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Disease outbreaks are a major impediment to aquaculture production, and vaccines are integral for disease management. Vaccines can be expensive, vary in effectiveness, and come with adjuvant-induced adverse effects, causing fish welfare issues and negative economic impacts. Three-dimensional biopolymer hydrogels are an appealing new technology for vaccine delivery in aquaculture, with the potential for controlled release of multiple immunomodulators and antigens simultaneously, action as local depots, and tunable surface properties. This research examined the intraperitoneal implantation of a cross-linked TEMPO cellulose nanofiber (TOCNF) hydrogel formulated with a Vibrio anguillarum bacterin in Atlantic salmon with macroscopic and microscopic monitoring to 600-degree days post-implantation. Results demonstrated a modified passive integrated transponder tagging (PITT) device allowed for implantation of the hydrogel. However, the Atlantic salmon implanted with TOCNF hydrogels exhibited a significant foreign body response (FBR) compared to sham-injected negative controls. The FBR was characterized by gross and microscopic external and visceral proliferative lesions, granulomas, adhesions, and fibrosis surrounding the hydrogel using Speilberg scoring of the peritoneum and histopathology of the body wall and coelom. Acutely, gross monitoring displayed rapid coagulation of blood in response to the implantation wound with development of fibrinous adhesions surrounding the hydrogel by 72 h post-implantation consistent with early stage FBR. While these results were undesirable for aquaculture vaccines, this work informs on the innate immune response to an implanted biopolymer hydrogel in Atlantic salmon and directs future research using cellulose nanomaterial formulations in Atlantic salmon for a new generation of aquaculture vaccine technology.
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Celulose Oxidada , Doenças dos Peixes , Nanofibras , Salmo salar , Animais , Hidrogéis , Antígenos , Adjuvantes Imunológicos , Vacinas Bacterianas , Celulose , AquiculturaRESUMO
Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species. Unfortunately, SFS provides no spatial information on a surface; small scale heterogeneities that may exist are averaged over the large areas typically probed. Over the past decade, this has begun to be addressed with the advent of SFS microscopy. Here we detail the construction and function of a total internal reflection (TIR) SFS spectral and confocal fluorescence imaging microscope directly amenable to surface investigations. This instrument combines, for the first time, sample scanning TIR-SFS imaging with confocal fluorescence microscopy.
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Microscopia Confocal/instrumentação , Microscopia de Fluorescência/instrumentação , Espectrometria de Fluorescência/instrumentação , Fluoreto de Cálcio/química , Etanolaminas/química , Ouro/química , Fosfatidilcolinas/química , Rodaminas/química , Propriedades de SuperfícieRESUMO
Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity.
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Carcinógenos Ambientais/toxicidade , Cobalto/toxicidade , Pulmão/efeitos dos fármacos , Mutagênicos/toxicidade , Transporte Biológico , Carcinógenos Ambientais/análise , Carcinógenos Ambientais/química , Carcinógenos Ambientais/metabolismo , Ciclo Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Cobalto/análise , Cobalto/química , Cobalto/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Humanos , Pulmão/química , Pulmão/metabolismo , Mutagênicos/análise , Mutagênicos/química , Mutagênicos/metabolismo , Concentração Osmolar , Óxidos/análise , Óxidos/química , Óxidos/metabolismo , Óxidos/toxicidade , Tamanho da Partícula , Fagocitose/efeitos dos fármacos , SolubilidadeRESUMO
Hexavalent chromium [Cr(VI)] is a known lung carcinogen and a driving mechanism in human lung cells for Cr(VI)-induced lung cancer is chromosome instability, caused by prolonged Cr(VI) exposure inducing DNA double-strand breaks, while simultaneously inhibiting the repair of these breaks. In North Atlantic right whales, Cr(VI) induces breaks but does not inhibit repair. It is unclear if this repair inhibition is specific to human lung cells or occurs in other species, as it has only been considered in humans and North Atlantic right whales. We evaluated these outcomes in rodent cells, as rodents are an experimental model for metal-induced lung carcinogenesis. We used a guinea pig lung fibroblast cell line, JH4 Clone 1, and rat lung fibroblasts. Cells were exposed to two different particulate Cr(VI) compounds, ranging from 0 to 0.5 ug/cm2, for 24 or 120 h and assessed for cytotoxicity, DNA double-strand breaks, and DNA double-strand break repair. Both particulate Cr(VI) compounds induced a concentration-dependent increase in cytotoxicity and DNA double-strand breaks after acute and prolonged exposures. Notably, while the repair of Cr(VI)-induced DNA double-strand breaks increased after acute exposure, the repair of these breaks was inhibited after prolonged exposure. These results are consistent with outcomes in human lung cells indicating rodent cells respond like human cells, while whale cells have a markedly different response.
