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The application of a liquid crystal (LC) in displays has driven the development of novel LC elements. In this Letter, polarization variable line-space (PVLS) gratings based on photoalignment are fabricated, and their variable-spacing properties are derived using the vector diffraction theory. Both transmissive and reflective PVLS gratings are fabricated to validate the correctness of the derivation. Experimental results indicate that PVLS gratings have a wider wavelength response bandwidth than that of polarization volume grating (PVG). PVLS gratings have angle selectivity, and a large incident angle causes wavelength blueshift. Additionally, the relationship between wavelength and focal length indicates its anomalous dispersion as a diffractive optical element. These results of photoalignment-based PVLS gratings provide valuable insights for the advancement of displays and have the potential to improve visual experiences.
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Significance: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy. Aim: We describe the development and applications of T-GECO1-a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1. Approach: We use protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices. Results: The Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1 cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold, and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled the detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant. Conclusions: T-GECO1 is a high-performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.
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The pineal gland has evolved from a photoreceptive organ in fish to a neuroendocrine organ in mammals. This study integrated multiple daytime single-cell RNA-seq datasets from the pineal glands of zebrafish, rats, and monkeys, providing a detailed examination of the evolutionary transition at single-cell resolution. We identified key factors responsible for the anatomical and functional transformation of the pineal gland. We retrieved and integrated daytime single-cell transcriptomic datasets from the pineal glands of zebrafish, rats, and monkeys, resulting in a total of 22 431 cells after rigorous quality filtering. Comparative analysis was then conducted to elucidate the evolution of pineal cells, their photosensitivity, their role in melatonin production, and the signaling processes within the glands of these species. Our analysis identified distinct cellular compositions of the pineal gland in zebrafish, rats, and monkeys. Zebrafish photoreceptors exhibited comprehensive phototransduction gene expression, while specific genes, including transducin (Gngt1, Gnb3, and Gngt2) and phosducin (Pdc), were consistently present in mammalian pinealocytes. We found transcriptional similarities between the pineal gland and retina, underscoring shared evolutionary and functional pathways. Zebrafish displayed unique light-responsive circadian gene activity compared to rats and monkeys. Key ligand-receptor interactions were identified, especially involving MDK and PTN, influencing melatonin synthesis across species. Furthermore, we observed species-specific GPCR (G protein-coupled receptors) expressions related to melatonin synthesis and their alignment with retinal expressions. Our findings also highlighted specific transcription factors (TFs) and regulatory networks associated with pineal gland evolution and function. Our study provides a detailed analysis of the pineal gland's evolution from fish to mammals. We identified key transcriptional changes and controls that highlight the gland's functional diversity. Notably, we found significant ligand-receptor interactions influencing melatonin synthesis and demonstrated parallels between pineal and retinal expressions. These insights enhance our understanding of the pineal gland's role in phototransduction, melatonin production, and circadian rhythms in vertebrates.
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Melatonina , Glándula Pineal , Animales , Ratas , Glándula Pineal/metabolismo , Melatonina/metabolismo , Pez Cebra/genética , Ligandos , Ritmo Circadiano/genética , Mamíferos/metabolismoRESUMEN
Wavefront coding (WFC) is an effective technique for extending the depth-of-field of imaging systems, including optical encoding and digital decoding. We applied physical prior information and frequency domain model to the wavefront decoding, proposing a reconstruction method by a generative model. Specifically, we rebuild the baseline inspired by the transformer and propose three modules, including the point spread function (PSF) attention layer, multi-feature fusion block, and frequency domain self-attention block. These models are used for end-to-end learning to extract PSF feature information, fuse it into the image features, and further re-normalize the image feature information, respectively. To verify the validity, in the encoding part, we use the genetic algorithm to design a phase mask in a large field-of-view fluorescence microscope system to generate the encoded images. And the experimental results after wavefront decoding show that our method effectively reduces noise, artifacts, and blur. Therefore, we provide a deep-learning wavefront decoding model, which improves reconstruction image quality while considering the large depth-of-field (DOF) of a large field-of-view system, with good potential in detecting digital polymerase chain reaction (dPCR) and biological images.
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Significance: Genetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy. Aim: We describe the development and applications of T-GECO1 - a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1. Approach: We used protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices. Results: The Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant. Conclusion: T-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.
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The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.
