Cloning and expression analysis of cytochrome P450 AsCYP71D1 from Aquilaria sinensis (Lour.) Gilg / 药学学报

Cytochrome P450 (CYP450) is a kind of superfamily oxidase containing heme, which is distributed in various aerobic organisms. They are widely involved in the biosynthesis of terpenoids, alkaloids, flavonoids, fatty acids, etc. In this study, the full-length cDNA sequence of a P450 was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) technology, with the specific primers that designed according to the sequence of a transcript annotated as P450 from the Aquilaria sinensis (Lour.) Gilg transcriptome database. The tissue expression and subcellular localization were also studied. The full-length cDNA of the cloned P450 gene is 1 920 bp, with 88 bp 5′-untranslated region (UTR), 344 bp 3′-UTR and a 21 bp polyA tail, and 1 488 bp open reading frame (ORF), encoding 495 amino acids. Sequence alignment revealed that the protein belonged to CYP71D family of cytochrome P450 family, and named AsCYP71D1. Tissue expression analysis indicated that AsCYP71D1 was mainly expressed in stem. Further subcellular localization of onion epidermis showed that AsCYP71D1 was expressed in cytoplasm, nucleus and cell membrane. This study will provide a foundation for further research on its function in agarwood sesquiterpene biosynthesis.

Cloning and expression analysis of JrGI gene in walnut / 生物工程学报

GI (GIGANTEA) is one of the output key genes for circadian clock in the plant. The JrGI gene was cloned and its expression in different tissues was analyzed to facilitate the functional research of JrGI. RT-PCR (reverse transcription-polymerase chain reaction) was used to clone JrGI gene in present study. This gene was then analyzed by bioinformatics, subcellular localization and gene expression. The coding sequence (CDS) full length of JrGI gene was 3 516 bp, encoding 1 171 amino acids with a molecular mass of 128.60 kDa and a theoretical isoelectric point of 6.13. It was a hydrophilic protein. Phylogenetic analysis showed that JrGI of 'Xinxin 2' was highly homologous to GI of Populus euphratica. The result of subcellular localization showed that JrGI protein was located in nucleus. The JrGI, JrCO and JrFT genes in female flower buds undifferentiated and early differentiated of 'Xinxin 2' were analyzed by RT-qPCR (real-time quantitative PCR). The results showed that the expression of JrGI, JrCO and JrFT genes were the highest on morphological differentiation, implying the temporal and special regulation of JrGI in the differential process of female flower buds of'Xinxin 2'. In addition, RT-qPCR analysis showed that JrGI gene was expressed in all tissues examined, whereas the expression level in leaves was the highest. It is suggested that JrGI gene plays a key role in the development of walnut leaves.

Emergence and clonal expansión of Klebsiella pneumoniae ST307, simultaneously producing KPC-3 and NDM-1

Abstract MDR Klebsiella pneumoniae ST307 is a high-risk clone, whose genetic features contribute to its adaptation to hospital environments and the human host. This study describesthe emergence and clonal dissemination of K. pneumoniae ST307, recovered during November2018 to February 2019 in a hospital in Buenos Aires city, which concurrently harbored KPC-3and NDM-1. These isolates were resistant to all -lactams and to the ceftazidime/avibactamcombination. Molecular studies showed that blaKPC-3was located in Tn4401a platform, whileblaNDM-1was surrounded upstream by ISKpn14 followed by a partial sequence of ISAba125 anddownstream by bleMBL-trpF, located in a 145.5 kb conjugative plasmid belonging to the Inc A/Cgroup. The dissemination of K. pneumoniae ST307 isolates co-producing KPC-3 and NDM-1 couldlead to a worrisome scenario due to the remarkable features of this clone and its resistanceprofile.

Resumen Klebsiella pneumoniae ST307 es un clon de alto riesgo, cuyas características genéticas contribuyen a su adaptación al entorno hospitalario y al huésped humano. Este estudio describe la emergencia y diseminación clonal de aislamientos de K. pneumoniae ST307 productores de KPC-3 y NDM-1, recuperados en un hospital de Buenos Aires. Estos aislamientos fueron resistentes a todos los p-lactámicos y a la combinación ceftacidima/avibactam. Los estudios moleculares evidenciaron que el contexto genético de blaKPC-3 se correspondió con el Tn4401a, mientras que blaNDM-1 estuvo flanqueado corriente arriba por ISKpn14 y una secuencia parcial de ISAba125 y corriente abajo por bleMBL - trpF, localizado a su vez en un plásmido conjugativo de 145.5 kb perteneciente al grupo Inc A/C. La emergencia de aislamientos de K. pneumoniae ST307 coproductores de KPC-3 y NDM-1 pone de manifiesto una situación altamente preocupante debido a las características de este clon y a su perfil de multirresistencia.

