RESUMO
Localization-based ultrasound imaging methods that use microbubbles or nanodroplets offer high-resolution imaging with improved sensitivity and reduced background signal. However, these methods require long acquisition times (typically seconds to minutes), preventing their use for real-time imaging and, thus, limiting their clinical translational potential. Here, we present a new ultrafast localization method using blinking ultrasound-responsive nanoparticles (BNPs). When activated with high frame rate (1 kHz) plane wave ultrasound pulses with a mechanical index of 1.5, the BNPs incept growth of micrometer-sized bubbles, which in turn collapse and generate a blinking ultrasound signal. We showed that background-free ultrasound images could be obtained by localizing these blinking events using acquisition times as low as 11 ms. In addition, we demonstrated that BNPs enable in vivo background-free ultrasound imaging in mice. We envision that BNPs will facilitate the clinical translation of localization-based ultrasound imaging for more sensitive detection of cancer and other diseases.
Assuntos
Piscadela , Nanopartículas , Camundongos , Animais , Meios de Contraste , Ultrassonografia/métodos , MicrobolhasRESUMO
Lysyl oxidase-2 (LOXL2) is a Cu2+ and lysine tyrosylquinone (LTQ)-dependent amine oxidase that catalyzes the oxidative deamination of peptidyl lysine and hydroxylysine residues to promote crosslinking of extracellular matrix proteins. LTQ is post-translationally derived from Lys653 and Tyr689, but its biogenesis mechanism remains still elusive. A 2.4 Å Zn2+-bound precursor structure lacking LTQ (PDB:5ZE3) has become available, where Lys653 and Tyr689 are 16.6 Å apart, thus a substantial conformational rearrangement is expected to take place for LTQ biogenesis. However, we have recently shown that the overall structures of the precursor (no LTQ) and the mature (LTQ-containing) LOXL2s are very similar and disulfide bonds are conserved. In this study, we aim to gain insights into the spatial arrangement of LTQ and the active site Cu2+ in the mature LOXL2 using a recombinant LOXL2 that is inhibited by 2-hydrazinopyridine (2HP). Comparative UV-vis and resonance Raman spectroscopic studies of the 2HP-inhibited LOXL2 and the corresponding model compounds and an EPR study of the latter support that 2HP-modified LTQ serves as a tridentate ligand to the active site Cu2. We propose that LTQ resides within 2.9 Å of the active site of Cu2+ in the mature LOXL2, and both LTQ and Cu2+ are solvent-exposed.
Assuntos
Lisina , Proteína-Lisina 6-Oxidase , Lisina/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Domínio Catalítico , Quinonas/químicaRESUMO
Addition of dioxygen at low temperature to the non-heme ferrous complex FeII(Me3TACN)((OSiPh2)2O) (1) in 2-MeTHF produces a peroxo-bridged diferric complex Fe2III(µ-O2)(Me3TACN)2((OSiPh2)2O)2 (2), which was characterized by UV-vis, resonance Raman, and variable field Mössbauer spectroscopies. Illumination of a frozen solution of 2 in THF with white light leads to homolytic O-O bond cleavage and generation of a FeIV(O) complex 4 (ν(Fe=O) = 818 cm-1; δ = 0.22 mm s-1, ΔEQ = 0.23 mm s-1). Variable field Mössbauer spectroscopy measurements show that 4 is a rare example of a high-spin S = 2 FeIV(O) complex and the first synthetic example to be generated directly from O2. Complex 4 is highly reactive, as expected for a high-spin ferryl, and decays rapidly in fluid solution at cryogenic temperatures. This decay process in 2-MeTHF involves C-H cleavage of the solvent. However, the controlled photolysis of 2 in situ with visible light and excess phenol substrate leads to competitive phenol oxidation, via the proposed transient generation of 4 as the active oxidant.
Assuntos
Compostos Férricos/química , Oxigênio/química , Modelos Moleculares , Estrutura Molecular , Fenóis/químicaRESUMO
Dinuclear iron centers with a bridging hydroxido or oxido ligand form active sites within a variety of metalloproteins. A key feature of these sites is the ability of the protein to control the structures around the Fe centers, which leads to entatic states that are essential for function. To simulate this controlled environment, artificial proteins have been engineered using biotin-streptavidin (Sav) technology in which Fe complexes from adjacent subunits can assemble to form [FeIII-(µ-OH)-FeIII] cores. The assembly process is promoted by the site-specific localization of the Fe complexes within a subunit through the designed mutation of a tyrosinate side chain to coordinate the Fe centers. An important outcome is that the Sav host can regulate the Fe···Fe separation, which is known to be important for function in natural metalloproteins. Spectroscopic and structural studies from X-ray diffraction methods revealed uncommonly long Fe···Fe separations that change by less than 0.3 Å upon the binding of additional bridging ligands. The structural constraints imposed by the protein host on the di-Fe cores are unique and create examples of active sites having entatic states within engineered artificial metalloproteins.
