Complete chloroplast genome of Stephania tetrandra (Menispermaceae) from Zhejiang Province: insights into molecular structures, comparative genome analysis, mutational hotspots and phylogenetic relationships.

BACKGROUND: The Stephania tetrandra S. Moore (S. tetrandra) is a medicinal plant belonging to the family Menispermaceae that has high medicinal value and is well worth doing further exploration. The wild resources of S. tetrandra were widely distributed in tropical and subtropical regions of China, generating potential genetic diversity and unique population structures. The geographical origin of S. tetrandra is an important factor influencing its quality and price in the market. In addition, the species relationship within Stephania genus still remains uncertain due to high morphological similarity and low support values of molecular analysis approach. The complete chloroplast (cp) genome data has become a promising strategy to determine geographical origin and understand species evolution for closely related plant species. Herein, we sequenced the complete cp genome of S. tetrandra from Zhejiang Province and conducted a comparative analysis within Stephania plants to reveal the structural variations, informative markers and phylogenetic relationship of Stephania species. RESULTS: The cp genome of S. tetrandra voucher ZJ was 157,725 bp, consisting of a large single copy region (89,468 bp), a small single copy region (19,685 bp) and a pair of inverted repeat regions (24,286 bp each). A total of 134 genes were identified in the cp genome of S. tetrandra, including 87 protein-coding genes, 8 rRNA genes, 37 tRNA genes and 2 pseudogene copies (ycf1 and rps19). The gene order and GC content were highly consistent in the Stephania species according to the comparative analysis results, with the highest RSCU value in arginine (1.79) and lowest RSCU value in serine of S. tetrandra, respectively. A total of 90 SSRs have been identified in the cp genome of S. tetrandra, where repeats that consisting of A or T bases were much higher than that of G or C bases. In addition, 92 potential RNA editing sites were identified in 25 protein-coding genes, with the most predicted RNA editing sites in ndhB gene. The variations on length and expansion extent to the junction of ycf1 gene were observed between S. tetrandra vouchers from different regions, indicating potential markers for further geographical origin discrimination. Moreover, the values of transition to transversion ratio (Ts/Tv) in the Stephania species were significantly higher than 1 using Pericampylus glaucus as reference. Comparative analysis of the Stephania cp genomes revealed 5 highly variable regions, including 3 intergenic regions (trnH-psbA, trnD-trnY, trnP) and two protein coding genes (rps16 and ndhA). The identified mutational hotspots of Stephania plants exhibited multiple SNP sites and Gaps, as well as different Ka/Ks ratio values. In addition, five pairs of specific primers targeting the divergence regions were accordingly designed, which could be utilized as potential molecular markers for species identification, population genetic and phylogenetic analysis in Stephania species. Phylogenetic tree analysis based on the conserved chloroplast protein coding genes indicated a sister relationship between S. tetrandra and the monophyletic group of S. japonica and S. kwangsiensis with high support values, suggesting a close genetic relationship within Stephania plants. However, two S. tetrandra vouches from different regions failed to cluster into one clade, confirming the occurrences of genetic diversities and requiring further investigation for geographical tracing strategy. CONCLUSIONS: Overall, we provided comprehensive and detailed information on the complete chloroplast genome and identified nucleotide diversity hotspots of Stephania species. The obtained genetic resource of S. tetrandra from Zhejiang Province would facilitate future studies in DNA barcode, species discrimination, the intraspecific and interspecific variability and the phylogenetic relationships of Stephania plants.

Low frequency pulsed electromagnetic field promotes the recovery of neurological function after spinal cord injury in rats.