Epidermal growth factor receptor mutations in adenocarcinoma lung: Comparison of techniques for mutation detection

Background: Targeted therapy using tyrosine kinase inhibitors in cases of non-small-cell lung carcinoma (NSCLC) that harbor epidermal growth factor receptor (EGFR) mutations has drastically improved the overall survival rate. The current study estimated the frequency of EGFR mutations in the Indian population by analyzing the diagnostic parameters of various techniques available for the detection of these mutations. Materials and Methods: A case series of 100 histologically diagnosed and immunohistochemically confirmed NSCLC with the adenocarcinoma phenotype comprises the study sample. EGFR mutations were detected using clone-specific immunohistochemistry (IHC), real-time polymerase chain reaction (PCR), and Sanger sequencing. Results: EGFR mutations were identified in 48% cases with 72.78% mutations involving exon 19. Clone-specific IHC had a low sensitivity of 46.43%, and the specificity was 79.17%. Sanger sequencing yielded interpretable results in 16% cases only, which were in concordance with the results of real-time PCR. Conclusion: EGFR mutations are increasingly being explored for targeted therapy and personalized medicine. Real-time PCR was found to be the best and the most accurate method for the detection of somatic EGFR mutations in adenocarcinoma primarily in the lungs.

Cloning and functional analysis of the phenylalaninammo-nialyase gene from Rhododendron fortunei / 生物工程学报

Phenylalaninammo-nialyase (PAL) is a key enzyme in the synthesis of methyl benzoate - a plant aroma compound. In order to understand the function of this enzyme in the formation of fragrance in the scented Rhododendron species-Rhododendron fortunei, we cloned a gene encoding this enzyme and subsequently examined the gene expression patterns and the profile of enzyme activity during development in various tissues. The full length of RhPAL gene was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The expression levels of RhPAL gene were measured by real-time quantitative reverse transcription PCR (qRT-PCR) and the amount of phenylalanine and cinnamic acid were assayed with LC-MS. The results showed that the ORF sequence of RhPAL gene amplified from the cDNA templates of flower buds had 2 145 bp, encoding 715 amino acids, and shared 90% homology to the PAL amino acid sequences from other species. qRT-PCR analysis showed that the expression of RhPAL in petals during flowering kept in rising even until the flowers wilted. The expression of RhPAL in pistil was much higher than that in stamen, while the expression in the younger leaves was higher than in old leaves. However, the expression level was relatively lower in petal and stamen compared to that in leaves. We also measured the PAL activity by Enzyme-linked immuno sorbent assay in the petals of flowers at different flowering stages. The results showed that PAL activity reached the highest at the bud stage and then decreased gradually to the lowest when the flowers wilted, which followed a similar trend in the emission of the flower fragrance. The phenylalanine and cinnamic acid contents measured by LC-MS were highly correlated to the expression level of RhPAL in various tissues and at different flowering stages, implying that RhPAL plays an important role in the formation of the flower fragrance. This work may facilitate the breeding and improvement of new fragrant Rhododendron cultivars.

Composição volátil das uvas ao espumante da cv. Chardonnay em diferentes condições de manejo no sul de Minas Gerais (Brasil) / Volatile composition from grapes to sparkling wine of Chardonnay vine in different management conditions in southern of Minas Gerais (Brazil)

