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Mackin, Robert D., Ruth A. Frey, Carmina Gutierrez, Ashley A. Farre, Shoji Kawamura, Diana M. Mitchell e Deborah L. Stenkamp. "Endocrine regulation of multichromatic color vision". Proceedings of the National Academy of Sciences 116, n.º 34 (5 de agosto de 2019): 16882–91. http://dx.doi.org/10.1073/pnas.1904783116.
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Vertebrate color vision requires spectrally selective opsin-based pigments, expressed in distinct cone photoreceptor populations. In primates and in fish, spectrally divergent opsin genes may reside in head-to-tail tandem arrays. Mechanisms underlying differential expression from such arrays have not been fully elucidated. Regulation of human red (LWS) vs. green (MWS) opsins is considered a stochastic event, whereby upstream enhancers associate randomly with promoters of the proximal or distal gene, and one of these associations becomes permanent. We demonstrate that, distinct from this stochastic model, the endocrine signal thyroid hormone (TH) regulates differential expression of the orthologous zebrafish lws1/lws2 array, and of the tandemly quadruplicated rh2-1/rh2-2/rh2-3/rh2-4 array. TH treatment caused dramatic, dose-dependent increases in abundance of lws1, the proximal member of the lws array, and reduced lws2. Fluorescent lws reporters permitted direct visualization of individual cones switching expression from lws2 to lws1. Athyroidism increased lws2 and reduced lws1, except within a small ventral domain of lws1 that was likely sustained by retinoic acid signaling. Changes in lws abundance and distribution in athyroid zebrafish were rescued by TH, demonstrating plasticity of cone phenotype in response to this signal. TH manipulations also regulated the rh2 array, with athyroidism reducing abundance of distal members. Interestingly, the opsins encoded by the proximal lws gene and distal rh2 genes are sensitive to longer wavelengths than other members of their respective arrays; therefore, endogenous TH acts upon each opsin array to shift overall spectral sensitivity toward longer wavelengths, underlying coordinated changes in visual system function during development and growth.
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FEI, YIJIAN, e THOMAS E. HUGHES. "Transgenic expression of the jellyfish green fluorescent protein in the cone photoreceptors of the mouse". Visual Neuroscience 18, n.º 4 (julho de 2001): 615–23. http://dx.doi.org/10.1017/s0952523801184117.
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The goal of this study was to determine whether the jellyfish green fluorescent protein (GFP) could be used in transgenic mice to label and purify cone photoreceptors from the living retina. We created a transgene containing the 5′ regulatory sequence of the human red pigment gene (pR6.5 lacZ clone; kindly provided by J. Nathans & Y. Wang), fused to the GFP coding sequence. This transgene was used to generate seven lines of PCR-positive founders. Three of the lines had bright green fluorescent cone photoreceptors. The GFP fills the entire cell. Two mouse lines had only a few (∼10–100) fluorescent cells per retina, and one line (R6.85933) had many thousands. In the latter, double labeling of the cones with RITC-conjugated peanut agglutinin reveals that in the ventral retina a small proportion of the cones express GFP, while in the dorsal retina the majority do. Cells dissociated from the retinae of line R6.85933 continue to fluoresce and can be readily detected and enriched with flow cytometry. The signal provides a log unit of separation between the fluorescent cone soma and the remaining retinal cells. Roughly 3% of the cells are this fluorescent, and it is possible to purify up to 30,000 cells from one mouse. RT-PCR analysis of the mRNA from these isolated cells detects both the middle and short wavelength opsins with little if any contamination from rhodopsin.
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Idzhilova, Olga S., Gulnur R. Smirnova, Lada E. Petrovskaya, Darya A. Kolotova, Mikhail A. Ostrovsky e Alexey Y. Malyshev. "Cationic Channelrhodopsin from the Alga Platymonas subcordiformis as a Promising Optogenetic Tool". Biochemistry (Moscow) 87, n.º 11 (novembro de 2022): 1327–34. http://dx.doi.org/10.1134/s0006297922110116.
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Abstract The progress in optogenetics largely depends on the development of light-activated proteins as new molecular tools. Using cultured hippocampal neurons, we compared the properties of two light-activated cation channels – classical channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) and recently described channelrhodopsin isolated from the alga Platymonas subcordiformis (PsChR2). PsChR2 ensured generation of action potentials by neurons when activated by the pulsed light stimulation with the frequencies up to 40-50 Hz, while the upper limit for CrChR2 was 20-30 Hz. An important advantage of PsChR2 compared to classical channelrhodopsin CrChR2 is the blue shift of its excitation spectrum, which opens the possibility for its application in all-optical electrophysiology experiments that require the separation of the maxima of the spectra of channelrhodopsins used for the stimulation of neurons and the maxima of the excitation spectra of various red fluorescent probes. We compared the response (generation of action potentials) of neurons expressing CrChR2 and PsChR2 to light stimuli at 530 and 550 nm commonly used for the excitation of red fluorescent probes. The 530-nm light was significantly (3.7 times) less efficient in the activation of neurons expressing PsChR2 vs. CrChR2-expressing neurons. The light at 550 nm, even at the maximal used intensity, failed to stimulate neurons expressing either of the studied opsins. This indicates that the PsChR2 channelrhodopsin from the alga P. subcordiformis is a promising optogenetic tool, both in terms of its frequency characteristics and possibility of its application for neuronal stimulation with a short-wavelength (blue, 470 nm) light accompanied by simultaneous recording of various physiological processes using fluorescent probes.
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Athanasiou, Dimitra, Maria Kosmaoglou, Naheed Kanuga, Sergey S. Novoselov, Adrienne W. Paton, James C. Paton, J. Paul Chapple e Michael E. Cheetham. "BiP prevents rod opsin aggregation". Molecular Biology of the Cell 23, n.º 18 (15 de setembro de 2012): 3522–31. http://dx.doi.org/10.1091/mbc.e12-02-0168.
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Mutations in rod opsin—the light-sensitive protein of rod cells—cause retinitis pigmentosa. Many rod opsin mutations lead to protein misfolding, and therefore it is important to understand the role of molecular chaperones in rod opsin biogenesis. We show that BiP (HSPA5) prevents the aggregation of rod opsin. Cleavage of BiP with the subtilase cytotoxin SubAB results in endoplasmic reticulum (ER) retention and ubiquitylation of wild-type (WT) rod opsin (WT–green fluorescent protein [GFP]) at the ER. Fluorescence recovery after photobleaching reveals that WT-GFP is usually mobile in the ER. By contrast, depletion of BiP activity by treatment with SubAB or coexpression of a BiP ATPase mutant, BiP(T37G), decreases WT-GFP mobility to below that of the misfolding P23H mutant of rod opsin (P23H-GFP), which is retained in the ER and can form cytoplasmic ubiquitylated inclusions. SubAB treatment of P23H-GFP–expressing cells decreases the mobility of the mutant protein further and leads to ubiquitylation throughout the ER. Of interest, BiP overexpression increases the mobility of P23H-GFP, suggesting that it can reduce mutant rod opsin aggregation. Therefore inhibition of BiP function results in aggregation of rod opsin in the ER, which suggests that BiP is important for maintaining the solubility of rod opsin in the ER.
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MAUCK, MATTHEW C., KATHERINE MANCUSO, JAMES A. KUCHENBECKER, THOMAS B. CONNOR, WILLIAM W. HAUSWIRTH, JAY NEITZ e MAUREEN NEITZ. "Longitudinal evaluation of expression of virally delivered transgenes in gerbil cone photoreceptors". Visual Neuroscience 25, n.º 3 (maio de 2008): 273–82. http://dx.doi.org/10.1017/s0952523808080577.
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Delivery of foreign opsin genes to cone photoreceptors using recombinant adeno-associated virus (rAAV) is a potential tool for studying the basic mechanisms underlying cone based vision and for treating vision disorders. We used an in vivo retinal imaging system to monitor, over time, expression of virally-delivered genes targeted to cone photoreceptors in the Mongolian gerbil (Meriones unguiculatus). Gerbils have a well-developed photopic visual system, with 11–14% of their photoreceptors being cones. We used replication deficient serotype 5 rAAV to deliver a gene for green fluorescent protein (GFP). In an effort to direct expression of the gene specifically to either S or M cones, the transgene was under the control of either the human X-chromosome opsin gene regulatory elements, i.e., an enhancer termed the locus control region (LCR) and L promoter, or the human S-opsin promoter. Longitudinal fluorescence images reveal that gene expression is first detectable about 14 days post-injection, reaches a peak after about 3 months, and is observed more than a year post-injection if the initial viral concentration is sufficiently high. The regulatory elements are able to direct expression to a subpopulation of cones while excluding expression in rods and non-photoreceptor retinal cells. When the same viral constructs are used to deliver a human long-wavelength opsin gene to gerbil cones, stimulation of the introduced human photopigment with long-wavelength light produces robust cone responses.
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Polans, A. S., L. G. Altman e D. S. Papermaster. "Immunocytochemical binding of anti-opsin N-terminal-specific antibodies to the extracellular surface of rod outer segment plasma membranes. Fixation induces antibody binding." Journal of Histochemistry & Cytochemistry 34, n.º 5 (maio de 1986): 659–64. http://dx.doi.org/10.1177/34.5.2939131.
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We have examined the binding of anti-opsin antibodies to the plasma membrane of frog retinal rod outer segments (ROS) by fluorescence light microscopy and electron microscopy. Polyclonal and monoclonal antibodies specific for the N-terminal domain of opsin were observed to bind to the extracellular surface of ROS plasma membrane of aldehyde-fixed but not of unfixed retinas. This reaction was found regardless of whether purified ROS, rhodopsin, opsin, or an N-terminal peptide of opsin was used as the immunogen. The fixation-induced binding of these antibodies contrasts with the more frequently noted loss of antigenicity upon fixation. Concanavalin A, however, binds to unfixed ROS plasma membranes. Its binding sites in the plasma membrane may be oligosaccharides in the N-terminal region of opsin. These results suggest that the N-terminal domain of opsin is latent in the native membrane and that changes in conformation may account for its detectability in fixed membranes.
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Ullrich, Sybille, Ronnie Gueta e Georg Nagel. "Degradation of channelopsin-2 in the absence of retinal and degradation resistance in certain mutants". Biological Chemistry 394, n.º 2 (1 de fevereiro de 2013): 271–80. http://dx.doi.org/10.1515/hsz-2012-0256.
