The insula and frontal operculum (primary taste area) are also involved in reward processing. The aim was to compare human gustatory evoked potentials (GEP) recorded in the primary and secondary gustatory cortices in a fasted state with those after food intake. Fifteen healthy volunteers were enrolled in this observational study. In each of two sessions, two GEP recordings were performed (at 11:00 am and 1:30 pm) in response to sucrose gustatory stimulation, and a sucrose-gustatory threshold was determined. During one session, a standard lunch was provided between the two GEP recordings. During the other session, subjects had nothing to eat. Hunger sensation, wanting, liking, and the perception of the solution's intensity were evaluated with visual analog scales. GEP latencies measured in the Pz (p < 0.001), Cz (p < 0.01), Fz (p < 0.001) recordings (primary taste area) were longer after lunch than in the pre-prandial condition. Fp1 and Fp2 latencies (secondary taste area) tended to be longer after lunch, but the difference was not significant. No difference was observed for the sucrose-gustatory threshold regardless of the session and time. Modifications in the primary taste area activity during the post-prandial period occurred regardless of the nature of the food eaten and could represent the activity of the frontal operculum and insula, which was recently shown to be modulated by gut signals  through vagal afferent neurons or metabolic changes of the internal milieu after nutrient absorption. This trial was registered at clinicalstrials.gov as NCT02472444.Previous functional Magnetic Resonance Imaging studies evaluated the role of satiety on cortical taste area activity and highlighted decreased activation in the orbito-frontal cortex when food was eaten until satiation. The modulation of orbito-frontal neurons (secondary taste area) by  Neuronal recording using electrophysiology in rodents and primates and functional neuroimaging in humans have been conducted to assess how food intake modulates brain activation. Earlier studies showed decreased activation following the consumption of food eaten to satiation Rolls, , 2015. TIn a different way, it has been observed that responses in the primary gustatory cortex (insula and opercular cortex) correlated with the subjective intensity, temperature, viscosity, and fat texture of foods . There were no significant gender differences for these parameters. All were non-smokers, and none had dental or neurological problems, or any remarkable medical history. We excluded subjects under current medical treatment, or who were overweight (BMI > 25 kg/m2) or underweight (BMI < 19 kg/m2).Fifteen healthy volunteers  were enrolled in this observational study. The mean age was 28 ± 7 years old (range: 22–35 years old), and the mean BMI was 22 ± 3 kg.mThe subjects agreed to participate with written consent after being informed about the nature and aims of the experiments. The study was approved by the regional Ethics Committee of Burgundy, France, in accordance with the latest revision of the Declaration of Helsinki and European Law (ISO EN 14155). This trial was registered at clinicalstrials.gov as NCT02472444.The taste delivery system is described in Figure 2, tested just after stimulation of the tongue by a methylene blue solution in a preliminary work). The same oral receptive fields were therefore activated in each subject. The flow rate of 100 mL/h of water was chosen because it did not induce somatosensory differences compared with the flow rate of 200 mL/h, and it meant that participants did not have swallow too often, which could lead to artifacts on the GEP recording. This was tested in a preliminary study and verified in the control recordings (stimulation by water through the two tubes). Air was purged from the taste delivery system to avoid a feeling of air on the tongue.Each subject put the two parallel tubes on the middle of his/her tongue in his/her mouth. The extremity of the semi-rigid tubes  was placed at 1.5 ± 0.5 cm from the dental arch on the midline of the tongue (same distance for each subject). Due to their rigidity, the tubes could not deviate from this position. Solutions were delivered to the tongue through a hole at the extremity of each tube. A taste solution was intermittently delivered through the first tube (flow rate = 200 mL/h). During the period without the taste solution, water was continuously delivered through the second tube (flow = 100 mL/h) to minimize the likelihood that subjects would feel different sensations between injections from the two tubes. The flow rate of 200 mL/h of taste solution was chosen because it allowed the uniform stimulation of a large lingual surface . The subjects also had to close their eyes to avoid light stimulation. Hence, no blink artifacts contaminated the recordings. Participants were instructed to swallow after receiving the taste stimulus. Control GEP recordings showed that swallowing or mouth movement did not lead to frontal electrode artifacts.The tasting stimulus was a solution of sucrose applied in one concentration: 10 g per 100 mL of water .ad libitum until satiation. No dessert was served to avoid sweet food before a sucrose GEP recording. The food was eaten in 15–20 min. In the fasting session, no lunch was served between the two GEP recordings. The two sessions were randomly assigned.Subjects were investigated in two parallel sessions separated by at least 1 day. Subjects were asked to eat their usual breakfast before 8:00 am and not to eat or drink anything except water until the first GEP recording. During one session, each subject had two GEP recordings, the first at 11:00 am, and the second at 1:30 pm Figure . In the Before each GEP recording, subjects were asked to rate their level of hunger and desire to eat (wanting) using a 10-cm visual analog scale Figure . After eElectroencephalographic (EEG) signals were recorded according to the international 10–20 system, using a conventional EEG recording. Nine sites were recorded for measurements in the primary gustatory cortex Pz, Cz, C3, C4, Fz), and the secondary gustatory cortex  with the following data: time constant 1 s, sampling frequency 2048 Hz, 200 Hz low-pass filter, 0.4 Hz high-pass filter, 50 Hz filter. Recordings were additionally filtered offline. GEPs were averaged after the recording for each session , amplitude (μV), and duration (ms) of the GEPs were noted for each recorded electrode. The initial latency was defined as the time between stimulus delivery and the onset of the increase in potential. The amplitude of each response was calculated from positive to negative peaks. The duration of the GEPs was calculated between the end and the beginning  of the GEP. The positive peak corresponded to the peak pointing down whereas the negative peak corresponded to the peak pointing up. GEP was defined by three peaks, as described in previous studies  spaced by 0.25 log units . The procedure was stopped when the participant correctly identified the stimulus sample at a given concentration three consecutive times. This concentration was called the sucrose-gustatory threshold for the individual participant.p-value below 0.01 was considered statistically significant .Initial latency, amplitude and duration of GEPs  were analyzed using means and standard errors of the mean (SEM). Mean differences in GEP latency, amplitude, and duration between data obtained in the morning and afternoon recordings, the results from the visual analog scales  and sucrose-gustatory thresholds were compared between the feeding and fasting sessions. The analyses were performed using a repeated measures ANOVA (with a single factor: feeding or fasting session). A SAS 9.2  was used for all analyses.The sucrose-gustatory threshold was similar regardless of the prandial states in all of the participants. It ranged from 0.0609 to 0.3423 g/100 mL of water. The median sucrose-gustatory threshold was 0.1925 g/100 mL of water for both the prandial states.p < 0.001) and significantly decreased after food intake (p < 0.001).The sensation of hunger and wanting significantly increased with fasting  is shown in Figure There was no statistical difference between male and female participants for GEP parameters.p < 0.001), Cz (p < 0.001), and Fz (p < 0.01) electrodes. No significant change was observed in the secondary gustatory area, although there was a slight trend toward longer latencies in Fp1 and Fp2.In the fasting sessions, the GEP latencies were not significantly different between the pre- and post-prandial conditions. However, in the feeding sessions, they were longer after food intake than before. These changes were significant in the Pz  for GEP amplitude and duration.This study highlighted the change in the primary gustatory area 1 h after food intake compared with the fasting state: GEP latencies in response to sucrose stimulation lengthened after lunch even though the meal did not contain sweet food. A similar, though non-significant trend, was present in the secondary taste cortex. In contrast, no decrease in GEP amplitudes was noted according to the prandial state.ad libitum food intake when compare with data in the literature. In fact, neurophysiological recordings in monkeys and rodents and functional neuroimaging studies in humans showed decreased activation of the secondary taste cortex. The neuronal cortical activity was found to be modified in the orbitofrontal cortex  participate in the perceived pleasantness of taste  were significant. This result demonstrates that activity in the primary taste area changes after food intake regardless of the food eaten. These neuronal changes could be due to modifications of the internal state and gut stimulation after food intake and could partly explain the mechanism related to negative alliesthesia. Further studies are needed to determine the mechanisms underlying these modifications.AJ, CL, LP, and LB designed the study. AJ, SG, TM, and SM conducted the research. VG provided essential materials necessary for the research. AJ performed statistical analyses. AJ, CL, LP, and LB analyzed data. AJ wrote the paper. AJ had primary responsibility for final content. All the authors reviewed and approved the final manuscript.This work was supported by CNRS, INRA, the University of Bourgogne Franche-Comté, the FEDER and the Dijon Association for Neurosciences (ADSNB), France.