O aroma é um dos fatores mais importantes na determinação da qualidade e do caráter do vinho. Isso se deve à presença de compostos voláteis que estão associados às suas características organolépticas ou diferentes proporções entre estes compostos que podem ser influenciadas por fatores vitícolas (clima, solo, cultivar, manejo) e enológicos (maturação da uva, fermentação, tratamentos pósfermentativos). A região do sul de Minas Gerais vem se destacando na produção de espumantes de qualidade, e, nesse contexto, o presente trabalho teve como objetivo conhecer a influência do manejo da videira no desenvolvimento do aroma, da baga até o espumante, a fim de estabelecer associações com a qualidade do produto final. Os experimentos foram realizados com a cultivar Chardonnay em diferentes condições de manejo, em que foram avaliados clones, porta-enxertos, sistemas de condução e densidades de plantio. Foram analisados os compostos voláteis livres por HS-SPME/GC-MS das bagas, mostos, vinhos base e espumantes nas safras 2016, 2017 e 2018. O trabalho foi dividido em quatro partes para a apresentação dos resultados. A primeira consistiu em verificar a influência do material genético na composição volátil da cv. Chardonnay com os experimentos de clones e portaenxertos; a segunda parte avaliou a composição volátil do clone 809 até o espumante; a terceira, em analisar as vinificações dos diferentes sistemas de condução; e a quarta, em avaliar a evolução dos compostos voláteis da baga ao espumante e analisar os aromas que as densidades de plantio podem conferir ao espumante. As principais classes de compostos aromáticos identificados nas matrizes foram: C6-C9 aldeídos, álcoois superiores, aldeídos ramificados, benzenoides, monoterpenoides, norisoprenoides, sesquiterpenoides, cetonas e ácidos graxos. Os resultados mostraram que os clones e os porta-enxertos apresentaram perfis voláteis diferentes, indicando que a variabilidade entre os clones e que a enxertia têm influência no metabolismo da baga; o clone 809 apresenta maior abundância de compostos monoterpenoides, confirmando o seu caráter moscato, das uvas aos espumantes; os diferentes sistemas de condução e densidades de plantio alteram o metabolismo da14 baga, refletindo no perfil volátil dos espumantes nas safras estudadas. Dessa forma, os dados indicam que a composição volátil sofre influência do manejo da videira ao espumante

Aroma is one of the most important factors in determining the quality and character of wine. This is due to the presence of volatile compounds that are associated with their organoleptic characteristics or different proportions among these compounds that can be influenced by viticultural (climate, soil, cultivar, management) and oenological factors (grape maturation, fermentation, post fermentation treatments). The southern region of Minas Gerais has been standing out in the production of quality sparkling wines, and in this context, the purpose of the present work was to learn about the influence of grapevine management on the development of aroma, from berry to sparkling wine, in order to establish associations with the quality of the final product. The experiments were carried out with the Chardonnay cultivar under different management conditions, in which clones, rootstocks, trellising systems and planting densities were evaluated. The free volatile compounds by HS-SPME/GC-MS of the berries, musts, base and sparkling wines in the 2016, 2017 and 2018 harvests were analyzed. The work was divided into four parts in order to present the results. The first part consisted of verifying the influence of genetic material on the volatile composition of the cv. Chardonnay with the experiments on clones and rootstocks; the second part evaluated the volatile composition of clone 809 up to the sparkling wine; the third one part analyzed the vinification of the different trainig systems; and the fourth part evaluated the evolution of the volatile compounds from the berry to the sparkling wine and analyzed the aromas that the planting densities can confer to the sparkling wine. The main classes of aromatic compounds identified in the matrices were: C6-C9 aldehydes, higher alcohols, branched aldehydes, benzenoids, monoterpenoids, norisoprenoids, sesquiterpenoids, ketones and fatty acids. The results showed that the clones and the rootstocks have different volatile profiles, indicating that variability among clones and that grafting have great relevance to the berry secondary metabolism; the 809 clone presents a greater abundance of monoterpenoid compounds, confirming its muscat character, from grapes to sparkling wines; the different training systems and planting densities alter the berry´s metabolism, reflecting16 in the volatile profile of sparkling wines in the studied harvests. The data indicate that the volatile composition is influenced by the management of the berry to the sparkling wine

Orange-fleshed Sweet Potato Chips: Processing Effect on Carotenoid Content and Resistant Starch and Sensory Acceptance

HIGHLIGHTS Chips from orange-fleshed sweet potato have a good acceptability. Drying process showed retention of carotenoids total content. Chips from drying or frying process showed high resistant starch content.

Abstract There is currently a great demand for industrialized products with functional properties, together with the increase in consumption of roots and sweet potato products. Sweet potatoes have a high content of resistant starch, while only the orange-fleshed roots also have a high content of carotenoids. Due to these, this work aimed to produce orange-fleshed sweet potato chips, by two processes: drying oven and immersion frying. The chips were evaluated for the content of resistant starch and carotenoids in nature and chips sweet potatoes, and evaluations of the physical attributes and sensory analysis of the chips. The drying process retained a greater content of total carotenoids. Fried chips can be considered high resistant starch content, even with a decrease in the content after this processing; they also showed more intense coloring and pleasant texture. There was a statistical difference between the varieties only regarding the content of carotenoids and resistant starch. Thereby, it can be concluded that the chips of both processing have good technological and functional qualities, and that the frying process presented best hardness which led to greater acceptability and purchase intention.

Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy / Identificação e Caracterização do ATG8, um marcador de autofagia em Eimeria tenella

Abstract Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Resumo A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.

Construction and identification of an infectious clone for CDV-3 strain of canine distemper virus / 生物工程学报

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 10⁷·⁶⁶⁷ TCID₅₀/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 10⁶·⁶⁶⁷ TCID₅₀/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.

Gene Cloning and Bioinformatics Analysis of Limonene-3-hydroxylase from Schizonepeta tenuifolia / 中国实验方剂学杂志

Objective:This paper aims to clone the cDNA sequence of<italic> limonene</italic>-3-<italic>hydroxylase</italic>（<italic>StL</italic>3<italic>OH</italic>） in <italic>Schizonepeta tenuifolia</italic> and analyze its sequence by bioinformatics. Method:Specific primers were designed based on sequences of<italic> StL</italic>3<italic>OH </italic>gene screened from transcriptome sequencing data of <italic>S. tenuifolia</italic> and the cDNA sequence of <italic>StL</italic>3<italic>OH </italic>gene was cloned by reverse transcription polymerase chain reaction （RT-PCR） and analyzed for its bioinformatics. Result:The <italic>StL3OH</italic> gene cDNA sequence length was 1 598 bp，containing a 1 497 bp long complete open reading frame which encoded 498 amino acids. StL3OH protein had a theoretical relative molecular mass of 56.40 kDa，with a hydrophilic and unstable nature. Bioinformatics analysis showed that StL3OH protein had no signal peptide but had a transmembrane domain which might be located in endoplasmic reticulum. Multiple sequence alignment and cluster analysis showed that the amino acid sequence of MsL3OH protein had a high similarity with StL3OH protein，both of which contained cytochrome P450 heme binding region，belonging to the D subfamily of cytochrome CYP71 family. Codon bias analysis showed that <italic>StL</italic>3<italic>OH</italic> gene preferred guanine/cytosine（G/C） ending codon，with 27 skewed codons， and Nicotiana benthamiana was proven to be the most suitable host for exogenous expression of <italic>StL</italic>3<italic>OH</italic> gene. Conclusion:The cDNA sequence of<italic> StL3OH</italic> gene was cloned from <italic>S. tenuifolia</italic> for the first time，which will provide a basis for further study on the structure and function of StL3OH protein and the regulation mechanism of <italic>StL3OH </italic>gene in the accumulation and biosynthesis of monoterpenes in<italic> S. tenuifolia</italic>.

Pathological features of cloned calves that died in the neonatal period / Aspectos patológicos de bezerros clonados com óbito no período neonatal

Somatic-cell nuclear transfer is a cloning technique that enables the creation of a viable embryo from a donor adult to produce a genetically identical individual. This technique opens numerous potential possibilities for medicine and animal reproduction. However, several reports have documented cloning-related issues. Embryo and fetal losses remain significantly higher than in other techniques, and there is a high incidence of dystocia and hydrops, which decreases efficiency and increases costs. Animals delivered at term often exhibit a syndrome known as macrosomia and experience difficulties in adapting to life outside the uterus, and death is a common outcome. In the present study, 41 cloned calves that died in the neonatal period were subjected to gross and histopathological examination. Most important gross lesions were found in the liver (enlargement, congestion, yellowish color), kidneys (brownish color at surface and cut, and cysts), lungs (atelectasis, parenchymal consolidation, and secretions in bronchi and bronchioles), and heart (concentric and eccentric hypertrophy, hematic cysts, persistence of ductus arteriosus). Primary microscopic findings were seen in the liver, kidneys, and lungs from neonatal calves. In the liver, 85% of the animals exhibited hepatic degeneration. The presence of a brownish pigment within the cortical tubules of the kidneys was found in approximately 90% of the samples; the presence of this pigment has not been previously reported in cloned calves. In the lungs, a large number of animals exhibiting lesions characteristic of pneumonia (55%). These changes were the pivotal causes of death, mainly due to problems in adapting to life outside the uterus and opportunistic infections in the neonatal period. Further investigation focusing on pathological anatomical changes is necessary to map these abnormalities in cloned animals.(AU)

