Gotowa bibliografia na temat „Photoactivatable systems”

Photoactivatable fluorophores switch from a nonemissive to an emissive state upon illumination at an activating wavelength and then emit after irradiation at an exciting wavelength. The interplay of such activation and excitation events can be exploited to switch fluorescence on in a defined region of space at a given interval of time. In turn, the spatiotemporal control of fluorescence translates into the opportunity to implement imaging and spectroscopic schemes that are not possible with conventional fluorophores. Specifically, photoactivatable fluorophores permit the monitoring of dynamic processes in real time as well as the reconstruction of images with subdiffraction resolution. These promising applications can have a significant impact on the characterization of the structures and functions of biomolecular systems. As a result, strategies to implement mechanisms for fluorescence photoactivation with synthetic fluorophores are particularly valuable. In fact, a number of versatile operating principles have already been identified to activate the fluorescence of numerous members of the main families of synthetic dyes. These methods are based on either the irreversible cleavage of covalent bonds or the reversible opening and closing of rings. This paper overviews the fundamental mechanisms that govern the behavior of these photoresponsive systems, illustrates structural designs for fluorescence photoactivation, and provides representative examples of photoactivatable fluorophores in actions.

This short review highlights some of the exciting new experimental and theoretical developments in the field of photoactivatable metal complexes and their applications in biotechnology and medicine. The examples chosen are based on some of the presentations at the Royal Society Discussion Meeting in June 2012, many of which are featured in more detail in other articles in this issue. This is a young field. Even the photochemistry of well-known systems such as metal–carbonyl complexes is still being elucidated. Striking are the recent developments in theory and computation (e.g. time-dependent density functional theory) and in ultrafast-pulsed radiation techniques which allow photochemical reactions to be followed and their mechanisms to be revealed on picosecond/nanosecond time scales. Not only do some metal complexes (e.g. those of Ru and Ir) possess favourable emission properties which allow functional imaging of cells and tissues (e.g. DNA interactions), but metal complexes can also provide spatially controlled photorelease of bioactive small molecules (e.g. CO and NO)—a novel strategy for site-directed therapy. This extends to cancer therapy, where metal-based precursors offer the prospect of generating excited-state drugs with new mechanisms of action that complement and augment those of current organic photosensitizers.

The development of controllable gene editing systems on the base of CRISPR/Cas is an actually problem of modern molecular biology and genetic enginery. Interesting variant of solution of this problem is modification of guide RNA by introduction of photocleavable linkers. We developed the approach to the synthesis of cyclic photocleavable guide crRNA for the CRISPR/Cas9 system with photolinker on the base of 1-(2-nitrophenyl)-1,2-ethanediol (PL). Upon irradiation by UV-light these guide RNA are linearized and CRISPR/Cas9 system is activated. Two chemical methods to the cyclization of RNA were tested: Michael reaction (thiol-maleimide condensation) and Cu-catalyzed azide-alkyne cycloaddition (CuAAC, click-chemistry reaction). For this purpose 5',3'-modified RNA containing reactive groups were prepared. The advantages of CuAAC reaction for cyclic RNA preparation was demonstrated. Effectivity of cyclic RNAs is depends from their secondary structure and ability of reactive groups to draw together. Series of photocleavable and control non-cleavable cyclic crRNA were obtained. It was shown that cyclic crRNAs guide nuclease Cas9 for plasmid cleavage less effective but linearization of photocleavable cyclic crRNA increases extent of plasmid cleavage. Developed approach permits prepare cyclic photocleavable RNA including spatiotemporal activation of guide RNA for gene editing. Photoregulation of gene editing will permit to lower the off-target effects and to carry out the editing more targeting.

Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type–specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.