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to determine whether metabolic syndrome (MetS) or chronic kidney disease (CKD) is associated with hearing thresholds in the general Korean population.2 or a dipstick proteinuria result of ≥1+. The hearing thresholds were measured at 0.5, 1, 2, 3, 4, and 6 kHz. Low-frequency (Freq) was defined as pure-tone averages at 0.5 and 1 kHz, while Mid-Freq and High-Freq were defined as the average thresholds at mid-frequency (2 and 3 kHz) and high frequency (4 and 6 kHz), respectively.A total of 16,554 participants were included in this study. MetS was defined using the National Cholesterol Education Program Adult Treatment Panel III guidelines, and CKD was defined as an estimated glomerular filtration rate <60 mL/min/1.73 mIn men, the hearing thresholds were 15.1 ± 14.5 dB, 22.2 ± 21.3 dB, and 37.3 ± 26.5 dB for Low-, Mid-, and High-Freq, respectively. In women, the hearing thresholds were 14.9 ± 15.3 dB, 16.6 ± 18.0 dB, and 26.1 ± 21.5 dB for Low-, Mid-, and High-Freq, respectively. The hearing thresholds for men were significantly higher than the hearing thresholds for women in all 3 threshold categories. Male and female subjects with MetS or CKD had higher hearing thresholds than the subjects that did not have these disorders. In the multivariate analysis, MetS was associated with increased hearing thresholds in women, and CKD was associated with increased hearing thresholds in men and women.MetS is associated with hearing thresholds in women, and CKD is associated with hearing thresholds in men and women. Therefore, patients with MetS or CKD should be closely monitored for hearing impairment. Hearing loss significantly affects quality of life . In a reThe incidence of metabolic syndrome (MetS) and chronic kidney disease (CKD) has increased significantly over the last decade, and it is expected to continue rising –8. MetS Several studies have reported an association between each component of the cardiometabolic risk factors and hearing loss –21. HoweData from the Korean National Health and Nutrition Examination Survey (KNHANES) 2009–2012 were used for the analyses. KNHANES is a nationwide, multi-stage stratified survey of a representative sample of the entire South Korean population that is conducted by the Korea Centers for Disease Control and Prevention. The total number of participants in KNHANES was 36,067. Participants were excluded from the present study based on lack of data regarding renal function  or hearing thresholds , external or middle ear disease (n = 836) or brain disorders such as stroke , or age <18 years old . As a result, 16,554 participants were included in this study. This study design was approved by the institutional review board of Yeungnam University Hospital (YUH-14–0487-O71). The board waived the need for informed consent, as the subjects’ records and information were anonymized and de-identified prior to the analysis.2), waist circumference (cm), serum creatinine levels (mg/dL), systolic blood pressure , diastolic blood pressure , fasting glucose levels (mg/dL), total cholesterol levels (mg/dL), high-density lipoprotein (HDL) cholesterol levels (mg/dL), triglyceride levels (mg/dL), smoking behavior, alcohol consumption, and estimated glomerular filtration rate .Clinical and laboratory data were collected from the subjects during the health examinations, including age, sex, body mass index  was calculated using the following equation:DM was defined as a self-reported diagnosis of DM or a fasting glucose level of ≥126 mg/dL. HTN was defined as a SBP of ≥140 mmHg, a DBP of ≥90 mmHg, a self-reported history of HTN, or the use of anti-hypertension medication. BMI was calculated as weight divided by height squared, and MetS was defined using the National Cholesterol Education Program Adult Treatment Panel III guidelines . Serum cP < 0.001). Low-Freq was defined as pure-tone averages at 0.5 and 1 kHz, and Mid-Freq and High-Freq were defined as average thresholds at mid-frequency (2 and 3 kHz) and high frequency (4 and 6 kHz), respectively.Ear examinations and pure-tone audiometry were performed as previously described . None oft tests or one-way analysis of variance, followed by a post-hoc Tukey comparison. Categorical variables were expressed as counts and percentages, and were compared using the Pearson χ2 test or Fisher exact test. Correlation analysis was performed to assess the strength of the relationship between continuous variables. Multivariate analyses using analyses of covariance or multiple regression analyses were used to determine the independent predictors of hearing thresholds. For MetS, the covariates were age, smoking, alcohol consumption, BMI, exposure to explosive noise, and exposure to occupational noise. For CKD, the covariates were age, smoking, alcohol consumption, BMI, DM, HTN, exposure to explosive noise, and exposure to occupational noise. The data for the multivariate analyses were expressed as mean ± standard error, and the level of statistical significance was set at P < 0.05.The data were analyzed using SPSS version 19 . Continuous variables were expressed as mean ± standard deviation, and were compared using P < 0.001).The mean age was 50.4 ± 16.6 years for men  and 49.2 ± 16.4 years for women  . BMI, waMale and female subjects with MetS had higher hearing thresholds than subjects without MetS . In men,P < 0.001) (P < 0.001)  . Similar< 0.001) . Among t< 0.001) . SubjectHOMA-IR was 2.58 ± 1.74 in men who were <65 years old, 2.60 ± 1.73 in men who were ≥65 years old, 2.33 ± 1.30 in women who were <65 years old, and 2.87 ± 1.90 in women who were ≥65 years old. There was no significant difference in the men’s HOMA-IR for each age group, the HOMA-IR results for elderly women were significantly higher than those for men or younger women. A significant correlation between HOMA-IR and the hearing thresholds was only observed in women.Previous studies have shown that men have poorer hearing thresholds compared to women and that the left ear has a consistently worse hearing threshold than the right ear . Thus, wMetS is a well-known condition that is linked to the development of various complications, such as DM, HTN, dyslipidemia, and cardiovascular disease ,6,17. InIn the present study, there was a significant association between MetS or HOMA-IR and hearing thresholds in women. MetS is a cause and result of insulin resistance, while HOMA-IR is a well-known index of insulin resistance. Our results revealed an association between these markers for insulin resistance and hearing thresholds in women, although not in men. While the cause for this gender-related difference is not yet clear, changes in estrogen and adiponectin levels may be relevant. For example, estrogen plays a major role in the auditory system, and baseline adiponectin levels are higher in women than in men ,32. Howe+, K+-ATPase); these mechanism are sensitive to blood pressure, blood supply, and vasoconstriction [Although the underlying mechanisms for the association between CKD and hearing are not clear, uremic neuropathy and fluid or electrolyte abnormalities are two possible causes ,23,33. Ctriction –39. Thertriction –42. Vilatriction . Althoug2 or a dipstick proteinuria result of ≥1+, and creatinine and proteinuria were evaluated using a single measurement. Unfortunately, repeated measurements are needed for a definite diagnosis, and these measurements were not performed in the present study. However, many studies in large-scale populations have analyzed results from a single measurement for proteinuria [The present study is the largest cross-sectional analysis to demonstrate the association between hearing threshold and CKD in an Asian population. However, it also has a number of limitations. First, the cross-sectional study design prevented us from evaluating the changes in hearing levels. Therefore, prospective analysis is needed to confirm whether there is a casual relationship between changes in renal function and hearing thresholds. Second, this study did not include an analysis of causative markers, such as adipokines, oxidative stress, or vasculopathy, which are known to be associated with the development of hearing impairment in MetS or CKD. Third, this study did not evaluate any sensitive components of hearing difficulties, such as speech discrimination. Fourth, CKD was defined as an eGFR of <60 mL/min/1.73 mteinuria –48. We bIn conclusion, MetS is associated with hearing thresholds in women, and CKD is associated with hearing thresholds in men and women. Therefore, patients with MetS or CKD should be closely monitored for hearing impairment.


In order to make objective and intelligent decisions regarding caloric intake for fat loss/physique enhancement, one’s resting metabolic rate should be measured. It is important that the device used to measure resting metabolic rate be valid and reliable. Therefore, the purpose of this study was to establish the inter- and intra-day test-retest reliability of the Cosmed Fitmate ProTM indirect calorimeter for resting metabolic rate (RMR).34 participants  volunteered to have their RMR measured a total of three times on two different days. On day 1, two 15-minute RMR tests were completed in back to back fashion. On day 2 (occurring within 7 days of day 1), another 15-minute RMR test was conducted. All RMR tests were conducted after an overnight fast in a rested condition. Reliability of RMR was evaluated using Pearson’s correlation, intraclass correlation coefficients (ICC), standard error of the measurement (SEM), and standard error of the measurement as a percentage of the mean (SEM%). Systematic error was examined using a one-way repeated measures ANOVA. Data was analyzed using SPSS version 22.0. Consent to publish the results was obtained from all participants.Average RMR values for each test are shown in Table The Cosmed Fitmate ProTM indirect calorimeter is a reliable instrument for measuring resting metabolic rate in non-overweight males and females. The instrument demonstrated test-retest reliability both within the day and between two different days. Relative consistency was acceptable with ICC values of 0.981 and 0.946 for intra and inter-day measures, respectively. Absolute consistency was also acceptable with SEM values (expressed as a percentage of the mean) of 4.13% and 6.89% for intra and inter-day measures, respectively. Further, the ANOVA results indicated no systematic error among tests and days.


