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PURPOSE: Transcorneal freezing is a common technique used in rabbits to induce damage to the corneal endothelium. Previous studies have been performed with a range of freezing temperatures, times, and rabbit ages. Here, we aimed to characterize the aged rabbit endothelium after transcorneal freezing to establish an innate corneal endothelial cell regrowth rate and propose it as a mechanism for evaluation of therapeutic efficacy in rabbit models. METHODS: Central corneas of anesthetized New Zealand White rabbits (n=3) aged 18-24 months were exposed to nitrous oxide cooled probes for 30 seconds. Animals were assessed by in vivo confocal microscopy, applanation tonometry, specular microscopy, optical coherence tomography, and histology. The contralateral eye acted as a control. Images were taken immediately before and after injury and on days 2, 4, 7, 11, and 14. RESULTS: Following transcorneal freezing, there was a significant decrease in corneal endothelium density and a temporary increase in corneal thickness. Endothelial density decreased by 95% immediately after injury compared to controls and showed linear recovery over 14 days, reaching a 38% reduction by day 14. There was a significant increase in pleomorphism across all time points post-injury. Conversely, corneal thickness increased two days post injury but recovered at all later time points. Intraocular pressure was not affected throughout. CONCLUSIONS: This corneal endothelium injury platform is ideal for injury and therapeutic research as it can be rapidly performed, and has minimal impact on corneal thickness and intraocular pressure. Due to innate rabbit endothelial regrowth, it is vital to establish corneal endothelial recovery rate before evaluating therapeutics for efficacy in this model system.
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Lesiones de la Cornea , Endotelio Corneal , Animales , Córnea , Células Endoteliales , Congelación , ConejosRESUMEN
Purpose: To determine the long-term biocompatibility of HyStem® hydrogel in the rabbit eye for use as a carrier for cell or drug delivery into the ocular space. Methods: HyStem hydrogel formulation solidifies â¼20 min after reconstitution, thus can potentially form a solid deposit after injection in situ. To study the ocular disposition of fluorescein-labeled HyStem, we delivered 50 µL/eye over 1 min into the vitreous space of the rabbit. We used 3 Dutch-Belted and 3 New Zealand-pigmented rabbits, all females, delivered the gel into the right eyes, and injected 50 µL BSS Plus into the left eyes as a control. Retinal morphology was assessed by optical coherence tomography (OCT) and white light fundus photography. Fluorescence fundus photography enabled measurement of the clearance of the labeled hydrogel from the posterior chamber. Visual function was evaluated using flash and flicker electroretinography (ERG) pre- and postinjection and at weekly intervals thereafter for 6 weeks. Retinal immunohistochemistry for microglial inflammatory markers was carried out with antiglial fibrillary acidic protein (GFAP) antibody, isolectin B4 (IB4), and 4',6-diamidino-2-phenylindole (DAPI). Results: The gel was successfully delivered into the vitreous space without the formation of a discrete retinal deposit. Fundus imaging, OCT measurements of retinal thickness, and immunohistochemical data indicated an absence of retinal inflammation, and ERG indicated no impact on retinal function. The half-time of HyStem clearance calculated from the loss of fundus fluorescence was 3.9 days. Conclusions: HyStem hydrogel appears to be biocompatible in the ocular space of a large eye and safe for long-term intraocular application.
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Materiales Biocompatibles/administración & dosificación , Ojo/efectos de los fármacos , Hidrogeles/administración & dosificación , Animales , Tolerancia a Medicamentos , Femenino , Inyecciones Intraoculares , ConejosRESUMEN
Purpose: Photoacoustics (optoacoustics) is a hybrid technology utilizing light excitation of acoustic responses in targets of interest. It has found numerous applications in biomedicine, including eye research, because of its ability to report both morphological and functional data about the interrogated tissue. This presentation will give an overview of current applications. Methods: Wavelength-dependent absorption of light in a tissue chromophore causes local heating, leading to a thermoelastic expansion-contraction cycle. If nanosecond pulses of light are used to excite this process, the resulting pressure wave is an ultrasound signal propagating through the tissue and detectable at the tissue surface. This is highly advantageous because of the known properties of ultrasound propagation in tissue and the ability to use standard, medical ultrasound equipment for detection. The time of arrival and amplitude of ultrasound signals provide information about the location and nature of the absorber. Results: Due to the wavelength dependence of the photoacoustic response, functional and physiological applications are possible. For example, retinal oximetry can be determined from the different optical absorption properties of oxy- and deoxyhemoglobin. Multispectral imaging of the posterior segment can identify pigments such as melanin or lipofuscin or the nature of foreign bodies. The technique can be combined with other imaging modalities such as ultrasound and optical coherence tomography to produce high-resolution images of retinal structures along with functional information. Conclusion: Photoacoustic technology is a powerful noninvasive tool for ocular research and to study ocular morphology, fundamental physiological parameters, cellular responses, and molecular expression.
