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The post-translational acetylation of lysine residues is found in many nonhistone proteins and is involved in a wide range of biological processes. Recently, we showed that the nucleoprotein of the influenza A virus is acetylated by histone acetyltransferases (HATs), a phenomenon that affects viral transcription. Here, we report that the PA subunit of influenza A virus RNA-dependent RNA polymerase is acetylated by the HATs, P300/CREB-binding protein-associated factor (PCAF), and general control nonderepressible 5 (GCN5), resulting in accelerated endonuclease activity. Specifically, the full-length PA subunit expressed in cultured 293T cells was found to be strongly acetylated. Moreover, the partial recombinant protein of the PA N-terminal region containing the endonuclease domain was also acetylated by PCAF and GCN5 in vitro, which facilitated its endonuclease activity. Mass spectrometry analyses identified K19 as a candidate acetylation target in the PA N-terminal region. Notably, the substitution of the lysine residue at position 19 with glutamine, a mimic of the acetyl-lysine residue, enhanced its endonuclease activity in vitro; this point mutation also accelerated influenza A virus RNA-dependent RNA polymerase activity in the cell. Our findings suggest that PA acetylation is important for the regulation of the endonuclease and RNA polymerase activities of the influenza A virus.

Background Prejudice and stigma against patients with depression may lead to adverse clinical consequences, such as delayed initiation and premature termination of therapy. However, available survey results on pharmacists' awareness showed a lack of confidence in dealing with patients with depression. Until recently, few studies addressed the relationship between pharmacists' perceptions of their social distance from and confidence in interacting with patients with depression. Objectives To determine pharmacists' social distance from patients with depression, and investigate the factors that influence and are influenced by it. Methods The target population of this survey study included 161 pharmacists employed at a drug chain who had participated in the Effects of Community Pharmacists' Medication Assistance for the Patients Taking Antidepressant study. The questionnaire included six questions on social distance, which were adopted from a previous nation-wide survey of mental illness stigma in Japan. Other questions related to the respondents' attributes, personal experience (living with depression, abusive behaviors inflicted by patients with depression), and confidence in interacting with patients with depression. Results Valid responses were obtained from 77 study participants. The mean total social distance score was 12.49. Greater total social distance scores were associated with greater difficulty in relationship building, information provision, and comprehension of condition. Pharmacists' personal experience of living with depression decreased their social distance from patients with depression, whereas their experience of being verbally or physically abused by patients with depression increased their social distance. Conclusion This study showed that community pharmacists in Japan had less social distance from patients with depression than the general population. A significant negative correlation was observed between community pharmacists' social distance from and their confidence in interacting with patients with depression.

In Japan, there has been a private-sector initiative to register dietitians in pharmacies. There is not yet an adequate amount of data on the attitudes of dietitians in pharmacies regarding their work.To assess the attitudes of pharmacists and dietitians working in community pharmacies in Japan about their work, and particularly about demonstrating expertise and overall job satisfaction.We administered a web-based questionnaire to pharmacists and dietitians working in pharmacy branches with registered dietitians at two pharmacy chains that agreed to cooperate. We used our own 7-item questionnaire and compared the scores for each item between pharmacists and dietitians.A total of 61 persons (22 pharmacists and 39 dietitians) answered the questionnaire. The item with the highest mean score (standard deviation) for pharmacists was "I feel that I am helpful to patients," at 3.50 (0.74), and for dietitians it was "I have colleagues at my workplace whom I can talk to when I have a problem," at 3.51 (1.00). Conversely, the items with the lowest mean score (standard deviation) were "I am satisfied with my current job" for pharmacists, at 3.14 (0.83), and "I can grow sufficiently as a professional" for dietitians, at 2.41 (0.97). As a result of factor analysis, the seven-item question could be divided into two factors: "demonstrating expertise" and "overall job satisfaction." The factor-score for "demonstrating expertise" was significantly lower for dietitians than for pharmacists (p0.01), but there was no significant difference in overall job satisfaction between the two professions (p=0.36). Further research is needed to understand why dietitians find "demonstrating expertise" difficult in pharmacies.The attitudes of pharmacists and dietitians working in pharmacies in Japan toward their jobs were shown to consist of "demonstration of expertise" and "overall job satisfaction. Dietitians had significantly lower scores on "demonstration of expertise" than pharmacists.

