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Electroactive microbes that can release or take up electrons are essential components of nearly every ecological niche and are powerful tools for the development of alternative energy technologies. Small-molecule mediators are critical for this electron transfer but remain difficult to study and engineer because they perform concerted two-electron transfer in native systems but only individual, one-electron transfers in electrochemical studies. Here, we report that electrode modification with ion- and electron-conductive polymers yields biosimilar, concerted two-electron transfer from Shewanella oneidensis via flavin mediators. S. oneidensis biofilms on these polymers show significantly improved per-microbe current generation and morphologies that more closely resemble native systems, setting a new paradigm for the study and optimization of these electron transfer processes. The unprecedented concerted electron transfer was found to be due to altered mediator electron transfer thermodynamics, enabling biologically relevant studies of electroactive biofilms in the lab for the first time. These important findings pave the way for a complete understanding of the ecological role of electroactive microbes and their broad application in sustainable technologies.
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Biopelículas , Polímeros , Shewanella , Termodinámica , Shewanella/metabolismo , Shewanella/química , Transporte de Electrón , Biopelículas/efectos de los fármacos , Polímeros/química , Fuentes de Energía Bioeléctrica , Electrodos , Conductividad Eléctrica , Electrones , Técnicas ElectroquímicasRESUMEN
BACKGROUND: With ideal mean arterial pressure (MAP) targets in resuscitated out-of-hospital cardiac arrest (OHCA) patients unknown, we performed a meta-analysis of randomised controlled trials (RCTs) to compare the effects of higher versus lower MAP targets. METHODS: We searched four databases until 1 May 2023 for RCTs reporting the effects of higher MAP targets (>70 mmHg) in resuscitated OHCA patients and conducted random-effects meta-analyses. The primary outcome was mortality while secondary outcomes were neurological evaluations, arrhythmias, acute kidney injury, and durations of mechanical ventilation and ICU stay. We conducted inverse-variance weighted strata-level meta-regression against a proportion of non-survivors to assess differences between reported MAPs. We also conducted a trial sequential analysis of RCTs. RESULTS: Four RCTs were included. Higher MAP was not associated with reduced mortality (OR: 1.09, 95%-CI: 0.84 to 1.42, p = 0.51), or improved neurological outcomes (OR: 0.99, 95%-CI: 0.77 to 1.27, p = 0.92). Such findings were consistent despite additional sensitivity analyses. Our robust variance strata-level meta-regression revealed no significant associations between mean MAP and the proportion of non-survivors (B: 0.029, 95%-CI: -0.023 to 0.081, p = 0.162), and trial sequential analysis revealed no meaningful survival benefit for higher MAPs. CONCLUSIONS: A higher MAP target was not significantly associated with improved mortality and neurological outcomes in resuscitated OHCA patients.
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Modification of electrodes with biomolecules is an essential first step for the development of bioelectrochemical systems, which are used in a variety of applications ranging from sensors to fuel cells. Gold is often used because of its ease of modification with thiolated biomolecules, but carbon screen-printed electrodes (SPEs) are gaining popularity due to their low cost and fabrication from abundant resources. However, their effective modification with biomolecules remains a challenge; the majority of work to-date relies on nonspecific adhesion or broad amide bond formation to chemical handles on the electrode surface. By combining facile electrochemical modification to add an aniline handle to electrodes with a specific and biocompatible oxidative coupling reaction, we can readily modify carbon electrodes with a variety of biomolecules. Importantly, both proteins and DNA maintain bioactive conformations following coupling. We have then used biomolecule-modified electrodes to generate microbial monolayers through DNA-directed immobilization. This work provides an easy, general strategy to modify inexpensive carbon electrodes, significantly expanding their potential as bioelectrochemical systems.
