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Starch and alcohol serve as pivotal indicators in assessing the quality of lees fermentation. In this paper, two hyperspectral imaging (HSI) techniques (visible-near-infrared (Vis-NIR) and NIR) were utilized to acquire separate HSI data, which were then fused and analyzed toforecast the starch and alcohol contents during the fermentation of lees. Five preprocessing methods were first used to preprocess the Vis-NIR, NIR, and the fused Vis-NIR and NIR data, after which partial least squares regression models were established to determine the best preprocessing method. Following, competitive adaptive reweighted sampling, successive projection algorithm, and principal component analysis algorithms were used to extract the characteristic wavelengths to accurately predict the starch and alcohol levels. Finally, support vector machine (SVM)-AdaBoost and XGBoost models were built based on the low-level fusion (LLF) and intermediate-level fusion (ILF) of single Vis-NIR and NIR as well as the fused data. The results showed that the SVM-AdaBoost model built using the LLF data afterpreprocessing by standard normalized variable was most accurate for predicting the starch content, with an R P 2 $\ R_P^2$ of 0.9976 and a root mean square error of prediction (RMSEP) of 0.0992. The XGBoost model built using ILF data was most accurate for predicting the alcohol content, with an R P 2 $R_P^2$ of 0.9969 and an RMSEP of 0.0605. In conclusion, the analysis of fused data from distinct HSI technologies facilitates rapid and precise determination of the starch and alcohol contents in fermented grains.
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Fermentación , Imágenes Hiperespectrales , Espectroscopía Infrarroja Corta , Almidón , Máquina de Vectores de Soporte , Almidón/análisis , Imágenes Hiperespectrales/métodos , Espectroscopía Infrarroja Corta/métodos , Grano Comestible/química , Alimentos Fermentados/análisis , Alcoholes/análisis , Análisis de Componente Principal , Algoritmos , Análisis de los Mínimos CuadradosRESUMEN
4,4-Dimethylsterols constitute a unique class of phytosterols responsible for regulating endogenous cannabinoid system (ECS) functions. However, precise mechanism through which 4,4-dimethylsterols affect fat metabolism and the linkage to the ECS remain unresolved. In this study, we identified that 4,4-dimethylsterols, distinct from 4-demethseterols, act as inhibitors of fatty acid amide hydrolases (FAAHs) both in vivo and in vitro. Genetic ablation of FAAHs (faah-1) abolishes the effects of 4,4-dimethylsterols on fat accumulation and locomotion behavior in a Caenorhabditis elegans model. We confirmed that dietary intervention with 4,4-dimethylsterols in a high-fat diet (HFD) mouse model leads to a significant reduction in body weight (>11.28%) with improved lipid profiles in the liver and adipose tissues and increased fecal triacylglycerol excretion. Untargeted and targeted metabolomics further verified that 4,4-dimethylsterols influence unsaturated fatty acid biosynthesis and elevate oleoyl ethanolamine levels in the intestine. We propose a potential molecular mechanism in which 4,4-dimethylsterols engage in binding interactions with the catalytic pocket (Ser241) of FAAH-1 protein due to the shielded polarity, arising from the presence of 2 additional methyl groups (CH3). Consequently, 4,4-dimethylsterols represent an unexplored class of beneficial phytosterols that coordinate with FAAH-1 activity to reduce fat accumulation, which offers new insight into intervention strategies for treating diet-induced obesity.