In addition, a few studies have demonstrated that rare occurrence of colorectal liver metastases among patients with fatty liver, cirrhosis or chronic hepatitis B and C virus infection. We performed a meta-analysis to verify the association between the incidences of colorectal liver metastases with chronically diseased livers.Relevant studies were identified by a search of electronic database PubMed, Cochrane Library, OVID, Web of Science and CNKI . Pooled odds ratio (OR) with 95% confidence interval (CI) was calculated using random- or fixed-effect models when appropriate. Meta-analysis and publication bias (Bgger's test) was evaluated with STATA 12.0.P = 0.000 fixed-effects model). The result of Begg's test  revealed no publication bias.A total of 10,349 colorectal cancer patients from 10 studies were included. The meta-analysis result showed there was a significant difference in the incidences of colorectal liver metastases between patients with normal and chronically diseased livers (OR = 0.32; 95% CI 95%: 0.26–0.38, The results of this meta-analysis demonstrated that patients with chronically diseased livers had significantly lower incidences of colorectal liver metastases than those with normal livers. Colorectal cancer (CRC) is a high morbidity malignancy tumor over the world, which is predicted will account for 8% of new cancer cases in the United States in 2014 During the past twenty years, several studies have discovered a low incidence of colorectal liver metastases in patients with liver diseases, including fatty liver We collected the potentially relevant studies through a search in electronic database PubMed, Cochrane Library, OVID, Web of Science and Chinese National Knowledge Infrastructure (CNKI). Chronically diseased liver included cirrhosis, fatty liver and hepatitis virus infection. The keywords used for the search including “colorectal cancer”, “diseased liver”, “hepatitis”, “fatty liver”, “cirrhosis” and “liver metastasis”. All non-English articles were translated in English and then analyzed. The latest search was updated on February 24, 2014.Inclusion criteria: (1) studies evaluating the association between colorectal liver metastases and chronically diseased livers; (2) only patients with advanced colorectal cancer were included in the study, because early-staged colorectal cancers such as a lesion confined to the mucosa or submucosa rarely metastasized to the liver; (3) all patients were closely followed up; (4) containing useful figures including numbers of the two groups  and numbers of liver metastases in each group. Exclusion criteria: (1) animal studies, pharmaceutical researches, case reports; (2) reports without usable data; (3) duplicate publications.Two investigators (CB and LK) extracted data from eligible studies independently, according to the inclusion and exclusion criteria above. For disagreements, a consensus was reached by a third investigator (ZS). The following information was collected from each study: first author, publication date, country of origin, ethnicity, type of liver diseases, number of each group , and number of liver metastases in each group.2I statistics were used. For the Q test, A P value less than 0.05 indicated significant heterogeneity; for the 2I statistics, an 2I value greater than 50% was considered severe heterogeneity. The potential publication bias was assessed using a “funnel plot” and the Begg's test. The fixed-effects model was adopted in the initial calculation of odds ratio with corresponding 95% CIs. If there was a significant statistical heterogeneity among the studies, the random-effects model was applied for the analysis. By comparing the incidences of colorectal liver metastases in normal and chronically diseased livers, we tried to explore the impact of liver diseases on colorectal liver metastases.Meta-analysis was performed by using Stata 12.0 software . To determine the statistical heterogeneity of the studies, Chi-square-based Q test and A total of 10 retrospective studies published between 1992 and 2013 were identified as eligible according to the selection criteria 2I = 37.4% P>0.05), and then the fixed-effects model was adopted. The odds ratio, expressed as diseased liver group versus normal liver group, was 0.32 . Through comparing the incidences of colorectal liver metastases between the diseased liver group and normal liver group, we found there was a significant difference in the incidences of colorectal liver metastases between the two groups. This result demonstrated that patients with chronically diseased livers had significantly lower incidences of colorectal liver metastases than those with normal livers.As P>0.05).This meta-analysis showed that colorectal cancer patients with chronically diseased livers had significantly lower incidences of colorectal liver metastases than those with normal livers. The reason for this phenomenon is still unknown. Studies have shown that clinicopathological characteristics of primary colorectal cancers were not altered by viral hepatitis Karube Cirrhosis, as defined by the World Health Organization (WHO), was “a diffuse process characterized by fibrosis and conversion of normal liver architecture into structurally abnormal nodules” Seitz It has been reported that hepatitis virus infection resulted in a high state of the immune response in livers The phenomenon of the rare occurrence of metastatic colorectal cancers in chronically diseased livers has been observed for more than 70 years, but few related articles had been reported. This meta-analysis collected all related reports so far, and verified that patients with diseased livers have significantly lower incidences of colorectal liver metastases than those with normal livers. Nevertheless, it is still necessary to conduct larger size and better design studies to confirm our results. Moreover, of the ten included studies, nine studies evaluated patients from Asia. Because of this, our finding may just represent cancer patients from Asia. In addition, we suggest particular attention should be given to the precise mechanism of this phenomenon, so as to provide a research strategy on basic research and clinical prevention of colorectal liver metastases.Checklist S1PRISMA Checklist.(PDF)Click here for additional data file.


Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP.Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV  Influenza A virus nucleoprotein (NP) is a highly conserved multifunctional protein that plays an essential role in infection by all subtypes of influenza A virus, making it an attractive target for new antiviral drugs. NP regulates viral polymerase activity and transport of the viral genome into/from the host cell nucleus by forming the viral ribonucleoprotein complex (vRNP). Because NP regulates replication and transcription of the viral genome in addition to its role in nuclear export , it is a promising drug target. Here, we used the antiviral compound RK424 to identify a novel pocket structure within NP. This structure encompassed three different functional domains that are involved in the above-mentioned replication steps. RK424 inhibits viral genome replication/transcription and nuclear export of NP by destabilizing the NP oligomer and inhibiting the binding of chromosome region maintenance 1 (CRM1) to NP via nuclear export signal (NES) 3, which is located in close proximity to the NP pocket. Taken together, these findings suggest that this small NP pocket is a novel antiviral target. Influenza A virus (IAV) causes periodic and widespread epidemics or pandemics, which take the form of respiratory diseases with cold-like symptoms; however, the virus can sometimes cause serious disease with high mortality rates . One of Recently, the viral nucleoprotein (NP) has attracted interest as a target for new antiviral drugs. NP is the major component of the viral ribonucleoprotein complex (vRNP), which comprises three polymerase subunit proteins  and viral RNA (vRNA) . The vRNin vitro and in vivo . Th. Thin viin vitro and in vivo.Taken together, these data suggest that RK424 has antiviral activity both In contrast to treatment with oseltamivir phosphate , treatment with RK424 led to a reduction in the number, but not the size, of viral plaques  and it win situ hybridization (FISH) analysis with 48 single Quasar 670-labeled DNA oligos against vRNA encoding the PB2 subunit [To confirm the inhibitory effect of RK424 on viral genome replication in infected cells, we next performed single-molecule fluorescence  subunit , 29  NP from the Protein Data Bank (PDB) and performed docking studies using AutoDock molecular modeling simulation software [To further examine how RK424 inhibits NP function, we used software . Three psoftware , the NP software  and NES3software   using a photo-cross-linking method  and exam50 = 0.43±0.15 μM) as RK424 (IC50 = 0.48±0.19 μM), but we could not obtain sufficient Analog #4 for further analyses. Analog #4 is synthesized by Wakunaga Pharmaceuticals and is not commercially provided by any supplier. Therefore, we used RK424 for further analysis. Taken