Slowing the production rate of the AHL-degrading enzyme, AiiA, generates stable fixed states, limit cycle oscillations and Turing patterns. Further tuning of signaling parameters determines local robustness and controls the range of unstable wavenumbers in the patterning regime. These findings provide a roadmap for optimizing spatial patterns of gene expression based on familiar quorum and gas sensitive E. coli promoters. The circuit design and predictions may be useful for (re)programming spatial dynamics in synthetic and natural gene expression systems.The Turing instability was proposed more than six decades ago as a mechanism leading to spatial patterning, but it has yet to be exploited in a synthetic biology setting. Here we characterize the Turing instability in a specific gene circuit that can be implemented  Self-organization and self-assembly govern the emergent properties of spatial structures from the molecular to the galactic scale , 2. In tOne mechanism by which ensembles of cells could self-organize is the Turing instability , 12, 13 et al. [Despite the ubiquity of Turing patterns at the multi-cellular scale, they have yet to be demonstrated in gene expression sytems. This is surprising given the many alternative pattern forming mechanisms found in natural , 4, 6 anet al.  have preet al.  has receIf it is possible, then Turing patterning of gene expression could be quite useful for basic science and biotechnology applications. These include self-organized spatial sequestration of gene expression and downstream metabolites (as is currently done inside cells to improve yield ), spatiaMotivated by this, we propose a realistic synthetic gene circuit that implements repressor-activated degradation using activating homoserine lactone and inhibitory peroxide gas, and demonstrate how this system can be switched between stable uniform states, limit cycles, and Turing patterns. We characterize the effects of experimentally relevant parameter changes on the presence and properties of the expected Turing patterns. We conclude by discussing our plans to experimentally implement the circuit and to refine the mathematical model that describes it.μm and dt = 10−5 min. We rescaled space by assuming DL = 5.6 × 10−3μm2/min. The fixed points, eigenvalues and unstable wavenumbers of the differential equations were computed using the symbolic and variable-precision solvers in Matlab . Bifurca2O2) as the long-range inhibitor signal .L, H, A, LR, P represent AHL, H2O2, Aiia, LuxR and the AHL-LuxR complex, respectively. Production is controlled by fi(Z) = (δi + Z)/(ci + Z), where c1 = kPL, c2 = kPH, c3 = kHA are the thresholds for production of each species. Degradation is first order for H2O2 and Aiia, but enzymatic for AHL, with g(L) = A/(kLD + L). Total LuxR is assumed to be constant, and the AHL-LuxR complex  is assumed to be relatively stable to degradation.The compound chemical reactions  underlying the proposed gene circuit are:x by Pm is the total amount of LuxR. The parameters of Eqs (k2)2 + B(λ)(k2) + C(λ) = 0. Finding the root of the discriminant of the quadratic, α3 = 3.1 min.−1 in Restricting AiiA dynamics to reasonable relative production and degradation rates , 27, we t in Eqs –4) show showα3, t in Eqs . The Turα3 = 3.5 min.−1  when undergoing the transition to the Turing instability. This means that the classic notion of Turing patterns arising from a stable fixed point can be extended to include some cases with an unstable point inside a limit cycle.Overall, the results indicate that slowing AiiA feedback on AHL degradation can lead to limit cycle oscillations before the onset of Turing patterns. This is consistent with previous studies showing that an increased time delay in a negative feedback signal produces limit cycle oscillations –47. It iWe next investigate how additional parameters can be used to experimentally tune spatial patterning in the circuit. In contrast to the single promoter circuit model that was proposed , increasα1, affecting the maximal rate of LuxI expression, and experimentally controlled by a regulatable hybrid promoter, plasmid copy number, or the strength of the promoter or ribosome binding site. Covarying this parameter with the AiiA production rate creates basins for stable, Hopf unstable and Turing unstable solutions  also increases. Raising α3 also tends to increase α1-robustness (Re(λ(k)) > 0 towards higher wavenumbers (shorter wavelengths). Thus, tuning the maximal AiiA production rate to achieve more α1-robust Turing patterning also results in shorter spatial periods in the resultant pattern.A more experimentally tuneable parameter is olutions . For α3<stigated  as α1 isbustness . Fig 4b Pm, representing the amount of LuxR. At the nominal parameter set  > 0, that determines how fast unstable solutions diverge from the fixed point. Although the high dimensionality of our model makes it challenging to derive analytical expressions for the pattern formation speed , or seq2 (as in ).2O2 have not been measured directly for bacterial monolayers in microfluidic devices. However, Danino and colleagues [V = 8.5-35 V = 0.17 DL = 2.5-42 × 103DL = 5.9-29 × 1032O2, DH = 72-120 × 103D = 4-12. However, Prindle et al. measured H2O2-mediated fluorescence conduction velocity V ≈ 700 D = 100, used in our model is reasonable. In addition, decreasing D as much as fourfold from D = 100 can still allow for patterning to occur , and regulation of the aiiA promoter , as well as Hpromoter ). All ofpromoter , 58 suggpromoter . Furthern system .E. coli), it would be prudent to further examine this system using stochastic simulations. Hsia and colleagues [Considering that protein numbers are low at the steady states analyzed Click here for additional data file.S1 FigD. (a) At the nominal parameter set  For α3 = 2.5, despite a Hopf instability at k = 0, Turing patterns are maintained when decreasing the ratio as low as D = 25.Dependence of the dispersion relation on the ratio of diffusion coefficients, eter set  except w(PDF)Click here for additional data file.S2 Figα1), and (b) amount of LuxR (Pm).Dependence of the dispersion relation on LuxI and LuxR parameters. Varying these parameters around the nominal parameter set  demonstr(PDF)Click here for additional data file.S1 Equations(PDF)Click here for additional data file.S3 Fig2O2 on plux, β. At the nominal parameter set Click here for additional data file.S1 Movie(MOV)Click here for additional data file.


Oncidesa orchids. Using 10,000 µmol mol−1 CO2 and a photosynthetic photon flux density of 60 µmol m−2 s−1, the number of leaves and roots and shoot and root fresh and dry weights could be considerably increased, but only when cultured on Kyoto medium rather than Vacin and Went medium. Super-elevated CO2 enrichment and growth under cold cathode fluorescent lamps can positively affect the efficiency and quality of commercial production of clonal Oncidesa plantlets.How can one increase the biomass of plants, particularly orchids, in an effective way? Photoautotrophic micropropagation is an effective means of increasing the biomass of  2) to maximize plant growth and productivity. The effect of super-elevated CO2 with cold cathode fluorescent lamps (CCFLs) on the photoautotrophic growth of Oncidesa in vitro has been studied using a gas-permeable film culture vessel, the ‘Vitron’. The growth of Oncidesa (formerly Oncidesa Gower Ramsey ‘U-1’) plantlets on Vacin and Went (VW) medium was stimulated by 10 000 μmol mol−1 CO2. In particular, increasing the photosynthetic photon flux density (PPFD) from 45 to 60 μmol m−2 s−1 under 10 000 μmol mol−1 CO2 in the growth chamber remarkably increased the number of leaves and roots, and shoot and root fresh and dry weights compared with plantlets under the same level of CO2 under low PPFD (45 μmol m−2 s−1). However, there was a remarkable decrease in photosynthetic capacity, and chlorosis and browning of leaves. In stark contrast, plantlets grown on Kyoto medium at 10 000 μmol mol−1 CO2 under high PPFD had a higher photosynthetic rate than plantlets grown on VW medium, and no chlorosis or browning was observed. Furthermore, shoot growth was remarkably enhanced. Therefore, super-elevated CO2 (10 000 μmol mol−1) enrichment and growth under CCFLs can positively affect the efficiency and quality of commercial production of clonal Oncidesa plantlets.As interest in how to increase biomass production through biotechnological means gains traction, focus is turning towards the use of photoautotrophic micropropagation under elevated levels of carbon dioxide (CO A similar trend was noticed for the Cymbidium hybrid but at a lower CO2 level (3000 μmol mol−1)  in film culture vessels. Thus, one key objective of our study was to ascertain whether super-elevated CO2 would have further positive effects on the photoautotrophic growth of Oncidesa plantlets in film culture vessels.Cymbidium and Phalaenopsis in vitro plantlets cultured under CCFLs showed enhanced photoautotrophic growth compared with plantlets cultured under conventional heat fluorescent lamps. Gerbera. Our present study is the first report on the use of CCFLs as a radiation source for Oncidesa plantlet growth in vitro.Light is also an essential factor for plant growth and development. Recently, the use of cold cathode fluorescent lamps (CCFLs) as a radiation source for plants has been attempted because of their attractive features, including their small diameter (1.6–3.0 mm), long life (50 000 h) and low heat generation, with an estimated 40 % reduction in electric energy consumption used for lighting and air conditioners during micropropagation . Those sOncidium hybrid (Oncidesa), by super-elevated CO2 enrichment under CCFLs on two different media.The aim of the present study was to achieve more efficient and higher-quality commercial clonal orchid plantlets, in this case, an Oncidesa   sugar-fr® Rockwool Multiblock™ AO 18/30, Grodania A/S, Denmark) with 180 mL of liquid medium. The rockwool was previously sterilized in a dry sterilizer , and placed in the ‘Vitron’. Then, sterilized liquid medium was poured evenly over the rockwool.The film culture vessel ‘Vitron’ (122 mm × 122 mm × 140 mm) consists of a three-dimensional injection-moulded polypropylene frame covered by a heat-sealed OTP film  on all sides, except the top . OTP is −2 s−1 (R/B ratio: 80 % red (∼660 nm) + 20 % blue (∼450 nm), a conventional CCFL light unit; NK System, Osaka, Japan). CO2 enrichment was 380 (ambient/control), 3000, 5000 or 10 000 μmol mol−1. Experiments were conducted under each CO2 concentration by placing the vessels in different transparent acrylic desiccation chambers  and a CO2 gas inlet