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BACKGROUND: Anorexia nervosa (AN) is associated with left ventricular (LV) atrophy and unexplained sudden death. Myocardial mechanics have not been well studied in adults with AN. Whether LV mass or illness duration, markers of AN severity, correlate with abnormal strain imaging is unknown. METHODS: We performed a prospective study among patients hospitalized with severe AN (n = 29) [body mass index (BMI) < 14.5 kg/m2] and sex/age-matched controls (n = 16) (BMI > 18.5 kg/m2). LV ejection fraction (LVEF) was calculated via modified-biplane method and LV mass was derived using the truncated ellipsoid formula. Apical 2-, 3-, and 4-chamber images were used to generate regional strain mapping and global longitudinal strain (GLS). N-terminal brain natriuretic peptide (NT-proBNP) levels were measured and linear regression was used to determine independent predictors of strain. RESULTS: Mean LVEF did not differ (65% ± 6.0 vs. 62% ± 4.4, p = 0.06), but LV mass was substantially reduced (61.6 ± 16.8 vs. 97.6 ± 19.1 g, p < .0001). GLS was similar (- 20.6 ± 3.8 vs. - 20.9 ± 2.8, p = 0.82), however, the basal strain was worse (-18.7 ± 4.8 vs. -21.9 ± 4.1, p = 0.03). Lower LV mass was associated with worsening GLS (r = - 0.40, p = 0.003), but not among controls (p = 0.89). Median (IQR) NT-proBNP (pg/ml) was higher in patients with AN [141 (59-257) vs. 35.5 (21-56.5) p = 0.0007]. Both increasing NT-proBNP and illness duration were associated with worsening strain patterns in AN (both p = .001). CONCLUSIONS: While LVEF and GLS did not differ, regional strain variation was noted among patients with AN. Elevated NT-proBNP may reflect increased wall tension from LV atrophy. Whether strain heterogeneity can identify patients with AN, at risk for sudden death, requires further study.
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We aimed to evaluate whether the content of eating/body image-related beliefs in individuals with anorexia nervosa (AN) was associated with important aspects of eating disorder (ED) psychopathology. Females with AN completed assessments within 96 hours of admission to an inpatient medical stabilization program. Study staff administered the Brown Assessment of Beliefs Scale and participants completed self-report measures. We derived belief content domains using an inductive approach and examined associations between beliefs and clinical variables. The following belief categories emerged (% with a belief in that category): body image beliefs (64%), food beliefs (30%), body function beliefs (20%), rejection of illness beliefs (12%), morality beliefs (10%), and control beliefs (6%). No one belief domain was significantly associated with greater delusional intensity. However, findings indicate that greater delusionality was generally associated with worse ED psychopathology.
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Anorexia Nerviosa , Trastornos de Alimentación y de la Ingestión de Alimentos , Adulto , Humanos , Femenino , Psicopatología , Imagen Corporal , HospitalizaciónRESUMEN
The composition of the gut microbiota in patients with anorexia nervosa (AN), and the ability of this microbial community to influence the host, remains uncertain. To achieve a broader understanding of the role of the intestinal microbiota in patients with AN, we collected fecal samples before and following clinical treatment at two geographically distinct eating disorder units (Center of Excellence for Eating Disorders [UNC-CH] and ACUTE Center for Eating Disorders [Denver Health]). Gut microbiotas were characterized in patients with AN, before and after inpatient treatment, and in non-eating disorder (non-ED) controls using shotgun metagenomic sequencing. The impact of inpatient treatment on the AN gut microbiota was remarkably consistent between eating disorder units. Although weight in patients with AN showed improvements, AN microbiotas post-treatment remained distinct from non-ED controls. Additionally, AN gut microbiotas prior to treatment exhibited more fermentation pathways and a lower ability to degrade carbohydrates than non-ED controls. As the intestinal microbiota can influence nutrient metabolism, our data highlight the complex microbial communities in patients with AN as an element needing further attention post inpatient treatment. Additionally, this study defines the effects of renourishment on the AN gut microbiota and serves as a platform to develop precision nutrition approaches to potentially mitigate impediments to recovery.
