Enhancing transcriptome analysis in medicinal plants: multiple unigene sets in Astragalus membranaceus.

Astragalus membranaceus is a medicinal plant mainly used in East Asia and contains abundant secondary metabolites. Despite the importance of this plant, the available genomic and genetic information is still limited. De novo transcriptome construction is recognized as an essential method for transcriptome research when reference genome information is incomplete. In this study, we constructed three individual transcriptome sets (unigene sets) for detailed analysis of the phenylpropanoid biosynthesis pathway, a major metabolite of A. membranaceus. Set-1 was a circular consensus sequence (CCS) generated using PacBio sequencing (PacBio-seq). Set-2 consisted of hybridized assembled unigenes with Illumina sequencing (Illumina-seq) reads and PacBio CCS using rnaSPAdes. Set-3 unigenes were assembled from Illumina-seq reads using the Trinity software. Construction of multiple unigene sets provides several advantages for transcriptome analysis. First, it provides an appropriate expression filtering threshold for assembly-based unigenes: a threshold transcripts per million (TPM) ≥ 5 removed more than 88% of assembly-based unigenes, which were mostly short and low-expressing unigenes. Second, assembly-based unigenes compensated for the incomplete length of PacBio CCSs: the ends of the 5`/3` untranslated regions of phenylpropanoid-related unigenes derived from set-1 were incomplete, which suggests that PacBio CCSs are unlikely to be full-length transcripts. Third, more isoform unigenes could be obtained from multiple unigene sets; isoform unigenes missing in Set-1 were detected in set-2 and set-3. Finally, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that phenylpropanoid biosynthesis and carbohydrate metabolism were highly activated in A. membranaceus roots. Various sequencing technologies and assemblers have been developed for de novo transcriptome analysis. However, no technique is perfect for de novo transcriptome analysis, suggesting the need to construct multiple unigene sets. This method enables efficient transcript filtering and detection of longer and more diverse transcripts.

Co-Expression Analysis Reveals Differential Expression of Homologous Genes Associated with Specific Terpenoid Biosynthesis in Rehmannia glutinosa.

Terpenoids are naturally occurring compounds involved in respiration, photosynthesis, membrane fluidity, and pathogen interactions and are classified according to the structure of their carbon skeleton. Although most terpenoids possess pharmacological activity, knowledge about terpenoid metabolism in medicinal plants is insufficient. Rehmannia glutinosa (R. glutinosa) is a traditional herb that is widely used in East Asia and has been reported to contain various terpenoids. In this study, we performed a comprehensive transcriptome analysis of terpenoid metabolism in R. glutinosa using two RNA sequencing platforms: Illumina and PacBio. The results show that the sterol, saponin, iridoid, and carotenoid pathways are active in R. glutinosa. Sterol and saponin biosynthesis were mevalonate pathway dependent, whereas iridoid and carotenoid biosynthesis were methylerythritol 4-phosphate pathway dependent. In addition, we found that the homologous genes of key enzymes involved in terpenoid metabolism were expressed differentially and that the differential expression of these genes was associated with specific terpenoid biosynthesis. The different expression of homologous genes encoding acetyl-CoA acetyltransferase, 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, mevalonate diphosphate decarboxylase, farnesyl pyrophosphate synthase, squalene synthase, and squalene epoxidase was associated with sterol and saponin biosynthesis. Homologous genes encoding 1-deoxy-D-xylulose 5-phosphate synthase were also differentially expressed and were associated with carotenoid and iridoid biosynthesis. These results suggest that the biosynthesis of specific terpenoids can be regulated by the homologous of key enzymes involved in plant terpenoid metabolism.

Alginate and fucoidan changes the bacterial community in different directions and the alginate or fucoidan degrading bacteria isolated from paddy soil promotes the plant growth.

Seaweed and its extracts have been developed as fertilizers because they possess plant-growth-promoting and antibacterial compounds. For use as fertilizers, the major carbohydrates in seaweed, including fucoidan and alginate, need to be efficiently digested in the soil. We isolated fucoidan/alginate degrading bacteria from paddy soil and verified its use as a biofertilizer. Results show that Stenotrophomonas pavanii has a high alginate degrading activity, and also stimulating melon, pepper, and tomato growth. The growth stimulation effect of the bacteria was enhanced by alginate treatment. Bacillus sp. was isolated as a fucoidan degrading bacterium and this bacterium was also able to stimulate melon growth. Using 16S ribosomal DNA analysis, fucoidan/alginate resistant or susceptible bacteria were successively selected. Bacteria with increased population due to fucoidan and alginate had specificity to each carbohydrate, whereas those with decreased population showed susceptibility to both carbohydrates. This report demonstrates some bacteria for their use as biofertilizers with seaweed and demonstrated that a high throughput method is efficient in identifying bacteria with specific properties.

Misinsertion of central venous catheter into the suspected vertebral vein: a case report.

We experienced a case in which a central venous catheter (CVC) was misplaced into the wrong vein, which was mistaken for the internal jugular vein (IJV), identified by chest x-ray and ultrasound. The vertebral vein passes through the transverse foramina from the atlas to the 6th cervical vertebra. After exiting the transverse foramen of the 6th vertebra, the vein subsequently runs anterolateral to the vertebral artery and posterior to the IJV and drains the innominate vein. In this case, chest x-ray and ultrasound revealed that the inserted CVC had a course very similar to the vertebral vein. The misplacement of a CVC into the vertebral vein might occur from excessive rotation of the patient's head, which leads to alterations in the cervical vascular anatomy, and from deep insertion of the puncture needle. Therefore, it is advised, for safe CVC insertion, to minimize a patient's head rotation and to make use of ultrasound when the anatomical structures cannot be clearly identified.