line  of 45 and 60 μmol min vitro were recorded after 90 days. The pseudobulb formation frequency was calculated as a percentage of the plantlets that formed a pseudobulb. Pseudobulb volume (Vp) was calculated as an ellipsoid:H, B and W are pseudobulb height, breadth and width, respectively  value of leaves of plantlets grown −2 s−1. The net photosynthetic rate was measured at 300 μmol m−2 s−1 (saturating or near-saturating PPFD). The CO2 concentration and temperature in the leaf chamber were adjusted to maintain 400 μmol mol−1 and 25 °C, respectively. The relative humidity in the leaf chamber was kept as close to 65–70 % as possible. The air flow rate was 200 mL min−1.The photosynthetic light response curve and net photosynthetic rate were measured in the second leaf, counting from the top downwards, of plantlets, in which a pseudobulb was not formed, after culturing for 90 days. It was measured in at least five plants using a portable infrared gas analyser . For obtaining the photosynthetic light response curve, the photon flux density that was provided from a red LED light source built into the top of the leaf chamber was changed from 300 to 0 μmol mt-test at P = 0.05.Means were separated by ANOVA and significant differences were assessed by Tukey's multiple range test and a Student's 2 increases the dry mass of plants  to 10 000 μmol mol−1 under both low and high PPFD; the maximum number of roots, root length, and root fresh and dry weights were obtained when plantlets were grown under 10 000 μmol mol−1, regardless of PPFD level  under 10 000 μmol mol−1 CO2 enrichment further enhanced the in vitro growth of both shoots and roots  with high PPFD (provided by CCFLs) also has a positive effect on the growth of both shoots and roots in Oncidesa.In 2 and super-elevated CO2 enrichment tend to cause foliar symptoms such as chlorosis, necrosis or bleaching in several plant species  compared with plantlets at the same CO2 concentration under low PPFD  at the end of the culture period. At low PPFD, even in plantlets grown at 10 000 μmol mol−1 CO2, a decrease in the net photosynthetic rate of single leaves did not occur, while at high PPFD, plantlets grown at the same high level of CO2 showed a significant decrease  under high PPFD emitted by CCFLs enhanced the photoautotrophic growth of Oncidesa plantlets in the ‘Vitron’, although the upper ‘threshold limit’ would be 5000 μmol mol−1 CO2 enrichment under high PPFD before a negative impact on photosynthesis would occur. This would help to maximize the productivity and quality of Oncidesa plantlets cultured in vitro. In addition, CCFLs have several advantages over existing lighting systems used for plant tissue culture (2 enrichment. Our results indicate that there is great hope for using super-elevated CO2 enrichment under CCFLs for more efficient and higher-quality commercial production of clonal orchid plantlets, which is a key objective of orchid biotechnology (We have shown in this study that super-elevated COchnology .All authors have made a substantial contribution to the manuscript and the research presented. M.T. and A.N. co-designed the experiment. M.T. and J.A.T.d.S. oversaw the experimental execution. A.N. conducted all research. A.N. and J.A.T.d.S. drafted the paper and made all edits for the revisions. All authors have seen and agreed to the submitted manuscript.None declared.


A wide array of wildlife species, including many animals, are used in traditional medicines across many medicinal systems, including in Traditional Chinese Medicine (TCM). Due to over-exploitation and habitat loss, the populations of many animals commonly used in TCM have declined and are unable to meet market demand. A number of measures have been taken to try to reduce the impact that this large and growing market for TCM may have on wild animal species. Consumer preferences and behavior are known to play an important role in the consumption and protection of wild animals used in traditional medicine, and thus are likely to be an important factor in the success of many of these mechanisms—particularly given the significant percentage of TCMs that are over-the-counter products (access to which is not mediated by practitioners). In this study we conducted questionnaires and designed stated preference experiments embodying different simulation scenarios using a random sample of the population in Beijing to elicit individuals’ knowledge, perceptions and preferences toward wild or farmed animal materials and their substitutes used in traditional Chinese medicine. We found that respondents had a stated preference for wild materials over farm-raised and other alternatives because they believe that the effectiveness of wild-sourced materials is more credible than that of other sources. However, we also found that, although respondents used TCM products, they had a poor understanding of the function or composition of either traditional Chinese medicines or proprietary Chinese medicines (PCM), and paid little attention to the composition of products when making purchasing decisions. Furthermore, awareness of the need for species protection, or “conservation consciousness” was found to play an important role in willingness to accept substitutions for wild animal materials, while traditional animal medicinal materials (TAMs) derived from well-known endangered species, such as bear bile and tiger bone, show relatively higher substitutability. These results suggest that there is still hope for conservation measures which seek to promote a transition to farmed animal, plant and synthetic ingredients and provide clear directions for future social marketing, education and engagement efforts. Chinese Pharmacopoeia recognizes 616 kinds of medicinal materials and cut crude drugs, of which 52 are derived from animal sources [A wide array of wildlife species, including many animals, are used in traditional medicines across many medicinal systems –3. In Ch sources . This is sources ,6–7.TCM accounts for approximately 80% of “all over-the-counter” (OTC) drugs in China .Key National Protected Wild Animal List [Chinese Pharmacopoeia in a series of revisions from 1963 to 2010 [A number of measures have been taken to try to reduce the impact that this large and growing market for TCM may have on wild animal species. The Chinese government has placed 161 wild animal species used in TCM on the mal List . Due to  to 2010 . In addi to 2010 –14, and  to 2010 –16.The success of many of these conservation mechanisms remains uncertain. In particular, while conservationists and TCM practitioners have recognized that pressure on wild populations may be relieved by captive breeding and the production of synthetic materials ,7,15, anThe factors influencing consumer behavior are highly complex and include consumption need and motivations, perceptions, behavioral learning, attitudes, and socio-cultural factors –20. TherWhat little research has examined consumer perspectives on conservation of animals used for TCM in fact suggests that consumer attitudes and beliefs may undermine a number of the current conservation mechanisms. For example, existing research suggests that people may prefer products produced from wild animals because they believe that wild materials are more potent than farm-raised ones ,16. AccoWe therefore conducted this study to investigate consumer perceptions of animal materials in TCM and in particular, to examine whether substitution of wild animal-based products may be feasible. We sought to determine whether, and if so why, consumers prefer wild-sourced animal materials and evaluate the acceptability of substitutes for wild-sourced animal species in TCM. Thus, we examined our data with a view to answering the following research questions:What are consumers’ knowledge and perceptions of medicinal animal materials?Do consumers prefer wild, farmed or substitute medicinal materials, and why?Can wild-sourced animals species used as TCM be substituted?jiangzg@ioz.ac.cn).This study was reviewed and approved by the Ethical Committee of the Institute of Zoology, Chinese Academy of Sciences, and written informed consent was obtained from all respondents (Permit Number is: IOZ11013). All procedures performed in this study were in accordance with the instructions and permission of the Ethical Committee. All researchers and investigators were certified before performing this study. All data collected through this study remains anonymous and confidential. The study design and study reporting were conducted in accordance with the consolidated criteria for reporting qualitative research (COREQ) framework . The cheThis study was conducted in Beijing in the summer of 2011. A stratified survey design was chosen to randomly select neighborhoods and households from a sample frame, according to random number table or using draw lots . InterviOnce a sample household was identified, a face-to-face interview was conducted at home with randomly selected household members who were 18 years of age or older and had lived in Beijing for at least one year . Before Members of our research group conducted the survey. In addition, we recruited and trained investigators with a bachelor’s degree or higher and at least one year of work experience in a professional market research company, (preferably in a conservation-related area) . To expeThe interviews were randomly checked by supervisors on the spot to assure sampling normalization. Interviews containing any omissions or errors were asked for a re-run. Interviews were not deemed suitable for a re-run if it was determined that knowledge of the survey content which respondents gain from the last round of survey would bias the results, for instance, respondents may have gotten the right answers to the questions from the last round of the survey. Supervisors randomly selected 30% of the questionnaires to validate by telephone return visit, to ensure that no fraud had taken place and eliminate unqualified questionnaires.The initial design of the questionnaire was modified based on the results of a pilot study on 11 Chinese postgraduate students at the Institute of Zoology, Chinese Academy of Sciences, and also on a total of 30 adult residents who were randomly chosen. The questionnaire consisted of 5 parts: cover letter, completion instructions, questions, answers, and coding . An objeChinese Pharmacopoeia [We conceived and designed the questionnaire for this investigation  see S3 . First, acopoeia . We alsoacopoeia .We also designed stated preference experiments embodying three different scenarios to elicit consumer preferences, and a choice experiment approach was also used to collect