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Anorexia Nerviosa , Microbioma Gastrointestinal , Microbiota , Humanos , Anorexia Nerviosa/terapia , Pacientes Internos , HecesRESUMEN
Objectives: To understand the presence of transient autophony symptoms in patients being treated for severe anorexia nervosa (AN), and whether those symptoms were due to patulous eustachian tube (PET). Methods: A prospective observational study was performed in patients requiring admission for treatment of severe AN. All enrolled patients completed The Eustachian Tube Dysfunction Questionnaire (ETDQ-7) and were screened for symptoms of autophony. If patients reported autophony and had a score of ≥14.5 on the ETDQ-7 they were asked to undergo comprehensive audiological testing and an evaluation with an otolaryngologist. Results: Of the 73 patients enrolled in the study, 35 patients (44%) reported autophony and 36 (49%) scored 14.5 or higher on the ETDQ-7. Of the 16 (22%) patients who had both autophony and an ETDQ-7 score of 14.5 or higher, 7 patient s (representing 11 symptomatic ears) underwent evaluations by audiology and otolaryngology. Every evaluation of a symptomatic ear revealed objective evidence of PET. Nine of 11 (81.8%) symptomatic ears had subjectively resolved within 12 days of admission after nutritional rehabilitation and weight gain. Conclusion: Transient autophony in severe AN patients is due to PET, and was present in at least 8% of patients within our cohort. Further study is warranted to understand the quality of life impact and pathophysiology of transient PET in this patient population.
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OBJECTIVE: To assess for the prevalence of autophony, a distressing auditory symptom commonly attributed to patulous eustachian tube, in a cohort of individuals with severe malnourishment due to an eating disorder. METHOD: A cross-sectional survey study was performed. Patients admitted for inpatient medical stabilization of an eating disorder, who were also at low body weight, were asked to complete a survey assessing aural symptoms present in the previous 24 hr, including autophony. Anthropometric data and prealbumin levels were collected. RESULTS: Of 101 patients enrolled, 43 (42.6%) reported symptoms of autophony. The presence of autophony was associated with lower serum prealbumin levels and lower body weight as measured by percentage of ideal body weight. DISCUSSION: Autophony is a commonly reported, albeit rarely discussed, symptom in individuals with severe eating disorders and correlates with degree of malnutrition.
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Anorexia Nerviosa , Trastorno de la Ingesta Alimentaria Evitativa/Restrictiva , Trastornos de Alimentación y de la Ingestión de Alimentos , Desnutrición , Anorexia Nerviosa/complicaciones , Estudios Transversales , Trastornos de Alimentación y de la Ingestión de Alimentos/complicaciones , Humanos , Pacientes Internos , Desnutrición/complicaciones , Desnutrición/epidemiología , Estudios RetrospectivosRESUMEN
OBJECTIVE: To investigate serum uric acid (UA) levels in patients with extreme forms of eating disorders, at admission and discharge, following weeks of nutritional rehabilitation and weight restoration. METHOD: This observational study enrolled 160 patients diagnosed with anorexia nervosa restricting subtype (AN-R), AN binge-purge subtype (AN-BP), or avoidant restrictive food intake disorder (ARFID). Serum UA levels were drawn on admission and discharge. RESULTS: Most of the cohorts were admitted with serum UA levels on the lower end of normal. Mean serum uric level for women was 4.3 mg/dl (SD: 2.3). Patients diagnosed with AN-BP had significantly higher UA levels on admission compared to patients with AN-R and ARFID; p < .0001, η2 = 0.13. High UA levels positively correlated with purging and admission serum blood urea nitrogen (r = .5, p = .009). DISCUSSION: Serum UA levels tended to be in the low-normal range in most patients with severe AN-R, but not in AN-BP. However, levels did increase with nutritional intake and weight gain. There may be clinical value in checking UA levels on admission for patients with eating disorders.
