The Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) Has Digestive Capacity to Degrade Complex Substrates: Functional Characterization and Heterologous Expression of an α-Amylase

Dendroctonus-bark beetles are natural agents contributing to vital processes in coniferous forests, such as regeneration, succession, and material recycling, as they colonize and kill damaged, stressed, or old pine trees. These beetles spend most of their life cycle under stem and roots bark where they breed, develop, and feed on phloem. This tissue is rich in essential nutrients and complex molecules such as starch, cellulose, hemicellulose, and lignin, which apparently are not available for these beetles. We evaluated the digestive capacity of Dendroctonus rhizophagus to hydrolyze starch. Our aim was to identify α-amylases and characterize them both molecularly and biochemically. The findings showed that D. rhizophagus has an α-amylase gene (AmyDr) with a single isoform, and ORF of 1452 bp encoding a 483-amino acid protein (53.15 kDa) with a predicted signal peptide of 16 amino acids. AmyDr has a mutation in the chlorine-binding site, present in other phytophagous insects and in a marine bacterium. Docking analysis showed that AmyDr presents a higher binding affinity to amylopectin compared to amylose, and an affinity binding equally stable to calcium, chlorine, and nitrate ions. AmyDr native protein showed amylolytic activity in the head-pronotum and gut, and its recombinant protein, a polypeptide of ~53 kDa, showed conformational stability, and its activity is maintained both in the presence and absence of chlorine and nitrate ions. The AmyDr gene showed a differential expression significantly higher in the gut than the head-pronotum, indicating that starch hydrolysis occurs mainly in the midgut. An overview of the AmyDr gene expression suggests that the amylolytic activity is regulated through the developmental stages of this bark beetle and associated with starch availability in the host tree.     

临床血清循环miRNAs定量技术的建立

目的建立可靠的临床血清(浆)循环miRNAs定量技术。方法常规收集血清(浆)标本,mirVana PARIS试剂盒法抽提血清(浆)总RNA,采用DNaseI消化总RNA提取液,以miRNAs特异性茎-环引物引导反转录,通过TaqMan实时荧光定量PCR对U6及靶miRNAs进行检测。结果10份新鲜血浆总RNA浓度介于3.5～35.4ng/μl之间。对常规收集的400μl临床血清标本中U6、miR-16、miR-224均能实现特异扩增及定量,相应的平均Ct值约为30、25及32。6份不同留置时间血清标本总RNA浓度分别10.24和4.46ng/μl,定量PCR结果显示其中相应miR-16和miR-224的丰度却相对稳定。结论血清(浆)总RNA抽提及循环miRNAs定量切实可行。     

Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop,Plukenetia volubilis,by Real-Time Quantitative PCR

Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔC_t), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.     

Real time quantitative PCR detection of histamine-producing lactic acid bacteria in cheese: Relation with histamine content

Biogenic amines (BAs) are low molecular weight organic bases with biological activity, mainly formed by the microbial decarboxylation of amino acids. The consumption of food containing large amounts of some BAs can have toxicological consequences. Histamine is the most active BA and the most frequently involved in food-borne intoxications. This article reports the concentrations of histamine found in 80 cheeses made from different types of milk and subjected to different ripening periods. Histamine was detected in 41.25% of the samples, with values ranging from 20 to over 1000 mg per Kg of cheese. The highest histamine concentrations were recorded in long ripening cheeses made from raw milk. The presence and quantification of histamine-producing lactic acid bacteria was determined by RT-qPCR, and a good association was obtained between the C_t values and histamine concentrations determined by HPLC.