Jiadifenolide has been reported to have neurotrophin-like activity in primary rat cortical neurons, and also possesses neurotrophic effects in neuronal precursor cells derived from human induced pluripotent stem cells (hiPSCs), as we have previously reported. However, the molecular mechanisms by which jiadifenolide exerts its neurotrophic effects in rat and human neurons are unknown. Thus, we aimed to investigate the molecular mechanisms and pathways by which jiadifenolide promotes neurotrophic effects. Here, we found that jiadifenolide activated cellular communication network factor (CCN) signaling pathways by up-regulating mRNA level expression of CCN genes in human neuronal cells. We also found that CCN2 (also known as connective tissue growth factor, CTGF) protein promotes neurotrophic effects through activation of the p44/42 mitogen-activated protein kinase signaling pathway. This is the first discovery which links neurotrophic activity with CCN signaling.

Novel antiviral agents for influenza, which poses a substantial threat to humans, are required. Cyclobakuchiols A and B have been isolated from Psoralea glandulosa, and cyclobakuchiol C has been isolated from P. corylifolia. The structural differences between cyclobakuchiol A and C arise due to the oxidation state of isopropyl group, and these compounds can be derived from (+)-(S)-bakuchiol, a phenolic isoprenoid compound present in P. corylifolia seeds. We previously reported that bakuchiol induces enantiospecific anti-influenza A virus activity involving nuclear factor erythroid 2-related factor 2 (Nrf2) activation. However, it remains unclear whether cyclobakuchiols A–C induce anti-influenza A virus activity. In this study, cyclobakuchiols A, B, and C along with cyclobakuchiol D, a new artificial compound derived from cyclobakuchiol B, were synthesized and examined for their anti-influenza A virus activities using Madin-Darby canine kidney cells. As a result, cyclobakuchiols A–D were found to inhibit influenza A viral infection, growth, and the reduction of expression of viral mRNAs and proteins in influenza A virus-infected cells. Additionally, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-β and myxovirus-resistant protein 1. In addition, cyclobakuchiols A–D upregulated the mRNA levels of NAD(P)H quinone oxidoreductase 1, an Nrf2-induced gene, in influenza A virus-infected cells. Notably, cyclobakuchiols A, B, and C, but not D, induced the Nrf2 activation pathway. These findings demonstrate that cyclobakuchiols have anti-influenza viral activity involving host cell oxidative stress response. In addition, our results suggest that the suitably spatial configuration between oxidized isopropyl group and phenol moiety in the structure of cyclobakuchiols is required for their effect.

Human influenza virus infections occur annually worldwide and are associated with high morbidity and mortality. Hence, development of novel anti-influenza drugs is urgently required. Rice Power® extract developed by the Yushin Brewer Co. Ltd. is a novel aqueous extract of rice obtained via saccharization and fermentation with various microorganisms, such as Aspergillus oryzae, yeast [such as Saccharomyces cerevisiae], and lactic acid bacteria, possessing various biological and pharmacological properties. In our previous experimental screening with thirty types of Rice Power® extracts, we observed that the 30th Rice Power® (Y30) extract promoted the survival of influenza A virus-infected Madin-Darby canine kidney (MDCK) cells. Therefore, to identify compounds for the development of novel anti-influenza drugs, we aimed to investigate whether the Y30 extract exhibits anti-influenza A virus activity. In the present study, we demonstrated that the Y30 extract strongly promoted the survival of influenza A H1N1 Puerto Rico 8/34 (A/PR/8/34), California 7/09, or H3N2 Aichi 2/68 (A/Aichi/2/68) viruses-infected MDCK cells and inhibited A/PR/8/34 or A/Aichi/2/68 viruses infection and growth in the co-treatment and pre-infection experiments. The pre-treatment of Y30 extract on MDCK cells did not induce anti-influenza activity in the cell. The Y30 extract did not significantly affect influenza A virus hemagglutination, and neuraminidase and RNA-dependent RNA polymerase activities. Interestingly, the electron microscopy experiment revealed that the Y30 extract disrupts the integrity of influenza A virus particles by permeabilizing the viral membrane envelope, suggesting that Y30 extract has a direct virucidal effect against influenza A virus. Furthermore, we observed that compared to the ethyl acetate (EtOAc) extract, the water extract of Y30 extract considerably promoted the survival of cells infected with A/PR/8/34 virus. These results indicated that more anti-influenza components were present in the water extract of Y30 extract than in the EtOAc extract. Our results highlight the potential of a rice extract fermented with A. oryzae and S. cerevisiae as an anti-influenza medicine and a drug source for the development of anti-influenza compounds.