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Enzima Convertidora de Angiotensina 2 , Rodopsina , Ratones , Animales , Potenciales de Acción/fisiología , Rodopsina/genética , Neuronas/fisiología , Mutación/genéticaRESUMEN
Rice protein was used as a starting material to provide rice protein hydrolysates (RPH) through enzyme-assisted extraction. RPH was further fractionated using ultrafiltration membrane (UF) and classified by molecular weight (MW; MW < 1 kDa, MW 1-10 kDa, and MW > 10 kDa). Peptides with MW < 1 kDa possessed superior antioxidant properties (p < 0.05). Therefore, UF demonstrated great efficacy in selectively separating antioxidant peptides. A Pearson correlation analysis revealed that the total phenolic concentration was correlated with oxygen radical absorbance capacity (ORAC; r = 0.999, p < 0.05). Amino acid contents had negative correlations with the scavenging activity (specifically, IC50) of 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radicals (r = - 0.986 to - 1.000). Reducing power was related to aromatic amino acid contents (r = 0.997, p < 0.05). In this study, enzymatic hydrolysis was discovered to be an effective method of extracting and isolating natural antioxidant proteins from broken rice, thus preserving the nutritional quality of rice and making those proteins more accessible in future applications.
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Oryza , Hidrolisados de Proteína , Antioxidantes , Peso Molecular , Capacidad de Absorbancia de Radicales de OxígenoRESUMEN
Calcium imaging with protein-based indicators1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators3-8. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.
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Señalización del Calcio , Calcio , Calmodulina , Neuronas , Óxido Nítrico Sintasa de Tipo III , Fragmentos de Péptidos , Calcio/análisis , Calcio/metabolismo , Calmodulina/metabolismo , Neuronas/metabolismo , Cinética , Óxido Nítrico Sintasa de Tipo III/química , Óxido Nítrico Sintasa de Tipo III/metabolismo , Factores de Tiempo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismoRESUMEN
In general, the shape of traditional holographic grating is fixed and immutable, the period will not change after fabrication, this means that the modulation effect on the light field is unalterable. However, traditional grating cannot satisfy all requirements of current optical systems. In order to increase the versatility of holographic grating, a flexible curved radius grating (FCRG) which consists of holographic polymer-dispersed liquid crystal (H-PDLC) was proposed. The FCRG has an important self-focusing property that it can be adjusted the focal length by changing its own radius of curvature correspondingly. In this paper, we use the scalar diffraction theory to analysis the interference and diffraction processes for FCRG under different conditions, then a relationship equation has been deduced to express quantitatively about FCRG between its radius and focal length. According to the relationship, a tunable holographic element is achieved for the function of mechanically-controlled self-focusing effect. Experiments show that the FCRG has two conjugated focusing effects on the positive first-order and negative first-order, both two effects can achieve focus-adjusted ability by changing their radius of curvature. The FCRG provides a way for the coupler of curved waveguide display system for augmented reality in the future.
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The glucagon receptor (GCGR) is a potential target for diabetes therapy. Several emerging GCGR antagonism-based therapies are under preclinical and clinical development. However, GCGR antagonism, as well as genetically engineered GCGR deficiency in animal models, are accompanied by α-cell hyperplasia and hyperglucagonemia, which may limit the application of GCGR antagonism. To better understand the physiological changes in α cells following GCGR disruption, we performed single cell sequencing of α cells isolated from control and gcgr-/- (glucagon receptor deficient) zebrafish. Interestingly, beyond the α-cell hyperplasia, we also found that the expression of gcga, gcgb, pnoca, and several glucagon-regulatory transcription factors were dramatically increased in one cluster of gcgr-/- α cells. We further confirmed that glucagon mRNA was upregulated in gcgr-/- animals by in situ hybridization and that glucagon promoter activity was increased in gcgr-/-;Tg(gcga:GFP) reporter zebrafish. We also demonstrated that gcgr-/- α cells had increased glucagon protein levels and increased granules after GCGR disruption. Intriguingly, the increased mRNA and protein levels could be suppressed by treatment with high-level glucose or knockdown of the pnoca gene. In conclusion, these data demonstrated that GCGR deficiency not only induced α-cell hyperplasia but also increased glucagon expression in α cells, findings which provide more information about physiological changes in α-cells when the GCGR is disrupted.