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Abstract Channelrhodopsin-2 is a light-gated cation channel from the green alga Chlamydomonas reinhardtii. It is functional in animal cells and therefore widely used for light-activated depolarization, especially in neurons. To achieve a fully functional protein, the chromophore all-trans-retinal is needed. It has not been investigated whether or not the apoprotein is stable without its cofactor until now. Here we show that channelopsin-2 (Chop2, protein without bound retinal) is much more prone to degradation than channelrhodopsin-2 (protein with retinal). Constructs of Chop2 fused to yellow fluorescent protein (Chop2::YFP) in the absence and presence of retinal confirm this observation by exhibiting strongly differing fluorescence. We present mutants of Chop2 with highly increased stability in the absence of retinal. Substitution of threonine 159 with aromatic amino acids causes enhanced resistance to degradation in the absence of retinal, which is confirmed by fluorescence intensity, the increase in photocurrents on the addition of retinal to previously expressed protein, and Western blot analysis. Exchanging threonine 159 with cysteine, however, increases photocurrents due to better binding of retinal, without obvious stabilization against degradation of the retinal-free opsin. We also show that the light-activated hyperpolarizing chloride pump halorhodopsin from Natronomonas pharaonis (NpHR) is not prone to retinal-dependent degradation.
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Lu, Xiaocen, Yi Shen e Robert E. Campbell. "Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators". International Journal of Molecular Sciences 21, n.º 18 (7 de setembro de 2020): 6522. http://dx.doi.org/10.3390/ijms21186522.
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Optogenetic (photo-responsive) actuators engineered from photoreceptors are widely used in various applications to study cell biology and tissue physiology. In the toolkit of optogenetic actuators, the key building blocks are genetically encodable light-sensitive proteins. Currently, most optogenetic photosensory modules are engineered from naturally-occurring photoreceptor proteins from bacteria, fungi, and plants. There is a growing demand for novel photosensory domains with improved optical properties and light-induced responses to satisfy the needs of a wider variety of studies in biological sciences. In this review, we focus on progress towards engineering of non-opsin-based photosensory domains, and their representative applications in cell biology and physiology. We summarize current knowledge of engineering of light-sensitive proteins including light-oxygen-voltage-sensing domain (LOV), cryptochrome (CRY2), phytochrome (PhyB and BphP), and fluorescent protein (FP)-based photosensitive domains (Dronpa and PhoCl).
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PERKINS, BRIAN D., PAMELA M. KAINZ, DONALD M. O'MALLEY e JOHN E. DOWLING. "Transgenic expression of a GFP-rhodopsin COOH-terminal fusion protein in zebrafish rod photoreceptors". Visual Neuroscience 19, n.º 3 (maio de 2002): 257–64. http://dx.doi.org/10.1017/s0952523802192030.
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To facilitate the identification and characterization of mutations affecting the retina and photoreceptors in the zebrafish, a transgene expressing green fluorescent protein (GFP) fused to the C-terminal 44 amino acids of Xenopus rhodopsin (Tam et al., 2000) under the control of the 1.3-kb proximal Xenopus opsin promoter was inserted into the zebrafish genome. GFP expression was easily observed in a ventral patch of retinal cells at 4 days postfertilization (dpf). Between 45–50% of the progeny from the F1, F2, and F3 generations expressed the transgene, consistent with a single integration event following microinjection. Immunohistochemical analysis demonstrated that GFP is expressed exclusively in rod photoreceptors and not in the UV, blue, or red/green double cones. Furthermore, GFP is localized to the rod outer segments with little to no fluorescence in the rod inner segments, rod cell bodies, or rod synapse regions, indicating proper targeting and transport of the GFP fusion protein. Application of exogenous retinoic acid (RA) increased the number of GFP-expressing cells throughout the retina, and possibly the level of expressed rhodopsin. When bred to a zebrafish rod degeneration mutant, fewer GFP-expressing rods were seen in living mutants as compared to wild-type siblings. This transgenic line will facilitate the search for recessive and dominant mutations affecting rod photoreceptor development and survival as well as proper rhodopsin expression, targeting, and transport.
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Schafer, Christopher T., Anthony Shumate e David L. Farrens. "Novel fluorescent GPCR biosensor detects retinal equilibrium binding to opsin and active G protein and arrestin signaling conformations". Journal of Biological Chemistry 295, n.º 51 (6 de outubro de 2020): 17486–96. http://dx.doi.org/10.1074/jbc.ra120.014631.
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Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.
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Kobayashi, Mao, Shokoku Shu, Kana Marunaka, Toshiyuki Matsunaga e Akira Ikari. "Weak Ultraviolet B Enhances the Mislocalization of Claudin-1 Mediated by Nitric Oxide and Peroxynitrite Production in Human Keratinocyte-Derived HaCaT Cells". International Journal of Molecular Sciences 21, n.º 19 (27 de setembro de 2020): 7138. http://dx.doi.org/10.3390/ijms21197138.
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A tight junction (TJ) makes a physical barrier in the epidermal cells of skin. Ultraviolet (UV) light may disrupt the TJ barrier, but the mechanism has not been well clarified. Weak UVB (5 mJ/cm2) caused mislocalization of claudin-1 (CLDN1), a component of the TJ strand, and disruption of TJ barrier in human keratinocyte-derived HaCaT cells. The UVB-induced mislocalization of CLDN1 was inhibited by monodansylcadaverine (MDC), a clathrin-dependent endocytosis inhibitor, suggesting that UVB enhances the internalization of CLDN1. Transepidermal electrical resistance and paracellular flux of lucifer yellow, a fluorescent hydrophilic marker, were rescued by MDC. UVB changed neither the total nor phosphorylation levels of CLDN1, but it increased both mono-ubiquitination and tyrosine nitration levels of CLDN1. Fluorescence measurements revealed that UVB increased intracellular free Ca2+, nitric oxide (NO), and peroxynitrite contents, which were inhibited by Opsin2 (OPN2) siRNA, suggesting that OPN2 functions as a UVB sensor. The effects of UVB were inhibited by an antagonist of transient receptor potential type vanilloid 1 (TRPV1) and Ca2+ chelator. Both NO donor and peroxynitrite donor induced the mislocalization of CLDN1 and disruption of TJ barrier, which were rescued by a NO synthase (NOS) inhibitor and a peroxynitrite scavenger. Weak UVB irradiation induced the disruption of TJ barrier mediated by mislocalization of CLDN1 in HaCaT cells. The OPN2/TRPV1/NOS signaling pathway may be a novel target for preventing destruction of the TJ barrier by UVB irradiation.
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Subach, Oksana M., Anna V. Vlaskina, Yuliya K. Agapova, Dmitriy A. Korzhenevskiy, Alena Y. Nikolaeva, Anna M. Varizhuk, Maksim F. Subach et al. "cNTnC and fYTnC2, Genetically Encoded Green Calcium Indicators Based on Troponin C from Fast Animals". International Journal of Molecular Sciences 23, n.º 23 (23 de novembro de 2022): 14614. http://dx.doi.org/10.3390/ijms232314614.
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NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca2+ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively.
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Wang, Lei, Yugo Iwasaki, Kiran K. Andra, Kalpana Pandey, Anant K. Menon e Peter Bütikofer. "Scrambling of natural and fluorescently tagged phosphatidylinositol by reconstituted G protein–coupled receptor and TMEM16 scramblases". Journal of Biological Chemistry 293, n.º 47 (4 de outubro de 2018): 18318–27. http://dx.doi.org/10.1074/jbc.ra118.004213.
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Members of the G protein–coupled receptor and TMEM16 (transmembrane protein 16) protein families are phospholipid scramblases that facilitate rapid, bidirectional movement of phospholipids across a membrane bilayer in an ATP-independent manner. On reconstitution into large unilamellar vesicles, these proteins scramble more than 10,000 lipids/protein/s as measured with co-reconstituted fluorescent nitrobenzoxadiazole (NBD)-labeled phospholipids. Although NBD-labeled phospholipids are ubiquitously used as reporters of scramblase activity, it remains unclear whether the NBD modification influences the quantitative outcomes of the scramblase assay. We now report a refined biochemical approach for measuring the activity of scramblase proteins with radiolabeled natural phosphatidylinositol ([3H]PI) and exploiting the hydrolytic activity of bacterial PI-specific phospholipase C (PI-PLC) to detect the transbilayer movement of PI. PI-PLC rapidly hydrolyzed 50% of [3H]PI in large symmetric, unilamellar liposomes, corresponding to the lipid pool in the outer leaflet. On reconstitution of a crude preparation of yeast endoplasmic reticulum scramblase, purified bovine opsin, or purified Nectria haematococca TMEM16, the extent of [3H]PI hydrolysis increased, indicating that [3H]PI from the inner leaflet had been scrambled to the outer leaflet. Using transphosphatidylation, we synthesized acyl-NBD-PI and used it to compare our PI-PLC–based assay with conventional fluorescence-based methods. Our results revealed quantitative differences between the two assays that we attribute to the specific features of the assays themselves rather than to the nature of the phospholipid. In summary, we have developed an assay that measures scrambling of a chemically unmodified phospholipid by a reconstituted scramblase.
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Lehmann, A. K., A. Halstensen, I. S. Aaberge, J. Holst, T. E. Michaelsen, S. Sørnes, L. M. Wetzler e H. K. Guttormsen. "Human Opsonins Induced during Meningococcal Disease Recognize Outer Membrane Proteins PorA and PorB". Infection and Immunity 67, n.º 5 (1 de maio de 1999): 2552–60. http://dx.doi.org/10.1128/iai.67.5.2552-2560.1999.
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ABSTRACT Human opsonins directed against specific meningococcal outer membrane structures in sera obtained during meningococcal disease were quantified with a recently developed antigen-specific, opsonin-dependent phagocytosis and oxidative burst assay. Outer membrane vesicles (OMVs) and PorA (class 1) and PorB (class 3) proteins purified from mutants of the same strain (44/76; B:15:P1.7.16) were adsorbed to fluorescent beads, opsonized with acute- and convalescent-phase sera from 40 patients with meningococcal disease, and exposed to human leukocytes. Flow cytometric quantitation of the resulting leukocyte phagocytosis products (PPs) demonstrated that disease-induced serum opsonins recognized meningococcal OMV components and both porins. The PPPorA and PPPorB values induced by convalescent-phase sera correlated positively with the PPOMV values. However, the PPPorB values were higher than the PPPorA values in convalescent-phase sera (medians [ranges] of 754 [17 to 1,057] and 107 [4 to 458], respectively) (P < 0.0001) and correlated positively with higher levels of immunoglobulin G against PorB than against PorA as evaluated by enzyme-linked immunosorbent assay. Extensive individual variations in the anti-OMV and antiporin serum opsonic activities between patients infected by serotypes and serosubtypes homologous and heterologous to the target antigens were observed. Simultaneously measured oxidative burst activity correlated with the opsonophagocytosis, an indication that both of these important steps in the in vitro phagocytic elimination of meningococci are initiated by opsonins directed against OMV components, including PorA and PorB. In conclusion, human patient opsonins against meningococcal OMV components and in particular PorB epitopes were identified by this new method, which might facilitate selection of opsonin-inducing meningococcal antigens for inclusion in future vaccines.