together, these results agree with those obtained from the docking model  of RK424 using RK424 analogs. Substituting the carboxyl group with an amide group  led to a partial reduction in the antiviral effect, whereas the loss of the biphenyl ring  and quinolone backbone  led to a complete loss of antiviral activity. By contrast, replacing the biphenyl ring with a diphenyl ether moiety  resulted in an antiviral effect comparable with that of RK424 . Analog ng model , i.e., tThe four amino acids residues that play a potential role in RK424 binding to NP reside within different functional domains. These domains are involved in (i) oligomerization of NP via NP-RNA and NP-NP interactions, and (ii) nuclear export of NP. We first used surface plasmon resonance  to examine the effect of inhibiting the NP-RNA interaction. A 24-mer RNA oligomer was biotinylated at the 5’-end (to facilitate immobilization) and methylated at the 2’-OH of ribose (to provide nuclease resistance) was immobilized on an SA sensor chip. We chose a 24-mer because studies show that this length approximates the binding distance between NP and vRNA ,34,35,36We next used a pull-down assay incorporating NP proteins fused to different tags to examine whether RK424 caused the NP-NP homo-oligomer to dissociate. In the absence of RK424, a purified recombinant wild-type NP protein fused to a HA tag (NP-HA) bound to FLAG agarose beads coupled to purified recombinant wild-type NP protein fused to mRFP-Flag-tag (NP-mRFP-Flag beads); however, NP-HA did not bind to control FLAG agarose beads coupled to mRFP-Flag protein (mRFP-Flag beads). RK424 inhibited the binding of NP-HA to NP-mRFP-Flag beads in a dose-dependent manner . These rin vitro transcription; NP shapes the main backbone and regulates the conformation of vRNP by interacting with RNA and with itself (NP-NP homodimers) ,34,37. Tin vitro nuclear export assay using digitonin-permeabilized, semi-intact HeLa cells ; however, binding was inhibited in the presence of RK424 (5 μM or 10 μM) and LMB (positive control) . RK424 iWhen developing new drugs, identifying potential off-target effects is very important. With this in mind, we superimposed the NP pocket on to other known binding pocket surfaces to identify structural similarities. To do this, we searched two comprehensive databases: Pocket Similarity Search using Multiple-sketches (PoSSuM)  and ProBrd passage, but no CPE occurred at the 4th passage. On the other hand, as the number of passages increased, the concentration of nuclozin at which CPE occurred was up to 10 μM by the 4th passage  with that of the initial WT viral titer used in the selection of the escape mutant. The titer of the RK424 3rd passage virus was 1.41×103 PFU/mL per HAU, which was lower than that of the WT (2.19×105 PFU/mL per HAU)  which were previously reported to be result in resistance to nucleozin [rd passage virus, which also support the result of sensitivity of RK424- and nucleozin-selected viruses to RK424 and nucleozin.Finally, to demonstrate the accuracy of our docking model and confirm that RK424 binds to the binding pocket within NP, we selected escape mutant viruses by passaging an IAV in the presence of RK424. In this experiment, we selected nucleozin as the positive control, because nucleozin exerts a potent antiviral effect via its ability to cross-link two NP molecules . The str passage . Further passage .These reper HAU) . These rucleozin . Nucloezucleozin , but no Therefore, we next used a reverse genetics approach to generate mutant viruses harboring an alanine substitution at each potential RK424 binding site within NP . We succin vivo co-administration (RK424 and oseltamivir phosphate) data and the effectiveness of RK424 against the oseltamivir resistant virus indicated that RK424 inhibited viral replication via a pathway different that of existing antiviral drugs (neuraminidase inhibitors). Further analyses showed that RK424 bound to the small pocket of NP and interfered with three core NP functions: i) the NP-RNA interaction, ii) the NP-NP interaction, and iii) the nuclear export of NP. In addition, we demonstrated that the NP pocket structure comprised three functional domains: the RNA binding groove, the dimer interface, and NES3. Previous reports [et al. showed that NP-NES3 is the most important NP-NES sequence for nuclear export of NP and viral replication [Here, we identified a novel small pocket structure within NP as a potential target for antiviral drugs. Moreover, the  reports , 34, 43 lication . Taken tlication ,18,34,43In silico docking studies incorporating RK424 and monomeric NP identified three potential binding sites for RK424  revealed that the NP pocket had the best fitting score. The carboxyl group of RK424 forms a hydrogen bond with R162 within the NP pocket and the phenyl ring derived from the quinolone backbone of RK424 interacts with the L264 side chain via Van der Waals forces. Furthermore, the biphenyl ring of RK424 forms a CH-π hydrogen bond with the Y487 side chain. The S165 side chain does not bind directly to RK424 in this model; however, other configuration models  suggested a direct interaction between residue S165 and the carboxyl group or the phenyl ring derived from the quinolone backbone of RK424. Moreover, other amino acid sequences, such as F488 and F489, were also predicted to interact with RK424 . Thus, iThe vRNP, comprising the viral genome, polymerase subunits PB2, PB1, and PA, and NP, regulates viral transcription and replication in the host cell nucleus, and is maintained via NP-NP and NP-RNA interactions ,34,37. APredicting possible side effects is an important issue when designing new drugs. The potential utility of a molecule as a targetable drug can be examined by comparing the structure of its target binding pocket with the structures of host proteins . Moleculin vitro against many different subtypes of IAVs including currently circulating A/California/7/2009 (H1N1) virus and the highly pathogenic avian influenza virus strains, A/Ck/Yamaguchi/7/2004 (H5N1) and A/Anhui/1/2013 (H7N9). These results support the findings that RK424 targets conserved amino acids in different virus strains. Indeed, although we were able to generate nucleozin-resistant virus, we were unable to generate the RK424-resistant virus, even after four passages , NP salt bridge inhibitors , and NP-aggregation inducers  ,23,24,25passages . The inain vitro, we also showed that RK424 exhibited antiviral effects in vivo. Remarkably, significant (~2 log) reductions in viral titers in the lung were observed after day 6 post-infection. However, mice body weight was lower after the administration of 10 mg/kg RK424 than after the administration of 10 mg/kg oseltamivir phosphate, which resulted in no weight loss, and recovery from morbidity was modest in the 10 mg/kg RK424-treated group. Therefore, viral titers may rebound after day 6 post-infection and thus mice may continue to succumb to viral infection. Another group has reported that administration of 50 mg/kg T-705 for 5 days, beginning 1 h post-infection, results in significantly lower(~2 log) viral titers (3.50 log10 PFU/mL) in the lung than in negative control group (5.47 log10 PFU/mL). This was observed after day 6 post-infection, but recovery from morbidity was only 20% of that of the negative control group [in vivo. The reason for this limited antiviral effect of RK424 in vivo may be because RK424 is highly lipophilic, a property that affects important parameters such as its solubility, absorbency, and stability, all of which affect PK [in vivo. RK424 has a high plasma protein binding rate because of its high lipophilicity. The efficacy of a drug is affected by the degree to which it exists in a form free from plasma proteins (free drug) within blood plasma. Unbound drugs can traverse cell membranes and become absorbed by target tissues, where they can interact with the drug target and exhibit pharmacologic effects. The plasma protein binding of RK424 is 99.6%, which means only 0.4% of the drug exists in free form in blood plasma. The Cmax of RK424 (39200 ng/mL) is more than 200 times higher than the IC50 concentration used in vitro (0.5 μM = 162.7 ng/mL), but the free drug maximum concentration (Cfree max) was 156.8 ng/mL because almost all of the drug was bound to plasma proteins. Taking into account this property, the free plasma exposure of RK424 was adjusted to obtain the in vitro IC50 (0.5 μM = 162.7 ng/mL) concentration by the intraperitoneal administration of 10 mg/kg RK424 twice per day, but the antiviral effect was limited. This may mean that it will be necessary to increase the plasma free drug concentration to arrive at the in vitro IC90 concentration (1.2 μM = 390.5 ng/mL). However, the intraperitoneal administration of RK424 at 10 mg/kg did not result in the in vitro IC90 concentration. We could not increase the dose of RK424 further, because RK424 precipitated from the viecle solution. The oral administration route provides a way to administer a drug with low solubility to animals. However, to achieve and maintain the in vitro IC90 concentration (1.2 μM = 390.5 ng/mL) in plasma via oral administration, multiple does of around 100 mg/kg RK424 would be needed because the bioavailability and t1/2 of oral administration are lower than those obtainable by intraperitoneal administration. Indeed, to design a multiple dosing regimen precisely, the PK study has to be done as a multiple dose of RK424. Moreover, the Vd (583mL/kg) parameter, which is lower than mouse total body water (725 mL/kg), suggests that RK424 may not be efficiently absorbed into target tissues. In conclusion, the PK properties of RK424 are insufficient to exert an antiviral effect in vivo. To acquire antiviral activity in vivo, the pharmacologic parameters of RK424 will need to be optimized by introducing changes in its chemical structure.In addition to demonstrating antiviral activity ol group . These rffect PK . Thus, tTo summarize, the results presented herein highlight a novel strategy for developing new drugs that target the NP pocket; such drugs may have multifunctional effects and reduce the emergence of drug-resistant viruses. One possible approach to developing a potent antiviral drug targeting the NP pocket is to optimize the structure of RK424. This could be achieved by replacing its aromatic rings, the cause of its high lipophilicity, with aromatic heterocyles or by introducing side chains modified with hydrophilic groups, as long as these changes do not affect its antiviral activity. In addition to this approach, target-oriented drug screening may be a more desirable approach because the NP pocket has a very unique surface structure and the amino acid residues within this pocket are highly conserved. These characteristics make the NP pocket an attractive target for drug development. Thus, another promising strategy is computational virtual screening, which is a useful way of identifying small molecular compounds with backbone structures that differ from those of RK424.RK424 was purchased from Enamine Ltd . RK424 analogs were synthesized by Wakunaga Pharmaceutical Co. Ltd.  and a stock solution (10 mM) was prepared in dimethyl sulfoxide (DMSO). Oseltamivir phosphate was purchased from Sundia MediTech Company, Ltd  and a stock solution was prepared in DMSO. Leptomycin B (LMB) was purchased from Sigma . LMB was dissolved in ethanol (EtOH) and then diluted in DMSO.2. Transfections were performed using FuGENE HD  according to the manufacturer's instructions. The influenza A viruses were propagated in MDCK cells at 37°C for 48 h in 5% CO2.Madin-Darby canine kidney (MDCK) cells, HeLa cells, and human embryonic kidney 293T (HEK293T) cells from the repository of our laboratory, were grown in Dulbecco’s modified Eagle’s medium  containing Pen Strep Glutamine  and 10% fetal bovine serum (Sigma). Cells were cultured at 37°C in 5% COCTCGAGATGGCGACCAAAGGCACCAA-3’ and NotI-HA-NP(R), 5’-TAGCGGCCGCTTAAGCGTAATCTGGAACATCGTATGGGTAATTGTCGTACTCCTCTGCAT-3’ (restriction enzyme sites underlined) and NP/pCAGGS as a template. The PCR product was cloned into the XhoI and NotI sites within pCAGGS. The plasmid encoding wild-type NP fused to glutathione S-transferase (GST) (NP-GST) was generated by PCR using primers EcoRI-NP (F), 5’-GGGAATTCCATGGCGACCAAA-3’ and Xho1-GST-NP (R), 5’-AACTCGAGTTAATTGTCGTACTCCTCTGC-3’ (restriction enzyme sites underlined) and NP/pCAGGS as the template. The PCR product was cloned into the EcoRI and XhoI sites within pGEX6p-3 . Plasmids NP/pHH21 and NP/pCAGGS harboring site specific mutations  were created by Prime STAR Max DNA Polymerase  using the following primers Y52H-(F), 5’-AGTGATCATGAGGGACGGCTGATTCAG-3’ and Y52H-(R), 5’-TCCCTCATGATCACTGAGTTTAAGTTC-3’; R162A-(F), 5’-GATCCCGCGATGTGCTCACTGATGCAG-3’ and R162A-(R), 5’-GCACATCGCGGGATCCATTCCTGTGCG-3’; S165A-(F), 5’-ATGTGCGCACTGATGCAGGGCTCAACC-3’ and S165A-(R), 5’-CATCAGTGCGCACATCCTGGGATCCAT-3’; L264A-(F), 5’-TCTGCAGCCATATTGAGAGGGTCAGTT-3’ and L264A-(R), 5’-CAATATGGCTGCAGACCGTGCTAAAAA-3’; Y289H-(F), 5’-AGTGGACACGACTTTGAAAGAGAGGGA-3’ and Y289H-(R), 5’-AAAGTCGTGTCCACTGGCTACGGCAGA-3’; and Y487A-(F), 5’-GGATCTGCTTTCTTCGGAGACAATGCA-3’ and Y487A-(R), 5’-GAAGAAAGCAGATCCTTCATTACTCAT-3’ (point mutated sites underlined).The mammalian expression vector, pCAGGS, encoding monomeric red fluorescent protein (mRFP) fused to a Flag tag (mRFP-Flag), wild-type NP fused to mRFP-Flag tag (NP-mRFP-Flag), and wild-type NP fused to a Flag tag (NP-Flag) have been described previously ,22. The A pCAGGS plasmid encoding a Flag-tagged mutant NP (mutant NP-Flag) harboring all R162A, S165A, L264A, and Y487A mutations was generated using the method described above. All mutations were confirmed by DNA sequencing.2 incubator. The number of plaques was counted and the IC50 calculated.The antiviral activity of the different compounds was measured in a plaque assay as described previously . Briefly50) was calculated by comparing the viability of RK424-treated cells with that of DMSO-treated cells.Cell toxicity was measured in a water soluble tetrazolium salt-1 (WST-1) (Takara Bio) assay as previously described . The 50%All animal experiments were approved by RIKEN Institutional Animal Use and Care Administrative Advisory Committees and were performed in accordance with RIKEN institutional guidelines and regulations (Certificate number: H23-2-101). All procedures were performed under pentobarbital treatment (30–50 mg/kg), and all efforts were made to minimize suffering. Mice were euthanized at the endpoint (14 days after virus inoculation) by intraperitoneal injection of pentobarbital . Mice were monitored and their body weight were measured daily during the study. Mice were euthanized if rapid and lasting weight loss (loss of more than 20% of body weight in a few days) was observed (a humane endpoint).50) of A/WSN/1933 (H1N1). Mice (eight per each RK424 treated-group) were intraperitoneally injected with RK424 2 h before virus exposure and then twice per day (2 × 5 mg/kg and 2 × 10 mg/kg) for 5 days beginning on the day of infection. Seven mice per positive control group also received 2 × 10 mg/kg of oseltamivir phosphate per day. Seven mice per negative control group received PBS. Bodyweight was measured daily and survival evaluated. Three mice from the negative control group and the 10 mg/kg RK424 group were euthanized on day 6 post-infection and virus titers in the lungs were measured in a plaque assay. Serial diluted oseltamivir phosphate was administered to three mice per each group  twice per day according to the same protocol mentioned above and appropriate doses of oseltamivir phosphate were estimated for co-administration with RK424. In the co-administration study, 2 × 0.02 mg/kg oseltamivir phosphate together with 2 × 5 mg/kg or 2 × 10 mg/kg of RK424 were also administered twice per day in the same way and synergetic antiviral effect was evaluated with respect to mono therapy (0.02 mg/kg of oseltamivir phosphate or 5 or 10 mg/kg RK424) and the negative control group (PBS). Seven mice per each group were tested in the co-administration study.RK424 was dissolved in polyethylene glycol 400  and then diluted with PBS to a final concentration of 1 mg/mL or 2 mg/mL. Six-week-old Balb/c mice  were infected intranasally with ten 50% lethal doses  and isotonic sodium chloride solution  and was intravenously administrated; RK424 sufficient to achieve 10 mg/kg was dissolved in PEG 400 (Sigma) and was intraperitoneally administrated; and RK424 sufficient to achieve 10 mg/kg was dissolved in 0.5% methyl cellulose  solution and was orally administrated to eight-week-old Balb/c mice . Three mice were assigned to each experimental group. Venous blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6 and 24 h after intraperitoneal administration and collected at 0, 0.25, 0.5, 1, 2, 6 and 24 h after intravenous and oral administration. The blood samples were deproteinized and subjected to LC/MS/MS  analyses. Pharmacokinetic parameters  were calculated using Winnolin Ver.6.1 .These studies were performed at Nemoto Science Co., Ltd. . RK424 sufficient to achieve 1 mg/kg was dissolved in These studies were performed at Wakunaga pharmaceutical Co., Ltd. .Mouse plasma was prepared from seven-week-old mouse blood samples . RK424 (50 μM) solution was prepared by mixing 5 mM RK424 in DMSO with mouse plasma and incubating the mixture at 37°C for 30 min. RK424 free from plasma protein was separated by centrifugal filtration using 10 kDa size cut filter . The filtrate sample was measured by ACQUITY Ultra Performance Liquid chromatography (UPLC) system  and the plasma protein binding rate was calculated.HEK293T cells were cultured in 24 well plates at 37°C for 24 h. Cells were transfected with plasmid  DNA (0.5 μg) and a luciferase RNA expression (vNP-luc/pHH21) vector. Cells were treated (or not) with the test compounds and luciferase activity was measured according to manufacturer’s instructions after incubation at 37°C for 48 h.5) and HEK-293T (6 × 105) cells was transfected with the eight viral genome-expressing plasmids or with the four viral protein-expressing plasmids. At 72 h post-transfection, recombinant viruses were harvested and the virus