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Prevention of limb ischemia in patients with venoarterial extracorporeal membrane oxygenation (VA-ECMO) is primarily achieved through the use of distal perfusion catheters (DPC). Our objective was to assess the role of DPC, and specifically the size of the catheter, in reducing the incidence of acute limb ischemia (ALI) through a meta-analysis. Seventeen studies met criteria for analysis. Pooled analysis included a total of 2,040 patients, of which 904 patients received ECMO with DPC and 1,136 patients underwent ECMO without DPC. Compared with ECMO alone, ECMO with DPC, regardless of size, significantly decreased ALI (relative risk [RR]: 0.49, 95% confidence interval [CI]: 0.31-0.77; p = 0.002). When comparing reactive versus prophylactic placement of DPC, prophylactic DPC was associated with significantly decreased ALI (RR: 0.41, 95% CI: 0.24-0.71; p = 0.02). No differences in mortality (RR: 0.89, 95% CI: 0.76-1.03; p = 0.12) and bleeding events (RR: 1.43, 95% CI: 0.41-4.96; p = 0.58) were observed between the two groups. This analysis demonstrates that the placement of DPC, if done prophylactically and regardless of size, is associated with a reduced risk of ALI versus the absence of DPC placement, but is not associated with differences in mortality or bleeding events.
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BACKGROUND: Ex vivo lung perfusion (EVLP) enables lung resuscitation before transplantation, and training is key, particularly in low-volume settings. To enable technique refinement and continuing education, we sought to demonstrate the value of a low-cost, high-fidelity EVLP simulator that would allow reproducible clinical scenarios. METHODS: In partnership with our EVLP manufacturer, we utilized the XPS™ Jensen Lung with our clinical system. The Jensen Lung has two simulated lung bladders and an in-line polymethylpentene fiber oxygenator. It allows titration of ventilator support which aids in accurate clinical simulation. For simulations, blood gases (BGs) were obtained and compared with integrated in-line perfusate gas monitors (PGMs). PaO2 , PCO2 , and pH were measured and compared. RESULTS: The PGM and BG values were not significantly different throughout the range of FiO2 and sweep gas flow rates evaluated. The "delta" PaO2 was measured between LA and PA and did not show any change between approaches. The pH measurement between BG and PGM was not significantly different. CONCLUSIONS: The XPS™ Jensen Lung simulator allows for a high-fidelity simulator of clinical EVLP. The correlation of the PGM and the BG measurement of the PaO2 and pH allow for a low-cost simulation, as the PGMs are in line in the circuit, and enable real-time tracking of perfusate gas parameters with the PGM. Implementation of a standardized clinical EVLP training program allows the maintenance of technique and enables clinical simulation training without the need for costly animal perfusions and the use of multiple BG measurements.
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Trasplante de Pulmón , Animales , Trasplante de Pulmón/métodos , Pulmón , Circulación Extracorporea/métodos , Perfusión/métodos , GasesRESUMEN
Posttranslational modifications (PTMs) play vital roles in cellular homeostasis and are implicated in various pathological conditions. This work uses two ion mobility spectrometry-mass spectrometry (IMS-MS) modalities, drift-tube IMS (DT-IMS) and trapped IMS (TIMS), to characterize three important nonenzymatic PTMs that induce no mass loss: l/d isomerization, aspartate/isoaspartate isomerization, and cis/trans proline isomerization. These PTMs are assessed in a single peptide system, the recently discovered pleurin peptides, Plrn2, from Aplysia californica. We determine that the DT-IMS-MS/MS can capture and locate asparagine deamidation into aspartate and its subsequent isomerization to isoaspartate, a key biomarker for age-related diseases. Additionally, nonenzymatic peptide cleavage via in-source fragmentation is evaluated for differences in the intensities and patterns of fragment peaks between these PTMs. Peptide fragments resulting from in-source fragmentation, preceded by peptide denaturation by liquid chromatography (LC) mobile phase, exhibited cis/trans proline isomerization. Finally, the effects of differing the fragmentation voltage at the source and solution-based denaturation conditions on in-source fragmentation profiles are evaluated, confirming that LC denaturation and in-source fragmentation profoundly impact N-terminal peptide bond cleavages of Plrn2 and the structures of their fragment ions. With that, LC-IMS-MS/MS coupled with in-source fragmentation could be a robust method to identify three important posttranslational modifications: l/d isomerization, Asn-deamidation leading to Asp/IsoAsp isomerization, and cis/trans proline isomerization.