A transferência nuclear de células somáticas ou clonagem é uma técnica que permite produzir um indivíduo geneticamente igual a um outro indivíduo adulto. Esta técnica abre inúmeras possibilidades para a medicina e para a reprodução animal. Porém, existem inúmeros relatos de problemas associados à clonagem. A taxa de perda nos períodos embrionário e fetal ainda é muito alta quando comparada a outras biotécnicas; além disso, há uma maior incidência de hidropsias e distocias, diminuindo a eficiência e aumentando o custo da técnica. Os animais que vem a termo frequentemente apresentam uma síndrome chamada de macrossomia, e apresentam dificuldades de adaptação à vida extrauterina e, por isso, o óbito é um desfecho comum. No presente trabalho realizou-se necropsia e coleta de fragmentos de órgãos para avaliação histopatológica de 41 bezerros com óbito neonatal. As lesões macroscópicas mais importantes foram encontradas no fígado (hepatomegalia, congestão e coloração amarelada), rins (coloração amarronzada na superfície e ao corte, e cistos), pulmões (atelectasia, parênquima consolidado, e secreções nos brônquios e bronquíolos), e coração (hipertrofia concêntrica e excêntrica, cistos hemáticos e persistência de ducto arterioso). As principais alterações microscópicas observadas foram presença de pigmento acastanhado no interior dos túbulos corticais renais (aproximadamente 90% dos animais), degeneração hepática (85% das amostras avaliadas) e lesões características de pneumonia (55% dos animais). A pigmentação acastanhada no interior dos túbulos corticais é uma alteração que ainda não havia sido relatada anteriormente em animais clonados. As alterações observadas nestes órgãos foram determinantes para o óbito, e devem ter ocorrido sobretudo devido a problemas na adaptação ao ambiente extrauterino e em decorrência de infecções adquiridas no período neonatal. Os achados encontrados no presente trabalho denotam a necessidade de investigação anatomopatológica detalhada de animais clonados inviáveis, na tentativa de mapear as anormalidades apresentadas por eles.(AU)

Rooting competence of mini-cuttings of Cabralea canjerana clones in different seasons / Enraizamento de miniestacas de clones de Cabralea canjerana em diferentes estações do ano

ABSTRACT: The objectives of this research were to evaluate the rooting competence of mini-cuttings throughout the four seasons and to estimate the adventitious rooting time of canjerana clones. A clonal mini-garden was established with 11 clones in a closed hydroponic system. Evaluations were performed throughout the four seasons for the number of mini-cuttings produced per mini-stump, percentage of survival and rooting of mini-cuttings, number of roots, average root length, and number of rooted mini-cuttings per mini-stump. Data were submitted to analysis of variance and means were compared. A rooting curve was estimated for clones 10SM05, 12SMI25, and 12SMI43 that exhibited high competence for adventitious rooting. Our results indicated that canjerana clones can be selected for adventitious rooting competence of mini-cuttings during different seasons, and that canjerana mini-cuttings should be cultivated for 63 days in a rooting chamber.

RESUMO: Os objetivos deste trabalho foram avaliar a competência ao enraizamento de miniestacas ao longo das quatro estações do ano e determinar o tempo de enraizamento adventício de clones de canjerana. O minijardim clonal foi estabelecido com 11 clones em um sistema fechado de cultivo. Foram realizadas avaliações do número total de miniestacas produzidas por minicepa, da percentagem de sobrevivência e de enraizamento das miniestacas, do número de raízes, do comprimento médio das raízes e contabilizado o número de miniestacas enraizadas por minicepa, ao longo das quatro estações do ano. Os dados foram submetidos à análise de variância e realizado teste de comparação de médias. Também foi elaborada a curva de enraizamento para os clones 10SM05, 12SMI25 e 12SMI43, com alta competência ao enraizamento adventício. Clones de canjerana podem ser selecionados para a competência ao enraizamento adventício das miniestacas nas diferentes estações do ano. Miniestacas de canjerana devem ser cultivadas em câmara úmida por 63 dias para o enraizamento.