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Model cellular membranes enable the study of biological processes in a controlled environment and reduce the traditional challenges associated with live or fixed cell studies. However, model membrane systems based on the air/water or oil/solution interface do not allow for incorporation of transmembrane proteins or for the study of protein transport mechanisms. Conversely, a phospholipid bilayer deposited via the Langmuir-Blodgett/Langmuir-Schaefer method on a hydrogel layer is potentially an effective mimic of the cross section of a biological membrane and facilitates both protein incorporation and transport studies. Prior to application, however, such membranes must be fully characterized, particularly with respect to the phospholipid bilayer phase transition temperature. Here we present a detailed characterization of the phase transition temperature of the inner and outer leaflets of a chitosan supported model membrane system. Specifically, the lateral diffusion coefficient of each individual leaflet has been determined as a function of temperature. Measurements were performed utilizing z-scan fluorescence correlation spectroscopy (FCS), a technique that yields calibration-free diffusion information. Analysis via the method of Wawrezinieck and co-workers revealed that phospholipid diffusion changes from raftlike to free diffusion as the temperature is increased-an insight into the dynamic behavior of hydrogel supported membranes not previously reported.
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Membranas Artificiais , Fosfolipídeos/química , Espectrometria de Fluorescência/métodos , DifusãoRESUMO
Importance: Older patients using many prescription drugs (hyperpolypharmacy) may be at increased risk of adverse drug effects. Objective: To test the effectiveness and safety of a quality intervention intended to reduce hyperpolypharmacy. Design, Setting, and Participants: This randomized clinical trial allocated patients 76 years or older who used 10 or more prescription medications to a deprescribing intervention or to usual care (1:1 ratio) at an integrated health system with multiple preexisting deprescribing workflows. Data were collected from October 15, 2020, to July 29, 2022. Intervention: Physician-pharmacist collaborative drug therapy management, standard-of-care practice recommendations, shared decision-making, and deprescribing protocols administered by telephone over multiple cycles for a maximum of 180 days after allocation. Main Outcomes and Measures: Primary end points were change in the number of medications and in the prevalence of geriatric syndrome (falls, cognition, urinary incontinence, and pain) from 181 to 365 days after allocation compared with before randomization. Secondary outcomes were use of medical services and adverse drug withdrawal effects. Results: Of a random sample of 2860 patients selected for potential enrollment, 2470 (86.4%) remained eligible after physician authorization, with 1237 randomized to the intervention and 1233 to usual care. A total of 1062 intervention patients (85.9%) were reached and agreed to enroll. Demographic variables were balanced. The median age of the 2470 patients was 80 (range, 76-104) years, and 1273 (51.5%) were women. In terms of race and ethnicity, 185 patients (7.5%) were African American, 234 (9.5%) were Asian or Pacific Islander, 220 (8.9%) were Hispanic, 1574 (63.7%) were White (63.7%), and 257 (10.4%) were of other (including American Indian or Alaska Native, Native Hawaiian, or >1 race or ethnicity) or unknown race or ethnicity. During follow-up, both the intervention and usual care groups had slight reductions in the number of medications dispensed (mean changes, -0.4 [95% CI, -0.6 to -0.2] and -0.4 [95% CI, -0.6 to -0.3], respectively), with no difference between the groups (P = .71). There were no significant changes in the prevalence of a geriatric condition in the usual care and intervention groups at the end of follow-up and no difference between the groups (baseline prevalence: 47.7% [95% CI, 44.9%-50.5%] vs 42.9% [95% CI, 40.1%-45.7%], respectively; difference-in-differences, 1.0 [95% CI, -3.5 to 5.6]; P = .65). No differences in use of medical services or adverse drug withdrawal effects were observed. Conclusions and Relevance: In this randomized clinical trial from an integrated care setting with various preexisting deprescribing workflows, a bundled hyperpolypharmacy deprescribing intervention was not associated with reduction in medication dispensing, prevalence of geriatric syndrome, utilization of medical services, or adverse drug withdrawal effects. Additional research is needed in less integrated settings and in more targeted populations. Trial Registration: ClinicalTrials.gov Identifier: NCT05616689.