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Ácido Aspártico , Ácido Isoaspártico , Secuencia de Aminoácidos , Ácido Aspártico/química , Espectrometría de Masas en Tándem , Péptidos/química , Prolina , IsomerismoRESUMEN
The effects of dipole interactions on magnetic nanoparticle magnetization and relaxation dynamics were investigated using five nanoparticle (NP) systems with different surfactants, carrier liquids, size distributions, inter-particle spacing, and NP confinement. Dipole interactions were found to play a crucial role in modifying the blocking temperature behavior of the superparamagnetic nanoparticles, where stronger interactions were found to increase the blocking temperatures. Consequently, the blocking temperature of a densely packed nanoparticle system with stronger dipolar interactions was found to be substantially higher than those of the discrete nanoparticle systems. The frequencies of the dominant relaxation mechanisms were determined by magnetic susceptibility measurements in the frequency range of 100 Hz-7 GHz. The loss mechanisms were identified in terms of Brownian relaxation (1 kHz-10 kHz) and gyromagnetic resonance of Fe3O4 (~1.12 GHz). It was observed that the microwave absorption of the Fe3O4 nanoparticles depend on the local environment surrounding the NPs, as well as the long-range dipole-dipole interactions. These significant findings will be profoundly important in magnetic hyperthermia medical therapeutics and energy applications.
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Both endovascular repair (EVR) and open repair (OR) surgery of thoraco-abdominal aortic aneurysms cause spinal cord (SC) injury that can lead to paraparesis or paraplegia. It has been assumed that mechanisms responsible for SC damage after EVR are similar to those after OR. This pilot study compared the pathophysiology of SC injury after EVR versus OR using a newly developed EVR dog model. An increasing number of stents similar to those used in patients were inserted in the aorta of three dogs to ensure thoracic or thoracic plus lumbar coverage. The aorta of OR dogs was cross-clamped for 45 min. Behavior assessment demonstrated unique patterns of proprioceptive ataxia and evolving paraparesis in EVR versus irreversible paraplegia in OR. MRI showed posterior signal in lumbar SC after EVR versus central cord edema after OR. Histopathology showed white matter edema in L3-L5 localized to the dorsal column medial lemniscus area associated with loss of myelin basic protein but not neurons after EVR, versus massive neuronal loss in the gray matter in L3-L5 after OR. Metabolome analysis demonstrates a distinctive chemical fingerprint of cellular processes in both interventions. Our results call for the development of new therapeutics tailored to these distinct pathophysiologic findings.
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Aneurisma de la Aorta Torácica/cirugía , Implantación de Prótesis Vascular/efectos adversos , Prótesis Vascular/efectos adversos , Procedimientos Endovasculares/efectos adversos , Procedimientos Endovasculares/métodos , Complicaciones Posoperatorias/etiología , Traumatismos de la Médula Espinal/etiología , Stents/efectos adversos , Animales , Conducta Animal , Angiografía por Tomografía Computarizada/métodos , Modelos Animales de Enfermedad , Perros , Imagen por Resonancia Magnética/métodos , Masculino , Metaboloma , Paraplejía/etiología , Proyectos Piloto , Complicaciones Posoperatorias/diagnóstico por imagen , Médula Espinal/diagnóstico por imagen , Traumatismos de la Médula Espinal/diagnóstico por imagen , Resultado del TratamientoRESUMEN
Single cell analysis strives to probe molecular heterogeneity in morphologically similar cell populations through quantitative or qualitative measurements of genetic, proteomic, or metabolic products. Here, we applied mass analysis of single neurons to investigate cell-cell signaling peptides. The multiplicity of endogenous cell-cell signaling peptides is a common source of chemical diversity among cell populations. Certain peptides can undergo post-translational isomerization of select residues, which has important physiological consequences. The limited number of single cell analysis techniques that are sensitive to peptide stereochemistry make it challenging to study isomerization at the individual cell level. We performed capillary electrophoresis (CE) with mass spectrometry (MS) detection to characterize the peptide content of single cells. Using complementary trapped ion mobility spectrometry (TIMS) separations, we measured the stereochemical configurations of three neuropeptide gene products derived from the pleurin precursor in individual neurons (N = 3) isolated from the central nervous system of Aplysia californica. An analysis of the resultant mobility profiles indicated >98% of the detectable pleurin-derived peptides exist as the nonisomerized, all-l forms in individual neuron cell bodies. However, we observed 44% of the Plrn2 peptide from the pleurin precursor was present as the isomerized, d-residue-containing form in the nerve tissue. These findings demonstrate an unusual distribution of isomerized peptides in A. californica and establish CE-TIMS MS as a powerful analytical tool for investigating peptide stereochemistry at the single cell level.
