Phytochemical Composition and Biological Activities of Extracts from Early, Mature, and Germinated Somatic Embryos of Cotyledon orbiculata L.

Cotyledon orbiculata L. (Crassulaceae)-round-leafed navelwort-is used worldwide as a potted ornamental plant, and it is also used in South African traditional medicine. The current work aims to assess the influence of plant growth regulators (PGR) on somatic embryogenesis (SE) in C. orbiculata; compare the metabolite profile in early, mature, and germinated somatic embryos (SoEs) by utilizing ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS); and determine the antioxidant and enzyme inhibitory potentials of SoEs. A maximum SoE induction rate of 97.2% and a mean number of SoEs per C. orbiculata leaf explant of 35.8 were achieved on Murashige and Skoog (MS) medium with 25 µM 2,4-Dichlorophenoxyacetic acid and 2.2 µM 1-phenyl-3-(1,2,3,-thiadiazol-5-yl)urea. The globular SoEs were found to mature and germinate best on MS medium with gibberellic acid (4 µM). The germinated SoE extract had the highest amounts of both total phenolics (32.90 mg gallic acid equivalent/g extract) and flavonoids (1.45 mg rutin equivalent/g extract). Phytochemical evaluation of SoE extracts by UHPLC-MS/MS reveals the presence of three new compounds in mature and germinated SoEs. Among the SoE extracts tested, germinated SoE extract exhibited the most potent antioxidant activity, followed by early and mature somatic embryos. The mature SoE extract showed the best acetylcholinesterase inhibitory activity. The SE protocol established for C. orbiculata can be used for the production of biologically active compounds, mass multiplication, and conservation of this important species.

Non-invasive administration of AAV to target lung parenchymal cells and develop SARS-CoV-2-susceptible mice.

Adeno-associated virus (AAV)-mediated gene delivery holds great promise for gene therapy. However, the non-invasive delivery of AAV for lung tissues has not been adequately established. Here, we revealed that the intratracheal administration of an appropriate amount of AAV2/8 predominantly targets lung tissue. AAV-mediated gene delivery that we used in this study induced the expression of the desired protein in lung parenchymal cells, including alveolar type II cells. We harnessed the technique to develop severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-susceptible mice. Three kinds of immune function-relevant gene knockout (KO) mice were transduced with AAV encoding human angiotensin-converting enzyme 2 (hACE2) and then injected with SARS-CoV-2. Among these mice, type I interferon receptor (IFNAR) KO mice showed increased viral titer in the lungs compared to that in the other KO mice. Moreover, nucleocapsid protein of SARS-CoV-2 and multiple lesions in the trachea and lung were observed in AAV-hACE2-transduced, SARS-CoV-2-infected IFNAR KO mice, indicating the involvement of type I interferon signaling in the protection of SARS-CoV-2. In this study, we demonstrate the ease and rapidness of the intratracheal administration of AAV for targeting lung tissue in mice, and this can be used to study diverse pulmonary diseases.

Evaluation of Photobiogoverning Role of Blue Light Irradiation on Viral Replication.

Most recently, severe acute respiratory syndrome coronavirus-2 has triggered a global pandemic without successful therapeutics. The goal of the present study was to define the antiviral effect and therapeutic action of blue light irradiation in SARS-CoV-2-infected cells. Vero cells were infected with SARS-CoV-2 (NCCP43326) or mock inoculum at 50 pfu/well. After blue light irradiation, the inhibitory effect was assessed by qPCR and plaque reduction assay. When Vero cells were irradiated to blue light ranging from 1.6 to 10 J cm-2 , SARS-CoV-2 replication was inhibited by up to 80%. The antiviral effect of blue light irradiation was associated with translation suppression via the phosphorylation of eIF2α by prolonging endoplasmic reticulum (ER) stress. The levels of LC3A/B and Beclin-1, which are key markers of autophagy, and the levels of PERK and PDI for ER stress were highly increased, whereas caspase-3 cleavage was inhibited after blue light irradiation in the later stage of infection. Our data revealed that blue light irradiation exerted antiviral and photo-biogoverning activities by prolonging ER stress and stimulating autophagy progression during viral infection. The findings increase our understanding of how photo-energy acts on viral progression and have implications for use in therapeutic strategies against COVID-19.

