Pro-inflammatory factors are important indicators for assessing inflammation severity and drug efficacy. Coptisine has been reported to inhibit LPS-induced TNF-α and NO production. In this study, we aim to build a pharmacokinetic-pharmacodynamic model to quantify the coptisine time course and potency of its anti-inflammatory effect in LPS-stimulated rats. The plasma and lung coptisine concentrations, plasma and lung TNF-α concentrations, plasma NO concentration, and lung iNOS expression were measured in LPS-stimulated rats after intravenous injection of three coptisine doses. The coptisine disposition kinetics were described by a two-compartment model. The coptisine distribution process from the plasma to the lung was described by first-order dynamics. The dynamics of plasma TNF-α generation and elimination followed zero-order kinetics and the Michaelis-Menten equation. A first-order kinetic model described the TNF-α diffusion process from the plasma to the lung. A precursor-pool indirect response model was used to describe the iNOS and NO generation induced by TNF-α. The inhibition rates of TNF-α production by coptisine  calculated from the simulation model were close to the decline rates of the plasma TNF-α AUC . Coptisine suppressed plasma TNF-α generation in a linear manner, resulting in a cascading reduction of iNOS and NO. The early term TNF-α response to stimulation is a key factor in the subsequent inflammatory cascade. In conclusion, this comprehensive PK-PD model provided a rational explanation for the interlocking relationship among TNF-α, iNOS and NO production triggered by LPS and a quantitative evaluation method for inhibition of TNF-α production by coptisine. Previous studies have confirmed that the dynamic profiles of these biomarkers are asynchronous. Lin, Nientsung and Lee, Ru Ping et al. examined the time course of the plasma TNF-α and NO concentrations in lipopolysaccharide (LPS)-stimulated rats  and found that the plasma TNF-α concentration peaked within 1 h5, whereas the plasma nitric oxide (NO) concentration culminated at 9 h6. Moreover, the inflammatory response shows spatial differences. Inflammatory cytokines can transmit inflammatory signals from the area of inflammation to other tissues, participate in upregulation of the inflammatory response and aggravate the damage. Inducible nitric oxide synthase (iNOS) mRNA expression has been reported to reach a peak in the lungs of rats at 3 hours and in the liver at 6 hours after a 10 mg/kg LPS infusion, whereas low levels are found in the spleen, heart and kidney5. Considering these temporal and spatial differences in the dynamic profiles of biomarkers, an integrated analysis based on physiology is a reasonable method for assessing inflammatory processes, interactions between inflammatory mediators and holistic effects of drugs.Inflammation is an immune response to harmful stimuli, such as trauma, bacteria, and viruses. Immune cells, including T cells and macrophages, are stimulated to excrete inflammatory cytokines, such as TNF-α, IL-6, IFN-γ and NO. These inflammatory factors have been widely used as biomarkers to evaluate the inflammation severity and effects of drugsAbhijit Chakraborty et al. established a comprehensive PK-PD model to capture the dynamics of regulation of plasma NO generation by TNF-α and IFN-γ and used it to evaluate the processes, efficacy and interaction of IL-10 and prednisolone7. Siddharth Sukumaran et al. built a mechanism-based model to describe the NO production process regulated by iNOS mRNA expression in LPS-stimulated rats and quantitatively assessed the holistic anti-inflammatory effects of methylprednisolone8.A pharmacokinetic-pharmacodynamic (PK-PD) model is a useful tool to analyze multiple biomarkers and has been broadly used to evaluate the effects of drugs on pathological processes. 9, has many biological activities in many diseases, including inflammation, infection, and cancer12. To date, many studies have found that coptisine affects inflammatory molecule expression in several cell types10 and in vivo14. In our previous study15, we found that coptisine inhibited NF-κB, MAPK, and PI3K/Akt activation, suppressed iNOS expression, and decreased NO and proinflammatory cytokine  levels. However, these studies on the anti-inflammatory effects of coptisine were performed at selected time points and did not consider the relationships among biomarkers in the network. Therefore, these assessments may cause deviation in evaluating the therapeutic effect of coptisine. Here, a comprehensive mathematical model including the expression of multiple biomarkers during the detailed inflammatory process can be established to capture the entire inflammatory phase from excitation to response. Recently, the coptisine plasma and urine concentration-time profiles and bioavailability in rats have been reported21. However, no detailed report has investigated the relationship between drug exposure and the pharmacodynamic response to coptisine.Coptisine, which is an isoquinoline alkaloid Fig. 9, has maIn this study, we established a pharmacokinetic model (PK) to describe the dynamics of coptisine. Simultaneously, we used inflammatory cytokines  as evaluation indicators and built a kinetic model to describe the detailed pathological process in LPS-stimulated rats. Then, the inhibitory effect of coptisine was quantitatively added to this kinetic model to obtain the overall anti-inflammatory effect. This mechanism-based PK-PD modeling approach provided a feasible method to assess and quantify the anti-inflammatory and dose-effect relationships of coptisine through a comprehensive analysis.0–12h of the plasma-drug concentration . The AUC0–12h values for lung coptisine were 5082.12 and 3123.74 ng·h/mL for the LPS + coptisine 7.74 mg/kg and LPS + coptisine 3.87 mg/kg groups, respectively. In these two dosing groups, the AUC ratios of lung coptisine and coptisine were 4.33 and 6.34, respectively. The lung-to-plasma AUC ratios were greater than one, which meant that coptisine was readily distributed to the lung. Following intravenous administration, the three coptisine doses were rapidly eliminated. The coptisine concentration in the blood declined with a t1/2 of 1.51 hours, which was close to that reported in previous research22.A two-compartment pharmacokinetic model was built to describe the kinetics of coptisine Fig. . Figure in vitro result. The in vitro study showed that the peak of iNOS expression appeared at 8 h in LPS-stimulated RAW264.7 cells  of the three coptisine doses inhibited plasma TNF-α production by approximately 54.73%, 26.49%, and 13.25%. The rate of decline calculated from the mathematical model was close to the descent rate of the plasma TNF-α AUC. The AUC0-12h values of plasma TNF-α were 5461.63, 2333.54, 3258.53, and 4097.53 pg·h/mL for the LPS, LPS + coptisine 7.74 mg/kg, LPS + coptisine 3.87 mg/kg, and LPS + 1.94 mg/kg groups, respectively. Compared with that of the LPS group, the ratios of the plasma TNF-α AUC decreased by 57.27%, 40.33%, and 24.98%, respectively. Thus, it could be confirmed that coptisine inhibited plasma TNF-α production in a linear manner.Coptisine inhibited TNF-α generation in the blood in a linear manner, and its inhibitory constant was calculated as 8.83 × 100–12h values of lung iNOS were 55.75, 28.2, and 30.72 . Compared with that of the LPS group, the ratios were decreased by 49.43% and 44.9%, respectively. The AUC0–12h plasma NO values for the four groups were 6447.87, 3924.59, 4072.68, and 7381.51 μmol·h/L . Compared to that of the LPS group, the ratios were reduced by 39.13%, 36.84% and a negative value, respectively. The AUCs of TNF-α, iNOS and NO showed the same trends with the coptisine doses, which proved that inhibition of TNF-α by coptisine could cause a cascading decrease in NO.The reduction of inflammatory cytokines in the later phase (iNOS and NO) should present linear dependence on the coptisine dose because the model is a tandem model. The Western blotting results for iNOS expression are shown in Fig. The visual prediction and evaluation of the final PK-PD model are presented in Fig. 23. Pro-inflammatory factors, such as TNF-α, upregulate the inflammatory response and cause tissue damage, even in the absence of infection. As biomarkers, generally inflammatory mediators are used to evaluate disease pathological processes and drug properties. The time courses of different biomarkers vary. Researchers found that the rat plasma TNF-α concentration peaked 1 h after endotoxin treatment, whereas the plasma NO concentration peaked at 9 h24. Ayse ER et al. found that the IL-1β concentration in the bronchoalveolar lavage fluid of rats after LPS-induced lung inflammation peaked at 1 h, whereas the peak IL-6 level occurred at 4 h25. Moreover, the inflammatory cytokine levels in various tissues are different. Inflammatory factors act as agents to transfer inflammatory signals from infected areas to other tissues and activate immune responses in uninfected areas. The experiment demonstrated that iNOS mRNA expression was different in various rat tissues after LPS infusion at a 10 mg/kg dose5. The peak of iNOS mRNA expression in the lung was detected at 3 h and the highest iNOS mRNA expression in the liver was found at 6 h, but iNOS mRNA was expressed at low levels in the spleen, heart and kidney. These findings indicate temporal and spatial variation and complex signal transfer for biomarkers.Endotoxin can cause uncontrolled activation of immune responses to generate inflammatory cytokines27. In vivo experiments also found that treatment with both anti-TNF-α and anti-IFN-γ antibodies almost completely inhibited the plasma NO production induced by LPS28. Furthermore, Abhijit Chakraborty et al. quantitatively evaluated the TNF-α- and IFN-γ-mediated NO production processes after LPS stimulation and found that TNF-α played a major role7. Thus, TNF-α served as the main mediator of iNOS and NO synthesis after LPS treatment. When LPS enters the vein, it is bound by macrophages in the blood, which rapidly produces large amounts of TNF-α. The quickly generated TNF-α binds to specific receptors and activates a secondary inflammatory reaction similar to ERK, JNK and MAPK pathway activation by LPS, leading to overexpression of iNOS and NO31.In fact, the proinflammatory cytokine TNF-α participates in signal transmission for endotoxin-induced NO generation and plays an important role in upregulating NO synthesis. Anti-TNF-α antibody administration had been reported to reduce LPS-induced NO release in mouse macrophages, which indicates that TNF-α is a significant mediator of NO synthesisin vitro and in vivo. Coptisine has an inhibitory effect on TLR4-mediated inflammatory signaling pathways. Our previous research confirmed that coptisine could prevent IκBα degradation and ERK, JNK, MAPK and PI3k/Akt phosphorylation in LPS-stimulated mice and macrophages. Coptisine effectively