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BACKGROUND: Wheat is one of the key ingredients used to make Chinese liquor, and its saccharification power and protein content directly affect the quality of the liquor. In pursuit of a non-destructive assessment of wheat components and the optimization of raw material proportions in liquor, this study introduces a precise predictive model that integrates hyperspectral imaging (HSI) with stacked ensemble learning (SEL). RESULTS: This study extracted hyperspectral information from 14 different varieties of wheat and employed various algorithms for preprocessing. It was observed that multiplicative scatter correction (MSC) emerged as the most effective spectral preprocessing method. The feature wavelengths were extracted from the preprocessed spectral data using three different feature extraction methods. Then, single models (support vector machine (SVM), backpropagation neural network (BPNN), random forest (RF), and gradient boosting tree (XGBoost)) and a SEL model were developed to compare the prediction accuracies of the SEL model and the single models based on the full-band spectral data and the characteristic wavelengths. The findings indicate that the MSC-competitive adaptive reweighted sampling-SEL model demonstrated the highest prediction accuracy, with Rp 2 (test set-determined coefficient) values of 0.9308 and 0.9939 for predicting the saccharification power and protein content and root mean square error of the test set values of 0.0081 U and 0.0116 g kg-1, respectively. CONCLUSION: The predictive model established in this study, integrating HSI and SEL models, accurately detected wheat saccharification power and protein content. This validation underscores the practical potential of the SEL model and holds significant importance for non-destructive component analysis of raw materials used in liquor. © 2024 Society of Chemical Industry.
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Imágenes Hiperespectrales , Triticum , Algoritmos , Redes Neurales de la Computación , Máquina de Vectores de Soporte , Análisis de los Mínimos CuadradosRESUMEN
Tumor microparticles (T-MPs) are considered as a tumor vaccine candidate. Although some studies have analyzed the mechanism of T-MPs as tumor vaccine, we still lack understanding of how T-MPs stimulate a strong anti-tumor immune response. Here, we show that T-MPs induce macrophages to release a key chemotactic factor CCL2, which attracts monocytes to the vaccine injection site and enhances endocytosis of antigen. Monocytes subsequently enter the draining lymph node, and differentiate into monocyte-derived DCs (moDCs), which present tumor antigens to T lymphocytes and deliver a potent anti-tumor immune response. Mechanically, T-MPs activate the cGAS-STING signaling through DNA fragments, and then induce monocytes to upregulate the expression of IRF4, which is a key factor for monocyte differentiation into moDCs. More importantly, monocytes that have endocytosed T-MPs acquire the ability to treat tumors. Collectively, this work might provide novel vaccination strategy for the development of tumor vaccines and facilitate the application of T-MPs for clinic oncotherapy. Supplementary Information: The online version contains supplementary material available at 10.1186/s12645-023-00190-x.
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Essential oils (EOs) have been proved as natural food preservatives because of their effective and wide-spectrum antimicrobial activity. They have been extensively explored for potential applications in food industry, and substantial progresses have been achieved. However well EOs perform in antibacterial tests in vitro, it has generally been found that a higher level of EOs is needed to achieve the same effect in foods. Nevertheless, this unsimilar effect has not been clearly quantified and elaborated, as well as the underlying mechanisms. This review highlights the influence of intrinsic properties (e.g., oils and fats, carbohydrates, proteins, pH, physical structure, water, and salt) and extrinsic factors (e.g., temperature, bacteria characteristics, and packaging in vacuum/gas/air) of food matrix systems on EOs action. Controversy findings and possible mechanism hypotheses are also systematically discussed. Furthermore, the organoleptic aspects of EOs in foods and promising strategies to address this hurdle are reviewed. Finally, some considerations about the EOs safety are presented, as well as the future trends and research prospects of EOs applications in foods. The present review aims to fill the evidenced gap, providing a comprehensive overview about the influence of the intrinsic and extrinsic factors of food matrix systems to efficiently orientate EOs applications.
Both intrinsic properties and extrinsic factors of food matrix affect the EOs action.EOs influence on the food organoleptic aspects were reviewed.Promising strategies for overcoming the organoleptic aspects of EOs were listed.Future research prospects are outlined to accelerate EOs application in foods.