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Glucagón , Receptores de Glucagón , Animales , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Hiperplasia , ARN MensajeroRESUMEN
BACKGROUND: The melanocortin receptor accessory proteins (MRAP1 and MRAP2) are well-known endocrine regulators for the trafficking and signalling of all five melanocortin receptors (MC1R-MC5R). The observation of MRAP2 on regulating several non-melanocortin G protein-coupled receptors (GPCRs) has been sporadically reported, whereas other endogenous GPCR partners of the MRAP protein family are largely unknown. METHODS: Here, we performed single-cell transcriptome analysis and drew a fine GPCR blueprint and MRAPs-associated network of two major endocrine organs, the hypothalamus and adrenal gland at single-cell resolution. We also integrated multiple bulk RNA-seq profiles and single-cell datasets of human and mouse tissues, and narrowed down a list of 48 GPCRs with strong endogenous co-expression correlation with MRAPs. RESULTS: 36 and 46 metabolic-related GPCRs were consequently identified as novel interacting partners of MRAP1 or MRAP2, respectively. MRAPs exhibited protein-protein interactions and varying pharmacological properties on the surface translocation, constitutive activities and ligand-stimulated downstream signalling of these GPCRs. Knockdown of MRAP2 expression by hypothalamic administration of adeno-associated virus (AAV) packed shRNA stimulated body weight gain in mouse model. Co-injection of corticotropinreleasing factor (CRF), the agonist of corticotropin releasing hormone receptor 1 (CRHR1), suppressed feeding behaviour in a MRAP2-dependent manner. CONCLUSIONS: Collectively, our study has comprehensively elucidated the complex GPCR networks in two major endocrine organs and redefined the MRAP protein family as broad-spectrum GPCR modulators. MRAP proteins not only serve as a vital endocrine pivot on the regulation of global GPCR activities in vivo that could explain the composite physiological phenotypes of the MRAP2 null murine model but also provide us with new insights of the phenotyping investigation of GPCR-MRAP functional complexes.
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Proteínas Portadoras , Receptores de Melanocortina , Animales , Humanos , Ratones , Receptores de Melanocortina/genética , Receptores de Melanocortina/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Melanocortinas/metabolismo , Glándulas Suprarrenales/metabolismo , Hipotálamo/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
2,2',7,7'-Tetrakis(N,N-di-p-methoxyphenyl-amine)-9,9'-spirobifluorene (spiro-OMeTAD), the most widely used hole transport material in high-efficiency perovskite solar cells (PSCs), still has serious defects, such as moisture absorption and poor long-term conductivity, which seriously restrict further improvement of the power conversion efficiency (PCE) and stability of the cell. Herein, to overcome these problems, inorganic salt PbSO4(PbO)4 quantum dots (QDs) are incorporated into spiro-OMeTAD as the hole transport layer (HTL) for the first time. The incorporated PbSO4(PbO)4 QDs significantly hinder the agglomeration of lithium bis(trifluoromethanesulfonyl)-imide and improve the long-term conductivity through the oxidative interaction between PbSO4(PbO)4 QDs and spiro-OMeTAD and hydrophobicity of the HTL. Furthermore, the spiro-OMeTAD:PbSO4(PbO)4 composite film can effectively passivate perovskite defects at the perovskite/HTL interface, resulting in suppressed interfacial recombination. As a result, the PSC based on the spiro-OMeTAD:PbSO4(PbO)4 HTL shows an improved PCE of 22.66%, which is much higher than that (18.89%) of the control device. PbSO4(PbO)4 also significantly improves the moisture stability for 50 days at room temperature (at RH â¼ 40-50%) without encapsulation. This work indicates that inorganic PbSO4(PbO)4 QDs are crucial materials that can be employed as an additive in spiro-OMeTAD to enhance the efficiency and stability of PSCs.
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PIWI-interacting small RNAs (piRNAs) protect the germline genome and are essential for fertility. piRNAs originate from transposable element (TE) RNAs, long non-coding RNAs, or 3´ untranslated regions (3´UTRs) of protein-coding messenger genes, with the last being the least characterized of the three piRNA classes. Here, we demonstrate that the precursors of 3´UTR piRNAs are full-length mRNAs and that post-termination 80S ribosomes guide piRNA production on 3´UTRs in mice and chickens. At the pachytene stage, when other co-translational RNA surveillance pathways are sequestered, piRNA biogenesis degrades mRNAs right after pioneer rounds of translation and fine-tunes protein production from mRNAs. Although 3´UTR piRNA precursor mRNAs code for distinct proteins in mice and chickens, they all harbor embedded TEs and produce piRNAs that cleave TEs. Altogether, we discover a function of the piRNA pathway in fine-tuning protein production and reveal a conserved piRNA biogenesis mechanism that recognizes translating RNAs in amniotes.
