addition the “mirror image” γ-hydroxy- and γ-oxo- α,β-alkenal phospholipids, can serve as high affinity ligands for biological receptors triggering pathology. Therefore, the mechanisms by which these aldehydes are generated in vivo are under intense scrutiny. We now report observations supporting the intermediacy of a unique pseudo symmetrical diepoxycarbinyl radical that accounts for the coproduction of HNE, ONE and their “mirror image” analogues 9-hydroxy-12-oxo-dodec-10-enoic acid (HODA), 9-keto-12-oxo-dodec-10-enoic acid (KODA) upon fragmentation of 13-hydroperoxy-cis-9,10-epoxyoctadeca-11-enoic acid (13-HP-Epo-Acid).
lipid peroxidation; linoleate-derived γ-hydroperoxy-α,β-unsaturated epoxide; HNE; ONE; HODA; KODA; pseudo symmetrical diepoxycarbinyl radical
The magnetic field dependence of the composite 1H2O NMR signal T1 was measured for excised samples of rat liver, muscle, and kidney over the field range from 0.7 to 7 T (35 MHz to 300 MHz) with a NMR spectrometer using sample-shuttle methods. Based on extensive measurements on simpler component systems, the magnetic field dependence of T1 of all tissues studied are readily fitted at Larmor frequencies above 1 MHz with a simple relaxation equation consisting of three contributions: a power law, A*ω−0.60 related to the interaction of water with long-lived-protein binding sites, a logarithmic term B*τd*log(1+1/(ωτd)2) related to water diffusion at macromolecular interfacial regions, and a constant term associated with the high frequency limit of water-spin-lattice relaxation. The parameters A and B include the concentration and surface area dependences respectively. The logarithmic diffusion term becomes significant at high magnetic fields and is consistent with rapid translational dynamics at macromolecular surfaces. The data are fitted well with translational correlation times of approximately 15 ps for human brain white matter, but with a B value three times larger than gray matter tissues. This analysis suggests that the water-surface translational correlation time is approximately three times longer than gray matter.
T1; spin-lattice relaxation; liver; kidney; muscle; magnetic relaxation dispersion; contrast field dependence
The oral pathogen, Streptococcus mutans, possesses inducible DNA repair defenses for protection against pH fluctuations and production of reactive oxygen metabolites such as hydrogen peroxide (H2O2), which are present in the oral cavity. DNA base excision repair (BER) has a critical role in genome maintenance by preventing the accumulation of mutations associated with environmental factors and normal products of cellular metabolism. In this study, we examined the consequences of compromising the DNA glycosylases (Fpg and MutY) and endonucleases (Smx and Smn) of the BER pathway and their relative role in adaptation and virulence. Enzymatic characterization of the BER system showed that it protects the organism against the effects of the highly mutagenic lesion, 7,8-dihydro-8-oxo-2’-deoxyguanine (8-oxo-dG). S. mutans strains lacking a functional Fpg, MutY, or Smn showed elevated spontaneous mutation frequencies; and, these mutator phenotypes correlated with the ability of the strains to survive killing by acid and oxidative agents. In addition, in the G. mellonella virulence model, strains of S. mutans deficient in Fpg, MutY and Smn showed increased virulence as compared to the parent strain. Our results suggest that, for S. mutans, mutator phenotypes, due to loss of BER enzymes, may confer an advantage to virulence of the organism.
Oral streptococci; DNA repair; base excision repair; stress response; mutagenesis
Background & Aims
Zebrafish mutants generated by ethylnitrosourea (ENU)-mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes, including organogenesis. One zebrafish mutant, “flotte lotte” (flo), displays striking defects in intestinal, liver, pancreas and eye formation at 78hpf. In this study we sought to identify the underlying mutated gene in flo and link the genetic lesion to its phenotype.
Positional cloning was employed to map the flo mutation. Sub-cellular characterization of flo embryos was achieved using histology, immunocytochemistry, bromodeoxyuridine incorporation analysis, confocal and electron microscopy.