Pyomelanin biosynthetic pathway in pigment-producer strains from the pandemic Acinetobacter baumannii IC-5

BACKGROUND Acinetobacter baumannii outbreaks have been associated with pandemic International Clones (ICs), but the virulence factors involved with their pathogenicity are sparsely understood. Pigment production has been linked with bacterial pathogenicity, however, this phenotype is rarely observed in A. baumannii. OBJECTIVES This study aimed to characterise the reddish-brown pigment produced by A. baumannii strains, and to determine its biosynthetic pathway by genomic approaches. METHODS Pigment characterisation and antimicrobial susceptibility were conducted by phenotypic tests. The clonal relationship was obtained by pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The genome of an A. baumannii was obtained for characterisation of genes involved with pigment production. FINDINGS The pyomelanin was the pigment produced by A. baumannii. Strains were extensively drug resistant and belonged to the IC-5/ST79. The pyomelanin biosynthetic pathway was determined and presented a particular architecture concerning the peripheral (tyrB, phhB and hpd) and central (hmgB, hmgC and hmgR) metabolic pathway genes. The identification of a distant HmgA homologue, probably without dioxygenase activity, could explain pyomelanin production. Virulence determinants involved with adherence (csuA/BABCDE and a T5bSS-carrying genomic island), and iron uptake (basABCDEFGHIJ, bauABCDEF and barAB) were characterised. MAIN CONCLUSION There is a biosynthetic pathway compatible with the pyomelanin production observed in persistent A. baumannii IC-5 strains.

Comparison of twopassage methods affecting the transfection efficiency of human embryonic stem cells / 中国组织工程研究

BACKGROUND: In the research of human embryonic stem cells, introducing exogenous molecules such as DNA into cells is a common research method, but the transfection efficiency is relatively low. It is crucial to answer the question of how to optimize the existing conditions to improve the transfection efficiency. OBJECTIVE: To compare the effects of two different passaging methods on H9 transfection efficiency, in order to optimize the conditions required for embryonic stem cell transfection. METHODS: Human embryonic stem cell lines H9 were cultured for 48 hours after small clone passaging or single-cell passaging. Lipofectamine 3000 was used to transfect pAdTrack-AKT1 fluorescent plasmid into human embryonic stem cells. After 2 days of transfection, the expression of fluorescent plasmids was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry. RT-qPCR and western blot were used to detect the mRNA and protein expression levels of AKT1 respectively. RESULTS AND CONCLUSION: Under the fluorescence microscopy, the number of cells expressing fluorescent plasmids in the single-cell passaging group was more than that in the small clone passaging group, and the flow cytometry analysis showed that the transfection efficiency of cells in the single-cell passaging group was (47.18±2.00)%, which was significantly higher than (19.52±0.86)% in the small clone passaging group (P < 0.01). RT-qPCR and western blot analysis showed that the expression levels of AKT1 mRNA and protein in the single-cell passaging group were significantly higher than those in the small clone passaging group (P < 0.01). These findings indicate that single-cell passaging can increase the contact area between cells and transfection reagent liposomes, and improve the transfection efficiency of human embryonic stem cells.

Cloning and expression of LAR gene and its correlation with phloridzin content in Lithocarpus polystachyus / 中草药

Objective: To clone the leucoanthocyanidin reductase (LAR) gene of Lithocarpus polystachyus, and analyze the relationship between LAR gene expression level and phloridzin content. Methods: According to the results of L. polystachyus transcriptome sequencing (unigene: DN30711_c0_g1_i1), the full-length cDNA sequence of LAR gene was amplified by PCR and the bioinformatics analysis was carried out. Its expression was detected by quantitative Real-time PCR (qRT-PCR). The phloridzin content of L. polystachyus was measured by UPLC method and the correlation between LAR gene expression and phloridzin content was analyzed by SPSS 18.0 software. Results: The full-length cDNA of the LAR gene was 1 053 bp and contained a complete open reading frame that encoded 350 amino acids. This protein did not exist a transmembrane domain and was localized in the cytoplasm. The LAR protein of L. polystachyus was the number of PCBER_SDR_a family and had a high similarity (95%) to the LAR protein of Quercus suber and their genetic relationship was close. The phloridzin content of L. polystachyus was positively correlated with the expression of LAR gene (P < 0.05). Conclusion: The LAR gene of L. polystachyus was cloned for the first time. It was confirmed that the content of phloridzin was positively correlated with the expression of LAR gene of L. polystachyus, which laid a theoretical and technical basis for revealing the biosynthesis mechanism of phloridzin of L. polystachyus.

