First report of konjac mosaic virus in Spuriopimpinella brachycarpa in Korea.

Spuriopimpinella brachycarpa Nakai (Common name, Chamnamul; family Apiaceae) is a plant whose leaves are consumed as a vegetable and used as a folk medicine in Korea (Kim et al., 2020). In February 2020, seven samples of S. brachycarpa leaf showing virus symptoms including yellowing, vein chlorosis, chlorotic lesions, and severe mottling were collected from a greenhouse in Busan, South Korea, to diagnose the potential disease (Fig. S1a, b). The disease incidence rate in the greenhouse was >10% (2,970 m2). To identify the causal virus, we analyzed leaf dip preparation and thin sections of the symptomatic leaves by transmission electron microscopy. Filamentous virus particles and pinwheel structures were observed, indicating the presence of a potyvirus (Fig. S1c, d). To confirm these results, the symptomatic leaf samples were further analyzed by reverse-transcription polymerase chain reaction (RT-PCR) using potyvirus universal primers (Table S2) and direct sequencing of the PCR products. All samples were positive for konjac mosaic virus (KoMV). To exclude the possibility of infection by multiple viruses, we performed high-throughput sequencing (HTS) on an Illumina NovaSeq 6000 system (Macrogen Inc., Seoul, South Korea). There were two contigs (9,267 and 2,851 nt) mapping to KoMV sequences. A large contig (9,267 nt; 705,967 mapped reads; mean read coverage of 11,351.4x) showed about 80% identity (93% coverage) with KoMV-F (GenBank accession no. NC_007913) isolated from Amorphophallus konjac in Japan (Nishiguchi et al., 2006). To isolate KoMV from S. brachycarpa, we mechanically inoculated leaf extracts from symptomatic samples onto Chenopodium quinoa as an assay host via three single-lesion passages, followed by propagation in Nicotiana benthamiana. In a bioassay of the KoMV isolate (KoMV-BS), we mechanically inoculated sap from infected N. benthamiana onto 31 indicator plants including Cryptotaenia japonica (Apiaceae), which is similar to S. brachycarpa (Table S3). KoMV-BS systemically induced vein chlorosis and/or leaf mottling in four Nicotiana species and C. japonica, and chlorotic local lesions in upper leaves of C. quinoa; no symptoms were observed in 25 other indicator plants. These results were confirmed by RT-PCR. Next, we obtained the complete genome sequence of KoMV-BS using HTS and 5' and 3' rapid amplification of cDNA ends, with newly designed primers (Table S2). The assembled full-length KoMV-BS genome sequence was 9,392 nt in length, excluding the poly(A) tail, and encoded a polyprotein composed of 3,060 amino acids. The sequence was deposited in GenBank (accession no. OR001914). BLAST analysis showed 84~88% and 90~98% identities at CP nucleotide and amino acid levels, respectively with the reported KoMV isolates, confirming the virus to be an isolate of KoMV (synonym; Japanese hornwort mosaic virus, zantedeschia mosaic virus) (Adams et al., 2005; Nishiguchi et al., 2006). KoMV infection was first reported in A. konjac from Japan (Shimoyama et al. 1992) and has been spread worldwide as one of the major causal agents of viral diseases in calla lily (Liao et al., 2020). To the best of our knowledge, this is the first report of KoMV infection in S. brachycarpa. To date, cucumber mosaic virus and tobacco mosaic virus have been reported to infect S. brachycarpa in Korea (Yoon et al., 2016; 2017). Our findings will be helpful for developing virus-management strategies to prevent yield and quality loss in S. brachycarpa.

Efficacy and safety of acupuncture for functional constipation: a randomised, sham-controlled pilot trial.