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Técnicas Biosensibles , Carbono , Carbono/química , Proteínas , ADN , Electrodos , Oro/química , Técnicas ElectroquímicasRESUMEN
Electrochemical biosensors are promising technologies for detection and monitoring in low-resource settings due to their potential for easy use and low-cost instrumentation. Disposable gold screen-printed electrodes (SPEs) are popular substrates for these biosensors, but necessary dopants in the ink used for their production can interfere with biosensor function and contribute to the heterogeneity of these electrodes. We recently reported an alternative disposable gold electrode made from gold leaf generated using low-cost, equipment-free fabrication. We have directly compared the surface topology, biorecognition element deposition, and functional performance of three disposable gold electrodes: our gold leaf electrodes and two commercial SPEs. Our leaf electrodes significantly outperformed the SPEs for reproducible and effective biosensing in a DNase I assay and are nearly an order of magnitude less expensive than the SPEs. Therefore, these electrodes are promising for further development as point-of-care diagnostics, especially in low-resource settings.
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BACKGROUND: There is compelling evidence implicating dysregulated inflammation in the mechanism of ventricular remodeling and heart failure (HF) after MI. The transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2, encoded by Nfe2l2) is a promising target in this context since it impedes transcriptional upregulation of pro-inflammatory cytokines and is anti-inflammatory in various murine models. OBJECTIVES: We aimed to investigate the contribution of Nrf2 to the inflammatory response after experimental myocardial infarction (MI). METHODS: We subjected Nrf2-/- mice and wild type (WT) controls to permanent left coronary artery (LCA) ligation. The inflammatory response was investigated with fluorescence-activated cell sorting (FACS) analysis of peripheral blood and heart cell suspensions, together with qRT-PCR of infarcted tissue for chemokines and their receptors. To investigate whether Nrf2-mediated transcription is a dedicated function of leukocytes, we interrogated publicly available RNA-sequencing (RNA-seq) data from mouse hearts after permanent LCA ligation for Nrf2-regulated gene (NRG) expression. RESULTS: FACS analysis demonstrated a profoundly inflamed phenotype in the hearts of global Nrf2-/- mice as compared to WT mice after MI. Moreover, infarcted tissue from Nrf2-/- mice displayed higher expression of mRNA coding for inflammatory cytokines, chemokines, and their receptors, including IL-6, Ccl2, and Cxcr4. RNA-seq analysis showed upregulated NRG expression in WT mice after MI compared to naive mice, which was significantly higher in bioinformatically isolated CCR2+ cells. CONCLUSIONS: Taken together, the results suggest that Nrf2 signalling in leukocytes, and possibly CCR2+ monocytes and monocyte-derived cardiac resident macrophages, may be potential targets to prevent post-MI ventricular remodeling.
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Infarto del Miocardio , Factor 2 Relacionado con NF-E2/metabolismo , Remodelación Ventricular , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Factor 2 Relacionado con NF-E2/genética , Remodelación Ventricular/fisiologíaRESUMEN
Myopia, or nearsightedness, is the most common form of refractive abnormality and is characterized by excessive ocular elongation in relation to ocular power. Retinal neurotransmitter signaling, including dopamine, is implicated in myopic ocular growth, but the visual pathways that initiate and sustain myopia remain unclear. Melanopsin-expressing retinal ganglion cells (mRGCs), which detect light, are important for visual function, and have connections with retinal dopamine cells. Here, we investigated how mRGCs influence normal and myopic refractive development using two mutant mouse models: Opn4-/- mice that lack functional melanopsin photopigments and intrinsic mRGC responses but still receive other photoreceptor-mediated input to these cells; and Opn4DTA/DTA mice that lack intrinsic and photoreceptor-mediated mRGC responses due to mRGC cell death. In mice with intact vision or form-deprivation, we measured refractive error, ocular properties including axial length and corneal curvature, and the levels of retinal dopamine and its primary metabolite, L-3,4-dihydroxyphenylalanine (DOPAC). Myopia was measured as a myopic shift, or the difference in refractive error between the form-deprived and contralateral eyes. We found that Opn4-/- mice had altered normal refractive development compared to Opn4+/+ wildtype mice, starting â¼4D more myopic but developing â¼2D greater hyperopia by 16 weeks of age. Consistent with hyperopia at older ages, 16 week-old Opn4-/- mice also had shorter eyes compared to Opn4+/+ mice (3.34 vs 3.42 mm). Opn4DTA/DTA mice, however, were more hyperopic than both Opn4+/+ and Opn4-/- mice across development ending with even shorter axial lengths. Despite these differences, both Opn4-/- and Opn4DTA/DTA mice had â¼2D greater myopic shifts in response to form-deprivation compared to Opn4+/+ mice. Furthermore, when vision was intact, dopamine and DOPAC levels were similar between Opn4-/- and Opn4+/+ mice, but higher in Opn4DTA/DTA mice, which differed with age. However, form-deprivation reduced retinal dopamine and DOAPC by â¼20% in Opn4-/- compared to Opn4+/+ mice but did not affect retinal dopamine and DOPAC in Opn4DTA/DTA mice. Lastly, systemically treating Opn4-/- mice with the dopamine precursor L-DOPA reduced their form-deprivation myopia by half compared to non-treated mice. Collectively our findings show that disruption of retinal melanopsin signaling alters the rate and magnitude of normal refractive development, yields greater susceptibility to form-deprivation myopia, and changes dopamine signaling. Our results suggest that mRGCs participate in the eye's response to myopigenic stimuli, acting partly through dopaminergic mechanisms, and provide a potential therapeutic target underling myopia progression. We conclude that proper mRGC function is necessary for correct refractive development and protection from myopia progression.
