Objective To establish a method for determination of copper, manganese and cadmium in human urine by transversely heated GFAAS.Methods Matrix modifiers were used and background absorption was deducted, copper, manganese and cadmium in human urine were determined by transversely heated GFAAS after digestion.Results 5 g/L Mg(NO3)2 was taken as the matrix modifier when copper and manganese were determined and 0.3 g/L Mg(NO3)2 and 5 g/L NH4H2PO4 was taken as the matrix modifier when cadmium was determined.Under the designed standard conditions, the detection limits of copper, manganese and cadmium were 0.028-0.060 ?g/L;RSDs were 1.1%-5.1%;Recovery rates were 94.2%-107.5%.Conclusion The method is simple, accurate and sensitive, and is applicable to the determination of copper, manganese and cadmium in human urine.

Objective To analyze the etiologic spectrum of hand, foot and mouth disease (HFMD) and the molecular characteristics of coxsackievirus A10 (CVA10) strains isolated in Qingdao from year 2010 to 2012.Methods Throat swab specimens were collected from patients with HFMD to detect to-tal enteroviruses ( EVs) , EV71 and CVA16 strains by multiplex real time RT-PCR.The EV-positive speci-mens were further detected by a semi-nested RT-PCR to amplify the sequence of viral genes encoding VP1. The serotypes of EVs were identified based on the sequences of genes encoding VP1.The full-length gene se-quences encoding VP1 of CVA10 isolates were amplified and sequenced. The phylogenetic analysis was con-ducted by using MEGA5.0 software package.Results A total of 1919 outpatients with mild HFMD and 1336inpatients with serious HFMD were recruited in this study .CVA16 strains were the predominant patho-gen for outpatients in year 2010 ( prevalence rate of 53%) and 2012 ( prevalence rate of 73%) .EV71 strains were the predominant pathogen for outpatients in year 2011 ( prevalence rate of 78%) and inpatients in year 2010 ( prevalence rate of 70%) and 2011 ( prevalence rate of 86%) . Some serotypes other than CVA16 or EV71 were the predominant pathogens for inpatients in year 2012 ( prevalence rate 44%) . CVA10 strains were identified in 12 patients with HFMD in year 2010and 17 patients with HFMD in 2012. The full-length gene sequences encoding VP1 of 23 CVA10 isolates were successfully amplified and se-quenced.The phylogenetic analysis showed that the 23 CVA10 isolates all belonged to genotype C and could be further divided into six clades.The genes encoding VP1 of the 23 CVA10 strains shared 97.3% to 1000.%homologies in amino acid sequences.The CVA10 isolates were also similar to their counterparts isolated from other regions during the same period.Conclusion CVA16 and EV71 strains were the preva-lent pathogens of HFMD in Qingdao from year 2010 to 2012, co-circulated with some other serotypes of EVs, especially CVA10 strains.The CVA10 strains belonged to genotype C and shared high homologies among them and their counterparts circulated in other regions during the same period.

The cyclization of 2,3-oxidosqualene is the key branch point of ergosterol and triterpenoid biosynthesis. Downregulation of 2,3-oxidosqualene metabolic flux to ergosterol in Saccharomyces cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway. In our study, primers were designed according to erg7 gene sequence of S. cerevisiae. Three fragments including 5' long fragment, 5' short fragment and erg7 coding region fragment were amplified by PCR. 5' long fragment consists of the promoter and a part of erg7 coding region sequence. 5' short fragment consists of a part of promoter and a part of erg7 coding region sequence. These fragments were inserted reversely into pESC-URA to construct antisense expression plasmids. The recombinant plasmids were transformed into S. cerevisiae INVSc1 and recombinant strains were screened on the nutritional deficient medium SD-URA. The erg7 expression level of recombinant strains, which harbored antisense expression plasmid of erg7 coding region, was similar to that of INVScl by semi-quantitative PCR detection. But erg7 expression level of recombinant strains, which harbored 5' long antisense fragment and 5' short antisense fragment, was significantly lower than that of the control. The results of TLC and HPLC showed that the ergosterol content of recombinant strains, which harbored 5' long antisense fragment, decreased obviously. The ergosterol contents of the others were almost equal to that of INVSc1. Lanosterol synthase gene expression was downregulated by antisense RNA technology in S. cerevisiae, which lays a foundation for reconstructing triterpenoid metabolic pathway in S. cerevisiae by synthetic biology technology.