inhibited the iNOS, IL-1β and IL-6 mRNA levels and iNOS, NO and cytokine production. In the supplementary materials  efficiently suppressed LPS-induced TNF-α, and IL-10 (IC50 = 0.057 ng/mL) was also effective at controlling TNF-α release. The inhibitory rates of the TNF-α AUC0–6h were 82% (prednisolone) and 93% (IL-10)7. To increase the rate of TNF-α inhibition, the maximum blood concentration of coptisine should be approximately 1200 ng/mL, and the intravenous dose of coptisine should be increased to 15 mg/kg. The median lethal dosage (LD50) of berberine through intravenous injection is 9.0386 mg/kg32. Coptisine has the same typical natural benzyl tetrahydroisoquinoline alkaloid skeleton as berberine. Thus, the LD50 of coptisine may be close to that of berberine. The maximum effective dose of coptisine is much higher than the LD50. In addition, the low content in herbs and the oral absolute bioavailability (<2%)33 cause a poor overall anti-inflammatory efficacy for coptisine. These results suggest that the efficacy of coptisine may need to be improved through pharmaceutics or structural modification.Considering the correlation among biomarkers, we established a mechanism-based PK-PD model to integrate TNF-α, iNOS and NO and to clarify the intrinsic connections among the biological and anti-inflammatory mechanisms of coptisine. Our results demonstrated that coptisine linearly suppressed the blood TNF-α production induced by LPS in a dose-dependent manner, resulting in cascade diminution of iNOS and NO expression. The plasma TNF-α AUCIn conclusion, early generation of TNF-α is a key factor in the subsequent inflammatory cascade. Coptisine decreased plasma TNF-α production in a linear manner, followed by a reduction in the iNOS and NO levels in this rat inflammation model. This integrated mechanism-based PK-PD model described the individual TNF-α and NO concentrations and iNOS expression and reasonably explained the lag and relationships among these inflammatory factors. This approach not only successfully modeled the experimental data but also provided a reasonable method to quantitatively clarify the process and target by which coptisine inhibits the inflammatory response. We believe that PK/PD studies may be a useful approach to explain the active mechanism and therapeutic response of coptisine and to screen experimental designs for preclinical and clinical research.15.Coptisine  and berberine chloride  were obtained from Must Biotechnology, Inc. . Lipopolysaccharide (Escherichia coli 055:B5) was purchased from Sigma-Aldrich Co., LLC. . Methanol and acetonitrile (HPLC-grade) were obtained from Thermo Fisher Scientific Inc. . The BCA protein assay kit was obtained from Thermo Fisher Scientific . The antibody to iNOS (Cat. NO. ab178945) was purchased from Abcam Company Ltd. . The antibody to GAPDH (Cat. NO. 200306-7E4) was obtained from Zen Bioscience . The coptisine injection was prepared in accordance with a previous studyMale Sprague–Dawley (SD) rats (200 ± 20 g) were obtained from the Institute of Laboratory Animals of Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital . The rats were acclimatized in a room with a stationary temperature (22 ± 2 °C) and light: dark cycle (12 h:12 h) for at least 1 week before the experimental procedures. The experiments were conducted under the experimental practices and standards approved by the Animal Welfare and Research Ethics Committee at Chengdu University of TCM. All experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. This study was approved by the Animal Experiment Committee of Chengdu University of TCM. We made every effort to minimize pain.Table Three rats were randomly divided into each of three groups . All of the rats received a 100 μg/kg LPS injection  via the tail vein (time = 0). The animals in groups 1B and 1C received coptisine  by intravenous injection in the tail after 0.5 h. Blood samples were collected with tubes  soaked with heparin sodium via the tail vein at the 0.5, 1, 2, 4, 6, 8, 12 and 24 h time points.2 gas, re-dissolved in 100 μL of acetonitrile/0.1% formic acid (40/60 v/v), and detected using a liquid chromatography (LC) system. Standard solutions and quality control were both taken into account. Berberine was used as an internal standard.All samples were centrifuged for 15 min at 3500 rpm and 4 °C. Plasma was preserved at eighty degrees below zero centigrade until analysis. At the time of analysis, plasma (50 μL) was mixed with acetonitrile (200 μL) to precipitate proteins. After high-speed centrifugation , the clear upper fluid was collected and evaporated with a flow NThe LC system was an Ultra-Fast Liquid Chromatography System (SHIMADZU Nexera UFLC LC-30A) with a Zorbox RRHD Eclipse Plus-C8  column . The mobile phase consisted of an ACN (A) and 0.1% formic acid (B) gradient elution as follows: 90%~60% B (0~10 min), 60% B (10~20 min), and 90% B (20~25 min). The mobile phase was delivered at a constant flow rate of 0.7 mL/min. The detection wavelength was 345 nm, the injection volume was 50 μL, and the oven temperature was maintained at 40 °C.SD rats (n = 12) were divided randomly into 4 groups . Each group received 100 μg/kg of LPS via the tail vein (time = 0). After 0.5 h (time = 0.5 h), the rats in groups 2B, 2 C and 2D were injected with coptisine . Sampling through the tail vein was carried out at the 0, 0.333, 0.667, 1, 2, 4, 6, 8, 12 and 24 h time points. The blood TNF-α concentration was measured using an ELISA kit  following the manufacturer’s instructions. The NO concentration was determined using nitrate reductase acquired from the Nanjing Jiancheng Bioengineering Institute .Animals (n = 74) were divided randomly into 4 groups . The rats in groups 3B, 3C, and 3D were injected with 100 μg/kg of LPS via the tail vein (time = 0) and then received vehicle (rats in group 3B) or coptisine  by intravenous injection in the tail after 0.5 h. Blood was sampled from the abdominal artery of rats anesthetized with urethane, and the lungs were collected immediately, frozen in liquid nitrogen and kept at minus eighty degrees centigrade. The corpses were disposed of properly. For animals in the control group (group 3A), samples were collected and denoted as 0 h. In groups 3B, 3C and 3D, blood was sampled at the 0.667, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12 h time points (n = 2 per group per time point).Study 2.The lung tissues were homogenized with RIPA (radioimmunoprecipitation assay buffer) including PMSF (phenylmethylsulfonyl fluoride) using a tissue homogenizer. After high-speed centrifugation, the treatment of the homogenate supernatants was similar to that of the blood samples. The coptisine concentrations in the lungs and plasma were detected by the LC system. Berberine was included as an internal standard. The mobile phase consisted of an ACN (A) and 0.1% formic acid (B) gradient elution as follows: 90~60% B (0~10 min), 60% B (10~20 min), and 90% B (20~25 min). The mobile phase was delivered at a constant flow rate of 0.7 mL/min. The detection wavelength was 345 nm, the injection volume was 50 μL, and the oven temperature was maintained at 40 °C. The TNF-α and NO concentrations in the lungs were quantified as described in iNOS0, group 3A, time = 0 h) and calculated as follows34:Pharmacodynamic research was established for iNOS expression in the lung as determined by western blotting. The total protein concentration in the lung was determined with the BCA protein assay kit. Samples with equal amounts of protein were separated using a 10% SDS polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane . The membrane was blocked with 5% BSA  dissolved in TBST . The membrane was incubated with a specific antibody (1:2000 dilution) overnight (4 °C) and then with TBST containing an HRP-conjugated secondary antibody (ZenBioScience) for 1 h at room temperature. The bands were scanned with the ChampChemi 610 Plus Imager system . The grayscale value of each band was detected by a computer image analysis system . GAPDH, which is considered a housekeeping protein, was measured simultaneously to standardize the iNOS level. The iNOS expression level at each time point were normalized to the control iNOS level (Study 3. Blood was sampled at the 0 (group 4A), 2, 4, 6, 7, 8, and 9 h time points (n=3 per group per time point), and the lungs were collected at the same time. The plasma and lung coptisine concentrations, plasma TNF-α and NO concentrations, and lung iNOS expression levels were measured.Animals (n = 57) were divided randomly into 4 groups . The rats in each group received the same treatment as described in Fig. Considering that the PK and PD data were all obtained from LPS-stimulated male rats with fewer biometric differences, we did not introduce covariates, such as weight or sex, into the PK or PD model. The estimation of the final parameters was conducted using mixed effects methods under no covariate, as described in detail below.The plasma coptisine concentration was evaluated after administration of the 1.94, 3.87 and 7.74 mg/kg doses. We developed a two-compartment pharmacokinetic model containing linear elimination.cop, cop2 is the peripheral compartment, k10 represents systemic clearance, and k12 and k21 are distribution rate constants between the plasma and peripheral compartment.The plasma coptisine concentration is coplung represents the coptisine concentration in the lung, and k13 and k31 represent the distribution of coptisine between the plasma and lung, respectively.Coptisine in the lung was distributed from the plasma, which we described using the following formula:7. Moreover, because the generation process is presented in two stages  in the blood. A similar model was reported by Abhijit Chakraborty et al.7. Because of the high elimination rate of TNF-α after 2 hours of LPS stimulation, the model with Michaelis–Menten elimination was considered to quantify the serum TNF-α concentration after comparison with the zero- and first-order eliminations. Thus, we described the dynamics of plasma TNF-α generation and elimination as follows:k0 represents the zero-order production rate constant of plasma TNF-α, which has different values at the two different time periods  injection via the rat tail vein , which causes their lungs to be insensitive to endotoxinein Fig. . The totein Fig. . Thus, wIn vitro results have demonstrated that an anti-TNF-a antibody can inhibit nitrite production in LPS-stimulated mouse macrophage cells27. Another in vivo study has clarified that NO production after LPS stimulation is mediated by both endogenous TNF-α and IFN-γ28. Mice treated with TNF-α and IFN-γ blockers together nearly avoid plasma NO production 6 hours after LPS injection. When pretreated with anti-TNF-α or anti-IFN-γ alone, a certain amount of NO production is still detected in the blood after endotoxin stimulation. Furthermore, Abhijit Chakraborty et al.7 established a model to