titer in the cell supernatants was determined in a plaque assay.Recombinant viruses were generated by DNA transfection as described previously . BrieflyHeLa cells were seeded on coverslips and grown to 60–70% confluence. Cells were then transfected with 0.5 μg of NP/pCAGGS or infected with A/WSN/1933 (H1N1) virus at an MOI of 10 in the presence or absence of the test compounds. After 48 h of transfection or 6 h of infection, cells were fixed with 4% paraformaldehyde for 20 min at room temperature (RT) and then further fixed with cold methanol for 10 min at −20°C. Cells were washed three times with PBS and then incubated with a primary anti-NP monoclonal antibody (MAb)  for 1 h at RT. The cells were then washed with PBS and incubated with an Alexa Fluor 488-conjugated goat secondary antibody against rabbit IgG  for 1 h at RT. The cells were then incubated with PBS containing Hoechst 33342 for 5 min and then washed three times with PBS. The excitation and emission wavelengths were set at 488 nm and 519 nm, respectively. Prepared samples were observed under a confocal laser scanning microscope .HeLa cells were attached to glass cover slips at 37°C for 24 h and then transiently transfected with NP-mRFP-Flag/pCAGGS. After 48 h, cells were washed twice with cold transport buffer (20 mM HEPES (pH 7.4), 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA (pH 7.4), 2 mM DTT, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin) and permeabilized with digitonin  in transport buffer for 5 min on ice. The cells were then washed with cold transport buffer and incubated with apyrase  for 5 min on ice. After washing twice with cold transport buffer, the cells were incubated in cold transport buffer for 10 min on ice before addition of a the reaction mixture , 10 mM ATP, 5 mM creatine phosphate, 0.64 U creatine kinase) containing (or not) the test compounds. The mixture was the incubated at 30°C for 1 h. After washing three times with cold transport buffer, mRFP fluorescence was observed under a confocal laser scanning microscope . The fluorescence intensity in the nuclei was analyzed with MetaMorph software . The fluorescence intensity of NP-mRFP-Flag was quantified in >100 nuclei in each of three independent experiments.HEK293T cells were transfected with 10 μg of NP-Flag/pCAGGS, NP-HA/pCAGGS, NP-mRFP-Flag/pCAGGS, or mRFP-Flag/pCAGGS for 48 h. The cells were then washed with PBS and lysed in 500 μL of lysis buffer (10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA and 1% NP-40) at 4°C for 1 h. The lysates were centrifuged at 20,000 × g for 10 min and the supernatants collected and mixed with ANTI-FLAG M2 agarose beads (Sigma) or ANTI-HA agarose beads (Sigma). After incubating at 4°C for 24 h, the beads were washed three times with wash buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.05% NP-40). The proteins were purified by adding 3 × Flag peptide (Sigma) or HA peptide (Sigma). The purified NP-Flag and NP-HA proteins were then subjected to 10% SDS-PAGE and analyzed by western blotting. FLAG agarose beads coupled to mRFP-Flag, NP-mRFP-Flag, and NP-Flag-bound were used to analyze the interactions between NP and CRM1, or between NP and NP, before elution with the 3 × Flag peptide.N-hydroxysuccinimide-activated beads were washed three times with 1 mM aqueous HCl followed by coupling solution (0.1 M NaHCO3 and 50% dioxane mixture). A solution of photoaffinity linker in coupling solution was then added to the beads, which were then incubated at 37°C for 2 h on a rotor. After washing five times with coupling solution, the beads were blocked with 1 M ethanolamine in 0.1 M Tris–HCl (pH 8.0) buffer at 37°C for 1 h on a rotor. The beads were then placed in a spin column and washed three times with Milli-Q water and methanol before being transferred to a glass sample vial. A methanol solution containing RK424 was then added to the beads and the mixture concentrated and dried in vacuo. The beads were then irradiated in a UV cross-linker at 365 nm (4 J/cm2) and washed with methanol to yield RK424-cross-linked affinity beads. Purified NP-Flag proteins were added to 20 μL of RK424-cross-linked affinity beads or uncross-linked beads. After incubating at 4°C for 24 h, the beads were washed five times with wash buffer and bound proteins eluted in 10% SDS-PAGE sample buffer at 100°C for 5 min. The proteins that bound to RK424 were then separated in 10% SDS–PAGE gels and detected by western blotting with an anti-Flag M2 MAb (Sigma).RK424 was cross-linked to Sepharose beads as previously described . BrieflyNP-Flag beads were incubated with HeLa cell lysate and different concentrations of RK424. After incubation at 4°C for 24 h, the NP-Flag beads were washed five times with wash buffer and the bound proteins separated in 10% SDS-PAGE gels. Binding of CRM1 to NP-Flag beads was detected by western blotting with an anti-CRM1 MAb . LMB was used as positive control (LMB inhibits the binding of NP to CRM1). EtOH and DMSO were used as vehicle controls for LMB and RK424, respectively.A biotinylated 2-O-methylated RNA oligonucleotide (5-UUU GUU ACA CAC ACA CAC GCU GUG-3) was purchased from Hokkaido System Science CO., Ltd.  and immobilized on an streptavidin-coated sensor chip  at a surface density of 100 resonance units (RU), according to manufacturer’s instructions The inhibitory effect of RK424 to NP-RNA binding was examined by incubating 10 ng/μL NP-FLAG proteins with RK424 at RT for 1 h. The RK424-treated samples were then injected onto the sensor chip surface and the SPR signal measured in a Biacore T-100 . Samples were injected in running buffer , and 0.05% Tween 20) at 25°C.FLAG agarose beads coupled to mRFP-Flag (mRFP-Flag beads), NP-mRFP-Flag (NP-mRFP-Flag beads), and purified NP-HA proteins were treated with RNase A  at RT for 10 min followed by a further incubation at 4°C overnight. The purified NP-HA proteins were then incubated with RK424 at 4°C for 1 h. Purified NP-HA proteins were then added to NP-mRFP-Flag beads or mRFP-Flag beads and incubated overnight at 4°C. The incubated beads were washed five times with wash buffer and then subjected to 10% SDS-PAGE. Binding of NP-HA to mRFP-Flag or NP-mRFP-Flag was detected by western blotting with an anti-HA Mab . An anti-Flag M2 Mab (Sigma) was used as a loading control.TAATACGACTCACTATATAGTTTTTTACTCCAGCTCTATGTTG-3’ and T7_WSNseg7-(R), 5’-AGCAAAAGCAGGTAGATATTG-3’ (T7 promoter sequence underlined).vRNA encoding the M gene was generated using a RiboMAX Large Scale RNA Production System-T7 . Briefly, approximately 1027 bp of the A/WSN/1933 (H1N1) virus negative-sense segment 7 genome was amplified from an M/pHH21 plasmid by PCR and the products purified using a QIAquick PCR purification kit . Five μg of the purified PCR product were added to a reaction mixture containing transcription buffer, rNTP mix , and T7 enzyme mix  and incubated at 37°C for 4 h. Synthesized vRNA was treated with 5 units of RQ1 RNAse-Free DNase I (Promega) at 37°C for 15 min. The following primers were used to amplify the negative-sense segment 7 genome: T7_WSN_seg7-(F), 5’-GACTCAGTE. coli strain BL21 CodonPlus (DE3)-RIL  harboring the GST-NP/pGEX6p-3 plasmid was cultured in the presence of 1 mM isopropyl-b-D-1 thiogalactopyranoside (IPTG) for 10 h at 22°C and then collected by centrifugation. The cell pellets were resuspended and sonicated in lysis buffer (50 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1% Triton X-100, 1 mg/mL lysozyme, and 0.15 mg/mL RNAse A). Glutathione Sepharose 4 Fast Flow beads  were then added to the cell lysate and rotated overnight at 4°C. After washing the beads, bound proteins were released from the beads using PreScission Protease . The GST-cleaved NP proteins were then incubated with Heparin Sepharose 6 Fast Flow beads  to dissociate RNA from NP. The bound NP proteins were then eluted with elution buffer (1.5 M NaCl and 50 mM Tris-HCl (pH 7.4)). The eluted samples were concentrated to 1 mg/mL and dialyzed into lower salt buffer (100 mM NaCl and 50 mM Tris-HCl (pH 7.4)) using an Amicon Ultra 50K device (Merck Millipore). The buffer exchanged proteins were maintained at 4°C to allow the equilibrium to shift from oligomers to monomers. The prepared NP proteins were then rotated with RK424 and vRNA at RT for 60 min. Samples were then loaded onto a Superdex 200 Increase 10/300 GL Column  attached to an ÄKTA purifier chromatography system . The size distribution data and integrated peak areas were calculated using the instruments software .5 cells/well) were seeded onto coverslips in 12-well plates and incubated overnight at 37°C. At 6 h post-infection with A/WSN/1933 (H1N1) at an MOI of 5 in the absence or presence of RK424, the cells were washed once with PBS and then fixed with 4% paraformaldehyde in PBS for 10 min at RT. After a brief wash with PBS, the cells were permeabilized in 0.5% Triton X-100 in PBS for 1 min at RT. Prior to hybridization, the cells were washed with PBS and incubated for 5 min in 2 × SSC  containing 10% formamide. To detect viral RNAs, 2 μM of labeled probes in 15 μl of hybridization buffer , 0.02% BSA, 50 mg