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BACKGROUND: The trajectory of blood pressure (BP) from childhood to early middle age suggested that individuals with elevated BP in early childhood were more likely to be affected by cardiovascular disease in adulthood. Exposure to metals may affect BP in children, and pregnancy is a sensitive time for metal exposure. This study assessed the relationship between different stages of prenatal exposure to metals or metal mixtures and BP in children aged 5-6 years. METHODS: The study included 2535, 2680, 2534 mother-child pairs in three trimesters, from the Ma'anshan birth cohort study (MABC). We collected maternal blood samples during pregnancy and measured the serum levels of four metals (arsenic, selenium, cadmium, and mercury). BP was measured in children aged 5-6 years. A linear regression model and Bayesian kernel machine regression (BKMR) were used to explore associations between prenatal exposure to metals at different stages and multiple metal exposure with BP in children aged 5-6 years. RESULTS: Associations were observed between the arsenic in the third trimester and children's diastolic blood pressure (DBP) (ß = 0.88, 95% CI: 0.44, 1.33), systolic blood pressure (SBP) (ß = 0.72, 95% CI: 0.19, 1.24) and mean arterial pressure (MAP) (ß = 0.83, 95% CI: 0.42, 1.23), as well as between the mercury and children's DBP (ß = 0.65, 95% CI: 0.13, 1.16) and MAP (ß = 0.60, 95% CI: 0.14, 1.07). The BKMR analysis showed that multiple metals had a significant positive joint effect on children's DBP, SBP and MAP. A potential interaction between arsenic and mercury was observed (ß = -0.85, 95% CI: -1.62, -0.08). CONCLUSIONS: Exposure to arsenic and mercury during pregnancy was associated with altered BP in children. The third trimester may represent an important window of opportunity to reduce the effects of metal exposure on children's blood pressure and long-term health.
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Arsénico , Mercurio , Efectos Tardíos de la Exposición Prenatal , Embarazo , Femenino , Humanos , Preescolar , Estudios de Cohortes , Presión Sanguínea , Arsénico/toxicidad , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/epidemiología , Teorema de Bayes , MetalesRESUMEN
OBJECTIVES: Barium is widely involved in drilling fluids, plastics, and personal care products. Although the neurodevelopmental toxicity of barium has been reported in animals, human data are scarce. This study aimed to investigate the effect of prenatal barium concentrations on preschoolers' intellectual function based on a birth cohort study. METHODS: A total of 2164 mother-child pairs from Ma'anshan city, China were included in this study. We measured serum barium concentrations in the first, second, and third trimesters and in cord blood. Intellectual function in children aged 3.0-6.0 years old was assessed using the Chinese version of the Wechsler Preschool and Primary Scale of Intelligence-Fourth Edition (WPPSI-IV). Linear regression models were used to analyze the association between averaged barium exposure during pregnancy and intellectual function. Multiple informant models were performed to jointly test for differences in associations between four repeated barium exposure and intellectual function. All models were further stratified by child sex. RESULTS: Collectively, we observed significant inverse associations of average maternal barium exposure levels with verbal comprehension index (VCI), visual spatial index (VSI), processing speed index (PSI), and full-scale intelligence quotient (FSIQ) scores. Maternal serum log10-transformed barium levels in the second trimester were inversely associated with VCI [-2.33 (95%CI: -4.02, -0.64)], VSI [-2.30 (95%CI: -4.08, -0.52)], working memory index (WMI) [-2.09 (95%CI: -3.71, -0.46)], PSI [-2.23 (95%CI: -3.82, -0.65)], and FSIQ scores [-2.73 (95%CI: -4.23, -1.22)]. Prenatal barium exposure was inversely associated with VCI, VSI, WMI, PSI, and FSIQ in girls, except for the fluid reasoning index (FRI). Additionally, inverse associations were found between prenatal barium exposure and VSI, PSI, and FSIQ in boys. CONCLUSIONS: Prenatal barium exposure had detrimental effects on intellectual function in preschoolers and girls drove these inverse associations more than boys. The second trimester may be the critical window of neurotoxicity to barium exposure.
