Cellulose synthase (CesA), one of the key enzymes in the biosynthesis of cellulose in plants, plays an important role in plant growth and plant resistance. In this study, a total of 21 AsCesA genes from Aquilaria sinensis were systematically identified and the physico-chemical characteristics were analyzed based on genome database and bioinformatical methods. The phylogenetic tree was constructed and the gene location on chromosome, cis-acting elements in the 2 000 basepairs upstream regulatory regions and conservative motifs were analyzed. The AsCesA proteins were mainly located on the plasma membrane. The number of amino acids of the proteins ranged from 390 to 1 261. The isoelectric point distributed from 5.67 to 8.86. All of the 21 AsCesA proteins possessed the transmembrane domains, the number of which was from 6 to 8. The genes were classified into 3 groups according to the phylogenetic relationship. Obvious differences were observed in motif composition in genes from different groups. However, motif2, motif6, motif7 and motif10 were observed in all of AsCesA proteins. Analysis of cis-acting elements indicated that AsCesA genes family has cis-acting elements related to plant hormones, abiotic stresses, and biological processes. Seven AsCesA genes with differential expression were selected according to the calli transcriptome data induced by NaCl at different times and their expression levels under different abiotic stresses were analyzed by quantitative real-time PCR. The results indicated that salt, low temperature, drought, and heavy metal stresses could affect the expression level of AsCesA genes, and the abundance of AsCesA1, AsCesA3 and AsCesA20 showed a significant change, implying their potential important roles to the abiotic stresses. The accumulation pattern of cellulose content under different abiotic stresses was similar to the expression trend of AsCesA genes. Our results provide valuable insights into the role of cellulose synthase in A.sinensis in plant defense.

Glyceryl phenylbutyrate(GPB)serves as a long-term management medication for Ornithine transcarbamylase deficiency(OTCD),effectively controlling hyperammonemia,but there is a lack of experience in using this medicine in China.This article retrospectively analyzes the case of a child diagnosed with OTCD at Shanghai Children's Medical Center,Shanghai Jiao Tong University School of Medicine,including a review of related literature.After diagnosis,the patient was treated with GPB,followed by efficacy follow-up and pharmacological monitoring.The 6-year and 6-month-old male patient exhibited poor speech development,disobedience,temper tantrums,and aggressive behavior.Blood ammonia levels peaked at 327 μmol/L;urine organic acid analysis indicated elevated uracil levels;cranial MRI showed extensive abnormal signals in both cerebral hemispheres.Genetic testing revealed de novo mutation in the OTC gene(c.241T＞C,p.S81P).Blood ammonia levels were approximately 43,80,and 56 μmol/L at 1,2,and 3 months after starting GPB treatment,respectively.During treatment,blood ammonia was well-controlled without drug-related adverse effects.The patient showed improvement in developmental delays,obedience,temperament,and absence of aggressive behavior.

Dihydroflavonol 4-reductase (DFR) plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color. Based on the transcriptome data of Cistanche tubulosa (Schenk) Wight, a full-length cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CtDFR contains an open reading frame (ORF) of 1 263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa. The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate (NADPH) binding domain and a specific substrate binding domain. The expression analysis indicated that CtDFR was highly expressed in red and purple flowers, and the relative expression levels were 4.04 and 19.37 times higher than those of white flowers, respectively. The recombinant CtDFR protein was expressed in E.coli BL21 (DE3) using vector pET-28a-CtDFR and was purified. In vitro enzyme activity analysis, CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol, dihydroquercetin, and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin. Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm. These results demonstrate that CtDFR plays an important role in regulation of flower color in C. tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C. tubulosa (Schenk) Wight flower color.

