Comparison of ONT and CCS sequencing technologies on the polyploid genome of a medicinal plant showed that high error rate of ONT reads are not suitable for self-correction.

BACKGROUND: Many medicinal plants are known for their complex genomes with high ploidy, heterozygosity, and repetitive content which pose severe challenges for genome sequencing of those species. Long reads from Oxford nanopore sequencing technology (ONT) or Pacific Biosciences Single Molecule, Real-Time (SMRT) sequencing offer great advantages in de novo genome assembly, especially for complex genomes with high heterozygosity and repetitive content. Currently, multiple allotetraploid species have sequenced their genomes by long-read sequencing. However, we found that a considerable proportion of these genomes (7.9% on average, maximum 23.7%) could not be covered by NGS (Next Generation Sequencing) reads (uncovered region by NGS reads, UCR) suggesting the questionable and low-quality of those area or genomic areas that can't be sequenced by NGS due to sequencing bias. The underlying causes of those UCR in the genome assembly and solutions to this problem have never been studied. METHODS: In the study, we sequenced the tetraploid genome of Veratrum dahuricum (Turcz.) O. Loes (VDL), a Chinese medicinal plant, with ONT platform and assembled the genome with three strategies in parallel. We compared the qualities, coverage, and heterozygosity of the three ONT assemblies with another released assembly of the same individual using reads from PacBio circular consensus sequencing (CCS) technology, to explore the cause of the UCR. RESULTS: By mapping the NGS reads against the three ONT assemblies and the CCS assembly, we found that the coverage of those ONT assemblies by NGS reads ranged from 49.15 to 76.31%, much smaller than that of the CCS assembly (99.53%). And alignment between ONT assemblies and CCS assembly showed that most UCR can be aligned with CCS assembly. So, we conclude that the UCRs in ONT assembly are low-quality sequences with a high error rate that can't be aligned with short reads, rather than genomic regions that can't be sequenced by NGS. Further comparison among the intermediate versions of ONT assemblies showed that the most probable origin of those errors is a combination of artificial errors introduced by "self-correction" and initial sequencing error in long reads. We also found that polishing the ONT assembly with CCS reads can correct those errors efficiently. CONCLUSIONS: Through analyzing genome features and reads alignment, we have found the causes for the high proportion of UCR in ONT assembly of VDL are sequencing errors and additional errors introduced by self-correction. The high error rates of ONT-raw reads make them not suitable for self-correction prior to allotetraploid genome assembly, as the self-correction will introduce artificial errors to > 5% of the UCR sequences. We suggest high-precision CCS reads be used to polish the assembly to correct those errors effectively for polyploid genomes.

Comparative and phylogenetic analyses of eleven complete chloroplast genomes of Dipterocarpoideae.

BACKGROUND: In South-east Asia, Dipterocarpoideae is predominant in most mature forest communities, comprising around 20% of all trees. As large quantity and high quality wood are produced in many species, Dipterocarpoideae plants are the most important and valuable source in the timber market. The d-borneol is one of the essential oil components from Dipterocarpoideae (for example, Dryobalanops aromatica or Dipterocarpus turbinatus) and it is also an important traditional Chinese medicine (TCM) formulation known as "Bingpian" in Chinese, with antibacterial, analgesic and anti-inflammatory effects and can enhance anticancer efficiency. METHODS: In this study, we analyzed 20 chloroplast (cp) genomes characteristics of Dipterocarpoideae, including eleven newly reported genomes and nine cp genomes previously published elsewhere, then we explored the chloroplast genomic features, inverted repeats contraction and expansion, codon usage, amino acid frequency, the repeat sequences and selective pressure analyses. At last, we constructed phylogenetic relationships of Dipterocarpoideae and found the potential barcoding loci. RESULTS: The cp genome of this subfamily has a typical quadripartite structure and maintains a high degree of consistency among species. There were slightly more tandem repeats in cp genomes of Dipterocarpus and Vatica, and the psbH gene was subjected to positive selection in the common ancestor of all the 20 species of Dipterocarpoideae compared with three outgroups. Phylogenetic tree showed that genus Shorea was not a monophyletic group, some Shorea species and genus Parashorea are placed in one clade. In addition, the rpoC2 gene can be used as a potential marker to achieve accurate and rapid species identification in subfamily Dipterocarpoideae. CONCLUSIONS: Dipterocarpoideae had similar cp genomic features and psbM, rbcL, psbH may function in the growth of Dipterocarpoideae. Phylogenetic analysis suggested new taxon treatment is needed for this subfamily indentification. In addition, rpoC2 is potential to be a barcoding gene to TCM distinguish.