data ,28. In oThe respondent was required to purchase 9 TCM products, including 3 types of TAMs, 2 TCM prescriptions, 2 PCMs, and 2 TCM health care products. Each TCM product contained TAMs from a wild animal, farmed animal, or substitute. It was explained that in this scenario these materials all had the same or very similar curative effects, and their prices were all affordable. To estimate consumer preferences, the respondent was asked to select their preference from 4 options: “wild”, “farmed”, “substitute”, or “whatever” materials. If “substitute” or “whatever” were selected, the respondent then selected among 5 types of substitute materials, including “wild animal”, “farmed animal”, “wild plant”, “farmed plant”, “synthetic”, or “whatever”. After making a choice, the respondent was then asked to explain their choice.The respondent was required to choose their preferred TAM from either “wild”, “farmed”, “other animal materials” as a substitute, “plant material” as a substitute, “synthetic” or “whatever” material under the following sets of conditions: (i) both the curative effects and prices of the TAMs were identical; (ii) the TAMs had identical curative effects but prices that decreased in the order presented above; and (iii) the TAMs had identical prices but curative effects that increased in the order presented above. These sets of conditions were then revisited in the context of purchasing 3 specific animal materials—musk, deer antler velvet, and bear bile.The overall setup of this scenario remained the same as in Scenario 1, except that respondents were asked beforehand which TAMs should be protected. The proportion of respondents who believed that a certain TAM must be protected was used as an indicator of the “conservation consciousness” of that respondent.Finally, we asked respondents how much attention they paid to the following aspects of TCM products: (i) curative effects, (ii) functions and indications, (iii) TAM or non-TAM as composition, (iv) price, (v) reputation, and/or (vi) side effects. The level of attention was measured using a seven-point Likert scale . We alsoField notes were made, and feedback from respondents was obtained during the interview by the interviewer . The queAn ordinal logistic regression model was used to estimate the relationship between the demographic variables and the use of TAMs. Unordered categorical variables including gender, career, birth place and urban/rural origin were transformed into dummy variables. As ordered categorical variables, age, income, and educational level were quantified as 1, 2, 3,…. The number of types of TAMs the respondents had used was divided into 3 levels , which were denoted as 1, 2, and 3, respectively.To explore consumer perceptions, variables including whether respondents had heard of or used TAMs and PCMs and their knowledge of TAM functions or PCM compositions were fitted using regression models and probability distribution models. Moreover, correlation and regression analyses were conducted to evaluate how having heard of or used TAMs and PCMs affected respondents’ perceptions of TAM functions and PCM composition to facilitate an understanding of respondents’ perceptions of TCM .u and v are the marginal distributions of the probability functions; θ is a parameter; and τ is the correlation coefficient, which was calculated as follows [In different scenarios, to explore the substitutability of wild TAMs, we established curvilinear regression models for the frequencies of choosing “wild”, “farmed”, and “substitutes” and a binary linear regression model among the frequencies of choosing these three sources. Curvilinear, binary logistic and nonlinear regression models were established to estimate the relationship between the frequencies of choosing different medicinal materials and “conservation consciousness”, “effect level”, and “price level” to explore how these factors impact consumer preferences . Moreove follows :t= and detrended canonical correspondence analysis (DCCA) were performed to analyze the relationship between consumers’ preferences for different medicinal materials and the reasons for those preferences to identify the major influencing factors.Akaike’s information criterion was used to determine the optimal model . Prior t2 = 96.586, df = 14, p<0.001), where the Cox & Snell R2 and Nagelkerke R2 were, respectively, 0.100 and 0.113, larger than 10%, and a proportional odds assumption was established . Age demonstrated a significant impact on the use of TAMs; the regression coefficient was 0.402 (p<0.01) . Other demographic variables, including gender, age, occupation, monthly income, native place, and urban/rural origin, showed no significant influence on the use of TAMs (p>0.05).A total of 1,100 questionnaires were issued, with 912 valid responses and a return rate of 82.91% (excluding incomplete or incorrectly completed questionnaires). The gender ratio was slightly biased toward women at 52.08%, compared to 48.38% females in the population in Beijing . The ord(p<0.01) . In partn = 912), there were significant differences among the numbers of TAMs that respondents had heard of (16.058±0.262), used (4.129±0.102), and knew the function of (2.209±0.077) . The numbers of TAMs that respondents had heard of, used, and knew the function of obeyed different function models  or used  (n = 34), the rate of respondents who knew the functions of TAMs was moderately positively correlated with the rate of those who had heard of  or used  them, as fit by linear  and logarithmic models , respectively (n = 912) , and the mean of intention to use was the highest (p<0.01), indicating that the respondents were willing to use TAMs.There was a low positive correlation between the numbers of TAMs for which respondents knew the function of and had heard of ( p<0.01) . Taking ectively . The pron = 912) had heard of (16.603±0.063), used (5.330±0.072), and knew the compositions of (0.595±0.037) . The numbers of PCMs that respondents had heard of and used, and the numbers of respondents who knew the compositions of PCMs obeyed different function models  knew the compositions of was slightly positively correlated with the numbers of PCMs that respondents had heard of  or used  (n = 21), the rate of respondents who knew the composition of PCMs was moderately positively correlated with the number who had heard of  or used PCMs  (n = 912) , indicating that the respondents were willing to use PCMs. In response to the question “How well do you know the functions and indications of PCMs?”, 48.46% responded “fully know”, 39.95% answered “a little”, and 11.59% responded “nothing”.The number of PCMs that respondents ( p<0.01) . Taking 185.520) . Howeverp<0.01). In particular, respondents paid more attention to curative effects (5.222±0.022), functions and indications (5.186±0.021), and side effects (5.272±0.023) than other indices (p<0.05) and less attention to the composition of TAMs (2.661±0.030) and non-TAMs (2.622±0.031) (p<0.05).There were also significant differences among the 7 attention indices when respondents purchased TCM products . More consumers chose “wild” source (0.576±0.009) than “farmed” (0.237±0.006)  and more “farmed” source than “substitute” (0.121±0.007) . The ratio of consumers that chose “whatever” was the smallest (0.066±0.002) (p<0.01) . “Synthetic” (0.293±0.011) and “whatever” (0.294±0.012) were chosen more often than “others” (p<0.01), but there was no significant difference between “synthetic” and “whatever” . The selection frequency of “wild animal” (0.060±0.006) was the lowest (p<0.01), and no significant difference  was observed between the choice of “farmed animal” (0.117±0.007), “wild plant” (0.123±0.007), and “farmed plant” (0.113±0.006) substitutes  . These rstitutes . These rrpearson = -0.735, p<0.01) and “substitute” , and the selection frequencies were fitted by the inverse model as ywild = 0.346+0.052/xfarmed  and the exponential model as ywild = 0.711e-1.798xsubstitute  (y) with “farmed” (x1) and “substitute” (x2) were fitted by the binary linear regression model as y = 0.920–0.956*x1-0.968*x2  (The selection frequency of “wild” was negatively correlated with that of “farmed” (334.718) , respect459.714) . These r2 = 421.865, df = 168, p<0.01). The selection frequencies of different sources and substitutes were fitted by probability distribution functions, which revealed that all the curves concentrated in different zones  and “wild-substitute” . The empirical accumulative probability fit the theoretical accumulative probability well. Compared with the probability function graph for “wild-substitute”, that for “wild-farmed” demonstrated larger y coordinates , “natural” (8.86%), “fewer side effects” (7.36%), and “more traditional” (2.97%). The reasons for choosing “farmed” were “protecting endangered animals” (6.04%), “more credible effects” (5.53%), “more hygienic” (4.80%) and “fewer side effects” (3.27%). The main reason for choosing “substitute” was “protecting endangered animals” (5.40%) .Higher selection frequencies were observed for different substitutes, approximately 29.91% for “synthetic” and 29.06% for “whatever”, followed by “wild plants” (12.41%), “farmed plants” (11.32%), “farmed animals” (11.27%), and “wild animals” (6.04%). The main reasons for choosing “synthetic” were “protecting endangered animals” (9.68%) and “protecting endangered plants” (7.07%). The reasons for choosing “wild plants” or “farmed plants” were “protecting endangered animals” and “acceptable medicinal materials”. The reasons for choosing “farmed animals” were “protecting endangered animals” (3.23%) and “acceptable TAMs” (2.75%) .The results of the PCA of respondent preferences for TCM products from different sources and substitutes indicated that the accumulative variance contributions of the first two principal components were more than 90.0%. The preference of the respondent for “wild”, “farmed”, and “substitute” increased along the direction of arrows a, b, and c, respectively . DiffereThe results of DCCA for respondents’ preferences and reasons showed that accumulative variance contributions of the first two principal axles were as high as 89.5%. Arrows FE , SE  and WA (which indicates that the reason for choosing “wild” was “credible effects”) were highly correlated with the first axis, and the correlation coefficients were 0.6032, 0.8790, and -0.8112, respectively, indicating that the factors that drove respondents’ preferences for “wild”, “farmed”, and “substitute” increased along the directions of arrows WA, FE and SE, respectively. Different TCM products located in different zones in the coordinate plane were influenced by