Cloning and analysis of CoDXR gene in Cornus officinalis / 中草药

Objective: To clone the full-length cDNA sequence of CoDXR, a key enzyme gene of Cornus officinalis, and provide a basis for further study of C. officinalis. Methods: In this study, we used the transcript sequence c147202_g1 from the transcriptome data of C. officinalis obtained in our laboratory as template, designed specific primers through Primer Premier 5.0, cloned the full-length cDNA sequence of C. officinalis DXR gene by RT-PCR technology, and the bioinformatics analysis and function prediction were carried out through the relevant bioinformatics software. Results: The results showed that the CoDXR gene was 1 505 bp in length and the ORF was 729 bp in length, encoding 242 amino acids. The results of predictive analysis of CoDXR protein by SignalP4.0Server and HMMTOP showed that the protein was a hydrophobic protein without signal peptide and transmembrane region. Phylogenetic tree analysis showed that the CoDXR protein had the highest similarity to the DXR protein sequence of Camellia sinensis. Conclusion: In this study, the key enzyme gene CoDXR was successfully cloned based on the sequencing of the C. officinalis transcriptome, and related bioinformatics analysis was carried out. The results of this study laid the foundation for further study on the function of CoDXR gene in the terpenoid synthesis pathway of C. officinalis.

Cloning and expression stability analysis of Actin, Tubulin and GAPDH genes in Dipsacus asper / 中草药

Objective: To clone and screen the stable internal reference genes from Dipsacus asper for qRT-PCR analysis correction, so as to provide a preliminary basis for future research on expression analysis and regulation mechanism of D. asper functional genes. Methods: The internal reference genes of Actin, Tubulin and GAPDH gene families were screened and cloned from D. asper transcriptome database. The D. asper plants from different origins, different tissues and different developmental stages were used to obtain expression information of each gene by qRT-PCR. The expression stability of each gene was analyzed by geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, and the best genes were synthetically evaluated and screened. Results: Ten core fragments for candidate internal reference genes were cloned, belonging to three gene families: Actin, Tubulin and GAPDH, with high homology among them. The results of stability analysis showed that the expression of DaACT103 was stable and relatively high in different regions and tissues, while the expression of DaTUB5 was stable and relatively low in different developmental stages. Conclusion:s DaACT103 and DaTUB5 are suitable as the internal reference genes for D. asper. DaACT103 is used as the internal reference gene with high abundances and DaACT105 is used as the internal reference gene with low abundances.

Cloning and expression analysis of tryptophan synthase gene BcTSB in Baphicacanthus cusia / 中草药

Objective: To clone the tryptophan synthase gene named as BcTSB (GenBank accession number AYM45644.1) involved in the synthesis pathway of indole alkaloids from Baphicacanthus cusia, meanwhile, the bioinformatics analysis and expression analysis were also performed. Method: The open reading frame (ORF) of BcTSB gene was obtained by the database of prophase Baphicacanthus cusia transcriptome. The function of the BcTSB gene was preliminarily predicted by a series of bioinformatics tools. The entire protein-coding cDNA of BcTSB was cloned into the prokaryotic expression vector pET32a, then the recombinant plasmid was transformed into E. coli BL21 (DE3) cells, with IPTG induction. SDS-PAGE was used to investigate the situation of expression. The expression of the gene in root, stem and leaf was determined by using real-time PCR (qRT-PCR). Results: The open reading frame (ORF) of cloned BcTSB gene was 1 452 bp, and encoding 483 amino acids, it was predicted by bioinformatics analysis as hydrophilic protein, being located in the chloroplasts. Bioinformatics analysis of the amino acid sequence showed that the molecular weight of encoded protein was 52 kDa, because prokaryotic expression vector pET32a contained 18 kDa label, SDS-PAGE results showed that a protein band at 70 000 was in consistent with molecular weight of the predicted protein. The QRT-PCR revealed that BcTSB gene was expressed in different tissues of B. cusia, the expression level of BcTSB in stems was much higher than that in roots and leaves. Conclusion: In this study, BcTSB gene of B. cusia was cloned and its expression was analyzed successfully, which laid an experimental foundation for further study on the function and regulation of the gene.