Assuntos
Materiais Biomiméticos/química , Ferro/química , Metaloproteínas/metabolismo , Biotina/metabolismo , Modelos Moleculares , Conformação Molecular , Estreptavidina/metabolismoRESUMO
A new nonheme iron(II) complex, FeII (Me3 TACN)((OSiPh2 )2 O) (1), is reported. Reaction of 1 with NO(g) gives a stable mononitrosyl complex Fe(NO)(Me3 TACN)((OSiPh2 )2 O) (2), which was characterized by Mössbauer (δ=0.52â mm s-1 , |ΔEQ |=0.80â mm s-1 ), EPR (S=3/2), resonance Raman (RR) and Fe K-edge X-ray absorption spectroscopies. The data show that 2 is an {FeNO}7 complex with an S=3/2 spin ground state. The RR spectrum (λexc =458â nm) of 2 combined with isotopic labeling (15 N, 18 O) reveals ν(N-O)=1680â cm-1 , which is highly activated, and is a nearly identical match to that seen for the reactive mononitrosyl intermediate in the nonheme iron enzyme FDPnor (ν(NO)=1681â cm-1 ). Complex 2 reacts rapidly with H2 O in THF to produce the N-N coupled product N2 O, providing the first example of a mononuclear nonheme iron complex that is capable of converting NO to N2 O in the absence of an exogenous reductant.
Assuntos
Compostos Ferrosos/química , Óxido Nítrico/química , Óxido Nitroso/química , Substâncias Redutoras/química , Conformação MolecularRESUMO
The LodA-like proteins make up a recently identified family of enzymes that rely on a cysteine tryptophylquinone cofactor for catalysis. They differ from other tryptophylquinone enzymes in that they are oxidases rather than dehydrogenases. GoxA is a member of this family that catalyzes the oxidative deamination of glycine. Our previous work with GoxA from Pseudoalteromonas luteoviolacea demonstrated that this protein forms a stable intermediate upon anaerobic incubation with glycine. The spectroscopic properties of this species were unique among those identified for tryptophylquinone enzymes characterized to date. Here we use X-ray crystallography and resonance Raman spectroscopy to identify the GoxA catalytic intermediate as a product Schiff base. Structural work additionally highlights features of the active site pocket that confer substrate specificity, intermediate stabilization, and catalytic activity. The unusual properties of GoxA are discussed within the context of the other tryptophylquinone enzymes.
Assuntos
Aminoácido Oxirredutases/química , Glicina/química , Bases de Schiff/química , Domínio Catalítico , Oxigênio/química , Pseudoalteromonas/enzimologia , Análise Espectral Raman , EstereoisomerismoRESUMO
High-valent ferryl species (e.g., (Por)FeIVâO, Cmpd-II) are observed or proposed key oxidizing intermediates in the catalytic cycles of heme-containing enzymes (P-450s, peroxidases, catalases, and cytochrome c oxidase) involved in biological respiration and oxidative metabolism. Herein, various axially ligated iron(IV)-oxo complexes were prepared to examine the influence of the identity of the base. These were generated by addition of various axial ligands (1,5-dicyclohexylimidazole (DCHIm), a tethered-imidazole system, and sodium derivatives of 3,5-dimethoxyphenolate and imidazolate). Characterization was carried out via UV-vis, electron paramagnetic resonance (EPR), 57Fe Mössbauer, Fe X-ray absorption (XAS), and 54/57Fe resonance Raman (rR) spectroscopies to confirm their formation and compare the axial ligand perturbation on the electronic and geometric structures of these heme iron(IV)-oxo species. Mössbauer studies confirmed that the axially ligated derivatives were iron(IV) and six-coordinate complexes. XAS and 54/57Fe rR data correlated with slight elongation of the iron-oxo bond with increasing donation from the axial ligands. The first reported synthetic H-bonded iron(IV)-oxo heme systems were made in the presence of the protic Lewis acid, 2,6-lutidinium triflate (LutH+), with (or without) DCHIm. Mössbauer, rR, and XAS spectroscopic data indicated the formation of molecular Lewis acid ferryl adducts (rather than full protonation). The reduction potentials of these novel Lewis acid adducts were bracketed through addition of outer-sphere reductants. The oxidizing capabilities of the ferryl species with or without Lewis acid vary drastically; addition of LutH+ to F8Cmpd-II (F8 = tetrakis(2,6-difluorophenyl)porphyrinate) increased its reduction potential by more than 890 mV, experimentally confirming that H-bonding interactions can increase the reactivity of ferryl species.