quantify the processes by which TNF-α and IFN-γ induced NO production after LPS challenge and found that the rate constant of TNF-α-induced NO production following LPS stimulation was much larger than that of IFN-γ-induced NO production . Accordingly, we took TNF-α as the most important mediator of iNOS and NO production in an inflammatory dynamic model induced by LPS. We depicted the effect of TNF-α in the lung on iNOS overexpression as follows:iNOS and sequential NO production have been proven to be mediated by TNF-α rather than directly induced by LPS. kiNOSmRNA, koutiNOSmRNA, and kiNOS are the intercompartmental rate constants, which describe iNOS production from TNF-α. iNOS and iNOSmRNA represent the iNOS and iNOS mRNA levels in the lung. koutiNOS is the first-order iNOS elimination rate. The western blotting analysis for iNOS in the lungs is presented in Fig. iNOSp represents the iNOS in the plasma, kiNOSlp is the first-order transfer rate from the lung to the plasma, and koutiNOSp is the elimination constant of iNOS in the plasma.In consideration of the lack of fluctuation in NO production in the lung Fig.  and the As soon as iNOS diffuses into the blood from the lung, NO is generated from L-arginine. The NO formation process was described with following formula.kiNOSNO indicates the NO produced by iNOS. NOplasma represents the amount of nitrite (NO2−) and nitrate (NO3−), and koutNO is the systemic clearance of NO. A similar model was adopted by Sukumaran et al.8.Based on these findings, we established a direct reaction model to describe the effect of coptisine.max, and sigmoid Emax models, we used a linear model marked as f(cop) to describe the inhibitory effect of coptisine on TNF-α formation. cop is the serum coptisine concentration. “InhibitCon” represents the linear inhibitory constant of the plasma coptisine concentration for the plasma TNF-α production rate.After comparison with several models, such as the log-linear, Ehttp://lixoft.com/products/monolix/). All model parameters were fitted simultaneously, and each of the between-animal variability (BAV) values was assumed to be log-normally distributed. The residual variability, which is the level of variance between the observations and their subject-specific predictions, is described with an additive model following a normal distribution with a zero mean and The PK-PD analysis was performed with the non-linear mixed effects (NLME) modeling program in the Monolix® 2016R1 software , visual predictive checks (VPCs) and a numerical predictive check (NPC) from 2000 model simulations.Supplementary Materials for Pharmacokinetic-Pharmacodynamic Modeling for Coptisine Challenge of Inflammation in LPS-Stimulated Rats


Sugarcane mosaic virus (SCMV) is the prevalent virus inducing maize dwarf mosaic and sugarcane mosaic diseases in China. According to the phylogenetic results of the complete genomic and coat protein gene sequences, SCMV was divided into four or five molecular groups, respectively. Previously, we detected SCMV isolates of group SO from Canna spp. in Ji’nan, Shandong province, China.Canna spp. in Ji’nan (Canna-Ji’nan) and Tai’an (Canna-Tai’an) of Shandong, China. Their complete genome sequences had genome of 9576 nucleotides and contained a large open reading frame encoding a polyprotein of 3063 amino acids. The phylogenetic analysis showed that the both Canna-Ji’nan and Canna-Tai’an were clustered into an independent group based on the complete genome sequence.In this study, we collected two SCMV isolates infecting Canna spp. from Ji’nan and Tai’an. This is the first report on SCMV belonging to SO group.In this study, we report the complete genome sequences of SCMV infecting The online version of this article (10.1186/s12985-018-1058-8) contains supplementary material, which is available to authorized users. Sugarcane mosaic virus (SCMV) is a species of the genus Potyvirus in the Potyviridae family and mainly infects plants of monocotyledons including maize, sorghum, sugarcane and canna. It is the major pathogen of sugarcane mosaic disease and maize dwarf mosaic disease and causes significant yield losses in China [Saccharum officinarum) in Vietnam and China [Cannas spp. [in China , 2. SCMVin China . SCMV isin China  . Howevein China . Isolatend China  and are nas spp. . But so Canna spp. showing symptoms of severe mosaic and dwarf were observed at incidence of 20%~ 30%. Two symptomatic leaf samples were collected in 2015 and 2016, respectively, from the parks of Ji’nan  and Tai’an , Shandong Province, China. Total RNA was extracted using the RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich) . The first-strand cDNA was synthesized using Moloney MLV reverse transcriptase  with primer 5’-GGTCGACTGCAGGATCCAAGCT15–3′ [Plant of Canna samples were positive to SCMV antiserum in ELISA detection. The SCMV infecting Canna plants in Ji’nan and Tai’an were designated as Canna-Ji’nan and Canna-Tai’an accordingly. The complete genome of SCMV Canna-Ji’nan and Canna-Tai’an have been deposited in the GenBank database and allocated the accession numbers KY548506 and KY548507, respectively. The complete genome of both SCMV canna isolates were 9576 nucleotides (nt) in length excluding the poly (A) tails. Their 5’-UTRs and 3’-UTRs were 148 nt and 238 nt, respectively. The conserved potyboxes ‘a’ and ‘b’ in 5’-UTR [19CAACAC25 and C38CAAGCA44. The first in-frame AUG149–151 situated in the context CGAGAUGGC was presumed to be the initiation codon of the polyprotein translation. The ORF of SCMV canna isolate was 9192 nt, encoding a polyprotein 3063 amino acids (aa). The polyprotein of SCMV is processed into ten mature functional proteins, and the nine putative protease cleavage sites were identified (Table Johnsongrass mosaic virus (JGMV). The cleavage site at VPg/NIa-Pro junction of other SCMV isolates was E/S, while that of Canna-Ji’nan and Canna-Tai’an was E/A. E/A was also found in the cleavage site at VPg/NIa-Pro junction of Tobacco vein banding mosaic virus (TVBMV) [2 686GAAAAAA2 693) motif was also identified within the P3 protein, producing PIPO with the + 2 reading frameshift.Both of the two The complete genome of SCMV Canna-Ji’nan and Canna-Tai’an shared nt identity of 98.0% and aa identity of 98.8%. At the complete genome level, Canna-Ji’nan shared the highest nt identities of 76.5% with isolates Henan (AF495510) and SX AY569692), aa identities of 85.5% with isolates Xiangshan (AJ310103) and Yuhang (AJ310104) from China and isolate sp. (AM110759) from Spain; while Canna-Tai’an shared the highest nt identities of 76.6% with isolates Beijing (AY042184) from China and JAL-1 (GU474635) from Mexcio, aa identities of 85.6% with isolates Xiangshan (AJ310103) and Yuhang (AJ310104). At the coat protein gene level, Canna-Ji’nan and Canna-Tai’an shared nt identities of 87–91% and 87–92% with isolates from Yunnan province, China and Vietnam (FM998893-FM998896 and DQ925427-DQ925428) in GenBank, which clustered into SO group based on cp gene. The most variable gene P1 shared the lowest nt identities of 56.7–62.2% and aa identities of 51.5–55.8%, while PIPO shared the highest nt identities of 87.1–91.2% and aa identities of 85.0–92.5% among the eleven mature proteins between SCMV canna isolates and other isolates. An additional WORD file shows the nt and aa identities in more detail  as the out-group. The results showed that these 31 SCMV isolates were clustered to five groups. Group I contained 12 SCMV isolates from maize and was divided into two subgroups. The isolates of subgroup IA were collected from Europe, Africa and America, while those of subgroup IB were from China. Group II contained 11 isolates and was also divided into two subgroups. Subgroup IIA contained four isolates from maize, three from Africa and one from America; subgroup IIB contained six isolates from sugarcane in America and Asia, the other one from maize in Iran was actually a recombinant . Group I691 as thCucumber mosaic virus (CMV) [Tomato aspermy virus (TAV) [Bean yellow mosaic virus (BYMV) [Canna yellow mottle virus (CaYMV) [Canna yellow streak virus (CaYSV) [Sugarcane mosaic virus (SCMV) [Canna plants by aphids and experienced distinct evolution events. To our knowledge, this is first complete genomic sequences of SCMV SO group and also the first complete genomic sequences of SCMV infecting canna.Canna is commonly cultivated as an important ornamental plant in the world. The canna virus disease has been increasing in the past years. The viruses reported to infect canna included us (CMV) , 13, Tomus (TAV) , Bean yes (BYMV) , Canna y (CaYMV) , Canna y (CaYSV)  and Sugas (SCMV) . We hypoAdditional file 1:SCMV primer sequences. (DOC 16 kb)Additional file 2:SCMV nucleotide and amino acid sequence identities. (DOC 102 kb)


The hantavirus envelope glycoproteins Gn and Gc mediate virion assembly and cell entry, with Gc driving fusion of viral and endosomal membranes. Although the X-ray structures and overall arrangement of Gn and Gc on the hantavirus spikes are known, their detailed interactions are not. Here we show that the lateral contacts between spikes are mediated by the same 2-fold contacts observed in Gc crystals at neutral pH, allowing the engineering of disulfide bonds to cross-link spikes. Disrupting the observed dimer interface affects particle assembly and overall spike stability. We further show that the spikes display a temperature-dependent dynamic behavior at neutral pH, alternating between ‘open’ and ‘closed’ forms. We show that the open form exposes the Gc fusion loops but is off-pathway for productive Gc-induced membrane fusion and cell entry. These data also provide crucial new insights for the design of optimized Gn/Gc immunogens to elicit protective immune responses. Hantaviruses infect rodents and other small mammals, but do not harm them. When transmitted to humans, often through rodent urine, feces or saliva, they can cause serious and even fatal diseases. Currently, there are no known methods that effectively prevent hantavirus infections or treat the diseases that they cause.During an infection, viruses invade the cells of their host. A hantavirus interacts with target cells through proteins on its surface called Gn and Gc glycoproteins. Previous work has shown that these glycoproteins are organized in bundles of four Gn and four Gc proteins, termed spikes, which project from the membrane that surrounds the virus. The Gc protein changes shape when it is activated and exposes a hidden region that can insert into the membrane of the target cell. The Gc proteins then change shape again to force the cell to fuse with the viral membrane. This process allows the virus to be taken up into the cell, where it can replicate.While the structures of each viral glycoprotein have been determined in isolation, it was not known how they interact within the Gn/Gc spike. Such information is crucial to understand how the viruses infect cells and