Ultra- and micro-structural changes of respiratory tracts in SARS-CoV-2 infected Syrian hamsters.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a global crisis. It is still unresolved. Although many therapies and vaccines are being studied, they are still in their infancy. As this pandemic continues, rapid and accurate research for the development of therapies and vaccines is needed. Therefore, it is necessary to understand characteristics of diseases caused by SARS-CoV-2 through animal models. Syrian hamsters are known to be susceptible to SARS-CoV-2. They were intranasally inoculated with SARS-CoV-2. At 2, 4, 8, 12, and 16 days post-infection (dpi), these hamsters were euthanized, and tissues were collected for ultrastructural and microstructural examinations. Microscopic lesions were prominent in the upper and lower respiratory tracts from 2 and 4 dpi groups, respectively. The respiratory epithelium in the trachea, bronchiole, and alveolar showed pathological changes. Inflammatory cells including neutrophils, lymphocytes, macrophages, and eosinophils were infiltrated in/around tracheal lamina propria, pulmonary vessels, alveoli, and bronchiole. In pulmonary lesions, alveolar wall was thickened with infiltrated inflammatory cells, mainly neutrophils and macrophages. In the trachea, epithelial damages started from 2 dpi and recovered from 8 dpi, consistent with microscopic results, High levels of SARS-CoV-2 nucleoprotein were detected at 2 dpi and 4 dpi. In the lung, lesions were most severe at 8 dpi. Meanwhile, high levels of SARS-CoV-2 were detected at 4 dpi. Electron microscopic examinations revealed cellular changes in the trachea epithelium and alveolar epithelium such as vacuolation, sparse micro-organelle, and poor cellular margin. In the trachea epithelium, the number of cytoplasmic organelles was diminished, and small vesicles were prominent from 2 dpi. Some of these electron-lucent vesicles were filled with virion particles. From 8 dpi, the trachea epithelium started to recover. Because of shrunken nucleus and swollen cytoplasm, the N/C ratio of type 2 pneumocyte decreased at 8 and 12 dpi. From 8 dpi, lamellar bodies on type 2 pneumocyte cytoplasm were increasingly observed. Their number then decreased from 16 dpi. However, there was no significant change in type 1 pneumocyte. Viral vesicles were only observed in the cytoplasm of type 2 pneumocyte. In conclusion, ultra- and micro-structural changes presented in this study may provide useful information for SARS-CoV-2 studies in various fields.

Potent antiviral activity of Agrimonia pilosa, Galla rhois, and their components against SARS-CoV-2.

Agrimonia pilosa (AP), Galla rhois (RG), and their mixture (APRG64) strongly inhibited SARS-CoV-2 by interfering with multiple steps of the viral life cycle including viral entry and replication. Furthermore, among 12 components identified in APRG64, three displayed strong antiviral activity, ursolic acid (1), quercetin (7), and 1,2,3,4,6-penta-O-galloyl-ß-d-glucose (12). Molecular docking analysis showed these components to bind potently to the spike receptor-binding-domain (RBD) of the SARS-CoV-2 and its variant B.1.1.7. Taken together, these findings indicate APRG64 as a potent drug candidate to treat SARS-CoV-2 and its variants.

Immunization with RBD-P2 and N protects against SARS-CoV-2 in nonhuman primates.