Assuntos
Elétrons , Heme/química , Ferro/química , Ácidos de Lewis/química , Imidazóis/química , Ligantes , Modelos Moleculares , Conformação MolecularRESUMO
The synthesis of a new nonheme iron NO binding complex, [FeII(CH3CN)(N3Py2PhSEtCN)](BF4)2 (1), is reported. Complex 1, which contains two sterically encumbering phenyl substituents, exhibits a high-spin (hs) FeII (S = 2) ground state in contrast to the S = 0 ground state for unsubstituted [FeII(CH3CN)(N3PySEtCN)(BF4)2. Reaction of 1 with NO(g) in CH3CN yields an {FeNO}7 (S = 3/2) complex 2, which slowly decays at 25 °C with loss of NO⢠to regenerate 1. One-electron reduction of 2 with Cr(C6H6)2 at -40 °C yields the metastable, S = 1â¯{FeNO}8 complex 3. The nitrosyl moieties in thioether-ligated 2 and 3 are significantly less activated than in thiolate-ligated [Fe(NO)(N3PyS)]+/0, a structurally analogous pair of hs {FeNO}7/8 complexes. Calculations reveal that reduction of 2 is iron-centered, which may be a general property of hs {FeNO}7/8 complexes.
RESUMO
FeBMbs are structural and functional models of native bacterial nitric oxide reductases (NORs) generated through engineering of myoglobin. These biosynthetic models replicate the heme-nonheme diiron site of NORs and allow substitutions of metal centers and heme cofactors. Here, we provide evidence for multiple NOR turnover in monoformyl-heme-containing FeBMb1 proteins loaded with FeII, CoII, or ZnII metal ions at the FeB site (FeII/CoII/ZnII-FeBMb1(MF-heme)). FTIR detection of the ν(NNO) band of N2O at 2231 cm-1 provides a direct quantitative measurement of the product in solution. A maximum number of turnover is observed with FeII-FeBMb1(MF-heme), but the NOR activity is retained when the FeB site is loaded with ZnII. These data support the viability of a one-electron semireduced pathway for the reduction of NO at binuclear centers in reducing conditions.
Assuntos
Materiais Biomiméticos/química , Mioglobina/química , Cobalto/química , Elétrons , Heme/química , Hidrazinas/química , Ferro/química , Óxido Nítrico/química , Doadores de Óxido Nítrico/química , Óxido Nitroso/síntese química , Oxirredução , Oxirredutases/química , Engenharia de Proteínas , Espectroscopia de Infravermelho com Transformada de Fourier , Zinco/químicaRESUMO
Metal nanoparticles can be sensitive molecular sensors due to enhanced absorption and scattering of light near a localized surface plasmon resonance (LSPR). Variations in both intrinsic properties such as the geometry and extrinsic properties such as the environment can cause heterogeneity in nanoparticle LSPR and impact the overall sensing responses. To date, however, few studies have examined LSPR and sensing heterogeneities, due to technical challenges in obtaining the full LSPR spectra of individual nanoparticles in dynamic assays. Here, we report multispectral LSPR (msLSPR), a wide-field imaging technique for real-time spectral monitoring of light scattering from individual nanoparticles across the whole field of view (FOV) at â¼0.5 nm spectral and â¼100 ms temporal resolutions. Using msLSPR, we studied the spectral and sensing properties of gold nanoparticles commonly used in LSPR assays, including spheres, rods, and bipyramids. Complemented with electron microscopy imaging, msLSPR analysis revealed that all classes of gold nanoparticles exhibited variations in LSPR peak wavelengths that largely paralleled variations in morphology. Compared with the rods and spheres, gold nanobipyramids exhibited both more uniform and stronger sensing responses as long as the bipyramids are structurally intact. Simulations incorporating the experimental LSPR properties demonstrate the negative impact of spectral heterogeneity on the overall performance of conventional, intensity-based LSPR assays and the ability of msLSPR in overcoming both particle heterogeneity and measurement noise. These results highlight the importance of spectral heterogeneity in LSPR-based sensors and the potential advantage of performing LSPR assays in the spectral domain.