Recently, opportunistic nosocomial infections caused by Acinetobacter baumannii have become increasingly prevalent worldwide. The pathogen often establishes biofilms that adhere to medical devices, causing chronic infections refractory to antimicrobial therapy. Clinical reports have indicated that some macrolide antibiotics are effective against chronic biofilm-related infections. In this study, we examined the direct anti-biofilm effects of seven macrolides (azithromycin, clarithromycin, erythromycin, josamycin, spiramycin, fidaxomicin, and ivermectin) on A. baumannii using a simple and newly established in vitro assay system for the swift and serial spectrophotometric determinations of two biofilm-amount indexes of viability and biomass. These macrolides were found to possess direct anti-biofilm effects exerting specific anti-biofilm effects not exclusively depending on their bacteriostatic/bactericidal effects. The anti-biofilm effect of azithromycin was found to be the strongest, while those of fidaxomicin and ivermectin were weak and limited. These results provide insights into possible adjunctive chemotherapy with macrolides for A. baumannii infection. Common five macrolides also interfered with the Agrobacterium tumefaciens NTL(pCF218) (pCF372) bioassay system of N-acyl homoserine lactones, providing insights into sample preparation for the bioassay, and putatively suggesting the actions of macrolides as remote signals in bacterial quorum sensing systems.

Histone acetylation plays crucial roles in transcriptional regulation and chromatin organization. Viral RNA of the influenza virus interacts with its nucleoprotein (NP), whose function corresponds to that of eukaryotic histones. NP regulates viral replication and has been shown to undergo acetylation by the cAMP-response element (CRE)–binding protein (CBP) from the host. However, whether NP is the target of other host acetyltransferases is unknown. Here, we show that influenza virus NP undergoes acetylation by the two host acetyltransferases GCN5 and P300/CBP-associated factor (PCAF) and that this modification affects viral polymerase activities. Western blot analysis with anti–acetyl-lysine antibody on cultured A549 human lung adenocarcinoma epithelial cells infected with different influenza virus strains indicated acetylation of the viral NP. A series of biochemical analyses disclosed that the host lysine acetyltransferases GCN5 and PCAF acetylate NP in vitro. MS experiments identified three lysine residues as acetylation targets in the host cells and suggested that Lys-31 and Lys-90 are acetylated by PCAF and GCN5, respectively. RNAi-mediated silencing of GCN5 and PCAF did not change acetylation levels of NP. However, interestingly, viral polymerase activities were increased by the PCAF silencing and were decreased by the GCN5 silencing, suggesting that acetylation of the Lys-31 and Lys-90 residues has opposing effects on viral replication. Our findings suggest that epigenetic control of NP via acetylation by host acetyltransferases contributes to regulation of polymerase activity in the influenza A virus.

The objective of this survey is investigating pharmacists' recognition of and belief in the importance of doing PRE-AVOID using the drug profile book (DPB). The main survey items were: 1. Awareness and self-confidence concerning PRE-AVOID using DPB, 2.Status of educating patients of the merits of the DPB, 3. Status of educating patients of the utilities of the DPB, 4. Status of educating patients of the roles of the DPB, 5. Guidance provided on methods of DPB usage, and 6. Interactions with patients. A multiple regression analysis was performed using "Awareness and self-confidence concerning PRE-AVOID using DPB" as a dependent variable. A regression equation including three items ("Educating the roles", "Blood test", and "Not using multiple DPBs at same time") was derived for "Self-confidence in PRE-AVOID work" (R2=0.20). The item most affecting "Self-confidence in PRE-AVOID work" was "Educating the roles" (R=0.28). A regression equation with "Awareness of PRE-AVOID work" as a dependent variable was not derived. The results of the study suggest that "Educating the roles" or "Not using multiple DPBs at same time" for patients, and asking the results of "Blood test" in a medication consultation may increase pharmacists belief in doing PRE-AVOID.