different causes. A coordinated point for four types of medicines containing tiger bone or bear bile  was located in the direction of arrow SE, indicating that respondents chose “substitute” for these materials mainly for the reason of “protecting endangered animals” .n = 29) was negatively correlated with the frequency of choosing “wild” sources  and was positively correlated with the frequency of choosing “substitute” sources . There was also a negative correlation between the frequencies of choosing “wild” and “substitute” . Comparing Scenario 3 with Scenario 1, the regression curve of “substitutes” and “conservation consciousness” shifted upward (z) with “conservation consciousness” (x) and “substitute” (y) were in accordance with function z = 0.737e-x/4.106e-y/0.883  in Scenario 1 and function z = 0.637e-x/1.606e-y/0.504  in Scenario 3 , and the selection frequency of “farmed”  or “substitute”  was smaller than that in Scenario 3. Joint probability distribution models were built for “wild-conservation consciousness” , “wild-farmed” , and “wild-substitute” . The probability function graph for Scenario 3 was shifted toward the direction of a higher selection frequency of “wild” compared to that for Scenario 1 (The selection frequency of “wild” in Scenario 1 was significantly higher than that in Scenario 3 (paired-sample t test: enario 1 .p<0.01). In sub-scenario (i), respondents preferred to choose “wild” (0.317±0.013) and “whatever” (0.284±0.022)  (Mann-Whitney U: p<0.01)  (p<0.01)  (p<0.01)  ; in sub-(p<0.01) ; and in (p<0.01) .2 test: χ2 = 220.347, df = 4, p<0.01) and between sub-scenario (iii) and sub-scenario (i) . The selection frequency was also significantly negatively correlated with price level in sub-scenario (ii) , whereas the selection frequency was positively correlated with effect level in sub-scenario (iii) . Binary logistic regression demonstrated that both curative effect and price had significant impacts on respondent choice (x) and the change rate of selection frequency (y): y = 2.301x-1.318–0.6979  (x) and the change rate of selection frequency (y): y = 8.309E-04x5.179–0.7141  (There were significant differences in consumer preference between sub-scenario (ii) and sub-scenario (i) (crosstabs χt choice . The cha-39.248) , as well-26.875) .Despite consuming TCMs, respondents in our study demonstrated minimal understanding of the function and composition of TCMs and PCMs. Although respondents claimed to pay attention to the curative effects, functions and indications of TCM products when making purchasing choices, they demonstrated little knowledge about TCMs, particularly their composition. This suggests that respondents preferred to simply take TCMs in a manner similar to using a computer simply by clicking the mouse without an understanding of the computer’s hardware configuration and computing principles.If respondents’ preferences for TCMs are solely determined by their curative effects, one might expect them to have no concern about the use of substitutes rather than original materials, so long as the curative effects are identical. However, our results demonstrated a stated preference amongst respondents for TAMs derived from wild animals. This preference was most commonly attributed to a belief that TAMs derived from wild animals are more effective than materials from other sources. In contrast, only a few respondents appeared to be motivated by conservation consciousness to choose substitutes or synthetic materials. This is consistent with existing research indicating that consumers of TCM prefer products made from wild sources and believe such products are more potent ,16. TCM In contrast to previous studies that measured consumer preferences based on demand functions ,37, our Indeed, our results indicate that TAMs have varying degrees of substitutability. More farmed or alternative materials were selected by consumers, indicating the better substitutability of wild materials. The degree of substitutability of varying animal materials changed following certain patterns and there was a probability for the substitution of each TAM . FurtherIt has previously been argued that conservation awareness has an important impact on consumer behavior in the context of wildlife trade ,38 and tPrice has the potential to significantly affect consumer preferences and choice behavior by impacting on consumer buying intention and satisfaction . HoweverWe believe that it is perhaps time to reframe our view of endangered wildlife from a supply-centric perspective to a demand-centric one which places a focus on consumer behavior change at the heart of our strategies to tackle the threat to endangered wildlife ,13. Our S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOC)Click here for additional data file.S3 Appendix(DOC)Click here for additional data file.S1 Fig(DOC)Click here for additional data file.S2 Fig(DOC)Click here for additional data file.


Litopenaeus vannamei) has been reported as a source of compounds with chemopreventive activities. In this study, shrimp lipids were extracted and then fractionated in order to isolate those compounds responsible for the antimutagenic activity. The antimutagenic activity was assessed by the inhibition of the mutagenic effect of aflatoxin B1 on TA98 and TA100 Salmonella tester strains using the Ames test. Methanolic fraction was responsible for the highest antimutagenic activity  and was further separated into fifteen different subfractions (M1–M15). Fraction M8 exerted the highest inhibition of AFB1 mutation  and, after further fractionation, four subfractions M8a, M8b, M8c, and M8d were obtained. Data from 1H and 13C NMR, and mass spectrometry analysis of fraction M8a (the one with the highest antimutagenic activity), suggest that the compound responsible for its antimutagenicity is an apocarotenoid.According to the World Health Organization, cancer is the main cause of mortality worldwide; thus, the search of chemopreventive compounds to prevent the disease has become a priority. White shrimp ( In economically developed countries, cancer, a disease considered preventable , has beeShrimp muscle has been reported as a rich source of high quality proteins and also low in fat content , 13 and  meso-zeaxanthin, β-carotene, zeaxanthin, α-carotene, and astaxanthin and its esters have been, individually or in combination, tested by the Ames test [The lipidic fraction of shrimp muscle contains different compounds including neutral lipids, phospholipids, glycolipids, and carotenoids . This frmes test  finding Litopenaeus vannamei) was purchased from the local market at Hermosillo, Sonora, Mexico, and transported in ice to the laboratory. Shrimp muscle was obtained, packed in self-sealing polyethylene bags, and stored at –20°C until their use. Shrimp muscle lipid fraction was extracted according to López-Saiz et al. [White shrimp , using 230–400-mesh silica gel . Subfraction M8 was poured onto the column and eluted using 500 mL of a series of mobile phases as follows: (A) hexane : ethyl acetate (8 : 2), (B) hexane : ethyl acetate (7 : 3), (C) hexane : ethyl acetate (2 : 3), (D) hexane : ethyl acetate (1 : 1), (E) ethyl acetate : hexane (4 : 1), (F) acetone, and finally (G) methanol. Silica gel-coated TLC testing plates, revealed with an iodide solution and observed under UV light, were used to monitor the eluents. Fractions providing similar signals were combined and used for further analyses. Salmonella typhimurium TA98 and TA100 tester strain cultures were stored at −80°C. Tester strains genetic characteristics were periodically confirmed according to Maron and Ames [Overnightand Ames . Salmonella/microsomal mutagenicity test [Theity test  was usedity test , using a1 inhibition according to the following equation:1R is number of aflatoxin B1-induced revertants/plate (positive control).Antimutagenic activity was reported as the percentage of AFBµL aliquot of CDCl3  was used to dissolve each fraction and tetramethylsilane (TMS) was also included as an internal standard. This mixture was placed into 5 mm diameter ultraprecision NMR sample tubes. Chemical shifts were registered as ppm units, employing TMS proton signals as internal standard.Analyses were carried out using Agilent Technologies 400/54 Premium Shielded (400 MHz) spectrometers. A 500 P ≤ 0.05.Data treatment was carried out using one-way analysis of variance (ANOVA) using Tukey-Kramer multiple comparison of means , Kaysville, UT, USA) with a significance level of Chloroform extraction from shrimp muscle yielded 1.860 ± 0.004% (dry basis), a value that falls within the lipid content (1-2% of its dry weight) that has been previously reported .1 (AFB1) as control mutagen. Shrimp muscle chloroform-extract inhibited AFB1 mutagenic potential in 94.6 ± 1.1 and 95.36 ± 2.41% in both Salmonella typhimurium TA98 and TA100 tester strains, respectively , which was comparable to that obtained for the chloroform-extract   without Isolation of the bioactive compounds was continued through M8 fractionation, which was subjected to a low-pressure chromatographic procedure (open column). Four new fractions were obtained, which were coded as M8a, M8b, M8c, and M8d. Polarity of sample decreased as follows: M8d > M8c > M8b > M8a; this last one exhibited a bright orange color; M8b and M8c showed a pale orange tone, whereas M8d had a pale yellow color.All of the M8 subfractions were highly antimutagenic and exerted a dose-response relationship . Since f1H NMR spectra (400 MHz) , which indicates that even though the color of the sample is orange, the compounds are not carotenoid. Signals observed at δ = 5.0–5.5 ppm may be attributed to protons involved in double bond, whereas signals at δ = 4.2 and 4.5 ppm are associated with protons adjacent to carbons attached to an ester bond (C–O). The signals found at signals at δ = 3.5 ppm are associated with protons in alcohol groups. Finally, chemical shifts that appear at high field (δ = 0–3.0 ppm) are attributed to methyl, methylene, and methine protons. All of these signals are characteristic of apocarotenoid compounds.According to the 400 MHz) , downfie13C NMR spectra (400 MHz)  , where dThe presence of bioactive compounds in shrimp has been previously reported; however, most of them were not extracted from shrimp muscle but from exoskeleton. Biological activities previously reported include antioxidant, which was found in crude extracts obtained from shrimp byproducts such as head , 15 and In all of these reports, bioactivity has been attributed to carotenoids, specifically to astaxanthin; nevertheless, all of these studies were carried out on crude extracts only, and their conclusions