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The use of ultrasound in the clinic has been long established for cancer detection and image-guided tissue biopsies. In addition, ultrasound-based methods have been widely explored to develop more effective cancer therapies such as localized drug delivery, sonodynamic therapy, and focused ultrasound surgery. Stabilized fluorocarbon microbubbles have been in use as contrast agents for ultrasound imaging in the clinic for several decades. It is also known that microbubble cavitation could generate thermal, mechanical, and chemical effects in the tissue to improve ultrasound-based therapies. However, the large size, poor stability, and short-term cavitation activity of microbubbles limit their applications in cancer imaging and therapy. This review will focus on an alternative type of ultrasound responsive material; gas-stabilizing nanoparticles, which can address the limitations of microbubbles with their nanoscale size, robustness, and high cavitation activity. This review will be of interest to researchers who wish to explore new agents to develop improved methods for molecular ultrasound imaging and therapy of cancer.
RESUMO
Recent studies have demonstrated that gas-stabilizing particles can generate cavitating micron-sized bubbles when exposed to ultrasound, offering excellent application potential, including ultrasound imaging, drug delivery, and tumor ablation. However, the majority of the reported gas-stabilizing particles are relatively large (>200 nm), and smaller particles require high acoustic pressures to promote cavitation. Here, this paper reports the preparation of sub-100 nm gas-stabilizing nanoparticles (GSNs) that can initiate cavitation at low acoustic intensities, which can be delivered using a conventional medical ultrasound imaging system. The highly echogenic GSNs (F127-hMSN) were prepared by carefully engineering the surfaces of â¼50 nm mesoporous silica nanoparticles. It was demonstrated that the F127-hMSNs could be continuously imaged with ultrasound in buffer or biological solutions or agarose phantoms for up to 20 min. Also, the F127-hMSN can be stored in phosphate-buffered saline for at least a month with no loss in ultrasound responsiveness. The particles significantly degraded when diluted in simulated body fluids, indicating possible biodegradation of the F127-hMSNs in vivo. Furthermore, at ultrasound imaging conditions, F127-hMSNs did not cause detectable cell death, supporting the potential safety of these particles. Finally, strong cavitation activity generation by the F127-hMSNs under high-intensity focused ultrasound insonation was demonstrated and applied to effectively ablate cancer cells.
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The coupling of the reaction of a tightly bound ubiquinone with the reduction of O2 in cytochrome bo3 of Escherichia coli was investigated. In the absence of the quinone, a strongly diminished rate of electrocatalytic reduction of oxygen is detected, which can be restored by adding quinones. The correlation of previous EPR data with the electrocatalytic study on mutations in the binding site at positions, Q101, D75, F93, H98, I102 and R71 reveal that the stabilization of the radical is not necessary for the oxygen reaction. The Q101 and F93 variants exhibit both well-defined catalytic i-V curves, whereas D75H, H98F, I102W and R71H exhibit broad i-V curves with large hysteresis pointing toward a strong alteration in their catalytic activity.
Assuntos
Benzoquinonas/metabolismo , Citocromos/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Oxigênio/metabolismo , Benzoquinonas/química , Sítios de Ligação/genética , Biocatálise , Cristalografia por Raios X , Grupo dos Citocromos b , Citocromos/química , Citocromos/genética , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutação , Oxigênio/química , Domínios Proteicos , Especificidade por SubstratoRESUMO
Metal oxide nanoparticles (MONPs) are used in a variety of applications including drug formulations, paint, sensors and biomedical devices due to their unique physicochemical properties. One of the major problems with their widespread implementation is their uncontrolled agglomeration. One approach to reduce agglomeration is to alter their surface chemistry with a proper functionality in an environmentally friendly way. In this study, the influence of hydrogen peroxide (H2O2) treatment on the dispersion of ZnO and TiO2 nanoparticle (NP) agglomerates as a function of temperature is studied. The H2O2 treatment of the MONPs increases the density of hydroxyl (-OH) groups on the NP surface, as verified with FTIR spectroscopy. The influence of heating on the dispersion of H2O2-treated ZnO and TiO2 NPs is investigated using dynamic light scattering. The untreated and H2O2-treated ZnO and TiO2 NP suspensions were heated from 30 °C to 90 °C at 5 °C intervals to monitor the breakdown of large aggregates into smaller aggregates and individual nanoparticles. It was shown that the combined effect of hydroxylation and heating enhances the dispersion of ZnO and TiO2 NPs in water.