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Holman, Holly A., Vy M. Tran, Mausam Kalita, Lynn N. Nguyen, Sailaja Arungundram, Balagurunathan Kuberan e Richard D. Rabbitt. "BODIPY-Conjugated Xyloside Primes Fluorescent Glycosaminoglycans in the Inner Ear of Opsanus tau". Journal of the Association for Research in Otolaryngology 17, n.º 6 (12 de setembro de 2016): 525–40. http://dx.doi.org/10.1007/s10162-016-0585-5.
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de Kleijn, Bertram J., Gijs T. N. Heldens, Jasmijn M. Herruer, Cornelis F. M. Sier, Cesare Piazza, Remco de Bree, Orlando Guntinas-Lichius et al. "Intraoperative Imaging Techniques to Improve Surgical Resection Margins of Oropharyngeal Squamous Cell Cancer: A Comprehensive Review of Current Literature". Cancers 15, n.º 3 (31 de janeiro de 2023): 896. http://dx.doi.org/10.3390/cancers15030896.
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Inadequate resection margins in head and neck squamous cell carcinoma surgery necessitate adjuvant therapies such as re-resection and radiotherapy with or without chemotherapy and imply increasing morbidity and worse prognosis. On the other hand, taking larger margins by extending the resection also leads to avoidable increased morbidity. Oropharyngeal squamous cell carcinomas (OPSCCs) are often difficult to access; resections are limited by anatomy and functionality and thus carry an increased risk for close or positive margins. Therefore, there is a need to improve intraoperative assessment of resection margins. Several intraoperative techniques are available, but these often lead to prolonged operative time and are only suitable for a subgroup of patients. In recent years, new diagnostic tools have been the subject of investigation. This study reviews the available literature on intraoperative techniques to improve resection margins for OPSCCs. A literature search was performed in Embase, PubMed, and Cochrane. Narrow band imaging (NBI), high-resolution microendoscopic imaging, confocal laser endomicroscopy, frozen section analysis (FSA), ultrasound (US), computed tomography scan (CT), (auto) fluorescence imaging (FI), and augmented reality (AR) have all been used for OPSCC. NBI, FSA, and US are most commonly used and increase the rate of negative margins. Other techniques will become available in the future, of which fluorescence imaging has high potential for use with OPSCC.
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Lehmann, A. K., A. R. Gorringe, K. M. Reddin, K. West, I. Smith e A. Halstensen. "Human Opsonins Induced during Meningococcal Disease Recognize Transferrin Binding Protein Complexes". Infection and Immunity 67, n.º 12 (1 de dezembro de 1999): 6526–32. http://dx.doi.org/10.1128/iai.67.12.6526-6532.1999.
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ABSTRACT Patient serum opsonins against transferrin binding protein A+B (TbpA+B) complexes from two Neisseria meningitidis strains (K454 and B16B6, with 85- and 68-kDa TbpB, respectively) were quantified by a functional phagocytosis and oxidative burst assay. TbpA+B complexes adsorbed to fluorescent beads were opsonized with individual acute and convalescent sera from 40 patients infected by a variety of meningococcal strains. Flow cytometric quantitation of leukocyte phagocytosis products (PP) demonstrated that disease-induced serum opsonins recognized TbpA+B, and the highest anti-TbpA+B serum opsonic activities were found between admission to hospital and 6 weeks later. The PP values obtained with TbpA+B from strain B16B6 (PPB16B6) were higher than those obtained with TbpA+B from strain K454 (PPK454), with both acute and convalescent sera (P < 0.0001), and correlated positively with higher immunoglobulin G enzyme-linked immunosorbent assay titers against TbpA+B from strain B16B6 than from strain K454 (P < 0.001). In spite of considerable variations between individuals, significant correlations were found between the PPB16B6 and PPK454 values, and the PP values did not depend on the variability of the TbpB proteins of the disease-causing strains. Simultaneously measured oxidative burst activity correlated closely with the PP values. We conclude that highly cross-reactive anti-TbpA+B serum opsonins are produced during meningococcal disease. The anti-TbpA+B opsonic activities were not affected by the variability of the TbpB proteins of the disease-causing strains, which further adds to the evidence for the vaccine potential of meningococcal TbpA+B complexes.
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Tam, Beatrice M., Orson L. Moritz, Lawrence B. Hurd e David S. Papermaster. "Identification of an Outer Segment Targeting Signal in the Cooh Terminus of Rhodopsin Using Transgenic Xenopus laevis". Journal of Cell Biology 151, n.º 7 (25 de dezembro de 2000): 1369–80. http://dx.doi.org/10.1083/jcb.151.7.1369.
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Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and α adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.
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Kennedy, Breandán N., Thomas S. Vihtelic, Lisa Checkley, Kevin T. Vaughan e David R. Hyde. "Isolation of a Zebrafish Rod Opsin Promoter to Generate a Transgenic Zebrafish Line Expressing Enhanced Green Fluorescent Protein in Rod Photoreceptors". Journal of Biological Chemistry 276, n.º 17 (18 de janeiro de 2001): 14037–43. http://dx.doi.org/10.1074/jbc.m010490200.
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Comar, William D., Sarah M. Schubert, Beata Jastrzebska, Krzysztof Palczewski e Adam W. Smith. "Time-Resolved Fluorescence Spectroscopy Measures Clustering and Mobility of a G Protein-Coupled Receptor Opsin in Live Cell Membranes". Journal of the American Chemical Society 136, n.º 23 (2 de junho de 2014): 8342–49. http://dx.doi.org/10.1021/ja501948w.
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Dewitt, Sharon, e Maurice B. Hallett. "Cytosolic free Ca2+ changes and calpain activation are required for β integrin–accelerated phagocytosis by human neutrophils". Journal of Cell Biology 159, n.º 1 (14 de outubro de 2002): 181–89. http://dx.doi.org/10.1083/jcb.200206089.
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Phagocytosis of microbes coated with opsonins such as the complement component C3bi is the key activity of neutrophils. However, the mechanism by which opsonins enhance the rate of phagocytosis by these cells is unknown and has been difficult to study, partly because of the problem of observing and quantifying the events associated with phagocytosis. In this study, C3bi-opsonized particles were presented to neutrophils with a micromanipulator, so that the events of binding, pseudopod cup formation, engulfment, and completion of phagocytosis were clearly defined and distinguished from those involved with chemotaxis. Using this approach in combination with simultaneous phase contrast and Ca2+ imaging, the temporal relationship between changes in cytosolic free Ca2+ concentration and phagocytosis were correlated. Here we show that whereas small, localized Ca2+ changes occur at the site of particle attachment and cup formation as a result of store release, rapid engulfment of the particle required a global change in cytosolic free Ca2+ which resulted from Ca2+ influx. This latter rise in cytosolic free Ca2+ concentration also liberated a fraction of β2 integrin receptors which were initially immobile on the neutrophil surface, as demonstrable by both fluorescence recovery after laser bleaching and by visualization of localized β2 integrin labelling. Inhibitors of calpain activation prevented both the Ca2+-induced liberation of β2 integrin and the rapid stage of phagocytosis, despite the persistence of the global Ca2+ signal. Therefore, we propose that Ca2+ activation of calpain causes β2 integrin liberation, and that this signal plays a key role in the acceleration of β2 integrin–mediated phagocytosis.
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de Busserolles, Fanny, Nathan S. Hart, David M. Hunt, Wayne I. Davies, N. Justin Marshall, Michael W. Clarke, Dorothee Hahne e Shaun P. Collin. "Spectral Tuning in the Eyes of Deep-Sea Lanternfishes (Myctophidae): A Novel Sexually Dimorphic Intra-Ocular Filter". Brain, Behavior and Evolution 85, n.º 2 (2015): 77–93. http://dx.doi.org/10.1159/000371652.
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Deep-sea fishes possess several adaptations to facilitate vision where light detection is pushed to its limit. Lanternfishes (Myctophidae), one of the world's most abundant groups of mesopelagic fishes, possess a novel and unique visual specialisation, a sexually dimorphic photostable yellow pigmentation, constituting the first record of a visual sexual dimorphism in any non-primate vertebrate. The topographic distribution of the yellow pigmentation across the retina is species specific, varying in location, shape and size. Spectrophotometric analyses reveal that this new retinal specialisation differs between species in terms of composition and acts as a filter, absorbing maximally between 356 and 443 nm. Microspectrophotometry and molecular analyses indicate that the species containing this pigmentation also possess at least 2 spectrally distinct rod visual pigments as a result of a duplication of the Rh1 opsin gene. After modelling the effect of the yellow pigmentation on photoreceptor spectral sensitivity, we suggest that this unique specialisation acts as a filter to enhance contrast, thereby improving the detection of bioluminescent emissions and possibly fluorescence in the extreme environment of the deep sea. The fact that this yellow pigmentation is species specific, sexually dimorphic and isolated within specific parts of the retina indicates an evolutionary pressure to visualise prey/predators/mates in a particular part of each species' visual field.
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BICKELMANN, CONSTANZE, JAMES M. MORROW, JOHANNES MÜLLER e BELINDA S. W. CHANG. "Functional characterization of the rod visual pigment of the echidna (Tachyglossus aculeatus), a basal mammal". Visual Neuroscience 29, n.º 4-5 (9 de julho de 2012): 211–17. http://dx.doi.org/10.1017/s0952523812000223.
