Optical substrates for drug-metabolizing enzymes: Recent advances and future perspectives

Drug-metabolizing enzymes (DMEs), a diverse group of enzymes responsible for the metabolic elimination of drugs and other xenobiotics, have been recognized as the critical determinants to drug safety and efficacy. Deciphering and understanding the key roles of individual DMEs in drug metabolism and toxicity, as well as characterizing the interactions of central DMEs with xenobiotics require reliable, practical and highly specific tools for sensing the activities of these enzymes in biological systems. In the last few decades, the scientists have developed a variety of optical substrates for sensing human DMEs, parts of them have been successfully used for studying target enzyme(s) in tissue preparations and living systems. Herein, molecular design principals and recent advances in the development and applications of optical substrates for human DMEs have been reviewed systematically. Furthermore, the challenges and future perspectives in this field are also highlighted. The presented information offers a group of practical approaches and imaging tools for sensing DMEs activities in complex biological systems, which strongly facilitates high-throughput screening the modulators of target DMEs and studies on drug/herb‒drug interactions, as well as promotes the fundamental researches for exploring the relevance of DMEs to human diseases and drug treatment outcomes.

Screening of Reference Genes of Andrographis paniculata Under MeJA and Abiotic Stresses by Real-time Fluorescence-based Quantitative PCR / 中国实验方剂学杂志

ObjectiveTo screen the appropriate reference genes for real-time fluorescence-based quantitative polymerase chain reaction（Real-time PCR）analysis of the Andrographis paniculata under methyl jasmonate（MeJA）and various abiotic stresses. MethodThe actin 1（ACT1），actin 2（ACT2），elongation factor（EF-1α），glyceraldehyde-3-phosphate dehydrogenase（GAPDH），tubulin（TUB），polyubiquitin（UBQ）， and 18S rRNA（18S）gene were selected as candidate reference genes based on the RNA-seq data of high temperature，drought， UV， and MeJA. The expression of seven candidate reference genes in the A. paniculata leaves was assessed by Real-time PCR，and the stability was analyzed by geNorm，NormFinder，BestKeeper， and Refinder. ResultThe results of stability evaluated by geNorm，NormFinder， and BestKeeper were not the same due to different indicators. As analyzed by Refinder， for the stability of the expression， the genes were ranked as UBQ>18S>EF-1α>ACT2>ACT1>GAPDH>TUB under high temperature stress， ACT1>UBQ>EF-1α>18S>ACT2>GAPDH>TUB under drought stress， EF-1α>TUB>ACT2>UBQ>18S>GAPDH>ACT1 under UV stress， and ACT1>EF-1α>UBQ>ACT2>18S>TUB>GAPDH under MeJA stress. Among them，18S gene was not suitable as an internal reference gene duo to its high expressive abundance. This study also verified the relative expression level of andrographolide synthesis-related gene hydroxy-methylglutaryl-CoA synthase （HMGS） in the four stresses on the basis of transcriptome data，and found that the Real-time PCR results of appropriate internal reference genes were accurate and reliable. ConclusionUBQ-ACT1-UBQ，EF-1α-TUB，and ACT1-EF-1α were the suitable combinations under stresses of high temperature，drought，UV， and MeJA. This study is expected to provide references for the research on function regulation and expression of genes in A. paniculata under high temperature，drought，UV， and MeJA stresses.

Glycyrrhetinic acid binds to the conserved P-loop region and interferes with the interaction of RAS-effector proteins

Members of the proto-oncogene superfamily are indispensable molecular switches that play critical roles in cell proliferation, differentiation, and cell survival. Recent studies have attempted to prevent the interaction of RAS/GTP with RAS guanine nucleotide exchange factors (GEFs), impair RAS-effector interactions, and suppress RAS localization to prevent oncogenic signalling. The present study aimed to investigate the effect of the natural triterpenoic acid inhibitor glycyrrhetinic acid, which is isolated from the roots of plant species, on RAS stability. We found that glycyrrhetinic acid may bind to the P-loop of RAS and alter its stability. Based on our biochemical tests and structural analysis results, glycyrrhetinic acid induced a conformational change in RAS. Meanwhile, glycyrrhetinic acid abolishes the function of RAS by interfering with the effector protein RAF kinase activation and RAS/MAPK signalling.