BACKGROUND: The prevalence of functional constipation (FC) is 3-27%, and FC has been reported to cause discomfort in daily life and various complications. The treatment for FC depends on laxatives, and thus, effective and non-toxic alternative treatments are needed. METHODS: We conducted a randomised, sham-controlled parallel-design, pilot trial. Participants with FC were randomly assigned to either the real acupuncture (RA) or sham acupuncture (SA) group. The RA consisted of eight fixed acupuncture points (bilateral ST25, ST27, BL52 and BL25) and four additional points targeted to the individual based on Traditional Korean medicine (TKM). SA consisted of shallow acupuncture insertion at 12 non-acupuncture points. Twelve sessions were provided over 4 weeks. The outcome measures were weekly defecation frequency (DF), spontaneous complete bowel movement (SCBM), Bristol stool scale (BSS) score and constipation assessment scale (CAS) score. The participants were followed for 4 weeks after the treatment. RESULTS: Thirty participants were enrolled (15:15). The mean DF were 5.86 ± 5.62, 5.43 ± 3.39 and 5.79 ± 3.64 in the RA group and 3.73 ± 1.62, 5.00 ± 1.77 and 5.40 ± 1.96 in the SA group at weeks 1, 5, and 9, respectively. The increases in weekly SCBMs were 2.50 ± 3.86 and 2.71 ± 4.01 with RA and 2.33 ± 2.74 and 1.93 ± 2.25 with SA at weeks 5 and 9, respectively (mean difference [MD] 0.78). The BSS scores were 0.57 ± 1.72 and 1.09 ± 1.30 with RA and 0.15 ± 1.06 and 0.14 ± 0.88 with SA at weeks 5 and 9, respectively (MD 0.95). The CAS score changes were - 3.21 ± 2.91 and - 3.50 ± 3.98 with RA and - 2.67 + ±2.82 and - 2.87 ± 2.95 with SA at weeks 5 and 9, respectively. Greater improvements were observed in subgroup analysis of participants with hard stool. The numbers of participants who developed adverse events (AEs) were equal in both groups (four in each group), and the AEs were not directly related to the intervention. CONCLUSIONS: This clinical trial shows feasibility with minor modifications to the primary outcome measure and comparator. Acupuncture showed clinically meaningful improvements in terms of SCBMs occurring more than 3 times per week and in these improvements being maintained for 4 weeks after treatment completion. As this is a pilot trial, future studies are warranted to confirm the efficacy and safety. TRIAL REGISTRATION: KCT0000926 (Registered on 14 November 2013).

Citrus aurantium flavonoids inhibit adipogenesis through the Akt signaling pathway in 3T3-L1 cells.

BACKGROUND: Obesity is a health hazard that is associated with a number of diseases and metabolic abnormalities, such as type-2 diabetes, hypertension, dyslipidemia, and coronary heart disease. In the current study, we investigated the effects of Citrus aurantium flavonoids (CAF) on the inhibition of adipogenesis and adipocyte differentiation in 3T3-L1 cells. METHODS: During adipocyte differentiation, 3T3-L1 cells were treated with 0, 10, and 50 µg/ml CAF, and then the mRNA and protein expression of adipogenesis-related genes was assayed. We examined the effect of CAF on level of phosphorylated Akt in 3T3-L1 cells treated with CAF at various concentrations during adipocyte differentiation. RESULTS: The insulin-induced expression of C/EBPß and PPARγ mRNA and protein were significantly down-regulated in a dose-dependent manner following CAF treatment. CAF also dramatically decreased the expression of C/EBPα, which is essential for the acquisition of insulin sensitivity by adipocytes. Moreover, the expression of the aP2 and FAS genes, which are involved in lipid metabolism, decreased dramatically upon treatment with CAF. Interestingly, CAF diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3ß (Ser9), which may reduce glucose uptake in response to insulin and lipid accumulation. Furthermore, CAF not only inhibited triglyceride accumulation during adipogenesis but also contributed to the lipolysis of adipocytes. CONCLUSIONS: In the present study, we demonstrate that CAF suppressed adipogenesis in 3T3-L1 adipocytes. Our results indicated that CAF down-regulates the expression of C/EBPß and subsequently inhibits the activation of PPARγ and C/EBPα. The anti-adipogenic activity of CAF was mediated by the inhibition of Akt activation and GSK3ß phosphorylation, which induced the down-regulation of lipid accumulation and lipid metabolizing genes, ultimately inhibiting adipocyte differentiation.