were based on absorbance observed at visible spectra wavelength (450–475 nm), attributing the bioactivity to carotenoids without any fractionation of the extract in order to isolate and identify the compound responsible for the bioactivity.1, has been evidenced; but also the fact that these compounds are not carotenoids has been demonstrated. Results of the present study suggest that products of the breakdown of this type of compounds called apocarotenoids are responsible for the antimutagenic activity found in white shrimp muscle.Recently, antimutagenic compounds present in fractions obtained after serial thin layer chromatography procedures have been reported . In the Carotenoid breakdown might be either enzymatic- or not enzymatic-type and can produce different kinds of compounds, depending on the reaction conditions. It has been previously reported that biological processes can be affected by these kinds of compounds instead of pure carotenoids, and they are solely responsible for the biological activity reported in carotenoids . Bixa orellana, which has been reported as an anticancer compound [Ditaxis heterantha), have also been reported as antiproliferative compounds in human malignant cells (HeLa and CaLo) [β-apo-8′-carotenal, which has been reported as an aflatoxin B1 inhibitor in rats [Apocarotenoids have previously been reported as bioactive compounds capable of showing bioactive properties; among those, bixin is an apocarotenoid isolated from the shrubcompound . Specificompound . Ditaxinnd CaLo) . Another in rats . Even th Litopenaeus vannamei muscle is a source of different antimutagenic compounds and even though astaxanthin is thought to be responsible for this activity, the present study demonstrated that the compounds that exerted the highest activity have an apocarotenoid chemical structure.The chloroform-soluble fraction from


Viola xwittrockiana “Nature Clear Lemon”) in a planter for 3 min. Artificial yellow pansies in a planter were used as the control. Heart rate variability was used as a physiological measurement and the modified semantic differential method was used for subjective evaluation. Compared with artificial pansies, visual stimulation with real flowers resulted in a significant decrease in the ratio of low- to high-frequency heart rate variability component, which reflects sympathetic nerve activity. In contrast, high frequency, which reflects parasympathetic nerve activity, showed no significant difference. With regard to the psychological indices, viewing real flowers resulted in “comfortable”, “relaxed”, and “natural” feelings. The findings indicate that visual stimulation with real pansies induced physiological and psychological relaxation effects in high school students.The relaxation effects of gardening have attracted attention; however, very few studies have researched its physiological effects on humans. This study aimed to clarify the physiological and psychological effects on high school students of viewing real and artificial pansies. Forty high school students  at Chiba Prefectural Kashiwanoha Senior High School, Japan, participated in this experiment. The subjects were presented with a visual stimulation of fresh yellow pansies ( Humans emerged millions of years ago ,2 and evronments . Our bodronments . Anotherronments .In cities, gardening enables easy contact with natural environments and has received considerable attention . School We have studied the effects of nature-derived stimulation, such as that of a forest ,34,35,36We focused on the stress state of minors (high school students) and studied the physiological effects of viewing flowers in a gardening activity. Some reports have stated that the numbers of high school students with stress and uneasiness have increased in Japan , the UniTherefore, to clarify the physiological effects of visual stimulation with pansies, which are the most wholesale variety of flower beds, we recruited Japanese high school students and recorded their HRV using fingertip acceleration pulse wave.We conducted this study in Chiba Prefectural Kashiwanoha Senior High School on October 30 and November 1, 2, and 5, 2012. The average temperature, humidity, and intensity of illumination in a classroom were 22.5 °C ± 1.1 °C, 50.9% ± 5.3%, and 1030 lx, respectively. The participants were 19 healthy high school boys (mean age ± standard deviation: 16.2 ± 0.7 years) and 21 healthy high school girls (mean age ± standard deviation: 16.6 ± 0.9 years).All subjects agreed to the study protocol and provided written informed consent. The study was conducted according to the regulations of the Ethics Committee of the Center for Environment, Health, and Field Sciences, Chiba University, Japan.Viola x wittrockiana, “Nature Clear Lemon”). The pansies’ scent was evaluated as “weak” in terms of the sense of strength and “slightly good” in the sensuality evaluation by the modified semantic differential (SD) method. The artificial flowers were yellow pansies made of polyester. Twelve real and 12 artificial flowering plants were set with gardening soil in a polypropylene planter .For visual stimulation, we used fresh yellow pansies  , and theEvaluation by the modified SD method  was perft test compared the differences in the mean physiological data, and a value of p < 0.05 was considered to indicate statistical significance. The Wilcoxon signed-rank test was used to analyze differences in the psychological indices, and values of p < 0.01 were considered to indicate statistical significance. A one-sided test was used in this study because of the hypothesis that humans would be relaxed by viewing real flowers.Statistical analysis was performed using SPSS 20.0 . A paired p < 0.05). The pre-value for real flowers and artificial flowers was 1.29 ± 0.19 and 1.23 ± 0.18, respectively, indicating no significant difference. In contrast, HF (which is considered to reflect parasympathetic nerve activity) and pulse rate did not show a significant difference.Time-dependent LF/HF, which reflects sympathetic nerve activity, changed every minute during visual stimulation with real flowers or control, as shown in p < 0.01), with the subjects feeling more comfortable with real flowers than with artificial flowers. In terms of “relaxed” feeling, the subjects felt “slightly relaxed” by real flowers. The difference between real flowers and artificial flowers was statistically significant (p < 0.01), with the subjects feeling more relaxed with real flowers than with artificial flowers. In terms of “natural” feeling, the subjects felt “slightly natural” to “moderately natural” with real flowers. In this respect, the difference between real flowers and artificial flowers was statistically significant (p < 0.01), with the subjects feeling “more natural” with real flowers than with artificial flowers.A modified SD method was used for subjective evaluation of “comfortable”, “relaxed”, and “natural” feelings . SubjectWe evaluated the physiological effects of visual stimulation with real and artificial pansies according to autonomic nerve activity. Compared with artificial flowers, real flowers significantly decreased sympathetic nerve activity. This result resembles that reported by a previous study on chrysanthemum transplantation for male university students with the Type A behavior pattern . In contWith regard to the psychological effects evaluated by the modified SD method, visual stimulation with real flowers resulted in “comfortable”, “relaxed,” and “natural” feelings. These results correspond with those of previous studies. In addition, few previous studies used the modified SD method on gardening activity, but chrysanthemum transplantation felt “comfortable”, “soothed”, and “natural” .Considerable attention is being paid to the effect of forest environment on humans. In addition to psychological indices, comprehensive evaluation using physiological indices has been performed for forests, indicating that the forest environment has relaxation effects ,34,35,36In conclusion, compared with artificial pansies, visual stimulation with fresh pansies decreased sympathetic nerve activity and the subjects felt comfortable, relaxed, and natural. Visual stimulation with fresh pansies induced physiological and psychological relaxation effects on high school students.This study has some limitations. First, high school students were the subjects in this study; studies with subjects from various ages and environments are required. Second, we used autonomic nerve activity as a physiological measurement. Other indices such as brain activity will be required from a perspective of systemic collaboration. Third, we focused on visual stimulation. However, we believe that the physiological effects of actually touching plants, such as transplantation, are important study themes.We believe that this study provides important findings about the relationship between humans and natural environments.Compared with artificial pansies, visual stimulation with fresh pansies decreased sympathetic nerve activity and the subjects felt comfortable, relaxed, and natural. The findings indicate that visual stimulation with fresh pansies induced physiological and psychological relaxation effects on high school students.