Background: Community pharmacists should be involved in diabetes care, while there has been less evidence about whether a brief lifestyle intervention is effective for diabetes care in community pharmacies. Objectives: To examine the effects of brief lifestyle intervention on glycemic control in patients with type 2 diabetes mellitus (T2D) by using a coaching style, provided by community pharmacists. Methods: A prospective, cluster-randomized, controlled trial was conducted in 50 groups of community pharmacies in Japan. In all, 132 patients with T2D (age, 20 - 75 years, ≥8.0% of hemoglobin A1c (HbA1c)) were assigned to the intervention group (n = 90) or the usual care group (n = 42). The intervention group (IG) underwent brief lifestyle coaching for self-care of T2D for 6 months. The standard care group (CG) received usual care by pharmacists and was given a general newsletter. The primary outcome was changes in HbA1c levels. Results: After 6 months, the IG had significantly improved HbA1c (IG: -0.6 ± 0.9 vs. CG: -0.2% ± 0.9%; p = 0.021 using the last observation carried forward analysis). Although the number of drugs reduced from 2.3 ± 0.8 to 2.0 ± 1.2 in the IG, the number increased from 2.3 ± 1.1 to 2.5 ± 1.1 in the CG (-0.2 ± 0.9 in IG vs. 0.2 ± 0.6 in CG; p = 0.023). Conclusions: The brief lifestyle intervention by community pharmacists improved glycemic control in patients with T2D. Community pharmacists may more positively participate as lifestyle coaches for diabetes care.

In Japanese pharmacies, Drug Profile Books (DPBs), which are a type of Personal Health Record (PHR), are incorporated in order to prevent duplicate medication and drug interactions in outpatients (patients) through the uniform management of drug administration information. In this study, we tried to clarify the effect on patient safety of brief interventions via DPBs by pharmacists. The study design was a randomized controlled trial on pharmacies as clusters. 65 pharmacies agreed to participate in the study (intervention group (IG): 33; control group (CG): 32). The primary outcomes were: rate of inquiry occurrence, rate of prescription change, and rates of duplicate medications & drug interactions. 56 pharmacies (IG: 29; CG: 27) completed the study. There was a higher tendency for prescription changes in the IG compared to the CG (IG: 0.03%; CG: 0.02%; P = 0.08). In addition, the rate of duplicate medications & drug interactions accounting for the inquiries was significantly higher in the IG than in the CG (IG: 89.2%; CG: 71.9%; P = 0.01). This implied that brief interventions by pharmacists using DPBs had an effect in raising patient safety.

Objectives To demonstrate how the confidence, attitude and job satisfaction of pharmacists who provide interventions and lifestyle support services to patients with hypertension are altered. Methods This study was a before-and-after study that used data from 50 pharmacists who participated in the Community Pharmacists Assist for Blood Pressure (COMPASS-BP) study, which was an interventional study that was conducted in Japan to evaluate the effects of lifestyle support services provided to hypertension patients. The pharmacists received 4 h of training before the study was initiated to learn how to quickly provide patients with guidance about diet and exercise and tobacco and alcohol consumption. During the interventional period, each pharmacist provided concise lifestyle guidance to 1–3 hypertension patients. The pharmacists met with the patients a total of 5 times. The parameters that were assessed were the pharmacists’ attitudes towards and confidence in their lifestyle support services, as well as their level of job satisfaction. We verified the scores of each parameter assessed before and after the study, as well as changes in the relationships among the different parameters. Key findings Significant increases from baseline to the end of the study were observed for Confidence (P < 0.01), Job satisfaction, including ‘Knowledge’ (P < 0.01), ‘Patient counselling management’ (P = 0.04) and ‘Sense of personal growth’ (P < 0.01). Additionally, path analysis results revealed that after the study, the causal relationship between ‘Knowledge’ and ‘Attitude’ disappeared, and a new causal relationship appeared between ‘Patient counselling and management’ and ‘Attitude’. Conclusions Providing lifestyle guidance improved pharmacists’ confidence and attitude. Also, ‘Attitude’ tended to improve as the ‘Patient counselling and management’ score increased.