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Ojo/diagnóstico por imagen , Imagen Óptica , Técnicas Fotoacústicas , HumanosRESUMEN
RATIONALE AND OBJECTIVES: In this study, we assessed the radiation dose to the lens and the impacts of various eye shields using either a fixed or modulated tube current. MATERIALS AND METHODS: Patients undergoing head computed tomography (CT) examinations were recruited, and each was randomly assigned to one of five imaging groups, either without a CT eye shield or with one of two types of shielding and topogram-based tube current modulation (TCM). The radiation dose at the eye lens was estimated using Gafchromic films. All CT images were analyzed for quality in the orbit and brain areas. Two radiologists also qualitatively assessed image artifacts and their impacts on image quality using three-point Likert scales. RESULTS: Both barium sulfate and bismuth-antimony shields significantly reduced radiation dose to the lens (by 28.60%-31.92% and 43.87%-47.00%, respectively) while significantly inducing image artifacts. The image quality of the intraocular structure, but not the intracranial structure, was significantly degraded by shielding. In addition, discriminating the periocular tissues was improved using a bismuth-antimony shield and topogram-based TCM. Compared to fixed tube current, topogram-based TCM provided better signal-to-noise and contrast-to-noise ratios in the intracranial structures when the bismuth-antimony and barium sulfate shields were applied, respectively. CONCLUSION: Artifacts resulting from the application of eye shields during head CT examinations can be reduced by using topogram-based TCM instead of a fixed tube current. This could be an alternative approach for maintaining image quality in CT scans that do not encompass organ-based TCM.
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Dispositivos de Protección de los Ojos , Ojo , Protección Radiológica , Tomografía Computarizada por Rayos X , Cabeza/diagnóstico por imagen , Humanos , Fantasmas de Imagen , Dosis de RadiaciónRESUMEN
Retinal organoid technology enables generation of an inexhaustible supply of three-dimensional retinal tissue from human pluripotent stem cells (hPSCs) for regenerative medicine applications. The high similarity of organoid-derived retinal tissue and transplantable human fetal retina provides an opportunity for evaluating and modeling retinal tissue replacement strategies in relevant animal models in the effort to develop a functional retinal patch to restore vision in patients with profound blindness caused by retinal degeneration. Because of the complexity of this very promising approach requiring specialized stem cell and grafting techniques, the tasks of retinal tissue derivation and transplantation are frequently split between geographically distant teams. Delivery of delicate and perishable neural tissue such as retina to the surgical sites requires a reliable shipping protocol and also controlled temperature conditions with damage-reporting mechanisms in place to prevent transplantation of tissue damaged in transit into expensive animal models. We have developed a robust overnight tissue shipping protocol providing reliable temperature control, live monitoring of the shipment conditions and physical location of the package, and damage reporting at the time of delivery. This allows for shipping of viable (transplantation-competent) hPSC-derived retinal tissue over large distances, thus enabling stem cell and surgical teams from different parts of the country to work together and maximize successful engraftment of organoid-derived retinal tissue. Although this protocol was developed for preclinical in vivo studies in animal models, it is potentially translatable for clinical transplantation in the future and will contribute to developing clinical protocols for restoring vision in patients with retinal degeneration.