Pichia pastoris is one of the most important systems used in the field of molecular biology for the expression of recombinant proteins. The system has advantages of high expression, high stability, high secretion, easy high-density fermentation and low cost. Many factors affect the expression of recombinant protein, such as gene copy number, codon usage preference, type of promoter, molecular chaperones, glycosylation, signal peptide and fermentation process. In this review, research advances of the above aspects are summarized, which lay a foundation for improving the expression of recombinant proteins in P. pastoris.

Traditional herbal medicines, Panax ginseng, Panax quinquefolium and Panax notoginseng, attract our attention for their extensive and powerful pharmacological activities. Ginsenosides are the main active constituents of these medicinal herbs. The related glycosyltransferases involved in ginsenoside biosynthesis are the key enzymes which catalyze the last important step. Modification of ginsenoside aglycones by glycosyltransferases produces the complexity and diversity of ginsenosides, which have more extensive pharmacological activity. At present, ginsenoside aglycones and compound K have been obtained by synthetic biology. As the last step of ginsenoside biosynthesis, glycosylation of ginsenoside aglycones has been studied intensively in recent years. This review summarizes the basic strategies and research advances in studies on glycosyltransferases involved in ginsenoside biosynthesis, which is expected to lay the theoretical foundation for the in-depth research of biosynthetic pathway of ginsenosides and their production by synthetic biology.

BmK AngM1 is a long-chain scorpion toxin purified from the venom of Buthus martensii Karsch. It has been reported to exhibit evident analgesic effect and low toxicity, and has the potential to be a novel analgesic drug. The BmKAngM1 gene was transformed into Pichiapastoris GS115. Mut+ and Mut(s) recombinant strains were screened by phenotype and Mut+ recombinant strains were used to detect BmK AngMl gene copy number in the real-time PCR. Expression of BmK AngM1 in the Mut+ recombinant strain was compared with that of the Mut(s) recombinant strain with the same single copy of BmK AngM1 gene under the same condition. The results indicated that the transcription level of BmK AngM1 gene in the Mut(s) recombinant strain was 2.7 fold of that in the Mut recombinant strain in the real-time PCR, and the expression of BmK AngM 1 in the Mut(s) recombinant strain was 1.5 fold of that in the Mut+ recombinant strain. Therefore, Mut(s) recombinant strain showed better ability to express BmK AngM1 than Mut+ recombinant strain.

Lanosterol synthase is encoded by the erg7 gene and catalyzes the cyclization of 2, 3-oxidosqualene, which is a rate-limiting step of the inherent mevalonate (MVA)/ergosterol metabolic pathway in Saccharomyces cerevisiae. The intermediate 2, 3-oxidosqualene is also the precursor of triterpenoids. Therefore, the cyclization of 2, 3-oxidosqualene is the key branch point of ergosterol and triterpenoids biosynthesis. Down-regulation of 2, 3-oxidosqualene metabolic flux to ergosterol in S. cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway reconstructed by the synthetic biology approach. To construct erg7 knockout cassette harboring the loxP-Marker-loxP element, long primers were designed, which were homologous to the sequences of both erg7 ORF and plasmid pUG66. The cassette was transformed into diploid wild strain INVSc1 by LiAc/SS Carrier DNA/PEG method and then erg7 gene haploid deficient mutant was obtained by homologous recombination. The results of semiquantitative PCR and real-time quantitative PCR revealed that erg7 expression level in erg7 gene haploid deficient mutant is one time lower than that in wild strain. The results of TLC and HPLC showed that the ergosterol content in deficient mutant decreased to 42% of that in wild strain.