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AbstractMonotremes are the most basal egg-laying mammals comprised of two extant genera, which are largely nocturnal. Visual pigments, the first step in the sensory transduction cascade in photoreceptors of the eye, have been examined in a variety of vertebrates, but little work has been done to study the rhodopsin of monotremes. We isolated the rhodopsin gene of the nocturnal short-beaked echidna (Tachyglossus aculeatus) and expressed and functionally characterized the protein in vitro. Three mutants were also expressed and characterized: N83D, an important site for spectral tuning and metarhodopsin kinetics, and two sites with amino acids unique to the echidna (T158A and F169A). The λmax of echidna rhodopsin (497.9 ± 1.1 nm) did not vary significantly in either T158A (498.0 ± 1.3 nm) or F169A (499.4 ± 0.1 nm) but was redshifted in N83D (503.8 ± 1.5 nm). Unlike other mammalian rhodopsins, echidna rhodopsin did react when exposed to hydroxylamine, although not as fast as cone opsins. The retinal release rate of light-activated echidna rhodopsin, as measured by fluorescence spectroscopy, had a half-life of 9.5 ± 2.6 min−1, which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 ± 0.1 min−1, even shorter than wild type. Our results show that with respect to hydroxylamine sensitivity and retinal release, the wild-type echidna rhodopsin displays major differences to all previously characterized mammalian rhodopsins and appears more similar to other nonmammalian vertebrate rhodopsins such as chicken and anole. However, our N83D mutagenesis results suggest that this site may mediate adaptation in the echidna to dim light environments, possibly via increased stability of light-activated intermediates. This study is the first characterization of a rhodopsin from a most basal mammal and indicates that there might be more functional variation in mammalian rhodopsins than previously assumed.
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Andreasen, Claire B., James R. Andreasen, Anita E. Sonn e Julie A. Oughton. "Comparison of the Effect of Different Opsonins on the Phagocytosis of Fluorescein-Labeled Staphylococcal Bacteria by Chicken Heterophils". Avian Diseases 40, n.º 4 (outubro de 1996): 778. http://dx.doi.org/10.2307/1592297.
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Grabner, R., e W. Meerbach. "Phagocytosis of surfactant by alveolar macrophages in vitro". American Journal of Physiology-Lung Cellular and Molecular Physiology 261, n.º 6 (1 de dezembro de 1991): L472—L477. http://dx.doi.org/10.1152/ajplung.1991.261.6.l472.
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Because of their localization and function as phagocytes, alveolar macrophages could take part in the catabolism of surfactant, including surfactants used for treatment. Conditions of the ingestion of the surfactant preparation AWD 56-02 by alveolar macrophages in vitro are described in this paper. The surfactant was labeled with rhodaminyl phosphatidylethanolamine and incubated with alveolar macrophages lavaged from rat lungs. Membrane binding and phagocytosis were evaluated by fluorescence microscopy and quantified fluorimetrically after extraction of the dye. The surfactant was phagocytosed only in the presence of rat serum. The opsonins in the serum are related to complement and Fc receptors as demonstrated by the heat lability of the factor and the inhibition by aggregated gamma-globulins. Furthermore, the phagocytosis depends on calcium and on a factor in the surfactant preparation. Experiments with inhibitors and competition with unlabeled surfactant show that the phagocytosis is a specific and energy-dependent process. Catabolism by alveolar macrophages might be an important step in the metabolism of surfactant, especially when administered in pathological conditions characterized by the presence of serum in the airspaces.
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Ndode-Ekane, Xavier Ekolle, Maria del Mar Puigferrat Pérez, Rossella Di Sapia, Niina Lapinlampi e Asla Pitkänen. "Reorganization of Thalamic Inputs to Lesioned Cortex Following Experimental Traumatic Brain Injury". International Journal of Molecular Sciences 22, n.º 12 (13 de junho de 2021): 6329. http://dx.doi.org/10.3390/ijms22126329.
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Traumatic brain injury (TBI) disrupts thalamic and cortical integrity. The effect of post-injury reorganization and plasticity in thalamocortical pathways on the functional outcome remains unclear. We evaluated whether TBI causes structural changes in the thalamocortical axonal projection terminals in the primary somatosensory cortex (S1) that lead to hyperexcitability. TBI was induced in adult male Sprague Dawley rats with lateral fluid-percussion injury. A virus carrying the fluorescent-tagged opsin channel rhodopsin 2 transgene was injected into the ventroposterior thalamus. We then traced the thalamocortical pathways and analyzed the reorganization of their axonal terminals in S1. Next, we optogenetically stimulated the thalamocortical relays from the ventral posterior lateral and medial nuclei to assess the post-TBI functionality of the pathway. Immunohistochemical analysis revealed that TBI did not alter the spatial distribution or lamina-specific targeting of projection terminals in S1. TBI reduced the axon terminal density in the motor cortex by 44% and in S1 by 30%. A nematic tensor-based analysis revealed that in control rats, the axon terminals in layer V were orientated perpendicular to the pial surface (60.3°). In TBI rats their orientation was more parallel to the pial surface (5.43°, difference between the groups p < 0.05). Moreover, the level of anisotropy of the axon terminals was high in controls (0.063) compared with TBI rats (0.045, p < 0.05). Optical stimulation of the sensory thalamus increased alpha activity in electroencephalography by 312% in controls (p > 0.05) and 237% (p > 0.05) in TBI rats compared with the baseline. However, only TBI rats showed increased beta activity (33%) with harmonics at 5 Hz. Our findings indicate that TBI induces reorganization of thalamocortical axonal terminals in the perilesional cortex, which alters responses to thalamic stimulation.
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Al-Busaidi, Hamed, Md Karim, Syafiq Abidin, Kyi Tha e Ezharul Chowdhury. "Magnesium Fluoride Forms Unique Protein Corona for Efficient Delivery of Doxorubicin into Breast Cancer Cells". Toxics 7, n.º 1 (22 de fevereiro de 2019): 10. http://dx.doi.org/10.3390/toxics7010010.
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Background: The efficacy of chemotherapy is undermined by adverse side effects and chemoresistance of target tissues. Developing a drug delivery system can reduce off-target side effects and increase the efficacy of drugs by increasing their accumulation in target tissues. Inorganic salts have several advantages over other drug delivery vectors in that they are non-carcinogenic and less immunogenic than viral vectors and have a higher loading capacity and better controlled release than lipid and polymer vectors. Methods: MgF2 crystals were fabricated by mixing 20 mM MgCl2 and 10 mM NaF and incubating for 30 min at 37 °C. The crystals were characterized by absorbance, dynamic light scattering, microscopic observance, pH sensitivity test, SEM, EDX and FTIR. The binding efficacy to doxorubicin was assessed by measuring fluorescence intensity. pH-dependent doxorubicin release profile was used to assess the controlled release capability of the particle-drug complex. Cellular uptake was assessed by fluorescence microscopy. Cytotoxicity of the particles and the drug-particle complex were assessed using MTT assay to measure cell viability of MCF-7 cells. Results and Discussion: Particle size on average was estimated to be <200 nm. The crystals were cubic in shape. The particles were pH-sensitive and capable of releasing doxorubicin in increasing acidic conditions. MgF2 nanocrystals were safe in lower concentrations, and when bound to doxorubicin, enhanced its uptake. The protein corona formed around MgF2 nanoparticles lacks typical opsonins but contains some dysopsonins. Conclusion: A drug delivery vector in the form of MgF2 nanocrystals has been developed to transport doxorubicin into breast cancer cells. It is pH-sensitive (allowing for controlled release), size-modifiable, simple and cheap to produce.
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Schott, A., J. Baik, S. Chung e F. Weber. "0071 A Medullary Circuit Controlling REM Sleep". Sleep 43, Supplement_1 (abril de 2020): A29. http://dx.doi.org/10.1093/sleep/zsaa056.069.
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Abstract Introduction Rapid eye movement (REM) sleep is a distinct brain state known for its association with vivid dreaming in humans, though it is also crucial for other mental processes such as memory consolidation and emotion regulation. REM sleep is punctuated by phasic neurophysiological events known as pontine (P)-waves, which are thought to contribute to the cognitive functions of REM sleep. However, little is known about the neural circuits regulating these P-waves, or those responsible for initiating REM sleep itself. Here, we show that a yet unstudied population of medullary neurons expressing corticotropin-releasing-hormone (CRH) are important for controlling both the induction of REM sleep and its phasic events. Methods To measure the endogenous activity of CRH+ neurons in the dorsomedial medulla (dmM), we injected the calcium indicator GCaMP6 in the dmM of CRH-Cre mice. To optogenetically manipulate dmM CRH+ neuron activity, we delivered either an excitatory (ChR2) or inhibitory (iC++) opsin to the dmM of CRH-Cre mice. To record P-waves, we implanted microelectrodes to record local field potentials in the subcoeruleus region of the pons. Results Fiber photometry recordings showed that dmM CRH+ neurons are selectively active during REM sleep, and optogenetic stimulation and inhibition of this population is sufficient to promote and reduce REM sleep, respectively. Additionally, dmM CRH+ neuron activity is correlated with P-waves in the pons, and optogenetic activation of dmM CRH+ cells reliably triggers P-waves during REM sleep. Finally, histological examination of fluorescently labeled dmM CRH+ axons revealed strong projections to several pontine areas involved in P-wave generation as well as modulation of the theta rhythm during REM sleep. Conclusion Our results suggest that dmM CRH+ neurons are involved in controlling REM sleep initiation as well as phasic events within REM sleep. These neurons thus constitute an important component of the brainstem circuitry regulating REM sleep. Support National Institutes of Health (R01 HL149133)
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Perry, S. F., e P. J. Walsh. "Metabolism of isolated fish gill cells: contribution of epithelial chloride cells". Journal of Experimental Biology 144, n.º 1 (1 de julho de 1989): 507–20. http://dx.doi.org/10.1242/jeb.144.1.507.
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Gill cell suspensions from freshwater (FW)- and seawater (SW)-adapted teleosts were obtained by density gradient centrifugation. The proportion of chloride cells (CCs) in the mixed cell suspensions was estimated using the fluorescent mitochondrial stain, DASPMEI, and ranged from less than 1% (FW-adapted tilapia) to approximately 13% (SW-adapted toadfish). The gill cells displayed relatively high viability based on Trypan Blue exclusion (greater than 75%), lactate dehydrogenase leakage (less than 6.5% h-1), oxygen consumption rates (5–15 mumol g-1 cell wet mass h-1) and ATP levels (1–3 mumol g-1 cell wet mass). There were no obvious differences between the viability of CCs and the other cell types present. An initial comparison of gill oxidative metabolism in SW-adapted tilapia (Oreochromis mossambicus) and toadfish (Opsanus beta) demonstrated that both species oxidized glucose and lactate at substantially greater rates than alanine or oleate. Metabolic rates were significantly higher in toadfish cell suspensions. Kinetic experiments revealed that toadfish gill cells displayed lower values of Km and higher values of Vm for both lactate and glucose, in comparison to tilapia. The elevated metabolism in toadfish gill cells was correlated with increased activities of the oxidative enzyme citrate synthase and Na+/K+-ATPase. The toadfish cell suspensions had a greater proportion of CCs and it is likely that the difference in CC numbers between the two species is the basis for the observed differences in enzyme activities and rates of oxidative metabolism. This idea is supported by the highly significant correlation between Na+/K+-ATPase activity (or CC numbers) and rates of lactate oxidation in gill cell suspensions from FW- and SW-adapted tilapia and toadfish, as well as SW-adapted tilapia chronically treated with cortisol to elevate CC numbers. Although it has been assumed widely that the high metabolic rate of gill tissue reflects, in part, the oxidative demands of the chloride cell, the results of this study provide the first experimental, albeit indirect, evidence for differential rates of metabolism in the various cell types that comprise the gill.