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Anorexia Nerviosa , Trastorno de la Ingesta Alimentaria Evitativa/Restrictiva , Trastorno por Atracón , Trastornos de Alimentación y de la Ingestión de Alimentos , Adulto , Anorexia Nerviosa/diagnóstico , Ingestión de Alimentos , Femenino , Humanos , Estudios Retrospectivos , Ácido ÚricoRESUMEN
OBJECTIVE: Eating disorder-related beliefs among individuals with anorexia nervosa (AN) often approach delusional intensity. Research to date on delusional beliefs in AN has been cross sectional. Thus, it is unknown how the intensity of delusional beliefs changes over time and if such change has prognostic value. METHOD: We assessed 50 individuals with severe to extreme AN (≥18 years old; M [SD] body mass index =12.7[1.3] kg/m2 ) at an inpatient medical stabilization facility within 96 hr of admission; 35 (70%) also completed the assessment at discharge (M[SD] = 25.53[13.21] days). Participants completed the Brown Assessment of Beliefs Scale and a battery of self-report measures of eating disorder-related psychopathology. RESULTS: The admission-to-discharge decrease in delusional intensity was not significant (p = .592; Hedges g = .10). Tests of predictive effects indicated that higher delusional intensity at intake predicted higher fear of fatness and restrictive eating, two hallmark features of AN, but not BMI, body checking, feared food avoidance, eating disorder-related impairment, depression, binge eating, or purging behavior at discharge. DISCUSSION: Although the delusional intensity of eating disorder beliefs did not significantly improve over this relatively brief interval, delusional intensity may be associated with the severity of central eating disorder attitudes and behaviors. Delusional intensity may therefore be a negative prognostic indicator, possibly warranting further treatment. Future research should examine changes in delusional intensity over longer intervals and test whether specifically targeting delusional beliefs improves treatment outcomes among individuals with AN.
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Anorexia Nerviosa , Trastorno por Atracón , Adolescente , Anorexia Nerviosa/terapia , Índice de Masa Corporal , Estudios Transversales , Humanos , PronósticoRESUMEN
BACKGROUND: Stimulant laxative abuse as a purging behavior can be profound in those with eating disorders. However, the psychopathology leading to stimulant laxative abuse is poorly understood. Furthermore, the medical impact of stimulant laxative abuse has not been studied in this population. METHODS: Six individuals abusing stimulant laxatives underwent a barium enema to assess for evidence of the cathartic colon syndrome and 29 individuals engaging in any purging behaviors completed the Tri-dimensional Personality Questionnaire-Short Form, Sensitivity to Punishment/Sensitivity to Reward Questionnaire-Short Form, Beck Depression Inventory, and the State Trait Anxiety Inventory questionnaires. RESULTS: Three of the six patients completing the barium enema had the radiographic findings consistent with cathartic colon. Individuals engaging in laxative abuse showed higher Novelty Seeking compared to those engaging in other forms of purging, and those engaging in any form of purging behavior showed greater Sensitivity to Punishment compared to Sensitivity to Reward. There was also the presence of greater Harm Avoidance than Reward Dependence in this population. CONCLUSION: There may be different psychopathology that contributes to the abuse of stimulant laxatives than that associated with other forms of purging. Regardless of the driving factor, further research is warranted to discover best therapeutic interventions given the potential to develop the cathartic colon syndrome with ongoing use of stimulant laxatives. Cathartic colon is a condition whereby the colon, or lower intestine, is converted into an inert tube incapable of propagating fecal matter. It is thought to develop due to over-use of stimulant laxatives. However, it is unclear if this condition truly exists and whether it contributes to the constipation experienced by individuals with eating disorders who have extensive past histories of abusing laxatives. It is also unclear if laxative abuse presents with different medical complications than other forms of purging. The purpose of this study is to determine whether radiographic evidence of cathartic colon can be found in eating disorder patients abusing stimulant laxatives, whether there are different medical complications with laxative abuse versus other forms of purging, and to examine the psychological composition of individuals who engage in severe laxative abuse. Specifically, the authors investigated the interrelationship between Harm Avoidance and Reward Dependence, with emphasis on gaining a better understanding of Reward Dependence by examining both Sensitivity to Reward and Sensitivity to Punishment in patients who engage in severe laxative abuse. Our findings suggest that stimulant laxative abuse may cause the development of cathartic colon changes and that there may be unique psychopathology that contributes to the abuse of stimulant laxatives. Given the higher Novelty Seeking personality-dimension in those abusing laxatives, it is possible that this purging behavior may be considered addiction-like in nature, which would have distinct treatment implications.