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Desprescrições , Humanos , Feminino , Idoso , Idoso de 80 Anos ou mais , Masculino , Conduta do Tratamento Medicamentoso , Alaska , HavaíRESUMO
The relevant photophysical properties of single fluorescent molecules and single SERS active surface-coated gold nanostars tagged with the Raman reporter molecule 4-mercaptopyridine are compared for imaging purposes. Mean count rate distributions are built from the single molecule/single probe level. The individually observed variance and count rates of both systems are compared as well as the behavior over multiple image acquisitions.
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Diffraction limits the biological structures that can be imaged by normal light microscopy. However, recently developed techniques are breaking the limits that diffraction poses and allowing imaging of biological samples at the molecular length scale. Fluorescence photoactivation localization microscopy (FPALM) and related methods can now image molecular distributions in fixed and living cells with measured resolution better than 30 nm. Based on localization of single photoactivatable molecules, FPALM uses repeated cycles of activation, localization, and photobleaching, combined with high-sensitivity fluorescence imaging, to identify and localize large numbers of molecules within a sample. Procedures and pitfalls for construction and use of such a microscope are discussed in detail. Representative images of cytosolic proteins, membrane proteins, and other structures, as well as examples of results during acquisition are shown. It is hoped that these details can be used to perform FPALM on a variety of biological samples, to significantly advance the understanding of biological systems.
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Microscopia de Fluorescência/métodos , Células/metabolismo , Interpretação Estatística de Dados , Recuperação de Fluorescência Após Fotodegradação , Corantes Fluorescentes , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/estatística & dados numéricos , Fotodegradação , Processos Fotoquímicos , Proteínas/metabolismoRESUMO
The application of nanoparticle technology is rapidly expanding. The reduced dimensionality of nanoparticles can give rise to changes in chemical and physical properties, often resulting in altered toxicity. People are exposed dermally to titanium dioxide (TiO2) nanoparticles in industrial and residential settings. The general public is increasingly exposed to these nanoparticles as their use in cosmetics, sunscreens and lotions expands. The toxicity of TiO2 nanoparticles towards human skin cells is unclear and understudied. We used a human skin fibroblast cell line to investigate the cytotoxicity and clastogenicity of TiO2 nanoparticles after 24 h exposure. In a clonogenic survival assay, treatments of 10, 50 and 100 µg/cm2 induced 97.8, 88.8 and 84.7% relative survival, respectively. Clastogenicity was assessed using a chromosomal aberration assay in order to determine whether TiO2 nanoparticles induced serious forms of DNA damage such as chromatid breaks, isochromatid lesions or chromatid exchanges. Treatments of 0, 10, 50 and 100 µg/cm2 induced 3.3, 3.0, 3.0 and 2.7% metaphases with damage, respectively. No isochromatid lesions or chromatid exchanges were detected. These data show that TiO2 nanoparticles are not cytotoxic or clastogenic to human skin cells.
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A recent iteration of fluorescence correlation spectroscopy (FCS), z-scan FCS, has drawn attention for its elegant solution to the problem of quantitative sample positioning when investigating two-dimensional systems while simultaneously providing an excellent method for extracting calibration-free diffusion coefficients. Unfortunately, the measurement of planar systems using (FCS and) z-scan FCS still requires extremely mechanically stable sample positioning, relative to a microscope objective. As axial sample position serves as the inherent length calibration, instabilities in sample position will affect measured diffusion coefficients. Here, we detail the design and function of a highly stable and mechanically simple inverted microscope stage that includes a temperature controlled liquid cell. The stage and sample cell are ideally suited to planar membrane investigations, but generally amenable to any quantitative microscopy that requires low drift and excellent axial and lateral stability. In the present work we evaluate the performance of our custom stage system and compare it with the stock microscope stage and typical sample sealing and holding methods.
