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Refrigeration is of vital importance for modern society-for example, for food storage and air conditioning-and 25 to 30 per cent of the world's electricity is consumed for refrigeration1. Current refrigeration technology mostly involves the conventional vapour compression cycle, but the materials used in this technology are of growing environmental concern because of their large global warming potential2. As a promising alternative, refrigeration technologies based on solid-state caloric effects have been attracting attention in recent decades3-5. However, their application is restricted by the limited performance of current caloric materials, owing to small isothermal entropy changes and large driving magnetic fields. Here we report colossal barocaloric effects (CBCEs) (barocaloric effects are cooling effects of pressure-induced phase transitions) in a class of disordered solids called plastic crystals. The obtained entropy changes in a representative plastic crystal, neopentylglycol, are about 389 joules per kilogram per kelvin near room temperature. Pressure-dependent neutron scattering measurements reveal that CBCEs in plastic crystals can be attributed to the combination of extensive molecular orientational disorder, giant compressibility and highly anharmonic lattice dynamics of these materials. Our study establishes the microscopic mechanism of CBCEs in plastic crystals and paves the way to next-generation solid-state refrigeration technologies.
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OBJECTIVE: This study aimed to explore the specific pathways by which HOX transcript antisense intergenic RNA contributes to the pathogenesis of unexplained recurrent spontaneous abortion. METHODS: Real-time quantitative PCR was employed to assess the differential expression levels of HOX transcript antisense intergenic RNA in chorionic villi tissues from unexplained recurrent spontaneous abortion patients and women with voluntarily terminated pregnancies. HTR-8/SVneo served as a cellular model. Knockdown and overexpression of HOX transcript antisense intergenic RNA in the cells were achieved through siRNA transfection and pcDNA3.1 transfection, respectively. Cell viability, migration, and invasion were evaluated using cell counting kit-8, scratch, and Transwell assays, respectively. The interaction among the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 axis was predicted through bioinformatics analysis and confirmed through in vitro experiments. Furthermore, the regulatory effects of the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 signaling axis on cellular behaviors were validated in HTR-8/SVneo cells. RESULTS: We found that HOX transcript antisense intergenic RNA was downregulated in chorionic villi tissues from unexplained recurrent spontaneous abortion patients. Overexpression of HOX transcript antisense intergenic RNA significantly enhanced the viability, migration, and invasion of HTR-8/SVneo cells, while knockdown of HOX transcript antisense intergenic RNA had the opposite effects. We further confirmed the regulatory effect of the HOX transcript antisense intergenic RNA /miR-1277-5p/fibrillin 2 signaling axis in unexplained recurrent spontaneous abortion. Specifically, HOX transcript antisense intergenic RNA and fibrillin 2 were found to reduce the risk of unexplained recurrent spontaneous abortion by enhancing cell viability, migration, and invasion, whereas miR-1277-5p exerted the opposite effects. CONCLUSION: HOX transcript antisense intergenic RNA promotes unexplained recurrent spontaneous abortion development by targeting inhibition of miR-1277-5p/fibrillin 2 axis.
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Aborto Habitual , Movimiento Celular , MicroARNs , ARN Largo no Codificante , Transducción de Señal , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Femenino , Aborto Habitual/genética , Aborto Habitual/metabolismo , Aborto Habitual/patología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Embarazo , Fibrilina-2/genética , Fibrilina-2/metabolismo , Adulto , Proliferación Celular , Línea Celular , Trofoblastos/metabolismo , Trofoblastos/fisiología , Vellosidades Coriónicas/metabolismoRESUMEN
X-ray standing-wave (XSW) excited photoelectron emission was used to measure the site-specific valence band (VB) for ½ monolayer (ML) Pt grown on a SrTiO_{3} (001) surface. The XSW induced modulations in the core level (CL), and VB photoemission from the surface and substrate atoms were monitored for three hkl substrate Bragg reflections. The XSW CL analysis shows the Pt to have a face-centered-cubic-like cube-on-cube epitaxy with the substrate. The XSW VB information compares well to a density functional theory calculated projected density of states from the surface and substrate atoms. Overall, this Letter represents a novel method for determining the contribution to the density of states by valence electrons from specific atomic surface sites.
