Preparation of an anti-cotinine monoclonal antibody and its application in immunological detection / 浙江大学学报·医学版

OBJECTIVE@#To prepare monoclonal antibody against cotinine (COT) and to establish immunoassay for detecting COT in human urinary samples.@*METHODS@#BALB/c mice were immunized with synthesized cotinine-bovine serum albumin (COT-BSA) to screen monoclonal antibody with technique of cell fusion. The monoclonal antibody was used for the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatographic strip assay for the detection of COT in human urine.@*RESULTS@#The monoclonal antibody against COT was identified by ic-ELISA with a 50%inhibitive concentration (IC@*CONCLUSIONS@#The ic-ELISA and colloidal gold immunochromatographic strip assay using the prepared monoclonal antibody against COT have been proved to be reliable for the rapid detection of COT in human urines, which may be used for monitoring of environmental tobacco smoke.

TSNAdb: A Database for Tumor-specific Neoantigens from Immunogenomics Data Analysis / 基因组蛋白质组与生物信息学报·英文版

Tumor-specific neoantigens have attracted much attention since they can be used as biomarkers to predict therapeutic effects of immune checkpoint blockade therapy and as potential targets for cancer immunotherapy. In this study, we developed a comprehensive tumor-specific neoantigen database (TSNAdb v1.0), based on pan-cancer immunogenomic analyses of somatic mutation data and human leukocyte antigen (HLA) allele information for 16 tumor types with 7748 tumor samples from The Cancer Genome Atlas (TCGA) and The Cancer Immunome Atlas (TCIA). We predicted binding affinities between mutant/wild-type peptides and HLA class I molecules by NetMHCpan v2.8/v4.0, and presented detailed information of 3,707,562/1,146,961 potential neoantigens generated by somatic mutations of all tumor samples. Moreover, we employed recurrent mutations in combination with highly frequent HLA alleles to predict potential shared neoantigens across tumor patients, which would facilitate the discovery of putative targets for neoantigen-based cancer immunotherapy. TSNAdb is freely available at http://biopharm.zju.edu.cn/tsnadb.

Chlorogenic acid inhibits non-enzymatic glycation and oxidation of low density lipoprotein / 浙江大学学报·医学版

OBJECTIVE@#: To investigate the effect of chlorogenic acid (CGA) on non-enzymatic glycation and oxidation of low density lipoprotein (LDL).@*METHODS@#: The non-enzymatic glycation incubation system of LDL-glucose was established. The contents of early glycation products (Amodori product) and intermediate products (dicarbonyl compound) were determined by ultraviolet-visible spectrophotometry, and the content of advanced glycation end products (AGEs) was determined by fluorescence spectrophotometry. The LDL oxidation incubation system was established. The contents of thiobarbituric acid reactive substances(TBARS) and conjugated diene were determined by ultraviolet-visible spectrophotometry. The tryptophan fluorescence quenching, and the content of lipofuscin, total fluorescence products, active aldehydes and malondialdehyde were determined by fluorescence spectrophotometry, and further verified by three-dimensional fluorescence spectroscopy.@*RESULTS@#: In the LDL glycation experiment, 150 μg/mL and 300 μg/mL CGA inhibited the formation of Amadori product, dicarbonyl compounds and AGEs. In the LDL oxidation experiment, 15 μg/mL and 25 μg/mL CGA inhibited the formation of TBARS effectively; 5 μg/mL and 10 μg/mL CGA inhibited tryptophan fluorescence quenching, and the formation of active aldehydes, malondialdehyde, total fluorescence products, lipofuscin and conjugated diolefine. And the three-dimensional fluorescence spectroscopy showed the same results.@*CONCLUSIONS@#: CGA can inhibit non-enzymatic glycation and oxidation of LDL.

