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As confusion mounts over RNA isoforms involved in phenotypic plasticity, aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity (ITH). Protease serine 3 (PRSS3), possessing four splice variants (PRSS3-SVs; PRSS3-V1-V4), is an indispensable trypsin that shows paradoxical effects on cancer development. Here, we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer, exhibiting opposing functions and clinical outcomes, namely, oncogenic PRSS3-V1 and PRSS3-V2 versus tumor-suppressive PRSS3-V3, by targeting different downstream genes. We identified an intragenic CpG island (iCpGI) in PRSS3. Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1 (MZF1) to regulate PRSS3 transcription. The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2'-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression. Epigenetic silencing of PRSS3-V3 via iCpGI methylation (iCpGIm) in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease. Thus, UHRF1/DNMT1-MZF1 axis-modulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs, conferring nongenetic functional ITH, with implications for early detection of lung cancer and targeted therapies.
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OBJECTIVE： To establish a method for simultaneous determination of 9 kinds of organic solvents residues in total flavonoids extracts from Abelmoschus manihot. METHODS： Headspace GC was adopted to determine the contents of 9 kinds of organic solvents residues in total flavonoids extracts from A. manihot， such as benzene， acrylonitrile， methyl methacrylate， toluene， 1，2-dichloroethane， xylene， chlorobenzene， styrene and divinylbenzene. The determination was performed on Agilent DB-WAX capillary column （30 m×0.25 mm， 0.25 μm） through temperature-programmed route. The inlet temperature and FID detector temperature were set at 250 ℃. The carrier gas was nitrogen with the flow rate of 1.2 mL/min. The split ratio was 10 ∶ 1. The containers of headspace injector were in equilibrium at 90 ℃ for 45 min and the sample size was 1 mL. RESULTS： The separation degree among the peaks of 9 components was greater than 1.5， and the blank solvent （10％ N-methyl pyrrolidone aqueous solution） had no interference. The linear ranges of them were 0.16-1.21， 0.80-6.03， 1.61-12.09，1.62-12.12， 0.16-1.21， 1.60-12.01， 0.81-6.11， 1.60-12.03， 0.80-6.03 μg/mL， respectively （r≥0.999 4）. The limits of quantitation were 0.162 08， 0.201 08， 0.080 6， 0.080 768， 0.161 92， 0.400 36， 0.040 712， 0.026 736， 0.013 395 μg/mL； the limits of detection were 0.040 52， 0.040 216， 0.026 87， 0.026 9，0.040 48， 0.080 072， 0.013 57， 0.008 912， 0.004 465 μg/mL， respectively. RSDs of precision （n＝5） and reproducibility （n＝6） tests were all lower than 5％. Average recoveries were 99.41％-111.27％（RSD＜9％， n＝9）. Above 9 residual solvents were not detected in 3 batches of total flavonoids extracts from A. manihot. CONCLUSIONS： Established method is simple， accurate and reliable， and can be applied for simultaneous detection of 9 kinds of organic solvents residues in total flavonoids extracts from A. manihot.
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Objective To compare the clinical effects of RPH combined with Milligan and PPH in the treatment of severe mixed hemorrhoids. Methods 168 patients with severe mixed hemorrhoids were assigned to a study group or a control group,84 patients for each group. The control group received PPH therapy,while the study group received RPH combined with Milligan procedure. Results The procedures were completed successfully in all the patients. The postoperative hospital stay and surgical duration were shorter and the amount of bleeding was smaller in the study group than in the control group(P<0.05). Three months after surgery,the rate of compli-cations including urinary retention,anal incontinence,anorectal stenosis,and secondary anal fissure was lower in the study group than in the control group(P < 0.05). The total effective rate was 97.6% in the study group and 85.7% in the control group,with a higher rate in the study group(P<0.05). Anal PSV and EDV values were lower in both groups three months after the procedures as compared with one day before the procedures(P<0.05),and the values were smaller in the study group than in the control group(P<0.05). Conclusions Milligan combined with RPH in the treatment of severe mixed hemorrhoids can reduce hemorrhoids blood flow. This procedure is mini-mally invasive and it can reduce the development of postoperative complications and improve efficacy.
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Objective: To establish an LC-MS/MS method for the determination of 4-( 4-amino-3-fluorophenoxy )-N-methylpyri-dine-2-carboxamide ( AFP-PMA) as a genotoxic impurity in regorafenib. Methods: The content of AFP-PMA was determined by an LC-MS/MS method. A Waters XBridge Shield RP18 column was adopted to separate the samples and the column temperature was 50℃. The mobile phase consisted of 5 mmol·L-1ammonium acetate aqueous (A)-acetonitrile (B) with gradient elution (0~9 min, 5%B→90%B) at a flow rate of 1. 0 ml·min-1. An electrospray ionization source (ESI) was used in a positive-ion and multiple reactions monitoring mode. The ion channel was m/z 262. 2→244. 1. Results:The standard curve was linear within the range of 2. 41-980. 90 ng·ml-1(r=0. 9998) and the limit of quantification was 8. 02 ng·ml-1. The limit of detection was 2. 41 ng·ml-1, which was e-quivalent to 0.000241% for the concentration of regorafenib. The average recovery was 100.95% and RSD was 2.37% (n=9). Conclusion:The method has good specificity, promising accuracy and high sensitivity, which can be used for determining the trace genotoxic impurity AFP-PMA in regorafenib.