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Islam, Rowshan Ara, Hamed Al-Busaidi, Rahela Zaman, Syafiq Asnawi Zainal Abidin, Iekhsan Othman e Ezharul Hoque Chowdhury. "Carbonate Apatite and Hydroxyapatite Formulated with Minimal Ingredients to Deliver SiRNA into Breast Cancer Cells In Vitro and In Vivo". Journal of Functional Biomaterials 11, n.º 3 (10 de setembro de 2020): 63. http://dx.doi.org/10.3390/jfb11030063.
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Introduction: Cancer is one of the top-ranked noncommunicable diseases causing deaths to nine million people and affecting almost double worldwide in 2018. Tremendous advancement in surgery, chemotherapy, radiation and targeted immunotherapy have improved the rate of cure and disease-free survival. As genetic mutations vary in different cancers, potential of customized treatment to silence the problem gene/s at the translational level is being explored too. Yet delivering therapeutics at the required dosage only to the affected cells without affecting the healthy ones, is a big hurdle to be overcome. Scientists worldwide have been working to invent a smart drug delivery system for targeted delivery of therapeutics to tumor tissues only. As part of such an effort, few organic nanocarriers went to clinical trials, while inorganic nanoparticles (NPs) are still in development stage despite their many customizable properties. Carbonate apatite (CA), a pH sensitive nanocarrier has emerged as an efficient delivery system for drugs, plasmids and siRNAs in preclinical models of breast and colon cancers. Like hydroxyapatite (HA) which serves as a classical tool for delivery of genetic materials such as siRNA and plasmid, CA is an apatite-based synthetic carrier. We developed simplified methods of formulating CA-in-DMEM and a DMEM-mimicking buffer and HA in a HEPES-buffered solution and characterized them in terms of size, stability, protein corona (PC) composition, cytotoxicity, siRNA delivery efficiency in breast cancer cells and siRNA biodistribution profile in a mouse model of breast cancer. Methods: Particle growth was analyzed via spectrophotometry and light microscopy, size was measured via dynamic light scattering and scanning electron microscopy and confirmation of functional groups in apatite structures was made by FT-IR. siRNA-binding was analyzed via spectrophotometry. Stability of the formulation solutions/buffers was tested over various time points and at different temperatures to determine their compatibility in the context of practical usage. Cellular uptake was studied via fluorescence microscopy. MTT assay was performed to measure the cytotoxicity of the NPs. Liquid chromatography—mass spectrometry was carried out to analyze the PC formed around all three different NPs in serum-containing media. To explore biodistribution of all the formulations, fluorescence-labeled siRNA-loaded NPs were administered intravenously prior to analysis of fluorescence intensity in the collected organs and tumors of the treated mice. Results: The size of NPs in 10% serum-containing media was dramatically different where CA-in-DMB and HA were much larger than CA-in-DMEM. Effect of media was notable on the PC composition of all three NPs. All three NPs bound albumin and some common protease inhibitors involved in bone metabolism due to their compositional similarity to our bone materials. Moreover, CA also bound heme-binding proteins and opsonins. Unlike CA, HA bound different kinds of keratins. Difference in PC constitution was likely to influence accumulation of NPs in various organs including those of reticuloendothelial system, such as liver and spleen and the tumor. We found 10 times more tumor accumulation of CA-in-DMB than CA-in-DMEM, which could be due to more stable siRNA-binding and distinct PC composition of the former. Conclusion: As a nanocarrier CA is more efficient than HA for siRNA delivery to the tumor. CA prepared in a buffer containing only the mere constituents was potentially more efficient than classical CA prepared in DMEM, owing to the exclusion of interference attributed by the inorganic ions and organic molecules present in DMEM.
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Sun, Haimin, Xinlei Chen, Jiang Zhu, Zhu Chen e Saijuan Chen. "Palladin Regulates Receptor Clustering and Actin Dynamics in Phagocytosis". Blood 128, n.º 22 (2 de dezembro de 2016): 2505. http://dx.doi.org/10.1182/blood.v128.22.2505.2505.
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Abstract BACKGROUND: Palladin is an actin microfilament associated protein, which together with myotilin and myopalladin form a novel cytoskeletal IgC2 domain protein family. However, little is known about the function of Palladin in myeloid cells. Here, we focus on the function of Palladin in phagocytosis. METHODS: We used ATRA induced differentiated NB4 cells as neutrophil-like cells, and lenti-viruses were used to build cell lines with palladin or ocrl knockdown and VAMP3-mcherry overexpression. Flow cytometry and immunofluorescence were used to detect the phagocytic ability under conditional opsonins, and the role of palladin knockdown on phagocytic events and F-actin dynamics were observed. CD32 antibodies followed with fluorescent secondary antibody were used as immune complex to stimulate FcγR clustering. We performed the mass spectrometry analysis on the immunoprecipitation (IP) lysate of differentiated NB4 cells, and the protein-protein interactions were confirmed by co-IP experiments; the colocalization and recruitment of different proteins or moleculars were observed under microscopy. RESULTS: Palladin was up-regulated during ATRA induced differentiation of several AML cell lines, as well as primary mouse bone marrow cells, and its upregulation correlated with increased phagocytic ability. Palladin defective cells showed impaired serum-mediated, IgG- or complement-mediated phagocytosis. The binding ability was measured at 4℃. After 1 hour incubation, ~17% of control cells bound with beads, whereas only ~7% palladin knockdown cells bound with beads, which suggests that Palladin regulated the particle binding. Using serum-opsonized zymosan-FITC as phagocytic targets, we found early phagosome formation, including the pseudopod extension and phagosome closure, was impaired in palladin knockdown cells. However, no significant effect was observed on the recruitment of VAMP3-mcherry, EEA1, Rab7 and LAMP1, so Palladin may not affect the focal exocytosis and phagosome maturation. The binding defect in Palladin-deficient cells was not attributable to difference in the cell surface expression of Fcγ receptor (FcγR). However, the FcγR clustering was much lower in Palladin-deficient cells. We explored how Palladin influenced the FcγR clustering, and our results showed that Palladin could regulate actin cytoskeleton dynamics and c-Src kinase activation, which resulted in the FcγR clustering. We also found that in palladin knockdown cells the actin remodeling was detained at both pseudopod extension stage and actin deploymerization stage. The Rac1 were accumulated in higher amount in the blocked cups formed in palladin KD cells, while no significant difference in Cdc42 recruitment. The recruitment intensity of Apr3 was higher in control cells than palladin KD cells. These results suggested that Palladin participated in the actin dynamics during phagocytic cup formation. We found OCRL is a new Palladin-interacted protein. Palladin interacted with OCRL's 5PPase domain through its third IgC2 domain. Palladin depletion caused a decrease of ~30% OCRL recruitment, retention of PI(4,5)P2, as well as more F-actin at phagosome. These observation suggested that Palladin regulated the recruitment of OCRL at sites of phagosome, and might play an essential role in regulating the hydrolysis of PI(4,5)P2, actin depolymerization and the completion of phagosome closure. CONCLUSIONS: We identify the role of Palladin in phagocytic receptor clustering and Palladin as an early coordinator in actin dynamics during phagosome formation in myeloid cells. Disclosures No relevant conflicts of interest to declare.
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Cogle, Christopher R., Guillermo Garcia-Manero, David L. Grinblatt, Rami S. Komrokji, Michael R. Savona, Bart L. Scott, Mikkael A. Sekeres et al. "Factors Associated with Early Therapy Initiation in Patients (pts) with Myelodysplastic Syndromes (MDS) in the Connect® MDS/AML Disease Registry". Blood 132, Supplement 1 (29 de novembro de 2018): 4731. http://dx.doi.org/10.1182/blood-2018-99-111145.