Effects of alendronate combined with calcitriol on bone metabolism in the treatment of postmenopausal osteoporosis patients / 中国生化药物杂志

Objective To investigate the effect of alendronate combined with calcitriol on bone metabolism in the treatment of postmenopausal osteoporosis patients.Methods66 postmenopausal osteoporosis patients from Cancer Hospital of Hangzhou were selected and randomly divided into the control group and the study group.33 cases in the control group were treated by compound calcium amino acid chelate.33 cases in the study group were treated by compound calcium amino acid chelate, alendronate and alfacalcidol.All patients were taking the drug for 48 weeks.The bone mineral density (BMD) of lumbar vertebrae between 4 and 1, Bone alkaline phosphatase (BALP) and bone metabolism index of left femur (BMD) Type I collagen C end peptide (CTX), acid phosphatase (TRAP), parathyroid hormone (PTH) and clinical curative effect were compared.ResultsAfter treatment, the clinical effective rate of the study group (93.94%) was higher than that of the control group (72.73%), the difference was statistically significant (P<0.05).Compared with the control group,the level of bone mineral density of lumbar spine was increased from 1 to 4, and the level of TRAP, CTX, PTH and BALP were increased,the difference was statistically significant (P<0.05).There was no difference in adverse effects between the two groups.ConclusionThe combination of the application of sodium hyaluronate and the combination of the method can effectively improve the bone mineral density and bone metabolism in postmenopausal women with osteoporosis, and improve the clinical efficacy.

Analysis of clinical features and genetic mutations in a Chinese family affected with Menkes disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To delineate the clinical features and potential mutation of the ATP7A gene in a family affected with Menkes disease.</p><p><b>METHODS</b>Clinical data of a patient and his family members were analyzed. Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) assays were performed to detect the mutation of the ATP7A gene.</p><p><b>RESULTS</b>The patient was admitted at the age of 5 months due to severe epilepsy and marked delayed psychomotor development. Significantly light complexion, pudgy cheeks and sparse fuzzy wooly hair were noted. Cranial magnetic resonance imaging and angiography revealed cortical atrophy, leukoencephalopathy and circuitous of intracranial vessels. The plasma ceruloplasmin was decreased. MLPA has identified a deletion spanning exons 8 to 12 of the ATP7A gene. His mother was found to be a heterozygous carrier of the same mutation.</p><p><b>CONCLUSION</b>The clinical features and a novel mutation of the ATP7A gene of the family have been delineated.</p>

Analysis of online Mendelian inheritance in man / 中国生化药物杂志

Objective To analyze the data from Online Mendelian Inheritance in Man (OMIM) to understand more about it, and provide reference to researchers using this database.Methods 19414 mutations which have definite relevant phenotypes from OMIM were obtained, then these mutations with three databases (1000 Genome Project,GO-ESP,ExAC) which record the mutation frequency in different population were compared.Results Most of the phenotype-related mutations from OMIM are rare mutations whose mutation frequency is less than 1%:18866 in 1000 Genome Project, 18981 in GO-ESP, 18979 in ExAC.The number of mutation whose frequency is more than 1% is 548433435 in 1000 Genome Project, GO-ESP, ExAC, respectively.And there are 320 mutations whose frequency is more than 1% in all databases.In all phenotypes, there are 127 polymorphism phenotypes, 584 susceptibility phenotypes, while in 320 ( 1.6%) phenotypes with common mutations, there are 62 polymorphism phenotypes, 88 susceptibility phenotypes and occupies 48.8%, 15.1%, respectively.Conclusion Approximately 97.5% mutations in OMIM are rare mutations.Polymorphism and susceptibility enrich in common mutations, especially in the mutation whose frequency is more than 10%.

Bibliometric analysis of antibody-drug conjugates / 中国生化药物杂志

Objective To retrieve and analyze domestic and international literatures about antibody-drug conjugates, and understand the recent progress and current situation.Methods PubMed, Web of Science and CNKI were searched to collect all the literatures connected with ADCs from the beginning to January, 2016.Endnote X7 was used to sort out and summarize.The type of literature, published year, first author, research institution, published journal, cited frequency, research contents and patent situation were analyzed with bibliometric methods.Results A total of 645 literatures were included, among which 495 were foreign articles and 150 were Chinese articles.The literatures greatly increased after the 21st century.The top one nation and journal which published the most articles were America and Clinical Cancer Research, respectively.Krop IE and Younes A published the most articles.Among them, the most frequently cited paper was cited up to 686 times.Selection of the targets, site-specific drug conjugation to antibodies and cytotoxic agents were frequently involved.Conclusion ADCs, which have made breakthrough progress, are the focus in the field of cancer research.However, there is still room for improvement, and we still need further exploration.