Since the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), various vaccines are being developed, with most vaccine candidates focusing on the viral spike protein. Here, we developed a previously unknown subunit vaccine comprising the receptor binding domain (RBD) of the spike protein fused with the tetanus toxoid epitope P2 (RBD-P2) and tested its efficacy in rodents and nonhuman primates (NHPs). We also investigated whether the SARS-CoV-2 nucleocapsid protein (N) could increase vaccine efficacy. Immunization with N and RBD-P2 (RBDP2/N) + alum increased T cell responses in mice and neutralizing antibody levels in rats compared with those obtained using RBD-P2 + alum. Furthermore, in NHPs, RBD-P2/N + alum induced slightly faster SARS-CoV-2 clearance than that induced by RBD-P2 + alum, albeit without statistical significance. Our study supports further development of RBD-P2 as a vaccine candidate against SARS-CoV-2. Also, it provides insights regarding the use of N in protein-based vaccines against SARS-CoV-2.

Effects of Acupuncture on Lowering Blood Pressure in Postmenopausal Women with Prehypertension or Stage 1 Hypertension: A Propensity Score-Matched Analysis.

Postmenopausal women have a higher prevalence of hypertension compared to premenopausal women. Hypertension is a risk factor for cardiovascular diseases, the prevalence of which is ever increasing. This study investigated the effects of long-term acupuncture on lowering the blood pressure of postmenopausal women with prehypertension and stage 1 hypertension. Participants were 122 postmenopausal women aged less than 65 years, diagnosed with prehypertension or stage 1 hypertension (systolic blood pressure 120-159 mmHg or diastolic blood pressure 80-99 mmHg). We used a propensity score-matched design. The experimental group (n = 61) received acupuncture for four weeks every six months over a period of two years. The control group (n = 61) received no intervention. An Analysis of covariance (ANCOVA) was performed for the primary efficacy analysis. Relative risk ratios were used to compare group differences in treatment effects. Acupuncture significantly reduced the participants' diastolic blood pressure (-9.92 mmHg; p < 0.001) and systolic blood pressure (-10.34 mmHg; p < 0.001) from baseline to follow-up. The results indicate that acupuncture alleviates hypertension in postmenopausal women, reducing their risk of developing cardiovascular diseases and improving their health and quality of life.

Expression Analyses of MicroRNAs in Hamster Lung Tissues Infected by SARS-CoV-2.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an infectious disease with multiple severe symptoms, such as fever over 37.5°C, cough, dyspnea, and pneumonia. In our research, microRNAs (miRNAs) binding to the genome sequences of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory-related coronavirus (MERS-CoV), and SARS-CoV-2 were identified by bioinformatic tools. Five miRNAs (hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-195-5p, hsa-miR-16-5p, and hsa-miR-196a-1-3p) were found to commonly bind to SARS-CoV, MERS-CoV, and SARS-CoV-2. We also identified miRNAs that bind to receptor proteins, such as ACE2, ADAM17, and TMPRSS2, which are important for understanding the infection mechanism of SARS-CoV-2. The expression patterns of those miRNAs were examined in hamster lung samples infected by SARS-CoV-2. Five miRNAs (hsa-miR-15b-5p, hsa-miR-195-5p, hsa-miR-221-3p, hsa-miR-140-3p, and hsa-miR-422a) showed differential expression patterns in lung tissues before and after infection. Especially, hsa-miR-15b-5p and hsa-miR-195-5p showed a large difference in expression, indicating that they may potentially be diagnostic biomarkers for SARS-CoV-2 infection.

Sphingosine kinase 2 restricts T cell immunopathology but permits viral persistence.

Chronic viral infections are often established by the exploitation of immune-regulatory mechanisms that result in nonfunctional T cell responses. Viruses that establish persistent infections remain a serious threat to human health. Sphingosine kinase 2 (SphK2) generates sphingosine 1-phosphate, which is a molecule known to regulate multiple cellular processes. However, little is known about SphK2's role during the host immune responses to viral infection. Here, we demonstrate that SphK2 functions during lymphocytic choriomeningitis virus Cl 13 (LCMV Cl 13) infection to limit T cell immune pathology, which subsequently aids in the establishment of virus-induced immunosuppression and the resultant viral persistence. The infection of Sphk2-deficient (Sphk2-/-) mice with LCMV Cl 13 led to the development of nephropathy and mortality via T cell-mediated immunopathology. Following LCMV infection, Sphk2-/- CD4+ T cells displayed increased activity and proliferation, and these cells promoted overactive LCMV Cl 13-specific CD8+ T cell responses. Notably, oral instillation of an SphK2-selective inhibitor promoted protective T cell responses and accelerated the termination of LCMV Cl 13 persistence in mice. Thus, SphK2 is indicated as an immunotherapeutic target for the control of persistent viral infections.

n-3 Polyunsaturated Fatty Acids Impede the TCR Mobility and the TCR-pMHC Interaction of Anti-Viral CD8+ T Cells.

