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1.
J Chromatogr A ; : 460471, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31471133

RESUMO

The influence of chlorine substituents in chiral separation of three racemic 2-(chlorophenyl)propanoic acids by countercurrent chromatography using hydroxypropyl-ß-cyclodextrin as a chiral additive were mainly investigated in the present paper, including 2-(2-chlorophenyl)propanoic acids, 2-(3-chlorophenyl)propanoic acids and 2-(4-chlorophenyl)propanoic acids. The influences of chromatographic conditions on the retention behavior were studied by enantioselective liquid-liquid extraction experiments using the methodology of response surface It was found that 2-(3-chlorophenyl)propanoic acids could be successfully chiral separated by countercurrent chromatography, while no resolution was achieved for racemic 2-(2-chlorophenyl)propanoic acids and 2-(4-chlorophenyl)propanoic acids under optimized separation conditions. The formation of 1:1 stoichiometric inclusion compounds between 2-(3-chlorophenyl)propanoic acids and HP-ß-CD was determined by UV spectra measurements. The inclusion constants for 2-(3-chlorophenyl)propanoic acids and HP-ß-CD were determined by the Benesi-Hildebrand equation. Meanwhile, the inclusion constants of 2-(3-chlorophenyl)-propanoic acid enantiomer and HP-ß-CD were obtained by the pesudophase retention equation in countercurrent chromatography. Furthermore, the inclusion interactions of the three racemates with HP-ß-CD were also investigated by the molecular docking. The results obtained from UV spectra measurements and molecular docking showed that the racemate with chlorine substituents in meta-position presented the highest enantiorecognition while the racemates with chlorine substituents in ortho-position had the lowest enantiorecognition. The above results further indicated that forming a stable inclusion complex between racemate and chiral selector is a prerequisite for a successful enantioseparation and at the same time, the difference in inclusion capacity between the two enantiomers is also essential for the enantioseparation.

2.
Med Sci Monit ; 25: 5666-5673, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31363077

RESUMO

BACKGROUND Epithelial ovarian cancer (EOC) is a gynecological malignancy that is associated with high mortality. Annexin A10 (ANXA10) is variably expressed in several types of human malignancy, but its role and clinical significance in EOC remain unknown. This study aimed to investigate the role of ANXA10 in EOC cells in vitro and to study the association between the protein expression levels of the ANXA10 in tumor tissue from patients with serous EOC and clinical outcome. MATERIAL AND METHODS The expression of ANXA10 was studied in 118 cases of serous EOC and in the ovarian cancer cell lines, SKOV-3, HO9810, HO8910PM, and OVCAR3 with immunohistochemistry and Western blot. Correlation between ANXA10 expression and clinicopathological variables and patient outcome were evaluated, including with Kaplan-Meier survival curves, univariate analysis with the log-rank test, and the multivariate analysis with the Cox-regression model. RESULTS ANXA10 was expressed by cells in the ovarian cancer cell lines. Patients with low expression and high expression of ANXA10 were 61.86% (73/118) and 38.14% (45/118), respectively. High expression of ANXA10 was correlated with poor response to chemotherapy (P=0.034), the presence of lymphatic invasion (P=0.043), and the International Federation of Gynecology and Obstetrics (FIGO) advanced stage (P=0.033), which were all associated with lower survival rates of serous EOC. Increased expression of ANXA10 was identified as an independent prognostic biomarker of serous EOC (HR=1.73; 95% CI, 1.01-2.98; P=0.046). CONCLUSIONS Increased expression of ANXA10 was an independent prognostic marker in patients with serous EOC.