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Miopía/metabolismo , Refracción Ocular/fisiología , Células Ganglionares de la Retina/metabolismo , Opsinas de Bastones/fisiología , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Longitud Axial del Ojo/patología , Córnea/patología , Modelos Animales de Enfermedad , Dopamina/metabolismo , Dopaminérgicos/farmacología , Femenino , Levodopa/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miopía/fisiopatología , Retina/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Vías Visuales/metabolismoRESUMEN
OBJECTIVES: This study aimed to review the evidence base regarding cognitive impairment and the development of dementia in patients with very late-onset schizophrenia-like psychosis (VLOSLP). METHODS: We conducted a systematic literature search of PubMed, PsycINFO and Web of Science according to Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines. Two reviewers independently screened records first by title and abstract and then by full text, resolving differences after each stage. Selected studies were assessed for quality using the GRADE system, and data on study design, participants, cognitive ability and rates of developing dementia were extracted and synthesised. RESULTS: Seventeen publications were identified for review. They were generally poor in quality and heterogenous in design. VLOSLP patients were found to have impaired global cognition compared to non-psychotic controls, but no difference was found between VLOSLP patients and aged early-onset schizophrenia (EOS) patients. No single cognitive domain was consistently affected. Patients with VLOSLP demonstrated significantly higher rates of dementia diagnosis (ranging from 4.4% over 3 years to 44.4% over 15 years) than controls, but no difference was found between VLOSLP patients and aged EOS patients. CONCLUSIONS: VLOSLP may not necessarily predict cognitive decline, but few studies have adequately investigated cohorts on a longitudinal basis. Heterogeneity between and within cohorts and varying selection criteria compromise the clinical generalisability of studies investigating the association between VLOSLP and neurodegenerative disease. Further studies on the clinical presentation, cognitive profile and neuropathology of VLOSLP with comparison to EOS/late-onset schizophrenia (LOS) and neurodegenerative disease are needed to better inform the diagnosis and management of VLOSLP.
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Glaucoma is the leading cause of irreversible blindness worldwide. Recently, estrogen deficiencies caused by early menopause, alterations in estrogen signaling via mutations in estrogen receptors, and polymorphisms along estrogen metabolic pathways have all been linked to an increased risk of developing glaucoma. Here, we examined how menopause and age impact visual function and retinal structure in an experimental model of glaucoma. Young (3-4 months) and aged (9-10 months) female Brown Norway rats were divided into pre- and post-menopausal cohorts by surgically inducing menopause via ovariectomy (OVX). After six weeks, ocular hypertension (OHT) was induced unilaterally for a period of eight weeks. Four cohorts were successfully followed to eight weeks: young sham (nâ¯=â¯8), young OVX (nâ¯=â¯9), aged sham (nâ¯=â¯10), and aged OVX (nâ¯=â¯11) animals. Intraocular pressure (IOP) was monitored weekly in all groups. Prior to inducing OHT (baseline) and at four and eight weeks after inducing OHT, we assessed visual acuity via the optomotor response (OMR) and retinal structure using optical coherence tomography (OCT). OHT decreased the OMR in all cohorts. We found that spatial frequency thresholds decreased by 54% in OVX animals after OHT compared to sham animals after OHT, regardless of age (pâ¯<â¯0.001). We also found thinning of the retinal nerve fiber layer (RNFL) and loss of total retinal thickness after induction of OHT. Aged animals had more thinning of the RNFL and loss of total retinal thickness compared to young animals (pâ¯<â¯0.001). Overall, OHT caused significant changes in visual function and retinal structure. Observing that OVX in young and aged animals further decreased spatial frequency thresholds after OHT suggests that an estrogen deficiency may intensify visual impairment after OHT.