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Sangre Fetal , Efectos Tardíos de la Exposición Prenatal , Embarazo , Masculino , Preescolar , Femenino , Humanos , Niño , Estudios de Cohortes , Bario , Cohorte de Nacimiento , Pruebas de Inteligencia , Exposición MaternaRESUMEN
Inhibition of α-glucosidase activity is closely related to the treatment of type 2 diabetes. However, the potential mechanism by which 4,4-dimethylsterols inhibit α-glucosidase has not been elucidated. In this work, the inhibitory activity and mechanism of 4,4-dimethylsterols against α-glucosidase were studied through kinetic analysis, fluorescence spectroscopy, ultraviolet spectroscopy, circular dichroism, and molecular docking. 4,4-Dimethylsterols showed higher inhibition activity against α-glucosidase than acarbose with an IC50 value of 0.71 mg/mL and a noncompetitive inhibition type. They could bind to α-glucosidase through van der Waals forces and hydrogen bonds and quench its endofluorescence with a static quenching mechanism. Changes in the secondary structure of α-glucosidase were induced by its binding interaction with 4,4-dimethylsterols. Molecular docking further indicated that a hydrogen bond was generated between OH at the C-3 position of 4,4-dimethylsterols and the α-glucosidase residue Arg-442. This study provides new insights into the potential utilization of 4,4-dimethylsterols as antidiabetic phytochemicals in dietary supplements.
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Inhibidores de Glicósido Hidrolasas/farmacología , Esteroles/farmacología , Dicroismo Circular , Cinética , Simulación del Acoplamiento Molecular , alfa-Glucosidasas/metabolismoRESUMEN
Consumption of 4-desmethylsterols has been claimed to have many beneficial effects, but the benefits of 4,4-dimethylsterols are less appreciated. We utilized a nematode model, Caenorhabditis elegans (C. elegans), to explore the anti-obesity effects of different classes of 4,4-dimethylsterols purified from rice bran oil (RST) and shea nut butter (SST). Both SST and RST significantly reduced fat deposition in C. elegans with smaller sizes and numbers of lipid droplets. But the food intake was not significantly affected. Metabolomics analysis indicated a significantly altered pathway after treatment with 4,4-dimethylsterols. Finally, it was found that 4,4-dimethylsterols targeted stearoyl-CoA desaturases (SCD) and nuclear hormone receptor-49 (NHR-49), resulting in a reduced desaturation index as proved by a lower ratio of oleic acid (C18:1n-9) to stearic acid (C18:0). Overall, 4,4-dimethylsterols can inhibit fat deposition via regulating the NHR-49/SCD pathway in C. elegans.
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Proteínas de Caenorhabditis elegans/metabolismo , Grasas/metabolismo , Fitosteroles , Aceites de Plantas/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Animales , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/metabolismo , Metaboloma/efectos de los fármacos , Fitosteroles/química , Fitosteroles/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
In this study, the 4,4-dimethylsterol oleates were efficiently synthesized through esterification of 4,4-dimethylsterols and oleoyl chloride. The impact of reaction parameters on the 4,4-dimethylsterol conversion were investigated. The 4,4-dimethylsterol conversion increased with pyridine dosage, molar ratio of oleoyl chloride to 4,4-dimethylsterols, and temperature. The highest conversion of 99.27% was obtained with molar ratio of 1.1:1 at 313â¯K for 60â¯min. A second-order kinetic model describing acyl chloride esterification featuring high correlation coefficients was established. Arrhenius-Van't Hoff plot suggested activation energy and pre-exponential factor were 15.54â¯kJâ¯mol-1 and 1.78â¯×â¯103 L mol-1 min-1, respectively. The molecular structure of 4,4-dimethylsterol oleates were finally identified by attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR), ultra-performance liquid chromatography system coupled with quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS), and nuclear magnetic resonance (NMR).