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Organoides/citología , Células Madre Pluripotentes/citología , Medicina Regenerativa/métodos , Retina/patología , Ingeniería de Tejidos/métodos , Ceguera , Diferenciación Celular , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Organoides/metabolismo , Retina/embriología , Degeneración Retiniana , Trasplante de Células Madre/métodos , Células Madre/citología , Temperatura , Andamios del TejidoRESUMEN
Purpose: Our study aimed to determine whether the altered expression of biomarkers linked to corneal injuries, such as the edema-regulating proteins aquaporin-1 and aquaporin-5 (AQP1 and AQP5), occurred following primary blast exposure. Methods: Adult male Dutch Belted rabbits were anesthetized and exposed to blast waves with peak overpressures of 142.5-164.1 kPa (20.4-23.4 psi). These exposure groups experienced peak blast overpressure-specific impulses (impulse per unit surface area) of 199.6-228.5 kPa-ms. Unexposed rabbits were included as controls. The animals were euthanized at 48 h post-exposure. Corneas obtained from the euthanized blast-exposed and control rabbits were processed for quantitative PCR and western blot to quantify mRNA and the protein expression of AQP1 and AQP5. Immunohistochemical analysis was conducted to determine the cellular localization of AQP1 and AQP5. Results: Corneal thickness increased up to 18% with the peak blast overpressure-specific impulses of 199.6-228.5 kPa-ms at 48 h after blast exposure. mRNA levels of AQP1 and AQP5 increased in the whole cornea lysates of blast-exposed rabbits relative to those of the controls. Western blot analyses of whole cornea lysates revealed that the expression levels of AQP1 and AQP5 were approximately 2- and 1.5-fold higher, respectively, in blast-exposed rabbits compared to controls. The extent of AQP1 immunostaining (AQP1-IS) increased in the epithelial cell layer after blast exposure. The AQP5-IS pattern changed from a mixed membrane and cytoplasmic expression in the controls to predominantly cytoplasmic expression in the basally located cornea epithelial cells of blast-exposed rabbits. Conclusions: Primary blast exposure resulted in edema-related changes in the cornea manifested by the altered expression of the edema-regulating proteins AQP1 and AQP5 with blast overpressure-specific impulses. These findings support potential acute corneal injury mechanisms in which the altered regulation of water permeability is caused by primary blast exposure.
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Acuaporina 1/genética , Acuaporina 5/genética , Traumatismos por Explosión/genética , Córnea/metabolismo , Lesiones de la Cornea/genética , Regulación de la Expresión Génica , Animales , Acuaporina 1/metabolismo , Acuaporina 5/metabolismo , Traumatismos por Explosión/patología , Córnea/patología , Lesiones de la Cornea/patología , Paquimetría Corneal , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Lámpara de HendiduraRESUMEN
Adipose tissue dysregulation, a hallmark of obesity, contributes to a chronic state of low-grade inflammation and is associated with increased risk and progression of several breast cancer subtypes, including claudin-low breast tumors. Unfortunately, mechanistic targets for breaking the links between obesity-associated adipose tissue dysfunction, inflammation, and claudin-low breast cancer growth have not been elucidated. Ovariectomized female C57BL/6 mice were randomized (n = 15/group) to receive a control diet, a diet-induced obesity (DIO) diet, or a DIO + resveratrol (0.5% wt/wt) diet. Mice consumed these diets ad libitum throughout study and after 6 weeks were orthotopically injected with M-Wnt murine mammary tumor cells, a model of estrogen receptor (ER)-negative claudin-low breast cancer. Compared with controls, DIO mice displayed adipose dysregulation and metabolic perturbations including increased mammary adipocyte size, cyclooxygenase-2 (COX-2) expression, inflammatory eicosanoid levels, macrophage infiltration, and prevalence of crown-like structures (CLS). DIO mice (relative to controls) also had increased systemic inflammatory cytokines and decreased adipocyte expression of peroxisome proliferator-activated receptor gamma (PPARγ) and other adipogenesis-regulating genes. Supplementing the DIO diet with resveratrol prevented obesity-associated increases in mammary tumor growth, mammary adipocyte hypertrophy, COX-2 expression, macrophage infiltration, CLS prevalence, and serum cytokines. Resveratrol also offset the obesity-associated downregulation of adipocyte PPARγ and other adipogenesis genes in DIO mice. Our findings suggest that resveratrol may inhibit obesity-associated inflammation and claudin-low breast cancer growth by inhibiting adipocyte hypertrophy and associated adipose tissue dysregulation that typically accompanies obesity.