3.
J BUON ; 24(3): 1027-1037, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31424657

RESUMO

PURPOSE: To evaluate the effect on breast cancer cell proliferation and apoptosis after silencing the HCCR-1 and to study its mechanism. METHODS: HCCR-1 siRNA was transfected into the breast cancer cell line MCF -7, and mRNA and protein level of HCCR-1 and Bax were evaluated by real-time quatitative PCR (qRT-PCR) and Western blotting, respectively. The cell proliferation and apoptosis were studied by MTT assay and flow cytometry. RESULTS: The apoptosis rate in the experimental, control and blank groups were 32.57±2.35%, 3.53±0.60% and 3.15±0.46% respectively. The apoptosis rate of MCF-7 cells was significantly increased in the experimental group, compared with the other two groups (p<0.05). The A490 value in the experimental, control and blank groups were: 24h: 0.78±0.06, 1.18±0.05, 1.24±0.05; 48h: 1.09±0.05, 1.48±0.02, 1.54±0.04; 72h: 1.29±0.01, 1.81±0.02, 1.84±0.04. The proliferation of MCF-7 cells was significantly decreased in the experimental group, compared with the other two groups (p<0.05). The mRNA levels of HCCR-1 and Bax in the experimental, control and blank groups were: HCCR-1 0.46±0.03, 1.01±0.11, 1.00; and Bax 4.40±0.99, 1.03±0.10, 1.00. The protein levels were: HCCR-1 0.62±0.07, 0.89±0.09, 0.94±0.17; and Bax 0.95±0.22, 0.67±0.19, 0.69±0.11. The expressions of mRNA and protein of HCCR-1 were significantly reduced, however the expressions of mRNA and protein of Bax were significantly increased in the experimental group compared with the other two groups (p<0.05). CONCLUSIONS: HCCR-1 siRNA transfection causes significant increase in the apoptosis and decreases in the proliferation of MCF-7 cells.These effects are related to the upregulation of the Bax expression in the MCF-7 cells.

4.
In Vitro Cell Dev Biol Anim ; 55(8): 586-597, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31367859

RESUMO

Rotavirus (RV) is the leading cause of viral gastroenteritis in neonates and VP6 protein has been discussed as a potential candidate vaccine. CRISPR/Cas9 was the latest generation of gene editing tools that can mediate the site-specific knock-in of exogenous genes, providing strong support for the expression of recombinant proteins. Here, seeking to design a rotavirus vaccine that would be suitable for both mammary-gland-based production and milk-based administration, rabbit ß-casein (CSN2) locus was chosen as the target site to integrate the VP6 gene. The efficiency of inducing mutations in different target sites of rabbit CSN2 locus was analyzed and g4 site seems to be the best one to generate mutations (g4 72.76 ± 0.32% vs g1 30.14 ± 1.93%, g2 38.53 ± 0.75%, g3 52.26 ± 1.16%, P < 0.05). We further compared the knock-in efficiency through cytoplasmic injection of two group mixtures (containing 100 ng/µL Cas9 mRNA or Cas9 protein, 20 ng/µL sgRNA4, and 100 ng/µL donor vector) in rabbit zygotes, though the Cas9 mRNA group induced an HDR efficiency as high as 20.0% ± 2.6% than Cas9 protein group (10.3% ± 3.1%), 37.5% of the knock-in events were partial integration in the target site, when Cas9 protein used in the CRISPR/Cas9 system, all of the positive blastocysts showed completely integrated, results showed that the use of Cas9 protein is better than Cas9 mRNA to integrate the correct exogenous gene into the target site. Moreover, the transgenic rabbit that harbored correct integration of VP6 gene was obtained using Cas9 protein group and was used to produce an experimental milk-based rotavirus vaccine. Our research provides a novel strategy to produce rotavirus subunit vaccine and make a foundation for building broader milk-based vaccine protection against other pathogens.