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OBJECTIVES: Prone position ventilation is a potentially life-saving ancillary intervention but is not widely adopted for coronavirus disease 2019 or acute respiratory distress syndrome from other causes. Implementation of lung-protective ventilation including prone positioning for coronavirus disease 2019 acute respiratory distress syndrome is limited by isolation precautions and personal protective equipment scarcity. We sought to determine the safety and associated clinical outcomes for coronavirus disease 2019 acute respiratory distress syndrome treated with prolonged prone position ventilation without daily repositioning. DESIGN: Retrospective single-center study. SETTING: Community academic medical ICU. PATIENTS: Sequential mechanically ventilated patients with coronavirus disease 2019 acute respiratory distress syndrome. INTERVENTIONS: Lung-protective ventilation and prolonged protocolized prone position ventilation without daily supine repositioning. Supine repositioning was performed only when Fio2 less than 60% with positive end-expiratory pressure less than 10 cm H2O for greater than or equal to 4 hours. MEASUREMENTS AND MAIN RESULTS: Primary safety outcome: proportion with pressure wounds by Grades (0-4). Secondary outcomes: hospital survival, length of stay, rates of facial and limb edema, hospital-acquired infections, device displacement, and measures of lung mechanics and oxygenation. Eighty-seven coronavirus disease 2019 patients were mechanically ventilated. Sixty-one were treated with prone position ventilation, whereas 26 did not meet criteria. Forty-two survived (68.9%). Median (interquartile range) time from intubation to prone position ventilation was 0.28 d (0.11-0.80 d). Total prone position ventilation duration was 4.87 d (2.08-9.97 d). Prone position ventilation was applied for 30.3% (18.2-42.2%) of the first 28 days. Pao2:Fio2 diverged significantly by day 3 between survivors 147 (108-164) and nonsurvivors 107 (85-146), mean difference -9.632 (95% CI, -48.3 to 0.0; p = 0·05). Age, driving pressure, day 1, and day 3 Pao2:Fio2 were predictive of time to death. Thirty-eight (71.7%) developed ventral pressure wounds that were associated with prone position ventilation duration and day 3 Sequential Organ Failure Assessment. Limb weakness occurred in 58 (95.1%) with brachial plexus palsies in five (8.2%). Hospital-acquired infections other than central line-associated blood stream infections were infrequent. CONCLUSIONS: Prolonged prone position ventilation was feasible and relatively safe with implications for wider adoption in treating critically ill coronavirus disease 2019 patients and acute respiratory distress syndrome of other etiologies.
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COVID-19/complicaciones , Evaluación de Procesos y Resultados en Atención de Salud , Posicionamiento del Paciente , Respiración Artificial/métodos , Síndrome de Dificultad Respiratoria/terapia , Insuficiencia Respiratoria/terapia , Centros Médicos Académicos , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Posición Prona , Síndrome de Dificultad Respiratoria/etiología , Insuficiencia Respiratoria/etiología , Estudios Retrospectivos , Estados Unidos/epidemiologíaRESUMEN
Deficiency of methyl donor nutrients folate, choline, and methionine (methyl deficiency) during gestation can impair fetal development and perturb DNA methylation. Here, we assessed genetic susceptibility to methyl deficiency by comparing effects in wildtype C57BL/6J (B6) mice to mutant mice carrying a 1.3 kb deletion at the H19/Igf2 Imprinting Control Region (ICR) (H19 ICRΔ2,3). The H19 ICRΔ2,3 mutation mimics microdeletions observed in Beckwith-Wiedemann syndrome (BWS) patients, who exhibit epimutations in cis that cause loss of imprinting and fetal overgrowth. Dams were treated during pregnancy with 1 of 4 methyl sufficient (MS) or methyl deficient (MD) diets, with or without the antibiotic commonly used to deplete folate producing gut microbes. As expected, after ~9 weeks of treatment, dams in MD and MD + antibiotic groups exhibited substantially reduced plasma folate concentrations. H19 ICRΔ2,3 mutant lines were more susceptible to adverse pregnancy outcomes caused by methyl deficiency (reduced birth rate and increased pup lethality) and antibiotic (decreased litter size and litter survival). Surprisingly, pup growth/development was only minimally affected by methyl deficiency, while antibiotic treatment caused inverse effects on B6 and H19 ICRΔ2,3 lines. B6 pups treated with antibiotic exhibited increased neonatal and weanling bodyweight, while both wildtype and mutant pups of heterozygous H19 ICRΔ2,3/+ dams exhibited decreased neonatal bodyweight that persisted into adulthood. Interestingly, only antibiotic-treated pups carrying the H19 ICRΔ2,3 mutation exhibited altered DNA methylation at the H19/Igf2 ICR, suggesting ICR epimutation was not sufficient to explain the altered phenotypes. These findings demonstrate that genetic mutation of the H19/Igf2 ICR increases offspring susceptibility to developmental perturbation in the methyl deficiency model, maternal and pup genotype play an essential role, and antibiotic treatment in the model also plays a key independent role.