Role of Ectodermal-neural cortex 1 protein in human glioma progression, identification of a peptide internalized by human glioblastoma cells and development of an alternative method to generate growth curves of adherent cultures / Papel da proteína Ectodermal-neural cortex 1 (ENC1) na progressão de glioma humano, identificação de um peptídeo internalizado por células de glioblastoma humano e desenvolvimento de um método alternativo para gerar curvas de crescimento celular

Gliomas are the most common form of primary intracranial malignancy, among which astrocytomas are the most frequent. Ectodermal-cortex protein 1 (ENC 1), also known as Nuclear Restricted Protein/Brain (NRP/B), was first characterized as a protein which interacts with the cytoskeleton by binding to actin through Kelch-like domains, being related to neural fate specification during development of the nervous system. The first chapter of this thesis confirms ENC1 as a tumor suppression properties by a genomic edition approach, analyses ENC1 expression in a set of patient glioma samples and describes the correlation these data with patients survival and progression-free survival, concluding that ENC1 expression may constitute a biomarker for glioma aggressiveness. The second chapter refers to the identification and in vitro characterization of the LHTNELQ peptide, which was selected by the Phage Display method using human glioblastoma cells. This new peptide is able to be internalized by these cells and features as a new tool for the development of glioma therapeutics. The third chapter report an alternative method to generate growth curves of adherent cell cultures, which is based on the CFSE fluorescence decay over time. It is an alternative method to determine growth curves of cultured cells, with smaller variation among technical replicates than that of counting-based methods

Gliomas são a forma mais comum de malignidades primárias intracranianas, dentre os quais os astrocitomas são os mais frequentes. A proteína Ectodermal-neural cortex 1 (ENC1), também conhecida como Nuclear Restricted Protein/Brain (NRP/B), foi primeiramente caracterizada como uma proteína que interage com o citoesqueleto por meio de ligação à actina através de domínios Kelch-like, sendo relacionada com diferenciação neuronal durante o desenvolvimento do sistema nervoso. O primeiro capítulo desta tese descreve confirmação da capacidade supressora tumoral de ENC1 por abordagem de edição genômica, analisa a expressão de ENC1 em um conjunto de amostras de pacientes com gliomas e correlaciona esses dados com tempo de sobrevida geral e sobrevida livre de progressão tumoral nos pacientes, concluindo que a expressão de ENC1 pode ser utilizada como um biomarcador da agressividade do glioma. O segundo capítulo apresenta a identificação e caracterização in vitro do peptídeo LHTNELQ, que foi selecionado pela metodologia de Phage display utilizandose de células de glioblastoma humano. Este novo peptídeo é capaz de internalizar-se nestas células e figura como uma nova ferramenta para o desenvolvimento de estratégias terapêuticas para glioblastomas. No terceiro capítulo propõe-se um método alternativo para gerar curvas de crescimento celular de cultura aderente, o qual é baseado no decaimento da fluorescência do reagente CFSE ao longo do tempo. Tratase de um método alternativo para a determinação de curvas de crescimento de culturas aderentes, com menor variação entre as réplicas técnicas do que os métodos baseados em contagem das células

Roles and regulation mechanism of miR-31 in human cutaneous squamous cell carcinoma growth / 中国肿瘤临床

Objective:To investigate the roles and regulation mechanism of miR-31 in human cutaneous squamous cell carcino-ma (cSCC) growth. Methods:cSCC cells were transfected with the antisense oligonucleotide (ASO) of miR-31, and the cSCC growth was tested by colony formation and in vivo tumor formation assays. The target gene of miR-31 was validated by Western blot and green fluorescent protein (GFP) reporter assay. The cells were then transfected with the siRNA of the target gene, and the effect of the target gene on cell growth was preformed by colony formation assay. Finally, real-time PCR and immunohistochemistry were used for analy-sis of the expression of miR-31 and its target gene. Results:miR-31 ASO resulted in a low number of cell colonies and small tumor vol-ume (P<0.05). Western blot showed that the cells with miR-31 ASO had a higher protein level of large tumor suppressor homolog 2 (LATS2) than the control. The 3' UTR of LATS2 had a binding site with miR-31, and miR-31 ASO increased the GFP intensity con-trolled by LATS2 3' UTR, whereas no effect was observed on the mutant LATS2 3' UTR. Western blot showed that LATS2 siRNA inhib-ited the expression of LATS2 protein by about 80%. Knocking down of LATS2 increased the colony number by about 70%or 1.3-fold in cSCC cells. Real-time PCR showed that miR-31 was overexpressed in most cSCC tissues, compared with normal tissues. An inverse relationship existed between miR-31 and LATS2 expression levels. Immunohistochemistry validated that LATS2 was downregulated in cSCC tissues. Conclusion:miR-31, which functions as an oncogene, promotes cSCC growth by suppressing LATS2 expression. Our da-ta suggest that miR-31 is a potential miRNA-based therapeutic target for cSCC growth.