The molecular lesion in flo is a nonsense mutation in the elys (embryonic large molecule derived from yolk sac) gene which encodes a severely truncated protein lacking the Elys C-terminal AT-hook DNA binding domain. Recently, ELYS has been shown to play a critical, and hitherto unsuspected, role in nuclear pore assembly. Though elys mRNA is expressed broadly during early zebrafish development, widespread early defects in flo are circumvented by the persistence of maternally-expressed elys mRNA until 24hpf. From 72hpf, elys mRNA expression is restricted to proliferating tissues, including the intestinal epithelium, pancreas, liver and eye. Cells in these tissues display disrupted nuclear pore formation; ultimately intestinal epithelial cells undergo apoptosis.
Our results demonstrate that Elys regulates digestive organ formation.
Pediatric gastrointestinal stromal tumors (GIST) are rare and occur
preferentially in females as multifocal gastric tumors, typically lacking
mutations in KIT and PDGFRA. As KIT
oncoprotein is consistently overexpressed in pediatric GIST, we sought to
investigate the activation of KIT downstream targets and alterations of
KIT/PDGFRA gene copy number, mine novel therapeutic
targets by gene expression, and test tyrosine kinase receptor activation by
Seventeen pediatric GISTs were investigated for
KIT/PDGFRA genotype and biochemical activation of KIT
downstream targets. The transcriptional profile of 13 nodules from 8
pediatric patients was compared with 8 adult wild-type (WT) GISTs, including
3 young adults. The drug sensitivity of second-generation kinase inhibitors
was tested in murine Ba/F3 cells expressing human WT KIT, as well as in
short-term culture of explants of WT GIST cells.
A KIT/PDGFRA WT genotype was identified in all 12
female patients, whereas two of five males had either a KIT
exon 11 or PDGFRA exon 18 mutation. KIT downstream targets
were consistently activated. Pediatric GISTs showed a distinct
transcriptional signature, with overexpression of BAALC, PLAG1,
IGF1R, FGF4, and NELL1. In vitro studies
showed that nilotinib, sunitinib, dasatinib, and sorafenib are more
effective than imatinib against WT KIT.
Rare cases of pediatric GIST may occur in male patients and harbor
activating KIT/PDGFRA mutations. Pediatric GISTs show
distinct transcriptional signature, suggesting a different biology than WT
GIST in adults. In vitro drug screening showed that
second-generation kinase inhibitors may provide greater clinical benefit in
In this communication, a translational clinical brain-machine interface (BMI) roadmap for an EEG-based BMI to a robotic exoskeleton (NeuroRex) is presented. This multi-faceted project addresses important engineering and clinical challenges: It addresses the validation of an intelligent, self-balancing, robotic lower-body and trunk exoskeleton (Rex) augmented with EEG-based BMI capabilities to interpret user intent to assist a mobility-impaired person to walk independently. The goal is to improve the quality of life and health status of wheelchair-bounded persons by enabling standing and sitting, walking and backing, turning, ascending and descending stairs/curbs, and navigating sloping surfaces in a variety of conditions without the need for additional support or crutches.
Brain-Machine Interface (BMI) systems allow users to control external mechanical systems using their thoughts. Commonly used in literature are invasive techniques to acquire brain signals and decode user’s attempted motions to drive these systems (e.g. a robotic manipulator). In this work we use a lower-body exoskeleton and measure the users brain activity using non-invasive electroencephalography (EEG). The main focus of this study is to decode a paraplegic subject’s motion intentions and provide him with the ability of walking with a lower-body exoskeleton accordingly. We present our novel method of decoding with high offline evaluation accuracies (around 98%), our closed loop implementation structure with considerably short on-site training time (around 38 sec), and preliminary results from the real-time closed loop implementation (NeuroRex) with a paraplegic test subject.