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Due to special cavity structure, cyclodextrin can form inclusion complex with a large number of compounds, which can be widely used in food industry, such as enhancing antibacterial activity, extending the storage period of food, increasing the solubility of food ingredients, removing cholesterol in food and so on. In this paper, the formation mechanism, classification and properties of cyclodextrin inclusion complex were reviewed, and the applications of cyclodextrin and its derivatives in food industry were discussed.
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Ciclodextrinas , Antioxidantes/química , Ciclodextrinas/química , Industria de Alimentos , SolubilidadRESUMEN
During redox reactions, oxide-supported catalytic systems undergo structural and chemical changes. Improving subsequent catalytic properties requires an understanding of the atomic-scale structure with chemical state specificity under reaction conditions. For the case of 1/2 monolayer vanadia on α-TiO2(110), we use X-ray standing wave (XSW) excited X-ray photoelectron spectroscopy to follow the redox induced atomic positional and chemical state changes of this interface. While the resulting XSW 3D composite atomic maps include the Ti and O substrate atoms and V surface atoms, our focus in this report is on the previously unseen surface oxygen species with comparison to density functional theory predictions.
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Transition metal carbides have attractive physical and chemical properties that are much different from their parent metals. Particularly, noble metal carbides are expected to be promising materials for a variety of applications, particularly as efficient catalysts. However, noble metal carbides have rarely been obtained because carbide phases do not appear in noble metal-carbon phase diagrams and a reasonable synthesis method to make noble metal carbides has not yet been established. Here, we propose a new synthesis method for noble metal carbides and describe the first synthesis of rhodium carbide using tetracyanoethylene (TCNE). The rhodium carbide was synthesized without extreme conditions, such as the very high temperature and/or pressure typically required in conventional carbide syntheses. Moreover, we investigated the electronic structure and catalytic activity for the hydrogen evolution reaction (HER). We found that rhodium carbide has much higher catalytic activity for HER than pure Rh. Our study provides a feasible strategy to create new metal carbides to help advance the field of materials science.
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BACKGROUND: GTPase-activating proteins (GAPs) with a TBC (Tre-2/Bub2/Cdc16) domain architecture serve as negative regulators of Rab GTPases. The related crystal structure has been studied and reported by other members of our research group in 2017 (Chen et al. in Protein Sci 26(4):834-846, 2017). The protein crystal structure and sequencing data accession numbers in Protein structure database (PDB) are 5TUB (Shark TBC1D15 GAP) and 5TUC (Sus TBC1D15 GAP), respectively. In this paper, we analyzed the Rab-GAP specificity of TBC1D15 in the evolution and influence of key amino acid residue mutations on Rab-GAP activity. RESULTS: Sequence alignment showed that five arginine residues of the TBC1D15-GAP domain are conserved among the species Sus/Mus/Homo but have been replaced by glycine or lysine in Shark. A fragment activity assay was conducted by altering the five residues of Shark TBC1D15-GAP to arginine, and the corresponding arginine in TBC1D15 GAP domains from Sus and Homo species were mutated to resemble Shark TBC1D15-GAP. Our data revealed that the residues of G28, K45, K119, K122 and K221 in the Shark TBC1D15-GAP domain had a key role in determining the specificity for Rab7 and Rab11. Mutation of the five residues significantly altered the Shark TBC1D15-GAP activity. CONCLUSIONS: These results revealed that the substrate specificity of TBC1D15 has had different mechanisms across the evolution of species from lower-cartilaginous fish to higher mammals. Collectively, the data support a different mechanism of Shark TBC1D15-GAP in substrate selection, which provides a new idea for the development of Marine drugs.
