Luminescent sensing and imaging of oxygen: Fierce competition to the Clark electrode

Luminescence‐based sensing schemes for oxygen have experienced a fast growth and are in the process of replacing the Clark electrode in many fields. Unlike electrodes, sensing is not limited to point measurements via fiber optic microsensors, but includes additional features such as planar sensing, imaging, and intracellular assays using nanosized sensor particles. In this essay, I review and discuss the essentials of (i) common solid‐state sensor approaches based on the use of luminescent indicator dyes and host polymers; (ii) fiber optic and planar sensing schemes; (iii) nanoparticle‐based intracellular sensing; and (iv) common spectroscopies. Optical sensors are also capable of multiple simultaneous sensing (such as O₂ and temperature). Sensors for O₂ are produced nowadays in large quantities in industry. Fields of application include sensing of O₂ in plant and animal physiology, in clinical chemistry, in marine sciences, in the chemical industry and in process biotechnology.     

铽(Ⅲ)离子及其复合物:从发光特性到传感应用

稀土元素也称镧系元素,因其独特的发光性质和配位性质,其发光复合物被广泛研究于生物技术领域。其中稀土铽(Ⅲ)离子复合物因具有优异的光谱特性,关于其研究呈现出快速的发展趋势。主要从其发光特性的角度出发,探讨了其发光机理,并对铽(Ⅲ)离子与不同有机化合物结合形成的发光铽配合物以及铽(Ⅲ)离子及其配合物与不同纳米材料形成的复合物进行了分类综述。此外,还详细地阐述了铽离子及其复合物在荧光探针、生物传感器、药物递送、细胞成像、癌症治疗等相关领域的应用。最后,对其今后发展趋势和潜在的研究价值进行了展望。     

Afferent connections of the adjoining nucleus with the amygdaloid body and dopaminergic mesencephalic structures of the cat brain

By means of the retrograde axonal transport of horseradish peroxidase and luminescent markers the data have been obtained on topical organization of projections of the basal nucleus of the amygdaloid body, of the ventral field of the operculum and of the substantia nigra nuclei to the adjoining nucleus. In the medial and lateral segments of the adjoining nucleus the terminal fields of these structures overlap and have collaterals in the nuclei of the striopallidum. The interaction of limbic and motor informations in the adjoining nucleus is discussed.     

Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles

Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.     

Responses of neurons in the pigeon's optic tectum to visual stimuli

The responses by neurons in various layers of the pigeon's optic tectum to visual stimuli of different sizes moving at various speeds in receptive fields (RF's) were recorded by means of microelectrodes. Analysis of the relationship between the characteristics of the RF's and the location of neurons in the optic tectum showed that with increase in the depth of the layer the structure of the RF's became more complex, their size increased, the effect of peripheral inhibition decreased, and the properties of directional selectivity were displayed more clearly. A wide convergence of signals of different modalities on the efferent neurons of the optic tectum, and their rapid habituation to repeated application of stimuli, were observed.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 3, No. 1, pp. 99–105, January–February, 1971.     

The intrinsic neuronal organisation of the nucleus of the basal optic root in the domestic chicken; a light and electron microscopic study using anterograde tracers and postembedding GABA-immunostaining

The intrinsic neuronal organisation in the nucleus of the basal optic root of chickens was investigated. The divergent connections with various areas and the functional complexity of the nucleus require a complex intrinsic structural arrangement. Therefore, an analysis of Golgi impregnated material, ultrastructure, GABA-immunocytochemistry and biotinylated dextran-amine anterograde tracer analysis of the nucleus was carried out. In the Golgi analysis, a characteristic dendritic ramification pattern of two types of putative projection neurons was observed. These neurons form dendritic nests with their overlapping dendritic terminal sections, that develop synaptic fields with the optic fibre terminals. These synaptic fields were confirmed by electron microscopy. GABA-immunopositive terminals synapse with distinct loci of the dendritic trees of projection neurons; they may therefore play an important role in the inhibitory-modulatory system of the nucleus of the basal optic root. The GABA-immunopositive terminals derive from small and/or elongated local circuit neurons which receive retinal afferents, and from myelinated fibres afferents to the nucleus of unknown origin.     

Encoding of motion in real time by the fly visual system.