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Espectrometria de Fluorescência/métodos , Temperatura , Bicamadas Lipídicas/química , Microscopia Confocal , Fosfatidilgliceróis/químicaRESUMO
There currently exists much debate as to the active state related to the "long afterglow" effect in europium doped oxide materials. Redox couples that consist of Eu(+/2+) and Eu(2+/3+) are discussed, but no common answer is currently accepted. Here, we present a comparison of the optical properties of a commercially available SrAl(2)O(4):Eu, Dy phosphor, as a function of nanoparticle size reduction via dry mechanical milling. X-ray and optical spectroscopic data indicate a significant decrease in phosphorescence efficiency and an increase in laser stimulated emission efficiency as near surface Eu(2+) ions are oxidized to Eu(3+) as a consequence of increased exposure during the milling process. These results show evidence only for Eu(2+/3+) oxidation states, suggesting the mechanism related to long afterglow effect does not arise from Eu(+) species. We also suggest that size reduction, as a rule, cannot be universally applied to improve optical properties of nanostructures.
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Európio/química , Nanoestruturas/química , Oxirredução , Óxidos/química , Espectrometria de Fluorescência , Síncrotrons , Espectroscopia por Absorção de Raios XRESUMO
This chapter describes the methodology by which mAb-F19-conjugated gold nanoparticles were prepared and used to label human pancreatic adenocarcinoma. Specifically, gold nanoparticles were coated with dithiol bearing hetero-bifunctional PEG (polyethylene glycol), and cancer-specific mAb F19 was attached by means of NHS-EDC coupling chemistry taking advantage of a carboxylic acid group on the heterobifunctional PEG. These conjugates were completely stable and were characterized by a variety of methods, including UV-Vis absorbance spectrometry, darkfield microscopy, DLS (dynamic light scattering), TEM (transmission electron microscopy), SEC (size-exclusion chromatography), and confocal microscopy. Nanoparticle bioconjugates were used to label sections of healthy and cancerous human pancreatic tissue. Labeled tissue sections were examined by darkfield microscopy and indicate that these nanoparticle bioconjugates may selectively bind to cancerous tissue and provide a means of optical contrast.
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Diagnóstico por Imagem/métodos , Ouro , Nanopartículas Metálicas/química , Nanomedicina/métodos , Neoplasias/diagnóstico , Anticorpos/metabolismo , Cromatografia em Gel , Humanos , Imuno-Histoquímica , Luz , Nanopartículas Metálicas/ultraestrutura , Neoplasias Pancreáticas/patologia , Polietilenoglicóis/química , Espalhamento de Radiação , Propriedades de SuperfícieRESUMO
Nanoparticles are being widely investigated for a range of applications due to their unique physical properties. For example, silver nanoparticles are used in commercial products for their antibacterial and antifungal properties. Some of these products are likely to result in silver nanoparticles reaching the aquatic environment. As such, nanoparticles pose a health concern for humans and aquatic species. We used a medaka (Oryzias latipes) cell line to investigate the cytotoxicity and genotoxicity of 30nm diameter silver nanospheres. Treatments of 0.05, 0.3, 0.5, 3 and 5microg/cm(2) induced 80, 45.7, 24.3, 1 and 0.1% survival, respectively, in a colony forming assay. Silver nanoparticles also induced chromosomal aberrations and aneuploidy. Treatments of 0, 0.05, 0.1 and 0.3microg/cm(2) induced damage in 8, 10.8, 16 and 15.8% of metaphases and 10.8, 15.6, 24 and 24 total aberrations in 100 metaphases, respectively. These data show that silver nanoparticles are cytotoxic and genotoxic to fish cells.