Adenovirus vectors (Adv) elicit innate immune responses via several pattern-recognition receptors. Although it has been suggested that various Adv-induced mechanisms play important roles in the induction of innate immunity in vitro , the impacts of these mechanisms in vivo remain unclear. Viral nucleic acids elicit innate immune responses through the recognition of cytosolic nucleic acid sensors and transduce intracellular signals to TANK-binding kinase (TBK) 1. In this study, to determine the impacts of viral nucleic acids on innate immune responses in vivo , we administered transgene-expressing Adv to Tbk1 -deficient mice. The systemic Adv administration failed to induce type I interferons (type I IFNs) in the spleen, but not the liver, of Tbk1 -deficient mice, resulting in the increase of transgene-expressing cells in the spleen, but not the liver. Moreover, Adv failed to induce type I IFNs in the bone-marrow-derived dendritic cells, but not the mouse embryonic fibroblasts, from Tbk1 -deficient mice in vitro . These results support the idea that Adv elicit innate immunity in immune cells and non-immune cells in a TBK1-dependent and TBK1-independent manner, respectively.

The COMmunity Pharamcists ASSist for Blood Pressure (COMPASS-BP) study aimed to assess the effectiveness of lifestyle support programs administered in community pharmacies on hypertension control. This open-label, two-armed parallel group, cluster-randomized controlled trial included 73 pharmacies (clusters) in Japan randomized to a control or intervention group. Eligible hypertensive patients (n = 125), aged 20-75 years, received the intervention (n = 64) or the control treatment (n = 61), as dictated by their pharmacy randomization. Patients in the intervention group received brochures and healthy lifestyle advice from pharmacists using motivational interviewing methods during pharmacy visits over a 12-week period, with their usual pharmacy care. Conversely, the control group just received usual care. The main outcome measure was a change in morning systolic blood pressure (SBP) from baseline to week 12. The intervention group exhibited a decrease in morning SBP that was 6.0 mmHg greater than that of the control group (95% confidence interval [CI]: -11.0 to -0.9, p = 0.021). In a mixed-effect model for repeated measures analysis, the intergroup difference in morning SBP decrease was -4.5 mmHg (95% CI: -8.5 to -0.6, p = 0.024). Our findings indicate that implementation of a lifestyle advice program in pharmacies is feasible and may lead to reduced blood pressure.

Few current vaccines can establish antigen (Ag)-specific immune responses in both mucosal and systemic compartments. Therefore, development of vaccines providing defense against diverse infectious agents in both compartments is of high priority in global health. Intramuscular vaccination of an adenovirus vector (Adv) has been shown to induce Ag-specific cytotoxic T lymphocytes (CTLs) in both systemic and gut-mucosal compartments. We previously found that type I interferon (IFN) signaling is required for induction of gut-mucosal, but not systemic, CTLs following vaccination; however, the molecular mechanism involving type I IFN signaling remains unknown. Here, we found that T helper 17 (Th17)-polarizing cytokine expression was down-regulated in the inguinal lymph nodes (iLNs) of Ifnar2-/- mice, resulting in the reduction of Ag-specific Th17 cells in the iLNs and gut mucosa of the mice. We also found that prior transfer of Th17 cells reversed the decrease in the number of Ag-specific gut-mucosal CTLs in Ifnar2-/- mice following Adv vaccination. Additionally, prior transfer of Th17 cells into wild-type mice enhanced the induction of Ag-specific CTLs in the gut mucosa, but not in systemic compartments, suggesting a gut mucosa-specific mechanism where Th17 cells regulate the magnitude of vaccine-elicited Ag-specific CTL responses. These data suggest that Th17 cells translate systemic type I IFN signaling into a gut-mucosal CTL response following vaccination, which could promote the development of promising Adv vaccines capable of establishing both systemic and gut-mucosal protective immunity.