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Tejido Adiposo/efectos de los fármacos , Antiinflamatorios no Esteroideos/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Obesidad/tratamiento farmacológico , Resveratrol/uso terapéutico , Tejido Adiposo/fisiopatología , Animales , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/fisiopatología , Modelos Animales de Enfermedad , Femenino , Ratones Endogámicos C57BL , Obesidad/complicaciones , Obesidad/fisiopatología , PosmenopausiaRESUMEN
BACKGROUND: Exposure of cells to very short (<1 µs) electric pulses in the megavolt/meter range have been shown to cause a multitude of effects, both physical and molecular in nature. Physically, nanosecond electrical pulses (nsEP) can cause disruption of the plasma membrane, cellular swelling, shrinking and blebbing. Molecularly, nsEP have been shown to activate signaling pathways, produce oxidative stress, stimulate hormone secretion and induce both apoptotic and necrotic death. We hypothesize that studying the genetic response of primary human dermal fibroblasts exposed to nsEP, will gain insight into the molecular mechanism(s) either activated directly by nsEP, or indirectly through electrophysiology interactions. METHODS: Microarray analysis in conjunction with quantitative real time polymerase chain reaction (qRT-PCR) was used to screen and validate genes selectively upregulated in response to nsEP exposure. RESULTS: Expression profiles of 486 genes were found to be significantly changed by nsEP exposure. 50% of the top 20 responding genes coded for proteins located in two distinct cellular locations, the plasma membrane and the nucleus. Further analysis of five of the top 20 upregulated genes indicated that the HDFa cells' response to nsEP exposure included many elements of a mechanical stress response. CONCLUSIONS: We found that several genes, some of which are mechanosensitive, were selectively upregulated due to nsEP exposure. This genetic response appears to be a primary response to the stimuli and not a secondary response to cellular swelling. GENERAL SIGNIFICANCE: This work provides strong evidence that cells exposed to nsEP interpret the insult as a mechanical stress.
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Optoacoustic (OA) microscopy using an all-optical system based on the probe beam deflection technique (PBDT) for detection of laser-induced acoustic signals was investigated as an alternative to conventional piezoelectric transducers. PBDT provides a number of advantages for OA microscopy including (i) efficient coupling of laser excitation energy to the samples being imaged through the probing laser beam, (ii) undistorted coupling of acoustic waves to the detector without the need for separation of the optical and acoustic paths, (iii) high sensitivity and (iv) ultrawide bandwidth. Because of the unimpeded optical path in PBDT, diffraction-limited lateral resolution can be readily achieved. The sensitivity of the current PBDT sensor of 22 µV/Pa and its noise equivalent pressure (NEP) of 11.4 Pa are comparable with these parameters of the optical micro-ring resonator and commercial piezoelectric ultrasonic transducers. Benefits of the present prototype OA microscope were demonstrated by successfully resolving micron-size details in histological sections of cardiac muscle.
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Nanosecond electrical pulse (nsEP) exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s) between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress.
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Electricidad , Nanotecnología/métodos , Línea Celular Tumoral , Membrana Celular/fisiología , Permeabilidad de la Membrana Celular/fisiología , Electroquímica , Regulación de la Expresión Génica/fisiología , Humanos , Células Jurkat , Estrés MecánicoRESUMEN
The objective of this study was to determine whether clinically significant ocular trauma can be induced by a survivable isolated primary blast using a live animal model. Both eyes of 18 Dutch Belted rabbits were exposed to various survivable low-level blast overpressures in a large-scale shock tube simulating a primary blast similar to an improvised explosive device. Eyes of the blast-exposed rabbits (as well as five control rabbits) were thoroughly examined before and after blast to detect changes. Clinically significant changes in corneal thickness arose immediately after blast and were sustained through 48 h, suggesting possible disruption of endothelial function. Retinal thickness (RT) increased with increasing specific impulse immediately after exposure. Intraocular pressure (IOP) was inversely correlated with the specific impulse of the blast wave. These findings clearly indicate that survivable primary blast causes ocular injuries with likely visual functional sequelae of clinical and military relevance.