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Espectrometría de Movilidad Iónica , Proteómica , Electroforesis Capilar , Espectrometría de Masas , PéptidosRESUMEN
The discovery of enzyme-derived d-amino acid-containing peptides (DAACPs) that have physiological importance in the metazoan challenges previous assumptions about the homochirality of animal proteins while simultaneously revealing new analytical challenges in the structural and functional characterization of peptides. Most known DAACPs have been identified though laborious activity-guided purification studies or by homology to previously identified DAACPs. Peptide characterization experiments are increasingly dominated by high throughput mass spectrometry-based peptidomics, with stereochemistry rarely considered due to the technical challenges of identifying l/d isomerization. This review discusses the prevalence of enzyme-derived DAACPs among animals and the physiological consequences of peptide isomerization. Also highlighted are the analytical methods that have been applied for structural characterization/discovery of DAACPs, including results of several recent studies using non-targeted discovery methods for revealing novel DAACPs, strongly suggesting that more DAACPs remain to be uncovered.
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Péptidos Catiónicos Antimicrobianos/fisiología , Conotoxinas/química , Oligopéptidos/fisiología , Péptidos Opioides/fisiología , Péptidos Cíclicos/fisiología , Aminoácidos/química , Aminoácidos/metabolismo , Anfibios/clasificación , Anfibios/fisiología , Animales , Péptidos Catiónicos Antimicrobianos/química , Artrópodos/clasificación , Artrópodos/fisiología , Mamíferos/clasificación , Mamíferos/fisiología , Moluscos/clasificación , Moluscos/fisiología , Oligopéptidos/química , Péptidos Opioides/química , Péptidos Cíclicos/química , Filogenia , EstereoisomerismoRESUMEN
Linaridins are members of the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products. Five linaridins have been reported, which are defined by the presence of dehydrobutyrine, a dehydrated, alkene-containing amino acid derived from threonine. This work describes the development of a linaridin-specific scoring module for Rapid ORF Description and Evaluation Online (RODEO), a genome-mining tool tailored toward RiPP discovery. Upon mining publicly accessible genomes available in the NCBI database, RODEO identified 561 (382 nonredundant) linaridin biosynthetic gene clusters. Linaridin BGCs with unique gene architectures and precursor sequences markedly different from previous predictions were uncovered during these efforts. To aid in data set validation, two new linaridins, pegvadin A and B, were detected through reactivity-based screening and isolated from Streptomyces noursei and Streptomyces auratus, respectively. Reactivity-based screening involves the use of a probe that chemoselectively modifies an organic functional group present in the natural product. The dehydrated amino acids present in linaridins as α/ß-unsaturated carbonyls were appropriate electrophiles for nucleophilic 1,4-addition using a thiol-functionalized probe. The data presented within significantly expand the number of predicted linaridin biosynthetic gene clusters and serve as a roadmap for future work in the area. The combination of bioinformatics and reactivity-based screening is a powerful approach to accelerate natural product discovery.
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Bacterias/genética , Péptidos/genética , Bacterias/metabolismo , Minería de Datos , Genómica/métodos , Familia de Multigenes , Péptidos/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
BACKGROUND: Survival rates for out-of-hospital cardiac arrest are very low and neurologic recovery is poor. Innovative strategies have been developed to improve outcomes. A collaborative extracorporeal cardiopulmonary resuscitation (ECPR) program for out-of-hospital refractory pulseless ventricular tachycardia (VT) and/or ventricular fibrillation (VF) has been developed between The Ohio State University Wexner Medical Center and Columbus Division of Fire. METHODS: From August 15, 2017, to June 1, 2019, there were 86 patients that were evaluated in the field for cardiac arrest in which 42 (49%) had refractory pulseless VT and/or VF resulting from different underlying pathologies and were placed on an automated cardiopulmonary resuscitation device; from these 42 patients, 16 (38%) met final inclusion criteria for ECPR and were placed on extracorporeal membrane oxygenation (ECMO) in the cardiac catheterization laboratory (CCL). RESULTS: From the 16 patients who underwent ECPR, 4 (25%) survived to hospital discharge with cerebral perfusion category 1 or 2. Survivors tended to be younger (48.0 ± 16.7 vs. 59.3 ± 12.7 years); however, this difference was not statistically significant (p=0.28) likely due to a small number of patients. Overall, 38% of patients underwent percutaneous coronary intervention (PCI). No significant difference was found between survivors and nonsurvivors in emergency medical services dispatch to CCL arrival time, lactate in CCL, coronary artery disease severity, undergoing PCI, and pre-ECMO PaO2, pH, and hemoglobin. Recovery was seen in different underlying pathologies. CONCLUSION: ECPR for out-of-hospital refractory VT/VF cardiac arrest demonstrated encouraging outcomes. Younger patients may have a greater chance of survival, perhaps the need to be more aggressive in this subgroup of patients.