The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA) function in the E. coli periplasm, while recombinant system III (CCHL) attaches heme to its cognate receptor in the cytoplasm of E. coli, which makes direct comparisons between the three systems difficult. Here we show that the human CCHL (with a secretion signal) attaches heme to the human cytochrome c (with a signal sequence) in the E.coli periplasm, which is bioenergetically (p-side) analogous to the mitochondrial intermembrane space. The human CCHL is specific for the human cytochrome c, whereas recombinant system II can attach heme to multiple non-cognate c-type cytochromes (possessing the CXXCH motif.) We also show that the recombinant periplasmic systems II and III use components of the natural E.coli periplasmic DsbC/DsbD thiol-reduction pathway.
cytochrome c assembly; CCHL; heme attachment; periplasmic; thioreduction
The Forkhead Box O (Foxo) proteins represent an evolutionarily conserved family of transcription factors that play an important role in regulating processes including metabolism, longevity, and cell death/survival. How is it that a single transcription factor can initiate such divergent cellular responses? We will review the evidence that specific patterns of post-translational modifications play a key role in directing Foxo into various transcriptional readouts. This regulation appears to take on a two tiered regulatory model; with a group of well defined post-translational modifications regulating nuclear localization and transcriptional activity while a second set of modifications regulate the transcriptional specificity of Foxo.
Foxo; post-translational modifications; phosphorylation; acetylation; ubiquitination
It is not well understood how people perceive the difficulty of performing brain-computer interface (BCI) tasks, which specific aspects of mental workload contribute the most, and whether there is a difference in perceived workload between participants who are able-bodied and disabled. This study evaluated mental workload using the NASA Task Load Index (TLX), a multi-dimensional rating procedure with six subscales: Mental Demands, Physical Demands, Temporal Demands, Performance, Effort, and Frustration. Able-bodied and motor disabled participants completed the survey after performing EEG-based BCI Fitts’ law target acquisition and phrase spelling tasks. The NASA-TLX scores were similar for able-bodied and disabled participants. For example, overall workload scores (range 0 – 100) for 1D horizontal tasks were 48.5 (SD = 17.7) and 46.6 (SD 10.3), respectively. The TLX can be used to inform the design of BCIs that will have greater usability by evaluating subjective workload between BCI tasks, participant groups, and control modalities.
BCI; Brain-computer interface; mental workload; NASA Task Load Index; NASA-TLX; Fitts’ law; EEG; electroencephalogram
We described a versatile spectrometer designed for the study of dynamic nuclear polarization (DNP) at low temperatures and high fields. The instrument functions both as an NMR spectrometer operating at 212 MHz (1H frequency) with DNP capabilities, and as a pulsed-EPR operating at 140 GHz. A coiled TE011 resonator acts as both an NMR coil and microwave resonator, and a double balanced (1H, 13C) radio frequency circuit greatly stabilizes the NMR performance. A new 140 GHz microwave bridge has also been developed, which utilizes a four-phase network and ELDOR channel at 8.75 GHz, that is then multiplied and mixed to obtain 140 GHz microwave pulses with an output power of 120 mW. Nutation frequencies obtained are as follows: 6 MHz on S = ½ electron spins, 100 kHz on 1H, and 50 kHz on 13C. We demonstrate basic EPR, ELDOR, ENDOR, and DNP experiments here. Our solid effect DNP results demonstrate an enhancement of 144 and sensitivity gain of 310 using OX063 trityl at 80 K and an enhancement of 157 and maximum sensitivity gain of 234 using Gd-DOTA at 20 K, which is significantly better performance than previously reported at high fields (>3 T).
To evaluate associations between ADHD and comorbid psychiatric disorders using research-identified incident cases of ADHD and population-based controls.