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Traumatismos por Explosión , Modelos Animales de Enfermedad , Lesiones Oculares , Animales , ConejosRESUMEN
The mechanism(s) responsible for the breakdown (nanoporation) of cell plasma membranes after nanosecond pulse (nsEP) exposure remains poorly understood. Current theories focus exclusively on the electrical field, citing electrostriction, water dipole alignment and/or electrodeformation as the primary mechanisms for pore formation. However, the delivery of a high-voltage nsEP to cells by tungsten electrodes creates a multitude of biophysical phenomena, including electrohydraulic cavitation, electrochemical interactions, thermoelastic expansion, and others. To date, very limited research has investigated non-electric phenomena occurring during nsEP exposures and their potential effect on cell nanoporation. Of primary interest is the production of acoustic shock waves during nsEP exposure, as it is known that acoustic shock waves can cause membrane poration (sonoporation). Based on these observations, our group characterized the acoustic pressure transients generated by nsEP and determined if such transients played any role in nanoporation. In this paper, we show that nsEP exposures, equivalent to those used in cellular studies, are capable of generating high-frequency (2.5 MHz), high-intensity (>13 kPa) pressure transients. Using confocal microscopy to measure cell uptake of YO-PRO®-1 (indicator of nanoporation of the plasma membrane) and changing the electrode geometry, we determined that acoustic waves alone are not responsible for poration of the membrane.
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Permeabilidad de la Membrana Celular , Membrana Celular/metabolismo , Electroporación/instrumentación , Electroporación/métodos , Animales , Benzoxazoles/metabolismo , Benzoxazoles/farmacocinética , Células CHO , Membrana Celular/química , Cricetinae , Cricetulus , Electricidad , Colorantes Fluorescentes/metabolismo , Colorantes Fluorescentes/farmacocinética , Análisis de Fourier , Microscopía Confocal , Porosidad , Presión , Compuestos de Quinolinio/metabolismo , Compuestos de Quinolinio/farmacocinética , Factores de TiempoRESUMEN
PURPOSE: A computational model of the porcine eye was developed to simulate primary blast exposure. This model facilitates understanding of blast-induced injury mechanisms. METHODS: A computational model of the porcine eye was used to simulate the effects of primary blast loading for comparison with experimental findings from shock tube experiments. The eye model was exposed to overpressure-time histories measured during physical experiments. Deformations and mechanical stresses within various ocular tissues were then examined for correlation with pathological findings in the experiments. RESULTS: Stresses and strains experienced in the eye during a primary blast event increase as the severity of the blast exposure increases. Peak stresses in the model occurred in locations in which damage was most often observed in the physical experiments. CONCLUSIONS: Blast injuries to the anterior chamber may be due to inertial displacement of the lens and ciliary body while posterior damage may arise due to contrecoup interactions of the vitreous and retina. Correlation of modeling predictions with physical experiments lends confidence that the model accurately represents the conditions found in the physical experiments. TRANSLATIONAL RELEVANCE: This computational model offers insights into the mechanisms of ocular injuries arising due to primary blast and may be used to simulate the effects of new protective eyewear designs.
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PURPOSE: The continued emergence of multidrug resistant bacterial infections and the decline in discovery of new antibiotics are major challenges for health care throughout the world. This situation has heightened the need for novel antimicrobial therapies as alternatives to traditional antibiotics. The combination of metallic nanoparticles and laser exposure has been proposed as a strategy to induce physical damage to bacteria, regardless of antibiotic sensitivity. The purpose of this study was to test the antibacterial effect of antibody-targeted gold nanoparticles combined with pulsed laser irradiation. METHODS: Gold nanoparticles conjugated to antibodies specific to Staphylococcus aureus peptidoglycan were incubated with suspensions of methicillin-resistant and methicillin-sensitive S. aureus (MRSA and MSSA). Bacterial suspensions were then exposed to 8 ns pulsed laser irradiation at a wavelength of 532 nm and fluences ranging from 1 to 5 J/cm(2). Viability of the bacteria following laser exposure was determined using colony forming unit assays. Scanning electron microscopy was used to confirm the binding of nanoparticles to bacteria and the presence of cellular damage. RESULTS: The laser-activated nanoparticle treatment reduced the surviving population to 31% of control in the MSSA population, while the survival in the MRSA population was reduced to 58% of control. Significant decreases in bacterial viability occurred when the laser fluence exceeded 1 J/cm(2), and this effect was linear from 0 to 5 J/cm(2) (r (2)=0.97). Significantly less bactericidal effect was observed for nonfunctionalized nanoparticles or functionalized nanoparticles without laser activation. CONCLUSION: Laser-activated nanoparticles targeted to S. aureus surface antigens significantly reduced the percentage of viable organisms and represents a promising new treatment modality that could be used either alone or as an adjunct to existing, conventional antibiotic therapy.