Recent advances of neoantigen in tumor therapy / 中国生化药物杂志

As the research work went further and more detailed, a variety of new treatments compete to come out.However, it remains unclear that how the antigen works to distinguish cancer cells and normal cells.Neoantigen, which is located in the tumor cell surface of a specific antigen, its presence makes human immunotherapy into new areas which may make personalized treatment possible in the near future.Emerging data suggest that the identification of such newantigens is a major factor in clinical immunotherapy.They can form a biomarker in cancer immunotherapy to provide targets for a variety of therapeutic approaches to attack, which allows T cells to selectively enhance the immune response against this class of antigens.

Analysis of somatic mutations in membrane proteins of tumor cells / 中国生化药物杂志

Objective To screen specific mutations on extracellular regions of membrane proteins ( extracellular membrane protein mutations ) in tumor cells and provide the reference information for target searching in cancer precision medicine .Methods Somatic mutations on extracellular regions of membrane proteins of 7042 tumor samples were collected to screen all specific extracellular membrane protein mutations, and the overall distribution of these mutations were obtained by statistical analysis.Genes, gene site and cancer types occured high frequency of extracellular membrane protein mutations were identified.Results 97193 specific extracellular membrane protein mutations were obtained from 4938362 somatic mutations in 7042 tumor samples (30 cancer types), the statistical analysis showed that 4347 genes and 65532 sites were involved in these specific mutations.The study further analyzed five genes (MUC16、LRP1B、CSMD3、RYR2、USH2A), one site (17:37868208) and six cancer types (including colorectal cancer, melanoma, uterine cancer, brain lower grade glioma, lung adenocarcinoma and stomach adenocarcinoma) which occured high frequency of extracellular membrane protein mutations.Conclusion An information library of specific mutations on extracellular regions of membrane proteins was established and the distribution of these specific mutations was obtained which can provide reference information for target detection in targeted cancer therapy and immunological therapy.

Introduction for clinical trials of aogliptin / 中国生化药物杂志

Alogliptin is a class of highly selective dipeptidyl peptidase-IV (DPP-IV) inhibitors.It can reduce the glucose level mainly through inhibiting the decomposition of dipeptidyl peptidase of glucagon peptide-1 (GLP-1), therefor promote insulin secretion.A large number of clinical trials have been conducted before and after algliptin get approved by Food and Drug Administration form different countries , which proves that alogliptin can remarkably reduce blood glucose without causing any serious risks.This article is mean to introduce most of the important clinical trials that has been conducted, from Phase I to Phase IV.

Gel immobilization of human genome / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To develop a solid phase PCR method by covalent single point immobilization for recycle utilization of human genome.</p><p><b>METHODS</b>Polymethacrylamide gel was selected as a solid PCR carrier based on DNA-hydrogel copolymer chemistry presented by Mirzabekov. (CH2)6NH2 amino-modified PCR product and randomly fractured formic acid-modified plasmid pGEM-T-HLA-G were used as templates. The specificity of the attachment chemistry was characterized by acrylamide gel electrophoresis, and the thermal stability of method was demonstrated by PCR. This method was applied for the recycle utilization of human genome. Sequencing was used to exclude the possibility of introduced mutations during modification and immobilization procedures.</p><p><b>RESULTS</b>The PCR detections of plasmid DNA and human genome DNA immobilized by polymethacrylamide gel was successful. The thermal stability of method was successfully demonstrated by performing PCR after 16 rounds of standard 36 PCR cycles. And the sequencing was found no mutation.</p><p><b>CONCLUSION</b>The DNA immobilization method with polymethacrylamide gel as a solid phase carrier is stable and specific, which can be a possible approach for realizing recycle utilization of human genome for whole-genome sequencing and SNP detection.</p>

Determination of several environmental contaminants in human body / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To detect common environmental pollutants in human body.</p><p><b>METHODS</b>Urine samples were collected from 80 healthy subjects. Chromatography mass spectrometry (GC-MS), HPLC and ELISA were applied to detect several common environmental pollutants in urine samples.</p><p><b>RESULTS</b>DBP and methylbenzene were present in 75.3% and 41.2% of urine samples. The methanal and AFM1 were found in most of urine samples (approximately 91≊97%). By contrast, PCBs, CPZ, 4, 5-DCC were found in less than 5 samples, but there was no TMT detected.</p><p><b>CONCLUSION</b>Some of the environmental pollutants including carcinogens are detected in urine samples in this study.</p>