The immune-suppressive effects of omega-3 (n-3) polyunsaturated fatty acids (PUFAs) on T cells have been observed via multiple in vitro and in vivo models. However, the precise mechanism that causes these effects is still undefined. In this study, we investigated whether n-3 PUFAs regulated T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) interactions. The expansion of anti-viral CD8+ T cells that endogenously synthesize n-3 PUFAs (FAT-1) dramatically decreased upon lymphocytic choriomeningitis virus (LCMV) infection in vivo. This decrease was not caused by the considerable reduction of TCR expression or the impaired chemotactic activity of T cells. Interestingly, a highly inclined and laminated optical sheet (HILO) microscopic analysis revealed that the TCR motility was notably reduced on the surface of the FAT-1 CD8+ T cells compared to the wild type (WT) CD8+ T cells. Importantly, the adhesion strength of the FAT-1 CD8+ T cells to the peptide-MHC was significantly lower than that of the WT CD8+T cells. Consistent with this result, treatment with docosahexaenoic acid (DHA), one type of n-3 PUFA, significantly decreased CD8+ T cell adhesion to the pMHC. Collectively, our results reveal a novel mechanism through which n-3 PUFAs decrease TCR-pMHC interactions by modulating TCR mobility on CD8+ T cell surfaces.

MERS-CoV Spike Protein Vaccine and Inactivated Influenza Vaccine Formulated with Single Strand RNA Adjuvant Induce T-Cell Activation through Intranasal Immunization in Mice.

The effectiveness of vaccines is enhanced by adding adjuvants. Furthermore, the selection of an inoculation route depends on the type of adjuvant used and is important for achieving optimum vaccine efficacy. We investigated the immunological differences between two types of vaccines-spike protein from the Middle East respiratory syndrome virus and inactivated influenza virus vaccine, in combination with a single-stranded RNA adjuvant-administered through various routes (intramuscular, intradermal, and intranasal) to BALB/c mice. Intramuscular immunization with the RNA adjuvant-formulated spike protein elicited the highest humoral immune response, characterized by IgG1 and neutralizing antibody production. Although intranasal immunization did not elicit a humoral response, it showed extensive T-cell activation through large-scale induction of interferon-γ- and interleukin-2-secreting cells, as well as CD4+ T-cell activation in mouse splenocytes. Moreover, only intranasal immunization induced IgA production. When immunized with the inactivated influenza vaccine, administration of the RNA adjuvant via all routes led to protection after viral challenge, regardless of the presence of a vaccine-specific antibody. Therefore, the inoculation route should depend on the type of immune response needed; i.e., the intramuscular route is suitable for eliciting a humoral immune response, whereas the intranasal route is useful for T-cell activation and IgA induction.

In Vitro Propagation of Gastrochilus matsuran (Makino) Schltr., an Endangered Epiphytic Orchid.

Gastrochilus matsuran (Makino) Schltr. (Orchidaceae) populations are declining quickly because of overexploitation, climatic changes, and deforestation; therefore, mass-production protocols are required for this orchid. Natural propagation of this species is often hampered by meager seed germination and slow growth. Thus, our aim was to establish an effective protocol for the in vitro propagation of G. matsuran and reduce the risk of its extinction. We investigated the impacts of culture media, coconut water (CW), and plant hormones (gibberellic acid (GA3), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA), and thidiazuron (TDZ)) on asymbiotic germination, multiplication and conversion of protocorms, and plantlet development. Maximal seed germination (93.3%) was achieved on ½ MS medium without vitamins plus 5% CW, 1 µM NAA, and 1.5 µM GA3. Secondary protocorm formation was best achieved on ½ MS medium without vitamins plus 2 µM TDZ. The conversion of protocorms into seedlings was maximized by supplementation with 2 µM IBA or 1 µM NAA. Acclimatized plantlets that exhibited exuberant growth on sphagnum moss were reintroduced to tree trunks in a natural habitat, with a 67% survival rate. This in vitro propagation procedure would be helpful for the mass production and conservation of this rare epiphytic orchid.