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Abstract Introduction: In prior studies, only 13-22% of lower-risk (LR) MDS pts and 11-69% of higher-risk (HR) MDS pts received potentially disease-modifying therapy despite the proven benefits of reduced blood transfusions and prolonged survival. Pt age, frailty, and comorbidities are commonly assumed to influence therapy choice; however, this has not been proven and other factors may govern treatment (tx) decisions. To identify factors associated with receiving first-line tx for MDS, health services data from the Connect® MDS/AML Registry, which includes MDS pts treated in US academic, community, and government-based centers, were analyzed. Methods: The Connect MDS/AML Registry (NCT01688011) is an ongoing, prospective observational cohort study of pts with newly diagnosed acute myeloid leukemia (AML) (aged ≥ 55 years) or MDS (aged ≥ 18 years). Local AML and MDS diagnoses are confirmed by an independent central review of all available diagnostic reports. Baseline demographics, disease characteristics, laboratory parameters, and ZIP-code-level per capita income were collected at enrollment on MDS pts from December 2013 to March 2018. Analyses were performed separately for LR-MDS pts, defined as having Low- or Intermediate (Int)-1-risk MDS according to the International Prognostic Scoring System (IPSS), and HR-MDS pts, defined as having Int-2- or High-risk MDS. Uni- and multivariable logistic regression analyses were used to identify factors associated with early use of first-line tx in MDS pts, defined as chemotherapy or biotherapy initiated ≤ 45 days after diagnosis. Univariable testing was performed for each potential predictor; those with an association of P < 0.15 were included in multivariable analysis. The final multivariable model was derived using a score-based selection method. Pts receiving first-line tx (regardless of intensity) were compared with a combined group of pts receiving best supportive care (BSC) or no tx. Results: As of March 8, 2018, data from 536 MDS pts (232 HR-MDS and 304 LR-MDS) from 130 institutions (20 academic and 110 community/government) were available for analysis. In the LR-MDS group, median age was 76 years (range 28-95), with 65% male, and 89% white; 19% had private insurance, and the average median ZIP-code-level per capita income was $28,000 ($10,000-$92,000). In the HR-MDS group, median age was 73 years (range 19-94), with 67% male and 86% white; 23% had private insurance, and the average median ZIP-code-level per capita income was $26,000 (range $14,000-$83,000). In LR-MDS pts, univariable predictors of early first-line therapy initiation included baseline transfusion dependency (defined as ≥ 1 transfusion episode in the 8 weeks prior to diagnosis), comorbidities (higher Adult Comorbidity Evaluation 27 [ACE-27] score), having fluorescence in situ hybridization (FISH) or molecular analyses performed, Int-1 IPSS risk score, primary vs secondary MDS, and higher bone marrow (BM) blast percentage. Multivariable logistic regression analysis identified transfusion dependency (P = 0.001), Int-1 IPSS risk score (P = 0.026), BM blast percentage ≥ 5% (P = 0.002), and having both FISH and molecular genetic testing performed at diagnosis (P = 0.022) as factors significantly associated with early initiation of first-line tx (Table 1). Univariable analysis of HR-MDS pts showed insurance status, comorbidities (ACE-27 score ≤ 2), High IPSS risk score, primary vs secondary MDS, having flow cytometry analysis performed, lower baseline frailty, and BM blast percentage as univariable correlates associated with early tx. Multivariable logistic regression analysis identified private insurance coverage (P = 0.031) and BM blast percentage ≥ 10% (P = 0.021) as factors significantly associated with early initiation of first-line tx (Table 2). Conclusions: Early first-line tx in MDS pts was significantly associated with disease severity as indicated by transfusion dependency, higher IPSS risk score, and higher BM blast percentage. However, counter to common assumptions, age, frailty, and comorbidities were not associated with receiving early tx in MDS. Access to care may be an important factor in the tx of MDS pts, as having private health insurance and undergoing genetic testing were also significantly associated with receiving early tx. Additional pt-centered research with prescribing MDS physicians as stakeholders is needed to verify and extend these findings. Disclosures Cogle: Celgene: Other: Steering Committee Member of Connect MDS/AML Registry. Grinblatt:Celgene: Membership on an entity's Board of Directors or advisory committees; Alexion: Speakers Bureau; AbbVie: Consultancy. Komrokji:Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding. Savona:Boehringer Ingelheim: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding. Scott:Celgene: Consultancy, Honoraria, Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Louis:Cellmedica: Patents & Royalties; Celgene: Employment. Flick:Celgene: Employment. Nifenecker:Celgene: Employment. Kiselev:Celgene: Employment, Equity Ownership. Swern:Celgene: Employment, Equity Ownership. Steensma:Takeda: Consultancy; Syros: Research Funding; Otsuka: Membership on an entity's Board of Directors or advisory committees; Onconova: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Kura: Research Funding; Janssen: Consultancy, Research Funding; H3 Biosciences: Research Funding; Celgene: Research Funding; Amphivena: Membership on an entity's Board of Directors or advisory committees; Acceleron: Consultancy.
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Stroh, A., e I. Diester. "Optogenetics: a new method for the causal analysis of neuronal networks in vivo". e-Neuroforum 18, n.º 4 (1 de janeiro de 2012). http://dx.doi.org/10.1007/s13295-012-0035-8.
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AbstractThe causal analysis of neuronal network function requires selective manipulations of genetically defined neuronal subpopulations in the intact living brain. Here, we highlight the method of optogenetics, which meets those needs. We cover methodological aspects, limitations, and practical applications in the field of neurosciences. The fundamentals of optogenetics are light-sensitive transmembrane channels and light-driven ion pumps, which can be genetically encoded, without requiring the application of exogenous cofactors. These opsins are expressed in neurons by means of viral gene transfer and cell-specific promoters. Light for stimulation can be non- or minimally invasively delivered by optical fibers. Illumination of opsins results in a depolarization or hyperpolarization of genetically modified neurons, depending on the type of optogenetic actuator. Strong expression levels and sufficient light densities provided, neuronal activity can be optically controlled in the intact network with millisecond precision. By applying fluorescent indicators of neuronal activity, an all-optical neurophysiological approach becomes reality.
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Zhou, Yang, Meiqi Ding, Georg Nagel, Kai R. Konrad e Shiqiang Gao. "Advances and prospects of rhodopsin-based optogenetics in plant research". Plant Physiology, 30 de agosto de 2021. http://dx.doi.org/10.1093/plphys/kiab338.
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Abstract Microbial rhodopsins have advanced optogenetics since the discovery of channelrhodopsins almost two decades ago. During this time an abundance of microbial rhodopsins has been discovered, engineered, and improved for studies in neuroscience and other animal research fields. Optogenetic applications in plant research, however, lagged largely behind. Starting with light-regulated gene expression, optogenetics has slowly expanded into plant research. The recently established all-trans retinal production in plants now enables the use of many microbial opsins, bringing extra opportunities to plant research. In this review, we summarize the recent advances of rhodopsin-based plant optogenetics and provide a perspective for future use, combined with fluorescent sensors to monitor physiological parameters.
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Richardson, Rachael T., Alex C. Thompson, Andrew K. Wise, Elise A. Ajay, Niliksha Gunewardene, Stephen J. O’Leary, Paul R. Stoddart e James B. Fallon. "Viral-mediated transduction of auditory neurons with opsins for optical and hybrid activation". Scientific Reports 11, n.º 1 (27 de maio de 2021). http://dx.doi.org/10.1038/s41598-021-90764-9.
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AbstractOptical stimulation is a paradigm-shifting approach to modulating neural activity that has the potential to overcome the issue of current spread that occurs with electrical stimulation by providing focused stimuli. But optical stimulation either requires high power infrared light or genetic modification of neurons to make them responsive to lower power visible light. This work examines optical activation of auditory neurons following optogenetic modification via AAV injection in two species (mouse and guinea pig). An Anc80 viral vector was used to express the channelrhodopsin variant ChR2-H134R fused to a fluorescent reporter gene under the control of the human synapsin-1 promoter. The AAV was administered directly to the cochlea (n = 33) or posterior semi-circular canal of C57BL/6 mice (n = 4) or to guinea pig cochleae (n = 6). Light (488 nm), electrical stimuli or the combination of these (hybrid stimulation) was delivered to the cochlea via a laser-coupled optical fibre and co-located platinum wire. Activation thresholds, spread of activation and stimulus interactions were obtained from multi-unit recordings from the central nucleus of the inferior colliculus of injected mice, as well as ChR2-H134R transgenic mice (n = 4). Expression of ChR2-H134R was examined by histology. In the mouse, transduction of auditory neurons by the Anc80 viral vector was most successful when injected at a neonatal age with up to 89% of neurons transduced. Auditory neuron transductions were not successful in guinea pigs. Inferior colliculus responses to optical stimuli were detected in a cochleotopic manner in all mice with ChR2-H134R expression. There was a significant correlation between lower activation thresholds in mice and higher proportions of transduced neurons. There was no difference in spread of activation between optical stimulation and electrical stimulation provided by the light/electrical delivery system used here (optical fibre with bonded 25 µm platinum/iridium wire). Hybrid stimulation, comprised of sub-threshold optical stimulation to ‘prime’ or raise the excitability of the neurons, lowered the threshold for electrical activation in most cases, but the impact on excitation width was more variable compared to transgenic mice. This study demonstrates the impact of opsin expression levels and expression pattern on optical and hybrid stimulation when considering optical or hybrid stimulation techniques for neuromodulation.
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Kilaru, Sreedhar, Elena Fantozzi, Stuart Cannon, Martin Schuster, Thomas M. Chaloner, Celia Guiu-Aragones, Sarah J. Gurr e Gero Steinberg. "Zymoseptoria tritici white-collar complex integrates light, temperature and plant cues to initiate dimorphism and pathogenesis". Nature Communications 13, n.º 1 (26 de setembro de 2022). http://dx.doi.org/10.1038/s41467-022-33183-2.
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AbstractTransitioning from spores to hyphae is pivotal to host invasion by the plant pathogenic fungus Zymoseptoria tritici. This dimorphic switch can be initiated by high temperature in vitro (~27 °C); however, such a condition may induce cellular heat stress, questioning its relevance to field infections. Here, we study the regulation of the dimorphic switch by temperature and other factors. Climate data from wheat-growing areas indicate that the pathogen sporadically experiences high temperatures such as 27 °C during summer months. However, using a fluorescent dimorphic switch reporter (FDR1) in four wild-type strains, we show that dimorphic switching already initiates at 15–18 °C, and is enhanced by wheat leaf surface compounds. Transcriptomics reveals 1261 genes that are up- or down-regulated in hyphae of all strains. These pan-strain core dimorphism genes (PCDGs) encode known effectors, dimorphism and transcription factors, and light-responsive proteins (velvet factors, opsins, putative blue light receptors). An FDR1-based genetic screen reveals a crucial role for the white-collar complex (WCC) in dimorphism and virulence, mediated by control of PCDG expression. Thus, WCC integrates light with biotic and abiotic cues to orchestrate Z. tritici infection.
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Cheney, Karen L., Jemma Hudson, Fanny de Busserolles, Martin Luehrmann, Abigail Shaughnessy, Cedric van den Berg, Naomi F. Green, N. Justin Marshall e Fabio Cortesi. "Seeing Picasso: an investigation into the visual system of the triggerfish Rhinecanthus aculeatus". Journal of Experimental Biology 225, n.º 7 (1 de abril de 2022). http://dx.doi.org/10.1242/jeb.243907.
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ABSTRACT Vision is used by animals to find food and mates, avoid predators, defend resources and navigate through complex habitats. Behavioural experiments are essential for understanding animals' perception but are often challenging and time-consuming; therefore, using species that can be trained easily for complex tasks is advantageous. Picasso triggerfish, Rhinecanthus aculeatus, have been used in many behavioural studies investigating vision and navigation. However, little is known about the molecular and anatomical basis of their visual system. We addressed this knowledge gap here and behaviourally tested achromatic and chromatic acuity. In terms of visual opsins, R. aculeatus possessed one rod opsin gene (RH1) and at least nine cone opsins: one violet-sensitive SWS2B gene, seven duplicates of the blue–green-sensitive RH2 gene (RH2A, RH2B, RH2C1-5) and one red-sensitive LWS gene. However, only five cone opsins were expressed: SWS2B expression was consistent, while RH2A, RH2C-1 and RH2C-2 expression varied depending on whether fish were sampled from the field or aquaria. Levels of LWS expression were very low. Using fluorescence in situ hybridisation, we found SWS2B was expressed exclusively in single cones, whereas RH2A and RH2Cs were expressed in opposite double cone members. Anatomical resolution estimated from ganglion cell densities was 6.8 cycles per degree (cpd), which was significantly higher than values obtained from behavioural testing for black-and-white achromatic stimuli (3.9 cpd) and chromatic stimuli (1.7–1.8 cpd). These measures were twice as high as previously reported. This detailed information on their visual system will help inform future studies with this emerging focal species.