Subjects included a birth cohort of all children born 1976–1982 remaining in Rochester, MN after age five (n = 5718). Among them we identified 379 ADHD incident cases and 758 age-sex matched non-ADHD controls, passively followed to age 19 years. All psychiatric diagnoses were identified and abstracted, but only those confirmed by qualified medical professionals were included in the analysis. For each psychiatric disorder, cumulative incidence rates for subjects with and without ADHD were estimated using the Kaplan-Meier method. Corresponding hazard ratios (HR) were estimated using Cox models adjusted for gender and mother’s age and education at the subject’s birth. The association between ADHD and the likelihood of having an internalizing or externalizing disorder was summarized by estimating odds ratios (OR).
ADHD was associated with a significantly increased risk of adjustment disorders (HR=3.88), conduct/oppositional defiant disorder (HR=9.54), mood disorders (HR=3.67), anxiety disorders (HR=2.94), tic disorders (HR=6.53), eating disorders (HR=5.68), personality disorders (HR=5.80), and substance-related disorders (HR=4.03). When psychiatric comorbidities were classified on the internalization-externalization dimension, ADHD was strongly associated with coexisting internalizing/externalizing (OR=10.6), or externalizing-only (OR=10.0) disorders.
This population-based study confirms that children with ADHD are at significantly increased risk for a wide range of psychiatric disorders. Besides treating the ADHD, clinicians should identify and provide appropriate treatment for psychiatric comorbidities.
ADD/ADHD; psychiatric practice; epidemiology
Several novel anti-influenza compounds are in various phases of clinical development. One of these, T-705 (favipiravir), has a mechanism of action that is not fully understood but is suggested to target influenza virus RNA-dependent RNA polymerase. We investigated the mechanism of T-705 activity against influenza A (H1N1) viruses by applying selective drug pressure over multiple sequential passages in MDCK cells. We found that T-705 treatment did not select specific mutations in potential target proteins, including PB1, PB2, PA, and NP. Phenotypic assays based on cell viability confirmed that no T-705-resistant variants were selected. In the presence of T-705, titers of infectious virus decreased significantly (P < 0.0001) during serial passage in MDCK cells inoculated with seasonal influenza A (H1N1) viruses at a low multiplicity of infection (MOI; 0.0001 PFU/cell) or with 2009 pandemic H1N1 viruses at a high MOI (10 PFU/cell). There was no corresponding decrease in the number of viral RNA copies; therefore, specific virus infectivity (the ratio of infectious virus yield to viral RNA copy number) was reduced. Sequence analysis showed enrichment of G→A and C→T transversion mutations, increased mutation frequency, and a shift of the nucleotide profiles of individual NP gene clones under drug selection pressure. Our results demonstrate that T-705 induces a high rate of mutation that generates a nonviable viral phenotype and that lethal mutagenesis is a key antiviral mechanism of T-705. Our findings also explain the broad spectrum of activity of T-705 against viruses of multiple families.
Highly pathogenic avian influenza (HPAI) virus H5N1 has been enzootic in Egypt since 2008. Virus-associated mortality (but not the number of cases) in humans and poultry seems to have decreased over time, but the reason for this remains unknown. We investigated the role of a single amino acid substitution in the hemagglutinin cleavage site on virus pathogenicity and transmission in chickens. The R325G substitution significantly reduced pathogenicity without altering the transmission efficiency of HPAI H5N1 virus.