Preparation of salbutamol polyclonal antibodies and development of indirect competitive enzyme-linked immunoassay / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare the antibodies against salbutamol (SAL) with high sensitivity and to develop an indirect competitive enzyme-linked immunoassay (ic-ELISA) for fast detection of SAL.</p><p><b>METHODS</b>The New Zealand white rabbits were immunized with SAL in a small dose and long period mode. The method of ic-ELISA was optimized and adopted for the detection of a series of SAL samples, then the standard curve of SAL was established. The precision and the recoveries of the method were determined.</p><p><b>RESULTS</b>The antibodies with high sensitivity towards SAL were prepared with a IC50 of 12.21 ng/ml. The ic-ELISA method for SAL measurement was established, the recoveries of measurement was between 95%-105% and the CV was <3%.</p><p><b>CONCLUSION</b>The antibodies against salbutamol have been prepared and an indirect competitive enzyme-linked immunoassay for fast and specific detection of SAL has been developed.</p>

Synthesis of artificial diethylstilbestrol antigen for preparation of polyclonal antibodies / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To synthesize artificial diethylstilbestrol (DES) antigen and to prepare DES polyclonal antibody with high titer and sensitivity.</p><p><b>METHODS</b>The derivative of DES (DES-HS) was synthesized from diethylstilbestrol, ethyl bromoacetate,bovine serum albumin (BSA) and chicken ovalbumin (OVA) with the nucleophilic substitution reaction; the compound was identified by electrospray ionization mass spectrometry(ESI-MS). The DES-HS and the carrier proteins (BSA, OVA) were cross-linked to prepare the artificial antigen; the UV absorption spectrophotometry and high performance liquid chromatography (HPLC) were used to identify the prepared artificial antigen. The rabbits were immunized with the DES artificial antigen to prepare the DES polyclonal antibodies.</p><p><b>RESULTS</b>The DES-HS was synthesized. The DES artificial antigen was prepared successfully with a coupling rate of 22:1. The DES polyclonal antibodies with a titer of 1:25 600 and IC50 of 10.81 ng/ml were prepared with DES artificial antigen.</p><p><b>CONCLUSION</b>A set of methods to synthesize DES artificial antigen and to prepare the DES polyclonal antibodies has been developed successfully.</p>

Optimization of tri-expression of human CYP3A4 with POR and cyt b5 in Sf 9 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the optimal conditions of tri-expression of CYP3A4, POR and cyt b5 in Sf 9 cells.</p><p><b>METHODS</b>The Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks. The optimized conditions, including the temperature and rotation speed, the culture volume, the amount of surfactant and the culture time were studied. The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined.</p><p><b>RESULTS</b>When the temperature and rotation speed of the shaker were 27 degree and 90 r/min, the cell density and culture volume were 5X105 cells/ml and 80-120 ml per 250 ml shaker flasks, respectively. When Pluronic F-68 was 0.1% and the culture time was 72 h, the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins. When the ratio of the volume of three added viruses was 1:1:1, the expression condition was optimal, under which the Km, Vmax, and CLint for testosterone metabolism were 119.6 μmol/L,0.52 μmol/(min*g protein) and 4.34 ml/(min*g protein), respectively.</p><p><b>CONCLUSION</b>The conditions of tri-expressing of CYP3A4, POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.</p>

Whole-genome sequencing: a new approach for understanding of pathogenesis and individualized treatment of cancer / 浙江大学学报·医学版

With the development of sequencing technology, the cost of whole-genome sequencing was significantly declined.Meanwhile, with the application of combined whole-genome sequencing with epigenetic analysis on methylation and histone acetylation, the comprehensive and systematic analysis of numerous samples became a reality and we are able to re-understand the genesis and development of cancer. New ideas are emerging in comparative genomics research methods, from comparison of genomes among different individuals to horizontal self-comparison of different tissues and vertical self-comparison of genomes recently.Individualized diagnosis and treatment of cancer has shown a bright future.

Heterogeneous expression of human CYP and its application in early stage of new drug research and development / 浙江大学学报·医学版

Human cytochrome P450 (CYP) has a pivotal role on metabolism of xenobiotics and endogenous substances in clinical practice. Since the CYP from human tissue is very complex, and the human tissue itself is not easy to obtain, investigators begin to use all kinds of expression system to heterogeneously express the CYP. The single CYP expressed was then used for drug metabolism and drug-drug interaction research, to improve the efficiency of high-throughput drug screening greatly. Besides, since the polymorphism of drug-metabolizing enzymes makes efficacy variance for some drugs in different population, the heterogeneous expression and drug metabolizing research of certain CYP mutants will be helpful to guide the optimization of therapeutic regimen and conduct the personalized medication.