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Regiones no Traducidas 3' , Fertilidad/genética , Biosíntesis de Proteínas , ARN Interferente Pequeño/genética , Ribosomas/genética , Espermatogénesis/genética , Animales , Secuencia de Bases , Pollos , Elementos Transponibles de ADN , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fase Paquiteno , ARN Interferente Pequeño/metabolismo , Ribosomas/metabolismo , Testículo/citología , Testículo/crecimiento & desarrollo , Testículo/metabolismoRESUMEN
As a member of the seven-transmembrane rhodopsin-like G protein-coupled receptor superfamily, the melanocortin-3 receptor (MC3R) is vital for the regulation of energy homeostasis and rhythms synchronizing in mammals, and its pharmacological effect could be directly influenced by the presence of melanocortin receptor accessory proteins (MRAPs), MRAP1 and MRAP2. The tetrapod amphibian Xenopus laevis (xl) retains higher duplicated genome than extant teleosts and serves as an ideal model system for embryonic development and physiological studies. However, the melanocortin system of the Xenopus laevis has not yet been thoroughly evaluated. In this work, we performed sequence alignment, phylogenetic tree, and synteny analysis of two xlMC3Rs. Co-immunoprecipitation and immunofluorescence assay further confirmed the co-localization and in vitro interaction of xlMC3Rs with xlMRAPs on the plasma membrane. Our results demonstrated that xlMRAP2.L/S could improve α-MSH-stimulated xlMC3Rs signaling and suppress their surface expression. Moreover, xlMC3R.L showed a similar profile on the ligands and surface expression in the presence of xlMRAP1.L. Overall, the distinct pharmacological modulation of xlMC3R.L and xlMC3R.S by dual MRAP2 proteins elucidated the functional consistency of melanocortin system during genomic duplication of tetrapod vertebrates.
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Diffuse large B cells in the cerebrospinal fluid (CSF-DLBCs) have offered great promise for the diagnostics and therapeutics of central nervous system lymphoma (CNSL) leptomeningeal involvement. To explore the phenotypic states of CSF-DLBCs, we analyzed the transcriptomes of more than one thousand CSF-DLBCs from six patients with CNSL diffuse large B-cell lymphoma (DLBCL) using Smart-seq2 single-cell RNA sequencing. CSF-DLBCs were defined based on abundant expression of B-cell markers, the active cell proliferation and energy metabolism properties, and immunoglobulin light chain restriction. We identified inherent heterogeneity of CSF-DLBCs, which were mainly manifested in cell cycle state, cancer-testis antigen expression, and classification based on single-cell germinal center B-cell signature. In addition, the 16 upregulated genes in CSF-DLBCs compared to various normal B cells showed great possibility in the homing effect of the CNS-DLBCL for the leptomeninges. Our results will provide insight into the mechanism research and diagnostic direction of CNSL-DLBCL leptomeningeal involvement.
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Droplet digital polymerase chain reaction (ddPCR) is an effective technique, with unparalleled sensitivity, for the absolute quantification of target nucleic acids. However, current commercial ddPCR devices for detecting the gene chip are time consuming due to complex image stitching. To address this issue, we propose a universal concentration determination system and realize one-time gene chip imaging with high resolution. All the functional units are controlled by self-developed software using the PyQt5 module in Python. Without stitching technology, images of the ddPCR chip (28 mm × 18 mm) containing 20 000 independent 0.81 nL micro chambers can be obtained in less than 15 seconds, which saves about 165 seconds. A white laser light source (2 mW cm-2) was employed as a substitute for the mercury lamp. Its wavelength matches well with typical fluorescent dyes (e.g., HEX, ROX and Cy5), and thus it can strengthen the fluorescence intensity for weak signals. The results also demonstrated that the correlation coefficient for the measured concentration and theoretical value was above 99%, by testing the ddPCR products with COVID-19 virus. Such a system can greatly reduce the time required for image acquisition and DNA concentration determination, and thus is able to speed up the lab-to-application process for ddPCR technology.