which areas are exposed to the immune system of the host – and so could be targeted by antiviral treatments.Bignon et al. have now identified the molecular contacts that occur between spikes and interconnect them into a grid-like lattice on the surface of the virus. Genetically altering specific sections of the Gc glycoprotein strengthened or weakened these contacts, which correspondingly increased or decreased how stable the spike was. Preventing the contacts from forming resulted in cells releasing fewer virus-like particles.Bignon et al. also show that at the body temperature of mammals, the shape of the spike fluctuates between an ‘open’ form that exposes the region of Gc that inserts into the cell membrane, and a ‘closed’ form that hides this region. However, when Gc is activated, the open form becomes unable to cause the viral and cell membranes to fuse together.Together, the results presented by Bignon et al. help us to understand how changes to the hantavirus surface enable the virus to infect cells. This knowledge will help researchers to design vaccines that protect against hantavirus infections. Bunyavirales, family Hantaviridae, genus Orthohantavirus) persistently infect rodents throughout the world. When transmitted to humans they can cause serious disease such as hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS) with case fatalities up to 10% and 40%, respectively  segment encodes a membrane-anchored polyprotein precursor GPC, which is processed by host cell signal peptidases to generate glyoproteins Gn and Gc 3 spikes at the surface of alphavirus particles   revealedarticles   and 5.6 Å (Gly187-Gly187) . Because4 spikes at 20˚C and neutral pH, the detergent-solubilized spike was identified as a single band recognized by both, anti-Gn and anti-Gc MAbs , of the Gc post-fusion homotrimer (see migration at acidic pH), and of the standard reference bands at 480 and 720 kDa. In contrast, in the detergent-solubilized G187C VLPs the band containing both, Gn and Gc, barely entered the gel at temperatures up to 40˚C, indicating a high molecular mass, as expected if a disulfide bond interconnects multiple adjacent spikes (We compared the spikes of the G187C mutant VLPs to the wild type VLPs in terms of their migration profile in blue native PAGE (BN-PAGE) combined with western blotting upon detergent-solubilization of the spikes. When we incubated Andes VLPs bearing wild type (Gn/Gc)-Gc MAbs . This bat spikes .2 per subunit, which is a relatively small contact patch  of 37.7 ± 0.4°C. In comparison, the Gn/Gc band of the G187C VLPs did not dissociate into homooligomeric Gn or Gc species up to temperatures of 45°C, revealing a 10˚C increase of its Tm to 48°C  species . The abs to 48°C . The dis to 48°C . With thnterface . In the We also monitored the effect of mutations at the Gc:Gc inter-spike interface on the pH required for spike activation for membrane fusion. Among all Gc:Gc interface mutants, only Gc E26Q induced syncytia formation of cells expressing the mutant Gn/Gc construct upon incubation at pH 5.5, retaining ~50% of the fusion activity of the wild type . All othIn order to monitor the activation step allowing interaction with membranes of wild type and Gc:Gc interface mutant VLPs, we carried out liposome co-flotation studies at pH values ranging from 5.0 to 6.4. For this purpose, we mixed fluorescently labeled liposomes with the VLPs at each pH, and loaded the mixture to the bottom of a sucrose step gradient. After centrifugation, we monitored each fraction for the presence of liposomes (by fluorescence) and VLPs (by western blot against Gc). At pH 6.2, the liposomes migrated to the top of the gradient while the wild type VLPs remained in the bottom fractions , but incWhen we analyzed the H303F mutant for pH-induced liposome coflotation, we found that it was more resistant to activation and required a 0.2 units lower pH for fusion activation compared to wild type , opposit> 37°C was specifically induced by the Gc fusion loops, and not by non-specific interactions, we tested liposome coflotation of Andes VLPs bearing the W115A/F250A mutations in Gc. These two substitutions of aromatic residues to alanine, respectively at the tip of the cd and ij loops (which are two of the three fusion loops of Gc), do not interfere with VLP formation but were previously shown to abolish insertion into target membranes at low pH  and incubated them with liposomes. We observed R18 dequenching upon dropping the pH to 5.5, which indicated lipid mixing between the labeled VLPs and the unlabeled liposomes, as described previously . In turnTo test the validity of our observation across hantaviruses, we produced Sin Nombre VLPs as well as Hantaan VLPs and labeled them with R18. The labeled VLPs gave a clear lipid mixing signal upon acidification , which wTo understand the molecular basis of the drop in viral infectivity and membrane fusion after treatment of hantavirus particles at high temperature, we tested whether the temperature treatment would still allow for Gc homo-trimerization towards the post-fusion form, as a measure of an early step in the virus-cell fusion mechanism. We thus incubated Andes VLPs at different temperatures and assayed them for low-pH induced Gc trimerization by sedimentation on a sucrose gradient. As expected, this method allowed the detection of Gc running as monomer at pH 7.4 and homotrimer at pH 5.5 when using untreated VLPs. But when we pre-treated the VLPs at 50°C and then incubated them at pH 5.5, we found Gc migrating predominantly as monomer. The temperature-treatment of the VLPs thus resulted in a form of Gc unable to undergo trimerization at acidic pH to induce membrane fusion . When VL4 hetero-octameric spikes associate laterally via 2-fold related Gc:Gc contacts contacts  akin to contacts . An asse pattern  and with pattern . Contrar pattern  or when  pattern . These o pattern . Therefoe family . Our rese family , implyin2 of the 2-fold related hantavirus Gc:Gc  (tegrins)  that couSimilar conformational dynamics have been observed for a number of unrelated viruses . It has ANDV isolate CHI-7913  kindly . All worFor ANDV Gn/Gc expression we used the plasmid pI.18/GPC that codes for the full length GPC of ANDV strain CHI-7913 under the control of the cytomegalovirus promotor . Site-diFor Gn/Gc expression, 293FT cells (Thermo Fisher Scientific) were grown in 100 mm plates and calcium-transfected with the corresponding GPC encoding plasmid. 48 h later, cell surface proteins were labeled with biotin in order to separate the biotinylated (surface proteins) from non-biotinylated (intracellular proteins) fractions using a cell surface protein isolation kit (Pierce). For protein detection by western blot, primary anti-Gc monoclonal antibody (MAb) 2H4/F6 , anti-GnMAX is the maximal dissociation value, Tm is the temperature at 50% Gn/Gc dissociation and b is the Hill’s slope of the curve, indicating its steepness. The curve was fitted using a sigmoidal four parameters equation in SigmaPlot 12.0, Systat Software.The discontinuous native protein gel electrophoresis was performed similar to as previously described . BrieflyLiposomes were prepared fresh by the freeze-thaw and extrusion method . PC pho. First, MAX is the maximal VLP coflotation percentage, Tm is the temperature at 50% VLP coflotation and b is the Hill’s slope of the curve. The curve was fitted using a sigmoidal four parameters equation in SigmaPlot 12.0, Systat Software.The western blot signal was quantified using the ImageJ software  and VLP-ANDV was incubated at different temperatures for 15 min and subsequently added to Vero E6 cells (MOI = 1). After 1 h of adsorption at 4°C or 37°C, the cells were washed in excess and next infection was allowed to proceed for 16 h by incubation in MEM 10% FBS at 37°C. Quantification of viral infection was performed as previously described  by detecFor the lipid mixing assay, VLPs were labeled with 1 μg/ml of R18 (Invitrogen). Labeled VLPs were then mixed with liposomes in a continuously stirred fluorimeter cuvette at 37°C and lipid mixing was monitored by the decrease in R18 fluorescence generated by the dilution of the R18 probe with the unlabeled phospholipids in the liposome membrane. Fluorescence was recorded continuously at 580 nm using a fluorescence spectrophotometer  at an excitation wavelength of 560 nm using a 10 nm slit width for excitation and emission. After a stable base line was established at pH 7, it was subtracted from recording and established as base line value corresponding to 0% lipid mixing. The reaction initiation time (t = 0) corresponds to the lowering of the pH to 5.5. The maximal extent (100%) of excimer dilution was defined by the addition of Triton X-100 0.1% (v/v) after lipid mixing of each condition had concluded.Acid-induced Gc homotrimerization was tested as established before . VLPs coThis three-color fluorescence assay was performed as previously described . Vero E6All statistical analyses were carried in GraphPad Prism, version 6, and SPSS software .For protein structure analyses and graphics PyMOL Molecular Graphics System Version 2.0  was used. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by Neil Ferguson as the Senior Editor, a Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your article "Molecular organization and dynamics of the fusion protein Gc at the hantavirus surface" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Guided by structural information, Bignon and colleagues subjected the Andes virus (ANDV) Gn/Gc spike to mutagenic analysis in order to identify Gc residues important for contact between spikes. They show that the interface between two Gc proteins in their pre-fusion form, as detected previously by crystallography, is the same one that mediates Gc:Gc contacts between adjacent spikes on the virion surface. Specifically, they demonstrate that disulfide bonds can be formed between certain residues located at this interface and that an intact interface is required for assembly of virus-like particles. Moreover, residues located at the interface are shown to affect spike stability and responsiveness to low pH. Finally, they demonstrate that the fusion loop in Gc can be reversibly exposed at elevated temperatures – the spike transits between an open and a closed form – and that the open form is not compatible with low pH induced membrane fusion activation.Essential revisions:All three referees agree that the study is of interest and that the study provides reasonably solid experimental support for the hypothesis that the Gc:Gc intermolecular contacts observed in the crystal structure of Hantaan virus Gc represent physiologically relevant intermolecular contacts that contribute to the assembly and stability of hantavirus particles. Moreover, the observations that thermal destabilization of Andes virus or VLPs from other hantaviruses causes a conformational change that exposes the fusion loop and