application-genetically-encoded-fluorescent-nitric-oxide-no-probes

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Medical University of Graz

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: Presumimos que cierre de herida en pacientes traumatizados con trastornos en la cicatrizaci&oacute;n se acelera por la suplementaci&oacute;n de micronutrientes antioxidantes y glutamina.

Tiempo de cierre en pacientes traumatizados con trastornos en la cicatrizaci&oacute;n de la herida se acorta por suplementos que contienen micronutrientes antioxidantes y glutamina: un PRCT.

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: We hypothesize that wound closure in trauma patients with disorders in wound healing is accelerated by supplementation of antioxidant micronutrients and glutamine.

Time to wound closure in trauma patients with disorders in wound healing is shortened by supplements containing antioxidant micronutrients and glutamine: a PRCT.

: Nous &eacute;mettons l&#39;hypoth&egrave;se que la fermeture des plaies de patients victimes de traumatismes avec troubles de la cicatrisation des plaies est acc&eacute;l&eacute;r&eacute;e par la suppl&eacute;mentation en micronutriments antioxydants et de la glutamine.

Temps de blesser la fermeture dans les patients victimes de traumatismes avec troubles de la cicatrisation des plaies est r&eacute;duit de suppl&eacute;ments contenant des micronutriments antioxydants et la glutamine : un PRCT.

: Wir vermuten, dass die Schlie&szlig;ung der Wunde bei Trauma-Patienten mit St&ouml;rungen in der Wundheilung durch Supplementierung von antioxidativen Mikron&auml;hrstoffen und Glutamin beschleunigt wird.

Zeit zum Verschluss bei Trauma-Patienten mit St&ouml;rungen in der Wundheilung Wunde ist durch Nahrungserg&auml;nzungsmittel mit antioxidativen Mikron&auml;hrstoffen und Glutamin verk&uuml;rzt: ein PRCT.

: Ipotizziamo che chiusura di ferita in pazienti con trauma con disturbi nella guarigione delle ferite &egrave; accelerato dalla supplementazione di micronutrienti antiossidanti e glutammina.

Tempo di chiusura della ferita in pazienti con trauma con disturbi nella guarigione delle ferite &egrave; accorciato di integratori alimentari contenenti micronutrienti antiossidanti e glutammina: un PRCT.

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Disorders in wound healing (DWH) are common in trauma patients, the reasons being not completely understood. Inadequate nutritional status may favor DWH, partly by means of oxidative stress. Reliable data, however, are lacking. This study should investigate the status of extracellular micronutrients in patients with DWH within routine setting.

Extracellular micronutrient levels and pro-/antioxidant status in trauma patients with wound healing disorders: results of a cross-sectional study.

Mitochondria contribute to cell signaling by controlling store-operated Ca(2+) entry (SOCE). SOCE is activated by Ca(2+) release from the endoplasmic reticulum (ER), whereupon stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER-plasma-membrane junctions and opens plasma membrane Ca(2+) channels. The mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study, we used an shRNA approach to investigate the direct involvement of mitochondrial Ca(2+) buffering in SOCE. We demonstrate that knockdown of either of two proteins that are essential for mitochondrial Ca(2+) uptake, the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca(2+) buffering or ER Ca(2+) depletion by sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca(2+) uptake. However, MCU-dependent mitochondrial sequestration of Ca(2+) entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus-specific contribution of mitochondrial Ca(2+) uptake to the SOCE machinery, likely through a role in shaping cytosolic Ca(2+) micro-domains.

IP3-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake.

To compare visual outcome and manual handling of additional multifocal sulcus-fixated intraocular lenses (IOLs) of different materials and lens concepts.

Silicone-diffractive versus acrylic-refractive supplementary iols: visual performance and manual handling.

Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.

Generation of Red-Shifted Cameleons for Imaging Ca²⁺ Dynamics of the Endoplasmic Reticulum.

Nitric oxide () is a free radical with a wide range of biological effects, but practically impossible to visualize in single cells. Here we report the development of novel multicoloured fluorescent quenching-based probes by fusing a bacteria-derived -binding domain close to distinct fluorescent protein variants. These genetically encoded probes, referred to as geNOps, provide a selective, specific and real-time read-out of cellular dynamics and, hence, open a new era of bioimaging. The combination of geNOps with a Ca(2+) sensor allowed us to visualize and Ca(2+) signals simultaneously in single endothelial cells. Moreover, targeting of the probes was used to detect signals within mitochondria. The geNOps are useful new tools to further investigate and understand the complex patterns of signalling on the single (sub)cellular level.

Sandra Blass

Institute of Molecular Biology and Biochemistry

Medical University of Graz

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells