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The presence of minute quantities of water in organic solvents can affect the progress of many reactions and cause unnecessary losses and even safety accidents in the chemical industry, especially in the productions process of organic fine chemicals. Therefore, it is necessary to carry out high-performance strategies for trace water detections in commonly used organic solvents. In this work, a fluorescent sensing system based on competitive binding of protons has been developed, demonstrating remarkable responses by UV-vis absorption and fluorescence two-modes toward a trace amount of water in organic solvents including 1,4-dioxane (Diox), tetrahydrofuran (THF), acetonitrile (MeCN), acetone (ACE), dimethylsulfoxide (DMSO) and mixed organic solvents (THF: MeCN=1: 1). The key component of the sensing system is a newly designed fluorophore NBD-PMA, which can be deprotonated to form a dynamic non-luminescent adduct, namely NBD-PMA-F, by an organic fluoride salt tetrabutylammonium fluoride (TBAF). NBD-PMA-F can be reprotonated via using trace water, exhibiting fluorescence turn on of the system. The as-prepared sensing system shows superior sensitivity, low detection limits (v/v, 0.0007 %), quick response speed (≤1.2 s) and good reversibility. Moreover, naked-eye visual rapid detection has also been successfully realized at ambient temperature, which demonstrated their practical applications value for trace water determinations.
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Copper(II) chloride anionic coordination complexes with different imidazole-derived ligands due to the potential cytotoxic activity play the important role in protein. By investigating the experimental electron paramagnetic resonance (EPR) and ultraviolet-visible (UV-vis) spectra of [CuCl(C6H10N2)4]Cl, [CuCl(C6H10N2)4]Cl, [CuCl2(C4H6N2)4], and [Cu2Cl2(C5H8N2)6]Cl2·2H2O, the local structure of the corresponding Cu2+ centers and the role of different ligands are obtained. Based on the well-agreed EPR parameters and the d-d transitions (10Dq), the four Cu2+ centers show tetragonal and orthorhombic distortion, corresponding to the different anisotropies of EPR signals. In addition, the general rules of governing the impact of methanol in imidazolylalkyl derivatives are also discussed, especially the influence on the local environment (symmetry, distortion, covalency, and crystal field) of above four copper(II) chloride anionic coordination complexes. Therefore, the obtained results in this study will be beneficial to provide a theoretical basis for the experimental design of desired copper-containing imidazolyl alkyl derivatives.
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INTRODUCTION: Dry eye disease (DED) is a multifactor-induced disease accompanied by increased osmolarity of the tear film and inflammation of the ocular surface. Traditional anti-inflammation agent corticosteroids applied in DED treatment could result in high intraocular pressure, especially in long-term treatment. Therefore, we explored a nano drug that aimed to block the formation pathway of DED which had anti-inflammatory, sustained release, and good biocompatibility characteristics in this study. METHODS: We prepared a novel nanomedicine (Tet-ATS@PLGA) by the thin film dispersion-hydration ultrasonic method and detected its nanostructure, particle size, and zeta potential. Flow cytometry was used to detect the cell survival rate of each group after 24 h of drug treatment on inflammed Statens Seruminstitut Rabbit Corneal (SIRC) cells. Observed and recorded corneal epithelial staining, tear film rupture time, and Schirmer test to detect tear secretion on the ocular surface of rabbits. The corneal epithelial thickness, morphology, and number of bulbar conjunctival goblet cells were recorded by H&E staining. Finally, we detected the expression of VEGF, IL-1ß, PGE2, and TNF-α by cellular immunofluorescence staining and enzyme-linked immunosorbent assay (ELISA). RESULTS: The encapsulation efficiency and drug loading of Tet-ATS@PLGA were 79.85% and 32.47%, respectively. At eye surface temperature, Tet can easily release from Tet-ATS@PLGA while that it was difficult to release at storage temperature and room temperature. After 2 weeks medication, Tet-ATS@PLGA can effectively improve the tear film rupture time and tear secretion time in a DED model (p <0.05). Compared with the normal group (62.34 ± 4.86 mm), the thickness of corneal epithelium in ATS (29.47 ± 3.21 mm), Tet-ATS (46.23 ± 2.87 mm), and Tet-ATS@PLGA (55.76 ± 3.95 mm) gradually increased. Furthermore, the flow cytometry indicated that Tet-ATS@PLGA can effectively promote the apoptosis of inflammatory SIRC cells, and the cellular immunofluorescence and ELISA experiments showed that the expression intensity of inflammatory factors such as VEGF, IL-1ß, PGE2, and TNF-α decreased in this process. Interestingly, Tet also had the effect of reducing intraocular pressure. CONCLUSION: Tet-ATS@PLGA can effectively promote the apoptosis of inflammatory corneal epithelial cells, thus inhibiting the expression of inflammatory factors to block the formation of DED and improve the secretion of tear on the ocular surface.
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Síndromes de Ojo Seco , Nanopartículas , Animales , Conejos , Ácido Poliglicólico/análisis , Ácido Poliglicólico/metabolismo , Ácido Poliglicólico/uso terapéutico , Factor de Necrosis Tumoral alfa , Dinoprostona/análisis , Dinoprostona/metabolismo , Dinoprostona/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/metabolismo , Síndromes de Ojo Seco/diagnóstico , Lágrimas/metabolismo , Córnea/metabolismo , Antiinflamatorios/uso terapéutico , Nanopartículas/químicaRESUMEN
The cell walls of pathogenic and acidophilic bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae, contain lipoarabinomannan and arabinogalactan. These components are composed of D-arabinose, the enantiomer of the typical L-arabinose found in plants. The unique glycan structures of mycobacteria contribute to their ability to evade mammalian immune responses. In this study, we identified four enzymes (two GH183 endo-D-arabinanases, GH172 exo-α-D-arabinofuranosidase, and GH116 exo-ß-D-arabinofuranosidase) from Microbacterium arabinogalactanolyticum. These enzymes completely degraded the complex D-arabinan core structure of lipoarabinomannan and arabinogalactan in a concerted manner. Furthermore, through biochemical characterization using synthetic substrates and X-ray crystallography, we elucidated the mechanisms of substrate recognition and anomer-retaining hydrolysis for the α- and ß-D-arabinofuranosidic bonds in both endo- and exo-mode reactions. The discovery of these D-arabinan-degrading enzymes, along with the understanding of their structural basis for substrate specificity, provides valuable resources for investigating the intricate glycan architecture of mycobacterial cell wall polysaccharides and their contribution to pathogenicity.
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Endometriosis , Mycobacterium tuberculosis , Animales , Femenino , Humanos , Galactanos , Lipopolisacáridos , MamíferosRESUMEN
BACKGROUND: Variants in the CASK gene result in a wide range of observed phenotypes in humans, such as FG Syndrome 4 and intellectual disabilities. Intellectual developmental disorder with microcephaly and pontine and cerebellar hypoplasia (MICPCH) is an X-linked disorder that affects females and is characterized by severely impaired intellectual development and variable degrees of pontocerebellar hypoplasia. Variants in CASK are the main genetic cause of MICPCH. Variants in CASK can explain most patients with MICPCH, but there are still some patients whose disease aetiology cannot be explained. CASE PRESENTATION: An 11-month-old female diagnosed with MICPCH exhibited general developmental delays, microcephaly, and cerebellar hypoplasia. Whole-exome sequencing (WES) was used to find a novel heterozygous missense variant (NM_003688.3: c.638T>G) of CASK in this patient. Strikingly, this variant reduced the expression of CASK at the protein level but not at the mRNA level. By using protein structure prediction analysis, this study found that the amino acid change caused by the variant resulted in further changes in the stability of the protein structure, and these changes caused the downregulation of protein expression and loss of protein function. CONCLUSION: In this study, we first reported a novel heterozygous pathogenic variant and a causative mechanism of MICPCH. The amino acid change cause by this variant led to changes in the protein structure and a decrease in its stability, which caused a loss of protein function. This study could be helpful to the genetic diagnosis of this disease.