Selective and ATP-competitive kinesin KIF18A inhibitor suppresses the replication of influenza A virus.

The influenza virus is one of the major public health threats. However, the development of efficient vaccines and therapeutic drugs to combat this virus is greatly limited by its frequent genetic mutations. Because of this, targeting the host factors required for influenza virus replication may be a more effective strategy for inhibiting a broader spectrum of variants. Here, we demonstrated that inhibition of a motor protein kinesin family member 18A (KIF18A) suppresses the replication of the influenza A virus (IAV). The expression of KIF18A in host cells was increased following IAV infection. Intriguingly, treatment with the selective and ATP-competitive mitotic kinesin KIF18A inhibitor BTB-1 substantially decreased the expression of viral RNAs and proteins, and the production of infectious viral particles, while overexpression of KIF18A enhanced the replication of IAV. Importantly, BTB-1 treatment attenuated the activation of AKT, p38 MAPK, SAPK and Ran-binding protein 3 (RanBP3), which led to the prevention of the nuclear export of viral ribonucleoprotein complexes. Notably, administration of BTB-1 greatly improved the viability of IAV-infected mice. Collectively, our results unveiled a beneficial role of KIF18A in IAV replication, and thus, KIF18A could be a potential therapeutic target for the control of IAV infection.

Nanoformulated Single-Stranded RNA-Based Adjuvant with a Coordinative Amphiphile as an Effective Stabilizer: Inducing Humoral Immune Response by Activation of Antigen-Presenting Cells.

As agonists of TLR7/8, single-stranded RNAs (ssRNAs) are safe and promising adjuvants that do not cause off-target effects or innate immune overactivation. However, low stability prevents them from mounting sufficient immune responses. This study evaluates the adjuvant effects of ssRNA derived from the cricket paralysis virus intergenic region internal ribosome entry site, formulated as nanoparticles with a coordinative amphiphile, containing a zinc/dipicolylamine complex moiety as a coordinative phosphate binder, as a stabilizer for RNA-based adjuvants. The nanoformulated ssRNA adjuvant was resistant to enzymatic degradation in vitro and in vivo, and that with a coordinative amphiphile bearing an oleyl group (CA-O) was approximately 100 nm, promoted effective recognition, and improved activation of antigen-presenting cells, leading to better induction of neutralizing antibodies following single immunization. Hence, CA-O may increase the efficacy of ssRNA-based adjuvants, proving useful to meet the urgent need for vaccines during pathogen outbreaks.

Endogenous n-3 Polyunsaturated Fatty Acids Are Beneficial to Dampen CD8+ T Cell-Mediated Inflammatory Response upon the Viral Infection in Mice.

Omega-3 (n-3) polyunsaturated fatty acids (PUFAs) have been known to exert anti-inflammatory effects on various disease states. However, its effect on CD8+ T cell-mediated immunopathology upon viral infection has not been well elucidated yet. In this study, we investigated the possible implication of n-3 PUFAs in CD8+ T cell responses against an acute viral infection. Infection of FAT-1 transgenic mice that are capable of synthesizing n-3 PUFAs from n-6 PUFAs with lymphocytic choriomeningitis virus (LCMV) resulted in significant reduction of anti-viral CD8+ T cell responses. Interestingly, expansion of adoptively transferred wild-type (WT) LCMV-specific T cell receptor (TCR) transgenic CD8+ (P14) T cells into FAT-1 mice was significantly decreased. Also, activation of anti-viral CD4+ helper T cells was reduced in FAT-1 mice. Importantly, P14 cells carrying the fat-1 gene that were adoptively transferred into WT mice exhibited a substantially decreased ability to proliferate and produce cytokines against LCMV infection. Together, n-3 PUFAs attenuated anti-viral CD8+ T cell responses against an acute viral infection and thus could be used to alleviate immunopathology mediated by the viral infection.