5.
Gen Comp Endocrinol ; 284: 113243, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31408625

RESUMO

The suppressor of cytokine signaling 1 (SOCS1) is an essential feedback regulator extensively involved in many different cytokine signaling pathways, such as regulation of the immune system and growth of organism. However, the molecular and functional information on socs1 genes in freshwater fish is unclear. In the present paper, we identified and characterized the full-length closely related but distinct socs1 genes (socs 1a and -1b) in blunt snout bream (Megalobrama amblycephala). The bioinformatic analysis results showed that duplicated socs1s shared majority conserved motifs with other vertebrates. Both socs1a and -1b mRNAs were detected throughout embryogenesis, and gradually increase and then constantly expressed after 16 hpf. Whole-mount in situ hybridization demonstrated that socs1a and socs1b mRNAs were detected in the brain at 12hpf and 24hpf, and in the notochord and brain at 36hpf. In adult fish, the socs1a mRNA were strongly expressed in the heart, eye, kidney, spleen and gonad, but were found to be relatively low in the intestine and liver. On the other hand, the expression of socs1b mRNA was significantly high in the muscle, eye and spleen, and relatively low in the intestine, liver, skin and heart. The results of hGH treatment experiment showed that socs1a and 1b mRNAs were upregulated markedly in the kidney, muscle and liver. Overexpression of socs1s significantly inhibit the GH and JAK/STAT factor stat3 and the inhibitory effect of SOCS1s on GH may be involved in JAK-STAT signaling pathway. These results indicate that SOCS1 plays an important role in regulating growth and development.

6.
Prostate ; 79(14): 1589-1596, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31376183

RESUMO

BACKGROUND: Molecular studies have tried to address the unmet need for prognostic biomarkers in prostate cancer (PCa). Some gene expression tests improve upon clinical factors for prediction of outcomes, but additional tools for accurate prediction of tumor aggressiveness are needed. METHODS: Based on a previously published panel of 23 gene transcripts that distinguished patients with metastatic progression, we constructed a prediction model using independent training and testing datasets. Using the validated messenger RNAs and Gleason score (GS), we performed model selection in the training set to define a final locked model to classify patients who developed metastatic-lethal events from those who remained recurrence-free. In an independent testing dataset, we compared our locked model to established clinical prognostic factors and utilized Kaplan-Meier curves and receiver operating characteristic analyses to evaluate the model's performance. RESULTS: Thirteen of 23 previously identified gene transcripts that stratified patients with aggressive PCa were validated in the training dataset. These biomarkers plus GS were used to develop a four-gene (CST2, FBLN1, TNFRSF19, and ZNF704) transcript (4GT) score that was significantly higher in patients who progressed to metastatic-lethal events compared to those without recurrence in the testing dataset (P = 5.7 × 10-11 ). The 4GT score provided higher prediction accuracy (area under the ROC curve [AUC] = 0.76; 95% confidence interval [CI] = 0.69-0.83; partial area under the ROC curve [pAUC] = 0.008) than GS alone (AUC = 0.63; 95% CI = 0.56-0.70; pAUC = 0.002), and it improved risk stratification in subgroups defined by a combination of clinicopathological features (ie, Cancer of the Prostate Risk Assessment-Surgery). CONCLUSION: Our validated 4GT score has prognostic value for metastatic-lethal progression in men treated for localized PCa and warrants further evaluation for its clinical utility.

7.
Artigo em Inglês | MEDLINE | ID: mdl-31274473

RESUMO

Background/Aim: The prognosis of hepatocellular carcinoma (HCC) is very dismal and the targeted drugs of HCC are limited. Studies of HCC prognostic biomarkers have made little progress, though many new techniques such as high-throughput sequencing have been applied. FOS-like antigen 1 (FOSL1) is generally accepted as a proto-oncogene but its clinical significance in HCC has never been elucidated. Materials and Methods: In our study, we investigated the expression of FOSL1 in 114 paraffin-embedded HCC tissues, and detected FOSL1 mRNA levels in 20 pairs of fresh HCC tissues and their corresponding tumor adjacent tissues. The correlations between FOSL1 expression and clinicopathological factors were analyzed and the prognostic significance of FOSL1 was evaluated with univariate and multivariate analysis. Moreover, we detected the function of FOSL1 in HCC proliferation with experiments in vitro. Results: FOSL1 mRNAs in HCCs were significantly higher than those in tumor adjacent tissues. The percentage of high expression and low expression of FOSL1 accounted for 46% (53/114) and 54% (61/114), respectively. High expression of FOSL1 was significantly associated with larger tumor size (P = 0.021), hepatitis B virus infection (P = 0.014), advanced T stage (P = 0.014), and tumor necrosis metastasis stage (P = 0.014). Moreover, high expression of FOSL1 was significantly correlated with poor prognosis of HCC and could be identified as an independent prognostic biomarker of HCC (hazard ratio = 5.60, 95% confidence interval = 3.00-10.45, P < 0.001). With in vitro function assay, we demonstrated that FOSL1 played an essential role in HCC proliferation. Conclusions: High expression of FOSL1 is an independent risk factor of HCC predicting unfavorable prognosis, indicating that FOSL1 detection could stratify patients with high risk, and anti-FOSL1 therapy may be a promising way to treat HCC.