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Secuencia Conservada , Evolución Molecular , Proteínas Activadoras de GTPasa/química , Tiburones/metabolismo , Proteínas de Unión al GTP rab/química , Secuencia de Aminoácidos , Animales , Arginina/química , Arginina/genética , Cristalografía por Rayos X , Glicina/química , Glicina/genética , Humanos , Lisina/química , Lisina/genética , Ratones , Mutación , Dominios Proteicos , Alineación de Secuencia , Tiburones/genética , Especificidad por Sustrato , Porcinos , Proteínas de Unión al GTP rab/genéticaRESUMEN
Borates have been regarded as a rich source of functional materials due to their diverse structures and wide applications. Therein, zincobrates have aroused intensive interest owing to the effective structural and functional regulation effects of the strong-bonded zinc cations. In recent decades, numerous zincoborates with special crystal structures were obtained, such as Cs3Zn6B9O21 and AZn2BO3X2 (A = Na, K, Rb, NH4; X = Cl, Br) series with KBe2BO3F2-type layered structures were designed via substituting Be with Zn atoms, providing a feasible strategy to design promising non-linear optical materials; KZnB3O6 and Ba4Na2Zn4(B3O6)2(B12O24) with novel edge-sharing [BO4]5- tetrahedra were obtained under atmospheric pressure conditions, indicating that extreme conditions such as high pressure are not essential to obtain edge-sharing [BO4]5--containing borates; Ba4K2Zn5(B3O6)3(B9O19) and Ba2KZn3(B3O6)(B6O13) comprise two kinds of isolated polyborate anionic groups in one borate structure, which is rarely found in borates. Besides, many zincoborates emerged with particular physicochemical properties; for instance, Bi2ZnOB2O6 and BaZnBO3F are promising non-linear optical (NLO) materials; Zn4B6O13 and KZnB3O6 possess anomalous thermal expansion properties, etc. In this review, the synthesis, crystal structure features and properties of representative zincoborates are summarized, which could provide significant guidance for the exploration and design of new zincoborates with special structures and excellent performance.
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Boratos/química , Dispositivos Ópticos , Zinc/química , Boratos/síntesis química , Cationes Bivalentes , Cristalización , Cristalografía por Rayos X , Humanos , Modelos MolecularesRESUMEN
Two new borate fluorides, Li2 BaSc(BO3 )2 F and LiBa2 Pb(BO3 )2 F, with layered structures featuring special Li-O/F configurations have been successfully prepared by the high-temperature solution method. Li2 BaSc(BO3 )2 F contains the first reported 2 ∞ [Li2 (BO3 )2 F] double layers that are evolved from 2 ∞ [Be2 BO3 F2 ] single layers in KBe2 BO3 F2 through substituting BeO4 with the LiO3 F tetrahedra. The adjacent double layers are tightly connected by strong Sc-O bonds, generating the strongest interlayer bond and smallest interlayer distance among KBe2 BO3 F2 derivatives with double-layered structures. LiBa2 Pb(BO3 )2 F features 2 ∞ [Li(BO3 )2 F] single layers built up of special LiO4 F2 octahedra and the BO3 triangles. To our best knowledge, the LiO4 F2 octahedron is observed for the first time in borates. Besides, the LiO4 F2 octahedra construct unique 1 ∞ [LiO4 F] straight chains through sharing apical F atoms, which is different from the configurations of the similar six-coordinated LiO6 octahedra in other borates.
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Metal-organic frameworks (MOFs) provide highly selective catalytic activity because of their porous crystalline structure. There is particular interest in metal nanoparticle-MOF composites (MNP@MOF) that could take advantage of synergistic effects for enhanced catalytic properties. We present an investigation into the local geometry and electronic properties of thermally decomposed Ni-MOF-74 calcined at different temperatures and time durations. Pair distribution function analysis using high-energy X-ray diffraction reveals the formation of fcc-Ni nanoparticles with a mixture of MOF phase in samples heated at 623 K for 12 h. Elevating the calcination temperature and lengthening the time duration assisted complete precipitation of Ni nanoparticles in the MOF matrix. Local structures and valence states were investigated using X-ray absorption fine structure spectroscopy. Evidence of ligand-to-metal charge transfer and gradual reduction of Ni2+ is apparent for those samples heated above 623 K for 12 h. In addition, the Ni lattice was found to be slightly compressed as a result of surface stresses in the nanosized particles or surface ligand environment. Electronic structure investigation using hard X-ray photoelectron spectroscopy shows a significant narrowing of the valence band and a decrease in the d-band center (toward the Fermi level) when the heating temperature is increased, thus suggesting promising catalytic properties for NiNP@MOF composite.