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Reanimación Cardiopulmonar , Oxigenación por Membrana Extracorpórea/métodos , Paro Cardíaco Extrahospitalario , Taquicardia Ventricular/complicaciones , Fibrilación Ventricular/complicaciones , Factores de Edad , Reanimación Cardiopulmonar/instrumentación , Reanimación Cardiopulmonar/métodos , Reanimación Cardiopulmonar/mortalidad , Servicios Médicos de Urgencia/métodos , Servicios Médicos de Urgencia/estadística & datos numéricos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Paro Cardíaco Extrahospitalario/etiología , Paro Cardíaco Extrahospitalario/mortalidad , Paro Cardíaco Extrahospitalario/terapia , Intervención Coronaria Percutánea/métodos , Intervención Coronaria Percutánea/estadística & datos numéricos , Tasa de SupervivenciaRESUMEN
This work describes the design and fabrication of free-standing carbon nanotube-palladium (CNT-Pd) composite sheets for hydrogen gas sensing. The CNT-Pd composites were made by electroplating palladium onto a solvent-densified and oxygen plasma-treated CNT sheet. The latter was prepared using high purity CNTs drawn from a dense, vertically aligned array grown by chemical vapor deposition on silicon substrates. The CNT-Pd sheets were characterized by energy-dispersive spectroscopy, scanning electron microscopy, and X-ray diffraction. The amount of palladium in the composite was 16.5 wt % as measured via thermogravimetric analysis. Thin strips of the CNT-Pd sheets were assembled as chemiresistor sensors and tested for hydrogen gas detection. The sensors demonstrated a limit of detection of 0.1 mol % and displayed signal reversibility without the need for oxygen removal or heat treatment. A decrease in signal reversibility was observed after multiple exposure cycles; however, redensification with ethanol significantly restored the original reversibility. The sensor showed the Freundlich adsorption isotherm behavior when exposed to hydrogen. The material's potential application toward a wearable, flexible sensor was demonstrated by integrating the chemiresistor onto a fabric material using hot-press processing and testing the composite for hydrogen sensitivity.
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d-Amino acid-containing peptides (DAACPs) make up a class of post-translationally modified peptides in animals that play important roles as cell-to-cell signaling molecules. Despite the functional importance of l- to d-residue isomerization, little is known about its prevalence, mostly due to difficulties associated with detecting differences in peptide stereochemistry. Prior efforts to discover DAACPs have been largely focused on pursuing peptides based on homology to known DAACPs or DAACP-encoding precursors. Here, we used a combination of enzymatic screening, mass spectrometry, and chromatographic analysis to identify novel DAACPs in the central nervous system (CNS) of Aplysia californica. We identified five new DAACPs from the pleurin precursor and three DAACPs from previously uncharacterized proteins. In addition, two peptides from the pleurin precursor, Plrn2 and Plrn3, exist as DAACPs with the d-residue found at position 2 or 3. These differentially modified forms of Plrn2 and Plrn3 are located in specific regions of the animal's CNS. Plrn2 and Plrn3 appear to be the first animal DAACPs in which the d-residue is found at more than one position, and this suggests that l- to d-residue isomerization may be a more variable/dynamic modification than previously thought. Overall, this study demonstrates the utility of nontargeted DAACP discovery approaches for identifying new DAACPs and demonstrates that isomerization is prevalent throughout the CNS of A. californica.