inhibits low pH-dependent cell entry will be interest to researchers studying hantaviruses and potential treatments or vaccines against them. However, for the study to be acceptable the following points need to be addressed:1) The authors claim that the Gc:Gc dimer interface is "required for VLP assembly" (subsection “The inter-spike Gc dimer contacts are required for VLP assembly”). However, in Figure 3, seven of the eleven Gc:Gc interface mutants tested positive for Gc in the "VLP" cell supernatant fraction, indicating that these mutants can still form VLPs. When corrected for actin expression, the expression of Gc in these mutants does not appear significantly impaired, contrary to the authors' claim. Thus, in order to substantiate the author's conclusions, immunoblots should be quantified and averages +/- SD should be presented. Additionally, of the four mutants that failed to generate VLPs, three showed no Gc expression at all, indicating that the mutations prevented Gc expression and/or folding rather than VLP assembly per se, as claimed in subsection “The inter-spike Gc dimer contacts are required for VLP assembly”. Only one mutant, H303E, was expressed in cells but did not produce VLPs. Hence the claim that "Gc dimer contacts are required for VLP assembly" is unconvincing and not supported by the data presented. The authors should remove this claim (and related claims) perhaps replacing it with "…contacts are required for Gn/Gc assembly, or glycoprotein spike assembly".Alternatively, the authors would need to provide additional evidence to support their original claims, such as one or more pairs of interacting charge reversal mutations that rescue VLP assembly whereas each individual charge reversal mutation from one any one pair is deficient for VLP assembly but not for Gc expression in cells.2) Discussion of the results and representation of the literature:In the Introduction the authors quite sparsely cite studies by other groups, and in the Discussion previous knowledge and hypotheses by other groups are not appropriately considered. For instance, the authors do not discuss the findings presented in Hepojoki et al. (2010), especially since it contains studies on covalent bonding between hantavirus glycoproteins. In addition to the work by other groups mentioned in the introduction, the authors may consider a recent paper by Sperber et al. (2019). Also, it is unclear why the authors did not present Gc docked into the 3D structure of the spike complex (which is available from database). Does this mean that the Gc crystal structure does not fit into the spike structure, and if so, is the orientation of the domains in respect to each other as the author claim? Generally, the authors may expand the Discussion to put the results in perspective regarding previous studies and focus more on hantaviruses. Essential revisions:1) The authors claim that the Gc:Gc dimer interface is "required for VLP assembly" (subsection “The inter-spike Gc dimer contacts are required for VLP assembly”). However, in Figure 3, seven of the eleven Gc:Gc interface mutants tested positive for Gc in the "VLP" cell supernatant fraction, indicating that these mutants can still form VLPs. When corrected for actin expression, the expression of Gc in these mutants does not appear significantly impaired, contrary to the authors' claim. Thus, in order to substantiate the author's conclusions, immunoblots should be quantified and averages +/- SD should be presented.We apologize for this omission and thank the referee for their helpful suggestions. We have now included the quantification of the immunoblots +/- SD for VLP release into the cell supernatant including statistical analyses . In this figure, the amounts of VLP release of each mutant can now be compared with the corresponding signal for wild type VLPs. To ensure equal amounts of properly folded GPC for VLP release, the amount of cell surface accumulation equivalent to wild type GPC was adjusted by the amount of GPC encoding plasmid used for transfection. The corresponding western blots have been moved to Figure 3—figure supplement 1. As can be seen, each experiment includes different mutants together with the wild type as a control . A significant decrease of VLP assembly can be observed in the case of the alanine mutant D28A and opposite charge mutants  while maintaining their trafficking to the plasma membrane . In the cellular fraction, equal amounts of proteins in of cellular fraction loaded in each lane of the western blots were normalized by total protein quantification. Anti-β-actin was only used to detect plasma membrane integrity, as cytosolic protein control. All corresponding modifications have been introduced throughout the text  and the corresponding figure legends.Additionally, of the four mutants that failed to generate VLPs, three showed no Gc expression at all, indicating that the mutations prevented Gc expression and/or folding rather than VLP assembly per se, as claimed in subsection “The inter-spike Gc dimer contacts are required for VLP assembly”. Only one mutant, H303E, was expressed in cells but did not produce VLPs.This is correct. To ease the interpretation of the results, in the revised Figure 3A we now only show VLP release of those mutants that were properly expressed and reached the plasma membrane. The original western blots indicating expression/folding and VLP release of all mutants have been moved to Figure 3—figure supplement 1A-C.Hence the claim that "Gc dimer contacts are required for VLP assembly" is unconvincing and not supported by the data presented. The authors should remove this claim (and related claims) perhaps replacing it with "…contacts are required for Gn/Gc assembly, or glycoprotein spike assembly".We respectfully disagree here with the reviewer, as we are not claiming that the Gc dimer interface is required for Gn/Gc spike assembly, but rather for formation of the surface lattice that makes the VLP, via inter-spike contacts. But the reviewer’s remark make us realize that we were not clear enough in the original version of the manuscript. Our data clearly shows that substitutions by alanine or by residues bearing opposite charges, significantly decreases VLP assembly compared to wild type . Based on the helpful reviewer’s suggestions to quantify our data, and to remove mutants from the analysis that are not properly expressed, we believe that Figure 3A now is clearer in showing that “inter-spike Gc dimer contacts are required for VLP assembly”. The revised text was accordingly adjusted, explaining the results better.Alternatively, the authors would need to provide additional evidence to support their original claims, such as one or more pairs of interacting charge reversal mutations that rescue VLP assembly whereas each individual charge reversal mutation from one any one pair is deficient for VLP assembly but not for Gc expression in cells.We agree with the reviewer that charge reversal of pairs of interacting residues rescuing VLP formation would be a nice validation of our model. However, such experiments would take a lot of time, and we have shown here that engineering a cysteine residue at position G187 leads to disulfide crosslinked spikes, as expected if the dimer interface displayed in Figure 1B is the one relating two spikes. In our view, the disulfide bond is already a very strong evidence, and the additional mutagenesis only corroborates this result, by indicating that mutation of the other residues displayed in Figure 1B impacts the ease with which VLPs are formed. In other words, we agree the results reported in this session  alone are not convincing evidence that this is the dimer interface, but in combination to the disulfide bond explained in the previous section , they are. We therefore believe that the additional experiments suggested are not necessary.2) Discussion of the results and representation of the literature:In the Introduction the authors quite sparsely cite studies by other groups, and in the Discussion previous knowledge and hypotheses by other groups are not appropriately considered. For instance, the authors do not discuss the findings presented in Hepojoki et al. (2010), especially since it contains studies on covalent bonding between hantavirus glycoproteins.We had cited the work of Hepojoki et al. (2010) in the Introduction of the original submission. To address this comment, we have now better highlighted the major findings of that paper in the revised Introduction:“The work of Hepojoki et al. (2010) revealed that Gn/Gc species can be covalently crosslinked on the surface of virions and suggested oligomeric models for spike assembly based on the characterization of detergent-solubilized spikes.”In addition to the work by other groups mentioned in the introduction, the authors may consider a recent paper by Sperber et al. (2019).We have now included this very recent paper in the Introduction of our manuscript as follows:“In this line, a central role of Gn in self-association has recently been confirmed by number and brightness analysis in live, single cells showing that separate Gn expression allows detection of Gn oligomers while separate Gc expression predominantly leads to Gc monomers and some Gc dimers .”Also, it is unclear why the authors did not present Gc docked into the 3D structure of the spike complex (which is available from database). Does this mean that the Gc crystal structure does not fit into the spike structure, and if so, is the orientation of the domains in respect to each other as the author claim?We thank the reviewers for raising this issue. In our paper, we limit ourselves to the functional validation of the data observed in the crystallographic structure. Although crystal packing contacts have to be taken with caution, our data are further supported by the structural homology of Gc with alphavirus spikes, which interact in a similar way via the glycoprotein E1. Crystal contacts have also been shown to be valid in other cases, such as the Ebola virus matrix protein, where contacts about a crystallographic 2-fold axis were later found to be indeed involved in particle assembly .Generally, the authors may expand the Discussion to put the results in perspective regarding previous studies and focus more on hantaviruses.We now included additional discussion of our results concerning the biochemical approach in the field  and hantavirus infectivity in dependence of time and temperature . The discussion also includes comparison with structural information , functional approaches  and cell entry studies . We believe that this literature comprises the most relevant previous hantavirus studies related to this work. Given the similarity of hantaviruses to other viruses with class II fusion proteins or with viruses with a highly dynamic surface behavior, the comparison with those additional models is properly justified and helps drawing of future directions.