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Envejecimiento/fisiología , Menopausia/fisiología , Retina , Animales , Femenino , Glaucoma , Presión Intraocular/fisiología , Ratas , Retina/patología , Retina/fisiopatología , Agudeza Visual/fisiologíaRESUMEN
The pathophysiology of refractive errors is poorly understood. Myopia (nearsightedness) in particular both blurs vision and predisposes the eye to many blinding diseases during adulthood. Based on past findings of diurnal variations in the dimensions of the eyes of humans and other vertebrates, altered diurnal rhythms of these ocular dimensions with experimentally induced myopia, and evolving evidence that ambient light exposures influence refractive development, we assessed whether disturbances in circadian signals might alter the refractive development of the eye. In mice, retinal-specific knockout of the clock gene Bmal1 induces myopia and elongates the vitreous chamber, the optical compartment separating the lens and the retina. These alterations simulate common ocular findings in clinical myopia. In Drosophila melanogaster, knockouts of the clock genes cycle or period lengthen the pseudocone, the optical component of the ommatidium that separates the facet lens from the photoreceptors. Disrupting circadian signaling thus alters optical development of the eye in widely separated species. We propose that mechanisms of myopia include circadian dysregulation, a frequent occurrence in modern societies where myopia also is both highly prevalent and increasing at alarming rates. Addressing circadian dysregulation may improve understanding of the pathogenesis of refractive errors and introduce novel therapeutic approaches to ameliorate myopia development in children.
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Relojes Circadianos/genética , Ojo/fisiopatología , Animales , Ritmo Circadiano/genética , Drosophila melanogaster , Ratones , Miopía/genética , Miopía/fisiopatología , Retina/fisiopatologíaRESUMEN
Retinal photoreceptors are important in visual signaling for normal eye growth in animals. We used Gnat2cplf3/cplf3 (Gnat2-/-) mice, a genetic mouse model of cone dysfunction to investigate the influence of cone signaling in ocular refractive development and myopia susceptibility in mice. Refractive development under normal visual conditions was measured for Gnat2-/- and age-matched Gnat2+/+ mice, every 2 weeks from 4 to 14 weeks of age. Weekly measurements were performed on a separate cohort of mice that underwent monocular form-deprivation (FD) in the right eye from 4 weeks of age using head-mounted diffusers. Refraction, corneal curvature, and ocular biometrics were obtained using photorefraction, keratometry and optical coherence tomography, respectively. Retinas from FD mice were harvested, and analyzed for dopamine (DA) and 3,4-dihydroxyphenylacetate (DOPAC) using high-performance liquid chromatography. Under normal visual conditions, Gnat2+/+ and Gnat2-/- mice showed similar refractive error, axial length, and corneal radii across development (pâ¯>â¯0.05), indicating no significant effects of the Gnat2 mutation on normal ocular refractive development in mice. Three weeks of FD produced a significantly greater myopic shift in Gnat2-/- mice compared to Gnat2+/+ controls (-5.40⯱â¯1.33 D vs -2.28⯱â¯0.28 D, pâ¯=â¯0.042). Neither the Gnat2 mutation nor FD altered retinal levels of DA or DOPAC. Our results indicate that cone pathways needed for high acuity vision in primates are not as critical for normal refractive development in mice, and that both rods and cones contribute to visual signalling pathways needed to respond to FD in mammalian eyes.