8.
Restor Neurol Neurosci ; 37(4): 397-407, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31306143

RESUMO

Stroke often leads to neuronal injury and neurological functional deficits. Whilst spontaneous neurogenesis and axon regeneration are induced by ischemic stroke, effective pharmacological treatments are also essential for the improvement of neuroplasticity and functional recovery after stroke. However, no pharmacological therapy has been demonstrated to be able to effectively improve the functional recovery after stroke. Bumetanide is a specific Na+-K+-Cl- co-transporter inhibitor which can maintain chloride homeostasis in neurons. Therefore, many studies have focused on this drug's effect in stroke recovery in recent years. Here, we first review the function of Na+-K+-Cl- co-transporter in neurons, then how bumetanide's role in reducing brain damage, promoting neuroplasticity, leading to functional recovery after stroke, is elucidated. Finally, we discuss current limitations of bumetanide's efficiency and their potential solutions. These results may provide new avenues for further exploring mechanisms of post-stroke functional recovery as well as promising therapeutic targets for functional disability rehabilitation after ischemic stroke.

9.
Exp Mol Med ; 51(7): 84, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337748

RESUMO

Focal cortical dysplasia type II (FCDII) is a cerebral cortex malformation characterized by local cortical structure disorganization, neuronal dysmorphology, and refractory epilepsy. Brain somatic mutations in several genes involved in the PI3K/AKT/mTOR pathway are associated with FCDII, but they are only found in a proportion of patients with FCDII. The genetic causes underlying the development FCDII in other patients remain unclear. Here, we carried out whole exome sequencing and targeted sequencing in paired brain-blood DNA from patients with FCDII and identified a brain somatic doublet mutation c.(A104T, C105A) in the Ras homolog, mTORC1 binding (RHEB) gene, which led to the RHEB p.Y35L mutation in one patient with FCDII. This RHEB mutation carrier had a dramatic increase of ribosomal protein S6 phosphorylation, indicating mTOR activation in the region of the brain lesion. The RHEB p.Y35L mutant protein had increased GTPλS-binding activity compared with wild-type RHEB. Overexpression of the RHEB p.Y35L variant in cultured cells also resulted in elevated S6 phosphorylation compared to wild-type RHEB. Importantly, in utero electroporation of the RHEB p.Y35L variant in mice induced S6 phosphorylation, cytomegalic neurons, dysregulated neuron migration, abnormal electroencephalogram, and seizures, all of which are found in patients with FCDII. Rapamycin treatment rescued abnormal electroencephalograms and alleviated seizures in these mice. These results demonstrate that brain somatic mutations in RHEB are also responsible for the pathogenesis of FCDII, indicating that aberrant activation of mTOR signaling is a primary driver and potential drug target for FCDII.

10.
J Integr Plant Biol ; 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31180179

RESUMO

High amylose starch can be produced by plants deficient in the function of branching enzymes (BEs). Here we report the production of transgenic cassava with starches containing up to 50% amylose due to the constitutive expression of hair-pin dsRNAs targeting the BE1 or BE2 genes. All BE1-RNAi plant lines (BE1i) and BE2-RNAi plant lines (BE2i) were grown up in the field, but with reduced total biomass production. Considerably high amylose content in the storage roots of BE2i plant lines was achieved. Storage starch granules of BE1i and BE2i plants had similar morphology as wild type (WT), however, the size of BE1i starch granules were bigger than that of WT. Comparisons of amylograms and thermograms of all three sources of storage starches revealed dramatic changes to the pasting properties and a higher melting temperature for BE2i starches. Glucan chain length distribution analysis showed a slight increase in chains of DP>36 in BE1i lines and a dramatic increase in glucan chains between DP 10-20 and DP>40 in BE2i lines. Furthermore, BE2i starches displayed a B-type X-ray diffraction pattern instead of the A-type pattern found in BE1i and WT starches. Therefore, cassava BE1 and BE2 function differently in storage root starch biosynthesis. This article is protected by copyright. All rights reserved.