Berberine combined with stachyose induces better glycometabolism than berberine alone through modulating gut microbiota and fecal metabolomics in diabetic mice.

Berberine (BBR), a small alkaloid, is used as a hypoglycemic agent in China. Stachyose (Sta), a Rehmannia glutinosa oligosaccharide, acts as a prebiotic. This study aimed to evaluate whether BBR combined with Sta produced better glycometabolism than BBR alone, and explored the effects on gut microbiota and metabolomics. Type-2 diabetic db/db mice were administered BBR (100 mg/kg), Sta (200 mg/kg), or both by gavage once daily. Glucose metabolism, the balance of α- and ß-cells, and mucin-2 expression were ameliorated by combined treatment of BBR and Sta, with stronger effects than upon treatment with BBR alone. The microbial diversity and richness were altered after combined treatment and after treatment with BBR alone. The abundance of Akkermansia muciniphila was increased by combined treatment compared to treatment with BBR alone, while the levels of the metabolite all-trans-heptaprenyl diphosphate were decreased and the levels of fumaric acid were increased, which both showed a strong correlation with A. muciniphila. In summary, BBR combined with Sta produced better glycometabolism than BBR alone through modulating gut microbiota and fecal metabolomics, and may aid in the development of a novel pharmaceutical strategy for treating Type 2 diabetes mellitus.

The complete chloroplast genome of the common self-heal, Prunella vulgaris (Lamiaceae).

Prunella vulgaris L. is an important medicinal herb widely used in China and western countries. Its natural distribution occurs in various habitats throughout northern hemisphere. In present study, we assembled and characterized its whole chloroplast (cp) genome based on Illumina pair-end sequencing data. The complete chloroplast genome size is 156,132 bp. It contained 134 genes, including 89 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. 8 gene species had two copies. The overall GC content of this genome was 37.9%. A further phylogenomic analysis of Lamiaceae, including 29 taxa, was conducted for the placement of P. vulgaris.

New Immunosuppressive Sphingoid Base and Ceramide Analogues in Wild Cordyceps.

A comprehensive identification of sphingoid bases and ceramides in wild Cordyceps was performed by integrating a sequential chromatographic enrichment procedure and an UHPLC-ultrahigh definition-Q-TOF-MS based sphingolipidomic approach. A total of 43 sphingoid bases and 303 ceramides were identified from wild Cordyceps, including 12 new sphingoid base analogues and 159 new ceramide analogues based on high-resolution MS and MS/MS data, isotope distribution, matching with the comprehensive personal sphingolipid database, confirmation by sphingolipid standards and chromatographic retention time rule. The immunosuppressive bioassay results demonstrated that Cordyceps sphingoid base fraction exhibits more potent immunosuppressive activity than ceramide fraction, elucidating the immunosuppressive ingredients of wild Cordyceps. This study represented the most comprehensive identification of sphingoid bases and ceramides from a natural source. The findings of this study provided an insight into therapeutic application of wild Cordyceps.

YiQiFuMai Powder Injection Ameliorates Cerebral Ischemia by Inhibiting Endoplasmic Reticulum Stress-Mediated Neuronal Apoptosis.