Cricket paralysis virus internal ribosome entry site-derived RNA promotes conventional vaccine efficacy by enhancing a balanced Th1/Th2 response.

An ideal adjuvant should increase vaccine efficacy through balanced Th1/Th2 responses and be safe to use. Recombinant protein-based vaccines are usually formulated with aluminum (alum)-based adjuvants to ensure an adequate immune response. However, use of alum triggers a Th2-biased immune induction, and hence is not optimal. Although the adjuvanticity of RNA has been reported, a systematic and overall investigation on its efficacy is lacking. We found that single strand RNA (termed RNA adjuvant) derived from cricket paralysis virus intergenic region internal ribosome entry site induced the expression of various adjuvant-function-related genes, such as type 1 and 2 interferon (IFN) and toll-like receptor (TLR), T cell activation, and leukocyte chemotaxis in human peripheral blood mononuclear cells; furthermore, its innate and IFN transcriptome profile patterns were similar to those of a live-attenuated yellow fever vaccine. This suggests that protein-based vaccines formulated using RNA adjuvant function as live-attenuated vaccines. Application of the RNA adjuvant in mouse enhanced the efficacy of Middle East respiratory syndrome spike protein, a protein-subunit vaccine and human papillomavirus L1 protein, a virus-like particle vaccine, by activating innate immune response through TLR7 and enhancing pAPC chemotaxis, leading to a balanced Th1/Th2 responses. Moreover, the combination of alum and the RNA adjuvant synergistically induced humoral and cellular immune responses and endowed long-term immunity. Therefore, RNA adjuvants have broad applicability and can be used with all conventional vaccines to improve vaccine efficacy qualitatively and quantitively.

Immunological Compatibility of Bone Tissues from Alpha-1,3-galactosyltransferase Knockout Pig for Xenotransplantation.

We investigated whether the lack of galactosyltransferase (α-Gal) expression in bone tissue is associated with reduced immune response of human peripheral blood mononuclear cells (PBMCs) against pig bone tissue. When human PBMC obtained from heparinized blood of healthy volunteers was stimulated with bone extracts of pigs with α-1,3-galactosyltransferase knock out (α-Gal KO), the proliferation of human PBMCs and production of proinflammatory cytokines such as TNF-α and IL-1ß were significantly reduced compared to those stimulated with bone extracts of wild type (WT) pigs. In addition, activation of CD4+ helper T cells and production of IL-2, IFN-γ, and IL-17 were reduced upon stimulation with bone tissue extracts from α-Gal KO pigs. This is possibly due to the lowered activities of the NF-κB, p38, ERK, and JNK signaling pathways. Our findings can be used to evaluate the compatibility of bone tissues from α-Gal KO pigs with human bone grafting as novel natural biomaterials, thereby increasing the feasibility of future clinical applications.