11.
Hypertension ; 74(2): 375-383, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31230546

RESUMO

Hypertensive disorders of pregnancy (HDP) are associated with low birth weight, shorter gestational age, and increased risk of maternal and offspring cardiovascular diseases later in life. The mechanisms involved are poorly understood, but epigenetic regulation of gene expression may play a part. We performed meta-analyses in the Pregnancy and Childhood Epigenetics Consortium to test the association between either maternal HDP (10 cohorts; n=5242 [cases=476]) or preeclampsia (3 cohorts; n=2219 [cases=135]) and epigenome-wide DNA methylation in cord blood using the Illumina HumanMethylation450 BeadChip. In models adjusted for confounders, and with Bonferroni correction, HDP and preeclampsia were associated with DNA methylation at 43 and 26 CpG sites, respectively. HDP was associated with higher methylation at 27 (63%) of the 43 sites, and across all 43 sites, the mean absolute difference in methylation was between 0.6% and 2.6%. Epigenome-wide associations of HDP with offspring DNA methylation were modestly consistent with the equivalent epigenome-wide associations of preeclampsia with offspring DNA methylation (R2=0.26). In longitudinal analyses conducted in 1 study (n=108 HDP cases; 550 controls), there were similar changes in DNA methylation in offspring of those with and without HDP up to adolescence. Pathway analysis suggested that genes located at/near HDP-associated sites may be involved in developmental, embryogenesis, or neurological pathways. HDP is associated with offspring DNA methylation with potential relevance to development.

12.
J Cell Biochem ; 120(10): 17709-17722, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31161607

RESUMO

This study was aimed to verify whether there existed any associations between long noncoding RNA MEG3/miR-219a-5p/EGFR axis and the development of ovarian cancer (OC). As a whole, we gathered 317 pairs of OC tissues and surgical marginal normal tissues and simultaneously acquired four OC cell lines (ie, A2780, Caov-3, OVCAR-3, and SKOV-3) and human normal ovarian surface epithelial cell line. Moreover, pcDNA3.1-MEG3, si-MEG3, miR-219a-5p mimic, miR-219a-5p inhibitor, pcDNA3.1-EGFR, and si-EGFR were, respectively, transfected into the OC cells, and their impacts on viability, proliferation, apoptosis, invasion, and migration of OC cells were assessed via conduction of MTT assay, colony formation assay, flow cytometry assay, transwell assay, and scratch assay. Ultimately, dual-luciferase reporter gene assay was performed to testify the targeted relationships among maternally expressed gene 3 (MEG3), miR-219a-5p, and estimated glomerular filtration rate (EGFR). It was indicated that underexpressed MEG3 and miR-219a-5p were significantly associated with unfavorable prognosis of patients with OC when compared with overexpressed MEG3 and miR-219a-5p (P < .05). In addition, the OC cells transfected with si-MEG3 or miR-219a-5p inhibitor exhibited stronger viability, proliferation, invasion, and migration than untreated cells (P < .05). Correspondingly, the apoptotic percentage of OC cells was reduced observably under treatments of si-MEG3 and miR-219a-5p inhibitor (P < .05). Moreover, MEG3 exerted modulatory effects on the expression of miR-219a-5p (P < .05), and there was a sponging relationship between them (P < .05). Finally, EGFR expression was modified by both MEG3 and miR-219a-5p significantly (P < .05), and raising EGFR expression could changeover the impacts of MEG3 and miR-219a-5p on the above-mentioned activity of OC cells (P < .05). Conclusively, MEG3 could serve as a promising biomarker for diagnosis and treatment of OC, considering its involvement with OC etiology via regulation of miR-219a-5p/EGFR axis.