YiQiFuMai (YQFM) powder injection as a modern preparation derived from Sheng Mai San, a traditional Chinese medicine, has been widely used in the treatment of cardiovascular and cerebrovascular diseases. However, its neuroprotective effect and underlying mechanism in cerebral ischemia remain to be explored. The present study was designed to investigate the neuroprotective effect of YQFM on endoplasmic reticulum (ER) stress-mediated neuronal apoptosis in the permanent middle cerebral artery occlusion- (MCAO-) injured mice and the oxygen-glucose deprivation- (OGD-) induced pheochromocytoma (PC12) cells. The results showed that single administration of YQFM (1.342 g/kg, i.p.) could reduce the brain infarction and improve the neurological deficits and the cerebral blood flow (CBF) after MCAO for 24 h in mice. Moreover, incubation with YQFM (100, 200, and 400 µg/mL) could increase the cell viability, decrease the caspase-3 activity, and inhibit the cell apoptosis in OGD-induced PC12 cells for 12 h. In addition, YQFM treatment could significantly modulate cleaved caspase-3 and Bcl-2 expressions and inhibit the expressions of ER stress-related marker proteins and signaling pathways in vivo and in vitro. In conclusion, our findings provide the first evidence that YQFM ameliorates cerebral ischemic injury linked with modulating ER stress-related signaling pathways, which provided some new insights for its prevention and treatment of cerebral ischemia diseases.

Advances in the pharmacological activities and mechanisms of diosgenin.

Diosgenin, a well-known steroid sapogenin derived from plants, has been used as a starting material for production of steroidal hormones. The present review will summarize published literature concerning pharmacological potential of diosgenin, and the underlying mechanisms of actions. Diosgenin has shown a vast range of pharmacological activities in preclinical studies. It exhibits anticancer, cardiovascular protective, anti-diabetes, neuroprotective, immunomodulatory, estrogenic, and skin protective effects, mainly by inducing apoptosis, suppressing malignant transformation, decreasing oxidative stress, preventing inflammatory events, promoting cellular differentiation/proliferation, and regulating T-cell immune response, etc. It interferes with cell death pathways and their regulators to induce apoptosis. Diosgenin antagonizes tumor metastasis by modulating epithelial-mesenchymal transition and actin cytoskeleton to change cellular motility, suppressing degradation of matrix barrier, and inhibiting angiogenesis. Additionally, diosgenin improves antioxidant status and inhibits lipid peroxidation. Its anti-inflammatory activity is through inhibiting production of pro-inflammatory cytokines, enzymes and adhesion molecules. Furthermore, diosgenin drives cellular growth/differentiation through the estrogen receptor (ER) cascade and transcriptional factor PPARγ. In summary, these mechanistic studies provide a basis for further development of this compound for pharmacotherapy of various diseases.

Ophiopogonin-D suppresses MDA-MB-435 cell adhesion and invasion by inhibiting matrix metalloproteinase-9.

Ophiopogonin-D is one of steroidal saponins isolated from the root of the Chinese medicinal plant Ophiopogon japonicas. It has been claimed to possess anti-inflammatory and anti-oxidant properties. The present study was the first to examine the anti-tumor metastasis properties of ophiopogonin-D. An MTT assay showed that ophiopogonin-D inhibited the proliferation of MDA-MB-435 melanoma cells, and decreased invasion was demonstrated using a Transwell invasion assay. Furthermore, adhesion of MDA-MB-435 cells to human umbilical vascular endothelial cells and to fibronectin was inhibited by ophiopogonin-D. Gelatin zymography and western blot analysis showed that ophiopogonin-D inhibited the expression and secretion of matrix metalloproteinase-9 (MMP-9), but not that of MMP-2. Inhibition of phosphorylation of p38 by ophiopogonin-D indicated its inhibition of the mitogen-activated protein kinase pathway. Overall, the results suggested that ophiopogonin-D may be considered as a candidate drug for treating or preventing tumor metastasis.