Objective:To investigate the antitumor effect and the mechanism of Dunhuang Pingweiwan and its decomposed recipes based on phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway in SCG-7901 gastric cancer-mice. Method:The subcutaneous tumor bearing model of SCG-7901 gastric cancer in mice was established， and the the mice were randomized into model group， Dunhuang Pingweiwan group （14.04 g·kg^-1·d^-1）， Huoxue Jiedu group （6.50 g·kg^-1·d^-1）， Wenzhong Sanhan group （3.64 g·kg^-1·d^-1） and cisplatin group （2 mg·kg^-1·d^-1）， with 8 mice in each group. From the 8^th day of inoculation， the mice were administered for 10 consecutive days. The mice were weighed and the general conditions were observed every other day. On the next day of the last administration， the mice were sacrificed， and the tumor was removed and weighed to calculate the anti-tumor rate. The histopathological changes were observed by hematoxylin-eosin （HE） staining， and the mRNA and protein expressions of PI3K， Akt， and mTOR in tumor tissues were detected by real-time polymerase chain reaction（Real-time PCR） and immunohistochemistry （IHC）， respectively. Result:From the 10^th day of inoculation， the mice in cisplatin group were generally in poor condition and their body mass decreased. The mice in model group， Dunhuang Pingweiwan group， Huoxue Jiedu group and Wenzhong Sanhan group were generally fair， and their body mass increased without significant difference among groups. The tumor inhibition rates of Dunhuang Pingweiwan， Huoxue Jiedu， Wenzhong Sanhan and cisplatin groups were 30.74%， 24.80%， 4.19% and 63.84%， respectively. Except for Wenzhong Sanhan group， tumor weight of the other treatment groups was significantly lower than that of the model group （P<0.01）， and there was no significant difference between the Dunhuang Pingweiwan and Huoxue Jiedu group. Dunhuang Pingweiwan and Huoxue Jiedu group could significantly reduce tumor cell density and cause tumor cell necrosis. Compared with the model group， the expressions of PI3K， Akt， and mTOR mRNA and protein in the Dunhuang Pingweiwan， Huoxue Jiedu and cisplatin groups significantly decreased （P<0.05， P<0.01）， and there was no significant difference between the Dunhuang Pingweiwan group and Huoxue Jiedu group. Conclusion:Dunhuang Pingweiwan and its decomposed recipes （Huoxue Jiedu） have a certain anti-tumor effect on the SCG-7901 gastric cancer-mice， and the mechanism may be related to the down-regulation of key molecules in the PI3K/Akt/mTOR signaling pathway.

Traditional Chinese medicine （TCM） and western medicine have their respective advantages and limitations in the diagnosis and treatment of common otorhinolaryngology head and neck diseases. Although the integrated TCM and western medicine exhibits definite curative effects， there is no consensus on the otorhinolaryngology head and neck diseases responding specifically to TCM or integrated TCM and western medicine， as well as the diagnosis and treatment schemes. The China Association of Chinese Medicine （CACM） thus organized the otorhinolaryngology head and neck specialists of both TCM and western medicine to discuss the etiology， pathogenesis， and clinical diagnosis and treatment methods of common otorhinolaryngology head and neck diseases with the results of multiple clinical trials taken into account. The acute pharyngitis， chronic pharyngolaryngitis， paraesthesia pharyngis， hysterical aphasia， allergic rhinitis， subjective tinnitus， and otogenic vertigo were confirmed to respond specifically to TCM or integrated TCM and western medicine. Then a mutually agreed diagnosis and treatment scheme and recommendation with integrated TCM and western medicine was formulated as a reference for clinical practice， thus benefiting more patients.

The MYB gene family comprises one of the richest groups of transcription factors in plants. The full length of two MYB genes were isolated through heterologous screening of Aquilaria sinensis calli transcriptome data, and the reverse transcription PCR was performed to obstain the corrected MYB clones, named AsMYB1, AsMYB2. The MYB transmembrane domain and phylogenetic analysis were predicted by different software to analyze the bioinformatics of MYB proteins. The transcript level of AsMYB1, AsMYB2 was performed by real-time quantitative RT-PCR in different tissues and in responds to abiotic stresses including salt, cold, metal and drought stress, and hormone treatments including abscisic acid (ABA), salicylic acid (SA), gibberellins (GA3) and methyl jasmonate (MeJA) treatment. The AsMYB1 cDNA sequence had an ORF of 1 063 nucleotides, encoding a protein of 353 amino acids. The largest AsMYB2 ORF was 1 081 nucleotides, and its predicted translation products consisted of 359 amino acids. Two MYB genes had a tissues-specific pattern in A. sinensis. Moreover, the expression level of AsMYB1 and AsMYB2 was regulated by different abiotic stresses and hormone treatments, suggesting the transcription factors AsMYB1 and AsMYB2 play an important role in plant defense and hormone signal transduction in A. sinensis.