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Cloruros , Ácido Oléico , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Esterificación , Espectrometría de MasasRESUMEN
In vitro models of the liver have a good simulation of the micro-liquid environment inside the human liver and the communication between cell tissues, which provides an important research tool for drug research and liver disease treatment. In this paper, we designed a 3D liver chip and real-time monitoring system based on microfluidic technology. The in vitro model of the liver on the chip was established by the three-dimensional microfluidic chip pipeline and the corresponding microwell array. Meanwhile, the culture medium is continuously injected on the chip, and the electrochemical impedance spectroscopy and near-infrared spectroscopy of the liver chip are recorded and analyzed from day one to day five. When the 3D cultured liver chip in vitro model reached a certain period and stabilized, paracetamol with varying gradients of concentration was applied to the cultured cells for drug resistance testing. The experimental results show that the liver chip and its monitoring system designed in this paper can maintain 100% cell viability of hepatocytes in vitro for a long time. Furthermore, it can meet the requirements of measurement technologies such as electrical impedance measurement and near-infrared spectroscopy in real-time, providing a stable culture platform for the further study of organ chips.
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BACKGROUND: Rice blast fungus is a worldwide disease, and it is one of the most serious rice diseases in the north and south rice fields in China. The initial symptoms of rice blast are not obvious, and the speed of transmission is fast. Manual identification is time-consuming and laborious. At present, it is a great challenge to realize rapid and accurate early identification of rice blast. RESULTS: In this paper, an identification method based on crop disease spores' diffraction fingerprint texture for rice blast was studied; this method utilizes the light field and texture features of diffraction images. To verify the reliability of the model that we proposed, we selected two methods of manual identification and machine recognition to compare and detect rice blast spores. The experimental results show that the identification of light diffraction characteristics is not only higher than the traditional manual recognition by microscope (increased by more than 0.3%), but also faster after neural network training (increased by more than 90%). The diffraction recognition method used in this study, based on crop disease spores' diffraction fingerprint texture, can be completed in a few seconds, and its test accuracy is 97.18%. CONCLUSION: The proposed method, a rapid rice blast detection and identification method based on crop disease spores' diffraction fingerprint texture, has certain advantages compared with the existing manual identification by microscope. This method can be applied to the recognition of rice blast in agricultural research. © 2020 Society of Chemical Industry.
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Microscopía/métodos , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Esporas/aislamiento & purificación , China , Hojas de la Planta/microbiología , Esporas/clasificación , Esporas/citologíaRESUMEN
Pesticides vary in the level of poisonousness, while a conventional rapid test card only provides a general "absence or not" solution, which cannot identify the various genera of pesticides. In order to solve this problem, we proposed a seven-layer paper-based microfluidic chip, integrating the enzyme acetylcholinesterase (AChE) and chromogenic reaction. It enables on-chip pesticide identification via a reflected light intensity spectrum in time-sequence according to the different reaction efficiencies of pesticide molecules and assures the optimum temperature for enzyme activity. After pretreatment of figures of reflected light intensity during the 15 min period, the figures mainly focused on the reflected light variations aroused by the enzyme inhibition assay, and thus, the linear discriminant analysis showed satisfying discrimination of imidacloprid (Y = -1.6525X - 139.7500), phorate (Y = -3.9689X - 483.0526), and avermectin (Y = -2.3617X - 28.3082). The correlation coefficients for these linearity curves were 0.9635, 0.8093, and 0.9094, respectively, with a 95% limit of agreement. Then, the avermectin class chemicals and real-world samples (i.e., lettuce and rice) were tested, which all showed feasible graphic results to distinguish all the chemicals. Therefore, it is feasible to distinguish the three tested kinds of pesticides by the changes in the reflected light spectrum in each min (15 min) via the proposed chip with a high level of automation and integration.