Awa-ban tea is a uniquely flavored, pickled and anaerobically fermented tea found only in the Tokushima prefecture in the Shikoku region of Japan. Diabetes is a major health problem worldwide. Here, we report that Awa-ban tea suppressed the increase in blood glucose levels after oral administration of maltose, sucrose, or glucose in mice (n = 9–54). Awa-ban tea suppressed the area under the curve (AUC) of blood glucose by 72–83%. Thus, we propose that Awa-ban tea can be used as an alternative functional beverage for diabetes prevention.

Few of the vaccines in current use can induce antigen- (Ag-) specific immunity in both mucosal and systemic compartments. Hence, the development of vaccines that realize both mucosal and systemic protection against various pathogens is a high priority in global health. Recently, it has been reported that intramuscular (i.m.) vaccination of an adenovirus vector (Adv) can induce Ag-specific cytotoxic T lymphocytes (CTLs) in both systemic and gut mucosal compartments. We previously revealed that type I IFN signaling is required for the induction of gut mucosal CTLs, not systemic CTLs. However, the molecular mechanism via type I IFN signaling is largely unknown. Here, we report that type I IFN signaling following i.m. Adv vaccination is required for the expression of type I IFN in the inguinal lymph nodes (iLNs), which are the draining lymph nodes of the administration site. We also showed that the type I IFN signaling is indispensable for the early activation of CTLs in iLNs. These data suggested that type I IFN signaling has an important role in the translation of systemic innate immune response into mucosal adaptive immunity by amplifying the innate immune signaling and activating CTLs in the iLN.

The varicella-zoster virus (VZV) Oka vaccine strain (vOka) is a highly immunogenic and safe live vaccine that has long been used worldwide. Because its genome is large, making it suitable for inserting foreign genes, vOka is considered a candidate vector for novel polyvalent vaccines. Previously, a recombinant vOka, rvOka-HN, that expresses mumps virus (MuV) hemagglutinin-neuraminidase (HN) was generated by the present team. rvOka-HN induces production of neutralizing antibodies against MuV in guinea pigs. MuV also expresses fusion (F) protein, which is important for inducing neutralizing antibodies, in its viral envelope. To induce a more robust immune response against MuV than that obtained with rvOka-HN, here an rvOka expressing both HN and F (rvOka-HN-F) was generated. However, co-expression of HN and F caused the infected cells to form syncytia, which reduced virus titers. To reduce the amount of cell fusion, an rvOka expressing HN and a mutant F, F(S195Y) were generated. Almost no syncytia formed among the rvOka-HN-F(S195Y)-infected cells and the growth of rvOka-HN-F(S195Y) was similar to that of the original vOka clone. Moreover, replacement of serine 195 with tyrosine had no effect on the immunogenicity of F in mice and guinea pigs. Although obvious augmentation of neutralizing antibody production was not observed after adding F protein to vOka-HN, the anti-F antibodies did have neutralizing activity. These data suggest that F protein contributes to induction of immune protection against MuV. Therefore this recombinant virus is a promising candidate vaccine for polyvalent protection against both VZV and MuV.

This survey aimed to examine how patient-pharmacist communication using the drug profile book relates patient's behavior regarding its use. Among patients who visited one of the five pharmacies during the 4 months between July and October of 2013, 245 patients who had been prescribed antihypertensives were asked to complete a questionnaire. Items included patient attributes, whether the patient thought the drug profile book was useful to them ("sense of utility"), whether the patient has ever been questioned by a pharmacist while showing the drug profile book ("experience of being questioned by a pharmacist while showing the drug profile book"), and whether the patient has ever shown the drug profile book to the physician ("experience of showing the drug profile book to the physician"). In addition, pharmacists counted the frequency of patients bringing the drug profile book, and if so, the frequency of the sticker affix during the last 5 visits. 34.3% of responding patients answered that they had the "experience of being questioned while showing the drug profile book". Response rates of "frequency of bringing the drug profile book", "sense of utility", and "experience of showing the drug profile book to the physician" in the group with "experience of being questioned while showing the drug profile book" were significantly higher than those in the group without such experience. This survey indicated that experience of being questioned by a pharmacist while showing the drug profile book related patient's behavior regarding its use.