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Miopía/fisiopatología , Retina/fisiopatología , Células Fotorreceptoras Retinianas Conos/patología , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Dopamina/metabolismo , Femenino , Proteínas de Unión al GTP Heterotriméricas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Miopía/metabolismo , Refracción Ocular/fisiología , Retina/metabolismo , Privación Sensorial , Tomografía de Coherencia Óptica , Agudeza Visual/fisiologíaRESUMEN
Purpose: Experimental myopia in animal models suggests that bright light can influence refractive error and prevent myopia. Additionally, animal research indicates activation of rod pathways and circadian rhythms may influence eye growth. In children, objective measures of personal light exposure, recorded by wearable light sensors, have been used to examine the effects of bright light exposure on myopia. The effect of time spent in a broad range of light intensities on childhood refractive development is not known. This study aims to evaluate dim light exposure in myopia. Methods: We reanalyzed previously published data to investigate differences in dim light exposure across myopic and nonmyopic children from the Role of Outdoor Activity in Myopia (ROAM) study in Queensland, Australia. The amount of time children spent in scotopic (<1-1 lux), mesopic (1-30 lux), indoor photopic (>30-1000 lux), and outdoor photopic (>1000 lux) light over both weekdays and weekends was measured with wearable light sensors. Results: We found significant differences in average daily light exposure between myopic and nonmyopic children. On weekends, myopic children received significantly less scotopic light (P = 0.024) and less outdoor photopic light than nonmyopic children (P < 0.001). In myopic children, more myopic refractive errors were correlated with increased time in mesopic light (R = -0.46, P = 0.002). Conclusions: These findings suggest that in addition to bright light exposure, rod pathways stimulated by dim light exposure could be important to human myopia development. Optimal strategies for preventing myopia with environmental light may include both dim and bright light exposure.
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Miopía/fisiopatología , Visión Nocturna/fisiología , Actividades Cotidianas , Adolescente , Niño , Ritmo Circadiano/fisiología , Visión de Colores/fisiología , Exposición a Riesgos Ambientales , Femenino , Humanos , Masculino , Visión Mesópica/fisiología , Células Fotorreceptoras de Vertebrados/fisiología , Refracción Ocular/fisiología , Pruebas de VisiónRESUMEN
Visual experience during the critical period modulates visual development such that deprivation causes visual impairments while stimulation induces enhancements. This study aimed to determine whether visual stimulation in the form of daily optomotor response (OMR) testing during the mouse critical period (1) improves aspects of visual function, (2) involves retinal mechanisms and (3) is mediated by brain derived neurotrophic factor (BDNF) and dopamine (DA) signaling pathways. We tested spatial frequency thresholds in C57BL/6J mice daily from postnatal days 16 to 23 (P16 to P23) using OMR testing. Daily OMR-treated mice were compared to littermate controls that were placed in the OMR chamber without moving gratings. Contrast sensitivity thresholds, electroretinograms (ERGs), visual evoked potentials, and pattern ERGs were acquired at P21. To determine the role of BDNF signaling, a TrkB receptor antagonist (ANA-12) was systemically injected 2 hours prior to OMR testing in another cohort of mice. BDNF immunohistochemistry was performed on retina and brain sections. Retinal DA levels were measured using high-performance liquid chromatography. Daily OMR testing enhanced spatial frequency thresholds and contrast sensitivity compared to controls. OMR-treated mice also had improved rod-driven ERG oscillatory potential response times, greater BDNF immunoreactivity in the retinal ganglion cell layer, and increased retinal DA content compared to controls. VEPs and pattern ERGs were unchanged. Systemic delivery of ANA-12 attenuated OMR-induced visual enhancements. Daily OMR testing during the critical period leads to general visual function improvements accompanied by increased DA and BDNF in the retina, with this process being requisitely mediated by TrkB activation. These results suggest that novel combination therapies involving visual stimulation and using both behavioral and molecular approaches may benefit degenerative retinal diseases or amblyopia.