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Khelashvili, George, Anoop Narayana Pillai, Joon Lee, Kalpana Pandey, Alexander M. Payne, Zarek Siegel, Michel A. Cuendet et al. "Unusual mode of dimerization of retinitis pigmentosa-associated F220C rhodopsin". Scientific Reports 11, n.º 1 (18 de maio de 2021). http://dx.doi.org/10.1038/s41598-021-90039-3.
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AbstractMutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wild-type and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.
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Yang, Jinglei, Li Yang, Rongfang Chen, Yun Zhu, Siyao Wang, Xueqin Hou, Bei Wei et al. "A role of color vision in emmetropization in C57BL/6J mice". Scientific Reports 10, n.º 1 (10 de setembro de 2020). http://dx.doi.org/10.1038/s41598-020-71806-0.
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Abstract Spectral composition affects emmetropization in both humans and animal models. Because color vision interacts the effects of chromatic defocus, we developed a method to bypass the effects of longitudinal chromatic aberration by placing a spectral filter behind the optics of the eye, using genetic tools. Newborn C57BL/6J (B6) mice were reared in quasi-monochromatic red (410–510 nm) or blue (585–660 nm) light beginning before eye-opening. Refractive states and ocular dimensions were compared at 4, 6, 8, and 10 weeks with mice reared in normal white light. Cre recombinase-dependent Ai9 reporter mice were crossed with Chx10-Cre to obtain Chx10-Cre;Ai9 mice, expressing red fluorescent protein in retinal Cre-positive cells. Ai9 offsprings, with and without Cre, were reared under a normal visual environment. Refraction and axial components were measured as described above. Expression levels of M and S opsin were quantified by western blotting at 10 weeks. Compared with those reared in white light, B6 mice reared in red light developed relative hyperopia, principally characterized by flattening of corneal curvature. Emmetropization was not affected by blue light, possibly because the reduction in vitreous chamber depth compensated for the increase in corneal curvature. Compared with Cre-negative littermates, the refraction and axial dimensions of Chx10-Cre;Ai9 mice were not significantly different at the follow-up timepoints. M opsin levels were higher in Chx10-Cre;Ai9 mice at 10 weeks while S opsin levels were not different. Red light induced a hyperopic shift in mouse refractive development. Emmetropization was not impacted in mice with perturbed color vision caused by intrinsic red-fluorescent protein, suggesting that color vision may not be necessary in mouse emmetropization when other mechanisms are present.
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Venturini, Giulia, Despina Kokona, Beatrice L. Steiner, Emanuele G. Bulla, Joel Jovanovic, Martin S. Zinkernagel e Pascal Escher. "In vivo analysis of onset and progression of retinal degeneration in the Nr2e3rd7/rd7 mouse model of enhanced S-cone sensitivity syndrome". Scientific Reports 11, n.º 1 (24 de setembro de 2021). http://dx.doi.org/10.1038/s41598-021-98271-7.
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AbstractThe photoreceptor-specific nuclear receptor Nr2e3 is not expressed in Nr2e3rd7/rd7 mice, a mouse model of the recessively inherited retinal degeneration enhanced S-cone sensitivity syndrome (ESCS). We characterized in detail C57BL/6J Nr2e3rd7/rd7 mice in vivo by fundus photography, optical coherence tomography and fluorescein angiography and, post mortem, by histology and immunohistochemistry. White retinal spots and so-called ‘rosettes’ first appear at postnatal day (P) 12 in the dorsal retina and reach maximal expansion at P21. The highest density in ‘rosettes’ is observed within a region located between 100 and 350 µM from the optic nerve head. ‘Rosettes’ disappear between 9 to 12 months. Non-apoptotic cell death markers are detected during the slow photoreceptor degeneration, at a rate of an approximately 3% reduction of outer nuclear layer thickness per month, as observed from 7 to 31 months of age. In vivo analysis of Nr2e3rd7/rd7 Cx3cr1gfp/+ retinas identified microglial cells within ‘rosettes’ from P21 on. Subretinal macrophages were observed in vivo and by confocal microscopy earliest in 12-months-old Nr2e3rd7/rd7 retinas. At P21, S-opsin expression and the number of S-opsin expressing dorsal cones was increased. The dorso-ventral M-cone gradient was present in Nr2e3rd7/rd7 retinas, but M-opsin expression and M-opsin expressing cones were decreased. Retinal vasculature was normal.
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Garita-Hernandez, Marcela, Antoine Chaffiol, Laure Guibbal, Fiona Routet, Hanen Khabou, Luisa Riancho, Lyes Toualbi et al. "Control of Microbial Opsin Expression in Stem Cell Derived Cones for Improved Outcomes in Cell Therapy". Frontiers in Cellular Neuroscience 15 (18 de março de 2021). http://dx.doi.org/10.3389/fncel.2021.648210.
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Human-induced pluripotent stem cell (hiPSC) derived organoids have become increasingly used systems allowing 3D-modeling of human organ development, and disease. They are also a reliable source of cells for transplantation in cell therapy and an excellent model to validate gene therapies. To make full use of these systems, a toolkit of genetic modification techniques is necessary to control their activity in line with the downstream application. We have previously described adeno-associated viruse (AAV) vectors for efficient targeting of cells within human retinal organoids. Here, we describe biological restriction and enhanced gene expression in cone cells of such organoids thanks to the use of a 1.7-kb L-opsin promoter. We illustrate the usefulness of implementing such a promoter to enhance the expression of the red-shifted opsin Jaws in fusion with a fluorescent reporter gene, enabling cell sorting to enrich the desired cell population. Increased Jaws expression after transplantation improved light responses promising better therapeutic outcomes in a cell therapy setting. Our results point to the importance of promoter activity in restricting, improving, and controlling the kinetics of transgene expression during the maturation of hiPSC retinal derivatives. Differentiation requires mechanisms to initiate specific transcriptional changes and to reinforce those changes when mature cell states are reached. By employing a cell-type-specific promoter we put transgene expression under the new transcriptional program of mature cells.
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Agus, Viviana, Tod A. Flak, Paola Picardi, Sara Pizzi, Lucia Rutigliano, Silvia Cainarca, Loredana Redaelli, Jean-Francois Rolland e Lia Scarabottolo. "Parallel All-Optical Assay to Study Use-Dependent Functioning of Voltage-Gated Ion Channels in a Miniaturized Format". SLAS DISCOVERY: Advancing the Science of Drug Discovery, 17 de dezembro de 2020, 247255522097608. http://dx.doi.org/10.1177/2472555220976083.
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Voltage-gated ion channels produce rapid transmembrane currents responsible for action potential generation and propagation at the neuronal, muscular, and cardiac levels. They represent attractive clinical targets because their altered firing frequency is often the hallmark of pathological signaling leading to several neuromuscular disorders. Therefore, a method to study their functioning upon repeated triggers at different frequencies is desired to develop new drug molecules selectively targeting pathological phenotype. Optogenetics provides powerful tools for millisecond switch of cellular excitability in contactless, physiological, and low-cost settings. Nevertheless, its application to large-scale drug-screening operations is still limited by long processing time (due to sequential well read), rigid flashing pattern, lack of online compound addition, or high consumable costs of existing methods. Here, we developed a method that enables simultaneous analysis of 384-well plates with optical pacing, fluorescence recording, and liquid injection. We used our method to deliver programmable millisecond-switched depolarization through light-activated opsin in concomitance with continuous optical recording by a fluorescent indicator. We obtained 384-well pacing of recombinant voltage-activated sodium or calcium channels, as well as induced pluripotent stem cell (iPSC)-derived cardiomyocytes, in all-optical parallel settings. Furthermore, we demonstrated the use-dependent behavior of known ion channel blockers by optogenetic pacing at normal or pathological firing frequencies, obtaining very good signal reproducibility and accordance with electrophysiology data. Our method provides a novel physiological approach to study frequency-dependent drug behavior using reversible programmable triggers. The all-optical parallel settings combined with contained operational costs make our method particularly suited for large-scale drug-screening campaigns as well as cardiac liability studies.
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Kim, Hye Jin, e Janet R. Sparrow. "Bisretinoid phospholipid and vitamin A aldehyde: Shining a light". Journal of Lipid Research, 5 de maio de 2020, jlr.TR120000742. http://dx.doi.org/10.1194/jlr.tr120000742.
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Vitamin A aldehyde covalently bound to opsin protein is embedded in a phospholipid-rich membrane that supports photon absorption and phototransduction in photoreceptor cell outer segments. Following absorption of a photon, the 11-cis-retinal chromophore of visual pigment in photoreceptor cells isomerizes to all-trans-retinal. To maintain photosensitivity 11-cis-retinal must be replaced. At the same time, however, all-trans-retinal has to be handled so as to prevent nonspecific aldehyde activity. Some molecules of retinaldehyde upon release from opsin are efficiently reduced to retinol. Other molecules are released into the lipid phase of the disc membrane where they form a conjugate (N-retinylidene-PE, NRPE) through a Schiff base linkage with phosphatidylethanolamine (PE). The reversible formation of NRPE serves as a transient sink for retinaldehyde that is intended to return retinaldehyde to the visual cycle. However, if instead of hydrolyzing to PE and retinaldehyde, NRPE reacts with a second molecule of retinaldehyde a synthetic pathway is initiated that leads to the formation of multiple species of unwanted bisretinoid fluorophores. We report on recently identified members of the bisretinoid family some of which differ with respect to the acyl chains associated with the glycerol backbone. We discuss processing of the lipid moieties of these fluorophores in lysosomes of retinal pigment epithelial (RPE) cells, their fluorescence characters and new findings related to light and iron-associated oxidation of bisretinoids.
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Bryl, Krzysztof. "Fluorescence Resonance Energy Transfer (FRET) as a Spectroscopic Ruler for Investigation of Protein Induced Lipid Membrane Curvature: Bacteriorhodopsin and Bacteriorhodopsin Analogues in Model Lipid Membranes". Applied Spectroscopy, 13 de outubro de 2022, 000370282211356. http://dx.doi.org/10.1177/00037028221135645.