To find a novel influenza inhibitor targeting the endonuclease activity of influenza A virus polymerase acidic protein (PA), which is essential for the acquisition of primers for viral mRNA transcription, seven Kampo extracts were tested in vitro for their ability to inhibit endonuclease activity of the recombinant PA protein that was expressed and purified from Escherichia coli. The Kampo medicines Kakkonto, Shosaikoto, Saikokeishito, Keishito, Maobushisaishinto, and Maoto, but not Chikujountanto, inhibited PA endonuclease activity in a dose-dependent manner. Our results indicate that Kampo medicines are good sources providing a structural lead for optimization of an influenza endonuclease inhibitor.

Background: Novel therapeutic approaches against influenza are required. Bakuchiol is a phenolic isoprenoid found in Babchi seeds. Results: Bakuchiol enantiomer-selectively inhibited influenza A viral infection and growth and activated the Nrf2 pathway. Conclusion: Bakuchiol showed novel enantiomer-selective anti-influenza viral activity. Significance: The study of bakuchiol will contribute to the development of novel approaches to influenza therapy., Influenza represents a substantial threat to human health and requires novel therapeutic approaches. Bakuchiol is a phenolic isoprenoid compound present in Babchi (Psoralea corylifolia L.) seeds. We examined the anti-influenza viral activity of synthetic bakuchiol using Madin-Darby canine kidney cells. We found that the naturally occurring form, (+)-(S)-bakuchiol, and its enantiomer, (−)-(R)-bakuchiol, inhibited influenza A viral infection and growth and reduced the expression of viral mRNAs and proteins in these cells. Furthermore, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-β and myxovirus-resistant protein 1. Interestingly, (+)-(S)-bakuchiol had greater efficacy than (−)-(R)-bakuchiol, indicating that chirality influenced anti-influenza virus activity. In vitro studies indicated that bakuchiol did not strongly inhibit the activities of influenza surface proteins or the M2 ion channel, expressed in Chinese hamster ovary cells. Analysis of luciferase reporter assay data unexpectedly indicated that bakuchiol may induce some host cell factor(s) that inhibited firefly and Renilla luciferases. Next generation sequencing and KeyMolnet analysis of influenza A virus-infected and non-infected cells exposed to bakuchiol revealed activation of transcriptional regulation by nuclear factor erythroid 2-related factor (Nrf), and an Nrf2 reporter assay showed that (+)-(S)-bakuchiol activated Nrf2. Additionally, (+)-(S)-bakuchiol up-regulated the mRNA levels of two Nrf2-induced genes, NAD(P)H quinone oxidoreductase 1 and glutathione S-transferase A3. These findings demonstrated that bakuchiol had enantiomer-selective anti-influenza viral activity involving a novel effect on the host cell oxidative stress response.

Background: To contribute to the development of novel anti-influenza drugs, we investigated the anti-influenza activity of crude extracts from 118 medicinal plants collected in Myanmar. We discovered that extract from the stems of Jatropha multifida Linn. showed anti-influenza activity. J. multifida has been used in traditional medicine for the treatment of various diseases, and the stem has been reported to possess antimicrobial, antimalarial, and antitumor activities. However, the anti-influenza activity of this extract has not yet been investigated. Methods: We prepared water (H2O), ethyl acetate (EtOAc), n-hexane (Hex), and chloroform (CHCl3) extracts from the stems of J. multifida collected in Myanmar, and examined the survival of Madin-Darby canine kidney (MDCK) cells infected with the influenza A (H1N1) virus, and the inhibitory effects of these crude extracts on influenza A viral infection and growth in MDCK cells. Results: The H2O extracts from the stems of J. multifida promoted the survival of MDCK cells infected with the influenza A H1N1 virus. The EtOAc and CHCl3 extracts resulted in similar, but weaker, effects. The H2O, EtOAc, and CHCl3 extracts from the stems of J. multifida inhibited influenza A virus H1N1 infection; the H2O extract possessed the strongest inhibitory effect on influenza infection in MDCK cells. The EtOAc, Hex, and CHCl3 extracts all inhibited the growth of influenza A H1N1 virus, and the CHCl3 extract demonstrated the strongest activity in MDCK cells. Conclusion: The H2O or CHCl3 extracts from the stems of J. multifida collected in Myanmar demonstrated the strongest inhibition of influenza A H1N1 viral infection or growth in MDCK cells, respectively. These results indicated that the stems of J. multifida could be regarded as an anti-influenza herbal medicine as well as a potential crude drug source for the development of anti-influenza compounds.