E. coli tRNA, 2 × SSC, and 10% formamide) was added to each sample. Hybridization was performed in a humidified chamber maintained at 37°C for 16 h. The samples were then washed twice for 30 min at 37°C with 10% formamide containing 2 × SSC supplemented with 2 mM VRC. Nuclei were stained with Hoechst 33342 and the cells were washed with PBS. Prepared samples were observed under a confocal laser scanning microscope .RNA FISH was performed according as previously described ,29. The st passage, 0.2–3 μM at 2nd passage, 0.3–3μM at 3rd passage and 0.4–3 μM at 4th passage. Nucleozin concentrations ranged from 0.1–2 μM at 1st passage, 0.3–3 μM at 2nd passage, 1–5 μM at 3rd passage and 3–10 μM at 4th passage. After the 4th passage, drug sensitivity was confirmed by using the plaque reduction assay with 10 μM RK424 or nucleozin as mentioned above.MDCK cells were cultured in 12 well plate at 37°C for 24 h. MDCK cells were infected with A/WSN/1933 (H1N1) strain at 100 PFU/well and cultured in the presence of serial dilutions of RK424 or nucleozin. Viral replication was monitored for one week to observe any cytopathic effect (CPE) in the culture plates. The supernatants were collected from wells treated with the highest concentration of compound in wells in which CPE was observed and then stored at -80°C for subsequent analysis. Virus was serially passaged by using 5 μL of viral stock from the preceding passage to infect fresh MDCK cells in the presence of increasing concentrations of compound. The compound concentrations used in the selection varied, depending on the highest concentration of compound at which CPE was observed in the preceding passage. These passages led to the generation of mutant viruses resistant to RK424 or nucleozin when mutations occurred in the viral protein targeted by the antiviral compound. The selection was carried out for a total of four passages with RK424 or nucleozin. RK424 concentrations ranged from 0.1–2 μM at 1rd passage with RK424 and 4th passage with nucleozin using Trizol LS reagent (Invitrogen) according to the manufacturer’s protocol. Reverse transcription (RT)-PCR was carried out with the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer’s protocol using a minus-strand-specific universal primer; Uni12 (F), 5’-agcaaaagcagg-3’. The coding region of the NP sequence in the RT product was amplified with the above described EcoRI-NP (F) and Xho1-GST-NP (R) primers by using KOD-Plus- according to the manufacturer’s protocol. The PCR-amplified product was directly sequenced on an ABI3730xl DNA Analyzer using an ABI PRISM BigDye Terminator v 3.1 Ready Reaction Cycle Sequencing Kit .Viral RNA was isolated from the plaque-purified viruses at 3Complete sequences for IAV NP were downloaded using the advanced database search at the NCBI’s Influenza Virus Resource . Avian, The two-tailed t-test and the log-rank test were used for statistical analysis. P-values <0.05 were considered statistically significant.In silico analysis of RK424 and NP binding was performed using AutoDock (version 4.2) [ion 4.2) . The steion 4.2) .The pocket structures of NP and PA were extracted from complete NP (PDB ID: 2IQH) and PA (PDB ID: 4E5E) structures by removing residues within 10 Å of the small pocket  and the endonuclease motif , respectively. Protein structure libraries (PoSSuM  or ProBiThe accession numbers for genes and proteins mentioned in this study are shown below. PB2 (GenBank Accession No. J0217), PB1 (GenBank Accession No.J02178), PA (GenBank Accession No. X17336), HA (GenBank Accession No. J02176), NP (GenBank Accession No. CY010791), NA (GenBank Accession No. L25817), M (GenBank Accession No. L25818), NS (GenBank Accession No. M12597), firefly luciferase (GenBank Accession No. AAA89082.1), mRFP (GenBank Accession No. AF506027), and GST (GenBank Accession No. AAB37352).S1 FigRK424 (10 mg/kg) was intraperitoneally or orally administrated to 8-week-old Balb/c mice. The plasma concentration of RK424 was determined at 0, 0.5, 1, 2, 4, 6, and 24 h after intraperitoneal administration and at 0, 0.5, 1, 2, 6, and 24 h after oral administration by LC/MS/MS.(TIF)Click here for additional data file.S2 FigSerial diluted Os was intraperitoneally administered to 6-week-old Balb/c mice 2 h prior to virus exposure and then twice per day for 5 days beginning on the day of infection. PBS was used as a negative control. Mice were infected intranasally with ten 50% lethal doses (LD50) of influenza A/WSN/1933 (H1N1) virus. (A) The body weight of three mice from each group was monitored and (B) survival rate was calculated.(TIF)Click here for additional data file.S3 FigMDCK cells were infected with influenza A/WSN/1933 (H1N1) for 1 h at 37°C before being overlaid with agarose containing oseltamivir phosphate (Os) or RK424. Plaque formation was visualized by staining with crystal violet. Three independent experiments were performed and one representative result is shown.(TIF)Click here for additional data file.S4 FigInfluenza A/WSN/1933 (H1N1) virus was exposed to RK424 for 1 h at 4°C. The virus was then used to infect MDCK cells in a plaque assay. The viral titer was calculated by counting of number of plaques formed by virus exposed to RK424 or dimethyl sulfoxide (DMSO) (control). Values represent the mean ± SD of three independent experiments.(TIF)Click here for additional data file.S5 FigRK424 (0.5 μM or 2.0 μM) was added at the indicated time points. After 2 h treatment with RK424 at each time point, MDCK cells were washed with PBS and resuspended in fresh media. Cultured supernatants were collected 12 h post-infection. The viral titers of collected supernatants were estimated by the plaque titration assay. Values represent the mean ± SD of three independent experiments.(TIF)Click here for additional data file.S6 FigIn silico docking analysis identified three potential binding sites for RK424 on NP. The configuration of each binding site was visualized using PyMol. Functional domains located in close proximity to binding site 1 are colored orange (RNA binding groove: amino acid (aa) 1–180), yellow (NES3: aa 256–266), and purple (dimer interface: aa 482–489). The binding energy for each potential configuration of RK424 is shown in the table.(TIF)Click here for additional data file.S7 FigThe docking models depict six different RK424 configurations . The side chains of the amino acid residues involved in the NP-RK424 interaction are colored red. Functional domains close to binding site 1 are colored orange (RNA binding groove: amino acid (aa) 1–180), yellow (NES3: aa 256–266), and purple (dimer inter-face: aa 482–489). The binding interactions between the six RK424 configurations and the amino acid residues at binding site l are listed in the table.(TIF)Click here for additional data file.S8 FigThe binding of RK424 configuration 4 to potential binding site 2, and of RK424 configuration 7 to potential binding site 3, is shown. The side chains of the amino acid residues involved in the NP-RK424 interaction are colored red. In contrast to the residues near to binding site 1 , these a(TIF)Click here for additional data file.S9 Figin vitro transcription and subjected to electrophoresis on an 0.8% agarose gel containing ethidium bromide. The vRNA was visualized under UV irradiation. (C) Size exclusion chromatogram showing different NP protein configurations under high salt buffer  and low salt buffer  conditions.(A) NP proteins purified on GSH affinity beads were subjected to electrophoresis in 10% SDS-PAGE gels and the purity checked by Coomassie Brilliant Blue (CBB) staining. (B) vRNA (M segment) was synthesized by (TIF)Click here for additional data file.S10 Fig(A) The pocket structures of NP and PA were extracted from complete NP (PDB ID: 2IQH) and PA (PDB ID: 4E5E) structures by removing residues within 10 Å of the small pocket  and the endonuclease motif , respectively. (B) The pocket structures of NP and PA (red) were compared with other potential pocket structures identified from the database (blue). (C) Significant hits (Z-score>1.96) against the PA pocket are shown together with the PDB ID, Chain ID, and Z-score.(TIF)Click here for additional data file.S11 FigThe structure of compound 3 (A) and nucleozin (B) are indicated. Compound 3 inhibits formation of the NP trimer by disrupting the salt bridge between E339 and R416 within the NP tail loop binding pocket  and nucl(TIF)Click here for additional data file.S1 Tablemax, Cfree max, Tmax, AUC0–24 h, t1/2, and Vd) were calculated using Winnolin Ver.6.1 and different plasma concentrations of RK424. Plasma protein binding was also measured by UPLC.PK parameters (C(TIF)Click here for additional data file.S2 Tablemax, Cfree max, Tmax, AUC0–24 h, t1/2, and Vd) were calculated using Winnolin Ver.6.1 and different plasma concentrations of RK424. Relative bioavailability was also calculated using intraperitoneal and oral AUC0–24 h.PK parameters (C(TIF)Click here for additional data file.S3 TableThe probes were designed by STELLARIS RNA FISH PROBE DESIGNER and purchased from Biosearch Technologies, Inc.(TIF)Click here for additional data file.