13.
J Sep Sci ; 42(16): 2734-2742, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31207064

RESUMO

Four stereoisomeric components were produced during the synthesis of the antidepressant drug (1S, 4S)-sertraline hydrochloride due to the two chiral carbon centers in its chemical structure, including (1S, 4S), (1R, 4R), (1S, 4R), and (1R, 4S)-isomer. Stereoselective separation of the target isomer (1S, 4S)-sertraline from the medicinal reaction mixtures by countercurrent chromatography using hydroxypropyl-ß-cyclodextrin as the stereoselective selector was investigated. A biphasic solvent system composed of n-hexane/0.20 mol/L phosphate buffer solution with pH 7.6 containing 0.10 mol/L of hydroxypropyl-ß-cyclodextrin (1:1, v/v) was selected for separation of cis-sertraline and trans-sertraline using reverse phase elution mode and (1S, 4S)-sertraline was separated with (1R, 4R)-sertraline using recycling elution mode. A fabricated in-house analytical countercurrent chromatographic apparatus was used for optimization of the separation conditions. Stationary phase retention and peak resolution were investigated for separation of cis-sertraline and trans-sertraline by the analytical apparatus.

14.
Aging (Albany NY) ; 11(11): 3811-3823, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186379

RESUMO

Long non-coding RNAs (LncRNAs) have attracted increasing attention for their important regulation functions in a wide range of malignancies. AGAP2-AS1 was demonstrated as an oncogene in several cancers, including glioblastoma (GBM). However, the biological mechanisms of AGAP2-AS1 in GBM progression are still unclear. Herein, we found that AGAP2-AS1 expression was up-regulated in GBM tissues and cells. High AGAP2-AS1 expression may predict a poor prognosis in GBM patients. Functionally, silencing of AGAP2-AS1 suppressed proliferation and invasion, while enhanced apoptosis in GBM cells. Overexpression of AGAP2-AS1 promoted cell proliferation and invasion. Mechanically, AGAP2-AS1 could interact with EZH2 and LSD1, recruiting them to TFPI2 promoter region to inhibit its transcription. Moreover, TFPI2 overexpression decreased proliferation and invasion, and facilitated apoptosis in GBM cells. Furthermore, the tumor-suppressive effects mediated by AGAP2-AS1 knockdown were greatly reversed following down-regulation of TFPI2. Also, suppression of AGAP2-AS1 impaired tumor growth of GBM in vivo. In summary, AGAP2-AS1 exerts oncogenic functions in GBM by epigenetically silencing TFPI2 expression through binding to EZH2 and LSD1, illuminating a novel mechanism of AGAP2-AS1 in GBM development and furnishing a prospective therapeutic method to combat GBM.

15.
Pharmazie ; 74(5): 295-300, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31109400

RESUMO

This study intended to investigate the role of lncRNA NR2F1-AS1 in endometrial cancer (EC). The expression level of NR2F1-AS1 in tumor tissues and EC cells was measured. After sh-NR2F1-AS1 transfection, the cell viability, apoptosis, migration and invasion of EC cells were analyzed. Luciferase reporter assay was conducted to investigate the target gene of miR-363. The expression levels of PI3K/AKT/GSK-3ß pathway-associated factors were assayed using western blot. NR2F1-AS1 was significantly overexpressed in EC tissues and cells. NR2F1-AS1 inhibition decreased EC cell viability, migration and invasion, while promoted cell apoptosis. miR-363 was negatively regulated by NR2F1-AS1. SOX4 was a target of miR-363. NR2F1-AS1 functioned on EC progression via PI3K/AKT/GSK-3ß pathway. The results demonstrated that NR2F1-AS1 was highly expressed in EC, which involved in the proliferation and migration of EC cells through downregulation of miR-363 to target SOX4 and regulating PI3K/AKT/GSK-3ß pathway.