Effect of alpha-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes.

Boar semen is extremely vulnerable to cold shock and sensitive to peroxidative damage due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and the relatively low antioxidant capacity of seminal plasma. The present study evaluated the influence of alpha-tocopherol supplementation at various concentrations in the boar semen extender during cryopreservation on post-thawed sperm motility characteristics (total sperm motility, MOT; local motility, LCM; curvilinear velocity, VCL; straight linear velocity, VSL; and average path velocity, VAP), sperm qualities (viability, acrosomal integrity and apoptosis), expression of stress protein (HSP70), and the expression of pro-apoptotic (Bax and Bak) and anti-apoptotic (Bcl-2l and Bcl-xl) genes. Semen collected from 10 Duroc boars was cryopreserved in lactose-egg yolk buffer supplemented with various concentrations of alpha-tocopherol (0, 100, 200, 400, 600 and 800 microM) using the straw-freezing procedure and stored at -196 degrees C for a minimum period of one month. In frozen-thawed groups, sperm motility was significantly (P<0.05) lower than that of fresh sperm. In fresh sperm, HSP70 immunoreactivity expression was observed in the equatorial region, but in frozen-thawed groups, expressions were mostly observed in the sperm head. Higher apoptosis rates were observed in 600 and 800 microM alpha-tocopherol supplemented frozen-thawed groups. In alpha-tocopherol supplemented frozen-thawed groups immediately after thawing, the expression was similar to that of fresh group. But after incubation at 37 degrees C for 3h, the expression in 200 and 800 microM alpha-tocopherol supplemented groups was higher than that of others. Expression of pro-apoptotic genes was significantly higher and anti-apoptotic genes was significantly (P<0.01) lower in alpha-tocopherol supplemented frozen-thawed groups compared to fresh sperm group. In conclusion, alpha-tocopherol, supplemented at 200 microM concentration in boar semen extender during cryopreservation had a positive effect on post-thawed sperm survivability.

Structural requirement of chalcones for the inhibitory activity of interleukin-5.

Novel chalcones were found as potent inhibitors of interleukin (IL)-5. 1-(2-Benzyloxy-6-hydroxyphenyl)-3-(4-hydroxyphenyl)-2-propen-1-one (2b, 78.8% inhibition at 50microM, IC(50)=25.3microM) was initially identified as a potent inhibitor of IL-5. This shows the compatible activity with budesonide or sophoricoside. To identify structural requirements, 26 chalcones were prepared and their inhibitory activities were tested against IL-5. Among them, compound 4-[(E)-3-(2-cyclohexylmethoxy-6-hydroxyphenyl)-3-oxoprop-1-enyl]benzenesulfonamide (2w, 99.5% inhibition at 50microM, IC(50)=1.8microM) shows the most potent activity. The important structural requirements of these chalcone analogs exhibiting the inhibitory activity against IL-5 were recognized as the following. (1) The hydrophobic group such as benzyloxy or cyclohexylmethoxy at 6-position of A ring is necessary. (2) The existence of phenolic hydroxyl at 6-position of A ring is critical. (3) Propenone unit as alpha,beta-unsaturated ketone is essential. (4) Electron withdrawing groups with hydrogen acceptor property at 4-position of B ring enhance the activity and quantitative structure-activity relationship of 2 regarding these substituents was determined.

Anti-inflammatory action of legume isoflavonoid sophoricoside through inhibition on cyclooxygenase-2 activity.

Soy is a main source of isoflavonoids which are of high dietary intake for the oriental population. In this study, the anti-inflammatory action of sophoricoside, an isoflavone glycoside isolated from immature fruits of Sophora japonica L. (Leguminosae), has been demonstrated. When administered orally at > 100 mg/kg or injected intravenously at > 10 mg/kg, sophoricoside showed significant reduction of carrageenin-induced paw edema in mice. Sophoricoside has been identified as a selective inhibitor of cyclooxygenase (COX)-2 activity with an IC50 value of 3.3 microM.