Direction-selective cells in the fly visual system that have large receptive fields play a decisive role in encoding the time-dependent optic flow the animal encounters during locomotion. Recent experiments on the computations performed by these cells have highlighted the significance of dendritic integration and have addressed the role of spikes versus graded membrane potential changes in encoding optic flow information. It is becoming increasingly clear that the way optic flow is encoded in real time is constrained both by the computational needs of the animal in visually guided behaviour as well as by the specific properties of the underlying neuronal hardware.     

Physiological and anatomical properties of optical input-fibres to the mushroom body in the bee brain

More than 150 neurones in the nushroom body area of the bee brain were recorded and stained intracellularly with either Lucifer Yellow or Cobalt-Hexamminochloride (III). Among them 12 neurones have been characterized physiologically and anatomically which connect the medulla and the lobula with the mushroom bodies. All neurones responded to stationary or moving light stimuli exclusively. Movement-sensitive neurones were all direction-selective. Excitatory and inhibitory responses occurred in response to moving stripe patterns in the preferred and null directions respectively. Anatomically, the neurones could be clearly distinguished as belonging to three types depending on their input features in the optic lobes: (a) Neurones with small dendritic fields (up to 100 μm) in the lobula; (b) Neurones with large dendritic fields (up to 400 μm) in the lobula; (c) Neurones with small dendritic fields (up to 100 μm) in the medulla. The axons of all three cell types run from the optic lobes on each side to the outer ring tract around the pedunculus-calyx-transition and arborize in the collar region of the ipsilateral calyces. Additional branches invading the basal ring of the calyces had been observed; endings in the lip region were not found. The endings in the calyces often exhibited bleb-like specializations indicating their presynaptic nature. Retinotopic organization of the optic inputs into the calyces could not be proven. The results are compared with the characteristics of multimodal mushroom body output fibres and are discussed in context with the complex information processing and storage functions ascribed to the mushroom bodies.     

Mesenchymal stem cell therapy in retinal and optic nerve diseases: An update of clinical trials

Retinal and optic nerve diseases are degenerative ocular pathologies which lead to irreversible visual loss. Since the advanced therapies availability, cell-based therapies offer a new all-encompassing approach. Advances in the knowledge of neuroprotection, immunomodulation and regenerative properties of mesenchymal stem cells(MSCs) have been obtained by several preclinical studies of various neurodegenerative diseases. It has provided the opportunity to perform the translation of this knowledge to prospective treatment approaches for clinical practice. Since 2008, several first steps projecting new treatment approaches, have been taken regarding the use of cell therapy in patients with neurodegenerative pathologies of optic nerve and retina. Most of the clinical trials using MSCs are in Ⅰ/Ⅱ phase, recruiting patients or ongoing, and they have as main objective the safety assessment of MSCs using various routes of administration. However, it is important to recognize that, there is still a long way to go to reach clinical trials phase Ⅲ-Ⅳ. Hence, it is necessary to continue preclinical and clinical studies to improve this new therapeutic tool. This paper reviews the latest progress of MSCs in human clinical trials for retinal and optic nerve diseases.     

The neurons of the first synaptic region of the optic neuropil of the firefly,Phausis splendidula L. (Coleoptera)

On the basis of Golgi preparations the neuronal elements of the lamina ganglionaris (first synaptic region of the visual system) of the firefly. Phausis splendidula L., are described. Of the set of 8 retinula fibres that originate from each ommatidium of the compound eye, at least 6 terminate in the esternal plexiform layer. At least one, probably two, retinula fibres per ommatidium penetrate this layer to end in the medulla, via the first optic chiasma. Five types (m1-m5) of monopolar cells can be distinguished. Only two of these, m1 and m3 have dendritic fields limited to one column of the lamina mosaic; all other monopolar cells have larger fields of up to 45 mum diameter. m2 and m4 have various field spreads in different strata of the external plexiform layer. m5 has process in only one stratum of the external plexiform layer. Medulla-to-lamina cells with arborisations associated with only a single column of the lamina mosaic were not observed; medulla-to-lamina cells whose fields coincide with the various strata of the external plexiform layer were found, however. The present observations are briefly compared with those made on another beetle, Hoplia farinosa L. Comparisons with other species of insects, and the relationship between structure of the eye and structure of the lamina are also discussed.     