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ZL-004, a promising small molecule that increases white blood cell counts, was developed for extended-release nanosuspensions to improve low solubility and compliance of patients. In vivo pharmacokinetic studies of nanosuspensions with different particle sizes and administration volumes were conducted. Unexpectedly, Cmax of NS-PC-L (1156 nm) was 1.3 fold higher than NS-PB-L (836 nm), and area under plasma concentration-time curve (AUC) was similar. It suggested that in vivo behavior of nanosuspensions was influenced significantly by the original dissolved drug, which did not only rely on the particle size but also the amount of the free stabilizers. In addition, smaller administration volume (0.1 mL) achieved significantly lower Cmax and AUC than the higher volume (0.5 mL), due to the reduced amount of dissolved drug. DSC and XPRD demonstrated that the crystal forms of nanosuspensions prepared by the precipitation method and high-pressure homogenization were similar; therefore, in vivo behaviors did not show significant differences. An additional 0.15% PEG 4000 enhanced the redispersity and maintained the particle size for 3 months. Finally, a nanosuspensions with the desired initial release was achieved, which lasted approximately 32 days steadily after a single dose. AUC and t1/2 were 161.2 fold and 22.9 fold higher than oral administration.
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Sistemas de Liberación de Medicamentos/métodos , Pirrolidinonas/química , Pirrolidinonas/farmacocinética , Suspensiones/química , Liberación de Fármacos , Tamaño de la Partícula , SolubilidadRESUMEN
Five new mixed-metal borate crystals, namely, K2 BaB16 O26 , K3 CsB20 O32 , Na2 Cs2 BaB18 O30 , Na2 Rb2 PbB18 O30 , and Na2 Cs2 PbB18 O30 , have been synthesized by the high-temperature solution method. These structures were characterized by single-crystal X-ray diffraction and their B-O configurations are shown to feature complicated three-dimensional (3D) open-framework structures incorporating different types of channels. For all five compounds, the frameworks can be reduced to four-connected (4-c) uninodal nets with sra-, dia- and nbo-type topologies by assigning the [B5 O10 ]5- and [B3 O7 ]5- clusters as basic nodes. First-principles calculations have shown that the multiple cations in mixed-metal borates influence the ground-state electronic structures and associated optical absorptions. In addition, the crystal structures, thermal stabilities, and IR spectra of the title compounds have also been performed.
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Liver regeneration is a complicated process, and understanding the regulatory mechanism will be helpful in the treatment of diseases associated with liver. In this study, the one-third liver resection model was established in Chiloscyllium plagiosum, and the whole transcriptome of the C. plagiosum was generated using the Illumina-Solexa sequencing platform. Differentially expressed genes were analyzed using bioinformatics methods and verified using quantitative real-time PCR (qRT-PCR). Using miRanda and TargetScan, we screened the microRNA library for miRNAs that target the glutathione S-transferase P1(GSTP1) gene. Dual-luciferase reporter assays were used to confirm binding between the miRNA and GSTP1. Finally, we used western blotting analysis to determine expression of the GSTP1 protein. As a result, 65,356 unigenes were obtained in normal and damaged liver tissues, with mean length of 955 bp. A total of 359 differentially expressed genes were acquired; 217 of which were upregulated, and 142 were downregulated, including the GSTP1 gene, following liver resection. The presence of the GSTP1 protein in C. plagiosum was shown for the first time. Luciferase reporter assay revealed that GSTP1 messenger RNA was targeted by ipu-miR-143. The discovery and differential expression analysis of GSTP1 in C. plagiosum will be a valuable resource to explain the molecular mechanism of GSTP1 regulation of liver repair.
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Regulación Enzimológica de la Expresión Génica/fisiología , Glutatión Transferasa/metabolismo , Regeneración Hepática/fisiología , Hígado/metabolismo , MicroARNs/metabolismo , Tiburones/fisiología , Regiones no Traducidas 3' , Animales , ADN/genética , Glutatión Transferasa/genética , MicroARNs/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , TranscriptomaRESUMEN
Four new layered pentaborates Rb4Ba2.5B20O34Br, Rb2Ba4B20O34Br2, Rb4Ba2.5B20O34Cl, and KBaB5O9 have been successfully synthesized via a high-temperature solution method; the former three are the first series of compounds reported in the Rb-Ba-B-O-X (X = halogen) system. Interestingly, the structures of the above compounds are composed of the same [B5O11]7- fundamental building block (FBB), which could be further linked to form 2∞[B10O17]4- double layers for the former three compounds and 2∞[B5O9]3- single layers for the last one. The structure comparisons among all the available anhydrous pentaborates reveal that the structures of the anionic framework are affected by the relationship between the orientation of [B5O11]7- FBB axes and the layer plane (parallel or perpendicular), which will produce the different number of terminal O atoms in the initial pentaborate blocks. The viewpoints give us a feasible way to investigate the layered structures and expand the structural diversity of borates. Furthermore, the infrared spectra, UV-vis-NIR diffuse reflectance spectra, and thermal behaviors of these compounds were also studied.