Low frequency pulsed electromagnetic field (LFPEMF) has been shown to provide anti-inflammatory and antioxidative effects. However, there are no reports on whether LFPEMF can treat spinal cord injury (SCI) and its therapeutic mechanism. Therefore, this study was conducted to investigate whether LFPEMF can promote the recovery of neurological function after SCI in rats and its therapeutic mechanism. Basso-Beattie-Bresnahan (BBB) score and transcranial magnetic motor-evoked potentials (tcMMEPs) were recorded to assess the recovery of neurological function. Hematoxylin and eosin (HE) staining and luxol fast blue (LFB) staining were performed to assess the severity of SCI. Immunofluorescence (IF) staining and western blotting (WB) were performed to assess the differentiation of oligodendrocyte precursor cells (OPCs) into oligodendrocytes (OLs). Toluidine blue (TB) staining was performed to assess remyelination. WB and enzyme-linked immunosorbent assays (ELISA) were performed to assess the expression of neurotrophins and inflammatory factors. Our results showed that following stimulation by LFPEMF, there were significant improvements in BBB scores, tcMMEP amplitudes, the extent of the damage, and reduced demyelination in rats after SCI. The mature OLs, the number of well-myelinated fibers, and the myelin sheath thickness significantly increased in rats stimulated by LFPEMF after SCI. The expression of neurotrophins significantly increased, and the expression of inflammatory factors significantly decreased in rats stimulated by LFPEMF after SCI. Therefore, we suggest that LFPEMF can promote the recovery of neurological function in rats after SCI by improving the differentiation of OPCs into OLs and promoting remyelination, as well as by inhibiting inflammation and promoting neurotrophic effects. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:449-456, 2019.

The complete chloroplast genome sequence and phylogenetic analysis of the medicinal plant Lysimachia hemsleyana.

Lysimachia hemsleyana Maxim. is an important medical plant in the Family Primulaceae. In this study, we determined the complete chloroplast genome of L. hemsleyana. It is 155,618 bp in length, containing a large single copy (LSC) region of 85,615 bp, a small single copy (SSC) region of 17,861 bp, which were separated by a pair of inverted repeat (IR) regions of 26,071bp. The complete chloroplast genome of L. hemsleyana encoded a total of 134 genes, including 89 protein-coding genes with the pseudogene of ycf1, 8 ribosomal RNA genes and 37 transfer RNA genes. Phylogenetic analysis revealed that L. hemsleyana was most closely related to the Korea endemic plant Lysimachia coreana with high bootstrap support value. This work provides basic molecular information that would be useful for further investigation on conservation genetics and evolutionary relationships of L. hemsleyana.

Co-delivery of doxorubicin and recombinant plasmid pHSP70-Plk1-shRNA by bacterial magnetosomes for osteosarcoma therapy.

To explore a novel combination of chemotherapy, gene therapy, and thermotherapy for osteosarcoma, a targeted heat-sensitive co-delivery system based on bacterial magnetosomes (BMs) was developed. The optimal culture conditions of magnetotactic bacteria (MTB) AMB-1 and characterization of BMs were achieved. A recombinant eukaryotic plasmid heat shock protein 70-polo-like kinase 1-short hairpin RNA (pHSP70-Plk1-shRNA) under transcriptional control of a thermosensitive promoter (human HSP70 promoter) was constructed for gene therapy. Doxorubicin (DOX) and pHSP70-Plk1-shRNA were included in the targeted thermosensitive co-delivery system, and in vitro DOX release activity, targeted gene silencing efficiency and in vitro antitumor efficacy were investigated. The results showed that the optimal culture conditions of MTB AMB-1 are an oxygen concentration of 4.0%, a pH value of 7.0, 20 µmol/L of ferrous sulfate, 800 mg/L of sodium nitrate, and 200 mg/L of succinic acid. The temperature of BMs reached 43°C within 3 minutes and could be maintained for 30 minutes by adjusting the magnitude of the alternating magnetic field (AMF). The diameters of BMs, BM-DOX, BM-recombinant eukaryotic plasmid pHSP70-Plk1-shRNA (shPlk1), and BM-DOX-shPlk1 were 43.7±4.6, 79.2±5.4, 88.9±7.8, and 133.5±11.4 nm, respectively. The zeta potentials of BMs, BM-DOX, BM-shPlk1, and BM-DOX-shPlk1 were -29.4±6.9, -9.5±5.6, -16.7±4.8, and -10.3±3.1 mV, respectively. Besides, the system exhibited good release behavior. DOX release rate from BM-DOX-shPlk1 was 54% after incubation with phosphate-buffered saline at 43°C and 37% after incubation with 50% fetal bovine serum, which was significantly higher than that at 37°C (P<0.05). In addition, the expressions of Plk1 mRNA and protein were significantly suppressed in cells treated with BM-DOX-shPlk1 following hyperthermia treatment under the influence of an AMF compared to other groups (P<0.05). Furthermore, evaluation of the effect of in vitro antitumor revealed that BM-DOX-shPlk1 following hyperthermia treatment under the influence of an AMF was significantly more effective than others in tumor inhibition. In conclusion, the new heat-sensitive co-delivery system represents a promising approach for the treatment of cancer.