Identification of a Novel Geminivirus in Fraxinus rhynchophylla in Korea.

Fraxinus rhynchophylla, common name ash, belongs to the family Oleaceae and is found in China, Korea, North America, the Indian subcontinent, and eastern Russia. It has been used as a traditional herbal medicine in Korea and various parts of the world due to its chemical constituents. During a field survey in March 2019, mild vein thickening (almost negligible) was observed in a few ash trees. High-throughput sequencing of libraries of total DNA from ash trees, rolling-circle amplification (RCA), and polymerase chain reaction (PCR) allowed the identification of a Fraxinus symptomless virus. This virus has five confirmed open reading frames along with a possible sixth open reading frame that encodes the movement protein and is almost 2.7 kb in size, with a nonanucleotide and stem loop structure identical to begomoviruses. In terms of its size and structure, this virus strongly resembles begomoviruses, but does not show any significant sequence identity with them. To confirm movement of the virus within the trees, different parts of infected trees were examined, and viral movement was successfully observed. No satellite molecules or DNA B were identified. Two-step PCR confirmed the virion and complementary strands during replication in both freshly collected infected samples of ash tree and Nicotiana benthamiana samples agro-inoculated with infectious clones. This taxon is so distantly grouped from other known geminiviruses that it likely represents a new geminivirus genus.

De Novo Transcriptome Assembly of Two Microsorum Fern Species Identifies Enzymes Required for Two Upstream Pathways of Phytoecdysteroids.

Microsorum species produce a high amount of phytoecdysteroids (PEs), which are widely used in traditional medicine in the Pacific islands. The PEs in two different Microsorum species, M. punctatum (MP) and M. scolopendria (MS), were examined using high-performance liquid chromatography (HPLC). In particular, MS produces a high amount of 20-hydroxyecdysone, which is the main active compound in PEs. To identify genes for PE biosynthesis, we generated reference transcriptomes from sterile frond tissues using the NovaSeq 6000 system. De novo transcriptome assembly after deleting contaminants resulted in 57,252 and 54,618 clean transcripts for MP and MS, respectively. The clean Microsorum transcripts for each species were annotated according to gene ontology terms, UniProt pathways, and the clusters of the orthologous group protein database using the MEGAN6 and Sma3s programs. In total, 1852 and 1980 transcription factors were identified for MP and MS, respectively. We obtained transcripts encoding for 38 and 32 enzymes for MP and MS, respectively, potentially involved in mevalonate and sterol biosynthetic pathways, which produce precursors for PE biosynthesis. Phylogenetic analyses revealed many redundant and unique enzymes between the two species. Overall, this study provides two Microsorum reference transcriptomes that might be useful for further studies regarding PE biosynthesis in Microsorum species.

Enhanced Anticancer Effect of Adding Magnesium to Vitamin C Therapy: Inhibition of Hormetic Response by SVCT-2 Activation.

l-Ascorbic acid (vitamin C, AA) is known as an antioxidant, but at high concentrations, AA can kill cancer cells through a prooxidant property. Sodium-dependent vitamin C transporter family-2 (SVCT-2) determines the cellular uptake of AA, and the activity of SVCT-2 is directly related to the anticancer activity of AA. Cancer cells that showed high SVCT-2 expression levels were more sensitive to AA treatment than cancer cells with low SVCT-2 expression levels. Cells with low SVCT-2 expression showed a hormetic response to a low dose of AA. Magnesium ions, which are known to activate SVCT-2, could increase the Vmax value of SVCT-2, so we investigated whether providing magnesium supplements to cancer cells with low SVCT-2 expression that had shown a hormetic response to AA would elevate the Vmax value of SVCT-2, allowing more AA to accumulate. To evaluate the effects of magnesium on cancer cells, MgSO4 and MgCl2 were screened as magnesium supplements; both forms showed synergistic anticancer effects with AA. Taken together, the results of this study suggest that magnesium supplementation enhanced the anticancer effect of AA by inhibiting the hormetic response at a low dose. This study has also demonstrated that AA treatment with magnesium supplementation provided more effective anticancer therapy than AA treatment alone.

Hormetic dose response to L-ascorbic acid as an anti-cancer drug in colorectal cancer cell lines according to SVCT-2 expression.

L-Ascorbic acid (vitamin C, AA) exhibits anti-cancer effects with high-dose treatment through the generation of reactive oxygen species (ROS) and selective damage to cancer cells. The anti-cancer effects of L-ascorbic acid are determined by sodium-dependent vitamin C transporter 2 (SVCT-2), a transporter of L-ascorbic acid. In this study, we demonstrate that L-ascorbic acid treatment showed efficient anti-cancer activity in cell lines with high expression levels of SVCT-2 for a gradient concentration of L-ascorbic acid from 10 µM -2 mM. However, in low SVCT-2 expressing cell lines, high-dose L-ascorbic acid (>1 mM) showed anti-cancer effects but low-dose (<10 µM) treatment induced cell proliferation. Such conflicting results that depend on the concentration are called a hormetic dose response. A hormetic dose response to low-dose L-ascorbic acid was also observed in high SVCT-2 expressing cell lines in the presence of a SVCT family inhibitor. Insufficient uptake of L-ascorbic acid in low SVCT-2 expressing cancer cell lines cannot generate sufficient ROS to kill cancer cells, resulting in the hormetic response. Molecular analysis confirmed the increased expression of cancer proliferation markers in the hormetic dose response. These results suggest that L-ascorbic exhibits a biphasic effect in cancer cells depending on SVCT-2 expression.

Therapeutic Strategy for the Prevention of Pseudorabies Virus Infection in C57BL/6 Mice by 3D8 scFv with Intrinsic Nuclease Activity.

3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and 10 µg 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 LD50 PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% (5 µg) and 47% (10 µg). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.