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Birefringent materials with high optical anisotropy have been identified as a research hotspot owing to their significant scientific and technological significance in modern optoelectronics for manipulating light polarization. Researchers studying borate systems have discovered that adding π-conjugated units placed in parallel can significantly increase the birefringence of crystalline solids; some examples include [BO3] units, [B2O5] units, and [B3O6] units. However, there are not many borates with strictly parallel configurations of π-conjugated [B2O5] units. In this study, a new bimetallic borate Sr2Cd4(B2O5)3 with near-parallel arrangement of π-conjugated [B2O5] units was discovered. Sr2Cd4(B2O5)3 possesses the maximum number density of [B2O5] units, shortest dihedral angle of [B2O5] units (between the two [BO3]), and largest degree of [CdO6] octahedral distortion among all the currently known Sr-Cd-B-O tetragonal system borates, making it demonstrate a large birefringence of 0.102 at 532 nm. Theoretical analysis proves that π-conjugated [B2O5] anions are the primary source of the large birefringence of Sr2Cd4(B2O5)3.
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The interfacial structure formed by Pt nanoparticles grown epitaxially on a SrTiO3 (001) surface by pulsed laser deposition was studied by X-ray standing-wave (XSW) excited core-level photoelectron emission. The XSW-generated 3D atomic map of the Pt and interfacial oxygens for the oxidized Pt/SrTiO3 interface differs significantly from that of the as-deposited interface. After oxidation, the Pt atoms shifted upward and their atomic occupation at fcc-like sites evolved as the oxidation temperature increased. Interfacial oxygen atoms were differentiated from bulk O atoms by the chemical shift in the binding energy of their 1s electrons. After oxidation, the interfacial oxygen atoms rearranged to form a TiO2 bilayer at the interface. These results provide a more complete description of the strong metal-support interaction process at the interface.
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Background: Sepsis is organ dysfunction due to the host's deleterious response to infection, and the kidneys are one of the organs damaged in common sepsis. Sepsis-associated acute kidney injury (SA-AKI) increases the mortality in patients with sepsis. Although a substantial volume of research has improved the prevention and treatment of the disease, SA-SKI is still a significant clinical concern. Purpose: Aimed to use weighted gene co-expression network analysis (WGCNA) and immunoinfiltration analysis to study SA-AKI-related diagnostic markers and potential therapeutic targets. Methods: Immunoinfiltration analysis was performed on SA-AKI expression datasets from the Gene Expression Synthesis (GEO) database. A weighted gene co-expression network analysis (WGCNA) analysis was performed on immune invasion scores as trait data, and modules associated with immune cells of interest were identified as hub modules. Screening hub geneset in the hub module using protein-protein interaction (PPI) network analysis. The hub gene was identified as a target by intersecting with significantly different genes screened by differential expression analysis and validated using two external datasets. Finally, the correlation between the target gene, SA-AKI, and immune cells was verified experimentally. Results: Green modules associated with monocytes were identified using WGCNA and immune infiltration analysis. Differential expression analysis and PPI network analysis identified two hub genes (AFM and GSTA1). Further validation using additional AKI datasets GSE30718 and GSE44925 showed that AFM was significantly downregulated in AKI samples and correlated with the development of AKI. The correlation analysis of hub genes and immune cells showed that AFM was significantly associated with monocyte infiltration and hence, selected as a critical gene. In addition, Gene single-enrichment analysis (GSEA) and PPI analyses results showed that AFM was significantly related to the occurrence and development of SA-AKI. Conclusions: AFM is inversely correlated with the recruitment of monocytes and the release of various inflammatory factors in the kidneys of AKI. AFM can be a potential biomarker and therapeutic target for monocyte infiltration in sepsis-related AKI.