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Inhibidores Enzimáticos/análisis , Dispositivos Laboratorio en un Chip , Óptica y Fotónica/métodos , Papel , Residuos de Plaguicidas/análisis , Análisis por Conglomerados , Ivermectina/análogos & derivados , Ivermectina/análisis , Ivermectina/química , Neonicotinoides/análisis , Neonicotinoides/química , Nitrocompuestos/análisis , Nitrocompuestos/química , Forato/análisis , Forato/química , Factores de TiempoRESUMEN
The low expression of exogenous transferred gene limited the application of transgenic animal technology. Suppressor of variegation 3 â¼ 9 homolog 1(SUV39H1) gene plays a prominent role on repressive heterochromatin and transcription. To understand if exogenous transgenic gene expression was affected by SUV39H1 epigenetic modification, in this paper, the effective shRNA fragments targeting SUV39H1 gene were first screened, their roles on expression of exogenous transgenic genes were determined by using Bcap-37 cell line with stable expressing IFNα-2b gene as a model, the preliminary regulation mechanism of SUV39H1 gene was investigated. The results showed that the designed shRNA1/2 fragments of SUV39H1 gene had an obvious inhibition effect on the expression of SUV39H1 gene, reached 53.07 and 31.28%, respectively by qRT-PCR analysis. Compared with the control group, the expression of IFNα-2b gene in transgenic Bcap-37 cells infected with shRNA1 and 2 viruses significantly increased by 96.25 and 121.08%, respectively (p < 0.05). In addition, the expression of DNMT1, HDAC1 and G9a gene in the shRNA infected cells reduced significantly, and the expression of the HAT1 gene increased significantly (p < 0.05). The above results indicated that the expression of exogenous transgenic gene could be promoted by suppressing SUV39H1 gene at the cell level.
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N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Interferón alfa-2/metabolismo , ARN Interferente Pequeño , Transgenes , Animales , Búfalos , Bovinos , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Expresión Génica , Células HEK293 , Histona Acetiltransferasas/metabolismo , Histona Desacetilasa 1/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , HumanosRESUMEN
Three water-soluble polysaccharide fractions (GSP-1, GSP-2 and GSP-3) were obtained from Gentiana scabra Bunge roots by DEAE-Sepharose CL-6B and Sepharose CL-6B column chromatography. Their chemical characterizations were determined by high performance gel permeation chromatography (HPGPC), high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and Fourier transform infrared (FT-IR) spectrometer. Moreover, their in vitro anticoagulant activities were evaluated by activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) assays. GSP-1 and GSP-2 were composed of rhamnose, arabinose, galactose, glucose and galacturonic acid, while GSP-3 consisted of rhamnose, arabinose, galactose and galacturonic acid with a weight-average molecular weight of 5.8×10(4)Da. In comparison with the control group (saline), GSP, GSP-1, GSP-2 and GSP-3 could prolong APTT and TT, but not PT. Overall, GSP-3 exhibited potent anticoagulant activity and would be expected to be a potential source of anticoagulant.
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Anticoagulantes/aislamiento & purificación , Anticoagulantes/farmacología , Gentiana/química , Raíces de Plantas/química , Polisacáridos/aislamiento & purificación , Polisacáridos/farmacología , Anticoagulantes/química , Humanos , Peso Molecular , Monosacáridos/química , Polisacáridos/química , ProtonesRESUMEN
Auxin response factors (ARFs) are an important family of proteins in auxin-mediated response, with key roles in various physiological and biochemical processes. To date, a genome-wide overview of the ARF gene family in citrus was not available. A systematic analysis of this gene family in citrus was begun by carrying out a genome-wide search for the homologs of ARFs. A total of 19 nonredundant ARF genes (CiARF) were found and validated from the sweet orange. A comprehensive overview of the CiARFs was undertaken, including the gene structures, phylogenetic analysis, chromosome locations, conserved motifs of proteins, and cis-elements in promoters of CiARF. Furthermore, expression profiling using real-time PCR revealed many CiARF genes, albeit with different patterns depending on types of tissues and/or developmental stages. Comprehensive expression analysis of these genes was also performed under two hormone treatments using real-time PCR. Indole-3-acetic acid (IAA) and N-1-napthylphthalamic acid (NPA) treatment experiments revealed differential up-regulation and down-regulation, respectively, of the 19 citrus ARF genes in the callus of sweet orange. Our comprehensive analysis of ARF genes further elucidates the roles of CiARF family members during citrus growth and development process.