Alterations in the morphology of ganglion cell dendrites in the adult rat retina after optic nerve transection and grafting of peripheral nerve segments

Summary Transected ganglion cell axons from the adult retina are capable of reinnervating their central targets by growing into transplanted peripheral nerve (PN) segments. Injury of the optic nerve causes various metabolic and morphological changes in the retinal ganglion cell (RGC) perikarya and in the dendrites. The present work examined the dendritic trees of those ganglion cells surviving axotomy and of those whose severed axons re-elongated in PN grafts to reach either the superior colliculus (SC), transplanted SC, or transplanted autologous thigh muscle. The elaboration of the dendritic trees was visualized by means of the strongly fluorescent carbocyanine dye DiI, which is taken up by axons and transported to the cell bodies and from there to the dendritic branches. Alternatively, retinofugal axons regrowing through PN grafts were anterogradely filled from the eye cup with rhodamine B-isothiocyanate. The transection of the optic nerve resulted in characteristic changes in the ganglion cell dendrites, particularly in the degeneration of most of the terminal and preterminal dendritic branches. This occurred within the first 1 to 2 weeks following axotomy. The different types of ganglion cells appear to vary in their sensitivity to axotomy, as reflected by a rapid degeneration of certain cell dendrites after severance of the optic nerve. The most vulnerable cells were those with small perikarya and small dendritic fields (type II), whereas larger cells with larger dendritic fields (type I and III) were slower to respond and less dramatically affected. Regrowth of the lesioned axons in peripheral nerve grafts and reconnection of the retina with various tissues did not result in a significant immediate recovery of ganglion cell dendrites, although it did prevent some axotomized cells from further progression toward posttraumatic cell death.     

Simultaneous multicolor detection system of the single-molecular microbial antigen with total internal reflection fluorescence microscopy

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystal QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies . Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.     

Improved sensitivity of firefly luminescent intermediate‐based protein interaction assay using Ser 440 mutant with lower adenylation activity

Protein–protein interaction assays are important in various fields including molecular biology, diagnostics, and drug screening. We recently designed a novel protein–protein interaction assay, the firefly luminescent intermediate‐based protein interaction assay (FlimPIA), that exploited the unique reaction mechanism of firefly luciferase (Fluc). Using two mutant Flucs, each impaired with one of the two half‐reactions, namely adenylation and subsequent oxidative luminescent steps, FlimPIA detects the proximity of the two proteins tethered to the mutant Flucs. Here, we found that introducing a mutation into a residue in the hinge region (S440) of the mutant with lowered adenylation activity (‘Acceptor’ Fluc) further improved the response of FlimPIA by lowering the residual adenylation activity. Mutants with bulkier residues showed greater inhibition, probably due to increased steric hindrance at the adenylation conformation. As a result, the FlimPIA with S440 L acceptor showed the best signal/background ratio for the detection of rapamycin‐induced FKBP12–FRB interactions.     

AlphaLISA技术的发展及应用

AlphaLISA技术是一种基于微珠的化学发光的新型均相检测技术,与传统的ELISA技术相比具有更高的敏感性、精确性、均一性、背景低、广泛的动态检测范围,而且无需洗涤及样本需求量极少等特点.该技术可用于多种组织、细胞来源的生物分子的检测,如细胞因子、抗原抗体的检测、蛋白相互作用及蛋白质与核酸相互作用的检测以及药物分析等研究.随着AlphaLISA技术的发展,该技术已在医学及分子生物学领域得到了广泛的应用.     

The Circadian System of Crustaceans

Crustaceans exhibit a variety of overt circadian rhythms. Observations on intact animals suggest the existence of more than one circadian pacemaker in the nervous system. Ablation experiments so far have been inconclusive in pinpointing the location of putative pacemakers. However, various structures, most notably the optic peduncle, have been shown to sustain circadian rhythmicity in vitro. Retinal sensitivity and neurosecretory activity display circadian rhythms in the isolated optic peduncle, but they are also responsive to synchronizing influences from other regions of the central nervous system, most notably the supra-esophageal ganglion. A model based on a number of circadian pacemakers distributed in the central nervous system best fits the experimental results at present. Coupling of rhythmicity between independent circadian pacemakers is likely to occur, and a neuroendocrine stage of integration has been proposed for several rhythms. Various entraining agents have been identified, and more than one may play a part in the synchronization of a given rhythm.     