16.
J Chromatogr A ; 1601: 266-273, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31130224

RESUMO

The combined use of pH-zone-refining counter-current chromatography (CCC) and conventional CCC was successfully employed for the preparative separation and purification of baicalin and wogonoside from the traditional Chinese medicinal herb Scutellaria Baicalensis Georgi. A more environmentally friendly biphasic solvent system composed of n-butanol-ethyl acetate-water (2:3:5, v/v) was used for pH-zone-refining CCC, in which trifluoroacetic acid was added to a final concentration of 10 mmol L-1 in the organic phase and ammonia was added to a final concentration of 10 mmol L-1 in the aqueous phase while a two-phase solvent system composed of ethyl acetate-ethanol-3 mmol L-1 hydrochloric acid (10:1:10, v/v) was used for the conventional CCC. Two elution modes including reverse displacement mode and normal displacement mode were investigated for pH-zone-refining CCC. The first run provided 186.7 mg of baicalin with a purity of 95.3% and 143.4 mg of a mixture of baicalin and wogonoside from 500 mg of the crude extracts of Scutellaria Baicalensis Georgi by pH-zone-refining CCC with reverse displacement elution mode. The mixture was further separated by means of a second run in conventional CCC, yielding 64.3 mg of baicalin with a purity of 98.2% and 46.1 mg of wogonoside with a purity of 98.9%.


Assuntos
Técnicas de Química Analítica/métodos , Distribuição Contracorrente , Flavanonas/isolamento & purificação , Flavonoides/isolamento & purificação , Glucosídeos/isolamento & purificação , Scutellaria baicalensis/química , Concentração de Íons de Hidrogênio , Solventes/química
17.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096671

RESUMO

ATP-binding cassette (ABC) transporters are a superfamily of proteins that transport nutrient substances and secondary metabolites through cell membranes. They also act as an uptake system for N,N'-diacetylchitobiose (GlcNAc)2 in Streptomyces coelicolor. (GlcNAc)2 is an important inducer of chitinase. However, whether the ABC transporter in Trichoderma spp. is also responsible for (GlcNAc)2 uptake and chitinase induction has not yet been confirmed. In this study, we applied RNA interference and overexpression technologies to alter the expression level of the ABC-B transporter in order to detect changes in its transportation ability and the expression level of inducible endo-chitinase ECH42-an important biocontrol enzyme in Trichoderma asperellum. The results revealed that, after interference with the expression of the ABC-B transporter, T. asperellum T4 was only able to grow normally when glucose was the only carbon source. Compared with the wild-type, the efficiency of (GlcNAc)2 by the overexpression strain evidently increased, along with the activity level of ECH42. In conclusion, one of the functions of the ABC-B transporter in T. asperellum is the uptake and transport of (GlcNAc)2 into cells, and chitobiose is a strong inducer of ECH42 in T. asperellum T4.

18.
BMC Genomics ; 20(1): 392, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113378

RESUMO

BACKGROUND: Peanut embryo development is a complex process involving a series of gene regulatory pathways and is easily affected by various elements in the soil. Calcium deficiency in the soil induces early embryo abortion in peanut, which provides an opportunity to determine the mechanism underlying this important event. MicroRNA (miRNA)-guided target gene regulation is vital to a wide variety of biological processes. However, whether miRNAs participate in peanut embryo abortion under calcium deficiency has yet to be explored. RESULTS: In this study, with the assistance of a recently established platform for genome sequences of wild peanut species, we analyzed small RNAs (sRNAs) in early peanut embryos. A total of 29 known and 132 potential novel miRNAs were discovered in 12 peanut-specific miRNA families. Among the identified miRNAs, 87 were differentially expressed during early embryo development under calcium deficiency and sufficiency conditions, and 117 target genes of the differentially expressed miRNAs were identified. Integrated analysis of miRNAs and transcriptome expression revealed 52 differentially expressed target genes of 20 miRNAs. The expression profiles for some differentially expressed targets by gene chip analysis were consistent with the transcriptome sequencing results. Together, our results demonstrate that seed/embryo development-related genes such as TCP3, AP2, EMB2750, and GRFs; cell division and proliferation-related genes such as HsfB4 and DIVARICATA; plant hormone signaling pathway-related genes such as CYP707A1 and CYP707A3, with which abscisic acid (ABA) is involved; and BR1, with which brassinosteroids (BRs) are involved, were actively modulated by miRNAs during early embryo development. CONCLUSIONS: Both a number of miRNAs and corresponding target genes likely playing key roles in the regulation of peanut embryo abortion under calcium deficiency were identified. These findings provide for the first time new insights into miRNA-mediated regulatory pathways involved in peanut embryo abortion under calcium deficiency.