Muscovite mica with an amino silane-modified surface is commonly used as a substrate in atomic force microscopy (AFM) studies of biological macromolecules. Herein, the efficiency of two different protein immobilization strategies employing either (N-hydroxysuccinimide ester)-based crosslinker (DSP) or benzophenone-based photoactivatable crosslinker (SuccBB) has been compared using AFM and mass spectrometry analysis. Two proteins with different physicochemical properties—human serum albumin (HSA) and horseradish peroxidase enzyme protein (HRP)—have been used as model objects in the study. In the case of HRP, both crosslinkers exhibited high immobilization efficiency—as opposed to the case with HSA, when sufficient capturing efficiency has only been observed with SuccBB photocrosslinker. The results obtained herein can find their application in commonly employed bioanalytical systems and in the development of novel highly sensitive chip-based diagnostic platforms employing immobilized proteins. The obtained data can also be of interest for other research areas in medicine and biotechnology employing immobilized biomolecules.

Cyclic nucleotides are important second messengers involved in cellular events, and analogues of this type of molecules are promising drug candidates. Some cyclic nucleotide analogues have become standard tools for the investigation of biochemical and physiological signal transduction pathways, such as the Rp-diastereomers of adenosine and guanosine 3′,5′-cyclic monophosphorothioate, which are competitive inhibitors of cAMP- and cGMP-dependent protein kinases. Next generation analogues exhibit a higher membrane permeability, increased resistance against degradation, and improved target specificity, or are caged or photoactivatable for fast and/or targeted cellular imaging. Novel specific nucleotide analogues activating or inhibiting cyclic nucleotide-dependent ion channels, EPAC/GEF proteins, and bacterial target molecules have been developed, opening new avenues for basic and applied research. This review provides an overview of the current state of the field, what can be expected in the future and some practical considerations for the use of cyclic nucleotide analogues in biological systems.

In higher eukaryotic cells, microtubules within metaphase and anaphase spindles undergo poleward flux, the slow, poleward movement of tubulin subunits through the spindle microtubule lattice. Although a number of studies have documented this phenomenon across a wide range of model systems, the possibility of poleward flux before nuclear envelope breakdown (NEB) has not been examined. Using a mammalian cell line expressing photoactivatable green fluorescent protein (GFP)-tubulin, we observe microtubule motion, both toward and away from centrosomes, at a wide range of rates (0.5–4.5 μm/min) in prophase cells. Rapid microtubule motion in both directions is dynein dependent. In contrast, slow microtubule motion, which occurs at rates consistent with metaphase flux, is insensitive to inhibition of dynein but sensitive to perturbation of Eg5 and Kif2a, two proteins with previously documented roles in flux. Our results demonstrate that microtubules in prophase cells are unexpectedly dynamic and that a subpopulation of these microtubules shows motion that is consistent with flux. We propose that the marked reduction in rate and directionality of microtubule motion from prophase to metaphase results from changes in microtubule organization during spindle formation.

Photodynamic therapy (PDT) is mainly used to destroy cancerous cells; it combines the action of three components: a photoactivatable molecule or photosensitizer (PS), the light of an appropriate wavelength, and naturally occurring molecular oxygen. After light excitation of the PS, the excited PS then reacts with molecular oxygen to produce reactive oxygen species (ROS), leading to cellular damage. One of the drawbacks of PSs is their lack of solubility in water and body tissue fluids, thereby causing low bioavailability, drug-delivery efficiency, therapeutic efficacy, and ROS production. To improve the water-solubility and/or drug delivery of PSs, using cyclodextrins (CDs) is an interesting strategy. This review describes the in vitro or/and in vivo use of natural and derived CDs to improve antitumoral PDT efficiency in aqueous media. To achieve these goals, three types of binding modes of PSs with CDs are developed: non-covalent CD–PS inclusion complexes, covalent CD–PS conjugates, and CD–PS nanoassemblies. This review is divided into three parts: (1) non-covalent CD-PS inclusion complexes, covalent CD–PS conjugates, and CD–PS nanoassemblies, (2) incorporating CD–PS systems into hybrid nanoparticles (NPs) using up-converting or other types of NPs, and (3) CDs with fullerenes as PSs.