Cloning and expression of Schistosoma japonicum VCP gene and its mRNA expression levels in different stages / 中国血吸虫病防治杂志

Objective To prokaryotically express the valosin-containing protein(VCP)of Schistosoma japonicum,and ana-lyze its VCP mRNA expressions in the cercaria,schistosomulum,adult worm(female and male worms)and egg. Methods RNA of S. japonicum eggs were extracted,and reversely transcribed into cDNA. The VCP gene of S. japonicum was amplified by using polymerase chain reaction(PCR),and subcloned into the prokaryotically expressed vector pET15b. The recombined plasmid was transformed into BL21 cells,and the expression of the target gene was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). The recombinant protein was yielded through the purification of inclusion body,and identified by using sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The RNA(s)of cercaria,schistosomulum,female adult worm,male adult worm,and egg of S. japonicum were extracted,digested with DNase,purified,and reversely transcribed into cDNA. The mRNA expressions of the VCP gene in various developmental stages of S. japonicum were determined by using fluorescence-based quantitative real-time PCR. Results The VCP gene of S. japonicum was yielded by PCR amplification,and the recombinant pro-tein was obtained through recombinant plasmid expression and purification of inclusion body. The highest VCP mRNA expression in S. japonicum cercaria was detected by the fluorescence-based quantitative real-time PCR,while low expressions were found in the schistosomulum,egg,female and male adult worms. Conclusion The recombinant protein encoded by the VCP gene of S. ja-ponicum is successfully obtained,and the VCP mRNA expression is determined in various developmental stages of S. japonicum.

Preliminary study effect of artificial liver support on HBV cccDNA in patients with fulminant hepatitis B / 重庆医科大学学报

Objective:To establish a new fluorescence-based quantitative PCR assay for detecting hepatitis B virus covalently closed circular DNA(HBV cccDNA).To explore the effect of artificial liver support system(ALSS)on total HBV DNA and HBV cccDNA in sera of patients with fulminant hepatitis B.Methods:The serum specimens from 50 patients of fulminant hepatitis B prior to and after treatment with ALSS were collected.Then HBV cccDNA was quantitatively detected by fluorescent PCR with specific primer set and Taqman probe.And the results were analyzed by SAS8.0 statistics software.Results:We successfully established a new fluorescence-based quantitative PCR method for HBV cccDNA.HBV cccDNA was detectable in sera of the patients with fulminant hepatitis B.The level of total HBV DNA and HBV cccDNA decreased significantly following plasma exchange in ALSS.The levels of total HBV DNA and HBV cccDNA had positive correlation with degree of PT,ALT,andAST.Total HBV DNA also had positive correlation with HBV cccDNA.Conclusion:The level of total HBV DNA and HBV cccDNA has positive correlation with degree of PT,ALT,and AST,which might be indication of hepatocyte damage.The finding that significant decrease in total HBV DNA and HBV cccDNA after plasma exchange in ALSS may provide a clue of further research about the fulminant hepatitis B treated with ALSS.

STUDY ON GENETIC INSTABILITY OF nm23H1 GENE IN CHINESE WITH SQUAMOUS CELL LUNG CARCINOMA / 解剖学报

0.05).Conclusion The increase in the amount of nm23H1 protein expression can play an important role in restraining metastasis of squamous cell lung carcinoma.The heredity instability(MSI and LOH) of nm23H1 gene may not be implicated in expression of the gene and pathogenesis and progression of squamous cell lung carcinoma.