Galβ and GlcNAcβ terminal substructures, without sialic acid branches, were associated with the binding of human-, swine-, and avian-origin viruses; sulfated Neu5Acα2–3 substructures were associated with the binding of human- and swine-origin viruses. Finally, through three-dimensional structure characterization, we revealed that the role of glycan chain shapes is more important than that of torsion angles or of overall structural similarities in virus host tropisms.Influenza A viruses can infect a wide variety of animal species and, occasionally, humans. Infection occurs through the binding formed by viral surface glycoprotein hemagglutinin and certain types of glycan receptors on host cell membranes. Studies have shown that the α2,3-linked sialic acid motif  in avian, equine, and canine species; the α2,6-linked sialic acid motif  in humans; and SA2,3Gal and SA2,6Gal in swine are responsible for the corresponding host tropisms. However, more detailed and refined substructures that determine host tropisms are still not clear. Thus, in this study, we applied association mining on a set of glycan microarray data for 211 influenza viruses from five host groups: humans, swine, canine, migratory waterfowl, and terrestrial birds. The results suggest that besides Neu5Acα2–6Galβ, human-origin viruses could bind glycans with Neu5Acα2–8Neu5Acα2–8Neu5Ac and Neu5Gcα2–6Galβ1–4GlcNAc substructures;  Influenza A viruses infect a wide range of hosts, such as humans, sea mammals, swine, bats, and avian, equine, and canine species123In humans, glycans with α2,6-linked sialic acid  are detected more plentifully in the upper respiratory tract than the lower respiratory tract. SA2,6Gal and SA2,3Gal  are heterogeneously distributed in the human nasopharynx and bronchi. The expression of SA2,3Gal is greater than that of SA2,6Gal in the respiratory tract of young children68910101213By presenting multiple glycans or glycoconjugates printed on a single slide, the glycan microarray technique has offered high-throughput analyses of the glycan-binding profile of influenza viruses15192021232425262722et al.et al.To better understand structural specificities for glycan binding, Cholleti The relationship between host receptors (glycan substructures) and influenza A viruses  can be naturally formulated as a computational problem of data integration plus association rule mining. Therefore, in this study, we firstly applied a PLA regression on individual glycan microarray data entries as normalization and then used association rule analysis on extracted glycan substructure features to identify motifs for influenza virus host tropisms. In addition to SA2,3Gal and SA2,6Gal, results showed that glycan substructures with SA2,8SA, non-sialic acid saccharides , and sulfated SA2,3Gal could contribute to influenza host tropism differently. Additional computational modeling demonstrated that, for trisaccharide substructures, a shape angle formed by mass centers of three residues, instead of linkage torsion angles, may determine the overall glycan chain shapes and thus distinguish glycans with SA2,3Gal from those with SA2,6Gal or SA2,8SA. These findings may imply a more general property caused by glycan terminals than just by sialic acid with different linkages during influenza – glycan binding.A computational script was written to automatically retrieve glycan microarray datasets from CFGIn the CFG database, the datasets were generated by using 11 versions of glycan microarrays, each of which had a different number of glycan entries. For example, version 1 had 200 glycans, whereas version 5.1 had 611 glycans. However, most glycans present in earlier glycan microarray version are present in later versions. To facilitate the data analyses across different datasets, we merged all microarray versions into one with 936 unique glycans  and geneGlycan substructures were defined as described elsewhereT were the data derived from influenza virus–specific glycan microarray entries, so m = 211; the substructure features X derived from glycans on the array by previous PLS-β selection and the labeled features Y with host origin will form I. Given a rule confidence is defined as sup (X) is the support of item set support was defined as the proportion of transactions in the dataset, which contains the item set. Another measurement, lift, is the ratio of the observed support and was defined as Apriori algorithm implemented in RX, contains more items than a previous rule, but still results in the same right hand side, Y. Last, we kept only satisfied rules, which were filtered by leaving only those with terminal saccharides on the substructure features.We formulated the detection of host-specific glycan substructures as an association-mining task  The root-mean-square deviation (RMSD). Given two glycan substructures, each containing the terminal saccharide, we superimposed the corresponding atoms on the six-membered rings of the terminal saccharides. From there, while keeping the terminal saccharides superimposed, the following two values of RMSD were measured: RMSD2 and RMSD3. Using the standard formula of calculating RMSD from two sets of six-membered ring atoms n = 6, RMSD2 was calculated for the two saccharides linked directly to their respective terminal saccharides. If both glycan substructures had a third saccharide, RMSD3 was then calculated for the third pair of saccharides.(2) ϕ and ψ torsion angles. We calculated the ϕ and ψ torsion angles for each linkage between two adjacent residues. Glycosidic torsions were defined by only heavy linkage atoms as n–Cn and ψ = C1–On–Cn–Cn-1(3) To understand the structural determinants for a specific glycan associated with certain influenza A virus, we compared the spatial relationship between six terminal trisaccharide features derived from data mining. These six features were (Neu5Acα2–6)--4GlcNAc PDB, Neu5Ac, Neu5Ac, Neu5Gc, Galβ1–, and Ga. The folN-acetyllactosamine], analogous to Neu5Acα2–6Galβ1–4GlcNAc, and 3SLN [α2–3-sialyl-N-acetyllactosamine], analogous to Neu5Acα2–3Galβ1–4GlcNAc) and highlighted conserved and diverse regions on the receptor-binding pocket . Substructures, (Neu5Acα2–8)–(8Neu5Acα2–8)–8Neu5Ac and –(4GlcNAcβ1–3)–3Gal, were also simulated against bound 6SLN and 3SLN to be docked at the receptor-binding pocket of the human-origin and the avian-origin viral HAs respectively. The HA-glycan docking was conducted by following three steps: 1) an initial structure was obtained by superposing the structures of HA and a glycan analog against a native HA 3D structure with glycan; 2) an energy minimization with 500 steps of conjugation and 500 additional steps of steepest descent was performed by using the AMBER force fieldTo demonstrate structural interactions between influenza viruses and glycan substructures at the molecular level, we used four HA protein crystal structures. These structures were from viruses with different host origins  and were obtained from PDBCertain saccharide residues are enriched at glycan substructures contributing to influenza virus binding. In the integrated dataset of glycan microarrays with 936 unique glycans, the glycans with influenza virus–binding affinities mostly consist of sialic acids (Neu5Ac and Neu5Gc), galactose , N-acetylgalactosamine , glucose (Glc), N-acetylglucosamine (GlcNAc), fucose (Fuc), and mannose (Man). A comparison of data in To understand the specific substructures associated with each influenza A virus, we performed associate rule analyses across 211 influenza virus–specific glycan microarray data. On the basis of their host origins, the 211 influenza A viruses were categorized into human (n = 154), canine (n = 6), swine (n = 21), waterfowl (n = 17), terrestrial , and avian . The association analysis results , which were detected multiple times GlcNAcβ1–3Gal . The GalIn In The objective of this study was to characterize the host-specific glycan substructure responding to influenza A virus infections. Glycan microarray data provide an opportunity to systematically study the factors that determine virus–glycan binding. However, such analyses have several limitations. The first limitation is that glycan microarray data are not quantitative because values from batch to batch are highly variable. The variability is caused by spot intensities dependent on immobilization efficiency and results in the misleading use of fluorescence intensities to quantify binding affinitiesIn this study, we expected association analysis to detect significantly nonrandom, but possibly infrequent, substructure features contributing to influenza A virus binding. To ensure better coverage of all potential substructures, hierarchical clusters  of substructure profiles were characterized and integrated into data mining, and our analyses focused on the terminal structures. To minimize the potential noise across different datasets due to variations in glycan microarray versions and experiments, we integrated the significant substructures extracted from each individual dataset by PLS regression. To identify the host-associated glycan substructures, we categorized 211 data entries into five categories  and then formulated glycan substructure problems as a typical association mining problem, where we treated glycan substructure features as products, virus host types as the only label of customs, and the glycan–virus binding signals in the dataset as transactions. Comparing to other methods, either statistical or mining strategies, our formulation of the problem benefits the novel observations in this study in two following ways. First, after the PLS regressions on individual glycan microarray entries, the binding transection definition was used to integrate all of them for a cross-array analysis, by which we overcame the challenges from the varying numbers of glycans on different version of arrays. Second, the association mining strategy avoided particular hypothesis before analyses and were able to detect rare but potentially significant rules.We have not been able to use this method to identify the specific substructures for glycan bindings when multiple terminal glycans are present. For example, glycans with different terminals  were observed frequently, but they may both be important players during influenza virus binding because they could bind influenza viruses simultaneously. To avoid this problem, in this study, we ignored the associated substructures with branch linkages, because they may be extracted from a glycan with other terminals and by themselves may not contribute to virus binding. To avoid such false-positives, we included in the results only terminal substructures without branches. Moreover, four substructure definitions  could lead to overlapped glycan features that were associated with the same virus host. For example, in Our results show that (1) human-origin influenza A viruses could bind glycans with Neu5Acα2–8Neu5Acα2–8Neu5Ac and Neu5Gcα2–6Galβ1–4GlcNAc substructures; (2) Galβ and GlcNAcβ terminal substructures, without any existing sialic acid terminals, are associated with the glycan binding of human-, swine-, and avian-origin influenza A viruses; (3) Sulfated Neu5Acα2–3 substructures are believed to be associated with the glycan binding of human- and swine-origin influenza A viruses. These observations, on one hand, are consistent with previously reported results about various types of host-origin influenza A virusesThe potential glycan receptors with α2,8-linked sialic acid were reported to be associated with influenza virus binding52Our three-dimensional structure analysis of representative host-specific substructures showed that for trisaccharides, the shape angle formed by mass centers of three residues could be the key feature that distinguishes α2,6-linked, α2,8-linked and α2,3-linked glycans and their virus host tropisms . Althoug29How to cite this article: Zhao, N. et al. Association analyses of large-scale glycan microarray data reveal novel host-specific substructures in influenza A virus binding glycans. Sci. Rep.5, 15778; doi: 10.1038/srep15778 (2015).