La silice mésoporeuse est un support polyvalent largement utilisé dans le domaine biomédical et de la catalyse en raison de ses propriétés intéressantes. Dans ce travail, la silice mésoporeuse a d'abord été utilisée comme support pour des applications de thérapie photodynamique (PDT). La PDT est réalisée par l'excitation d'un photosensibilisateur (PS) avec de la lumière visible à certaines longueurs d'onde, puis le PS excité peut soit conduire des réactions de transfert d'électrons vers/depuis des molécules biologiques (mécanisme de type I) ou, alternativement, transférer son énergie à l'oxygène moléculaire, générant ainsi de l'oxygène singulet (1O2, mécanisme de type II).Le bleu de méthylène (MB) est un photosensibilisateur connu qui a la capacitéde générer de l'oxygène singulet. La principale espèce active est la forme monomérique. Cependant, ce colorant a tendance à s'auto-agréger pour former des dimères et des agrégats plus importants, qui sont moins actifs pour la génération de 1O2. Deux stratégies différentes (synthèse directe et post-synthèse) ont été conçues pour incorporer le bleu de méthylène dans des nanoparticules de silice (NPs). La conception de la synthèse est un paramètre clé pour éviter à la fois le relargage du colorant et son auto-agrégation. La matrice de silice chargée négativement permet l'incorporation du colorant, qui est cationique, sans relargage, tandis que la présence de fonctions phényle dans la matrice favorise la forme monomérique du MB. Nous observons que les formes dimériques et monomériques de MB peuvent générer des espèces de 1O2 en solution aqueuse lorsqu'elles sont confinées dans une matrice de silice, ce qui améliore l'activité par rapport au colorant nu en solution.L'effet de la matrice sur la génération de 1O2 a ensuite été étudié avec une série de NPs de silice mésoporeuse d'un diamètre de 80-100 nm avec différentes structures poreuses. La procédure de préparation des NPs de silice a été optimisée en tenant compte des exigences de stabilité colloïdale élevée en milieu aqueux pour l'application en PDT. La fonctionnalisation par zwitterion et l'enrobage lipidique des NPs de silice ont été tentés pour améliorer la stabilité colloïdale, mais ces deux stratégies n'ont pas répondu aux attentes. Les résultats ont montré que la stabilité colloïdale de toutes les NPs de silice conservées dans des solutions aqueuses était meilleure que celle des NPs de silice préalablement séchées après extraction de tensioactifs, puis redispersées dans l'eau. L'incorporation de MB a ensuite été réalisée sur des échantillons aqueux sans tensioactif en utilisant un rapport molaire fixe entre MB et silice. L'eau et le méthanol ont été utilisés comme solvants pour les tests de génération de 1O2, et tous les échantillons ont montré une activité photocatalytique.Des tests préliminaires sur l'activité photodynamique ont été réalisés avec les nanoparticules fonctionnalisées avec du MB, en utilisant des lignées cellulaires de cancer du poumon A549. L'effet sur la destruction de cellules cancéreuses est légèrement amélioré par rapport au bleu de méthylène libre à la même concentration.Des tests antibactériens préliminaires ont également été réalisés avec Staphylocoque (S.) doré positif et Pseudomonas (P.) aeruginosa négatif. Les résultats ont montré que la silice elle-même et les échantillons contenant de la MB avaient une activité antimicrobienne contre S. doré
Mesoporous silica is a versatile support widely applied in biomedical and catalysis field because of its desirable properties. In this work, mesoporous silica was first utilized as support for photodynamic therapy (PDT) applications. PDT is achieved by the excitation of a photosensitizer (PS) with visible light at certain wavelengths, and then the excited PS may either drive electron-transfer reactions to/from biological molecules (type I mechanism) or, alternatively, transfer its energy to molecular oxygen, thus generating singlet oxygen (1O2, type II mechanism).Methylene blue (MB) is a known photosensitizer with desirable ability to generate singlet oxygen. The main active species is the monomeric form. However, this dye tends to self-aggregate to form dimers and higher aggregates, which are less active for 1O2 generation. Two different strategies (one-pot and post-synthesis) were designed to incorporate methylene blue into silica nanoparticles (NPs). The design of the synthesis is a key parameter to avoid both leaching of the dye and its self-aggregation. The negatively charged silica matrix allows the incorporation of the dye, which is cationic, with no leaching, whereas the presence of phenyl functions in the matrix favors the monomeric form of MB. We observe that both dimeric and monomeric forms of MB can generate 1O2 species in aqueous solution when confined inside a silica matrix, improving the activity compared to the bare dye in solution.Matrix effect in 1O2 generation was then investigated with a series of mesoporous silica NPs of 80-100 nm diameter with various morphologies. The procedure of preparation of silica NPs was optimized considering the requirements of high colloidal stability in aqueous media for the application in PDT. Zwitterion functionalization and lipid-coating of silica NPs were attempted to improve colloidal stability but these two strategies did not meet the expectation. The results showed that the colloidal stability of all the silica NPs kept in aqueous solutions was better than that of the silica NPs previously dried after surfactant extraction and then redispersed in water. The incorporation of MB was then performed on aqueous surfactant-free samples using a fixed molar ratio between MB and silica. Water and methanol were used as solvents for 1O2 generation tests, and all the samples displayed photocatalytic activity.Initial exploratory tests on photodynamic activity were carried out with the MB-functionalized nanoparticles, using A549 lung cancer cell lines. The killing effect is slightly improved when compared to the free methylene blue at the same concentration.Preliminary antibacterial tests were also performed with positive Staphylococcus (S.) aureus and negative Pseudomonas (P.) aeruginosa. The results showed that silica itself and MB-containing samples had antimicrobial activity against S. aureus.Finally, the mesoporous silica systems here prepared were also used as a support for Ru complexes, to build heterogeneous catalysts for different organic transformations. Preliminary heterogeneous catalytic tests were conducted on alcohol coupling reactions of the borrowing hydrogen type and also on photoactivated alcohol oxidation. The ruthenium functionalized nanoparticles show in some cases improved efficiency when compared to the analogous homogeneous catalysts