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Bacteriorhodopsin (bR) is a light-driven proton pump existing in the purple membranes (PM) of Halobacterium salinarum. The effects associated with changes in proton distribution (proton gradient, membrane electric potential) play a key role in ATP-ase stimulation. However, how the bioenergetic modulus (bR-PM-ATPase) functions remains unclear. One can find indications that hydrophobic matching and the curvature of the lipid membrane may form a functional link between bR and ATP-ase.
To verify whether an interaction between bR and lipids can lead to curvature of the lipid membrane, a spectroscopic ruler – a fluorescence resonance energy transfer (FRET) tool – was used. The distances from fluorescent lipid probes [octadecyl rhodamine B chloride (RhB), 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI), 16-(9-anthroyloxy) palmitic acid (16AP)] and hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH), to the retinal chromophore of bR incorporated into phospholipid vesicles, were measured. The incorporation of retinal analogues with changed shape and/or altered electronic properties into the binding site of a bR or bR mutant were used to strengthen the feedback between the protein surrounding and chromophore. The experiments were performed with wild-type and D96N-mutated bR carrying retinal or 14-(12-,10-, 13,14-bi-) Fluororetinal.
As far as it is known, this is the first time that results obtained by the FRET method show that bR can induce a change in lipid structure interpreted as hydrophobically induced curving of the lipid membrane. Evidence was provided that the chromophore contributed to this effect. The extent of contribution was dependent on the chromophore structure in close vicinity to the place of its link with opsin. The implications of these findings for bR-PM-ATPase module functioning are also discussed.
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Xiang, Xuaner, Yuzhang Chen, Ke-Xin Li, Jianqiao Fang, Philip E. Bickler, Zhonghui Guan e Wei Zhou. "Neuroanatomical Basis for the Orexinergic Modulation of Anesthesia Arousal and Pain Control". Frontiers in Cellular Neuroscience 16 (26 de abril de 2022). http://dx.doi.org/10.3389/fncel.2022.891631.
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Hypothalamic orexin (hypocretin) neurons play crucial roles in arousal control. Their involvement in anesthesia and analgesia remains to be better understood. In order to enhance our view on the neuroanatomy, we systematically mapped the projections of orexin neurons with confocal microscope and light sheet microscope. We specifically expressed optogenetic opsins tagged with fluorescence markers in orexin neurons through adeno-associated viral infection in the mouse brain. The imaging results revealed fine details and novel features of the orexin projections throughout the brain, particularly related to the nuclei regulating arousal and pain. We then optogenetically activated orexin neurons in the lateral hypothalamus to study the effects on anesthesia-related behaviors. cFos staining showed that optogenetic stimulation can activate orexin neurons in the ChR2-mCherry group, but not the control mCherry group (62.86 ± 3.923% vs. 7.9 ± 2.072%; P < 0.0001). In behavior assays, optogenetic stimulation in the ChR2-mCherry group consistently elicited robust arousal from light isoflurane anesthesia (9.429 ± 3.804 s vs. 238.2 ± 17.42 s; P < 0.0001), shortened the emergence time after deep isoflurane anesthesia (109.5 ± 13.59 s vs. 213.8 ± 21.77 s; P = 0.0023), and increased the paw withdrawal latency in a hotplate test (11.45 ± 1.185 s vs. 8.767 ± 0.7775; P = 0.0317). The structural details of orexin fibers established the neuroanatomic basis for studying the role of orexin in anesthesia and analgesia.
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Xiang, Xuaner, Yuzhang Chen, Ke-Xin Li, Jianqiao Fang, Philip E. Bickler, Zhonghui Guan e Wei Zhou. "Neuroanatomical Basis for the Orexinergic Modulation of Anesthesia Arousal and Pain Control". Frontiers in Cellular Neuroscience 16 (26 de abril de 2022). http://dx.doi.org/10.3389/fncel.2022.891631.
Texto completo da fonteResumo:
Hypothalamic orexin (hypocretin) neurons play crucial roles in arousal control. Their involvement in anesthesia and analgesia remains to be better understood. In order to enhance our view on the neuroanatomy, we systematically mapped the projections of orexin neurons with confocal microscope and light sheet microscope. We specifically expressed optogenetic opsins tagged with fluorescence markers in orexin neurons through adeno-associated viral infection in the mouse brain. The imaging results revealed fine details and novel features of the orexin projections throughout the brain, particularly related to the nuclei regulating arousal and pain. We then optogenetically activated orexin neurons in the lateral hypothalamus to study the effects on anesthesia-related behaviors. cFos staining showed that optogenetic stimulation can activate orexin neurons in the ChR2-mCherry group, but not the control mCherry group (62.86 ± 3.923% vs. 7.9 ± 2.072%; P < 0.0001). In behavior assays, optogenetic stimulation in the ChR2-mCherry group consistently elicited robust arousal from light isoflurane anesthesia (9.429 ± 3.804 s vs. 238.2 ± 17.42 s; P < 0.0001), shortened the emergence time after deep isoflurane anesthesia (109.5 ± 13.59 s vs. 213.8 ± 21.77 s; P = 0.0023), and increased the paw withdrawal latency in a hotplate test (11.45 ± 1.185 s vs. 8.767 ± 0.7775; P = 0.0317). The structural details of orexin fibers established the neuroanatomic basis for studying the role of orexin in anesthesia and analgesia.
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Xue, Yuntian, Andrew W. Browne, William C. Tang, Jeffrey Delgado, Bryce T. McLelland, Gabriel Nistor, Jacqueline T. Chen et al. "Retinal Organoids Long-Term Functional Characterization Using Two-Photon Fluorescence Lifetime and Hyperspectral Microscopy". Frontiers in Cellular Neuroscience 15 (10 de dezembro de 2021). http://dx.doi.org/10.3389/fncel.2021.796903.
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Pluripotent stem cell-derived organoid technologies have opened avenues to preclinical basic science research, drug discovery, and transplantation therapy in organ systems. Stem cell-derived organoids follow a time course similar to species-specific organ gestation in vivo. However, heterogeneous tissue yields, and subjective tissue selection reduce the repeatability of organoid-based scientific experiments and clinical studies. To improve the quality control of organoids, we introduced a live imaging technique based on two-photon microscopy to non-invasively monitor and characterize retinal organoids’ (RtOgs’) long-term development. Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the metabolic trajectory, and hyperspectral imaging was applied to characterize structural and molecular changes. We further validated the live imaging experimental results with endpoint biological tests, including quantitative polymerase chain reaction (qPCR), single-cell RNA sequencing, and immunohistochemistry. With FLIM results, we analyzed the free/bound nicotinamide adenine dinucleotide (f/b NADH) ratio of the imaged regions and found that there was a metabolic shift from glycolysis to oxidative phosphorylation. This shift occurred between the second and third months of differentiation. The total metabolic activity shifted slightly back toward glycolysis between the third and fourth months and stayed relatively stable between the fourth and sixth months. Consistency in organoid development among cell lines and production lots was examined. Molecular analysis showed that retinal progenitor genes were expressed in all groups between days 51 and 159. Photoreceptor gene expression emerged around the second month of differentiation, which corresponded to the shift in the f/b NADH ratio. RtOgs between 3 and 6 months of differentiation exhibited photoreceptor gene expression levels that were between the native human fetal and adult retina gene expression levels. The occurrence of cone opsin expression (OPN1 SW and OPN1 LW) indicated the maturation of photoreceptors in the fourth month of differentiation, which was consistent with the stabilized level of f/b NADH ratio starting from 4 months. Endpoint single-cell RNA and immunohistology data showed that the cellular compositions and lamination of RtOgs at different developmental stages followed those in vivo.
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Gu, Yangguang, Yu Wang, Yinghua Lan, Jianglong Feng, Wen Zeng, Wei Zhang e Hongguang Lu. "Expression of Retinal G Protein-Coupled Receptor, a Member of the Opsin Family, in Human Skin Cells and Its Mediation of the Cellular Functions of Keratinocytes". Frontiers in Cell and Developmental Biology 10 (4 de abril de 2022). http://dx.doi.org/10.3389/fcell.2022.787730.
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Background: Photoreceptive proteins play critical physiological roles in human skin cells. The retinal G protein-coupled receptor (RGR) is a photoisomerase in the human retina, but its expression and cellular functions in human skin cells have not been reported.Objectives: We aimed to detect RGR expression in various skin cells and evaluate its regulation of the cellular functions of keratinocytes.Methods: The expression, distribution, and subcellular location of the RGR in normal human epidermal keratinocytes and cells with pathological conditions including psoriasis, seborrheic keratosis, and squamous cell carcinoma were determined using microscopic tools (immunohistochemical staining, immunofluorescence staining, and immunoelectron microscopy) and Western blotting (WB). The protein levels of the RGR in primary human melanocytes, keratinocytes, and fibroblasts isolated from the neonatal foreskin were measured by WB. The expression and subcellular localization of the RGR in these cells were detected by immunofluorescence staining under a fluorescence microscope and laser scanning confocal microscope. Additionally, the levels of RGR expression in normal keratinocytes exposed to ultraviolet (UV)-A or total ultraviolet radiation (UVR) in the presence or absence of all-trans-retinal were measured by WB. Furthermore, the effects of the RGR on human keratinocyte functions including proliferation, migration, and apoptosis were evaluated using the Cell Counting Kit 8, wound healing, and Transwell assays after reducing the RGR mRNA level in keratinocytes using small interfering RNA technology.Results: The RGR was primarily located in the epidermal basal and spinous layers and skin appendages. Its expression increased in psoriatic lesions, seborrheic keratosis, and squamous cell carcinoma. Confocal microscopy showed that the RGR was located in the cell membrane and nucleus of keratinocytes, melanocytes, and fibroblasts. Keratinocytes had a higher expression of the RGR than melanocytes and fibroblasts, as well as nuclear expression, according to nuclear/cytoplasmic fractionation. Colloidal gold immunoelectron microscopy technology further confirmed that the RGR is mainly located in the nucleoplasm and mitochondria and is scattered in the cytoplasm and other organelles in the epidermal keratinocytes. Notably, RGR knockdown in keratinocytes led to the inhibition of cell proliferation and migration, augmenting cell apoptosis.Conclusions: This study is the first to demonstrate the presence of RGR in the human skin. Our findings indicate that the RGR may play a critical role in the physiological function of epidermal keratinocytes.