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Antibacterianos , Anticuerpos Antibacterianos , Oro , Nanopartículas del Metal/química , Staphylococcus aureus , Antibacterianos/química , Antibacterianos/farmacología , Anticuerpos Antibacterianos/química , Anticuerpos Antibacterianos/farmacología , Oro/química , Oro/farmacología , Rayos Láser , Viabilidad Microbiana/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/efectos de la radiaciónRESUMEN
Accumulating evidence suggests that ubiquitin E3 ligases are involved in cancer development as their mutations correlate with genomic instability and genetic susceptibility to cancer. Despite significant findings of cancer-driving mutations in the BRCA1 gene, estrogen receptor (ER)-positive breast cancers progress upon treatment with DNA damaging-cytotoxic therapies. In order to understand the underlying mechanism by which ER-positive breast cancer cells develop resistance to DNA damaging agents, we employed an estrogen receptor agonist, Erb-041, to increase the activity of ERß and negatively regulate the expression and function of the estrogen receptor α (ERα) in MCF-7 breast cancer cells. Upon Erb-041-mediated ERα down-regulation, the transcription of an ERα downstream effector, BCA2 (Breast Cancer Associated gene 2), correspondingly decreased. The ubiquitination of chromatin-bound BCA2 was induced by ultraviolet C (UVC) irradiation but suppressed by Erb-041 pretreatment, resulting in a blunted DNA damage response. Upon BCA2 silencing, DNA double-stranded breaks increased with Rad51 up-regulation and ataxia telangiectasia mutated (ATM) activation. Mechanistically, UV-induced BCA2 ubiquitination and chromatin binding were found to promote DNA damage response and repair via the interaction of BCA2 with ATM, γH2AX and Rad51. Taken together, this study suggests that Erb-041 potentiates BCA2 dissociation from chromatin and co-localization with Rad51, resulting in inhibition of homologous recombination repair.
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In this work, the assembly of gold nanoparticles of (AuNPs) is used to detect the presence of the biomolecule glutathione (GSH) using a novel technique called "all-optical photoacoustic spectroscopy" (AOPAS). The AOPAS technique coupled with AuNPs forms the basis of a biosensing technique capable of probing the dynamic evolution of nano-bio interfaces within a microscopic volume. Dynamic Light Scattering (DLS) and ultraviolet-visible (UV-vis) spectra were measured to describe the kinetics governing the interparticle interactions by monitoring the AuNPs assembly and evolution of the surface plasmon resonance (SPR) band. A comparison of the same dynamic evolution of AuNPs assembly was performed using the AOPAS technique to confirm the validity of this method. The fundamental study is complemented by a demonstration of the performance of this biosensing technique in the presence of cell culture medium containing fetal bovine serum (FBS), which forms a protein corona on the surface of the AuNPs. This work demonstrates that the in vitro monitoring capabilities of the AOPAS provides sensitive measurement at the microscopic level and low nanoparticle concentrations without the artifacts limiting the use of conventional biosensing methods, such as fluorescent indicators. The AOPAS technique not only provides a facile approach for in vitro biosensing, but also shed a light on the real-time detection of thiol containing oxidative stress biomarkers in live systems using AuNPs.
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Glutatión/análisis , Glutatión/química , Oro/química , Nanopartículas del Metal/química , Microquímica/instrumentación , Técnicas Fotoacústicas/instrumentación , Análisis Espectral/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Oxidación-ReducciónRESUMEN
The goal of this paper is to demonstrate the unique capability of measuring the vector or angular information of propagating acoustic waves using an optical sensor. Acoustic waves were generated using photoacoustic interaction and detected by the probe beam deflection technique. Experiments and simulations were performed to study the interaction of acoustic emissions with an optical sensor in a coupling medium. The simulated results predict the probe beam and wavefront interaction and produced simulated signals that are verified by experiment.