Major histocompatibility complex class I (MHCI) molecules were recently identified as novel regulators of synaptic plasticity. These molecules are expressed in various brain areas, especially in regions undergoing activity-dependent synaptic plasticity, but their role in the nucleus accumbens (NAc) is unknown. In this study, we investigated the effects of genetic disruption of MHCI function, through deletion of β2-microblobulin, which causes lack of cell surface expression of MHCI. First, we confirmed that MHCI molecules are expressed in the NAc core in wild-type mice. Second, we performed electrophysiological recordings with NAc core slices from wild-type and β2-microglobulin knock-out mice lacking cell surface expression of MHCI. We found that low frequency stimulation induced long-term depression in wild-type but not knock-out mice, whereas high frequency stimulation induced long-term potentiation in both genotypes, with a larger magnitude in knock-out mice. Furthermore, we demonstrated that knock-out mice showed more persistent behavioral sensitization to cocaine, which is a NAc-related behavior. Using this model, we analyzed the density of total AMPA receptors and their subunits GluR1 and GluR2 in the NAc core, by SDS-digested freeze-fracture replica labeling. After repeated cocaine exposure, the density of GluR1 was increased, but there was no change in total AMPA receptors and GluR2 levels in wild-type mice. In contrast, following repeated cocaine exposure, increased densities of total AMPA receptors, GluR1 and GluR2 were observed in knock-out mice. These results indicate that functional deficiency of MHCI enhances synaptic potentiation, induced by electrical and pharmacological stimulation. Major histocompatibility complex class I (MHCI) molecules consist of a heavy chain (α1–α3) and a light chain, β2-microglobulin (β2m). MHCI proteins are expressed on the surface of most nucleated cells and are best known for their roles in cellular-mediated immunity. In the central nervous system, MHCI proteins are widely expressed and their expression level is particularly high in regions undergoing activity-dependent synaptic plasticity −/−), the activity-dependent remodeling of retinal ganglion cell axons projecting to the developing lateral geniculate nucleus is impaired, and as a result, inappropriate projections that are normally eliminated during development persist By knocking-out both β2m and Tap1, a protein required for loading of peptides on MHCI, most cell-surface MHCI expression is eliminated The nucleus accumbens (NAc) is involved in reward processing and reinforcement learning, and synaptic plasticity in this brain region has been extensively studied −/−) mice in which a majority of cell-surface MHCI expression is eliminated −/− mice treated repeatedly with cocaine.First, we examined whether MHCI is expressed in the NAc of wild-type (WT) mice, by immunostaining methods. Second, to examine the involvement of MHCI in synaptic plasticity induced by electrical stimulation, we prepared NAc slices from β2m knock-out (β2m−/− mice (maintained on a C57BL/6J background) were purchased from a commercial supplier . WT and β2m−/− mice were generated by heterozygous mutants, and we used WT littermates or WT C57Bl/6J mice  as a control group for the β2m−/− mice. All the mice were housed in specific pathogen-free (SPF) facility and behavioral analysis was also done there. In-house monitoring was performed every 3 months using a Monilisa IVA kit  that detects four major organisms, Sendai virus, mouse hepatitis virus, mycoplasma and Tyzzer’s organism β2mAll procedures were approved by the Hamamatsu University School of Medicine Animal Care and Use Committee, and carried out in accordance with National Institute of Health general guidelines for the Care and Use of Laboratory animals (NIH Publications No. 86-23). All efforts were made to minimize animal suffering and the number of animals used.Mice were deeply anesthetized with diethyl ether and gently perfused transcardially with PBS followed by 4% paraformaldehyde. Then, whole brains were dissected and kept in the same solution at 4°C overnight. After cryoprotection with 30% sucrose in PBS solution, coronal slices at 60 µm thickness were prepared by a cryostat . MHCI was stained with mouse monoclonal antibody against MHCI  in PBS with 3% BSA at 4°C overnight. For the negative control, the primary antibody was omitted. Then rabbit polyclonal antibody  was incubated for the staining of Neurogranin in PBS with 3% BSA and 0.5% Triton x-100 at room temperature for 1 hr. Those slices were labeled with anti-Rabbit Alexa fluor 488  for staining of Neurogranin and anti-Mouse Alexa fluor 546  for staining of MHCΙ. After being stained, the slices were embedded in ProLong Gold antifade reagents  for observations. Images were taken by a confocal microscope .−/− mice were decapitated and their brains were quickly removed and submerged in a cutting solution , bubbled by 95% oxygen and 5% carbon dioxide over 20 minutes. Parasagittal slices (350 µm thickness) were prepared by a vibratome slicer  and trimmed to include the NAc and prefrontal cortex. Slices were transferred to a recovery chamber filled with artificial cerebrospinal fluid (ACSF)  that was continuously bubbled by 95% oxygen and 5% carbon dioxide, and recovered over 1 h at room temperature (25–29°C). Then, the slices were transferred to the recording dish in a multi electrode array system  and constantly perfused by bubbled ACSF containing 50 µM picrotoxin at 37°C. The recording dish in the multi electrode array system had 60 titanium nitride electrodes, a 30 µm diameter at its base, and was arranged in 8×8 matrix with a 200 µm inter-electrode interval. Using these electrodes, we stimulated the tissue electrically and recorded extracellular potentials WT and β2m−/− mice were given intraperitoneal injections of either saline (0.9% NaCl) or cocaine (20 mg/kg) at a volume of 10 ml/kg. Immediately after each injection, locomotor activity was measured for 60 min in eight identical activity monitors measuring 30×30 cm with pyroelectric infrared sensors attached on the lid . It detects temperature changes caused by locomotion at time resolution of 200 ms and counts them up. After 3 days of saline injections, mice were divided into four groups that received seven daily injections of either saline or cocaine. The groups comprised WT mice treated with saline  or cocaine (WT-Coc) and β2m−/− mice treated with saline  or cocaine (β2m−/−-Coc). Following 14 days of withdrawal, all groups received a challenge injection of cocaine (20 mg/kg) and locomotor activity was assessed.WT and β2mSDS-FRL can be used to observe the two dimensional structure of cell membranes and the distribution of membrane proteins We used three different primary antibodies and performed single labeling on each separated replica. These antibodies were the pan-AMPA antibody raised in rabbit, which binds to GluR1, GluR2, GluR3 and GluR4 receptors  with repeated measures. Locomotor responses to cocaine challenge in the four groups were compared using two-way ANOVA. Post-hoc comparisons were performed using the Bonferroni test. Statistical calculations were made using the statistical software SPSS (version 17.0) for Windows. The level of significance was set at p<0.05.Recent studies revealed that MHCI molecules are expressed in numerous brain areas Synaptic plasticity in the NAc is dependent on the stimulation frequency, with high frequencies producing LTP −/− accumbal slices. HFS at 100 Hz resulted in a significant increase in the slope of the fEPSP curve at 45–50 min after tetanus (−/− mice was significantly higher than in WT mice (t(16) = 2.548, p<0.05). Additionally, there was no significant change in the fEPSP slope upon 1 Hz stimulation  = 0.349, p = 0.731), 50 msec  = 0.355, p = 0.727) and 100 msec  = 0.164, p = 0.872). This suggests that enhanced LTP we observed in β2m−/− mice is unlikely to result from altered probability of glutamate release.Next, we tested whether β2m contributes to LTP and/or LTD at accumbal synapses. As in WT mice, we examined the effects of three different stimulation frequencies on synaptic plasticity in β2mmulation . There w−/− mice. After 3 days of saline injections to acclimatize animals to the activity test box, acute locomotor responses to the drug increased dramatically across multiple days of testing in both WT  and β2m−/− mice  (−/− mice showed a much larger increase in locomotor sensitization to cocaine than WT mice (p<0.05). To test whether this method produced long-lasting sensitization, we administered a challenge dose of cocaine (20 mg/kg) to both saline- and cocaine-treated animals of WT and β2m−/− groups followed by a 14-day withdrawal. Mice pretreated with chronic cocaine exhibited a much greater locomotor response to the drug than did saline-treated animals in both WT  and β2m−/− mice  . These findings indicate that behavioral sensitization after 7 days of exposure to cocaine is enhanced in β2m−/− mice compared with WT mice, and this sensitization lasts for at least 2 weeks.To induce behavioral sensitization, we gave repeated cocaine injections to WT and β2m, n = 6) . Furtherp<0.001) . Again, −/− mice, and confirmed that there was no difference in the basal expression of AMPA receptors between these genotypes  = 0.82, p = 0.43; GluR1: t(10) = 0.44, p = 0.67; GluR2: t(10) = 0.69, p = 0.51; Student’s t-test). Next, we analyzed the effects of repeated cocaine exposure on AMPA receptor expression in the NAc of WT mice  = 2.28, p<0.05; GluR1: t(10) = 2.71, p<0.05; GluR2: t(10) = 2.93, p<0.05). This result indicates that β2m levels influence cocaine-induced AMPA receptor expression during synaptic adaptation.Chronic cocaine administration elicits long-lasting alteration of synaptic excitatory transmission in the NAc, likely contributing to behavioral sensitization enotypes . The den WT mice . The denvaluated . The den−/− mice deficient of cell-surface expression of MHCI. We performed electrophysiological recordings from accumbal slices and found that high frequency stimulation induced LTP and low frequency stimulation induced LTD in WT mice. In contrast, in β2m−/− mice, LTP was enhanced and LTD was not observed. Next, we found that β2m−/− mice showed significantly higher behavioral sensitization than WT mice in response to repeated cocaine exposure. Finally, when we analyzed the densities of AMPA receptors and their subunits on excitatory synapses of NAc core, we found that repeated exposure to cocaine significantly increased only GluR1 expression in WT mice, whereas all pan-AMPA, GluR1 and GluR2 receptors showed significantly higher expression in β2m−/− mice. Taken together, these findings demonstrate that β2m−/− mice show clearly augmented synaptic potentiation induced by electrical and pharmacological stimulation in comparison with WT mice.In this study, we first found by immunohistochemical staining that MHCI molecules are expressed in NAc-core neurons in wild-type mice. This indicates that MHCI may have a role in modulation of synaptic plasticity in the NAc. To investigate this possibility, we analyzed β2mMice deficient in MHCI function show enhanced LTP and abolished LTD in the hippocampus −/− mice show greater behavioral sensitization than WT mice following repeated cocaine injections. One important brain area underlying behavioral sensitization is the NAc, and glutamatergic efferents from prefrontal cortex to NAc core are critical for this form of sensitization . In our SDS-FRL experiments, the density of the AMPA receptors, which is related to the strength of synaptic connection, was increased in WT mice repeatedly treated with cocaine, and this result is consistent with the above-mentioned report . In WT mice, we found that repeated cocaine exposure significantly increased the density of GluR1 on synapses of neurons in the NAc. A previous study by Boudreau and Wolf (2005) reported that repeated cocaine exposure increased cell-surface expression of not only GluR1 but also GluR2/3 in the NAc following 10–21 days of withdrawal. Although the protein cross-linking assay used in this report can distinguish between cell surface and intracellular AMPA receptor, the method cannot discriminate extra-synaptic receptors from synaptic receptors that are directly involved in synaptic transmission. We directly analyzed