Objective To investigate the etiology spectrum of hand , foot and mouth disease ( HFMD) and to analysis the molecular characteristics of three predominant human enterovirus stains in Qingdao in 2013.Methods The total enterovirus (EV) strains and strains of EV71, CVA16 and CVA6 in throat swabs of HFMD cases were detected by using multiplex real time RT-PCR.The full-length of the viral VP1 genes of the EV strains were amplified and sequenced .The sequences were phylogenetically analyzed by using the MEGA5.0 software package .Results A total of 841 patients with mild HFMD and 107 patients with serious HFMD were recruited in this study and 64 .3%of them were positive for EV .The predominant pathogens were EV71 (44.8%), CVA6 (28.2%) and CVA16 (9.5%) in 2013.CVA6 replaced CVA16 as the second most common pathogen for HFMD , accounting for 42.7% of all pathogens in children aged less than 3 years and 22.2%of all pathogens in the serious patients .The proportions of CVA6 in the etiology spectrum showed a downtrend along with the increasing age of the patients (P<0.001).Phylogenetic analy-sis of the complete VP1 gene sequences showed that all of the EV 71 strains identified in this study belonged to the subgenotype C4 (evolutionary branch C4a) and all of the CVA16 strains belonged to the subgenotype B1 (evolutionary branches B1a and B1b).There were 6 genogroups (A to F) regarding to the VP1 gene of CVA6 and all of the CVA6 strains identified in this study belonged to genogroups A and D .Among the CVA6 strains isolated in Qingdao in 2013, 83.9% belonged to genogroup A, while the rest 16.1% belonged to genogroup D.66.7%of the CVA6 strains isolated in 2012 belonged to genogroup A, while the rest 33.3%belonged to genogroup D .All of the CVA6 strains isolated from year 2008 to 2011 in Qingdao belonged to genogroup D.Conclusion EV71, CVA6 and CVA16 were the prevalent pathogens responsible for the de-velopment of HFMD in Qingdao in 2013.The proportions of CVA6 strains in the etiology spectrum showed a downtrend with the increasing age in children .C4a was the major subtype of EV71 strains circulating in Qingdao in 2013, while B1a and B1b were the major subtypes of CVA16 strains.The pattern of endemic cir-culation of CVA6 strains showed a trend of changing from genogroup D to A from year 2008 to 2013 .

In hygienic examination experiment teaching,Science inquisition is the important practice for students to gain knowledge and solve problems. In order to enhance students’scientific accomplishment and raise their scientific research ability,inquisition subject design should be highlighted and inquisition horizontal level should be well designed and science inquisition method education should be valued in experiment teaching.

Objective:To investigate the whole genome characteristics of coxsackievirus A4 (CVA4) circulating in Qingdao city.Methods:Four CVA4 isolates circulating in Qingdao city during 2013 to 2015 were selected. Whole genome sequences of these strains were amplified by one-step reverse transcription-polymerase chain reaction (RT-PCR). Sequence alignment and phylogenetic analysis were performed using MEGA7.0 software package. Genetic recombination analysis was performed using similarity plots 3.5.1 software package.Results:Phylogenetic analysis showed that based on the sequences of the whole genome and P1, P2 and P3 regions, HS312/QD/CHN/2013 and HS605/QD/CHN/2014 strains together with the early domestic isolates belonged to the same clade, while FY218/QD/CHN/2015 strain and CV-A4/P1033/2013/China strain collected in Wenzhou in 2013 formed another clade in each phylogenetic tree. HS144/QD/CHN/2014 strain belonged to the same clade as HS312/QD/CHN/2014, HS605/QD/CHN/2014 and the early domestic CVA4 isolates in the phylogenetic tree based on the P1 region, but formed a separate clade in the phylogenetic trees based on the whole genome, P2 region and P3 region. Genetic recombination analysis revealed that there was genetic recombination between HS144/QD/CHN/2014 strain and the CVA2 strain of CV-A2/P373/2013/China isolated in mainland China in 2013 in the region of 2C-3D (5 081-7 301); FY218/QD/CHN/2015 and CV-A4/P1033/2013/China strains were highly homologous and recombination signal sequences were detected in the region of 2A-2B (3 821-4 161) between the two strains and the CVA2 strain of CV-A2/P373/2013/China.Conclusions:The CVA4 isolates circulating in Qingdao city presented obvious genetic diversity at the genome-wide level.