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Aminoácidos/química , Aplysia/química , Neuropéptidos/química , Fragmentos de Péptidos/química , Procesamiento Proteico-Postraduccional/genética , Secuencia de Aminoácidos , Animales , Aplysia/genética , Sistema Nervioso Central/química , Cromatografía Líquida de Alta Presión , Proteómica , Estereoisomerismo , Espectrometría de Masas en TándemRESUMEN
Transcriptional repression involves a class of proteins called corepressors that link transcription factors to chromatin remodeling complexes. In plants such as Arabidopsis thaliana, the most prominent corepressor is TOPLESS (TPL), which plays a key role in hormone signaling and development. Here we present the crystallographic structure of the Arabidopsis TPL N-terminal region comprising the LisH and CTLH (C-terminal to LisH) domains and a newly identified third region, which corresponds to a CRA domain. Comparing the structure of TPL with the mammalian TBL1, which shares a similar domain structure and performs a parallel corepressor function, revealed that the plant TPLs have evolved a new tetramerization interface and unique and highly conserved surface for interaction with repressors. Using site-directed mutagenesis, we validated those surfaces in vitro and in vivo and showed that TPL tetramerization and repressor binding are interdependent. Our results illustrate how evolution used a common set of protein domains to create a diversity of corepressors, achieving similar properties with different molecular solutions.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Co-Represoras/genética , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Co-Represoras/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Multimerización de ProteínaRESUMEN
Electrowetting and electrocapillarity of liquid metals have a long history, and a recent explosion of renewed interest. Liquid metals have electromagnetic properties and surface tensions (>500 mN/m) that enable new forms of reconfigurable devices. However, the only nontoxic option, gallium alloys, suffer from immediate formation of a semirigid surface oxide. Although acids or electrochemical reduction can remove this oxide, these approaches surround the gallium alloy in a fluid that is also electrically conducting, diminishing electromagnetic effectiveness and precluding electrowetting actuation. Reported here are acidified siloxanes that remove and prevent oxide formation. Importantly, the siloxane oil associatively incorporates hydrochloric or hydrobromic acids, is electrically insulating, is chemically stable, removes etching byproducts (including water), and allows robust electrowetting. This work opens up new opportunities for liquid metal reconfiguration, and is of fundamental interest due to the unexpected chemical stability of the acidified siloxanes and their application to other materials and surfaces.
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Nanoparticle mediated photothermal ablation of cancerous tissue shows promising results and applicability as a highly efficacious treatment method. As a majority of the photothermal work has been conducted with minimal attenuation of the laser before reaching the nanoparticles within surface seeded tumors in-vivo or through buffered media in-vitro, it is important to understand the effects of greater laser attenuation on photothermal efficacy mediated by changes in the scattering and absorption of the laser. Photothermal efficacy using a near infrared (NIR) 785nm laser irradiating polystyrene (PS) stabilized magnetite (Fe3O4) nanoparticles (PS-Fe3O4) is examined on MDA-MB-231 human mammary gland adenocarcinoma in-vitro. Agarose gel columns of various heights were created to simulate soft tissue and subsequently used for NIR laser attenuation. Polystyrene was found to significantly improve magnetite nanoparticle stability in serum containing media and modified Hank's Balanced Salt Solution and was able to induce significant hyperthermic ablation at mass concentrations which also did not elicit significant innate toxicity. Furthermore it was found that the polystyrene coating significantly reduced innate toxicity over 48h compared to uncoated magnetite. Agar gel layers provided similar optical attenuation in the NIR region to skin and prostate.
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Hipertermia Inducida/métodos , Nanopartículas de Magnetita/química , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Medios de Cultivo , Femenino , Óxido Ferrosoférrico/química , Humanos , Rayos Infrarrojos , Rayos Láser , Nanopartículas de Magnetita/toxicidad , Nanopartículas de Magnetita/ultraestructura , Microscopía Electrónica de Transmisión , Tamaño de la Partícula , Poliestirenos/químicaRESUMEN
This case report describes the intraoperative use of extracorporeal life support (ECLS) for an elective thoracoscopic maze procedure in which the patient could not tolerate one-lung ventilation because of hypoxia. Potential pitfalls associated with the anesthetic management of elective intraoperative ECLS include managing native cardiac ejection and ECLS flows to provide optimal oxygenation and cardiac output. Particular attention must be paid to cardiac and respiratory physiology when ECLS is used in a patient with normal cardiac function.