Jasmonic acid (JA) is an important signal molecule involved in plant resistance, and allene oxide synthase (AOS) is a key enzyme in the biosynthesis of jasmonates. In this study, a full-length cDNA of AsAOS1 gene was cloned from Aquilaria sinensis. Meanwhile, the sequence analysis, prokaryotic expression, purification, tissue-specific expression analysis and expression analysis under different abiotic stresses and hormone treatments were performed. The open reading frame (ORF) of AsAOS1 gene was 1 575 bp, encoding a protein of 524 amino acid residues, with a predicted molecular mass of 58.70 kDa. AsAOS1 protein possessed the conserved sequences of cytochrome P450 (CYP450). The phylogenetic analysis indicated that AsAOS1 protein had the highest level of homology with AOS protein of Citrus sinensis. The recombinant AsAOS1 protein was successfully expressed in Escherichia coli BL21(DE3) cells using the prokaryotic expression vector pET28a-AsAOS1 and the recombinant AsAOS1 was purified by Ni²⁺ affinity chromatography. Expression analysis results in different tissues showed that AsAOS1 was primarily observed in stems, and then roots, followed by leaves. AsAOS1 transcript level was significantly induced after 12 h treatment of NaCl, cold temperature and CdCl₂. Furthermore, AsAOS1 expression level was enhanced upon methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) treatment. However, mannitol and gibberellin (GA₃) treatments had little influence on the expression level of AsAOS1. These results provides valuable insights into the role of JA in the mechanism of agarwood formation and plant resistance.

BACKGROUND:Immortalized cervical epithelial cels H8 can become cancerous under the induction of carcinogenic agent, and may cause cervical cancer when there is a cofactor interaction. However, there is stil a lack of effective intervention for female patients with precancerous lesions, and this treatment is blank in the clinic. OBJECTIVE: To explore the effects and mechanism of astragalus injection on apoptosis of human immortalizedcervical epithelial cels H8. METHODS: This study contained two groups: astragalus drug group and the blank control group. (1) Enzyme linked immunosorbent assay (ELISA) was used to detect DNA fragments of apoptotic H8 after astragalus injection. (2) Enzyme-labeling instrument was used to analyze the changes in caspase-3 and caspase-9 activities a fter astragalus injection. (3) Western blot assay was used to detect the protein expression changes of caspase-3, caspase-9 and PARP in H8 cels after astragalus injection. RESULTS AND CONCLUSION: (1) ELISA results showed that at 0, 6, 12 and 24 hours after 20 g/L astragalus injection, DNA fragments were gradualy increased with time prolonged in a time-dependent effect (P < 0.05). (2) Enzyme-labeling instrument demonstrated that at 0, 6, 12 and 24 hours after 20 g/L astragalus injection, caspase-3 and caspase-9 activities increased in a time-dependent manner (P < 0.05). (3) At 0, 6, 12 and 24 hours after 20 g/L astragalus injection, the expression of cleaved caspase-3 and cleaved caspase-9 were gradualy increased in H8 cels (P < 0.05). Cleaved PARP protein expression was gradualy decreased (P < 0.05). These findings indicate that astragalus injection could obviously induce H8 apoptosis, which may be associated with the upregulated protein expression of caspase-3 and caspase-9.