Assuntos
Arachis/embriologia , Arachis/genética , Cálcio/fisiologia , Regulação da Expressão Gênica de Plantas , MicroRNAs/metabolismo , Arachis/anatomia & histologia , Arachis/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , RNA Mensageiro/metabolismo , Sementes/anatomia & histologia , Sementes/genética , Sementes/metabolismo
19.
Life Sci ; 226: 98-106, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980849

RESUMO

AIMS: The acquired drug resistance has been regarded as a main barrier for the effective treatment of temozolomide (TMZ) in glioblastoma (GBM). MiR-126-3p is commonly down-regulated and exerts tumor-suppressive roles in kinds of human cancers, including GBM. This study was designed to investigate the functions and mechanisms of miR-126-3p in regulating TMZ resistance in GBM. MATERIALS AND METHODS: qRT-PCR analysis was used to measure the expressions of miR-126-3p and SOX2 mRNA in GBM tissues and cells. Cell viability, colony forming ability and apoptosis were detected to evaluate the effect of miR-126-3p or SOX2 on TMZ resistance. Luciferase reporter experiments were applied to identify the target genes of miR-126-3p. Western blot analysis was performed to determine the protein levels associated with Wnt/ß-catenin signaling. TOP/FOP Flash assays were conducted to determine the effects of miR-126-3p or SOX2 on Wnt/ß-catenin signaling. KEY FINDINGS: miR-126-3p expression was decreased in TMZ-resistant GBM tissues and cells. High levels of miR-126-3p enhanced TMZ sensitivity by inhibiting cell viability, reducing colony forming potential and inducing apoptosis. Additionally, SOX2 was identified as a downstream target of miR-126-3p. On the contrary, SOX2 overexpression conferred TMZ resistance of GBM cells. Moreover, miR-126-3p-mediated TMZ sensitivity was reversed following increased expression of SOX2. Furthermore, miR-126-3p-induced inactivation of Wnt/ß-catenin signaling was greatly abrogated by SOX2 up-regulation. SIGNIFICANCE: MiR-126-3p sensitizes GBM cells to TMZ possibly by repressing SOX2 expression and blocking Wnt/ß-catenin signaling. This study provides novel targets to overcome TMZ resistance in GBM chemotherapy.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , MicroRNAs/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Temozolomida/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Antineoplásicos Alquilantes/farmacologia , Apoptose/fisiologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , MicroRNAs/biossíntese , MicroRNAs/genética , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética
20.
Artigo em Inglês | MEDLINE | ID: mdl-30998462

RESUMO

A key problem in co-saliency detection is how to effectively model the interactive relationship of a whole image group and the individual perspective of each image in a united data-driven manner. In this paper, we propose a group-wise deep co-saliency detection approach to address the co-saliency object discovery problem based on the fully convolutional network (FCN). The proposed approach captures the group-wise interaction information for group images by learning a semantics-aware image representation based on a convolutional neural network, which adaptively learns the group-wise features for co-saliency detection. Furthermore, the proposed approach discovers the collaborative and interactive relationships between group-wise feature representation and single image individual feature representation, and model this in a collaborative learning framework. Then, we set up a unified deep learning scheme to jointly optimize the process of group-wise feature representation learning and the collaborative learning, leading to more reliable and robust co-saliency detection results. Finally, we present a graph Laplacian regularized nonlinear regression model for saliency refinement. Experimental results demonstrate the effectiveness of our approach in comparison with the state-of-the-art approaches.

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