Doxorubicin (DOX) is a commonly used life-saving antineoplastic agent that also causes dose-dependent cardiotoxicity. Because ATP is absolutely required to sustain normal cardiac contractile function and because impaired ATP synthesis through creatine kinase (CK), the primary myocardial energy reserve reaction, may contribute to contractile dysfunction in heart failure, we hypothesized that impaired CK energy metabolism contributes to DOX-induced cardiotoxicity. We therefore overexpressed the myofibrillar isoform of CK (CK-M) in the heart and determined the energetic, contractile and survival effects of CK-M following weekly DOX (5mg/kg) administration using in vivo31P MRS and 1H MRI. In control animals, in vivo cardiac energetics were reduced at 7 weeks of DOX protocol and this was followed by a mild but significant reduction in left ventricular ejection fraction (EF) at 8 weeks of DOX, as compared to baseline. At baseline, CK-M overexpression (CK-M-OE) increased rates of ATP synthesis through cardiac CK (CK flux) but did not affect contractile function. Following DOX however, CK-M-OE hearts had better preservation of creatine phosphate and higher CK flux and higher EF as compared to control DOX hearts. Survival after DOX administration was significantly better in CK-M-OE than in control animals (p<0.02). Thus CK-M-OE attenuates the early decline in myocardial high-energy phosphates and contractile function caused by chronic DOX administration and increases survival. These findings suggest that CK impairment plays an energetic and functional role in this DOX-cardiotoxicity model and suggests that metabolic strategies, particularly those targeting CK, offer an appealing new strategy for limiting DOX-associated cardiotoxicity.
Carboxyethylpyrrole (CEP) adducts are oxidative modifications derived from docosahexaenoate-containing lipids that are elevated in ocular tissues and plasma in age-related macular degeneration (AMD) and in rodents exposed to intense light. The goal of this study was to determine whether light-induced CEP adducts and autoantibodies are modulated by pretreatment with AL-8309A under conditions that prevent photo-oxidative damage of rat retina. AL-8309A is a serotonin 5-HT1A receptor agonist.
Albino rats were dark adapted prior to blue light exposure. Control rats were maintained in normal cyclic light. Rats were injected subcutaneously 3x with 10 mg/kg AL-8309A (2 days, 1 day and 0 hours) before light exposure for 6 h (3.1 mW/cm2, λ=450 nm). Animals were sacrificed immediately following light exposure and eyes, retinas and plasma were collected. CEP adducts and autoantibodies were quantified by Western analysis or ELISA.
ANOVA supported significant differences in mean amounts of CEP adducts and autoantibodies among the light + vehicle, light + drug and dark control groups from both retina and plasma. Light-induced CEP adducts in retina were reduced ~20% following pretreatment with AL-8309A (n = 62 rats, p = 0.006) and retinal CEP immunoreactivity was less intense by immunohistochemistry. Plasma levels of light-induced CEP adducts were reduced at least 30% (n = 15 rats, p = 0.004) by drug pretreatment. Following drug treatment, average CEP autoantibody titer in light exposed rats (n = 22) was unchanged from dark control levels, and ~20% (p = 0.046) lower than in vehicle-treated rats.
Light-induced CEP adducts in rat retina and plasma were significantly decreased by pretreatment with AL-8309A. These results are consistent with and extend previous studies showing AL-8309A reduces light-induced retinal lesions in rats and support CEP biomarkers as possible tools for monitoring the efficacy of select therapeutics.
There remains considerable controversy in the management of eosinophilic disorders, mainly due to a paucity of information regarding the clinical interpretation of total blood eosinophil counts versus surface activation markers versus eosinophil-derived or eosinophil-influencing mediator levels. Regrettably, few tests have been validated that define a unique clinical or prognostic phenotype that is more useful than simply monitoring total blood eosinophil counts. In this manuscript, phenotypic (cell surface) markers, along with serum and tissue-based markers that have been examined in the context of disease activity, are reviewed. We also report the development of a novel assay for detecting soluble Siglec-8 (sSiglec-8), a protein likely derived largely from eosinophils, as a potential serum biomarker. The assay consists of a competitive ELISA using a recombinant Siglec-8-Fc fusion protein. The goal of this preliminary study was to determine if sSiglec-8 is a useful biomarker that differentiates among patients with various eosinophil-associated diseases. In the final analysis, it is fair to say that further research is sorely needed to fully understand and validate the utility of various biomarkers, including sSiglec-8, before their use in clinical practice can be recommended with confidence.
Eosinophil; granule proteins; ELISA; soluble Siglec-8