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Factor Neurotrófico Derivado del Encéfalo/metabolismo , Estimulación Luminosa , Retina/metabolismo , Agudeza Visual , Animales , Cromatografía Líquida de Alta Presión , Sensibilidad de Contraste , Dopamina/metabolismo , Electrorretinografía , Potenciales Evocados Visuales , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
Plasma administration has been recommended in calves older than 48h with failure of passive immunity (FPI) to provide immunity consistent with adequate colostral ingestion. However, the protective serum immunoglobulin G (IgG) concentrations (≥1000mg/dL) of plasma derived IgG only lasts up to 12h. In addition to IgG, maternally derived colostral cells also confer immunity. The objective of the study was to determine the effect of intravenous plasma transfusion on granulocyte and monocyte oxidative and phagocytic activity in calves with FPI. Twenty-seven, one day-old, Jersey calves were assigned into 3 groups. The colostral (CL, N=9) group received 3L of colostrum once by oroesophageal tubing. Two other groups of calves received 1L of colostrum once by oroesophageal tubing and were assigned based on their health status (sick or non-sick) at 4days of age, as the sick-group (SG, N=7) or the non-sick (NG, N=11) groups. At 4days of age, the SG and NG groups were administered plasma intravenously at 30mL/kg. Granulocyte and monocyte oxidative and phagocytic activity was determined by flow cytometry. There was no significant difference in the granulocyte and monocyte oxidative or phagocytic activity among the 3 groups (P>0.05). Plasma administration had no significant effect on the oxidative or phagocytic activity of granulocytes or monocytes. In clinical practice, plasma administration for enhancing oxidative or phagocytic activity of granulocytes or monocytes, alone, might not be justified in calves with FPI.
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Bovinos/inmunología , Granulocitos/metabolismo , Inmunidad Materno-Adquirida/inmunología , Monocitos/metabolismo , Estrés Oxidativo , Fagocitos/metabolismo , Animales , Femenino , Infusiones Intravenosas/veterinaria , Plasma/químicaRESUMEN
BACKGROUND: IgE cross-linking triggers many cellular processes that drive allergic disease. While the role of IgE in mediating allergic responses is best described on basophils and mast cells, expression of the high-affinity IgE receptor on other innate immune cells, including monocytes, suggests that it may affect the function of these cells in allergic environments. OBJECTIVE: To determine the effect of IgE cross-linking on the function of human monocytes. METHODS: Monocytes purified from healthy donor blood samples were cultured for 4 to 96 hours with media alone, a cross-linking anti-IgE antibody or control IgG. Surface CD14 and CD64 expression and secreted cytokine concentrations were determined. Monocyte function was determined by assessing (1) phagocytosis of Escherichia coli or apoptotic HEp2 cells and (2) killing of intracellular E coli. Select experiments were performed on monocytes obtained from participants with elevated versus normal serum IgE concentrations. RESULTS: IgE cross-linking on monocytes increased CD14 expression and induced secretion of TNF-α, IL-6, and autoregulatory IL-10. These effects were greatest in individuals with elevated serum IgE concentrations. In contrast, IgE cross-linking reduced CD64 expression and significantly impaired phagocytic function without disrupting the capacity of monocytes to kill bacteria. CONCLUSIONS: IgE cross-linking drives monocyte proinflammatory processes and autoregulatory IL-10 in a serum IgE-dependent manner. In contrast, monocyte phagocytic function is critically impaired by IgE cross-linking. Our findings suggest that IgE cross-linking on monocytes may contribute to allergic disease by both enhancing detrimental inflammatory responses and concomitantly crippling phagocytosis, a primary mechanism used by these cells to resolve inflammation.
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Inmunoglobulina E/inmunología , Monocitos/inmunología , Fagocitosis/inmunología , Adolescente , Adulto , Antígenos de Superficie/inmunología , Basófilos/inmunología , Basófilos/fisiología , Células Cultivadas , Escherichia coli/inmunología , Escherichia coli/fisiología , Femenino , Humanos , Inflamación/inmunología , Inflamación/fisiopatología , Interleucina-10/inmunología , Interleucina-6/inmunología , Receptores de Lipopolisacáridos/inmunología , Masculino , Receptores de IgE/inmunología , Receptores de IgG/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Adulto JovenRESUMEN
During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3' polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5'-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs. Three representative 20-nt RNAs from the PPT region were tested for primer extension. Each primer had activity in the absence of NC, but less than the PPT. NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected. RNase H cleavage and zinc coordination by NC were required for maximal inhibition of mispriming. Biophysical properties, including thermal stability, helical structure and reverse transcriptase (RT) binding affinity, showed significant differences between PPT and non-PPT duplexes and the trends were generally correlated with the biochemical data. Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NC's observed effects on mispriming efficiency. Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.
