Construction of PR domain eukaryotic expression vector and its inhibitory effect on esophageal cancer cells

Objective：PR domain is responsible for the tumor suppressing activity of RIZ1.The study aimed to construct human PR domain eukaryotic expression vectors,transfect human esophageal cancer cells （TE13）,and evaluate the anticancer activity of PR domain on human esophageal cancer TE13 cells.Methods：First,mRNA was extracted from human esophageal cancer tissue by RT-PCR,then reversetranscribed to cDNA.After amplifying from the DNA template,PR domain was linked to T vector.Second,after extraction,PR domain was cut using enzyme and linked to pcDNA3.1（＋）.Then,the plasmid was transfered to Trans1-T1 phage resistant competent cells,following by extracting the ultrapure plasmid,and transfecting into TE13 cells.In the end,the protein expression of pcDNA3.1（＋）/PR domain in TE13 was detected by Western blot,and the apoptosis of TE 13 by technique of flow cytometry.Results：More than 5,000 bp purposed band of pcDNA3.1（＋）/PR domain plasmid was found by agarose gel electrophoresis.After transfection,the PR domain （molecular weight of about 28 Da） was found only in 3,4 and 5 groups by Western blot.Flow cytometry assay showed apoptosis in experimental group was significantly more than that in the control group （P＜0.05）.Conclusions：The PR domain eukaryotic expression vector was constructed successfully.The protein of the PR domain could be expressed in esophageal cancer TE13 cells firmly after transfection,and a single PR domain could promote apoptosis of TE13 cells.

Genome-Wide Identification and Expression Analysis of the Phytocyanin Gene Family in Nicotiana tabacum

Phytocyanin(PC)is a class of plant-specific blue copper proteins involved in electron transport,plant growth,development,and stress resistance.However,PC proteins have not been systematically evaluated in tobacco plants.We determined the whole-genome sequences of the PC family in the tobacco cultivar‘K326.’The transcriptome data were used to analyze the expression of the NtPC family at different development stages and tissue-specific genes.Real-time fluorescence quantitative analysis was used to analyze the expression of the NtPC gene family under low temperature and methyl jasmonate stress.The tobacco NtPC family contained 110 members and was divided into four subfamilies:early nodulin-like protein(NtENODL),uclacyanin-like protein,stellacyanin1-like protein,and plantacyanin-like protein.According to phylogenetic and structural analyses,the NtPC family could be divided into eight structural types.Fifty-three NtPCs were randomly distributed on 22 of 24 tobacco chromosomes.Collinearity analysis revealed 33 pairs of genes belonging to the NtPC family.Gene ontology analysis showed that the PC genes are components of the plasma membrane and may participate in plasma membrane-related functions.The NtPC family contained numerous elements related to hormonal and abiotic stress responses and was specifically expressed in the tobacco prosperous,maturation,and budding periods.Tissue-specific expression analysis showed that some genes were tissue specific.The expression of NtENODL58 and other genes was significantly induced by low-temperature and methyl jasmonate stress.Thus,the NtPC gene family plays an important role in plant stress response.

Ginsenoside Rb1 Reduces D-GalN/LPS-induced Acute Liver Injury by Regulating TLR4/NF-κB Signaling and NLRP3 Inflammasome

Background and Aims:The effect of ginsenoside Rb1 on D-galactosamine(D-GalN)/lipopolysaccharide(LPS)-induced acute liver injury(ALI)is unknown.The aim of this study was to evaluate the effect of ginsenoside Rb1 on ALI and its underlying mechanisms.Methods:Mice were pretreated with ginsenoside Rb1 by intraperitoneal injection for 3 days before D-GalN/LPS treatment,to induce ALI.The survival rate was monitored every hour for 24 h,and serum biochemical parameters,hepatic index and histopathological analysis were evaluated to measure the degree of liver injury.ELISA was used to detect oxidative stress and inflammatory cytokines in hepatic tissue and serum.Immunohistochemistry staining,RT-PCR and western blotting were performed to evaluate the expression of toll-like receptor 4(TLR4),nuclear factorkappa B(NF-κB),and NLR family,pyrin domain-containing 3 protein(NLRP3)in liver tissue and Kupffer cells(KCs).Results:Ginsenoside Rb1 improved survival with D-GalN/LPS-induced ALI by up to 80%,significantly ameliorated the increased alanine and aspartate transaminase,restored the hepatic pathological changes and reduced the levels of oxidative stress and inflammatory cytokines altered by D-GalN/LPS.Compared to the control group,the KCs were increased in the D-GalN/LPS groups but did not increase significantly with Rb1 pretreatment.D-GalN/LPS could upregulate while Rb1 pretreatment could downregulate the expression of interleukin(IL)-1β,IL-18,NLRP3,apoptosis associated specklike protein containing CARD(ASC)and caspase-1 in isolated KCs.Furthermore,ginsenoside Rb1 inhibited activation of the TLR4/NF-κB signaling pathway and NLRP3 inflammasome induced by D-GalN/LPS administration.Conclusions:Ginsenoside Rb1 protects mice against D-GalN/LPS-induced ALI by attenuating oxidative stress and the inflammatory response through the TLR4/NF-κB signaling pathway and NLRP3 inflammasome activation.

Novel functional food from an invasive species Polygonum cuspidatum:safety evaluation,chemical composition,and hepatoprotective effects

Accidentally,we found that the shoots of Polygonum cuspidatum(SPC)have been consumed for centuries as a traditional vegetable in the Shennongjia region of China.Local residents believe that SPC has biological effects such as antibacterial,anti-aging,and antioxidant.To provide scientific support for the use of SPC as a functional food,SPC was evaluated in terms of safety,chemical composition,and antioxidant activity both in vivo and in vitro.In the first,SPC exhibited no adverse cytotoxic effects or acute toxicity in mice.Then the chemical composition of SPC was determined by ultrahigh performance liquid chromatography–electrospray ionization–quadrupole time of flight tandem mass spectrometry(UHPLC-ESI-QTOF-MS/MS).Twenty-two compounds were identified from the SPC extracts,including phenolic,flavonoid,stilbene,and anthra-quinone.Finally,an acute ethanol-induced oxidative stress model in mice showed hepatoprotective effects.In brief,our study indicated that SPC is a safe,multifunctional food with antioxidant and hepatoprotective activities.Importantly,the consumption of SPC as a functional food provides a novel strategy for the efficient utilization of the invasive plant.

Comprehensive treatment of acute-on-chronic liver failure in a patient with hepatitis B： a case report

The clinical data of a patient with acute-on-chronic liver failure were analyzed retrospectively. The patient has suffered from hepatitis B for 30 years. His liver function deteriorated, yielding Child-Pugh grade C and reaching a model for end-stage liver disease score of 33 points within a short period; this condition was complicated with highly active variceal bleeding and coagulation system failure （PT 〉 100 s）. The patient also presented hepatocellular carcinoma. Comprehensive treatments included effective inhibition of hepatitis B virus replication and intensive care support. Piggyback orthotopic liver transplantation was performed as the final treatment. The patient recovered uneventfully and was discharged after surgery.