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Previous work demonstrated that pre-exposure to ozone primes innate immunity and increases Toll-like receptor-4 (TLR4)-mediated responses to subsequent stimulation with LPS. To explore the pulmonary innate immune response to ozone exposure further, we investigated the effects of ozone in combination with Pam3CYS, a synthetic TLR2/TLR1 agonist. Whole-lung lavage (WLL) and lung tissue were harvested from C57BL/6 mice after exposure to ozone or filtered air, followed by saline or Pam3CYS 24 hours later. Cells and cytokines in the WLL, the surface expression of TLRs on macrophages, and lung RNA genomic expression profiles were examined. We demonstrated an increased WLL cell influx, increased IL-6 and chemokine KC (Cxcl1), and decreased macrophage inflammatory protein (MIP)-1α and TNF-α in response to Pam3CYS as a result of ozone pre-exposure. We also observed the increased cell surface expression of TLR4, TLR2, and TLR1 on macrophages as a result of ozone alone or in combination with Pam3CYS. Gene expression analysis of lung tissue revealed a significant increase in the expression of genes related to injury repair and the cell cycle as a result of ozone alone or in combination with Pam3CYS. Our results extend previous findings with ozone/LPS to other TLR ligands, and suggest that the ozone priming of innate immunity is a general mechanism. Gene expression profiling of lung tissue identified transcriptional networks and genes that contribute to the priming of innate immunity at the molecular level.
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Inmunidad Innata/efectos de los fármacos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Ozono/toxicidad , Receptor Toll-Like 2/agonistas , Animales , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Citocinas/metabolismo , Expresión Génica/efectos de los fármacos , Inmunidad Innata/genética , Mediadores de Inflamación/metabolismo , Lipoproteínas/farmacología , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Previous publications from our and other groups identified E2F1 as a transcription factor involved in the regulation of inflammatory response to Toll-like receptor ligands including LPS. In this study, we challenged E2F1-deficient mice with LPS systemically and demonstrated decreased survival despite attenuated inflammatory response compared with controls. Gene expression profiling of liver tissue identified a dampened transcriptional response in the coagulation cascade in B6;129(E2F1-/-) compared with B6x129 F2 mice. These data were further corroborated by increased prothrombin time, activated partial thromboplastin time, and fibrin split products in the blood of E2F1-deficient mice, suggesting disseminated intravascular coagulation as a consequence of uncontrolled sepsis providing at least a partial explanation for their decreased survival despite attenuated inflammatory response. To identify novel miRNAs involved in the innate immune response to LPS, we also performed miRNA profiling of liver tissue from B6;129(E2F1-/-) and B6x129 F2 mice treated with LPS systemically. Our analysis identified a set of miRNAs and their mRNA targets that are significantly differentially regulated in E2F1-deficient but not control mice including let-7g, miR-101b, miR-181b, and miR-455. These miRNAs represent novel regulators of the innate immune response. In summary, we used transcriptional and miRNA profiling to characterize the response of E2F1-deficient mice to systemic LPS.
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Factor de Transcripción E2F1/fisiología , Regulación de la Expresión Génica , Inmunidad Innata/genética , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Animales , Sitios de Unión , Factores de Coagulación Sanguínea/genética , Factores de Coagulación Sanguínea/metabolismo , Factor de Transcripción E2F1/deficiencia , Factor de Transcripción E2F1/genética , Perfilación de la Expresión Génica , Inflamación/genética , Inflamación/inmunología , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Bazo/inmunología , Bazo/patología , Trombosis/genéticaRESUMEN
Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These in vitro studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets immunized orally with rmSEB formulations experienced no significant decrease in food consumption and no weight loss during the vaccination regimen. Oral vaccination with 1-mg doses of rmSEB on days 0, 7, 14, and 24 resulted in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion of cholera toxin adjuvant in vaccine formulations containing rmSEB did not result in increased antibody responses compared to formulations using the immunogen alone. Taken together, these studies provide additional evidence for the potential use of nontoxic forms of SEB as vaccines.