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Discapacidad Intelectual , Microcefalia , Aminoácidos/genética , Cerebelo/anomalías , Discapacidades del Desarrollo/complicaciones , Discapacidades del Desarrollo/genética , Femenino , Guanilato-Quinasas/química , Guanilato-Quinasas/genética , Humanos , Lactante , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/genética , Discapacidad Intelectual Ligada al Cromosoma X , Microcefalia/complicaciones , Microcefalia/genética , Malformaciones del Sistema Nervioso , FenotipoRESUMEN
Although bFGF is highly expressed in the melanoma tissues, its specific role in melanoma progression is still not completely clarified. Here, we investigated the consequent cellular responses in melanoma B16 cells after bFGF blocking by using a neutralizing monoclonal antibody (mAb). Results showed that bFGF mAb concentration dependent inhibited tumor cell growth. Meanwhile, cell viability suppression was accompanied by reduced levels of proangiogenic factors in low-concentration bFGF mAb-treated cancer cells and increased levels of proangiogenic factors in high-concentration bFGF mAb-treated cells. Furthermore, low-concentration bFGF mAb induced autophagy but not apoptosis; conversely, high-concentration bFGF mAb led to activation of autophagy and apoptosis. Finally, we found that different degrees of bFGF blockade-induced autophagy play distinct roles in promoting cell survival and cell death. Our findings revealed different adaptive responses to bFGF blockade in melanoma cells, which should be taken seriously when developing bFGF-targeting agents for melanoma treatment.
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Factor 2 de Crecimiento de Fibroblastos , Melanoma , Humanos , Proliferación Celular , Células Tumorales Cultivadas , Apoptosis , Melanoma/tratamiento farmacológico , Melanoma/patología , Anticuerpos Monoclonales/farmacologíaRESUMEN
Genetic modification of industrial yeast strains often faces more difficulties than that of laboratory strains. Thus, new approaches are still required. In this research, the Angel Yeast-derived haploid strain Kα was genetically modified by multiple rounds of δ-integration, which was achieved via URA3 recycling. Three δ-integrative plasmids, pGδRU, pGδRU-BGL, and pGδRU-EG, were first constructed with two 167 bp δ sequences and a repeat-URA3-repeat fragment. Then, the δ-integrative strains containing the bgl1 or egl2 gene were successfully obtained by one-time transformation of the linearized pGδRU-BGL or pGδRU-EG fragment, respectively. Their counterparts in which the URA3 gene was looped out were also easily isolated by selection for growth on 5´-fluoroorotic acid plates, although the ratio of colonies lacking URA3 to the total number of colonies decreased with increasing copy number of the corresponding integrated cellulase-encoding gene. Similar results were observed during the second round of δ-integration, in which the δ-integration strain Kα(δ::bgl1-repeat) obtained from the first round was transformed with a linearized pGδRU-EG fragment. After 10 rounds of cell growth and transfer to fresh medium, the doubling times and enzyme activities of Kα(δ::bgl1-repeat), Kα(δ::egl2-repeat), and Kα(δ::bgl1-repeat)(δ::egl2-repeat) showed no significant change and were stable. Further, their maximum ethanol concentrations during simultaneous saccharification and fermentation of pretreated corncob over a 7-day period were 46.35, 33.13, and 51.77 g/L, respectively, which were all substantially higher than the parent Kα strain. Thus, repetitive δ-integration with URA3 recycling can be a feasible and valuable method for genetic engineering of Angel Yeast. These results also provide clues about some important issues related to δ-integration, such as the structural stability of δ-integrated genes and the effects of individual integration-site locations on gene expression. Further be elucidation of these issues should help to fully realize the potential of δ-integration-based methods in industrial yeast breeding.
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Celulasa/genética , Cromosomas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Celulasa/metabolismo , Cromosomas/metabolismo , Ingeniería Genética , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
RsgA plays an important role in maturation of 30S subunit in many bacteria that assists in the release of RbfA from the 30S subunit during a late stage of ribosome biosynthesis. Here, we genetically characterized functional roles of RsgA in Ralstonia solanacearum, hereafter designated RsRsgA. Deletion of R. solanacearum rsgA or rbfA resulted in distinct deficiency of 16S ribosomal RNA, significantly slowed growth in broth medium, and diminished growth in nutrient-limited medium, which are similar as phenotypes of rsgA mutants and rbfA mutants of Escherichia coli and other bacteria. Our gene-expression studies revealed that RsRsgA is important for expression of genes encoding the type III secretion system (T3SS) (a pathogenicity determinant of R. solanacearum) both in vitro and in planta. Compared with the wild-type R. solanacearum strain, proliferation of the rsgA and rbfA mutants in tobacco leaves was significantly impaired, while they failed to migrate into tobacco xylem vessels from infiltrated leaves, and hence, these two mutants failed to cause any bacterial wilt disease in tobacco plants. It was further revealed that rsgA expression was highly enhanced under nutrient-limited conditions compared with that in broth medium and RsRsgA affects T3SS expression through the PrhN-PrhG-HrpB pathway. Moreover, expression of a subset of type III effectors was substantially impaired in the rsgA mutant, some of which are responsible for R. solanacearum GMI1000 elicitation of a hypersensitive response (HR) in tobacco leaves, while RsRsgA is not required for HR elicitation of GMI1000 in tobacco leaves. All these results provide novel insights into understanding various biological functions of RsgA proteins and complex regulation on the T3SS in R. solanacearum.