Understanding the spatial dynamics of oral polio vaccine (OPV) transmission will improve resource targeting. Mexico provides a natural laboratory, as it uses inactivated polio vaccine routinely as well as OPV bi-annually.Using geospatial maps, we measured the distance and density of OPV vaccinees’ shedding in the areas nearest to unvaccinated households in 3 Mexican villages. Comparison of transmission to unvaccinated households utilized a mixed effects logistic regression with random effects for household and time, adjusted for age, gender, area, and running water.The median distance from an unvaccinated household to its nearest OPV-shedding household was 85 meters  and the median number of vaccinees shedding OPV within 200 m was 3 (2–6). Transmission to unvaccinated households occurred by day 1. There was no association  between the distance from OPV shedding and the odds of transmission. The number of OPV vaccinees shedding within 200 m came close to a significant association with unvaccinated transmission , but this was not the case for households 100 or 500 m apart. Results were consistent across the 3 villages.Geospatial analysis did not predict community transmission from vaccinated to unvaccinated households, because OPV use resulted in rapid, low transmission levels. This finding supports the global cessation of OPV. The goal of global polio eradication may soon be reached. In 2016, there were 37 cases of wild-type polio, a decrease from the 87 cases in 2015 [To date, the spatial characteristics of OPV transmission are not well characterized. Understanding the pattern and extent of geographic variation in OPV circulation could help to predict and prepare for the risk of OPV reintroduction, especially in undervaccinated communities. Areas of increased risk could be detected, and resources accordingly deployed to reduce or prevent prolonged OPV circulation. In the current global setting of polio transmission, this is particularly important for serotype 2, which was declared eradicated as of September 2015 by the World Health Organization .In this study, we had the opportunity to identify household and community transmission of OPV in Mexico, where inactivated polio vaccine (IPV) is provided for routine immunization at 2, 4, 6, and 18 months of age and OPV is only administered in 2 National Health Weeks (NHW) to children 5 years of age and under. We were able to investigate spatial transmission at a household level within 3 villages that received different levels of OPV vaccinations during the February 2015 NHW. Furthermore, we collected stool samples that allowed for the detection of OPV transmission from vaccinated children to their households and community contacts. Spatial analyses of polio transmission have been performed as far back as 1967 . This stIn this paper, we explore 2 aspects of between-household transmission of polio. First, we consider whether living near someone who is shedding poliovirus affects an individual’s chance of shedding poliovirus. Second, we determine how the number of people shedding near an unvaccinated contact impacts that individual’s chance of acquiring and shedding poliovirus. We refer to the distance and density of shedding, collectively, as proximity to shedding.The study has already been described in detail elsewhere  but, in After enrollment, 10 stool samples were scheduled for collection from each member of all enrolled households: 1 baseline sample before vaccination and 1 sample each at 1, 4, 7, 10, 14, 21, 28, 51, and 71 days after vaccination. During each visit, health information, travel and visit details, and records for any vaccines received during the study period for children <5 were collected via follow-up surveys. Exclusion criteria for follow up were individuals that refused to participate, changes in residence during the study period, or absence during follow-up visits.Viral ribonucleic acid (RNA) was extracted from frozen stool samples utilizing the MagNA Lyser (Roche) and KingFisher Duo Prime (Fisher Scientific), using the bacteriophage MS2 as an internal control for extraction efficiency. Viral RNA then underwent quantitative, reverse-transcription polymerase chain reaction (rt-QPCR) in order to detect and quantify any Sabin OPV present in the samples. The probes and primers were adopted and adapted from Kilpatrick et al.  and the We used 2 types of participants from the study: vaccinated individuals and individuals who lived in unvaccinated households, referred to here as unvaccinated individuals. We were interested in the outcomes of unvaccinated individuals and their proximity to vaccinated individuals. Therefore, vaccinated participants were not analyzed directly; instead, we created spatial variables for unvaccinated participants by comparing the locations of vaccinated and unvaccinated households. For instance, we measured the distance from an unvaccinated person to their nearest vaccinated household. We did not include unvaccinated people who lived in vaccinated households, as the focus of our analyses was between-household transmission. Within-household transmission is being analyzed separately . VaccinaMaps were used to visualize the spatial distribution of vaccination and shedding. We represented the density and location of vaccinated shedding over time using contour plots and overlaid the position of unvaccinated participants.The distributions of key variables were assessed graphically and through the calculation of summary statistics. Spatial variables were summarized using medians and interquartile ranges, as they were skewed. Coverage of the vaccine was considered important to adjust for, as higher coverage led to both a smaller distance to the nearest shedding household and a higher proportion of shedding.Stool samples were collected from participants at 10 points in time. We only analyzed data from the first 28 days of the study, as almost all the shedding occurred before this point. We first looked at the presence of poliovirus in stool samples and the proximity of unvaccinated partipants to vaccinated participants’ shedding at any time point in the first 28 days. Second, we considered the presence of poliovirus and the proximity of households at each given point in time.For the first analysis, the outcome was a binary variable for shedding at each point in time throughout the study period. We used a spatial variable approach, as identified in a recent systematic review . The appFor the second analyses, we calculated the same spatial variables, but within each point in time; therefore, the distance to the nearest vaccinated, shedding household could change depending on the study day. We also calculated the spatial variables using calendar days after initiation of the NHW OPV administration, and found no differences in the outcomes. In addition to this, we examined whether the amount of the viral load surrounding an unvaccinated household affected the likelihood of shedding. Here, we used the sum of the log viral load, where a greater number represents a higher viral load and a lower number a lower viral load.The study is a cluster-randomized trial with a hierarchical structure, giving multiple observations per individual per household. Mixed-effects logistic regression with a random effect for household was used to assess the association of the distance and density of vaccinated shedding with unvaccinated shedding over the 28-day period. A mixed-effects logistic regression with a random effect for household and an autoregressive lag 1 random effect for study day was used to look at the impact of the distance and density of OPV shedding and viral loads. The autoregressive random effect allows the previous study period to provide information to the next period. We adjusted for participant age and gender and for access to running water within the household.The spatial variables were included as continuous variables. Non-linearity was explored using quadratic terms and treating the variables as categorical; there was no suggestion of non-linear effects. The analyses were repeated by Sabin type: this increased the sparsity of the data and those results are not included, as they were consistent with the main analyses. The models were fitted using integrated, nested Laplace approximation,  and all There are 1145 unvaccinated individuals included in the analyses, from 286 households. The age distribution was positively skewed, with a median age of 17 years and a range from 1 month to 95 years. Female participants made up 58.1% of the study. Further details, stratified by community, can be seen in In unvaccinated individuals, there were 89 (0.9%) positive samples, which came from 80 (7.0%) individuals; only 6 people contributed more than 1 positive sample. The number of positive samples varied over time, with 24 positive samples observed at day 7, 2 at day 10, and 8 at day 14. More details at each point in time by locality can be found in the In the original study, 155 children were vaccinated. Of these, 137 (88.4%) had GPS data that was used to calculate the spatial variables; the remaining 18 did not have GPS data and were not used in the analyses, as we do not know their locations. The locations of the households, as well as the participants intervention and outcome statuses, are displayed in The vaccinated children provided 1237 samples, of which 342 (27.6%) were positive. There were 108 vaccinated children who shed OPV at any point in the study.An analysis of the samples collected at baseline confirmed that no children <5 were shedding before the study began. Shedding began quickly, and between-household transmission occurred on the first study day in some cases. In addition, the vaccinated children tended to shed more consistently throughout the study, with 36 (26.3%) individuals providing 4 or more positive samples and 2 individuals providing 8 positive stool samples. This can be seen in The median distance from unvaccinated participants to vaccinated, shedding participants during the first 28 days was 85.0 m (46.0 to 145.0 m), with a breakdown by village given in After adjusting for age, gender, and access to running water, there was very little suggestion  of an association between distance (per 100 meters) from a vaccinated, shedding household and the odds of shedding for an unvaccinated individual. This was consistent when considering non-linear effects of distance. Incorporating time into the analysis with an autoregressive lag of 1 resulted in comparable results, with an OR of 1.04 (CrI 0.92–1.16). There was also no indication that the density of vaccinated household shedding within 200 m of an individual affected their odds of shedding . When including time, there was some suggestion that the number of shedding, vaccinated individuals within 200 m may have had some effect on the shedding of unvaccinated individuals . Results for other distances are displayed in Through visualization of transmission onto maps, we were able to determine the dynamics of geospatial OPV transmission in a community with primary, IPV-induced immunity. We found that shedding due to the introduction of an oral polio vaccine (OPV) occurred rapidly and was associated with between-household transmission on the first day of OPV vaccination. We found little evidence to suggest that living nearer to a household with a person who was shedding OPV affected the likelihood of shedding OPV, up to the village dimensions of 850 meters. Indeed, there were no statistical differences in OPV acquisition among unvaccinated individuals based on their distance from vaccinated individuals. In addition, the threshold for OPV dispersion appeared to be low: between-household transmission in the 10% and 70% vaccination coverage communities were similar and, therefore, only a small amount of OPV appeared to be needed for community transmission of OPV. This raises important implications regarding the impact of using OPV in future outbreaks and vaccination campaigns, especially as the transmission of OPV would usually be undetected, at least in highly-vaccinated communities.There are several strengths of this study. First, the study included a large amount of individual-level data, with multiple observations per person. To the authors’ knowledge, previous spatial analyses have only been conducted at an aggregated level on wild-type polio, where the detection of cases was not through stool samples. As no other children were vaccinated with OPV in these communities until May 2015, we know the precise sources of OPV in these communities during the study period, giving us insight into what happens when OPV is administered a single time. Second, we were also able to consider the impact of variation of coverage, as 3 separate villages had 10%, 30%, and 70% vaccinated cover of children. Third, as a requirement of the study was that all vaccinated children had up-to-date IPV vaccinations, this data mimics the transmission environment in future settings, as the Polio Endgame requires at least 1 dose of IPV in routine immunization schedules globally [Using household location to represent a person’s location is, at best, an imprecise average of their movement throughout the day. Furthermore, it necessitates grouping people who live together to the same location. Information from contact tracing might have been useful to measure the proximity of individuals, but might not be available in practice. It seems clear that the transmission of polio is not purely spatial; when only household location is available, it appears to have a limited ability to predict transmission.We tried to minimize any misclassification of within-household transmission as between-household transmission by only using vaccinated individuals as point sources and unvaccinated households in the analyses. We excluded individuals who were unvaccinated and living with vaccinated individuals. Misclassification cannot be removed entirely, due to the propagation of between- and within-household transmission. We also attempted to reduce the number of false positives for stool samples by using a 2-step laboratory process to identify OPV by RT-QPCR; however, it is likely that there were small numbers of false negative and false positive samples. However, given the large number of individuals and stool samples collected, it is unlikely that a small number of incorrectly-identified samples would have affected the conclusions of this paper.Our results show that vaccinated children shed as early as 1 day post-vaccination. This result is supported by prior OPV trials, where most vaccinated children shed within 1 week of vaccination . That OPHousehold locations and spatial distribution appear to have limited use in predicting the transmission of poliovirus shedding. Therefore, in order to understand how OPV shedding occurs within a community, alternative information, such as contact patterns, should be analyzed. The mechanism for predicting shedding is not well understood, including the role of number of prior IPV and OPV doses and the viral loads seen after vaccination.The use of a live poliovirus vaccine results in rapid dispersion and persistent transmission of the poliovirus throughout a community, up to at least 71 days. The only way to avoid this is to not use OPV or to have strong controls around those vaccinated, such as quarantine or strict hygiene protocols. At present, what we observed in this study would not be detected through any clinical screening, since all transmissions were asymptomatic and detected by the analyses of prospectively-collected stool samples. Therefore, better methods, such as the collection and analysis of sewage samples, are critically needed to ensure shedding has stopped within a community. Without this, any conclusions about the eradication of circulating polioviruses are overconfident at best. These results further support the Polio Eradication and Endgame Strategic Plan 2013–2018’s intention to withdraw all OPV vaccines by 2020 [Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary_Table_1Click here for additional data file.


Climate change can have a pronounced impact on the physiology and behaviour of fishes. Notably, many climate change stressors, such as global warming, hypoxia and ocean acidification (OA), have been shown to alter the kinematics of predator–prey interactions in fishes, with potential effects at ecological levels. Here, we review the main effects of each of these stressors on fish escape responses using an integrative approach that encompasses behavioural and kinematic variables. Elevated temperature was shown to affect many components of the escape response, including escape latencies, kinematics and maximum swimming performance, while the main effect of hypoxia was on escape responsiveness and directionality. OA had a negative effect on the escape response of juvenile fish by decreasing their directionality, responsiveness and locomotor performance, although some studies show no effect of acidification. The few studies that have explored the effects of multiple stressors show that temperature tends to have a stronger effect on escape performance than OA. Overall, the effects of climate change on escape responses may occur through decreased muscle performance and/or an interference with brain and sensory functions. In all of these cases, since the escape response is a behaviour directly related to survival, these effects are likely to be fundamental drivers of changes in marine communities. The overall future impact of these stressors is discussed by including their potential effects on predator attack behaviour, thereby allowing the development of potential future scenarios for predator–prey interactions. Lay summary: Climate change in the aquatic environment is leading to global warming, OA and an increase in hypoxic areas and events. Here, we review the effects of these three major stressors of climate change on the escape response of fishes and their implications for predator–prey relationships. The indirect effects are related to the well-known relationship between temperature and the general condition of the fishes, i.e. growth rate (i.e. size), condition factor, muscle content, development and reproductive status . All of Carassius auratus) tend to show greater compensation in escape swimming  than species with a narrow thermal range (e.g. killifish Fundulus heteroclitus) after 4 weeks of acclimation (Poecilia reticulata) and Mediterranean grey mullets (Liza aurata), the escape speeds of fish acclimated  to each temperature did not differ, as a result of complete compensation (Cyprinus carpio), 2 weeks of acclimation can induce changes at the anaerobic muscle fibre level and extend the range of temperature for escape swimming but do not result in full compensation (Macquaria ambigua) tested within a range of 10–25°C after 3 weeks of acclimation, escape swimming performance decreased significantly at temperatures below approximately 15°C but was relatively constant between 15 and 25°C (Gambusia holbrooki) escape speeds did not differ between spring and summer fish at their natural water temperatures of 15 and 25°C, respectively , while acclimation can compensate for the effect of temperature on escape swimming to a large degree when temperatures change slowly, such as during seasonal temperature shifts in mean temperature (limation . Similarectively  and can C. auratus) (77 mm in body length) were not affected by increases in viscosity at constant temperature, while escape speeds in smaller guppies (Poecilia reticulata) (22 mm in body length) were reduced (Brachydanio rerio) (3.3 mm in body length), by significantly decreasing displacement, maximum velocity and acceleration during stage 1 (i.e. the first muscle contraction) of the escape response; however, the viscosity values used were outside the range of viscosity variation caused by temperature changes in nature  for 4 weeks at 5, 15 or 25°C and found that the higher temperature yielded the shorter latencies, suggesting that these effects were due, at least in part, to higher conduction velocity in the Mauthner axons of warm-acclimated populations (C. auratus) (C. auratus) to an approaching threat compared to the control level (15°C), likely as a result of the malfunctioning of the neurosensory system  . These sauratus) . Similary system .Pomacentrus moluccensis and Pomacentrus amboinensis), directionality returned to control levels after a longer term (90 days) acclimation only in P. moluccensis  exposure to high temperature was found to decrease directionality away from the threat in two species of coral reef fishes  on fish prey in coral reef species (Pomacentrus wardi)  had higher attack rates on mosquitofish (Gambusia holbrooki) than cold acclimated ones, and higher prey capture success. Interestingly, laboratory trials showed that the burst swimming performances of acclimated seabass did not differ between warm and cold temperatures, indicating that muscle function was maintained through acclimation, thus low capture rates at low temperature were likely to be due to decreased motivation to catch prey as a result of lower food requirement (Plectropomus leopardus), from the Great Barrier Reef —brown trout  interactions suggests that changes in relative speed performance were at the basis of the effect of long-term temperature exposure (3 months) on the outcome of the interactions  feeding in the winter economize energy expenditure by halving the time spent at sea, and halving the number but doubling the mass of each prey fish taken (Mirounga leonina) may target cold waters to facilitate the capture of fish prey that are less active at low temperatures  may impact other aspects of swimming (e.g. aerobic swimming), which can trade off with anaerobic swimming , cumulative distance and maximum swimming speeds were found to be reduced following a few hours of exposure to acute hypoxia  and their performance did not differ from that of fish in normoxia (Dicentrarchus labrax) (C. carassius)   or cruciCtenopharyngodon idellus) showed a decrease in maximum escape velocity only in normoxia-acclimated fish exposed to hypoxia, while no change was observed when fish were reared in similar hypoxic conditions (Marcusenius victoria) for 8 weeks to high and low dissolved oxygen (i.e. 20% of air saturation) before their escape swimming was tested under both these normoxic and hypoxic conditions. The results showed that the hypoxia-acclimated fish had shorter escape displacement when tested under hypoxia than in normoxia, while displacement of normoxia-acclimated fish was not affected by the test condition (Pseudocrenilabrus multicolor victoriae) (D. labrax and L. aurata exposed to an acute decrease of oxygen whether this latter species had access to the surface or not (Solea solea) facing a progressive decrease in oxygen down to 15% air saturation over c. 1.5 h (Ctenopharyngodon idellus) (Haemulon plumieri), even if hypoxic conditions were less severe  showed no effect when exposed to acute or chronic hypoxia, suggesting that the species-specific differences in hypoxia tolerance are an important factor  and air breathing (AB) are performed by some fish species in order to avoid hypoxic conditions in the water column . IrregulScomberomorus niphonius) (Morone saxatilis) and adult naked goby (Gobiosoma bosc) (Chrysaora quinquecirrha) (Aurelia aurita) (Egretta garzetta) were observed to take advantage of the ASR behaviour some lagoon fish undertake in the morning, when their surfacing in hypoxic waters (i.e. due to daily fluctuation in oxygen levels) makes them easy prey for birds  . Indeed,While these potential scenarios are generally supported by previous work on predator–prey interactions in hypoxia, it is worth noting that hypoxia can also be associated to some behavioural strategies by the predators or the prey to circumvent the constraints imposed by hypoxia. For example, while predation by fish tends to decrease in hypoxia, certain fish species living in stratified waters routinely undertake brief foraging forays into hypoxic habitats to catch their invertebrate prey, suggesting that foraging in hypoxic waters may be common in fish when prey abundance is low in surface waters , and it 2 will exceed 900 ppm by 2100 , despite the finding of an increase in aerobic scope with increased CO2 (2 (~900 μatm) decreased escape locomotor performance (in terms of escape distance within a fixed time) (2 negatively affected the escape swimming (mean and maximum speed) of the cinnamon clownfish (Amphiprion melanopus)  [exposure period of 21 days post-hatch (~1000 μatm)] (2 exposure (~900 μatm for 7 days) on escape distance, but there was a significant effect on escape speed in the Ward’s damselfish (P. wardi) (2 exposure on the maximum bending angle and the duration of stage 1 in the escape response of marine medaka (Oryzias melastigma) following a 8–16-day embryonic exposure to two CO2 levels (~1100 and 1800 μatm)  . Similar2 exposure on escape responses appears to be related to the sensory performance that underpins successful escapes. If we consider the initial phase of a predator–prey interaction, which is often triggered by a visual cue, any process that alters this can alter the outcome of the interaction. High CO2 is known to affect the performance of various sensory channels including vision (Acanthochromis polyacanthus) after a 6–7-day exposure to high CO2 (~900 μatm)  showed no effect of high CO2 on either reaction distance or ALT  larvae showed no effect on escape latency but did exhibit a lower responsiveness to a mechano-sensory stimuli when reared in high CO2 compared to larvae reared under control conditions (2 may be due to an effect on the development of the ear (e.g. otoliths), the afferent connections in the VIIIth nerve and the inputs to the Mauthner cells.The most significant impact of high COg vision . For exa in sensu in P. amortality , althoug2 impairment of the escape response as found in some of the species investigated is unknown. It has been hypothesized that OA may induce changes in the sensory responsiveness of the Mauthner cell or other reticulospinal cells responsible for escapes  reduced the negative effects of increased CO2 exposure on fish escape responses (i.e. both locomotor and non-locomotor traits), suggesting that trans-generational acclimation may occur. However, acclimation was not complete in escape speed and absent in directionality, suggesting that parental exposure does not completely compensate for the effects of high CO2 on escape performance  can reduce associated behavioural impairments in coral reef fish (2 levels is in the olfactory behaviour of species from multiple taxa (fish and invertebrates), across multiple ecosystems, responding maladaptively to predator odours and chemical alarm cues  to elevated CO2 and on a large number of species and species interactions are needed in order to increase our predictive power of the effect of high CO2 on predator–prey interactions. Increasing our understanding of the effects of high CO2 on predator–prey interactions is fundamental because these effects can cascade and result in changes in the species composition of communities, potentially disrupting the stability of the ecosystem  and temperature (15 and 19°C) of adult Japanese anchovy Engraulis japonicus acclimated for 1 month , with the exception of the turning rate that was significantly higher at 19°C than at 15°C. 2) to test the combined effects of projected climate change on the escape response of larval kingfish . Here, embryos and larvae were reared under the different environmental conditions until testing at 21 days post-hatch. High temperature improved performance by increasing responsiveness and maximum speed and decreasing escape latency. However, these effects were largely driven by a temperature-related increase in body mass and thus a more developmentally advanced stage for those individuals exposed to the higher temperature. On the other hand, high CO2 decreased distance covered and average speed during the escape but had no effect on responsiveness nor escape latency  and predatory dottybacks (P. fuscus) to control or elevated CO2 (~405 and 930 μatm) and temperature (27 and 30°C) conditions for 7 days in a 2 × 2 design. Detailed examination of the