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VIH-1/genética , Chaperonas Moleculares/metabolismo , ARN Viral/química , ARN/química , Transcripción Reversa , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Dicroismo Circular , ADN/química , ADN/metabolismo , Cartilla de ADN/química , Transcriptasa Inversa del VIH/metabolismo , Chaperonas Moleculares/química , Desnaturalización de Ácido Nucleico , Purinas/análisis , ARN/metabolismo , ARN Viral/metabolismo , Ribonucleasa H/metabolismo , Zinc/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
It has been reported that intermittent hypoxia treatment prevents oxidative injuries to the brain and protects the heart against ischemia-reperfusion injury. Both anti-oxidative defensive systems and prevention of free intracellular calcium overload might be the result of intermittent hypoxia. Thus, the purpose of this study was to explore the effects of intermittent hypoxia (8 h at 12 % O2 per day) for 0, 7 or 14 days on inducible nitric oxide synthase (iNOS) expression in the spleen and on splenic calcium response to the mitogen phytohemagglutinin (PHA). The results demonstrated that administration of intermittent hypoxia for 7 days caused severe hemolysis of erythrocytes in the spleen and the hemolytic condition was ameliorated by intermittent hypoxia for 14 days. However, a significant decline in splenic weight and an increase in plasma total bilirubin levels appeared in rats after hypoxia for 14 days. No calcium response to PHA was observed in splenocytes obtained from rats after intermittent hypoxia for 7 days. After intermittent hypoxia for 14 days, the calcium response to PHA was restored to the level of the controls. Intermittent hypoxia for 7 days was able to induce higher iNOS expression in splenic tissues than hypoxia for 14 days. These results suggested that intermittent hypoxia for 14 days appeared to involve acclimatization that protects the rats from oxidative injury through less hemolysis and iNOS expression in splenic tissues and by the presence of more bilirubin in the plasma. The increase in plasma total bilirubin levels might be the cause of induced adaptation to chronic intermittent hypoxia.
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Bilirrubina/sangre , Calcio/metabolismo , Hipoxia/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Bazo/metabolismo , Animales , Masculino , Fitohemaglutininas , Ratas , Ratas Sprague-DawleyRESUMEN
The enzymes in the alpha-ketoglutarate (alphaKG) dependent dioxygenase superfamily represent the largest class of non-heme iron oxidases and have important medical, ecological, and biotechnological roles. One such enzyme, taurine/alpha-ketoglutarate dioxygenase (TauD), catalyzes the conversion of 2-aminoethanesulfonate (taurine) to sulfite and aminoacetaldehyde while decomposing alphaKG to succinate and CO(2). This alphaKG dependent dioxygenase is expressed in Escherichia coli under sulfur starvation conditions and allows the cell to utilize taurine, and other similar sulfonates in the environment, as an alternative sulfur source. In this work, we report the structures of the apo and holo forms of TauD to 1.9 A resolution (R(cryst) = 21.2%, R(free) = 24.9%) and 2.5 A resolution (R(cryst) = 22.5%, R(free) = 27.8%), respectively. The models reported herein provide significant new insight into the substrate orientations at the active site and the conformational changes that are induced upon taurine binding. Furthermore, analysis of our crystallographic data coupled with reanalysis of the crystallographic model (resolution = 3.0 A, R(cryst) = 28.1, R(free) = 32.0) presented by Elkins et al. (Biochemistry (2002) 41, 5185-5192) reveals an alternative oligomeric arrangement for the enzyme that is consistent with the conserved primary and secondary structure elements of other alphaKG dependent dioxygenases.