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Proteínas Bacterianas/genética , GTP Fosfohidrolasas/genética , Ralstonia solanacearum , Ribosomas/metabolismo , Sistemas de Secreción Tipo III , Enfermedades de las Plantas/microbiología , Ralstonia solanacearum/genética , Nicotiana/microbiología , VirulenciaRESUMEN
Intestinal ischemia/reperfusion (I/R), which is associated with high morbidity and mortality, is also accompanied with abnormal energy metabolism and liver injury. Irisin, a novel exercise-induced hormone, can regulate adipose browning and thermogenesis. The following study investigated the potential role of dexmedetomidine in liver injury during intestinal I/R in rats. Adult male Sprague-Dawley rats underwent occlusion of the superior mesenteric artery for 90 min followed by 2 h of reperfusion. Dexmedetomidine or irisin-neutralizing antibody was intravenously administered for 1 h before surgery. The results demonstrated that severe intestine and liver injuries occurred during intestinal I/R as evidenced by pathological scores and an apparent increase in serum diamine oxidase (DAO), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels. In addition, the hepatic irisin, cleaved caspase-3, Bax, and NLRP3 inflammasome components (including NLRP3, ASC, and caspase-1), protein expressions, apoptotic index, reactive oxygen species (ROS), malondialdehyde (MDA), myeloperoxidase (MPO), tumor necrosis factor- (TNF-) α, and interleukin- (IL-) 6 levels increased; however, the serum irisin level and hepatic Bcl-2 protein expression and superoxide dismutase (SOD) activity decreased after intestinal I/R. Interestingly, dexmedetomidine could reduce the above listed changes and increase the irisin levels in plasma and the liver in I/R rats. Dexmedetomidine-mediated protective effects on liver injury and NLRP3 inflammasome activation during intestinal I/R were partially abrogated via irisin-neutralizing antibody treatment. The results suggest that irisin might contribute to the hepatoprotection of dexmedetomidine during intestinal ischemia/reperfusion.
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Anticuerpos Neutralizantes/uso terapéutico , Dexmedetomidina/uso terapéutico , Fibronectinas/metabolismo , Intestinos/efectos de los fármacos , Intestinos/patología , Isquemia/tratamiento farmacológico , Daño por Reperfusión/tratamiento farmacológico , Alanina Transaminasa/metabolismo , Animales , Aspartato Aminotransferasas/metabolismo , Fibronectinas/antagonistas & inhibidores , Fibronectinas/sangre , Isquemia/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Malondialdehído/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/sangre , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
LysR-type transcriptional regulators (LTTRs) are ubiquitous and abundant amongst bacteria and control a variety of cellular processes. Here, we investigated the effect of Rsc1880 (a putative LTTR, hereafter designated as PrhO) on the pathogenicity of Ralstonia solanacearum. Deletion of prhO substantially reduced the expression of the type III secretion system (T3SS) both in vitro and in planta, and resulted in significantly impaired virulence in tomato and tobacco plants. Complementary prhO completely restored the reduced virulence and T3SS expression to that of the wild-type. Moreover, PrhO-dependent T3SS and virulence were conserved amongst R. solanacearum species. However, deletion of prhO did not alter biofilm formation, swimming mobility and in planta growth. The expression of some type III effectors was significantly reduced in prhO mutants, but the hypersensitive response was not affected in tobacco leaves. Consistent with the key regulatory role of HrpB on T3SS, PrhO positively regulated the T3SS through HrpB. Furthermore, PrhO regulated hrpB expression via two close paralogues, HrpG and PrhG, which are two-component response regulators and positively regulate hrpB expression in a parallel manner. However, deletion of prhO did not alter the expression of phcA, prhJ and prhN, which are also involved in hrpB regulation. In addition, PrhO was expressed in a cell density-dependent manner, but negatively repressed by itself. No regulation was observed for HrpB, PhcA and PrhN on prhO expression. Taken together, we genetically demonstrated that PrhO is a novel virulence regulator of R. solanacearum, which positively regulates T3SS expression through HrpG, PrhG and HrpB and contributes to virulence.