Objective Toinvestigatethestatusoftick-borneRickettsiaeinfectionsamongmurine-likeanimalsin differentareasofZhejiangprovince.Methods Liverandspleensamplesofmurine-likeanimalscapturedthroughnight trapping method were collected from Anji,Jinhua and Tiantai County according to their geographic locations and historical detection of Rickettsiae .Nest-PCR tests were used to determine the presence of the 16S rRNA genes of Anaplasma and Ehrlichia ,and the heat shock protein genes (groEL)of Rickettsiae (including typhus and spotted fever group)and Orientiainthesesamples.Results Atotalof851murine-likeanimalsbelongingto14specieswerecaptured.The predominant species were Rattus confucianus (30.32%),Apodemus agrarius (18.80%) and Thallomys paedulcus (1 1.75%)and they were significantly different among three areas (P<0.05 ).48 Rickettsia positive were found in 562 tested samples with the positive rate of 8.54%,among which the percentage of Anaplasma,typhus group Rickettsia, Orientia,Ehrlichia and spotted fever group Rickettsia were 3.38%,1.78%,1.78%,1.07% and 0.53% respectively. The positive rates of Anaplasma in Jindong (4.76%)and Anji (4.27%)were significantly higher than that in Tiantai (P<0.05 )while the spotted fever group Rickettsia were found only in Tiantai County.Moreover,Rattus confucianus-the predominant species of Zhejiang Province-had the highest infection rate of tick-borne Rickettsiae up to 14.97%.Co-infections with several Rickettsiae were existed among the same species.Conclusion Rickettsiae infections exist widely among different areas of Zhejiang province and the positive rates are significantly different among species.

OBJECTIVETo investigate the plasma amino acid spectrum in infants more than 1-year-old with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) in order to identify potential diagnostic markers of NICCD.

METHODSInfants less than 1 year of age who had been referred to our hospital for investigation of suspected conjugated hyperbilirubinemia between June 2003 and June 2009 were eligible for enrollment. A total of 182 infants were enrolled and divided into three groups: infants diagnosed with NICCD (n = 24), according to gene testing and/or western blotting results; infants diagnosed with biliary atresia (BA; n = 20), according to intra-operative cholangiography findings; and infants diagnosed with idiopathic neonatal intrahepatic hepatitis (INH; n = 138), according to exclusionary findings for diseases affecting the extrahepatic biliary system and no positive serology results to indicate infections with hepatitis B, C, A or E, toxoplasmosis, rubella, herpes simplex, human immunodeficiency virus-1, or syphilis. The plasma amino acid spectrum of each infant was analyzed by tandem mass spectrometry (MS/MS). The concentrations of 18 amino acids, as well as the ratio of each amino acid to total amino acids, were compared among the three groups. Selected ratios of amino acids were analyzed by receiver operating characteristic (ROC) curve analysis.

RESULTSCompared with the BA and INH groups, the NICCD group had significantly lower levels of alanine (Ala; 175.7 and 205.7 vs. 136.3 mumol/L, P = 0.0001), aspartic acid (Asp; 47.5 and 43.1 vs. 31.55 mumol/L, P = 0.0041), glutamic acid (Glu; 276.16 and 263.24 vs. 175.71 mumol/L, P = 0.0075) and tryptophan (Trp; 41.90 and 47.97 vs. 28.51 mumol/L, P = 0.0003), but significantly higher levels of methionine (Met; 28.24 and 29.35 vs. 71.40 mumol/L, P = 0.0390), tyrosine (Tyr; 55.8 and 57.02 vs. 116.81 mumol/L, P = 0.0072) and citrulline (Cit; 15.09 and 15.65 vs. 97.42 mumol/L, P = 0.0001). The ratio of each amino acid to total amino acids showed the same trends for the NICCD group. The calculated areas under the ROC curves of the ratios of Cit, Tyr, and Met to the total amino acids were 0.874 (95% CI: 0.752 - 0.996), 0.814 (95% CI: 0.706 - 0.923), and 0.705 (95% CI: 0.535 - 0.875) respectively. The calculated area under the ROC curve of the ratio of Cit to Ala was 0.893 (95% CI: 0.781 - 1.005), and when the cut-off value of the ratio of Cit to Ala was 0.14 for diagnosis of NICCD, the sensitivity and specificity were 75% and 95% respectively.

CONCLUSIONThe plasma amino acid spectrum may represent a diagnostic indicator for NICCD, particularly the ratio of Cit to Ala.