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BACKGROUND: Cluster of differentiation 14 (CD14) functions as a co-receptor for Toll-like receptor (TLR)-4 and myeloid differentiation factor (MD)-2 in detecting bacterial lipopolysaccharide. Together, these complexes promote the phagocytosis and digestion of Gram-negative bacteria, and initiate immune responses. To date, much of our understanding of CD14 function during Gram-negative bacterial inflammation comes from studies on mouse knockout models and cell transfection. To identify the effect of CD14 knockdown in this process in large livestock animals, we established a mouse model expressing bovine CD14 short hairpin (sh) RNA. shRNA fragments targeting bovine CD14 were screened by co-transfection in HEK 293 cells, and the most effective CD14 shRNA fragment was cloned into the eukaryotic expression vector pSilencer4.1-CD14 shRNA-IRES (internal ribosome entry site) and transferred into mouse zygotes by pronuclear microinjection to obtain transgenic mice. Expression of the enhanced green fluorescent protein (EGFP) reporter and genes related to the TLR4 signaling pathway was detected by immunohistochemistry (IHC) and quantitative polymerase chain reaction (PCR), respectively. RESULTS: One effective shRNA fragment (shRNA-674) targeting bovine CD14 was obtained, the sequence of which was shown to be conserved between cows, buffalos, sheep, and humans. Thirty-seven founder pups were obtained by pronuclear microinjection, of which three were positive for the transgene. In the F(1) generation, 11 of 33 mice (33%) were positive for the transgene as detected by PCR. IHC analysis detected exogenous EGFP expression in the liver, kidney, and spleen of transgenic F(1) mice, indicating that they were chimeric. The expression of endogenous CD14 mRNA in the heart, liver, spleen, lung, and kidney of transgenic F(1) mice was decreased 8-, 3-, 19.5-, 6-, and 11-fold, respectively. The expression patterns of endogenous MD-2, TLR4, interleukin-6 and tumor necrosis factor-α genes in transgenic mice also varied. CONCLUSIONS: This study confirms that transgenic mice expressing bovine CD14 shRNA can be generated by pronuclear microinjection, and demonstrates inhibited endogenous mouse CD14 expression that alters gene expression related to the TLR4 signaling pathway.
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Receptores de Lipopolisacáridos/genética , ARN Interferente Pequeño/genética , Animales , Bovinos/genética , Femenino , Expresión Génica , Técnicas de Transferencia de Gen/veterinaria , Células HEK293 , Humanos , Masculino , Ratones/genética , Ratones Transgénicos/genéticaRESUMEN
The crude tea polysaccharides were extracted from the leaves of Camellia sinensis using deionized water. The tea polysaccharides (TPS) were further separated and purified by anion exchange chromatograph on DEAE sepharose CL-6B column to afford TPS-1, TPS-2, TPS-3 and TPS-4. The high performance gel permeation chromatograph analysis showed that the average molecular weight of polysaccharides (TPS-1, TPS-2 and TPS-3) were 20,760, 24,230 and 250,643, respectively. TPS-4 was 689, 113 and 4150, suggesting it was heterogeneous. Monosaccharide analysis detected fucose, glucosamine, rhamnose, arabinose, galactosamine, galactose, glucose, xylose, mannose, ribose, galacturonic acid and glucuronic acid in the four polysaccharide fractions. Anticoagulant activities in vitro tests showed TPS-4 could significantly prolong APTT and TT, but not PT. The result indicated TPS-4 in the regulation of coagulation initiated via the intrinsic pathway. With current findings TPS-4 should be explored as a natural potential anticoagulant.