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PURPOSE: We qualitatively describe the anatomic features of primary blast ocular injury observed using a postmortem porcine eye model. Porcine eyes were exposed to various levels of blast energy to determine the optimal conditions for future testing. METHODS: We studied 53 enucleated porcine eyes: 13 controls and 40 exposed to a range of primary blast energy levels. Eyes were preassessed with B-scan and ultrasound biomicroscopy (UBM) ultrasonography, photographed, mounted in gelatin within acrylic orbits, and monitored with high-speed videography during blast-tube impulse exposure. Postimpact photography, ultrasonography, and histopathology were performed, and ocular damage was assessed. RESULTS: Evidence for primary blast injury was obtained. While some of the same damage was observed in the control eyes, the incidence and severity of this damage in exposed eyes increased with impulse and peak pressure, suggesting that primary blast exacerbated these injuries. Common findings included angle recession, internal scleral delamination, cyclodialysis, peripheral chorioretinal detachments, and radial peripapillary retinal detachments. No full-thickness openings of the eyewall were observed in any of the eyes tested. Scleral damage demonstrated the strongest associative tendency for increasing likelihood of injury with increased overpressure. CONCLUSIONS: These data provide evidence that primary blast alone (in the absence of particle impact) can produce clinically relevant ocular damage in a postmortem model. The blast parameters derived from this study are being used currently in an in vivo model. We also propose a new Cumulative Injury Score indicating the clinical relevance of observed injuries.
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Segmento Anterior del Ojo/patología , Traumatismos por Explosión/patología , Modelos Animales de Enfermedad , Lesiones Oculares/patología , Heridas no Penetrantes/patología , Animales , Traumatismos por Explosión/diagnóstico por imagen , Lesiones Oculares/diagnóstico por imagen , Microscopía Acústica , Fotograbar , Enfermedades de la Esclerótica/diagnóstico por imagen , Enfermedades de la Esclerótica/patología , Porcinos , Índices de Gravedad del Trauma , Grabación en Video , Heridas no Penetrantes/diagnóstico por imagenRESUMEN
The signaling pathways via mTOR (mammalian target of rapamycin) and AMPK (AMP-activated protein kinase) play key roles in transcription, translation and carcinogenesis, and may be activated by light exposure. These pathways can be modulated by naturally occurring compounds, such as the triterpenoid, ursolic acid (UA). Previously, the transcription factors p53 and NF-κB, which transactivate mitochondrial apoptosis-related genes, were shown to be differentially modulated by UA. UA-modulated apoptosis, following exposure to UV-VIS radiation (ultraviolet to visible light broadband radiation, hereafter abbreviated to UVR), is observed to correspond to differential levels of oxidative stress in retinal pigment epithelial (RPE) and skin melanoma (SM) cells. The cellular response to this phytochemical was characterized using western blot, flow cytometry, microscopy with reactive oxidative species probes MitoTracker and dihydroethidium, and membrane permeability assay. UA pretreatment potentiated cell cycle arrest and UVR-induced apoptosis selectively in SM cells while reducing photo-oxidative stress in the DNA of RPE cells presumably by antioxidant activity of UA. Mechanistically, the nuclear transportation of p65 and p53 was reduced by UA administration prior to UVR exposure while the levels of p65 and p53 nuclear transportation in SM cells were sustained at a substantially higher level. Finally, the mitochondrial functional assay showed that UVR induced the collapse of the mitochondrial membrane potential, and this effect was exacerbated by rapamycin or UA pretreatment in SM preferentially. These results were consistent with reduced proliferation observed in the clonogenic assay, indicating that UA treatment enhanced the phototoxicity of UVR, by modulating the activation of p53 and NF-κB and initiating a mitogenic response to optical radiation that triggered mitochondria-dependent apoptosis, particularly in skin melanoma cells. The study indicates that this compound has multiple actions with the potential for protecting normal cells while sensitizing skin melanoma cells to UV irradiation.