碩士
國立清華大學
生物科技研究所
95
Recent advances in sensory neuroscience using Drosophila olfaction as a model system have revealed partial map for the representation of the external world within its brain. Currently, three levels of wiring in antenna lobe (AL) are known. Odorants are detected by the olfactory sensory neurons (OSNs), located on the antenna (Ant) and maxillary palp (MP), sending axons via the antennal nerve (AN) and the labial nerve (LN), respectively, to AL (Level 1); where the projection neurons (PNs) receive and calculate the information (Level 2) and then relay to the mushroom body (MB) and the lateral horn (LH) (Level 3). Projection neurons convey the information via three tracks: the inner antenna-cerebrum track (iACT), the medial ACT (mACT), the outer ACT (oACT). In order to investigate the detailed anatomy along these tracks, we generate a transgenic fly, UAS-Photoactivatable GFP (PaGFP), which produces PaGFP under GAL4 control. In this study, the first part is to characterize PaGFP in fly brain, including photoactivation and diffusion. The second part is to demonstrate the anatomical applications of PaGFP, such as revealing single glomerulus or the whole AL, following its activation by a two-photon laser at 820 nm. These results illustrate that circuits tracing is feasible in any region we are interested in Drosophila brains. In this way, we discover some new circuits in addition to the typical three tracts of olfactory circuits and find two regions, medial superior (MS) and ventral lateral (VL), which may also connect with AL. Therefore, PaGFP provides us a novel tool tracing neuronal circuitry and mapping the complete network in Drosophila brains.