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COVID-19 , Ácidos Nucleicos , Humanos , Microfluídica , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , SARS-CoV-2RESUMEN
Plant-derived protein hydrolysates have potential applications in nutrition. Rice protein hydrolysates (RPHs), an excellent source of proteins, have attracted attention for the development of cosmeceuticals. However, few studies have reported the potential application of RPH in analysis, and this study examined their antioxidant activities and the inhibitory activities of skin aging enzymes. The results indicated that the total phenolic and flavonoid concentrations were 2.06 ± 0.13 mg gallic acid equivalent/g RPHs and 25.96 ± 0.52 µg quercetin equivalent/g RPHs, respectively. RPHs demonstrated dose-dependent activity for scavenging free radicals from 1,1-diphenyl-2-picrylhydrazyl [half-maximal inhibitory concentration (IC50) = 42.58 ± 2.1 mg/g RPHs] and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (IC50 = 2.11 ± 0.88 mg/g RPHs), dose-dependent reduction capacity (6.95 ± 1.40 mg vitamin C equivalent/g RPHs) and oxygen radical absorbance capacity (473 µmol Trolox equivalent/g RPHs). The concentrations of the RPH solution required to achieve 50% inhibition of hyaluronidase and tyrosinase activities were determined to be 8.91 and 107.6 mg/mL, respectively. This study demonstrated that RPHs have antioxidant, antihyaluronidase, and antityrosinase activities for future cosmetic applications.
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Hidrolisados de Proteína/química , Hidrolisados de Proteína/farmacología , Envejecimiento/efectos de los fármacos , Animales , Antioxidantes/farmacología , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacología , Blanqueadores/química , Blanqueadores/metabolismo , Flavonoides/farmacología , Depuradores de Radicales Libres/química , Ácido Gálico/farmacología , Ratones , Oryza/química , Oryza/enzimología , Oryza/metabolismo , Oxidación-Reducción , Fenoles/farmacología , Picratos/química , Picratos/farmacología , Extractos Vegetales/química , Quercetina/farmacología , Células RAW 264.7 , Ácidos Sulfónicos/química , Ácidos Sulfónicos/farmacología , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
Physiological modulation of melanocortin-4 receptor (MC4R) signaling by MRAP2 proteins plays an indispensable role in appetite control and energy homeostasis in the central nervous system. Great interspecies differences of the interaction and regulation of melanocortin receptors by MRAP protein family have been reported in several diploid vertebrates but never been investigated in the tetrapod amphibian Xenopus laevis. Here, we performed phylogenetic and synteny-based analyses to explore the evolutionary aspects of dual copies of X. laevis MC4R (xlMC4R) and MRAP2 (xlMRAP2) proteins. Our data showed that xlMRAPs directly interacted with xlMC4Rs on the cell surface as a functional antiparallel dimeric topology and pharmacological studies suggested a homology specific regulatory pattern of the melanocortin system in X. laevis.
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Melanocortinas/metabolismo , Receptor de Melanocortina Tipo 4/metabolismo , Receptores de Melanocortina/metabolismo , Xenopus laevis/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Regulación del Apetito/fisiología , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Homeostasis/fisiología , Receptor de Melanocortina Tipo 4/genética , Transducción de Señal/fisiologíaRESUMEN
With the rapid development of digital precision medicine, the digital polymerase chain reaction (dPCR) deoxyribonucleic acid (DNA) gene chip integrates more channels with smaller size and larger area, which leads to a higher technical requirement for commercial optical fluorescence microscopy. The multitime image splicing method is widely used for DNA detection. However, it consumes time and has visible seamless image results. This work has demonstrated the design and fabrication of a three channel reversed and reduced fluorescence microscopic imaging system with high-resolution and large field of view for one-time imaging. We introduced the super ultra-thin dichroic mirror into the space between the objective lens and the gene chip to achieve a uniform illumination and a strong signal for the large area gene chip. The fabricated new fluorescence microscopy can take a one-time imaging for the 28×18mm dPCR gene chip with more than 20,000 multi micro-droplets within FAM, HEX, and ROX fluorescence channels. The optical system was designed with a numerical aperture (NA) of 0.106. Modulation transfer function (MTF) is higher than 0.675 at 70 lp/mm, and the function resolution capability is 10 µm with the whole magnification of -0.65times. The fly's eye lens-based illumination system was tested with a uniform output of over 90% in the whole Ï34.7mm chip area. The design was tested, and the experimental results showed that this new system provides a fast, efficient, and professional optical imaging method for detection of the new emerged digital PCR gene chip, which has larger area and more channels.
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Dispositivos Laboratorio en un Chip , Microscopía Fluorescente/instrumentación , Sistemas de Atención de Punto , Reacción en Cadena de la Polimerasa/instrumentación , Procesamiento de Señales Asistido por Computador/instrumentación , ADN/genética , Diseño de Equipo , Microquímica/instrumentación , Imagen ÓpticaRESUMEN
Imaging membrane voltage from genetically defined cells offers the unique ability to report spatial and temporal dynamics of electrical signaling at cellular and circuit levels. Here, we present a general approach to engineer electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with positive-going fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transform the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further apply this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.