Heterologous prime-boost vaccination with adenoviral vector and protein nanoparticles induces both Th1 and Th2 responses against Middle East respiratory syndrome coronavirus.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic and zoonotic virus with a fatality rate in humans of over 35%. Although several vaccine candidates have been developed, there is still no clinically available vaccine for MERS-CoV. In this study, we developed two types of MERS-CoV vaccines: a recombinant adenovirus serotype 5 encoding the MERS-CoV spike gene (Ad5/MERS) and spike protein nanoparticles formulated with aluminum (alum) adjuvant. Next, we tested a heterologous prime-boost vaccine strategy, which compared priming with Ad5/MERS and boosting with spike protein nanoparticles and vice versa, with homologous prime-boost vaccination comprising priming and boosting with either spike protein nanoparticles or Ad5/MERS. Although both types of vaccine could induce specific immunoglobulin G against MERS-CoV, neutralizing antibodies against MERS-CoV were induced only by heterologous prime-boost immunization and homologous immunization with spike protein nanoparticles. Interestingly, Th1 cell activation was induced by immunization schedules including Ad5/MERS, but not by those including only spike protein nanoparticles. Heterologous prime-boost vaccination regimens including Ad5/MERS elicited simultaneous Th1 and Th2 responses, but homologous prime-boost regimens did not. Thus, heterologous prime-boost may induce longer-lasting immune responses against MERS-CoV because of an appropriate balance of Th1/Th2 responses. However, both heterologous prime-boost and homologous spike protein nanoparticles vaccinations could provide protection from MERS-CoV challenge in mice. Our results demonstrate that heterologous immunization by priming with Ad5/MERS and boosting with spike protein nanoparticles could be an efficient prophylactic strategy against MERS-CoV infection.

Effects of acupuncture in postmenopausal women with prehypertension or stage 1 hypertension: study protocol for a prospective, comparative, interventional cohort study.

BACKGROUND: Hypertension is a major cause of cardiovascular disease and associated mortality, and postmenopausal women are at a high risk of hypertension. We aim to investigate the hypotensive effect and safety of acupuncture, focusing on postmenopausal women with prehypertension and stage 1 hypertension. In addition, we aim to investigate whether the effect of acupuncture treatment differed, depending on Sasang Constitution and cold-heat pattern. METHODS: This study is designed as an intervention cohort study. Two hundred postmenopausal women aged <65 years with prehypertension or stage 1 hypertension living in Daejeon city in Korea will be recruited, and randomly assigned to either an acupuncture or no-treatment control group. The intervention will consist of four sessions; one session will include acupuncture performed 10 times for 4 weeks. There will be a 20-week observation period after each session, and the total study duration will be 96 weeks. Acupuncture will be applied at the bilateral Fengchi (GB20), Quchi (LI11), Zusanli (ST36), and Sameumgyo (SP6) acupoints. The effect of acupuncture will be evaluated by comparing the change in systolic and diastolic blood pressure between the acupuncture and control groups every 4 weeks until the end of the study. DISCUSSION: To evaluate the success of blood pressure management, long-term observation is required, but no long-term studies have been conducted to evaluate the effect of acupuncture on blood pressure in postmenopausal women. To our knowledge, this study will be the first long-term study to investigate this issue for more than 6-8 weeks.

In vitro propagation of Cymbidium goeringii Reichenbach fil. through direct adventitious shoot regeneration.

The influence of 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and thidiazuron (TDZ) on direct rhizome induction and shoot formation from rhizome explants of Cymbidium goeringii was explored. Rhizome segments obtained from in vitro seed cultures of C. goeringii were placed on Murashige and Skoog (MS) medium incorporated with 5, 10, 20, or 40 µM 2,4-D and 1, 2, 4, or 8 µM BA or TDZ alone or in combination with 20 µM 2,4-D. The explants developed only rhizomes on MS medium with or without 2,4-D. The highest percent of rhizome formation (100%) was obtained on MS medium incorporated with 20 µM of 2,4-D. The morphology and number of rhizomes varied with the level of 2,4-D in the medium. Direct adventitious shoot formation was achieved on medium incorporated with BA or TDZ. The adventitious shoots produced per explant significantly increased with the supplementation of 2,4-D to cytokinin-containing medium. The highest mean of 21.8 ± 1.8 shoot buds per rhizome segment was obtained in medium fortified with 20 µM 2,4-D and 2 µM TDZ. The greatest percent of root induction (100%) and the mean of 5.3 ± 1.1 roots per shoot were achieved on ½ MS medium incorporated with 2 µM of α-naphthaleneacetic acid. About 97% of the in vitro-produced plantlets acclimatized in the greenhouse. An efficient in vitro propagation protocol was thus developed for C. goeringii using rhizome explants.