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Background Retinal vein occlusion is a common retinal vascular diseases.Thromblysis and anticoagulation therapies are main approaches.However,systemic thrombolysis is relatively inefficient,and it often enhances the risk of hemorrhage.Objective This study was to investigate the therapeutic effects of PLM-ΔK,a kringle deficiency mutant of plasmin,on photochemically induced branch retinal vein occlusion (BRVO) after intravitreal injection.Methods BRVO models were established by the combination of caudal vein injection of Rose Bengal with argon laser radiation of periphery area of retinal veins in SD rats.Forty model rats were randomized into balance salt solution (BSS) group and 0.01 U,0.02 U,0.03 U PLM-ΔK group,and 10 μl corresponding drug was intravtreally injected 12 hours after modeling.Ophthalmoscopy and fundus fluorescein angiography (FFA) were performed to observe the change of retinal veins.The animals were sacrificed 3 days after intravitreal injection,and hematoxylin and eosin staining was used for the histopathological and ultrastructural examination of retinas.The retina of the rats was isolated for the stretched preparation of retina.The expressions of fibronectin (FN) and laminin (LN) in eyeball wall were assayed by immunofluorescence technology.The use and care of the animals complied with Statement of the Association for Research in Vision and Ophthalmology.Results The revascularization of over 2 retinal veins was found in 0,3,6 and 8 rats in the BBS group and 0.01 U,0.02 U,0.03 U PLM-ΔK group 3 days after intravitreal injection,respectively,showing a significant difference among the groups (x2=9.635,P =0.022),and the rat number with revascularization in 0.01 U PLM-ΔK group was not significantly different from that in BSS group (Z=-1.558,P =0.119),but the difference between 0.03 U PLM-ΔK group and 0.01 U PLM-ΔK group was significant (Z=-2.762,P=0.006).In the third day after intravitreal injection,retinal vein thrombus were found in the BSS group under the light microscope,and angiogenesis was seen on the retinal flatmount nuclear.In the 0.03 U PLM-ΔK group,posterior vitreal detachment was exhibited under the light microcope,and no retinal new vessel and cell damage were seen.FN was strongly expressed in the inner limiting membrane (ILM) layer,photocyte layer,outer limiting membrane (OLM) layer,choroid and scleral layer,and LN was expressed mainly in the ILM,OLM and scleral layer in the BSS group.However,the expression intensities of FN and LN were obviously weakened in the 0.03 U PLM-ΔK group.Conclusions Intravitreal injection of PLM-ΔK can enhance the reperfusion of occluded branch retinal vein and serve as a potential therapeutic drug for BRVO.Also it can permeate into choroid after intravitreal injection to degradate FN and LN.
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Objective:To establish a method for the determination of residual triethylamine in tenofovir disoproxil fumarate. Methods:The residual treithylamine was determined by GC with an Agilent DB-624 capillary column(30 m × 0. 53 mm,3 μm)and an FID detector. The carrier gas was nitrogen and the flow rate was 2. 0 ml · min-1 . The oven temperature was programming in-creased. The initial column temperature was 50℃,and then raised to 220℃ at a rate of 10℃·min-1 ,and maintained 4 min. Trieth-ylamine was quantified by an external standard. Results:The calibration showed a good linearity within the range of 53. 02-742. 34 μg ·ml-1 for triethylamine. The correlation coefficient was 0. 999 8. The recoveries were within the range of 96. 52%-102. 27%. The relative standard deviation(RSD)was 2. 42%(n=9). Conclusion:The method has good specificity,repeatability and sensitivity, which can be used in determining the content of residual trietbylamine in tenofovir disoproxil fumarate.
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Objective To describe the mastery of knowledge about antihypertensive drugs of clinical nurses in one tertiary comprehensive hospital of Beijing.Methods Purposive sampling was used to recruit 195 clinical nurses who were working in one tertiary comprehensive hospital in Beijing.The demographic questionnaire,knowledge of antihypertensive drugs questionnaire were filled.Results The score of knowledge of antihypertensive drugs Questionnaire was (45.63 ± 3.79) points;different education,wishes to take part in hypertension related knowledge training had significant differences (t =2.007,2.049,P ＜ 0.05).Conclusion Knowledge of antihypertensive drugs of clinical nurses in Beijing was at medium level.Those who indicated that it was not matter whether to attend hypertension related knowledge training and had low level of education had more problems in knowledge of antihypertensive drugs.
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Protozoan ciliates are a group of unicellular eukaryotes. The special characteristics of stop codons usage in termination of protein biosynthesis in ciliates cells makes them an ideal model to study the mechanism of stop codon recognition of polypeptides release factors. To localize the functional positions of biomolecules in ciliates cell, we constructed a macronuclear artificial chromosome containing a gene encoding red fluorescence protein (EoMAC_R) based on the structural characteristics of ciliates chromosome. Three factors, L11, eRF1a, and eRF3 that are involved in termination process of protein synthesis were colocalized in Euplotes octocarinatus cells by using novel EoMAC_R and the previously constructed EoMAC_G. The results indicated that protein synthesis mainly occurred inside the "C" shape macronucleus, suggesting that EoMAC could be a useful tool for localizing biomolecules in ciliates cell.