Detection of somatic mutations in deteriorated cell of peritoneal mesothelioma by whole genome sequencing / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To detect the somatic mutations in peritoneal mesothelioma with whole genome sequencing technique.</p><p><b>METHODS</b>Surgically resected cancer and pericancerous tissue samples from one patient with peritoneal mesothelioma were obtained. The whole genome sequences of tumor tissue and pericancerous tissue were examined by the second generation sequencing technique and compared with reference sequences from human genome database.</p><p><b>RESULTS</b>There were 639 717 single nucleotide variations (Single Nucleotide Variation SNV) found in both tumor and pericancerous tissue cells; while 20 302 SNVs were unique for tumor cells and 2 185 SNVs unique for pericancerous tissue, but still 223 SNVs found in cancer and pericancerous tissue were differed from those in human genome database.</p><p><b>CONCLUSION</b>The preliminary results indicate that merely comparing the gene sequences of cancer and pericancerous tissue samples in an individual with the human genome reference sequence can not accurately locate all somatic mutations in pathological cells. For those individualized diseases caused by random somatic mutations, it is suggested to sequence the whole genome at birth or at least to reserve a DNA sample at early age for both research and clinical needs.</p>

CYP2D6*1, CYP2D6*10 co-expressed with CYPOR in Bac-to-Bac expression system and activity determination / 药学学报

CYP2D6 is an important drug-metabolizing enzyme. The polymorphism of CYP2D6 leads to metabolism difference and the different reactions of drugs in the individuals and different races are normal phenomenon in clinical medication. CYP2D6*10 is an important subtype in Asian people and 51.3% Chinese are classified with this subtype. To obtain recombinant active CYP2D6*1/CYP2D6*10 in baculovirus system by optimizing coexpression with CYPOR, and detect their activity to catalyze dextromethorphan, three recombinants pFastBac-CYP2D6*1, pFastBac-CYP2D6*10 and pFastBac-CYPOR were constructed and transformed into DH10Bac cell to obtain the recombinant Bacmid-CYPOR, Bacmid-CYP2D6*1 and Bacmid-CYP2D6*10. And then the recombinant CYP2D6*1 and CYP2D6*10 virus were obtained by transfecting Sf9. Then homogenate protein activity was determined with dextromethorphan as substrate. The multiple of infection (MOI) and its ratio of recombinant CYP2D6 virus to CYPOR virus were adjusted by detecting the activity of the homogenate protein. The Km and Vmax are 26.67 +/- 2.71 micromol x L(-1) (n=3) and 666.7 +/- 56.78 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*1 to catalyze dextromethaphan. The Km and Vmax are 111.36 +/- 10.89 micromol x L(-1) (n=3) and 222.2 +/- 20.12 pmol x nmol(-1) (CYP2D6) x min(-1) (n=3) for CYP2D6*10 to catalyze dextromethorphan. There is significant difference between CYP2D6*1 and CYP2D6*10 for Vmax and Km (P < 0.01). The clearance ratio of CYP2D6*1 is 25.0 and the clearance ratio of CYP2D6*10 is 2.0. The expressed CYP2D6*1 and CYP2D6*10 are useful tools to screen the metabolism profile of many xenobiotics and endobiotics in vitro, which are benefit to understand individual metabolism difference.

The role of ADME evaluation in translation research of innovative drug / 药学学报

New Chemical Entities (NCEs) development is a systematic long-term project that involves multiple disciplines. The translation research will help to build an advanced R&D system from the basic laboratory research, preclinical studies and clinical evaluation to clinical application of drug, for the purpose of shortening the R&D cycle and accelerate the launch of new drugs. In new drug R&D and its clinical application, drug disposition (absorption, distribution, metabolism, excretion, ADME) properties are important criteria for assessing drug-likeness of candidates. ADME evaluation of NCEs plays an important role in the translation research throughout innovative drug R&D process. Therefore, ADME evaluation at the early stage of drug design and development will be helpful to improve the success rate and reduce costs, and further access to safe, effective drugs.