Expression of valosin-containing protein in epithelial ovarian cancer tissue and its clinical significance / 吉林大学学报(医学版)

Objective: To observe the expression of valosin-containing protein CVCP) in the epithelial ovarian cancer (EOC) tissue and its relationships with the clinicopathological features of the EOC patients∗ and to provide the basis for the molecular treatment of EOC. Methods: The expressions of VCP in 94 EOC tissue samples, 13 ovarian borderline tumor tissue samples, 36 ovarian benign tumor tissue samples and 8 normal ovarian tissue samples were measured by immunohistochemical method. The relationships between the VCP expression and the clinicopathological parameters (age, FIGO stage, pathological type, histological grade, lymph node metastasis or not, ascites or not, preoperative CA125 level) of the EOC patients were analyzed. The expressions of VCP in human ovarian epithelial HOSEPIC cells and human EOC SKOV-3 cells were detected by immunofluorescence technique and Western blotting method. The SKOV-3 cells were divided into control group (without CB-5083) and treatment group (with CB-5083)∗ the migration rates of the SKOV-3 cells in two groups were analyzed by scratch experiment, and the clone formation rates of the SKOV-3 cells in two groups were analyzed by plate clone formation experiment. The expressions of VCP in the cells in two groups were analyzed by immunofluorescence technique. The expression levels of VCP, NF-kB/P65» IieBa∗ and p-Iiefta proteins in the SKOV-3 cells in two groups were detected by Western blotting method. Results: The positive expression rates of VCP in EOC, borderline ovarian tumor, benign ovarian tumor and normal ovarian tissues had significant difference ( P0. 05). The VCP expression level in the SKOV-3 cells was higher than that in HOSEpiC cells ( P<0. 05). The migration rate of the SKOV-3 cells in treatment group was lower than that in control group ( P<0.05), the cloning formation rate of the SKOV-3 cells in treatment group was lower than that in control group ( P<0. 05). The expression levels of VCP and NF-kB/P65 proteins of the SKOV-3 cells in treatment group were lower than those in control group (P'<0. 05) ∗ and the expression level of p-IieBa protein was higher than that in control group ( P< 0. 05). Conclusion: VCP is highly expressed in the EOC tissue and cells∗ and high-expression VCP can promot the tumor migration and proliferation.

Study on the mechanism of llinc00052 regulating Wnt pathway affecting invasion and metastasis of gastric cancer / 中国基层医药

Objective To explore the mechanism of llinc00052 regulating Wnt pathway affecting invasion and metastasis of gastric cancer.Methods SGC-7901 cells were selected from gastric cancer cell lines ,and siRNAs related to INC0005 and had invasion and metastasis effects in gastric cancer cells were screened by binding of IncRNA expression profiles to qRT -PCR.Ilinc00052 and miRNA expression changes were studied by in vitro cell transfection experiments.Through molecular experiments ,the expression of llinc00052 and the effect on Wnt／β-catenin expression were investigated to explore whether llinc 00052 could affect the invasion and metastasis of gastric cancer cells by regulating miRNAs affecting Wnt／β-catenin signaling pathway.Results Transwell chamber test showed that the number of transmembrane cells in the untransfected plasmid group was (134.10 ±4.29),and the number of transmembrane cells in the overexpressed llinc 000522 plasmid group was (169.24 ±6.99)(t＝8.956,P＝0.001). The scratch test showed that the migration distance in the llinc 000522 overexpression transfection plasmid group was significantly higher than that in the no -load plasmid transfection group (r＝0.907,P＜0.01).The clone formation rate of llinc000522 overexpressed transfected plasmid group was significantly higher than that of the empty plasmid group[(92.75 ±6.32)% vs.(73.34 ±9.14)%] (t＝5.998,P＜0.05).Compared with the transfection of blank plasmid,the expressions of Wnt1,Wnt3a,Wnt2 and β-catenin mRNA in the llinc000522 overexpression transfection group were significantly up -regulated(P＜0.05),while the miRNA transfection group had no significant effect on the expression.The expressions of Wnt1,Wnt3a,Wnt2,and β-catenin mRNA were significantly increased [(1.82 ± 0.11),(1.52 ±0.15),(1.42 ±0.21),(1.71 ±0.19)] ( P＜0.05),but their expressions were still lower than those of the genes transfected with llinc000522 alone.Compared with the blank plasmid transfection ,the expressions of Wnt1,Wnt3a,Wnt2 and β-catenin protein in the llinc000522 overexpression transfection group were significantly up-regulated(all P＜0.05),while the miRNA transfection group had no significant effect on its expression.The protein expressions of Wnt1,Wnt3a,Wnt2 and β-catenin were also significantly increased [(1.53 ±0.09),(1.4 ±0.21), (1.33 ±0.07),(1.47 ±0.19)](P＜0.05),but their expressions were still lower than those of the gene transfected with llinc000522 alone.Conclusion In gastric cancer cells, llinc00052 can affect the invasion and metastasis of gastric cancer by regulating the level of miRNA and affecting the Wnt ／β-catenin pathway.