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Lesión Renal Aguda , Sepsis , Humanos , Lesión Renal Aguda/genética , Bases de Datos Factuales , Riñón , Monocitos , Sepsis/complicacionesRESUMEN
Nervonic acid benefits the treatment of neurological diseases and the health of brain. In this study, we employed the oleaginous yeast Yarrowia lipolytica to overproduce nervonic acid oil by systematic metabolic engineering. First, the production of nervonic acid was dramatically improved by iterative expression of the genes ecoding ß-ketoacyl-CoA synthase CgKCS, fatty acid elongase gELOVL6 and desaturase MaOLE2. Second, the biosynthesis of both nervonic acid and lipids were further enhanced by expression of glycerol-3-phosphate acyltransferases and diacylglycerol acyltransferases from Malania oleifera in endoplasmic reticulum (ER). Third, overexpression of a newly identified ER structure regulator gene YlINO2 led to a 39.3% increase in lipid production. Fourth, disruption of the AMP-activated S/T protein kinase gene SNF1 increased the ratio of nervonic acid to lignoceric acid by 61.6%. Next, pilot-scale fermentation using the strain YLNA9 exhibited a lipid titer of 96.7 g/L and a nervonic acid titer of 17.3 g/L (17.9% of total fatty acids), the highest reported titer to date. Finally, a proof-of-concept purification and separation of nervonic acid were performed and the purity of it reached 98.7%. This study suggested that oleaginous yeasts are attractive hosts for the cost-efficient production of nervonic acid and possibly other very long-chain fatty acids (VLCFAs).
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Yarrowia , Yarrowia/genética , Ingeniería Metabólica , Ácidos Grasos/metabolismo , Aciltransferasas/metabolismoRESUMEN
The objective of this study was to explore an innovative sustained release technology and design a new microporous resin-based polymer device (RPD) for controlled release of glipizide (GZ). Photocurable resin was applied to prepare the resin layer to control GZ release. The impact of formulation parameters consisting of the type and amount of pore formers and pH modifiers, photocurable curing time as well as the weight of resin layer on GZ release were examined. The GZ-RPD was fabricated applying 24 mg of resin layer with PEG400 (100 % of the resin weight) as pore former and 10 mg of Na2CO3 as pH modifier. Scanning electron microscopy (SEM) demonstrated resin particles presenting a porous structure constituted the resin layer. The GZ-RPD possessed prolonged Tmax and reduced Cmax relative to commercial tablets. The relative bioavailability of the RPDs as well as commercial tablets was 93.65 % since the AUC0-24 h were 6111.05 ± 238.89 ng·h/mL and 6525.09 ± 760.59 ng h/mL, respectively. The release mechanism of the GZ-RPD was discussed. This paper provided an innovative concept to produce controlled GZ release oral formulation fabricated by photocurable resin, which demonstrated both excellent in vitro release and in vivo pharmacokinetics.
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Glipizida , Polímeros , Glipizida/química , Glipizida/farmacocinética , Preparaciones de Acción Retardada/química , Comprimidos , Excipientes/químicaRESUMEN
The exploration of A-Mo-P-O (A = Rb, Cs) systems has allowed several new Mo(V) phosphates, Rb2MoP2O9, Rb6Mo2P4O19 and Cs6Mo2P4O19, to be synthesized through the spontaneous nucleation method. Single-crystal X-ray diffraction analysis reveals that the identical stoichiometry compounds Rb2MoP2O9 and Cs2MoP2O9 belong to different space groups C2/c and Pbca, respectively. Both compounds consist of dissimilar 1D [Mo-O-P]∞ chains with different repeated building units, while monovalent cations fill in spaces to form 3D structures. However, Rb6Mo2P4O19 and Cs6Mo2P4O19 are isostructural and crystallize in the same space group of P21/c. They exhibit a 3D framework structure with 0D Mo2O5O6P4O8 groups, which are separated by Rb/Cs atoms. Interestingly, structural relationships between the different monophosphates of the A-Mo-P-O (A = Rb, Cs) systems are presented in which distinct polyanionic configurations appear owing to the A/P ratios, as well as the size of the univalent cations. Further, detailed structural comparisons, optical properties and theoretical calculations are also discussed.