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Células/efeitos dos fármacos , Nanosferas/toxicidade , Oryzias , Prata/toxicidade , Poluentes Químicos da Água/toxicidade , Aneuploidia , Animais , Linhagem Celular , Aberrações Cromossômicas/induzido quimicamente , Testes de MutagenicidadeRESUMO
Mesoporous silica membranes fabricated by the surfactant-templated sol-gel process have received attention because of the potential to prepare membranes with a narrow pore size distribution and ordering of the interconnected pores. Potential applications include ultrafiltration, biological separations and drug delivery, and separators in lithium-ion batteries. Despite advancements in synthesis and characterization of these membranes, a quantitative description of the membrane microstructure remains a challenge. Currently the membrane microstructure is characterized by the combination of results from several techniques, i.e., gas permeance testing, x-ray diffraction scanning electron microscopy, transmission electron microscopy, and permporometry. The results from these ensemble methods are then compiled and the data fitted to a particular flow model. Although these methods are very effective in determining membrane performance, general pore size distribution, and defect concentration, they are unable to monitor molecular paths through the membrane and quantitatively measure molecular interactions between the molecular specie and pore network. Single-molecule imaging techniques enable optical measurements that probe materials on nanometer length scales through observation of individual molecules without the influence of averaging. Using single-molecule imaging spectroscopy, we can quantitatively characterize the interaction between the probe molecule and the interior of the pore within mesoporous silica membranes. This approach is radically different from typical membrane characterization methods in that it has the potential to spatially sample the underlying pore structure distribution, the surface energy, and the transport properties. Our hope is that this new fundamental knowledge can be quantitatively linked to both the preparation and the performance of membranes, leading to the advancement of membrane science and technology. Fluorescent molecules, 1,1-dioctadecyl-3,3,3,3-tetramethylindo-carbocyanine perchlorate, used to interrogate the available free volume in their vicinity, were loaded into the mesoporous silica membranes at subnanomolar concentrations. The mesoporous silica films were prepared using a nonionic ethylene oxide-propylene oxide-ethylene oxide triblock copolymer surfactant, Pluronic P123, on single crystal silicon substrates using dip coating of a silica sol. Membranes were prepared resulting in an average pore diameter of approximately 5 nm as measured by helium, nitrogen permeance, and porosimetry. Fluorescent images and time transient experiments were recorded using a custom built single-molecule scanning confocal microscope at differing temperatures (10, 20, 30, 40, and 50 degrees C). Time-dependent polarization anisotropy was used to obtain the enthalpy of adsorption and Henry's law constant of the probe molecule.
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Membranas Artificiais , Microscopia de Fluorescência/métodos , Modelos Químicos , Modelos Moleculares , Dióxido de Silício/química , Tensoativos/química , Simulação por Computador , Conformação Molecular , Porosidade , Propriedades de SuperfícieRESUMO
Polarization anisotropy is investigated in single porous silicon nanoparticles containing multiple chromophores. Two classes of nanoparticles, low current density and high current density, are studied. Low current density samples exhibit red-shifted spectra and contain only one or two chromophores. High current density particles, on average, contain less than four chromophores and display a blue-shifted spectrum. We utilize single-molecule spectroscopy to probe the polarization effects of the particles, and we show that both classes of particles are influenced by a polarized excitation source. These results are exciting at the fundamental level for understanding coupled quantum dot emitters as well as for applications involving single-photon sources or silicon-based polarization-sensitive detectors.
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Cristalização/métodos , Medições Luminescentes/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Nanotecnologia/métodos , Silício/química , Titânio/química , Anisotropia , Luz , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Tamanho da Partícula , Refratometria , Espalhamento de Radiação , Propriedades de SuperfícieRESUMO
In this study, we describe optical detection of antibody-conjugated nanoparticles bound to surgically resected human pancreatic cancer tissue. Gold nanoparticles stabilized by heterobifunctional polyethylene glycol (PEG) were prepared using approximately 15 nm spherical gold cores and covalently coupled to F19 monoclonal antibodies. The heterobifunctional PEG ligands contain a dithiol group for stable anchoring onto the gold surface and a terminal carboxy group for coupling of antibodies to the outside of the PEG shell. The nanoparticle-antibody bioconjugates form highly stable dispersions and exhibit long-term resistance to agglomeration. This has been demonstrated by dynamic light scattering, size exclusion chromatography, and transmission electron microscopy. The nanoparticle bioconjugates were used to label tumor stroma in approximately 5 mum thick sections of resected human pancreatic adenocarcinoma. After rinsing away nonbound nanoparticles and fixation, the tissue samples were imaged by darkfield microscopy near the nanoparticle resonance scattering maximum (approximately 560 nm). The images display pronounced tissue features and suggest that this novel labeling method could provide for facile identification of cancer tissue. Tumor samples treated with gold nanoparticles conjugated to nonspecific control antibodies and noncancerous pancreatic tissue treated with mAb-F19-conjugated gold nanoparticles both exhibited correctly negative results and showed no tissue staining.