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Adenovirus vector (Adv) vaccination at a systemic site, such as intramuscular (i.m.) immunization, can induce antigen-specific CD8(+) T cell responses in both systemic and mucosal compartments. It remains unclear, however, how antigen-specific CD8(+) T cell response is induced in the mucosa. In this study, we found that type-I IFN signaling is required for the induction of mRNA expression of retinal dehydrogenase in the draining lymph nodes following the i.m. Adv vaccination. We show that type-I IFN signaling is required for the induction of antigen-specific CD8(+) T cell response in the gut-mucosal compartment following the i.m. Adv vaccination.

Adenovirus vector-based vaccine is a promising approach to protect HIV infection. However, a recent phase IIb clinical trial using the vector did not show its protective efficacy against HIV infection. To improve the vaccine, we explored the transgene protein expression and its immunogenicity using optimized codon usage, promoters and adaptors. We compared protein expression and immunogenicity of adenovirus vector vaccines carrying native or codon usage-optimized HIV-1 clade C gag and env genes expression cassettes driven by different promoters (CMV, CMVi, and CA promoters) and adapters (IRES and F2A). The adenovirus vector vaccine containing optimized gag gene produced higher Gag protein expression and induced higher immune responses than the vector containing native gag gene in mice. Furthermore, CA promoter generated higher transgene expression and elicited higher immune responses than other two popularly used promoters (CMV and CMVi). The second gene expression using F2A adaptor resulted in higher protein expression and immunity than that of using IRES and direct fusion protein. Taken together, the adenovirus vector containing the expression cassette with CA promoter, optimized HIV-1 clade C gene and an F2A adaptor produced the best protein expression and elicited the highest transgene-specific immune responses. This finding would be promising for vaccine design and gene therapy.

The H1N1 influenza A virus, which originated in swine, caused a global pandemic in 2009, and the highly pathogenic H5N1 avian influenza virus has also caused epidemics in Southeast Asia in recent years. Thus, the threat from influenza A remains a serious global health issue, and novel drugs that target these viruses are highly desirable. Influenza A RNA polymerase consists of the PA, PB1, and PB2 subunits, and the N-terminal domain of the PA subunit demonstrates endonuclease activity. Fullerene (C60) is a unique carbon molecule that forms a sphere. To identify potential new anti-influenza compounds, we screened 12 fullerene derivatives using an in vitro PA endonuclease inhibition assay. We identified 8 fullerene derivatives that inhibited the endonuclease activity of the PA N-terminal domain or full-length PA protein in vitro. We also performed in silico docking simulation analysis of the C60 fullerene and PA endonuclease, which suggested that fullerenes can bind to the active pocket of PA endonuclease. In a cell culture system, we found that several fullerene derivatives inhibit influenza A viral infection and the expression of influenza A nucleoprotein and nonstructural protein 1. These results indicate that fullerene derivatives are possible candidates for the development of novel anti-influenza drugs.

Streptomyces carzinostaticus produces the antitumor protein, NCS, and at the same time a related protein which antagonizes the antimicrobial effect of NCS. This has been designated pre-neocarzinostatin (pre-NCS). The physicochemical properties of pre-NCS are very close to those of NCS, except for a difference in isoelectric point. Production of pre-NCS in the culture filtrate precedes production of NCS. Pre-NCS has no antimicrobial and antitumor effect but antagonizes NCS activity.Pre-NCS, when given 2 hours prior to NCS, markedly diminishes the NCS-induced inhibition of Sarcina lutea growth and DNA synthesis in HeLa cells. However, the antagonistic action of pre-NCS is lost when pre-NCS is washed out from HeLa cell culture after 2 hours incubation. When pre-NCS was given intraperitoneally to mice bearing Sarcoma 180 ascites tumor, subsequent treatment with NCS failed to inhibit the tumor growth. On the other hand, the acute toxicity of NCS given intravenously was not abolished by preceding intravenous injection of pre-NCS.The acute toxicity of pre-NCS by intravenous injection was greater than 100mg/kg in mice.