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Fibrilación Atrial/cirugía , Ablación por Catéter/métodos , Oxigenación por Membrana Extracorpórea/métodos , Procedimientos Quirúrgicos Mínimamente Invasivos/métodos , Cirugía Torácica Asistida por Video/métodos , Anciano , Fibrilación Atrial/complicaciones , Gasto Cardíaco , Procedimientos Quirúrgicos Electivos , Humanos , Hipoxia/etiología , Masculino , Ventilación Unipulmonar/efectos adversos , Oxígeno/sangre , Insuficiencia Respiratoria/sangre , Insuficiencia Respiratoria/terapiaRESUMEN
Iron oxide exhibits fascinating physical properties especially in the nanometer range, not only from the standpoint of basic science, but also for a variety of engineering, particularly biomedical applications. For instance, Fe3O4 behaves as superparamagnetic as the particle size is reduced to a few nanometers in the single-domain region depending on the type of the material. The superparamagnetism is an important property for biomedical applications such as magnetic hyperthermia therapy of cancer. In this review article, we report on some of the most recent experimental and theoretical studies on magnetic heating mechanisms under an alternating (AC) magnetic field. The heating mechanisms are interpreted based on Néel and Brownian relaxations, and hysteresis loss. We also report on the recently discovered photoluminescence of Fe3O4 and explain the emission mechanisms in terms of the electronic band structures. Both optical and magnetic properties are correlated to the materials parameters of particle size, distribution, and physical confinement. By adjusting these parameters, both optical and magnetic properties are optimized. An important motivation to study iron oxide is due to its high potential in biomedical applications. Iron oxide nanoparticles can be used for MRI/optical multimodal imaging as well as the therapeutic mediator in cancer treatment. Both magnetic hyperthermia and photothermal effect has been utilized to kill cancer cells and inhibit tumor growth. Once the iron oxide nanoparticles are up taken by the tumor with sufficient concentration, greater localization provides enhanced effects over disseminated delivery while simultaneously requiring less therapeutic mass to elicit an equal response. Multi-modality provides highly beneficial co-localization. For magnetite (Fe3O4) nanoparticles the co-localization of diagnostics and therapeutics is achieved through magnetic based imaging and local hyperthermia generation through magnetic field or photon application. Here, Fe3O4 nanoparticles are shown to provide excellent conjugation bases for entrapment of therapeutic molecules, fluorescent agents, and targeting ligands; enhancement of solid tumor treatment is achieved through co-application of local hyperthermia with chemotherapeutic agents.
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Medios de Contraste/química , Colorantes Fluorescentes/química , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/uso terapéutico , Fotoquimioterapia/métodos , Animales , Medios de Contraste/uso terapéutico , Humanos , Hipertermia Inducida/métodos , Nanopartículas de Magnetita/ultraestructura , Microscopía Fluorescente/métodosRESUMEN
Activated forms of jasmonic acid (JA) are central signals coordinating plant responses to stresses, yet tools to analyse their spatial and temporal distribution are lacking. Here we describe a JA perception biosensor termed Jas9-VENUS that allows the quantification of dynamic changes in JA distribution in response to stress with high spatiotemporal sensitivity. We show that Jas9-VENUS abundance is dependent on bioactive JA isoforms, the COI1 co-receptor, a functional Jas motif and proteasome activity. We demonstrate the utility of Jas9-VENUS to analyse responses to JA in planta at a cellular scale, both quantitatively and dynamically. This included using Jas9-VENUS to determine the cotyledon-to-root JA signal velocities on wounding, revealing two distinct phases of JA activity in the root. Our results demonstrate the value of developing quantitative sensors such as Jas9-VENUS to provide high-resolution spatiotemporal data about hormone distribution in response to plant abiotic and biotic stresses.
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Técnicas Biosensibles/métodos , Ciclopentanos/análisis , Ciclopentanos/metabolismo , Oxilipinas/análisis , Oxilipinas/metabolismo , Plantas/metabolismo , Cotiledón/metabolismo , Raíces de Plantas/metabolismoRESUMEN
The photothermal effect of magnetite (Fe3O4) nanoparticles was characterized by photonic absorption in the near-infrared (NIR) region. Upon laser irradiation at 785 nm, the Fe3O4 nanoparticles generate localized hyperthermia in tumorous lesions, which is an effective strategy for cancer therapy; however, uncoated magnetite possesses an innate toxicity which can lead to drawbacks in the clinical setting. To reduce innate toxicity, a poly(acrylic acid) (PAA) coating on the nanoparticles was investigated in order to determine the alterations to stability and the degree of toxicity in an attempt to create a higher utility vector. It was found that the PAA coating significantly reduced the innate toxicity of the uncoated magnetite. Furthermore, the efficacy of PAA-coated magnetite nanoparticles (PAA-Fe3O4) was investigated for treating MDA-MB-231 (human mammary gland adenocarcinoma) cultures in viable concentration ranges (0.1-0.5mg/ml). An appropriate PAA-Fe3O4 concentration range was then established for inducing significant cell death by hyperthermic ablation, but not through innate toxicity.