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Anticorpos Monoclonais/química , Carcinoma/patologia , Ouro/química , Nanopartículas Metálicas/química , Nanotecnologia/métodos , Neoplasias Pancreáticas/patologia , Polietilenoglicóis/química , Carcinoma/metabolismo , Humanos , Ligantes , Luz , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Conformação Molecular , Neoplasias Pancreáticas/metabolismo , Espalhamento de Radiação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Depleted uranium (DU) is commonly used in military armor and munitions, and thus, exposure of soldiers and non-combatants is potentially frequent and widespread. DU is considered a suspected human carcinogen, affecting the bronchial cells of the lung. However, few investigations have studied DU in human bronchial cells. Accordingly, we determined the cytotoxicity and clastogenicity of both particulate (water-insoluble) and soluble DU in human bronchial fibroblasts (WTHBF-6 cells). We used uranium trioxide (UO3) and uranyl acetate (UA) as prototypical particulate and soluble DU salts, respectively. After a 24 h exposure, both UO3 and UA induced concentration-dependent cytotoxicity in WTHBF-6 cells. Specifically, 0.1, 0.5, 1, and 5 microg/cm2 UO3 induced 99, 57, 32, and 1% relative survival, respectively. Similarly, 100, 200, 400, and 800 microM UA induced 98, 92, 70, and 56% relative survival, respectively. When treated with chronic exposure, up to 72 h, of either UO3 or UA, there was an increased degree of cytotoxicity. We assessed the clastogenicity of these compounds and found that at concentrations of 0, 0.5, 1, and 5 microg/cm2 UO3, 5, 6, 10, and 15% of metaphase cells exhibit some form of chromosome damage. UA did not induce chromosome damage above background levels. There were slight increases in chromosome damage induced when we extended the UO3 treatment time to 48 or 72 h, but no meaningful increase in chromosome damage was observed with chronic exposure to UA.
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Fibroblastos/efeitos dos fármacos , Mutagênicos/toxicidade , Compostos Organometálicos/toxicidade , Material Particulado/toxicidade , Compostos de Urânio/toxicidade , Brônquios , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Aberrações Cromossômicas/induzido quimicamente , Relação Dose-Resposta a Droga , Fibroblastos/patologia , HumanosRESUMO
Biological structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resolution. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small numbers of them at a time, at low excitation intensity. To control the number of visible fluorophores in the field of view and ensure that optically active molecules are separated by much more than the width of the point spread function, photoactivatable fluorescent molecules are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable molecules, the activation rate is controlled by the activation illumination intensity; nonfluorescent inactive molecules are activated by a high-frequency (405-nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the molecules are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small number of molecules are visible at a given time, their positions can be determined precisely; with only approximately 100 detected photons per molecule, the localization precision can be as much as 10-fold better than the resolution, depending on background levels. Heterogeneities on length scales of the order of tens of nanometers are observed by FPALM of PA-GFP on glass. FPALM images are compared with images of the same molecules by widefield fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with atomic force microscopy and show that the full width at half-maximum of features approximately 86 +/- 4 nm is significantly better than the expected diffraction-limited optical resolution. The number of fluorescent molecules and their brightness distribution have also been determined using FPALM. This new method suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.
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Polarização de Fluorescência/métodos , Microscopia de Polarização/métodos , Óxido de Alumínio/química , Argônio/química , Biofísica/instrumentação , Biofísica/métodos , Simulação por Computador , Cristalização , Polarização de Fluorescência/instrumentação , Corantes Fluorescentes/farmacologia , Análise de Fourier , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Íons , Luz , Microscopia de Polarização/instrumentação , Microscopia de Vídeo , Fotodegradação , Espectrometria de FluorescênciaRESUMO
We report a method in which temperature dependent single-molecule fluorescence measurements are used to study the kinetics and thermodynamics of the acid-base interaction in films of photoresist polymer. We use the two distinct fluorescent prototropic forms of Coumarin 6 (C6-->C6+) to indicate the state of the acid-base system. Data are analyzed using a statistical model of the intensity probability distributions, yielding temperature dependent proton exchange rates, which is confirmed through agreement with a simple two-state Monte Carlo model. The temperature dependent rates are used to calculate the activation enthalpy for proton exchange.