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Anticuerpos Antibacterianos/sangre , Antitoxinas/sangre , Enterotoxinas/administración & dosificación , Enterotoxinas/inmunología , Inmunidad Mucosa , Vacunas Estafilocócicas/administración & dosificación , Vacunas Estafilocócicas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Administración Oral , Sustitución de Aminoácidos/genética , Animales , Animales Recién Nacidos , Toxina del Cólera/administración & dosificación , Enterotoxinas/toxicidad , Heces/química , Femenino , Humanos , Inmunización Secundaria/métodos , Inmunoglobulina A/análisis , Inmunoglobulina G/sangre , Masculino , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/inmunología , Proteínas Mutantes/toxicidad , Mutación Missense , Vacunas Estafilocócicas/efectos adversos , Vacunas Estafilocócicas/toxicidad , Superantígenos/administración & dosificación , Superantígenos/inmunología , Superantígenos/toxicidad , Porcinos , Factores de Tiempo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/toxicidadRESUMEN
Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of enteric diseases affecting livestock and humans. Edible transgenic plants producing E. coli fimbrial subunit proteins have the potential to vaccinate against these diseases, but have not reached their full potential as a renewable source of oral vaccines due in part to insufficient levels of recombinant protein accumulation. Previously, we reported that cytosol targeting of the E. coli K99 fimbrial subunit antigen resulted in FanC accumulation to approximately 0.4% of total soluble protein in soybean leaves (Piller et al. in Planta 222:6-18, 2005). In this study, we report on the subcellular targeting of FanC to chloroplasts. Twenty-two transgenic T1 progeny derived from seven individual T0 transformation events were characterized, and 17 accumulated transgenic FanC. All of the characterized events displayed relatively low T-DNA complexity, and all exhibited proper targeting of FanC to the chloroplast. Accumulation of chloroplast-targeted FanC was approximately 0.08% of total soluble leaf protein, or approximately 5-fold less than cytosol-targeted FanC. Protein analysis of leaves at various stages of maturity suggested stability of chloroplast-targeted FanC throughout leaf maturation. Furthermore, mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing chloroplast-targeted FanC developed significant antibody titers against FanC. This is the first report of subcellular targeting of a vaccine subunit antigen in soybean.
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Antígenos Bacterianos/genética , Cloroplastos/metabolismo , Escherichia coli/metabolismo , Proteínas Fimbrias/metabolismo , Glycine max/genética , Antígenos Bacterianos/metabolismo , Escherichia coli/genética , Proteínas Fimbrias/genética , Regulación de la Expresión Génica de las Plantas , Plantas Modificadas Genéticamente , Transporte de Proteínas , Glycine max/metabolismoRESUMEN
The upper 50-kDa region of myosin may be critical for coupling between the nucleotide- and actin-binding regions. We introduced a tetracysteine motif in the upper 50-kDa domain (residues 292-297) of myosin V containing a single IQ domain (MV 1IQ), allowing us to label this site with the fluorescein biarscenical hairpin-binding dye (FlAsH) (MV 1IQ FlAsH). The enzymatic properties of MV 1IQ FlAsH were similar to those of unlabeled MV 1IQ except for a 3-fold reduced ADP-release rate. MV 1IQ FlAsH was also capable of moving actin filaments in the in vitro motility assay. To examine rotation of the upper 50-kDa region, we determined the difference in the degree of energy transfer from N-methylanthraniloyl (mant)-labeled nucleotides to FlAsH in both steady-state and transient kinetic experiments. The energy transfer efficiency was higher with mant-ATP (0.65 +/- 0.02) compared with mant-ADP (0.55 +/- 0.02) in the absence of actin. Stopped-flow measurements suggested that the energy transfer efficiency decreased with phosphate release (0.04 s(-1)) in the absence of actin. In contrast, upon mixing MV 1IQ FlAsH in the ADP.P(i) state with actin, a decrease in the energy transfer signal was observed at a rate of 13 s(-1), similar to the ADP release rate. Our results demonstrate there was no change in the energy transfer signal upon actin-activated phosphate release and suggest that actin binding alters the dynamics of the upper 50-kDa region, which may be critical for the ability of myosin to bind tightly to both ADP and actin.