the density of AMPA receptor in the synapse and found that only GluR1 expression was increased in synapses. Interestingly, in β2m−/− mice repeatedly treated with cocaine, the densities of accumbal pan-AMPA, GluR1 and GluR2 on excitatory synapses were all higher than in saline-treated animals. In a previous study using primary culture of hippocampal neurons, immunostaining revealed significantly increased surface GluR1 in the neurons of MHCI-deficient mice, but not WT mice, and there was no significant change in GluR2 in either genotype, shortly following NMDA treatment As mentioned above, for the first time, we report that β2m−/− mice purely resulted from the absence of β2m in the nucleus accumbens. Recently, Lee et al. bDb knock-out (H2-KbDb−/−) mice. By crossing these mice with NSEDb+ mice in which H2-Db expression is regulated under the neuron-specific enolase (NSE) promoter, Lee et al. rescued their H2-Db expression exclusively to neurons while H2-KbDb−/− remains in the rest of the body. As a result, they found that neuronal H2-Db expression was enough for rescuing normal synaptic plasticity. This would be one supportive example that impaired immune system in the body has no effect on synaptic plasticity in the brain.In this study, we did not evaluate any effects of an impaired immune system itself on the parameters measured here. Therefore we should be careful in our interpretation that the changes observed in β2mIn conclusion, we found that MHCI proteins are expressed in the NAc of WT mice. We also found that LTP is enhanced whereas LTD is abolished in functional MHCI-deficient mice. We finally observed that baseline of AMPA receptor expression is normal, but there is an increase in AMPA receptor density including both GluR1 and GluR2 following repeated cocaine administration in functional MHCI-deficient mice, whereas only GluR1 is increased in WT mice. Thus, the present study indicates that MHCI proteins may play a role in lowering the strength of synaptic connections in the NAc. Furthermore, an interesting study has recently reported the persistence of abolished LTD in rats that displayed typical addiction-like behavior


There is growing evidence that mild traumatic brain injury (concussion) can affect locomotor characteristics for prolonged periods of time even when physical signs and symptoms are absent. While most locomotor deficits post-concussion have involved straight walking, turning gait has received little attention despite its pervasiveness in everyday locomotion and athletic competition.n = 4) and healthy matched control athletes (n = 4) to examine potential deficits during single and dual-task turning gait over the initial 6 weeks post-injury, with a one-year follow-up. Turning path kinematics , stride kinematics , and inclination angles were calculated from motion capture of participants walking around an obstacle.This study longitudinally examined kinematic characteristics during preplanned turning in a small sample of recently concussed athletes  contains supplementary material, which is available to authorized users. Sport-related mild traumatic brain injuries, commonly called concussions, affect 1.6–3.8 million people in the United States annually , making The use of a dual-task (DT) paradigm has been particularly successful at identifying residual deficits post-concussion . During Some studies have reported greater cognitive DTC (cDTC) during obstacle circumvention  and hockTurning gait is a common  complex Eight NCAA Division I varsity athletes  7 (0), 16 (2), 23 (2), 29 (1), 37 (2), and 45 (3) days following the concussed participant’s concussion. To control for potential musculoskeletal afflictions from recent games or practices that might have influenced gait, each control participant was also tested up to six times and, when possible, on the same days as their competition. Three concussed participants and two control participants also participated in a one-year follow-up testing session occurring at a mean (SD) of 363 (42) days post-concussion. Because of scheduling conflicts, graduations, and transfers, not all participants completed all sessions  and consisting of several turns of ~90°  were stationed around one ~90° turn on the course  was estimated using the three-dimensional mean location of the xiphoid process and T9 markers [UB to the pylon (dmin). The length of the horizontal trajectory, Lpath, was computed starting when the athlete proceeded below the pylon in the y direction, and ending when the athlete advanced above the pylon in the y direction  occurred closest to the point of maximum COMion Fig. . Stride ion Fig. .Fig. 3IlUB (vcom) was calculated over the turning stride. The mean curvature, k, of the COMUB was calculated as the mean of the previously described instantaneous curvature over the turning stride to compare the relationship between velocity and curvature.The strategy used within each turn was characterized using the apex limb. A “step turn” was identified if the apex limb was contralateral to the turn direction , whereas a “spin turn” was identified if the apex limb was ipsilateral to the turn direction  . The meaR = 1/k) and velocity has previously been defined for turning gait in both outlined [R is the radius of curvature, α is a constant coefficient, and A is the piecewise velocity gain factor that can vary with shape [vcom, and corresponding radii of curvature, 1/k, were compared for each group using the α and A coefficients determined from linear statistical models.A power law relationship between the radius of curvature  at an angle of θ , following from Eq. p < 0.10 were retained in the final models.The inclination angles If significant group*day interaction effects were present in any of the final models, a linear recovery timeline was constructed using the predicted values from the mixed model. The time from injury to full recovery was then estimated using the time when the model estimates of the concussed and control groups crossed. For all models, assumptions were validated using residuals. All statistical analyses were performed in SAS 9.4  using a two-tailed 0.05 significance level.vcom, k, Lpath, dmin is given in Table k was found; the concussed group increased their curvature over time, while controls maintained a constant curvature over time. Path length (Lpath) and COM clearance (dmin) did not differ between groups when adjusting for turning velocity. The group*task*day interaction was significant for vcom, as the concussed group slowed more than the control group during the DT; the associated lmDTC increased over time (greater difference between ST and DT over time) in the concussed group. Using the mixed model results, the estimated linear recovery time until concussed athletes returned to healthy curvature levels was 398 days.Concussed athletes utilized spin turns at similar frequencies (45 %) to controls 46 %) during both ST and DT turning. A summary of statistical results for  % duringLongitudinal results for SWidth, SLength, and STime, stratified by strategy, are provided in Figs. vcom and log 1/k yielded estimated mean (SE) of −0.24 (0.06) for parameter A in concussed athletes within 6 weeks of their concussion, and −0.08 (0.04) in controls , indicating that A significantly decreased in concussed athletes during the first 6 weeks. No significant interaction was found, indicating that α was similar between concussed and controls, whose respective values were 0.24 (0.03) and 0.21 (0.02).Linear fits between log ols Fig. . The dif−1(vcom2k) term , a group*day*task interaction was found, indicating an increase in the lmDTC difference between concussed and control groups as time increased. The estimated linear recovery time for concussed athletes was 115 and 123 days during step turns for θ2 and θ3, respectively, 239 days for θ1 during spin turns, and 222 days for θ2 during spin turns. As the DTC differences increased for θ3 over time, no recovery date could be estimated.Longitudinal results for each angle are provided in Figs. rm Table , signifiθ1, θ2, and θ3) and curvature – showed different patterns over time between groups when adjusted for turning speed, which suggests that turning gait may recover slower than other symptoms. Overall, these differences between groups over time describe the effects of concussion on turning gait in three domains: path trajectory (k), stride characteristics , and body orientation .This study prospectively examined single- and dual-task turning kinematics in recently concussed and healthy control athletes. It provides the first preliminary evidence of kinematic differences during ST and DT preplanned turning gait in a small sample of recently concussed athletes. Despite all athletes being medically cleared for competition, concussed athletes showed greater lmDTCs in SWidth, and STime compared to healthy controls. Additionally, several outcomes – including SWidth, STime, ML inclination angles (A) in concussed athletes relative to healthy controls. While both groups exhibited similar power law relationships between vcom and 1/k, the concussed group walked with slower velocities across the range of curvatures. Similar modulations of A have been reported as individuals transition to different outlined trajectories [α values between the concussed and control groups, suggesting that this long term coupling remains present post-concussion. However, the lower A values post-concussion indicate that the relationship has shifted to a slower speed, consistent with previously described decreases in gait speed post-concussion [α between groups, though, it is possible that some of the navigational deficits reported in concussed individuals [One key finding of this preliminary study is a decreased velocity gain factor  or minimum clearance (dmin). The present results, as evidenced by low R2 values from the velocity-curvature relationship , though overall conclusions are limited due to the small sample size. However, this appears to contradict Fait et al. [We did not find a significant difference between groups in minimum clearance . Based on a simplistic model of a continuous rolling rigid body, the inclination angle should be directly proportional to tan−1(vcom2k). Given that the concussed athletes generally walked slower across similar curvatures, lower magnitudes of θ in concussed athletes were expected. However, this group difference was present, though it resolved over time, even when adjusting the model for tan−1(vcom2k). For a given velocity and curvature, concussed athletes exhibited less inclination towards the inside of the turn . One may interpret this decreased inclination as another cautious adaptation of the concussed group in order to preserve stability [Recently concussed athletes increased their inclination towards the turn over time, nearing the inclination angles used by controls, after adjusting for the tantability , 21, 49 tability , 28, 49.θ introduces a new coronal plane moment if velocity, curvature, and mass are held constant .The small sample size of this preliminary study is a primary limitation and prompts caution when generalizing these results to an entire population. Nevertheless, significantly different lmDTC and changes in kinematic variables over time were found between concussed and healthy controls. Additionally, despite the small sample, this presents the first investigation into turning kinematics post-concussion and provides the only longitudinal data spanning up to one year. Variations in a few data points, such as the one-year follow-up data among controls, likely had particularly strong influence on the recovery timeline, potentially leading to over- or underestimated recovery in some instances  that could contribute to the higher musculoskeletal injury rates post-concussion , 54. TurA, power law gain factor between velocity and radius of curvature; BOS, base of support; C, concussed athletes; cDTC, cognitive dual-task cost; COM, whole-body center of mass; COMUB, center of mass of the upper body; DT, dual-task; DTC, dual-task cost; H, healthy matched controls; lmDTC, locomotor dual-task cost; ML, mediolateral; mTBI, mild traumatic brain injury; NCAA, National Collegiate Athletic Association; RTP, return to play; SD, standard deviation; SE, standard error; ST, single-task; α, power law exponent between velocity and radius of curvature; μ, coefficient of friction