interaction between predator and prey found that high temperature had an overwhelming effect on the escape behaviour of the prey  compared with the combined exposure to elevated CO2 and high temperature (significant interaction effect on escape speed), or the independent effect of elevated CO2 (which affected only escape speed)  . Exposur2, CO2 or temperature. In the only study to date to attempt to investigate the effect of diel cycles of CO2 on fish anti-predator performance (Acanthochromis polyacanthus) was examined. Fish were reared for 11 weeks in two stable (450 and 1000 μatm) and two diel-cycling elevated CO2 treatments (1000 ± 300 and 1000 ± 500 μatm) at both current-day (29°C) and projected future temperatures (31°C). No interaction between CO2 and temperature for any performance characteristics was found, though survival was affected. Survival was lower in the two cycling CO2 treatments at 31°C compared with 29°C, but did not differ between temperatures in the two stable CO2 treatments. While temperature had significant independent effects on escape performance traits, there was no effect of stable or cycling CO2 levels nor was there an interaction between temperature and CO2.These findings support the idea that water temperature, confined within realistic near-future projections, has the greatest impact on escape performance compared to elevated CO2  within a more slowly changing average  temperature signal. The capacity of an organism to cope with and respond to acute changes in environmental variables may then be just as important as their capacity to adjust through acclimation to seasonally driven cycles.2 conditions is linked to the local conditions experienced (Empirical studies on various taxa that have been sampled across a species geographic range have shown that sensitivity to elevated CO2 levels on predator–prey interactions is a challenging but a much needed task in order to increase our predictive ability of the ecological effects of climate change. The importance of these effects in shaping marine communities is highlighted by the differential outcomes that climate change may have on various species and taxa of predators and prey (Modelling the effects of changes in temperature, oxygen and CO


Arabidopsis thaliana stress-induced BTB protein 1 (AtSIBP1), which encodes a nucleus protein with a BTB domain in its C-terminal side and is induced by salt and other stresses. The expression of the β-glucuronidase (GUS) gene driven by the AtSIBP1 promoter was found to be significantly induced in the presence of NaCl. The sibp1 mutant that lost AtSIBP1 function was found to be highly sensitive to salt stress and more vulnerable to salt stress than the wild type WT, while the overexpression of AtSIBP1 transgenic plants exhibited more tolerance to salt stress. According to the DAB staining, the sibp1 mutant accumulated more reactive oxygen species (ROS) than the WT and AtSIBP1 overexpression plants after salt stress. In addition, the expression levels of stress-induced marker genes in AtSIBP1 overexpression plants were markedly higher than those in the WT and sibp1 mutant plants. Therefore, our results demonstrate that AtSIBP1 was a positive regulator in salinity responses in Arabidopsis.Because they are sessile organisms, plants need rapid and finely tuned signaling pathways to adapt to adverse environments, including salt stress. In this study, we identified a gene named  In nature, plants must cope with different kinds of abiotic stress, such as high temperatures, cold, salt, drought and osmotic stresses; salt stress is one of the most common and severe stresses . High saArabidopsis, there are more than 1400 genes that are predicted to encode E3 ubiquitin ligase enzymes [In  enzymes , which c enzymes . Among t enzymes ,8. The c enzymes . The BTB enzymes . A BTB d enzymes . The BTB enzymes ,13.Arabidopsis NPR1 (non-expressor of pathogenesis related genes 1) protein that contains the BTB domain is a key regulator at the intersection of the ISR (induced systemic resistance) and SAR (systemic acquired resistance) signaling pathways [Arabidopsis [Arabidopsis BTB/POZ-MATH (meprin and TRAF homology) proteins (BPMs) have been reported to participate in ABA responses such as germination and stomatal closing [BTB plays an important role in plant growth, development, stress resistance, protein ubiquitination and degradation, cytoskeletal composition, ion channel, and the regulation of the cell cycle ,15. Prevpathways ,17,18. Mbidopsis . Six Ara closing . ATHB6,  closing . The dis closing . TherefoAtSIBP1  that belongs to the Skp1 or ElonginC families. The expression of AtSIBP1 was induced by salt and other stresses. The overexpression of AtSIBP1 exhibited an enhanced tolerance to NaCl, while the sibp1 mutant was sensitive to NaCl. The expression levels of stress-responsive genes were significantly increased in AtSIBP1 overexpression lines. Our analysis demonstrates that AtSIBP1 acts as a positive regulator in salt stress responses. Here, we found a novel BTB domain-containing gene called Arabidopsis eFP (electronic Fluorescent Pictograph) browser, we noticed a novel gene (At1g55760) that is predicted to respond to salt and other stresses and is also a BTB-containing protein  treatment . In this study, we focused on the functions of AtSIBP1 in response to the salt stress.Most BTB family members are involved in abiotic and biotic stress responses. In the  protein . Subsequreatment . TherefoAtSIBP1 in the response of Arabidopsis to salt stress, we isolated a homozygous AtSIBP1 T-DNA insertion mutant (SALK_075267) and generated two independent AtSIBP1-overexpression lines (abbreviated as OE1 and OE2). The mutant and the insertion site of AtSIBP1 was confirmed by PCR and sequencing, respectively   c,d. AtSIBP1, the tissue-specific expression of AtSIBP1 was first analyzed. Transgenic lines carrying proAtSIBP1::GUS (β-glucuronidase) were generated, and the T3 homozygous seeds were used for the subsequent staining experiments. Histochemical staining showed that GUS activity could be monitored at all developmental stages , while the OE lines accumulated less ROS after salt stress, and there was no difference in plants under normal conditions  sharply increases and then impairs plant growth . The DABnditions d. In addt stress e. An anaith NaCl e. Taken AtSIBP1 response to salt stress in Arabidopsis, eight-day-old seedlings were treated with 200 mM of NaCl for six hours, and then the transcript levels of the stress-inducible genes were analyzed, including the responsive to desiccation 29A (RD29A), cold-regulated 15A (COR15A), and ascorbate peroxidase 2 (APX2) genes. The expression levels of them were significantly increased in the overexpression of AtSIBP1 transgenic plants, while the expression level of APX2 was decreased in the sibp1 mutant compared to the WT after the treatment with salt stress   .Arabidopsis microarray database and the results of qRT-PCR indicated that AtSIBP1 was induced by salt stress  and osmotic-stress related genes in transgenic tobacco [AtSIBP1 overexpression lines and was accumulated in the sibp1 mutant  and ABREs (abscisic acid-responsive elements), which have the potential to confer abiotic stress resistance. In our study, salt stress induced the expression of RD29A and COR15A in AtSIBP1-overexpressions to increase the tolerance of salinity [APX2, an ascorbate peroxidase, is one of the most important antioxidant enzymes in plants and detoxifies hydrogen peroxide by using ascorbate as a reductant [AtSIBP1 was found to result in a higher expression level of APX2, indicating an improved antioxidant capacity of plants during salt stress. Therefore, we conclude that the overexpression of AtSIBP1 leads to increased tolerance of plants to high salinity through the up-regulation of APX2 expression, and then mediates the reduction of ROS accumulation. The nuclear localization of the BTB protein may be a prerequisite for its activation and the regulation of plant immune response . Here, w nucleus c; thus, and APX2 . Previousalinity ,29. APX2eductant . The oveAtSIBP1 positively regulated the responses of plants to salt stress. AtSIBP1, as a potential substrate receptor for the CRL3 complex, is involved in the tolerance to salt stress. Thus, we infer that its degraded target protein may be a negative regulator in the salt signaling pathway. However, the substrates of AtSIBP1 have yet to be identified, and the details how AtSIBP1 works in response to salinity and other stresses need to be further explored in Arabidopsis.The functions of BTB proteins in biotic stress have been highly clarified, but their roles in abiotic stress are far from elucidated. In this study, we demonstrated that Arabidopsis used in this study were of the Columbia (Col-0) ecotype. The T-DNA insertion mutant At1g55760 (SALK_075267) was obtained from the Arabidopsis Biological Resource Center (ABRC). All plants were grown in pots containing a mixture of vermiculite and soil  in a greenhouse at 22 °C under 70% relative humidity with 16 h light/8 h dark photoperiod. All seeds were sterilized with NaClO  for 15min, were washed with sterile water six times, and then stored at 4 °C for 3 d to break dormancy. Surface-sterilized seeds were sown on a Murashige and Skoog (MS) medium containing 2% (w/v) sucrose and 0.8% (w/v) agar, pH 5.8. All AtSIBP1-overexpressing and the proAtSIBP1::GUS constructs, the full length CDS (Coding sequence) (990bp) and the promoter of AtSIBP1 (1521bp) were amplified and cloned into pCAMBIA1302 and pCAMBIA1301, respectively. The constructed plasmids were transformed into Agrobacterium tumefaciens (GV3101) and then infiltrated into the WT (wild type) using the floral dip method [AtSIBP1 were identified with a qRT-PCR assay. The third generation homozygous seeds of the transgenic plants (T3) were used for further analysis. The primers that were used in this assay were listed in To generate the aciens GV01 and thAtSIBP1 was cloned into pBI221 containing a 35S promoter for a green fluorescence protein (eGFP) by homologous recombination. The constructed plasmid 35S::AtSIBP1-eGFP and the control of 35S::eGFP were transfected into mesophyll protoplasts from tobacco leaves using the PEG-calcium  transfection method, as described in [pBI221-eGFP vector was used as a control. To confirm the location of AtSIBP1, 4’, 6’-diamidino-2-phenylindole (DAPI) was used to stain the nucleus of the cell. After transfection at 22 °C for 16 h in the dark, the fusion fluorescence proteins were observed and captured using a confocal laser-scanning microscope [The CDS of ribed in . The pBIcroscope  , then vacuumed for 5 min. After 6 h of incubation in the dark with gentle shaking, the leaves were immersed in a bleaching solution  and boiled for 20 min to decolorize the leaves (except for the deep brown polymerization product that was produced by the reaction of DAB with H2O2), and then the images were captured.The histochemical GUS staining of the homozygous T3 transgenic lines harboring escribed . For DABArabidopsis seedlings were used for gene expression level identification with or without NaCl treatment. The total RNA was extracted from plants with RNAiso Plus reagents (Takara) according to the manufacturer’s instruction. The cDNA was synthesized using 2 μg of RNA by a Prime Script RT reagent Kit with a gDNA Eraser (Takara). For semi-quantitative RT-PCR, the semi-quantitative primers were used to test the expression of AtSIBP1. For the qRT-PCR analysis, the TB Green Premix Ex TaqII kit and an Applied Biosystems 7500 real-time PCR system were used for reaction according to the manufacturer’s instruction. ACTIN2 was used as the internal control. All primers are shown in Eight-day-old AtSIBP1 was obtained from SMART (SIMPLE MODULAR ARCHITECTURE RESEARCH TOOL) and NCBI . Multiple alignment results were edited in the DNAMAN8.0 program. The phylogenetic analysis was performed with the MEGA7.0 program.The putative protein structure of n = 3). Statistical analysis was performed using Student’s t-test. The values of p < 0.05 were considered to be significant, and Values of p < 0.01 were considered as more significant. The data are represented as means ± SD (Standard Deviation) (