GJB2. The mutation spectrum and prevalence of mutations vary significantly among ethnic groups, and the relationship between p.V37I mutation in GJB2 and the hearing phenotype is controversial. Among the 3,864 patients in this study, 106 (2.74%) had a homozygous p.V37I variation or a compound p.V37I plus other GJB2 pathogenic mutation, a frequency that was significantly higher than that in the control group . The hearing loss phenotype ranged from mild to profound in all patients with the homozygous p.V37I variation or compound p.V37I plus other GJB2 pathogenic mutation. There was no difference in the distribution of the hearing level in the group with the homozygous p.V37I variation and the group with the compound p.V37I variation plus pathogenic mutation. Most patients (66.04%) with the V37I-homozygous variation or p.V37I plus other pathogenic mutation had a mild or moderate hearing level. This study found a definite relationship between p.V37I and deafness, and most patients who carried the pathogenic combination with p.V37I mutation had mild or moderate hearing loss. Therefore, otolaryngologists should consider that the milder phenotype might be caused by the GJB2 p.V37I mutation.The most common cause of nonsyndromic autosomal recessive hearing loss is mutations in  GJB2 gene [GJB2 have also been reported to cause dominant nonsyndromic or syndromic hearing loss [Hearing impairment is the most common neurosensory disorder in humans, with reported incidences ranging from 1 in 300 to 1 in 1,000 children. Genetics plays a role in approximately half of the cases, including syndromic and nonsyndromic forms. Nonsyndromic deafness accounts for 60–70% of inherited hearing impairment cases, with autosomal recessive being the most common type of inheritance. For many populations, the most common cause of nonsyndromic autosomal recessive hearing loss is mutation in connexin 26, a gap junction protein encoded by the ing loss ,5. ConneGJB2 gene. The mutation spectrum and prevalence of mutations vary significantly among ethnic groups. Three mutations, c.235delC, c.35delG, and c. 167delT, are the most frequent mutations in Asian, Caucasian and Ashkenazi Jewish populations respectively [GJB2 is highly prevalent in East Asian deafness, but there is a controversial relationship between some mutations, including p.V37I(c.109G>A), and the hearing phenotype. Few systematic studies have examined the role of p.V37I in the pathogenesis of hearing loss. In the present study, 3,864 patients with nonsyndromic sensorineural hearing loss and 600 normal-hearing individuals were included to investigate the nature of the p.V37I variant and provide possible genetic testing and counseling for hearing loss patients with this variant in China.To date, more than 150 mutations, polymorphisms, and unclassified variants have been described in the GJB2. High-resolution temporal bone computed tomography (CT) was available for all of the recruited patients to rule out inner ear malformations, including enlarged vestibular aqueduct syndrome patients. Additionally, all of the patients had no skin problems or other clinical abnormalities and no m.A1555G or m.C1494T mutation in mitochondrial 12S rRNA.In total, 3,864 patients with nonsyndromic sensorineural hearing loss (from unrelated families) were reached through the Otolaryngology Department of the Chinese PLA General Hospital and were included in the current study. A complete history, physical and otoscopic examinations, and audiological testing, including pure tone audiometry, tympanometry, or auditory brainstem response, were carried out in all of the patients, and diagnosed as sensorineural hearing loss. All 3,864 patients were sequence analyzed for the exon 2 of GJB2. The age ranged from 2 to 60 years, only 42 individuals were aged less than 18 years, and the median age was 32 years. Audiological testing was carried out in all of the individuals, revealing normal hearing or conductive hearing loss but normal bone conduction threshold.Fully informed written consent was obtained from all subjects or, in the case of children, from their guardians. The study was approved by the Chinese PLA General Hospital Research Ethics Committee. To identify the p.V37I prevalence in normal individuals, 600 individuals were included as the control group and were sequence analyzed for the exon 2 of No patient was found to have any goiter symptom or sign or syndromic symptoms in other systems. The pure-tone average (PTA) was calculated as the average of the threshold measured at 0.5, 1.0, 2.0, and 4.0 kHz and was used to compare subgroups of patients. The level of hearing loss, in terms of PTA, was described as follows: normal hearing, < 20 dB; mild hearing impairment, 21–40 dB; moderate hearing impairment, 41–70 dB; severe hearing impairment, 71–90 dB; and profound hearing impairment, > 90 dB.Chi-squared test (R × C table) was used to compare the distribution of the p.V37I mutation in the control and patient groups. The hearing level between the different groups was included in the nonparametric randomized block design information. The Friedman rank-sum test (M-test) was used to identify significant differences in hearing level among the groups. Statistical analyses were performed using the SPSS  16.0 software. A P-value of 0.05 or less was deemed to indicate statistical significance.GJB2 pathogenic mutation (GJB2 pathogenic mutation. The distribution of p.V37I homozygous and compound variation in the patient groups was significantly higher than that in the control group (P<0.005), suggesting that the presence of p.V37I homozygous and compound variation was more frequently associated with hearing loss , and the mean age of the 314 patients was 19.6 years (range: 7 months to 54 years). Heterozygous p.V37I variation, homozygous p.V37I variation, and compound p.V37I plus other mutation  were deting loss .GJB2 pathogenic mutation had a mild or moderate hearing level   .GJB2 is highly prevalent in East Asian deaf patients—11.1% in Thai deafness [GJB2, reports have increasingly considered p.V37I to be pathogenic with a mild-to-moderate hearing impairment phenotype. Dai’s study[The c.109G>A variation in deafness , 16.5% ideafness , 19.4% ii’s study concludei’s study assessedi’s study found thGJB2 pathogenic mutation (2.75%) in the patient groups was significantly higher than that in the control group (0%)  patients had all types of p.V37I variation; this frequency was slightly higher than that in Dai’s study (6.7%), likely because most patients in Dai’s study were severe-to-profound hearing impairment. The distribution of homozygous p.V37I variation or compound p.V37I plus other oup (0%) , suggestGJB2 mutations containing at least one p.V37I variation. Although there was no difference in the distribution of the hearing level in the patients with homozygous p.V37I variation or compound p.V37I plus other GJB2 pathogenic mutation, most patients with the p.V37I -homozygous variation or compound p.V37I plus other GJB2 pathogenic mutation had a mild or moderate hearing level , but did not reveal a major modifier gene, and it suggested that the phenotypic variability in c.35delG homozygous patients could not be explained by the effect of one major modifier gene and the significantly associated SNPs might reflect a small modifying effect on the phenotype. In addition, some papers reported that sensorineural hearing loss could be caused by mutations in two alleles of both different genes. There are reports of patients with sensorineural hearing loss caused by co-existing mutations in GJB2 or SLC26A4 and the mitochondrial gene[According to this study and previous reports, there is a broad spectrum of hearing phenotypes caused by the p.V37I variation , 13, 15,terature , which pterature  performerial gene, and onerial gene. So therBecause of the higher carriage rate of p.V37I in normal individuals, study of its role in pathogenesis will have a marked effect on the diagnosis strategy. To confirm the pathogenicity of p.V37I, comparative studies and epidemiological research with a large sample size are necessary. By evaluating subjects with the p.V37I variation and other mutations, this study found a definite relationship between p.V37I and deafness, and the majority of patients who carried the p.V37I mutation had mild or moderate hearing loss. The results of this study will facilitate analysis of the molecular etiology of patients with deafness of various phenotypes.S1 Text(DOC)Click here for additional data file.S2 Text(DOC)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.