Temporal and spatial effects of Sonic hedgehog signaling in chick eye morphogenesis

Proper dorsal--ventral pattern formation of the optic cup is essential for vertebrate eye morphogenesis and retinotectal topographic mapping. Previous studies have suggested that midline tissue-derived Sonic hedgehog (Shh) molecules play critical roles in establishing the bilateral eye fields and in determining the proximal--distal axis of the eye primordium. Here, we have examined the temporal requirements for Shh during the optic vesicle to optic cup transition and after early optic cup formation in chick embryos. Both misexpressing Shh by virus and blocking Shh activity by antibodies resulted in disruption of ventral ocular tissues. Decreasing endogenous Shh signals unexpectedly revealed a sharp morphological boundary subdividing dorsal and ventral portions of the optic cup. In addition, Shh signals differentially influenced expression patterns of genes involved in ocular tissue specification (Pax6, Pax2, and Otx2) and dorsal--ventral patterning (cVax) within the ventral but not dorsal optic cup. Ectopic Shh suppressed expression of Bone Morphogenetic Protein 4 (BMP4) in the dorsal retina, whereas reducing endogenous Sonic hedgehog activity resulted in a ventral expansion of BMP4 territory. These results demonstrate that temporal requirements for Shh signals persist after the formation of the optic cup and suggest that the early vertebrate optic primordium may be subdivided into dorsal and ventral compartments. We propose a model in which ventrally derived Shh signals and dorsally restricted BMP4 signals act antagonistically to regulate the growth and specification of the optic primordium.     

Visual fields of butterfly interneurons

A relatively novel and precise technique was employed for the detailed plotting of the extent and nature of the fields of fibres in the visual pathway of 11 species of butterflies. For these experiments, a moving, flashing spot of light on the face of an oscilloscope was used as a photostimulator. Some 160 fibres were analysed by this technique. In many cases, the response to light adaptation was observed. Unlike many previous reports, units were found to have exceedingly complex fields, not easily described.Two major categories of optic lobe units were detected. These included: (1) fibres with large diffuse fields with many ‘holes’ and channels within their substance, which were markedly altered by light adaptation, and (2) relatively uncommon, smaller visual fields, which were somewhat more uniform in responsiveness.Four categories of visual fields were recorded in the protocerebrum. These included: (1) units with a large, diffuse field with a sharp boundary corresponding to the horizon, (2) binocular fields with much, little, or no overlap, (3) large diffuse fields, not unlike those discovered in the optic lobe, and (4) fibres demonstrating an inhibitory influence and resulting decrease in the size of the visual field as a result of contralateral stimulation.     

Blocking gastrin and CCK-B autocrine loop affects cell proliferation and apoptosis in vitro

Since its initial discovery as Ca²⁺/calmodulin (CaM)-dependent serine/threonine protein phosphatase, calcineurin (CaN) has been extensively studied in many mammalian tissues. CaN has been shown to be involved in various biological and Ca²⁺-dependent signal transduction pathways. Over the last decade, our laboratory has been interested and has carried out numerous experiments on this specific protein phosphatase. While, a lot of research has been performed studying CaN’s involvement in ischemia, the immune system, and various mammalian tissues, not much is known about the potential role of CaN in various eye diseases. This review focuses on the studies that have been carried out in our laboratory on CaN, and specifically CaN’s involvement in the eye. We demonstrated that CaN is localized in various eye tissues (cornea, iris, ciliary body, vitreous body, retina, choroid, sclera, and optic nerve) and that both its protein expression and activity were observed in high amounts in the retina, optic nerve and cornea. Recently, we have cloned and characterized the CaN A and B subunits in the bovine retina. These initial findings suggest that CaN may play a potential role in visual transduction and various ocular diseases, including cancer.     

Database of natural luminescent bacteria

The database of luminescent bacteria stored in the IBSO collection is one of the metasections of BIOLUMBASE. A logical schema of the metasection “Natural luminescent organisms”, classification of entities, and methods of attribute presentation have been developed. The database of luminescent bacteria maintained in the IBSO collection is being widened by findings of the collection staff as well as by information from scientific literature. The expectant contents of the database will be useful for resolving various problems of microbial ecology and biotechnology which deal with luminescent bacteria, luminescent system derived from them, and lux-genes cloned to other organisms. A potential user would be able not only to access cataloged data on strains but also to get information on properties, functions, use, and bibliography and to perform an attribute-match search of a strain.